A morphogen gradient patterns the dorsal surface of the Drosophila embryo. Decapentaplegic (Dpp) and Screw (Scw), two BMP-type ligands, are known to be involved, with Dpp thought to play the primary role. Shimmi et al. show that morphogen activity is actually composed of two separate components, Dpp homodimers and Dpp/Scw heterodimers. These two activities are sorted along the embryonic dorsoventral axis by differential binding to two transporter proteins, Tsg and Sog. Using a computational approach, the authors show that heterodimers can buffer biological systems against changes in gene dose, thereby providing a selective advantage for using heterodimers as morphogens.
The difference between radically different destinies often reflects disarmingly small variations in timing or circumstance. If the motorcade takes a different route through Dallas, if Newton sits under a different tree, or if Ilsa picks another gin joint to walk into, the story is radically altered. In biology, perhaps the most profound example of the effects of small differences is the development of an adult organism from a single fertilized egg, a journey from inner cell mass to fully differentiated structure. The challenge of developmental biology lies in defining the influences and factors that contribute to this final destination.
Synthetic mRNAs can be injected to achieve transient gene expression even for 'non-model' organisms in which genetic approaches are not feasible. Here, we have used this technique to express proteins that can serve as lineage tracers or reporters of cellular events in embryos of the glossiphoniid leech Helobdella robusta (phylum Annelida). As representatives of the proposed super-phylum Lophotrochozoa, glossiphoniid leeches are of interest for developmental and evolutionary comparisons. Their embryos are suitable for microinjection, but no genetic approaches are currently available. We have injected segmentation stem cells (teloblasts) with mRNAs encoding nuclear localized green fluorescent protein (nGFP) and its spectral variants, and have used tandem injections of nGFP mRNA followed by antisense morpholino oligomer (AS MO), to label single blast cell clones. These techniques permit high resolution cell lineage tracing in living embryos.
In order to define the molecular steps involved in learning-related synaptic growth, Udo et al. demonstrate that the signaling pathway of ApCdc42, amember of the Rho GTPases, is required for the long-term changes in synaptic strength and structure induced by 5-HT at the sensory-motor neuron connection in Aplysia. 5-HT-induced activation of ApCdc42 in sensory neurons depends on the PI3 kinase and PLC pathways and recruits N-WASP and PAK to regulate the presynaptic actin network leading to the outgrowth of filopodia, which are precursors for the formation of new sensory neuron synapses associated with long-term facilitation.
MicroRNAs (miRNAs) are small RNAs that regulate gene expression post-transcriptionally. To block all miRNA formation in zebrafish, we generated maternal-zygotic dicer (MZdicer) mutants that disrupt the Dicer RNaseIII and dsRNA-binding domains. Mutant embryos do not process precursor-miRNAs into mature miRNAs, but injection of pre-processed miRNAs restores gene silencing, indicating that the disrupted domains are dispensable for later steps in silencing. MZdicer mutants undergo axis formation and differentiate multiple cell types but display abnormal morphogenesis during gastrulation, brain formation, somitogenesis and heart development. Injection of miR-430 miRNAs rescues the brain defects in MZdicer mutants, revealing essential roles for miRNAs during morphogenesis.
Decapentaplegic (Dpp) is a signaling molecule that controls growth and patterning of the developing Drosophila wing. Mutant cells lacking Dpp signal transduction have been shown to activate c-Jun amino-terminal kinase (JNK)–dependent apoptosis and to be lost from the wing disc epithelium. These observations have led to the hypothesis that Dpp promotes cell survival by preventing apoptosis. Here, we show that in the absence of JNK-dependent apoptosis, mutant cells lacking Dpp signal transduction can survive; however, they are still lost from the wing disc epithelium. This loss correlates with extensive cytoskeletal changes followed by basal epithelial extrusion. We propose that Dpp promotes cell survival within disc epithelia by affecting cytoskeletal organization.
During animal development, epithelial cell fates are specified according to spatial position by extracellular signaling pathways. Among these, the transforming growth factor ß/bone morphogenetic protein (TGF-ß/BMP) pathways are evolutionarily conserved and play crucial roles in the development and homeostasis of a wide range of multicellular tissues. Here we show that in the developing Drosophila wing imaginal epithelium, cell clones deprived of the BMP-like ligand Decapentaplegic (DPP) do not die as previously thought but rather extrude from the cell layer as viable cysts exhibiting marked abnormalities in cell shape and cytoskeletal organization. We propose that in addition to assigning cell fates, a crucial developmental function of DPP/BMP signaling is the position-specific control of epithelial architecture.
The mammalian intestine harbors a beneficial microbiota numbering approximately 1012 organisms per gram of colonic content. The host tolerates this tremendous bacterial load while maintaining the ability to efficiently respond to pathogenic organisms. In this study, we show that the Bacteroides use a mammalian-like pathway to decorate numerous surface capsular polysaccharides and glycoproteins with L-fucose, an abundant surface molecule of intestinal epithelial cells, resulting in the coordinated expression of this surface molecule by host and symbiont. A Bacteroides mutant deficient in the ability to cover its surface with L-fucose is defective in colonizing the mammalian intestine under competitive conditions.
Type VII collagen defects cause recessive dystrophic epidermolysis bullosa (RDEB), a blistering skin disorder often accompanied by epidermal cancers. To study the role of collagen VII in these cancers, we examined Ras-driven tumorigenesis in RDEB keratinocytes. Cells devoid of collagen VII did not form tumors in mice, whereas those retaining a specific collagen VII fragment (the amino-terminal noncollagenous domain NC1) were tumorigenic. Forced NC1 expression restored tumorigenicity to collagen VII–null epidermis in a non–cell-autonomous fashion. Fibronectin-like sequences within NC1 (FNC1) promoted tumor cell invasion in a laminin 5–dependent manner and were required for tumorigenesis. Tumor-stroma interactions mediated by collagen VII thus promote neoplasia, and retention of NC1 sequences in a subset of RDEB patients may contribute to their increased susceptibility to squamous cell carcinoma.
We show that the specific subcellular distribution of H- and Nras guanosine triphosphate–binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization.
Sex differences in brain and behavior are ubiquitous in sexually reproducing species. One cause of sexual dimorphisms is developmental differences in circulating concentrations of gonadal steroids. Neonatal testes produce androgens; thus, males are exposed to both testosterone and estradiol, whereas females are not exposed to high concentrations of either hormone until puberty. Classically, the development of neural sex differences is initiated by estradiol, which activates two processes in male neonates; masculinization, the development of male-type behaviors, and defeminization, the loss of the ability to display female-type behaviors. Here, we test the hypothesis that defeminization is regulated by estrogen receptor (ER). Adult male ER knockout and WT mice were gonadectomized, treated with female priming hormones, and tested for receptive behavior. Indicative of incomplete defeminization, male ER knockout mice showed significantly higher levels of female receptivity as compared with WT littermates. Testes-intact males did not differ in any aspects of their male sexual behavior, regardless of genotype. In olfactory preference tests, males of both genotypes showed equivalent preferences for female-soiled bedding. Based on these results, we hypothesize that ER is involved in defeminization of brain and behavior. This aspect of ER function may lead to developments in our understanding of neural-based sexually dimorphic human behaviors.
Homozygous deletions of recessive cancer genes and fragile sites are known to occur in human cancers. We identified 281 homozygous deletions in 636 cancer cell lines. Of these deletions, 86 were homozygous deletions of known recessive cancer genes, 17 were of sequenced common fragile sites, and 178 were in genomic regions that do not overlap known recessive oncogenes or fragile sites ("unexplained" homozygous deletions). Some cancer cell lines have multiple homozygous deletions whereas others have none, suggesting intrinsic variation in the tendency to develop this type of genetic abnormality (P < 0.001). The 178 unexplained homozygous deletions clustered into 131 genomic regions, 27 of which exhibit homozygous deletions in more than one cancer cell line. This degree of clustering indicates that the genomic positions of the unexplained homozygous deletions are not randomly determined (P < 0.001). Many homozygous deletions, including those that are in multiple clusters, do not overlap known genes and appear to be in intergenic DNA. Therefore, to elucidate further the pathogenesis of homozygous deletions in cancer, we investigated the genome landscape within unexplained homozygous deletions. The gene count within homozygous deletions is low compared with the rest of the genome. There are also fewer short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs), and low-copy-number repeats (LCRs). However, DNA within homozygous deletions has higher flexibility. These features may signal the presence of currently unrecognized zones of susceptibility to DNA rearrangement. They may also reflect a tendency to reduce the adverse effects of homozygous deletions by minimizing the number of genes removed.
In human breast cancer, overexpression of the protooncogene MET is strongly associated with poor prognosis and high risk of metastasis. It stands out as a reliable prognostic indicator of survival and defines a set of tumors exclusive of those that express HER2 or hormone receptors. Studies have shown that overexpression of mutant forms of MET cause cancer in mice. However, MET mutations have not been found in human breast cancer, and the consequences of overexpression of normal MET are unknown. To investigate the role of MET and other putative oncogenes in breast cancer, we developed an experimental system that involves retroviral delivery of genes into primary mammary epithelial cells, followed by transplantation of the transduced cells into mammary fat pads. Using this approach, we found that overexpression of wild-type MET leads to the development of nonprogressive neoplasms. The lesions progressed to mammary adenocarcinoma when a second protooncogene, MYC, was overexpressed, indicating that MET and MYC cooperate in mammary tumorigenesis. Both the nonprogressive neoplasms and adenocarcinomas display characteristics consistent with transformation and expansion of mammary progenitor cells. The approach described here should provide a useful model with which to efficiently test effects of various genes on tumor development in the breast.
