e shtunë, janar 29, 2005

Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs

Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Although a preventive vaccine is available, the therapeutic options for chronically infected patients are limited. It has been shown that RNA interference can prevent HBV gene expression and replication in vivo when HBV expression vectors are delivered simultaneously with small interfering RNA (siRNA) or siRNA expression constructs. However, the therapeutic potential of siRNAs to interrupt ongoing HBV replication in vivo has not been established. Here, we show that expression of HBV-specific siRNAs in the liver of HBV transgenic mice by recombinant adenoviruses can suppress preexisting HBV gene expression and replication to almost undetectable levels for at least 26 days. These results demonstrate that efficiently delivered siRNAs should be able to silence HBV in chronically infected patients.

Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans

Chronic infection with hepatitis B virus (HBV) is associated with the majority of hepatocellular carcinoma (HCC). The diagnosis of HCC is usually made in the late stages of the disease, when treatment options are limited and prognosis is poor. We therefore have developed a method of glycoproteomic analysis in an attempt to discover serum markers that can assist in the early detection of HBV-induced liver cancer. Briefly, a comparative method for analysis of oligosaccharides released from serum glycoproteins and for recovery and identification of proteins with aberrant glycosylation, as a function of cancer diagnosis, is described. The model we have used is the woodchuck (Marmota monax), which shares similarities in the glycosylation pattern associated with liver proteins in human HCC. In this report, we show that woodchucks diagnosed with HCC have dramatically higher levels of serum-associated core -1,6-linked fucose, as compared with woodchucks without a diagnosis of HCC. The coupling of this methodology with 2D gel proteomics has permitted the identification of several glycoproteins with altered glycosylation as a function of cancer. One such glycoprotein, Golgi Protein 73 (GP73), was found to be elevated and hyperfucosylated in animals with HCC. Further, the study showed GP73 to be elevated in the serum of people with a diagnosis of HCC, providing a validation of our approach. The potential of this technology for biomarker discovery and the implications of increased levels of GP73 in liver cancer are discussed.

Prion protein NMR structures of cats, dogs, pigs, and sheep

The NMR structures of the recombinant cellular form of the prion proteins (PrPC) of the cat (Felis catus), dog (Canis familiaris), and pig (Sus scrofa), and of two polymorphic forms of the prion protein from sheep (Ovis aries) are presented. In all of these species, PrPC consists of an N-terminal flexibly extended tail with 100 amino acid residues and a C-terminal globular domain of 100 residues with three -helices and a short antiparallel -sheet. Although this global architecture coincides with the previously reported murine, Syrian hamster, bovine, and human PrPC structures, there are local differences between the globular domains of the different species. Because the five newly determined PrPC structures originate from species with widely different transmissible spongiform encephalopathy records, the present data indicate previously uncharacterized possible correlations between local features in PrPC three-dimensional structures and susceptibility of different mammalian species to transmissible spongiform encephalopathies.

In vitro characterization of IroB, a pathogen-associated C-glycosyltransferase

Pathogenic strains of Escherichia coli and Salmonella enterica modify the tricatecholic siderophore enterobactin (Ent) by glucosylation of three aryl carbon atoms, a process controlled by the iroA locus [Hantke, K., Nicholson, G., Rabsch, W. & Winkelmann, G. (2003) Proc. Natl. Acad. Sci. USA 100, 3677–3682]. Here, we report the purification of the IroB protein and its characterization as the Ent C-glucosyltransferase. IroB transfers glucosyl groups from uridine-5'-diphosphoglucose to C5 of one, two, or three of the 2,3-dihydroxybenzoyl units of Ent to yield monoglucosyl-C-Ent (MGE), diglucosyl-C-Ent (DGE), and triglucosyl-C-Ent (TGE). DGE, also known as salmochelin S4, and macrolactone-opened derivatives have been isolated from the culture broths of S. enterica and uropathogenic E. coli [Bister, B., Bischoff, D., Nicholson, G. J., Valdebenito, M., Schneider, K., Winkelmann, G., Hantke, K. & Sussmuth, R. D. (2004) Biometals 17, 471–481], but MGE and TGE have not been reported previously. IroB has a kcat of 10 min-1 for the first C-glucosylation and is distributive, with sequential conversion and buildup of MGE and then DGE. The C5 to C1' regio-selectivity of the 2,3-dihydroxybenzoyl-glucose linkage at all three rings of TGE suggests a C5 carbanion, para to the C2 phenolate oxygen, as the carbon nucleophile in this novel enzymatic C-glucosylation.

