Twenty AdoMet-dependent methyltransferases (MTases) have been characterized structurally by X-ray crystallography and NMR. These include seven DNA MTases, five RNA MTases, four protein MTases and four small molecule MTases acting on the carbon, oxygen or nitrogen atoms of their substrates. The MTases share a common core structure of a mixed seven-stranded beta-sheet (6 downward arrow 7 upward arrow 5 downward arrow 4 downward arrow 1 downward arrow 2 downward arrow 3 downward arrow) referred to as an 'AdoMet-dependent MTase fold', with the exception of a protein arginine MTase which contains a compact consensus fold lacking the antiparallel hairpin strands (6 downward arrow 7 upward arrow). The consensus fold is useful to identify hypothetical MTases during structural proteomics efforts on unannotated proteins. The same core structure works for very different classes of MTase including those that act on substrates differing in size from small molecules (catechol or glycine) to macromolecules (DNA, RNA and protein). DNA MTases use a 'base flipping' mechanism to deliver a specific base within a DNA molecule into a typically concave catalytic pocket. Base flipping involves rotation of backbone bonds in double-stranded DNA to expose an out-of-stack nucleotide, which can then be a substrate for an enzyme-catalyzed chemical reaction. The phenomenon is fully established for DNA MTases and for DNA base excision repair enzymes, and is likely to prove general for enzymes that require access to unpaired, mismatched or damaged nucleotides within base-paired regions in DNA and RNA. Several newly discovered MTase families in eukaryotes (DNA 5mC MTases and protein arginine and lysine MTases) offer new challenges in the MTase field.

Six non-clonally related enterobacterial isolates producing a same extended-spectrum beta-lactamase CTX-M-15 were isolated in 1999 from patients hospitalized in a New Delhi hospital. CTX-M-15 differed from CTX-M-3 by an asparagine to glycine substitution in position ABL238. Its gene was located on large plasmids varying in size. In each case, a same insertion sequence ISEcp1 was identified upstream of the 5' end of bla(CTX-M-15). Typical -35 and -10 promoter sequences of Enterobacteriaceae were identified in the 3' end of ISEcp1. The location of ISEcp1 upstream of plasmid-mediated CTX-M-type beta-lactamase genes may contribute to their spread or/and their expression.

Different short-chain dehydrogenases are distantly related, constituting a protein family now known from at least 20 separate enzymes characterized, but with extensive differences, especially in the C-terminal third of their sequences. Many of the first known members were prokaryotic, but recent additions include mammalian enzymes from placenta, liver and other tissues, including 15-hydroxyprostaglandin, 17 beta-hydroxysteroid and 11 beta-hydroxysteroid dehydrogenases. In addition, species variants, isozyme-like multiplicities and mutants have been reported for several of the structures. Alignments of the different enzymes reveal large homologous parts, with clustered similarities indicating regions of special functional/structural importance. Several of these derive from relationships within a common type of coenzyme-binding domain, but central-chain patterns of similarity go beyond this domain. Total residue identities between enzyme pairs are typically around 25%, but single forms deviate more or less (14-58%). Only six of the 250-odd residues are strictly conserved and seven more are conserved in all but single cases. Over one third of the conserved residues are glycine, showing the importance of conformational and spatial restrictions. Secondary structure predictions, residue distributions and hydrophilicity profiles outline a common, N-terminal coenzyme-binding domain similar to that of other dehydrogenases, and a C-terminal domain with unique segments and presumably individual functions in each case. Strictly conserved residues of possible functional interest are limited, essentially only three polar residues. Asp64, Tyr152 and Lys156 (in the numbering of Drosophila alcohol dehydrogenase), but no histidine or cysteine residue like in the completely different, classical medium-chain alcohol dehydrogenase family. Asp64 is in the suggested coenzyme-binding domain, whereas Tyr152 and Lys156 are close to the center of the protein chain, at a putative inter-domain, active-site segment. Consequently, the overall comparisons suggest the possibility of related mechanisms and domain properties for different members of the short-chain family.

Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. The first and rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL, previously known as gamma-glutamylcysteine synthetase). GCL is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits that are expressed from different genes. GCLC catalyzes a unique gamma-carboxyl linkage from glutamate to cysteine and requires ATP and Mg(++) as cofactors in this reaction. GCLM increases the V(max) and K(cat) of GCLC, decreases the K(m) for glutamate and ATP, and increases the K(i) for GSH-mediated feedback inhibition of GCL. While post-translational modifications of GCLC (e.g. phosphorylation, myristoylation, caspase-mediated cleavage) have modest effects on GCL activity, oxidative stress dramatically affects GCL holoenzyme formation and activity. Pyridine nucleotides can also modulate GCL activity in some species. Variability in GCL expression is associated with several disease phenotypes and transgenic mouse and rat models promise to be highly useful for investigating the relationships between GCL activity, GSH synthesis, and disease in humans.