Faced with the problem of controlling a physical system, an engineer will identify a model for the system, and then use this model to design a process for automatically actuating some of the system's input signals in order that the behavior of the system follows a desired profile. The most common control processes use feedback. Consider the example of designing an autopilot function that controls an aircraft to fly at constant altitude (see Fig. 1). A mathematical model of the aircraft dynamics (the plant) is designed, which describes how the aircraft's altitude, pitch, and roll angles (the outputs) change when the elevators, ailerons, and throttle (the inputs) are manipulated. By using sensors to measure the outputs, the engineer uses the model to calculate how these measurements should be employed to automatically adjust the inputs, so that if the aircraft deviates off course, it is guided, quickly and smoothly, back to the desired altitude. This function, which maps the measurements to the input adjustment through the feedback path, is known as the controller. The signal that carries the desired profile is fed into the system in the feedforward path. The entire system composed of the functional parts plant, feedback, feedforward, and controller is referred to as the closed loop system. The hallmark of a good feedback control design is a resulting closed loop system that is stable and robust to modeling errors and parameter variation in the plant and achieves a desired output value quickly without unduly large actuation signals at the plant input. Some insightful recent papers advocate a similar modular decomposition of biological systems according to the well defined functional parts used in engineering (1–5) and, specifically, engineering control theory (6–10). Yi et al. (6) reported that adaptation
LADII (leukocyte adhesion deficiency type II)/CDGIIc (congenital disorder of glycosylation type IIc) is a rare autosomal recessive disease characterized by leukocyte adhesion deficiency as well as severe neurological and developmental abnormalities. It is caused by mutations in the Golgi GDP-fucose transporter, resulting in a reduction of fucosylated antigens on the cell surface. A recent study using fibroblasts from LADII/CDGIIc patients suggested that although terminal fucosylation of N-glycans is reduced severely, protein O-fucosylation is generally unaffected (Sturla, L., Rampal, R., Haltiwanger, R. S., Fruscione, F., Etzioni, A., and Tonetti, M. (2003) J. Biol. Chem. 278, 26727–26733). A potential explanation for this phenomenon is that enzymes adding O-fucose to proteins localize to cell organelles other than the Golgi apparatus. In this study, we investigated the subcellular localization of protein O-fucosyltransferase 1 (O-FucT-1), which is responsible for adding O-fucose to epidermal growth factor-like repeats. Our analysis reveals that, unlike all other known fucosyltransferases, O-FucT-1 is a soluble protein that localizes to the endoplasmic reticulum (ER). In addition, it appears that O-FucT-1 is retained in the ER by a KDEL-like sequence at its C terminus. Our results also suggest that enzymatic addition of O-fucose to proteins occurs in the ER, suggesting that a novel, ER-localized GDP-fucose transporter may exist. The fact that O-FucT-1 recognizes properly folded epidermal growth factor-like repeats, together with this unique localization, suggests that it may play a role in quality control.
George Wells Beadle (1903–1989) grew up on a 40-acre farm near the small town of Wahoo, Nebraska. Beadle might have become a farmer himself had it not been for the influence of his high school science teacher, Bess MacDonald, who persuaded him to enroll at the University of Nebraska College of Agriculture. After earning a B. S. in 1926, Beadle remained at Nebraska to obtain an M. A. with Franklin D. Keim. Through his work with Keim, Beadle became interested in fundamental genetics and was persuaded to apply to graduate school at Cornell University rather than return to the farm.
Listeria monocytogenes causes a variety of diseases, the most severe being meningoencephalitis. The bacterium is phagocytosed into cells and multiplies after escaping from the phagolysosomes. It then moves to the plasma membrane and forms a filopod that is ingested by an adjacent cell, and the cycle is repeated. Actin assembly is essential for the movement of the bacteria through the cytoplasm and formation of the filopod. Generally, in motile non-muscle cells, an accumulation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), converted from phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), is associated with the formation of new actin filaments. It is known that ActA, the bacterial surface protein that induces actin assembly, can bind to both PtdIns(4,5)P2 and PtdIns(3,4,5)P3, but the physiological significance of the inositide binding site has not been examined.
The innate immune response relies on CD14 to recognize several microbial and cellular products including lipopolysaccharide (LPS), a glycolipid found on the outer membrane of Gram-negative bacteria. Once CD14 binds to LPS or another molecule, it presents the ligand to toll-like receptors, which initiate a strong pro-inflammatory response that stimulates host defenses. Jung-In Kim and colleagues have now solved the crystal structure of CD14, providing crucial insights into how the receptor binds to its ligands.
Some pathogenesis-related genes are expressed in fungi only when the pathogen is in the host, but the host signals that trigger these gene expressions have not been identified. Virulent Nectria haematococca infects pea plants and requires either pelA, which is induced by pectin, or pelD, which is induced only in planta. However, the host signal(s) that trigger pelD expression was unknown. Here we report the isolation of the host signals and identify homoserine and asparagine, two free amino acids found in uniquely high levels in pea seedlings, as the pelD-inducing signals. N. haematococca has evolved a mechanism to sense the host tissue environment by using the high levels of two free amino acids in this plant, thereby triggering the expression of pelD to assist the pathogenic process.
Lactobacillus acidophilus NCFM is a probiotic bacterium that has been produced commercially since 1972. The complete genome is 1,993,564 nt and devoid of plasmids. The average GC content is 34.71% with 1,864 predicted ORFs, of which 72.5% were functionally classified. Nine phage-related integrases were predicted, but no complete prophages were found. However, three unique regions designated as potential autonomous units (PAUs) were identified. These units resemble a unique structure and bear characteristics of both plasmids and phages. Analysis of the three PAUs revealed the presence of two R/M systems and a prophage maintenance system killer protein. A spacers interspersed direct repeat locus containing 32 nearly perfect 29-bp repeats was discovered and may provide a unique molecular signature for this organism. In silico analyses predicted 17 transposase genes and a chromosomal locus for lactacin B, a class II bacteriocin. Several mucus- and fibronectin-binding proteins, implicated in adhesion to human intestinal cells, were also identified. Gene clusters for transport of a diverse group of carbohydrates, including fructooligosaccharides and raffinose, were present and often accompanied by transcriptional regulators of the lacI family. For protein degradation and peptide utilization, the organism encoded 20 putative peptidases, homologs for PrtP and PrtM, and two complete oligopeptide transport systems. Nine two-component regulatory systems were predicted, some associated with determinants implicated in bacteriocin production and acid tolerance. Collectively, these features within the genome sequence of L. acidophilus are likely to contribute to the organisms' gastric survival and promote interactions with the intestinal mucosa and microbiota.
Metabolic pathways play an important role in insecticide resistance, but the full spectra of the genes involved in resistance has not been established. We constructed a microarray containing unique fragments from 230 Anopheles gambiae genes putatively involved in insecticide metabolism [cytochrome P450s (P450s), GSTs, and carboxylesterases and redox genes, partners of the P450 oxidative metabolic complex, and various controls]. We used this detox chip to monitor the expression of the detoxifying genes in insecticide resistant and susceptible An. gambiae laboratory strains. Five genes were strongly up-regulated in the dichlorodiphenyltrichloroethane-resistant strain ZAN/U. These genes included the GST GSTE2, which has previously been implicated in dichlorodiphenyltrichloroethane resistance, two P450s, and two peroxidase genes. GSTE2 was also elevated in the pyrethroid-resistant RSP strain. In addition, the P450 CYP325A3, belonging to a class not previously associated with insecticide resistance, was expressed at statistically higher levels in this strain. The applications of this detox chip and its potential contribution to malaria vector insecticide resistance management programs are discussed.
The progenitors of the gametes, the primordial germ cells (PGCs) are typically specified early in the development in positions, which are distinct from the gonad. These cells then migrate toward the gonad where they differentiate into sperms and eggs. Here, we study the role of the germ cells in somatic development and particularly the role of the germ line in the sex differentiation in zebrafish. To this end, we ablated the germ cells using two independent methods and followed the development of the experimental fish. First, PGCs were ablated by knocking down the function of dead end, a gene important for the survival of this lineage. Second, a method to eliminate the PGCs using the toxin–antitoxin components of the parD bacterial genetic system was used. Specifically, we expressed a bacterial toxin Kid preferentially in the PGCs and at the same time protected somatic cells by uniformly expressing the specific antidote Kis. Our results demonstrate an unexpected role for the germ line in promoting female development because PGC-ablated fish invariably developed as males.
The effect of weak selection driving genome evolution has attracted much attention in the last decade, but the task of measuring the strength of such selection is particularly difficult. A useful approach is to contrast the evolution of X-linked and autosomal genes in two closely related species in a whole-genome analysis. If the fitness effect of mutations is recessive, X-linked genes should evolve more rapidly than autosomal genes when the mutations are advantageous, and they should evolve more slowly than autosomal genes when the mutations are deleterious. We found synonymous substitutions on the X chromosome of human and chimpanzee to be less frequent than those on the autosomes. When calibrated against substitutions in the intergenic regions and pseudogenes to filter out the differences in the mutation rate and ancestral population size between X chromosomes and autosomes, X-linked synonymous substitutions are still 10% less frequent. At least 90% of the synonymous substitutions in human and chimpanzee are estimated to be deleterious, but the fitness effect is weaker than the effect of genetic drift. However, X-linked nonsynonymous substitutions are 30% more frequent than autosomal ones, suggesting the fixation of advantageous mutations that are recessive.
We produced XXXY chimeras by using embryos whose X chromosomes were tagged with EGFP (X*), making the fluorescent green female (XX*) germ cells easily distinguishable from their nonfluorescent male (XY) counterparts. Taking advantage of tagging with EGFP, the XX* "prospermatogonia" were isolated from the testes, and the status of their genomic imprinting was examined. It was shown that these XX cells underwent a paternal imprinting, despite their chromosomal constitution. As previously indicated in sex-reversal XXsxr testes, we also found a few green XX* germ cells developed as "eggs" within the seminiferous tubules of XX*XY chimeric testes. These cells were indistinguishable from XX* prospermatogonia at birth but resumed oogenesis in a testicular environment. The biological nature of the "testicular eggs" was examined by recovering the eggs from chimeric testes. The testicular eggs not only formed an egg-specific structure, the zona pellucida, but also were able to fuse with sperm. The collected testicular eggs were indicated to undergo maternal imprinting, despite the testicular environment. The genomic imprinting did not always follow the environmental conditions of where the germ cells resided; rather, it was defined by the sex that was chosen by the germ cells at early embryonic stage.