The carboligation reaction of acetohydroxyacid synthase II: Steady-state intermediate distributions in wild type and mutants by NMR

The thiamin diphosphate (ThDP)-dependent enzyme acetohydroxyacid synthase (AHAS) catalyzes the first common step in branched-chain amino acid biosynthesis. By specific ligation of pyruvate with the alternative acceptor substrates 2-ketobutyrate and pyruvate, AHAS controls the flux through this branch point and determines the relative rates of synthesis of isoleucine, valine, and leucine, respectively. We used detailed NMR analysis to determine microscopic rate constants for elementary steps in the reactions of AHAS II and mutants altered at conserved residues Arg-276, Trp-464, and Met-250. In Arg276Lys, both the condensation of the enzyme-bound hydroxyethyl-ThDP carbanion/enamine (HEThDP) with the acceptor substrates and acetohydroxyacid release are slowed several orders of magnitude relative to the wild-type enzyme. We propose that the interaction of the guanidinium moiety of Arg-264 with the carboxylate of the acceptor ketoacid provides an optimal alignment of substrate and HEThDP orbitals in the reaction trajectory for acceptor ligation, whereas its interaction with the carboxylate of the covalent HEThDP-acceptor adduct plays a similar role in product release. Both Trp-464 and Met-250 affect the acceptor specificity. The high preference for ketobutyrate in the wild-type enzyme is lost in Trp464Leu as a consequence of similar forward rate constants of carboligation and product release for the alternative acceptors. In Met250Ala, the turnover rate is determined by the condensation of HEThDP with pyruvate and release of the acetolactate product, whereas the parallel steps with 2-ketobutyrate are considerably faster. We speculate that the specificity of carboligation and product liberation may be cumulative if the former is not completely committed.

O-fucosylation of notch occurs in the endoplasmic reticulum

LADII/CDG 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 reduction of fucosylated antigens on the cell surface. A recent study using fibroblasts from LADII/CDG IIc patients suggested that while 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) Differential terminal fucosylation of N-linked glycans versus protein O-fucosylation in Leukocyte adhesion deficiency type II (CDG IIc) 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), responsible for adding O-fucose to EGF repeats. Our analysis reveals that, unlike all other known fucosyltransferases, O-FucT-1 is a soluble protein that localizes to the 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 EGF repeats, together with this unique localization, suggests that it may play a role in quality cotrol.

Novel polyLacdiNAc and fucosylated polyLacdiNAc N-glycans from mammalian cells expressing 1,4-GalNAcT and 1,3-FucT

Glycans containing the GalNAc1-4GlcNAc (LacdiNAc or LDN) motif are expressed by many invertebrates, but this motif also occurs in vertebrates, and is found on several mammalian glycoprotein hormones. This motif contrasts with the more commonly occurring Gal1-4GlcNAc (LacNAc or LN) motif. To better understand LDN biosynthesis and regulation, we stably expressed the cDNA encoding the C. elegans 1,4N-acetylgalactosaminyltransferase (GalNAcT), which generates LDN in vitro, in Chinese hamster ovary (CHO) Lec8 cells, to establish L8-GalNAcT CHO cells. The glycan structures from these cells were determined by mass spectrometry and linkage analysis. The L8-GalNAcT cell line produces complex-type N-glycans quantitatively bearing LDN structures on their antennae. Unexpectedly, most of these complex-type N-glycans contain novel "polyLDN" structures consisting of repeating LDN motifs [-3GalNAc1-4GlcNAc1-]n. These novel structures are in contrast to the well-known poly-LN structures consisting of repeating LN motifs [-3Gal1-4GlcNAc1-]n. We also stably expressed human 1,3-fucosyltransferase IX (FucT 9) in the L8-GalNAcT cells to establish a new cell line L8-GalNAcT-FucT. These cells produce complex-type N-glycans with 1,3-fucosylated LDN (LDNF) GalNAc1-4(Fuc1-3)GlcNAc1-R, as well as novel "polyLDNF" structures [-3GalNAc1-4(Fuc1-3)GlcNAc1-]n. The ability of these cell lines to generate glycoprotein hormones with LDN-containing N-glycans was studied by expressing a recombinant form of the common -subunit in L8-GalNAcT cells. The -subunit N-glycans carried LDN structures, which were further modified by co-expression of the human GalNAc 4-sulfotransferase I, which generates SO4-4GalNAc1-4GlcNAc-R. Thus, the generation of these stable mammalian cells will facilitate future studies on the biological activities and properties of LDN-related structures in glycoproteins.