BACKGROUND

The hypofunction of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors has been implicated in the pathophysiology of schizophrenia. Several lines of evidence suggest that D-serine may function as an endogenous agonist of the glycine site of the NMDA receptor. The aim of this study was to examine whether serum levels of D- and L-serine in patients with schizophrenia are different from those of healthy controls.

METHODS

Forty-two patients with schizophrenia and 42 age- and sex-matched healthy controls were enrolled in this study. Symptoms were assessed using the Brief Psychiatric Rating Scale. Serum levels of total serine and D- and L-serine were measured by high-performance liquid chromatography.

RESULTS

Serum levels of D-serine in the patients with schizophrenia were significantly (z = -3.30, P =.001) lower than those of healthy controls. In contrast, serum levels of total (D and L) serine (z = -2.40, P =.02) and L-serine (z = -2.49, P =.01) in the schizophrenic patients were significantly higher than those of controls. In addition, the percentage of D-serine in the total serine in the schizophrenic patients was significantly (z = -4.78, P<.001) lower than that of controls, suggesting that the activity of serine racemase, an enzyme catalyzing the formation of D-serine from L-serine, may have been reduced in the schizophrenic patients.

CONCLUSIONS

Reduced levels of D-serine may play a role in the pathophysiology of schizophrenia, and serum D- and L-serine levels might provide a measurable biological marker for schizophrenia.

MN is a transmembrane glycoprotein that has been detected in HeLa cells and in some human carcinomas. The expression of MN protein in HeLa cells is regulated by cell density. In HeLa x fibroblast cell hybrids its expression correlates with tumorigenicity. Using a specific monoclonal antibody we have identified a cDNA clone coding for MN. Analysis of the deduced amino acid sequence revealed strong structural homology between the central region of the MN protein and carbonic anhydrases (CA). MN sequence retains the conserved zinc-binding site as well as the enzyme's active center. In accord with these findings, MN protein from HeLa cells was found to bind zinc and to have carbonic anhydrase activity. The N-terminal region of MN shares some similarity with DNA binding proteins of the helix-loop-helix (HLH) family, and the protein was found to have affinity for DNA by DNA-cellulose chromatography. The region between the CA-like domain and the putative HLH domain is rich in imperfect repeats of serine, proline, glycine and acidic residues with few hydrophobic amino acids, resembling thus an activation region of transcription factors. The fact that MN protein is detectable in several types of human carcinomas, but not in corresponding non-cancerous tissues, suggests its possible role in neoplasia. In addition, the analysis of biological consequences of MN expression of NIH3T3 cells provides the evidence in favour of MN protein involvement in control of cell proliferation and transformation.

Metabolomic profiling and the integration of whole-genome genetic association data has proven to be a powerful tool to comprehensively explore gene regulatory networks and to investigate the effects of genetic variation at the molecular level. Serum metabolite concentrations allow a direct readout of biological processes, and association of specific metabolomic signatures with complex diseases such as Alzheimer's disease and cardiovascular and metabolic disorders has been shown. There are well-known correlations between sex and the incidence, prevalence, age of onset, symptoms, and severity of a disease, as well as the reaction to drugs. However, most of the studies published so far did not consider the role of sexual dimorphism and did not analyse their data stratified by gender. This study investigated sex-specific differences of serum metabolite concentrations and their underlying genetic determination. For discovery and replication we used more than 3,300 independent individuals from KORA F3 and F4 with metabolite measurements of 131 metabolites, including amino acids, phosphatidylcholines, sphingomyelins, acylcarnitines, and C6-sugars. A linear regression approach revealed significant concentration differences between males and females for 102 out of 131 metabolites (p-values<3.8×10(-4); Bonferroni-corrected threshold). Sex-specific genome-wide association studies (GWAS) showed genome-wide significant differences in beta-estimates for SNPs in the CPS1 locus (carbamoyl-phosphate synthase 1, significance level: p<3.8×10(-10); Bonferroni-corrected threshold) for glycine. We showed that the metabolite profiles of males and females are significantly different and, furthermore, that specific genetic variants in metabolism-related genes depict sexual dimorphism. Our study provides new important insights into sex-specific differences of cell regulatory processes and underscores that studies should consider sex-specific effects in design and interpretation.

Collagen-rich extracellular matrices are abundant and ubiquitous in the mammalian body. Collagens are not only essential for the mechanical stability of tissues, but are also intimately involved in controlling cell behaviour. The hallmark of collagens is a triple helix made up of polypeptide chains containing glycine-X-Y repeats. A structurally and functionally diverse group of cell surface receptors mediates the recognition of triple-helical collagen: integrins, discoidin domain receptors, glycoprotein VI, leukocyte-associated IG-like receptor-1, and members of the mannose receptor family. In this review, we discuss the structure and function of these receptors, focussing on the principles involved in collagen recognition.