Gastrointestinal nematode infection is known to alter host T cell activation and has been used to study immune and inflammatory reactions in which nitric oxide (NO) is a versatile player. We previously demonstrated that Trichinella spiralis infection inhibits host inducible NO synthase (NOS-2) expression. We now demonstrate that (i) an IL-4 receptor -subunit (IL-4R)/Stat6-dependent but T cell-independent pathway is the key for the nematode-induced host NOS-2 inhibition; (ii) endogenous IL-4 and IL-13, the only known IL-4R ligands, are not required for activating the pathway; and (iii) treatment of RAW264.7 cells with parasite-cultured medium inhibits NOS-2 expression but not cyclooxygenase 2 expression. We propose that a yet-unidentified substance is released by the nematode during the host–parasite interaction.
Several lines of evidence indicate that some glycoconjugates are efficient effectors of the cellular prion protein (PrPC) conversion into its pathogenic (PrPSc) isoform. To assess how glycoconjugate glycan moieties participate in the biogenesis of PrPSc, an exhaustive comparative analysis of the expression of about 200 glycosylation-related genes was performed on prion-infected or not, hypothalamus-derived GT1 cells by hybridization of DNA microarrays, semiquantitative RT-PCR, and biochemical assays. A significant up- (30-fold) and down- (17-fold) regulation of the expression of the ChGn1 and Chst8 genes, respectively, was observed in prion-infected cells. ChGn1 and Chst8 are involved in the initiation of the synthesis of chondroitin sulfate and in the 4-O-sulfation of non-reducing N-acetylgalactosamine residues, respectively. A possible role for a hyposulfated chondroitin in PrPSc accumulation was evidenced at the protein level and by determination of chondroitin and heparan sulfate amounts. Treatment of Sc-GT1 cells with a heparan mimetic (HM2602) induced an important reduction of the amount of PrPSc, associated with a total reversion of the transcription pattern of the N-acetylgalactosamine-4-O-sulfotransferase 8. It suggests a link between the genetic control of 4-O-sulfation and PrPSc accumulation.
Crude brain homogenates of terminally diseased hamsters infected with the 263K strain of scrapie (PrPSc) and purified prion fibrils were heated or pressurized at 800 megapascals and 60 °C for 2 h in different buffers and in water. Prion proteins (PrP) were analyzed for their proteinase K resistance in immunoblots and for their infectivity in hamster bioassays. A notable decrease in the proteinase K resistance of unpurified prion proteins, probably because of pressure-induced changes in the protein conformation of native PrPSc or the N-truncated PrP-(27–30), could be demonstrated when pressurized at initially neutral conditions in several buffers and in water but not in a slightly acidic pH. A subsequent 6–7 log10 reduction of infectious units/g in phosphate-buffered saline buffer, pH 7.4, was found. The proteinase K-resistant core was also not detectable after purification of prions extracted from pressurized samples, confirming pressure effects at the level of the secondary structure of prion proteins. However, opposite results were found after pressurizing purified prions, arguing for the existence of pressure-sensitive -structures (PrPScPsen) and extremely pressure-resistant -structures (PrPScPres). Remarkably, after the first centrifugation step at 540,000 x g during isolation, prions remained proteinase K-resistant when pressurized in all tested buffers and in water. It is known that purified fibrils retain infectivity, but the isolated protein (full and N-truncated) behaved differently from native PrPSc under pressure, suggesting a kind of semicrystalline polymer structure.
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays an essential role in cell growth control. mTOR stimulates cell growth by phosphorylating p70 ribosomal S6 kinase (S6K) and eukaryote initiation factor 4E-binding protein 1 (4EBP1). The mTOR pathway is regulated by a wide variety of cellular signals, including mitogenic growth factors, nutrients, cellular energy levels, and stress conditions. Recent studies have proposed several mechanisms to explain how mTOR is regulated by growth factors and cellular energy levels. However, little is known as to how mTOR is regulated by stress conditions. We observed that two stress-induced proteins, RTP801/Redd1 and RTP801L/Redd2, potently inhibit signaling through mTOR. Our data support that RTP801 and RTP801L work downstream of AKT and upstream of TSC2 to inhibit mTOR functions. These results add a new dimension to mTOR pathway regulation and provide a possible molecular mechanism of how cellular stress conditions may regulate mTOR function.
Severo Ochoa (1905–1993) was born in Luarca, Spain and became interested in biology while in high school. Because there were few graduate programs in the biological sciences in Spain at that time, he had to enroll in the Medical School at the University of Madrid to study biology. Fortunately, during his second year, Ochoa was offered the opportunity to do research. His first experiment, isolating creatinine from urine, stimulated his interest in the function and metabolism of creatine and creatinine in the body. As a result, he and José G. Valdecasas came up with a simple micromethod for the determination of creatine in muscle. They submitted a paper on this method to the Journal of Biological Chemistry (JBC), and to their delight, it was published in 1929 (1). Said Ochoa, "Not even in my wildest dreams could I have dreamt that, years later, I would become a member of the editorial board of the JBC and serve as President of the American Society of Biological Chemists (now the American Society for Biochemistry and Molecular Biology)" (2).1
Plant ADP-glucose pyrophosphorylase consists of two subunits, a large modulatory subunit and a small catalytic subunit. By resurrecting the enzymatic activity of the modulatory subunit, Miguel Ballicora and colleagues proved that it descended from a catalytic ancestor. The amino acid similarity between the subunits (50% identity) suggested that the two proteins might share a common ancestry.
When immune cells are exposed to pro-inflammatory cytokines or bacterial endotoxin they initiate transcription of inducible nitric-oxide synthase (iNOS). This results in an increase in cellular nitric oxide (NO), with cytostatic and cytotoxic effects that contribute to facilitate host defense. However, too much NO can be a bad thing; sustained overproduction of NO can cause septic shock resulting in systemic hypotension, organ failure, and even death.
We describe two male-specific olfactory receptors (ORs) in the silk moth, Bombyx mori, that are mutually exclusively expressed in a pair of adjacent pheromone-sensitive neurons of male antennae: One is specifically tuned to bombykol, the sex pheromone, and the other to bombykal, its oxidized form. Both pheromone ORs are coexpressed with an OR from the highly conserved insect OR subfamily. This coexpression promotes the functional expression of pheromone receptors and confers ligand-stimulated nonselective cation channel activity. The same effects were also observed for general ORs. Both odorant and pheromone signaling pathways are mediated by means of a common mechanism in insects.
Notch proteins are receptors for a conserved signaling pathway that affects numerous cell fate decisions. We found that in Drosophila, Protein O-fucosyltransferase 1 (OFUT1), an enzyme that glycosylates epidermal growth factor–like domains of Notch, also has a distinct Notch chaperone activity. OFUT1 is an endoplasmic reticulum protein, and its localization was essential for function in vivo. OFUT1 could bind to Notch, was required for the trafficking of wild-type Notch out of the endoplasmic reticulum, and could partially rescue defects in secretion and ligand binding associated with Notch point mutations. This ability of OFUT1 to facilitate folding of Notch did not require its fucosyltransferase activity. Thus, a glycosyltransferase can bind its substrate in the endoplasmic reticulum to facilitate normal folding.
Appropriate cell number and organ size in a multicellular organism are determined by coordinated cell growth, proliferation, and apoptosis. Disruption of these processes can cause cancer. Recent studies have identified the Large tumor suppressor (Lats)/Warts (Wts) protein kinase as a key component of a pathway that controls the coordination between cell proliferation and apoptosis. Here we describe growth inhibitory functions for a Mob superfamily protein, termed Mats (Mob as tumor suppressor), in Drosophila. Loss of Mats function results in increased cell proliferation, defective apoptosis, and induction of tissue overgrowth. We show that mats and wts function in a common pathway. Mats physically associates with Wts to stimulate the catalytic activity of the Wts kinase. A human Mats ortholog (Mats1) can rescue the lethality associated with loss of Mats function in Drosophila. As Mats1 is mutated in human tumors, Mats-mediated growth inhibition and tumor suppression is likely conserved in humans.
Physical, genetic, and chemical-genetic interactions centered on the conserved chaperone Hsp90 were mapped at high resolution in yeast using systematic proteomic and genomic methods. Physical interactions were identified using genome-wide two hybrid screens combined with large-scale affinity purification of Hsp90-containing protein complexes. Genetic interactions were uncovered using synthetic genetic array technology and by a microarray-based chemical-genetic screen of a set of about 4700 viable yeast gene deletion mutants for hypersensitivity to the Hsp90 inhibitor geldanamycin. An extended network, consisting of 198 putative physical interactions and 451 putative genetic and chemical-genetic interactions, was found to connect Hsp90 to cofactors and substrates involved in a wide range of cellular functions. Two novel Hsp90 cofactors, Tah1 (YCR060W) and Pih1 (YHR034C), were also identified. These cofactors interact physically and functionally with the conserved AAA+-type DNA helicases Rvb1/Rvb2, which are key components of several chromatin remodeling factors, thereby linking Hsp90 to epigenetic gene regulation.
Illustrations of molecular models are widely used for the study and dissemination of molecular structure and function. Several metaphors are commonly used to create these illustrations, and each captures a relevant aspect of the molecule and omits other aspects. Effective tools are available for rendering atomic structures by using several standard representations, and the research community is highly sophisticated in their use. Molecular properties, such as electrostatics, and large complex molecular and cellular systems currently pose challenges for representation.
The medical application of antibiotics dramatically reduced human infant mortality in the previous century. A new study indicates that ground nesting wasps exploit Streptomyces strains that they rear in their antennae for the same purpose.