Imaging bacterial infections with radiolabeled 1-(2'deoxy-2'-fluoro--D-arabinofuranosyl)-5-iodouracil

Bacterial infections provide diagnostic dilemmas that could be enlightened by modern imaging technologies. We have developed a simple method for imaging bacterial infections in mice that relies on the phosphorylation and trapping of the thymidine kinase (TK) substrate 1-(2'-deoxy-2'-fluoro--D-arabinofuranosyl)-5-[125I]iodouracil ([125I]FIAU) within bacteria. FIAU was found to inhibit the growth of WT Escherichia coli but not a TK- strain, indicating that WT E. coli could metabolize this compound. In silico analyses demonstrated that all pathogenic strains of bacteria whose genomes have been sequenced contain a TK gene highly homologous to the E. coli TK. Accordingly, we demonstrated that localized infections caused by representatives of five genera of bacteria could be readily imaged with [125I]FIAU. Such imaging provides a general method for the diagnosis of localized bacterial infections that could be translatable to the clinic.

Genetic and molecular identification of genes required for female gametophyte development and function in Arabidopsis

The plant life cycle involves an alternation of generations between sporophyte and gametophyte. Currently, the genes and pathways involved in gametophytic development and function in flowering plants remain largely unknown. A large-scale mutant screen of Ds transposon insertion lines was employed to identify 130 mutants of Arabidopsis thaliana with defects in female gametophyte development and function. A wide variety of mutant phenotypes were observed, ranging from defects in different stages of early embryo sac development to mutants with apparently normal embryo sacs, but exhibiting defects in processes such as pollen tube guidance, fertilization or early embryo development. Unexpectedly, nearly half of the mutants isolated in this study were found to be primarily defective in post-fertilization processes dependent on the maternal allele, suggesting that genes expressed from the female gametophyte or the maternal genome play a major role in the early development of plant embryos. Sequence identification of the genes disrupted in the mutants revealed genes involved in protein degradation, cell death, signal transduction and transcriptional regulation required for embryo sac development, fertilization and early embryogenesis. These results provide a first comprehensive overview of the genes and gene products involved in female gametophyte development and function within a flowering plant

Transcriptional program controlled by the floral homeotic gene AGAMOUS during early organogenesis

Floral organs, whose identity is determined by specific combinations of homeotic genes, originate from a group of undifferentiated cells called the floral meristem. In Arabidopsis, the homeotic gene AGAMOUS (AG) terminates meristem activity and promotes development of stamens and carpels. To understand the program of gene expression activated by AG, we followed genome-wide expression during early stamen and carpel development. The AG target genes included most genes for which mutant screens revealed a function downstream of AG. Novel targets were validated by in situ hybridisation and binding to AG in vitro and in vivo. Transcription factors formed a large fraction of AG targets, suggesting that during early organogenesis, much of the genetic program is concerned with elaborating gene expression patterns. The results also suggest that AG and other homeotic proteins with which it interacts (SEPALLATA3, APETALA3, PISTILLATA) are coordinately regulated in a positive-feedback loop to maintain their own expression, and that AG activates biosynthesis of gibberellin, which has been proposed to promote the shift from meristem identity to differentiation.

e premte, janar 28, 2005

Metabolism in the Era of Molecular Biology

Richard W. Hanson

Prologue
I have spent a lifetime in the area of intermediary metabolism and have seen it move from the center of biochemistry to the backwater of our science in a very short period, only to be re-discovered by a new generation of biologists. By 1970, the writing was on the wall for metabolism; it was largely considered a "mature area," lacking excitement; molecular biology was the area of the future. A sure sign of this was that graduate students in biochemistry (always first to spot the trends) almost never selected their thesis research in metabolism. The course in intermediary metabolism that I taught was dropped from the curriculum of our graduate education program; our students were expected to learn all they needed to know about metabolism as undergraduates before they attended graduate school. After all, as a graduate student once said to me, "the great problems in metabolic research have been solved." As one could easily predict, there is currently a shortage of scientists who truly understand metabolism and its regulation.