The percentages of 22 14C-labeled amino acids remaining in rat brain 15 s after carotid injection were measured relative to a simultaneously injected diffusible internal standard, 3HOH. The injected solution also contained a nondiffusible internal standard, [113mIn]EDTA to correct for incomplete brain blood compartment washout. Self-inhibition and cross-inhibition was demonstrated by inclusion of unlabeled amino acids and carboxylic acids. All amino acids tested, excluding proline, alanine, and glycine, could be assigned to one, and only one, blood-brain barrier carrier system. The neutral carrier system transported phenylalanine, leucine, tyrosine, isoleucine, methionine, tryptophane, valine, DOPA, cysteine, histidine, threonine, glutamine, asparagine, and serine. Affinity for a basic amino acid carrier system was demonstrated for arginine, ornithine, and lysine. A third, low-capacity independent carrier system transporting aspartic and glutamic acids was demonstrated.

Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7c. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH2-coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. We conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c (or P3-2 and P3-6a) and not in P2-X.

Nuclear-architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as ageing. It is therefore plausible that diseases whose manifestations correlate with ageing might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of ageing-associated disorders by focusing on a leucine-rich repeat kinase 2 (LRRK2) dominant mutation (G2019S; glycine-to-serine substitution at amino acid 2019), which is associated with familial and sporadic Parkinson's disease as well as impairment of adult neurogenesis in mice. Here we report on the generation of induced pluripotent stem cells (iPSCs) derived from Parkinson's disease patients and the implications of LRRK2(G2019S) mutation in human neural-stem-cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in nuclear-envelope organization, clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in Parkinson's disease iPSCs and were recapitulated after targeted knock-in of the LRRK2(G2019S) mutation in human embryonic stem cells. Analysis of human brain tissue showed nuclear-envelope impairment in clinically diagnosed Parkinson's disease patients. Together, our results identify the nucleus as a previously unknown cellular organelle in Parkinson's disease pathology and may help to open new avenues for Parkinson's disease diagnoses as well as for the potential development of therapeutics targeting this fundamental cell structure.

Tetrahydrofolate (THF) polyglutamates are a family of cofactors that carry and chemically activate one-carbon units for biosynthesis. THF-mediated one-carbon metabolism is a metabolic network of interdependent biosynthetic pathways that is compartmentalized in the cytoplasm, mitochondria, and nucleus. One-carbon metabolism in the cytoplasm is required for the synthesis of purines and thymidylate and the remethylation of homocysteine to methionine. One-carbon metabolism in the mitochondria is required for the synthesis of formylated methionyl-tRNA; the catabolism of choline, purines, and histidine; and the interconversion of serine and glycine. Mitochondria are also the primary source of one-carbon units for cytoplasmic metabolism. Increasing evidence indicates that folate-dependent de novo thymidylate biosynthesis occurs in the nucleus of certain cell types. Disruption of folate-mediated one-carbon metabolism is associated with many pathologies and developmental anomalies, yet the biochemical mechanisms and causal metabolic pathways responsible for the initiation and/or progression of folate-associated pathologies have yet to be established. This chapter focuses on our current understanding of mammalian folate-mediated one-carbon metabolism, its cellular compartmentation, and knowledge gaps that limit our understanding of one-carbon metabolism and its regulation.

Reduced glutathione (L-gamma-glutamyl-L-cysteinyl-glycine, GSH) is the prevalent low-molecular-weight thiol in mammalian cells. It is formed in a two-step enzymatic process including, first, the formation of gamma-glutamylcysteine from glutamate and cysteine, by the activity of the gamma-glutamylcysteine synthetase; and second, the formation of GSH by the activity of GSH synthetase which uses gamma-glutamylcysteine and glycine as substrates. While its synthesis and metabolism occur intracellularly, its catabolism occurs extracellularly by a series of enzymatic and plasma membrane transport steps. Glutathione metabolism and transport participates in many cellular reactions including: antioxidant defense of the cell, drug detoxification and cell signaling (involved in the regulation of gene expression, apoptosis and cell proliferation). Alterations in its concentration have also been demonstrated to be a common feature of many pathological conditions including diabetes, cancer, AIDS, neurodegenerative and liver diseases. Additionally, GSH catabolism has been recently reported to modulate redox-sensitive components of signal transduction cascades. In this manuscript, we review the current state of knowledge on the role of GSH in the pathogenesis of human diseases with the aim to underscore its relevance in translational research for future therapeutic treatment design.