The nervous system processes sensory information and controls behavior by performing an enormous number of computations. These computations occur both within cells and between cells, but it is intercellular information processing, involving complex neural networks, that provides the nervous system with its remarkable functional capacity. The principal cells involved in information processing are neurons, of which there are hundreds, if not thousands of individual cell types based on morphology, location, connectivity and chemistry [1]. In addition to neurons, the other major kind of cell in the nervous system is the glia, which play critical support roles, but which are increasingly seen to function in some aspects of information processing.
Paramount among human cognitive abilities is the capacity to reason about what others think, want, and see—a capacity referred to as a theory of mind (ToM). Despite its importance in human cognition, the extent to which other primates share human ToM capacities has for decades remained a mystery. To date, primates [1, 2] have performed poorly in behavioral tasks that require ToM abilities, despite the fact that some macaques are known to encode social stimuli at the level of single neurons [3–5]. Here, we presented rhesus macaques with a more ecologically relevant ToM task in which subjects could “steal” a contested grape from one of two human competitors. In six experiments, monkeys selectively retrieved the grape from an experimenter who was incapable of seeing the grape rather than an experimenter who was visually aware. These results suggest that rhesus macaques possess an essential component of ToM: the ability to deduce what others perceive on the basis of where they are looking. These results converge with new findings illustrating the importance of competitive paradigms in apes [6]. Moreover, they raise the possibility that, in primates, cortical cells thought to encode where others are looking [7] may encode what those individuals see as well.
Traditional models of how cooperative strategies succeed in evolution have largely focused on social interactions among individuals and selection acting at kin and group levels. A recent study at the genetic level suggests that cooperation may also be promoted by the evolution of gene–trait relationships that limit the range of possible cheating mechanisms that can evolve.
In Drosophila, the patterns of homeotic gene transcription that specify body segment identity are maintained by the polycomb group of repressors and the trithorax group (trxG) of activators. On p. 1623, Srinivasan and colleagues report that the trxG protein Kismet maintains homeotic gene transcription by facilitating an early step in transcriptional elongation by RNA polymerase II (Pol II). kismet, which is needed for segmentation and for body segment specification, encodes the isoforms KIS-L and KIS-S. The researchers show that KIS-L associates with virtually all sites of transcriptionally active chromatin on salivary gland polytene chromosomes taken from Drosophila larvae. This distribution pattern largely overlaps with that of Pol II. Furthermore, the levels of elongating Pol II and of several elongation factors are reduced on polytene chromosomes from kis mutant larvae. The researchers conclude that KIS-L, unexpectedly given its specialised roles in development, plays a global role in transcription by Pol II.
Hox proteins are transcriptional regulators that specify segmental identity along the anteroposterior axis of multicellular animals. In Drosophila, Hox proteins bind to DNA in association with the Extradenticle (Exd) and Homothorax (Hth) co-factors. The Hox DNA-binding selectivity model proposes that distinct Hox/Exd/Hth complexes select different consensus DNA-binding sites to initiate different developmental programs. On p. 1591, Ebner and colleagues question this model. By screening the Drosophila genome for the consensus Lab/Exd/Hth-binding sequence, the authors have discovered a new target gene regulated by the Hox protein Labial (Lab). Surprisingly, the regulation of this gene by Lab does not depend on the Lab/Exd-binding consensus site but on a strongly divergent sequence. The researchers conclude that more complexity needs to be built into the Hox DNA-binding selectivity model to accommodate their findings. On p. 1567, Hersh and Carroll consider how Hox genes regulate development from an evolutionary point of view. The researchers identify the knot gene as a direct target of the Hox protein Ultrabithorax (Ubx) in the developing Drosophila haltere and find that the minimal element for knot repression by Ubx is not conserved between Drosophila species. They conclude that Hox cis-regulatory regions, the evolutionary selection of which underlies how Hoxproteins direct the production of different metazoan body forms, are more diffuse and larger than previously thought.
Hold a leaf up to the light and its complex vascular system or venation stands out clearly. Little is known about how leaf vascular development is spatially regulated, except that auxin signalling is involved. Now, on p. 1699, Koizumi andcolleagues report that vesicle transport mediated by VAN3, an ADP-ribosylation factor-GTPase activating protein (ARF-GAP), is involved in leaf vein pattern formation in Arabidopsis. The Arabidopsis mutant van3 has a discontinuous vein pattern, and, using double-mutant analyses, the researchers show that VAN3 is involved in auxin signal transduction. They identify VAN3 as a gene encoding a unique type of ARF-GAP and localise VAN3 to a subpopulation of the trans-Golgi network (TGN). The researchers conclude that VAN3 functions in vein pattern formation by regulating auxin signalling via a TGN-mediated transport system, thus uncovering the first developmental role for an ARF-GAP in plants.
Feathers are a defining feature of birds but their size, colour, shape and arrangement varies widely between species. Feather morphogenesis involves reciprocal signalling between the dermis and epidermis of the feather bud, but it is unclear how species-specific feather differences are generated. Now, Eames and Schneider report that premigratory neural crest cells transplanted from quails into ducks form dermis that instructs the host epidermis to form quail-like feather buds, and vice-versa (see p. 1499). Quail and duck have distinct feather patterns and divergent growth rates. The researchers show that in quail-duck chimeras, donor neural-crest-derived dermis alters the spatial pattern and time of formation of host cranial feathers by altering the expression of members and targets of the bone morphogenetic protein, sonic hedgehog and delta/notch pathways. The researchers suggest that this marked spatiotemporal plasticity in feather development might facilitate feather evolution.
During evolution, new gene functions are created by the duplication and functional diversification of existing genes. Diversification can be at the level of transcriptional regulation or protein function. Kirik and co-workers now describe how for two paralogous MYB-related transcription factors – MYB23 and GL1 – functional diversification at both levels is involved in the development of branching hair-like structures on Arabidopsis leaves called trichomes (see p. 1477). Their study of trichomes in myb23 and gl1 single and double mutants reveals that MYB23 controls trichome branching and trichome initiation at leaf edges, the latter redundantly with GL1. Promoter and protein-coding-region swap experiments indicate that, for trichome initiation, the diversification of gl1 and myb23 gene functions is determined only by how their expression is regulated. By contrast, the diversification of their functions with respect to trichome branching involves differences both in the regulation of the two genes and in the proteins they encode.
Tardigrades are poorly studied aquatic arthropods. Now a 3D time-lapse recording of the development of the tardigrade Thulinia stephaniae from embryogenesis to hatching provides new information on the ancestral mode of early development in Arthropoda (see p. 1349). By tracking the fate of individual cells in living embryos, Hejnol and Schnabel reveal that T. stephaniae embryos undergo an irregular indeterminate cleavage pattern. Furthermore, early embryogenesis is highly regulative; even after laser ablation of half the embryo at the two- or four-cell stage, normal juveniles develop, a degree of recovery from blastomere ablation not previously seen in protostome embryos. Because tardigrades are a basal lineage within the Arthropoda, a comparison of these new details of tardigrade development with those of other protostomes, such as crustaceans, suggests that indeterminate cleavage and regulatory development are part of the ground pattern of Arthropoda and probably all Ecdysozoa
The spatial regulation of neuorogenesis is a mysterious but crucial process in vertebrate central nervous system development. In zebrafish embryos, proneuronal domains, which produce early neurons, are separated by inter-proneuronal domains, which do not undergo neurogenesis until later in development. On p. 1375, Bae and colleagues report that the hairy- and enhancer of split-related genes her3 and her9 link positional information in the posterior neuroectoderm to the spatial regulation of neurogenesis. The expression of her3 and her9 is restricted to inter-proneuronal domains, and inhibiting either of their functions with antisense morpholinos causes the ectopic expression of proneural genes in these domains. Inhibiting both proteins' functions abolishes inter-proneuronal domain formation. Other results indicate that her3 and her9 expression is regulated by positional information in which Bmp signalling is involved. The researchers conclude that her3 and her9 spatially control neurogenesis by functioning as prepattern genes.
Mutations in the APC (adenomatous polyposis coli) gene occur in most sporadic colorectal cancers. But although APC loss is important in tumour initiation, it is unclear whether tumours form in the epithelial stem cells of the intestinal crypts or in the differentiated cells of the villus. On p. 1443, Andreu and colleagues report that Apc loss in the mouse intestine rapidly induces crypt-restricted premalignant changes. Using a conditional gene-ablation approach, they examined the effects of Apc loss along the crypt-villus axis of the mouse small intestine. Apc loss activates ß-catenin signalling in both compartments but only the crypt cells respond with increased proliferation and apoptosis, and impaired cell migration. The activation of ß-catenin signalling also alters crypt cell fate, promoting their commitment to the Paneth cell lineage. Overall, these results indicate that tumorigenesis caused by the loss of Apc in the colon occurs among the stem cells in the crypt base.
The human congenital malformation cleft lip with or without cleft palate (CL/P) seems to be genetically distinct from isolated cleft palate. However, recent data indicate that the bone morphogenetic protein (Bmp) signalling pathway may be involved in both forms of orofacial clefting. Liu and co-workers now report that Bmp signalling has distinct functions in lip and palate fusion in mice (see p. 1453). The researchers show that conditional inactivation of the type 1 Bmp receptor Bmpr1a in the facial primordia produces mice with bilateral CL/P. Diminished cell proliferation in the maxillary process mesenchyme underlies the palate defects, but increased apoptosis in the medial nasal process is responsible for the cleft lip. Furthermore, conditional inactivation of Bmp4 produces isolated cleft lip, indicating that a Bmp4-Bmpr1a genetic pathway functions in lip fusion. These results will aid in unravelling the mechanisms underlying human clefting conditions.