A Clock by Another Mechanism

Science, Vol 307, 14 January, 2005

The circadian clock enables diverse organisms to adapt their life to light-dark alterations of the day. It has been thought that a universal clock mechanism that generates and maintains self-sustainable oscillations, or rhythms, is based on a translation-transcription autoregulatory feedback model of core clock elements. Tomita et al. (p. 251) demonstrate that this model does not apply to cyanobacteria, the simplest organism known to show a circadian rhythm. Oscillation in the phosporylation of a core clock protein KaiC persisted in the dark, in the absence of any translation-transcription loop. The basis of the rhythm lies in the autoregulation of phosphorylation by KaiC itself. Thus, the clock model in eukaryotes may not apply to cyanobacteria under certain conditions.

Self-Propagating, Molecular-Level Polymorphism in Alzheimer's ß-Amyloid Fibrils

Science, Vol 307, Issue 5707, 262-265 , 14 January 2005

Amyloid fibrils commonly exhibit multiple distinct morphologies in electron microscope and atomic force microscope images, often within a single image field. By using electron microscopy and solid-state nuclear magnetic resonance measurements on fibrils formed by the 40-residue ß-amyloid peptide of Alzheimer's disease (Aß1–40), we show that different fibril morphologies have different underlying molecular structures, that the predominant structure can be controlled by subtle variations in fibril growth conditions, and that both morphology and molecular structure are self-propagating when fibrils grow from preformed seeds. Different Aß1–40 fibril morphologies also have significantly different toxicities in neuronal cell cultures. These results have implications for the mechanism of amyloid formation, the phenomenon of strains in prion diseases, the role of amyloid fibrils in amyloid diseases, and the development of amyloid-based nano-materials.

The Genome of the Basidiomycetous Yeast and Human Pathogen Cryptococcus Neoformans

Science, 14 Januar

Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis and an opportunistic human pathogen of global importance. We have sequenced its ~20 Mb genome, which contains ~6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes.

The Selective Cause of an Ancient Adaptation

Science online 13 January 2005
Guoping Zhu 1, G. Brian Golding 2, Antony M. Dean 3*

Phylogenetic analysis reveals that NADP use by prokaryotic isocitrate dehydrogenase arose around the time eukaryotic mitochondria first appeared, some 3.5 billion years ago. We replaced the wildtype gene encoding the NADP-dependent isocitrate dehydrogenase of Escherichia coli by an engineered one possessing the ancestral NAD-dependent phenotype. The engineered enzyme is disfavored during competition for acetate. The selection intensifies in genetic backgrounds where other sources of reduced NADP have been removed. A survey of sequenced prokaryotic genomes reveals that those encoding isocitrate lyase, which is essential for growth on acetate, always have an NADP-dependent isocitrate dehydrogenase. Those with only an NAD-dependent isocitrate dehydrogenase never have isocitrate lyase. Hence, the NADP-dependence of prokaryotic isocitrate dehydrogenase is an ancient adaptation to anabolic demand for reduced NADP during growth on acetate.

e enjte, janar 27, 2005

Drosophila synapse formation

BMC Biology 2005, 3:1

Discs-large, the Drosophila relative of mammalian MAGUKs proteins, is localized postsynaptically only when presynaptic neurons contact the postsynaptic cell, and controls postsynaptic glutamate receptor composition by stabilizing the receptor's GluRIIB subunits.


Histone Demethylation Mediated by the Nuclear Amine Oxidase Homolog LSD1

Benoit Coulombe: "This article reports for the first time the isolation of a protein, LSD1, that can demethylate histone methyl lysine residues. LSD1 is specific for H3-K4 demethylation, can only act on mono- or di-methylated lysines and is involved in transcriptional repression. Likely, this discovery will open the way to the identification of other histone demethylases having important regulatory functions."


Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, Casero RA, Shi Y, Cell 2004, Dec 29 119(7):941-53

Abstract

Posttranslational modifications of histone N-terminal tails impact chromatin structure and gene transcription. While the extent of histone acetylation is determined by both acetyltransferases and deacetylases, it has been unclear whether histone methylation is also regulated by enzymes with opposing activities. Here, we provide evidence that LSD1 (KIAA0601), a nuclear homolog of amine oxidases, functions as a histone demethylase and transcriptional corepressor. LSD1 specifically demethylates histone H3 lysine 4, which is linked to active transcription. Lysine demethylation occurs via an oxidation reaction that generates formaldehyde. Importantly, RNAi inhibition of LSD1 causes an increase in H3 lysine 4 methylation and concomitant derepression of target genes, suggesting that LSD1 represses transcription via histone demethylation. The results thus identify a histone demethylase conserved from S. pombe to human and reveal dynamic regulation of histone methylation by both histone methylases and demethylases.

Identification of arthritis genes

Arthritis Res Ther 2005, 7:R196-R207

Investigation of the entire mouse genome by DNA mircoarray technology has led to identification of 37 arthritis 'signature' genes, which are expressed prior to the onset of clinical symptoms.

Computational prediction of human metabolic pathways from the complete human genome

Pedro Romero1, 3 , Jonathan Wagg1 , Michelle L Green1 , Dale Kaiser2 , Markus Krummenacker1 and Peter D Karp1

Abstract
We present a computational pathway analysis of the human genome that assigns enzymes encoded therein to predicted metabolic pathways. Pathway assignments place genes in their larger biological context, and are a necessary first step toward quantitative modeling of metabolism.

Our analysis assigns 2,709 human enzymes to 896 bioreactions; 622 of the enzymes are assigned roles in 135 predicted metabolic pathways. The predicted pathways closely match the known nutritional requirements of humans. This analysis identifies probable omissions in the human genome annotation in the form of 203 pathway holes (missing enzymes within the predicted pathways). We have identified putative genes to fill 25 of these holes. The predicted human metabolic map is described by a Pathway/Genome Database called HumanCyc, which is available at http://HumanCyc.org/. We describe the generation of HumanCyc, and present an analysis of the human metabolic map. For example, we compare the predicted human metabolic pathway complement to the pathways of Escherichia coli and Arabidopsis thaliana and identify 35 pathways that are shared among all three organisms.

Our analysis elucidates a significant portion of the human metabolic map, and also indicates probable unidentified genes in the genome. HumanCyc provides a genome-based view of human nutrition that associates the essential dietary requirements of humans with a set of metabolic pathways whose existence is supported by the human genome. The database places many human genes in a pathway context, thereby facilitating analysis of gene expression, proteomics, and metabolomics datasets through a publicly available online tool called the Omics Viewer.

Natural selection in humans

A chromosomal inversion conveys a reproductive advantage in Iceland, researchers report | By Charles Q Choi
For the first time in humans, researchers have discovered a large chromosomal rearrangement that bears the mark of natural selection, they report in the February issue of Nature Genetics.
The rearrangement, a 900-kilobase inversion polymorphism, appears in two distinct lineages, H1 and H2, that have diverged for as long as 3 million years with no evidence of having recombined. The H2 lineage—which is rare in Africans, almost nonexistent in East Asians, but found in 20% of Europeans—appears to undergo positive selection in Iceland, with carrier females having 3.2% more children per generation and higher recombination rates.
"This raises the question of how many such inversions remain to be discovered in the genome and what their effects might be," study co-author Kari Stefansson, chief executive officer of biopharmaceutical company deCODE Genetics in Reykjavik, Iceland, told The Scientist. The mechanisms for the increased fertility and recombination rates remain uncertain and point for continued study, he added.
"If you told me beforehand this story without showing me the data, I'd think there was a 10,000 to 1 chance of it being correct, but the data are very, very strong," David Reich of Harvard Medical School, who did not participate in this study, told The Scientist. "They carefully attempted to control for environment, for differences between people with few children and many, between north and south, age. It's difficult to imagine this can be an artifact."