During organ development, a delicate balance between proliferation and terminal differentiation of progenitor cells ensures that the final organ has the correct size and structure. On p. 1363, Okubo and co-workers report that Nmyc is required to maintain this balance in developing mouse lungs. They show that Nmyc expression in mouse embryonic lung is restricted to a distal population of undifferentiated epithelial cells, many of which are in the S phase of the cell cycle. Overexpression of Nmyc in the epithelium of the developing lung increases the domain of S phase cells and inhibits differentiation; deletion of one or two copies of Nmyc produces the opposite outcomes. Thus,the researchers suggest, Nmyc's role in lung development is to maintain a distal population of undifferentiated, proliferating progenitor cells, a discovery that may help us to understand disorders such as lung cancer.
Many internal organs in vertebrates have conserved left-right (LR) orientations that are established by asymmetric gene expression during early development. The nodal flow model proposes that this `lopsided' gene expression is initiated in mice by cilia on cells producing a flow of extracellular fluid in the late gastrula node. Now, Joseph Yost's group report that a similar mechanism initiates LR asymmetry in zebrafish (see p. 1247). Using videomicroscopy, the researchers show that cilia on the dorsal forerunner cells, which form a ciliated epithelium inside the fluid-filled Kupffer's vesicle, are motile and create a fluid flow just before asymmetric gene expression starts in the embryo. Other results provide the first direct evidence that impairing cilia function in derivatives of the dorsal organizer affects LR development and identify genes upstream of cilia function. Kupffer's vesicle, the researchers conclude, is a transient embryonic organ of asymmetry in teleosts.
Vertebrate collagenases, members of the matrix metalloproteinase (MMP) family, initiate interstitial fibrillar collagen breakdown. It is essential in many biological processes, and unbalanced collagenolysis is associated with diseases such as arthritis, cancer, atherosclerosis, aneurysm, and fibrosis. These metalloproteinases are secreted from the cell as inactive precursors, procollagenases (proMMPs). To gain insights into the structural basis of their activation mechanisms and collagen binding, we have crystallized recombinant human proMMP-1 and determined its structure to 2.2 Å resolution. The catalytic metalloproteinase domain and the C-terminal hemopexin (Hpx) domain show the classical MMP-fold, but the structure has revealed new features in surface loops and domain interaction. The prodomain is formed by a three-helix bundle and gives insight into the stepwise activation mechanism of proMMP-1. The prodomain interacts with the Hpx domain, which affects the position of the Hpx domain relative to the catalytic domain. This interaction results in a "closed" configuration of proMMP-1 in contrast to the "open" configuration observed previously for the structure of active MMP-1. This is the first evidence of mobility of the Hpx domain in relation to the catalytic domain, providing an important clue toward the understanding of the collagenase-collagen interaction and subsequent collagenolysis.
Hsp70/Hsp90 organizing protein (Hop) coordinates Hsp70 and Hsp90 interactions during assembly of steroid receptor complexes. Hop is composed of three tetratricopeptide repeat (TPR) domains (TPR1, TPR2a, and TPR2b) and two DP repeat domains (DP1 and DP2); Hsp70 interacts directly with TPR1 and Hsp90 with TPR2a, but the function of other domains is less clear. Human Hop and the Saccharomyces cerevisiae ortholog Sti1p, which share a common domain arrangement, are functionally interchangeable in a yeast growth assay and in supporting the efficient maturation of glucocorticoid receptor (GR) function. To gain a better understanding of Hop structure/function relationships, we have extended comparisons to the Hop ortholog from Drosophila melanogaster (dHop), which lacks DP1. Although dHop binds Hsp70 and Hsp90 and can rescue the growth defect in yeast lacking Sti1p, dHop failed to support GR function in yeast, which suggests a novel role for Hop in GR maturation that goes beyond Hsp binding. Chimeric Hop constructs combining human and Drosophila domains demonstrate that the C-terminal domain DP2 is critical for this previously unrecognized role in steroid receptor function.
A few discrete portions of the Saccharomyces cerevisiae genome, such as the chromosome ends within 5–10 kilobase pairs (kb) of telomeres and the silent mating-type regions named HMR and HML, are defined as transcriptionally silent because they cause repression of an RNA polymerase II gene placed within them (reviewed comprehensively in Ref. 1). S. cerevisiae silencing is a form of transcriptional repression that is long range, occurring over distances larger than a single gene. Silencing occurs in only certain chromosomal regions and depends more on a gene's position in the genome than its promoter sequence. Another defining characteristic is a stable inheritance pattern during cell division, indicating that the repressive state is duplicated along with the underlying DNA sequences during DNA replication. These features make S. cerevisiae transcriptional silencing functionally akin to position-effect-variegation in Drosophila melanogaster and X-chromosome inactivation in female mammals
Three receptor activity-modifying proteins (RAMPs 1, 2, and 3) are required for plasma membrane expression and ligand binding of several G-protein-coupled receptors. For example, if RAMP1 binds to the calcitonin receptor-like receptor (CRLR), then a cell surface calcitonin generelated peptide 1 receptor is produced. In contrast, the binding of RAMP2 or -3 to CRLR produces an adrenomedullin peptide receptor. Jennifer M. Bomberger and colleagues now show that in addition to these roles, RAMP3 binding to the CRLR is required for recycling of internalized adrenomedullin receptors.
In yeast, chitin is laid down at three locations in the cell wall during the cell cycle: in a ring at the mother-bud neck, in the partition that forms between mother and daughter cells at cytokinesis, and in the cell wall of the daughter cell. Chitin is bound to the cell wall structure by two types of linkages: to the non-reducing end of (1–3)glucan and to side chains of (1–6)glucan. Enrico Cabib and Angel Durán hypothesized that the chitin in the ring at the mother-daughter neck might be linked differently from the chitin that dispersed throughout the cell wall.
Konrad Emil Bloch (1912–2000) was born in Neisse, eastern Germany (now Nysa in Poland). Growing up he was more interested in engineering and natural sciences than chemistry, but an organic chemistry course taught by future Nobel laureate Hans Fischer at the Technische Hochschule in Munich provided a turning point. Bloch said of Fischer's class, "As he presented it, the subject matter was fascinating, the organization superb, and the delivery monotonous" (1). Despite Fischer's monotone delivery, Bloch was influenced enough to become a chemistry student in his laboratory.1
Members of the mammalian ß1,4-galactosyltransferase family are among the best studied glycosyltransferases, but the requirements for all members of this family within an animal have not previously been determined. Here, we describe analysis of two Drosophila genes, ß4GalNAcTA (CG8536) and ß4GalNAcTB (CG14517), that are homologous to mammalian ß1,4-galactosyltransferases. Like their mammalian homologs, these glycosyltransferases use N-acetylglucosamine as an acceptor substrate. However, they transfer N-acetylgalactosamine rather than galactose. This activity, together with amino acid sequence similarity, places them among a group of recently identified invertebrate ß1,4-N-acetylgalactosaminyltransferases. To investigate the biological functions of these genes, null mutations were generated by imprecise excision of a transposable element (ß4GalNAcTA) or by gene-targeted homologous recombination (ß4GalNAcTB). Flies mutant for ß4GalNAcTA are viable and fertile but display behavioral phenotypes suggestive of essential roles for GalNAc-ß1,4-GlcNAc containing glycoconjugates in neuronal and/or muscular function. ß4GalNAcTB mutants are viable and display no evident morphological or behavioral phenotypes. Flies doubly mutant for both genes display only the behavioral phenotypes associated with mutation of ß4GalNAcTA. Thus Drosophila homologs of the mammalian ß4GalT family are essential for neuromuscular physiology or development but are not otherwise required for viability, fertility, or external morphology.
Deep-sea life requires adaptation to high pressure, an extreme yet common condition given that oceans cover 70% of Earth's surface and have an average depth of 3800 meters. Survival at such depths requires specific adaptation but, compared with other extreme conditions, high pressure has received little attention. Recently, Photobacterium profundum strain SS9 has been adopted as a model for piezophily. Here we report its genome sequence (6.4 megabase pairs) and transcriptome analysis. The results provide a first glimpse into the molecular basis for life in the largest portion of the biosphere, revealing high metabolic versatility.
Bacteria that selectively kill males ("male-killers") were first characterized more than 50 years ago in Drosophila and have proved to be common in insects. However, the mechanism by which sex specificity of virulence is achieved has remained unknown. We tested the ability of Spiroplasma poulsonii to kill Drosophila melanogaster males carrying mutations in genes that encode the dosage compensation complex. The bacterium failed to kill males lacking any of the five protein components of the complex.
Ascidian embryos develop with a fixed cell lineage into simple tadpoles. Their lineage is almost perfectly conserved, even between the evolutionarily distant species Halocynthia roretzi and Ciona intestinalis, which show no detectable sequence conservation in the non-coding regions of studied orthologous genes. To address how a common developmental program can be maintained without detectable cis-regulatory sequence conservation, we compared in both species the regulation of Otx, a gene with a shared complex expression pattern. We found that in Halocynthia, the regulatory logic is based on the use of very simple cell line-specific regulatory modules, the activities of which are conserved, in most cases, in the Ciona embryo. The activity of each of these enhancer modules relies on the conservation of a few repeated crucial binding sites for transcriptional activators, without obvious constraints on their precise number, order or orientation, or on the surrounding sequences. We propose that a combination of simplicity and degeneracy allows the conservation of the regulatory logic, despite drastic sequence divergence. The regulation of Otx in the anterior endoderm by Lhx and Fox factors may even be conserved with vertebrates.
Mammalian polynucleotide kinase (PNK) is a key component of both the base excision repair (BER) and nonhomologous end-joining (NHEJ) DNA repair pathways. PNK acts as a 5Åå-kinase/3Åå-phosphatase to create 5Åå-phosphate/3Åå-hydroxyl termini, which are a necessary prerequisite for ligation during repair. PNK is recruited to repair complexes through interactions between its N-terminal FHA domain and phosphorylated components of either pathway. Here, we describe the crystal structure of intact mammalian PNK and a structure of the PNK FHA bound to a cognate phosphopeptide. The kinase domain has a broad substrate binding pocket, which preferentially recognizes double-stranded substrates with recessed 5Åå termini. In contrast, the phosphatase domain efficiently dephosphorylates single-stranded 3Åå-phospho termini as well as double-stranded substrates. The FHA domain is linked to the kinase/phosphatase catalytic domain by a flexible tether, and it exhibits a mode of target selection based on electrostatic complementarity between the binding surface and the phosphothreonine peptide.
A fundamental principle in the field of olfaction is that each olfactory receptor neuron expresses a single odorant receptor. Goldman et al. have constructed a receptor-to-neuron map for an entire olfactory organ of the fruit fly Drosophila and find that one class of neuron coexpresses two receptors. Both receptors are functional in an in vivo expression system, are encoded by unlinked genes, and are only 16% identical in amino acid sequence. Coexpression of multiple receptors in a cell could provide an additional degree of freedom for odor coding.
Previous work suggests that the subgenual cingulate area of the brain has an important role in major depression. This study reports the first use of chronic deep brain stimulation (DBS) to modulate the activity of this brain region in depressed patients who have failed to respond to all other therapies. Mayberg et al. find that chronic stimulation of this area through implanted electrodes connected to a “pacemaker-like” pulse generator reverses overactive subgenual cingulate activity and normalizes other dysfunctional areas. The brain changes seen with stimulation are associated with striking clinical improvement in four of six patients. These results suggest that DBS can be used to treat depressed patients who are otherwise treatment resistant.
In this issue of Neuron, Fang and He demonstrate that viewing a face or object from a particular viewpoint leads to an aftereffect whereby later neutral views of the same stimulus appear to be biased away from the initial viewpoint. This new aftereffect suggests that the human brain contains populations of neurons tuned to the angle from which an object is viewed.
Fragile X syndrome is the most common heritable cause of mental retardation. Previous work has suggested that overactive signaling by group I metabotropic glutamate receptors (mGluRs) may be a mechanism underlying many of the disease symptoms. As a test of this theory, McBride et al. show that in a Drosophila model for Fragile X syndrome, treatment with mGluR antagonists can rescue short-term memory, courtship, and mushroom body defe
Proneural bHLH transcription factors are key mediators of Notch signaling, but their transcriptional targets and thus the mechanisms by which they contribute to neurogenesis are largely unknown. Reeves and Posakony undertook a systematic approach to proneural bHLH function, by FACS isolating cells of the Drosophila peripheral nervous system (PNS). Their analysis of PNS proneural-dependent gene expression not only identifies a large number of proneural targets and validates their functional importance in the PNS, but also defines a new transcriptional code highlighting Notch-independent proneural bHLH functions. This code may identify new classes of proneural bHLH targets in other organisms as well.
Gastrulation, the period during the early development of animals when major cell and tissue movements remodel an initially unstructured group of cells, requires coordinated control of different types of cellular activities in different cell populations. A hierarchy of genetic control mechanisms, involving cell signaling and transcriptional regulation, sets up the embryonic axes and specify the territories of the future germ layers. Cells in these territories modulate their cytoskeleton and their adhesive behavior, resulting in shape changes and movement. Similarities among different species in patterning and cell biological mechanisms are beginning to allow us to recognize general, conserved principles and speculate on possible ancestral mechanisms of gastrulation.
The small GTPase Rap1 was originally thought to function as an antagonist of Ras. A recent paper by Mishra et al. (2005) provides evidence that Ras and Rap1 function in parallel to activate Raf downstream of the Torso receptor tyrosine kinase in Drosophila.
Vertebrate eggs prevent parthenogenetic development by producing cytostatic factor (CSF), which blocks exit from metaphase of meiosis II until fertilization. CSF was never purified but recently suspected to inhibit the anaphase-promoting complex (APC), an ubiquitin ligase required for entry into anaphase. In a recent paper in Genes & Development, Schmidt et al. describe the Xenopus APC inhibitor Erp1, which seems to be the best candidate yet for the downstream effector of CSF activity.
The recent finding that the ObgE GTPase acts as a replication checkpoint protein in Escherichia coli has important implications. It reveals the existence of a new pathway of replication control by the nucleotide pool and suggests unsuspected links between replication, proteins synthesis, and cellular differentiation.
The origin of definitive hematopoiesis poses a fundamental biological question. In this issue of Developmental Cell, two groups have independently found a novel hematopoietic stem cell (HSC) niche in the extraembryonic placenta, in addition to previously identified alternative locations of hematopoiesis at different developmental stages.
Regulation of cell growth and proliferation has a fundamental role in animal and plant development and in the progression of cancer. In the context of development, it is important to understand the mechanisms that coordinate growth and patterning of tissues. Imaginal discs, which are larval precursors of fly limbs and organs, have provided much of what we currently know about these processes. Here, we consider the mechanism that is responsible for the observed uniformity of growth in wing imaginal discs, which persists in the presence of gradients in growth inducing morphogens in spite of the stochastic nature of cell division. The phenomenon of "cell competition," which manifests in apoptosis of slower-growing cells in the vicinity of faster growing tissue, suggests that uniform growth is not a default state but a result of active regulation. How can a patch of tissue compare its growth rate with that of its surroundings? A possible way is furnished by mechanical interactions. To demonstrate this mechanism, we formulate a mathematical model of nonuniform growth in a layer of tissue and examine its mechanical implications. We show that a clone growing faster or slower than the surrounding tissue is subject to mechanical stress, and we propose that dependence of the rate of cell division on local stress could provide an "integral-feedback" mechanism stabilizing uniform growth. The proposed mechanism of growth control is not specific to imaginal disc growth and could be of general relevance. Several experimental tests of the proposed mechanism are suggested.
Neoplastic progression in human tissues appears to be paralleled by a series of genetic and epigenetic alterations. In human colorectal cancers, defect Wnt/-catenin/T-cell factor and RAS/RAF signaling pathways have a major contributing role in tumor initiation and progression. To date, much of the research on the consequences of -catenin activation has been focused on genes whose expression is believed to be activated by -catenin-associated T-cell factor-dependent transcription. Little is known about genes whose expression may be down-regulated secondary to -catenin activation. Using a subtractive suppression hybridization approach, we identified a gene with markedly decreased expression in rat RK3E epithelial cells neoplastically transformed by -catenin. Because expression of this gene was also down-regulated in RK3E transformed by several other oncogenes, the gene was named DRO1 for "down-regulated by oncogenes 1." Compared with corresponding normal tissues, DRO1 expression was found to be very reduced in colon and pancreatic cancer cell lines as well as in most colorectal cancer specimens. The predicted DRO1 protein contains three repetitive elements with significant similarity to the carboxyl-terminal regions of the predicted proteins from DRS/SRPX/ETX1 and SRPUL genes, suggesting the existence of a new protein family. Ectopic expression of DRO1 in neoplastically transformed RK3E or colorectal and pancreatic cancer cell lines lacking endogenous DRO1 expression resulted in substantial inhibition of growth properties. DRO1 was found to suppress anchorage independent growth and to sensitize cells to anoikis and CD95-induced apoptosis. Our findings suggest that inhibition of DRO1 expression may be an important event in the development of colorectal and pancreatic cancers.
-Site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, A, and is implicated in triggering the pathogenesis of Alzheimer disease. We previously reported that BACE1 cleaved rat -galactoside 2,6-sialyltransferase (ST6Gal I) that was overexpressed in COS cells and that the NH2 terminus of ST6Gal I secreted from the cells (E41 form) was Glu41. Here we report that BACE1 gene knock-out mice have one third as much plasma ST6Gal I as control mice, indicating that BACE1 is a major protease which is responsible for cleaving ST6Gal I in vivo. We also found that BACE1-transgenic mice have increased level of ST6Gal I in plasma. Secretion of ST6Gal I from the liver into the plasma is known to be up-regulated during the acute-phase response. To investigate the role of BACE1 in ST6Gal I secretion in vivo, we analyzed the levels of BACE1 mRNA in the liver, as well as the plasma levels of ST6Gal I, in a hepatopathological model, i.e. Long-Evans Cinnamon (LEC) rats. This rat is a mutant that spontaneously accumulates copper in the liver and incurs hepatic damage. LEC rats exhibited simultaneous increases in BACE1 mRNA in the liver and in the E41 form of the ST6Gal I protein, the BACE1 product, in plasma as early as 6 weeks of age, again suggesting that BACE1 cleaves ST6Gal I in vivo and controls the secretion of the E41 form.
Art v 1, the major allergen of mugwort (Artemisia vulgaris) pollen contains galactose and arabinose. As the sera of some allergic patients react with natural but not with recombinant Art v 1 produced in bacteria, the glycosylation of Art v 1 may play a role in IgE binding and human allergic reactions. Chemical and enzymatic degradation, mass spectrometry, and 800 MHz 1H and 13C nuclear magnetic resonance spectroscopy indicated the proline-rich domain to be glycosylated in two ways. We found a large hydroxyproline-linked arabinogalactan composed of a short 1,6-galactan core, which is substituted by a variable number (5–28) of -arabinofuranose residues, which form branched side chains with 5-, 2,5-, 3,5-, and 2,3,5-substituted arabinoses. Thus, the design of the Art v 1 polysaccharide differs from that of the well known type II arabinogalactans, and we suggest it be named type III arabinogalactan.
Tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal exopeptidase that sequentially removes tripeptides from the N termini of polypeptides and shows a minor endoprotease activity. Mutations in TPP I lead to classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disease. TPP I proenzyme is converted in lysosomes into a mature enzyme with the assistance of another protease and is able to autoactivate in acidic pH in vitro via a unimolecular mechanism. Because autoactivation in vitro at the pH values reported for lysosomes generated inactive enzyme, we intended to determine whether physiologically relevant factors can modify this process to also make it plausible in vivo. Here, we report that high ionic strength and glycosaminoglycans (GAGs) increase yields (ionic strength) or yields and rates (GAGs) of activation, enhance degradation of liberated TPP I prosegment fragments, and switch effective autoactivation of TPP I proenzyme toward less acidic pH values (up to pH 6.0).
3'-(-Chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxyvinblastine (KAR-2) is a potent anti-microtubular agent that arrests mitosis in cancer cells without significant toxic side effects. In this study we demonstrate that in addition to targeting microtubules, KAR-2 also binds calmodulin, thereby countering the antagonistic effects of trifluoperazine. To determine the basis of both properties of KAR-2, the three-dimensional structure of its complex with Ca2+-calmodulin has been characterized both in solution using NMR and when crystallized using x-ray diffraction. Heterocorrelation (1H-15N heteronuclear single quantum coherence) spectra of 15N-labeled calmodulin indicate a global conformation change (closure) of the protein upon its binding to KAR-2. The crystal structure at 2.12-Å resolution reveals a more complete picture; KAR-2 binds to a novel structure created by amino acid residues of both the N- and C-terminal domains of calmodulin. Although first detected by x-ray diffraction of the crystallized ternary complex, this conformational change is consistent with its solution structure as characterized by NMR spectroscopy. It is noteworthy that a similar tertiary complex forms when calmodulin binds KAR-2 as when it binds trifluoperazine, even though the two ligands contact (for the most part) different amino acid residues. These observations explain the specificity of KAR-2 as an anti-microtubular agent; the drug interacts with a novel drug binding domain on calmodulin. Consequently, KAR-2 does not prevent calmodulin from binding most of its physiological targets.
Since the completion of the human and mouse genomes, the focus in mammalian biology has been on assessing gene function. Tools are needed for assessing the phenotypes of the many mouse models that are now being generated, where genes have been "knocked out," "knocked in," or mutated, so that gene expression can be understood in its biological context. Metabolic profiling of cardiac tissue through high resolution NMR spectroscopy in conjunction with multivariate statistics has been used to classify mouse models of cardiac disease. The data sets included metabolic profiles from mouse models of Duchenne muscular dystrophy, two models of cardiac arrhythmia, and one of cardiac hypertrophy. The metabolic profiles demonstrate that the strain background is an important component of the global metabolic phenotype of a mouse, providing insight into how a given gene deletion may result in very different responses in diverse populations. Despite these differences associated with strain, multivariate statistics were capable of separating each mouse model from its control strain, demonstrating that metabolic profiles could be generated for each disease. Thus, this approach is a rapid method of phenotyping mouse models of disease.
We recently reported the importance of Synoviolin in quality control of proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) system and its involvement in the pathogenesis of arthropathy through its anti-apoptotic effect. For further understanding of the role of Synoviolin in vivo, we generated in this study synoviolin-deficient (syno–/–) mice by genetargeted disruption. Strikingly, all fetuses lacking syno died in utero around embryonic day 13.5, although Hrd1p, a yeast orthologue of Synoviolin, is non-essential for survival. Histologically, hypocellularity and aberrant apoptosis were noted in the syno–/– fetal liver. Moreover, definitive erythropoiesis was affected in non-cell autonomous manner in syno–/– embryos, causing death in utero. Cultured embryonic fibroblasts derived from syno–/– mice were more susceptible to endoplasmic reticulum stress-induced apoptosis than those from syno+/+ mice, but the susceptibility was rescued by overexpression of synoviolin. Our findings emphasized the indispensable role of the Synoviolin in embryogenesis. a
Mammalian spermatogenesis is a highly ordered process that occurs in mitotic, meiotic, and postmeiotic phases. The unique mechanisms responsible for this tightly regulated developmental process suggest the presence of an intrinsic genetic program composed of spermatogenic cell-specific genes. In this study, we analyzed the mouse round spermatid UniGene library currently containing 2124 gene-oriented transcript clusters, predicting that 467 of them are testis-specific genes, and systematically identified 28 novel genes with evident testis-specific expression by in silico and in vitro approaches. We analyzed these genes by Northern blot hybridization and cDNA cloning, demonstrating the presence of additional transcript sequences in five genes and multiple transcript isoforms in six genes. Genomic analysis revealed lack of human orthologues for 10 genes, implying a relationship between these genes and male reproduction unique to mouse. We found that all of the novel genes are expressed in developmentally regulated and stage-specific patterns, suggesting that they are primary regulators of male germ cell development.
William H. Stein (1911–1980) graduated from Harvard in 1929 with a major in chemistry. He then spent a year as a graduate student in chemistry at Harvard but transferred to the Department of Biological Chemistry at the College of Physicians and Surgeons, Columbia University, to study biochemistry. He completed his thesis research on the amino acid composition of elastin in 1937 and joined Max Bergmann at The Rockefeller Institute for Medical Research in New York. Stein's initial project with Bergmann was to improve gravimetric methods of amino acid determination. In 1939, Stanford Moore (1913–1982), a graduate of Vanderbilt University who had just earned his Ph.D. from the University of Wisconsin, joined the Bergmann laboratory. In what marked the beginning of one of the longest and most fruitful collaborations in science, the two postdoctoral fellows pooled their efforts to develop the gravimetric methods based on the solubility product of salts of the amino acids into a practical analytical procedure.
Simian virus 40 encodes the oncogenic large T antigen whose functions include the inactivation of tumor suppressor genes. The most well known targets of the T antigen are the retinoblastoma protein and p53. Markus Welcker and Bruce E. Clurman now report that the T antigen also binds to another tumor suppressor: the F-box substrate recognition subunit (Fbw7) of a Skp1/Cullin/F-box protein ubiquitin ligase complex. Interestingly, the F-box protein binds to the T antigen via a decoy phospho-epitope on the antigen's C terminus that mimics a consensus sequence found in the substrates of the F-box. However, unlike its bona fide substrates, the F-box protein does not degrade the T antigen. The authors propose that the T antigen may bind and titrate out the F-box protein, leading to enhanced levels of substrates normally degraded by the ubiquitin ligase complex, such as cyclin E
Advances in understanding aging processes and their consequences are leading to the development of therapies to slow or reverse adverse changes formerly considered to be “normal” aging and processes that underlie multiple age-related conditions. Estimating the effectiveness of candidate aging therapies, whose effects on human aging may require many years to determine, is a particular challenge. Strategies for identifying candidate interventions can be developed through multiple approaches, including the screening of molecular targets and pathways in vitro and in animal models, informed as well by evidence from human genetic and epidemiologic data. A number of recently established programs and networks can serve as resources for such research. For all these research approaches, from in vitro molecular studies to clinical trials, contributions of cell and molecular biology are crucial and offer the prospect of therapeutic advances that address fundamental biological processes as well as the clinically important challenges of aging.
Aging can be defined as progressive functional decline and increasing mortality over time. Here, we review evidence linking aging to nuclear DNA lesions: DNA damage accumulates with age, and DNA repair defects can cause phenotypes resembling premature aging. We discuss how cellular DNA damage responses may contribute to manifestations of aging. We review Sir2, a factor linking genomic stability, metabolism, and aging. We conclude with a general discussion of the role of mutant mice in aging research and avenues for future investigation.
A cost of reproduction, where lifespan and fecundity are negatively correlated, is of widespread occurrence. Mutations in insulin/IGF signaling (IIS) pathways and dietary restriction (DR) can extend lifespan in model organisms but do not always reduce fecundity, suggesting that the link between lifespan and fecundity is not inevitable. Understanding the molecular basis of the cost of reproduction will be informed by elucidation of the mechanisms by which DR and IIS affect these two traits.
We investigated the role played by 2-containing neuronal nicotinic receptors [nicotinic acetylcholine receptors (nAChRs)] in mediating nicotine's side effects in the fetus and newborn. Pregnant WT and mutant mice lacking the 2 nAChR subunit were implanted with osmotic minipumps that delivered either water or a controlled dose of nicotine. Subsequently, we compared the development of the sympathoadrenal system and breathing and arousal reflexes of offspring shortly after birth, a period of increased vulnerability to nicotine exposure. Newborn WT pups exposed to nicotine exhibited all of the deficits associated with maternal tobacco and nicotine use, and linked to poor neonatal outcome: growth restriction, unstable breathing, and impaired arousal and catecholamine biosynthesis. Remarkably similar deficits were detected in pups lacking 2-containing nAChRs. Loss-of-function of these nAChRs consequently reproduces with astonishing fidelity many of the abnormalities caused by perinatal nicotine exposure. We propose that the underlying mechanisms of nicotine's detrimental side effects on a range of crucial defensive reflexes involve loss of function of nAChR subtypes, possibly via activity-dependent desensitization.
Unraveling cause-and-effect relationships in the nervous system is challenging because some biological processes begin stochastically, take a significant amount of time to unfold, and affect small neuronal subpopulations that can be difficult to isolate and measure. Single-cell approaches are slow, subject to user bias, and sometimes too laborious to achieve sample sizes large enough to detect important effects. Here, we describe an automated imaging and analysis system that enables us to follow the fates of individual cells and intracellular proteins over time. Observations can be quantified in a high-throughput manner with minimal user bias. We have adapted survival analysis methods to determine whether and how factors measured during longitudinal analysis predict a particular biological outcome. The ability to monitor complex processes at single-cell resolution quickly, quantitatively, and over long intervals should have wide applications for biology.
Several brightness illusions indicate that borders can affect the perception of surfaces dramatically. In the Cornsweet illusion, two equiluminant surfaces appear to be different in brightness because of the contrast border between them. Here, we report the existence of cells in monkey visual cortex that respond to such an "illusory" brightness. We find that luminance responsive cells are located in color-activated regions (cytochrome oxidase blobs and bridges) of primary visual cortex (V1), whereas Cornsweet responsive cells are found preferentially in the color-activated regions (thin stripes) of second visual area (V2). This colocalization of brightness and color processing within V1 and V2 suggests a segregation of contour and surface processing in early visual pathways and a hierarchy of brightness information processing from V1 to V2 in monkeys.
Human cytomegalovirus (HCMV) persists as a subclinical, lifelong infection in the normal human host, but reactivation from latency in immunocompromised subjects results in serious disease. Latency and reactivation are defining characteristics of the herpesviruses and are key to understanding their biology; however, the precise cellular sites in which HCMV is carried and the mechanisms regulating its latency and reactivation during natural infection remain poorly understood. Here we present evidence, based entirely on direct analysis of material isolated from healthy virus carriers, to show that myeloid dendritic cell (DC) progenitors are sites of HCMV latency and that their ex vivo differentiation to a mature DC phenotype is linked with reactivation of infectious virus resulting from differentiation-dependent chromatin remodeling of the viral major immediate-early promoter. Thus, myeloid DC progenitors are a site of HCMV latency during natural persistence, and there is a critical linkage between their differentiation to DC and transcriptional reactivation of latent virus, which is likely to play an important role in the pathogenesis of HCMV infection.
Thyroid hormone controls remodeling of the tadpole intestine during the climax of amphibian metamorphosis. In 8 days, the Xenopus laevis tadpole intestine shortens in length by 75%. Simultaneously, the longitudinal muscle fibers contract by about the same extent. The radial muscle fibers also shorten as the diameter narrows. Many radial fibers undergo programmed cell death. We conclude that muscle remodeling and contraction play key roles in the shortening process. Shortening is accompanied by a temporary "heaping" of the epithelial cells into many layers at climax. Cells that face the lumen undergo apoptosis. By the end of metamorphosis, when the epithelium is folded into crypts and villi, the epithelium is a single-cell layer once again. Throughout this remodeling, DNA replication occurs uniformly throughout the epithelium, as do changes in gene expression. The larval epithelial cells as a whole, rather than a subpopulation of stem cells, are the progenitors of the adult epithelial cells.
In mammalian cells, the nuclear export receptor, Exportin 5 (Exp5), exports pre-microRNAs (pre-miRNAs) as well as tRNAs into the cytoplasm. In this study, we examined the function of HASTY (HST), the Arabidopsis ortholog of Exp5, in the biogenesis of miRNAs and tRNAs. In contrast to mammals, we found that miRNAs exist as single-stranded 20- to 21-nt molecules in the nucleus in Arabidopsis. This observation is consistent with previous studies indicating that proteins involved in miRNA biogenesis are located in the nucleus in Arabidopsis. Although miRNAs exist in the nucleus, a majority accumulate in the cytoplasm. Interestingly, loss-of-function mutations in HST reduced the accumulation of most miRNAs but had no effect on the accumulation of tRNAs and endogenous small interfering RNAs, or on transgene silencing. In contrast, a mutation in PAUSED (PSD), the Arabidopsis ortholog of the tRNA export receptor, Exportin-t, interfered with the processing of tRNA-Tyr but did not affect the accumulation or nuclear export of miRNAs. These results demonstrate that HST and PSD do not share RNA cargos in nuclear export and strongly suggest that there are multiple nuclear export pathways for these small RNAs in Arabidopsis.
In human breast cancer, overexpression of the protooncogene MET is strongly associated with poor prognosis and high risk of metastasis. It stands out as a reliable prognostic indicator of survival and defines a set of tumors exclusive of those that express HER2 or hormone receptors. Studies have shown that overexpression of mutant forms of MET cause cancer in mice. However, MET mutations have not been found in human breast cancer, and the consequences of overexpression of normal MET are unknown. To investigate the role of MET and other putative oncogenes in breast cancer, we developed an experimental system that involves retroviral delivery of genes into primary mammary epithelial cells, followed by transplantation of the transduced cells into mammary fat pads. Using this approach, we found that overexpression of wild-type MET leads to the development of nonprogressive neoplasms. The lesions progressed to mammary adenocarcinoma when a second protooncogene, MYC, was overexpressed, indicating that MET and MYC cooperate in mammary tumorigenesis. Both the nonprogressive neoplasms and adenocarcinomas display characteristics consistent with transformation and expansion of mammary progenitor cells. The approach described here should provide a useful model with which to efficiently test effects of various genes on tumor development in the breast.
Through a genetic screen using myosin-like protein strains mlp1 mlp2 and biochemical purification, we identified a complex of eight proteins, each required for growth and DNA repair in Saccharomyces cerevisiae. Among the subunits are Mms21 that contains a putative Siz/PIAS (protein inhibitor of activated signal transducer and activator of transcription) RING domain characteristic of small ubiquitin-like modifier (SUMO) ligases, two structural-maintenance-of-chromosome (Smc) proteins, Smc5 and Smc6, and a protein that contains an ubiquitin ligase signature domain. We show that these proteins colocalized to several distinct nuclear foci. Biochemical and genetic data demonstrated that Mms21 indeed functions as a SUMO ligase and that this activity requires the Siz/PIAS (protein inhibitor of activated signal transducer and activator of transcription) RING domain. The substrates for this SUMO ligase include a subunit of the octameric complex, Smc5, and the DNA repair protein Yku70. We further show that the abolition of the SUMO E3 activity of Mms21 leads to such disparate phenotypes as DNA damage sensitivity, defects in nucleolar integrity and telomere clustering, silencing, and length regulation. We propose that Mms21 sumoylates proteins involved in these diverse processes and that the other members of the complex, particularly Smc5/6, facilitate proper substrate sumoylation by localizing Mms21 to specific chromosomal regions.
p53, the tumor suppressor protein, functions as a dimer of dimers. However, how the tetramer binds to the DNA is still an open question. In the crystal structure, three copies of the p53 monomers (containing chains A, B, and C) were crystallized with the DNA-consensus element. Although the structure provides crucial data on the p53-DNA contacts, the active oligomeric state is unclear because the two dimeric (A-B and B-C) interfaces present in the crystal cannot both exist in the tetramer. Here, we address the question of which of these two dimeric interfaces may be more biologically relevant. We analyze the sequence and structural properties of the p53-p53 dimeric interfaces and carry out extensive molecular dynamics simulations of the crystal structures of the human and mouse p53 dimers. We find that the A-B interface residues are more conserved than those of the B-C. Molecular dynamics simulations show that the A-B interface can provide a stable DNA-binding motif in the dimeric state, unlike B-C. Our results indicate that the interface between chains A-B in the p53-DNA complex constitutes a better candidate for a stable biological interface, whereas the B-C interface is more likely to be due to crystal packing. Thus, they have significant implications toward our understanding of DNA binding by p53 as well as p53-mediated interactions with other proteins.
Replication of DNA requires helicase and primase activities as part of a primosome assembly. In bacteriophage T4, helicase and primase are separate polypeptides for which little structural information is available and whose mechanism of association within the primosome is not yet understood. Three-dimensional structural information is provided here by means of reconstructions from electron microscopic images. Structures have been calculated for complexes of each of these proteins with ssDNA in the presence of MgATPS. Both the helicase (gp41) and primase (gp61) complexes are asymmetric hexagonal rings. The gp41 structure suggests two distinct forms that have been termed "open" and "closed." The gp61 structure is clearly a six-membered ring, which may be a trimer of dimers or a traditional hexamer of monomers. This structure provides conclusive evidence for an oligomeric primase-to-ssDNA stoichiometry of 6:1.
Medical scientists have always sought to uncover fundamental mechanistic explanations for human disease and to use this information to predict patient outcome and devise specific therapeutics. Although monogenetic diseases have been elucidated, the more common disorders often have complex or heterogeneous origins and involve the failure of multiple systems before disease is manifested. Breast cancer is an example. Many factors and genes have been implicated in the initiation of the disease (e.g., BRCA1, PTEN, P53, hormone exposure, irradiation, free radicals, etc.), but mortality is due to metastatic disease that requires invasion, evasion of immune surveillance, implantation in ectopic sites, continuous replication, cell migration, and angiogenesis (1). Capturing all of the genetic components that support these cellular processes has been a challenge for cancer cell biology.
Sensitivity to chemotherapeutic agents depends on the specific molecular state of the circadian clock.
Temporal organization of biological processes is a critical feature of life. In addition to orderly, carefully timed developmental processes, precise temporal organization is also observed on a daily time scale. On Earth, organisms ranging from bacteria to humans have evolved internal timekeeping mechanisms called circadian clocks that control biochemistry, physiology, and behavior to coordinate with the 24-hour day (1–3). Circadian clocks have long been known to drive rhythms in such things as activity, sleep, and hormone secretion, but in recent years it has become clear that these clocks influence the normal homeostasis of organisms on a profound scale, controlling processes as diverse as cell cycle regulation, cell signaling, metabolic events, and cognitive function. Disruption of clock function in mammals results in abnormalities in many physiological functions, resulting in increases in risks for cardiovascular and gastrointestinal diseases, sleep abnormalities, and cancer (reviewed in refs. 1 and 4). In a recent issue of PNAS, Gorbacheva et al. (5) describe a mechanistic link between the circadian clock and sensitivity to the chemotherapeutic agent cyclophosphamide (CY).
Many mammalian and plant pathogenic bacteria inject virulence effector proteins into host cells by means of the type III secretion system (TTSS) (1). Effector proteins attack the host innate immune system, modify cytoskeleton and membranes, or alter vesicle trafficking (2, 3). The collective action of these proteins promotes bacterial entry into, growth and movement within, and dissemination from infected host cells/tissues. The full complement of TTSS effectors is not known for most bacterial pathogens; their identification remains a crucial step toward a comprehensive understanding of bacterial pathogenesis, host range, and pathogen evolution. In a recent issue of PNAS, Chang et al. (4) describe a powerful method for discovering TTSS effectors. Using this method, those authors identified two nearly complete repertoires of TTSS effectors from two pathovars (specific to different plant species) of the plant pathogen Pseudomonas syringae. Here, we discuss this article in the context of earlier efforts to discover TTSS effectors in plant pathogens.