Several CNS disorders associated with specific antibodies to ion channels, receptors, and other synaptic proteins have been recognised over the past 10 years, and can be often successfully treated with immunotherapies. Antibodies to components of voltage-gated potassium channel complexes (VGKCs), NMDA receptors (NMDARs), AMPA receptors (AMPARs), GABA type B receptors (GABA(B)Rs), and glycine receptors (GlyRs) can be identified in patients and are associated with various clinical presentations, such as limbic encephalitis and complex and diffuse encephalopathies. These diseases can be associated with tumours, but they are more often non-paraneoplastic, and antibody assays can help with diagnosis. The new specialty of immunotherapy-responsive CNS disorders is likely to expand further as more antibody targets are discovered. Recent findings raise many questions about the classification of these diseases, the relation between antibodies and specific clinical phenotypes, the relative pathological roles of serum and intrathecal antibodies, the mechanisms of autoantibody generation, and the development of optimum treatment strategies.

Glycine N-methyltransferase (GNMT) is the main enzyme responsible for catabolism of excess hepatic S-adenosylmethionine (SAMe). GNMT is absent in hepatocellular carcinoma (HCC), messenger RNA (mRNA) levels are significantly lower in livers of patients at risk of developing HCC, and GNMT has been proposed to be a tumor-susceptibility gene for liver cancer. The identification of several children with liver disease as having mutations of the GNMT gene further suggests that this enzyme plays an important role in liver function. In the current study we studied development of liver pathologies including HCC in GNMT-knockout (GNMT-KO) mice. GNMT-KO mice have elevated serum aminotransferase, methionine, and SAMe levels and develop liver steatosis, fibrosis, and HCC. We found that activation of the Ras and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways was increased in liver tumors from GNMT-KO mice coincidently with the suppression of the Ras inhibitors Ras-association domain family/tumor suppressor (RASSF) 1 and 4 and the JAK/STAT inhibitors suppressor of cytokine signaling (SOCS) 1-3 and cytokine-inducible SH2-protein. Finally, we found that methylation of RASSF1 and SOCS2 promoters and the binding of trimethylated lysine 27 in histone 3 to these 2 genes was increased in HCC from GNMT-KO mice.

CONCLUSIONS

These data demonstrate that loss of GNMT induces aberrant methylation of DNA and histones, resulting in epigenetic modulation of critical carcinogenic pathways in mice.

Lysis of trypsinized Group A streptococcal cell walls with phage-associated lysin releases into solution dialyzable and non-dialyzable mucopeptide fractions composed of N-acetylglucosamine, N-acetylmuramic acid and alanine, glutamic acid, lysine, and glycine in addition to the characteristic group-specific carbohydrate. The latter substance contains appreciable amounts of N-acetylmuramic acid and the amino acids as well as N-acetylglucosamine and rhamnose. Hot formamide extraction of the cell walls results in a soluble fraction of group-specific carbohydrate and an insoluble residue. The Group A carbohydrate in this instance is composed of rhamnose and N-acetylglucosamine. The composition of the insoluble residue is similar to that of the mucopeptide fractions released from the cell wall by phage-associated lysin. This residue was shown by electron microscopy to be composed of discrete discs which appear similar in structure to the intact cell wall. The specific carbohydrate obtained by hot formamide extraction of Group A-variant cell walls was composed almost exclusively of rhamnose. The residue fraction was similar to that of Group A. The residue of cell walls extracted with hot formamide is extensively solubilized not only by phage-associated lysin and S. albus enzyme, but also by lysozyme, which has no measurable effect on the intact streptococcal cell wall.

The entire set of six closely related Drosophila actin genes was isolated using recombinant DNA methodology, and the structures of the respective coding regions were characterized by gene mapping techniques and by nucleotide sequencing of selected portions. Structural comparisons of these genes have resulted in several unexpected findings. Most striking is the nonconservation of the positions of intervening sequences within the protein-encoding regions of these genes. One of the Drosophila actin genes, DmA4, is split within a glycine codon at position 13; none of the remaining five genes is interrupted in the analogous position. Another gene, DmA6, is split within a glycine codon at position 307; at least two of the Drosophila actin genes are not split in the analogous position. Additionally, none of the Drosophila actin genes is split within codon four, where the yeast actin gene is interrupted. The six Drosophila actin genes encode several different proteins, but the amino acid sequence of each is similar to that of vertebrate cytoplasmic actins. None of the genes encodes a protein comparable in primary sequence to vertebrate skeletal muscle actin. Surprisingly, in each of these derived actin amino acid sequences in the initiator methionine is directly followed by a cysteine residue, which in turn precedes the string of three acidic amino acids characteristic of the amino termini of mature vertebrate cytoplasmic actins. We discuss these findings in the context of actin gene evolution and function.

Introduction by site-directed mutagenesis of three amino acids from the MII segment of glycine or gamma-aminobutyric acid (GABAA) receptors into the MII segment of alpha 7 nicotinic receptor was sufficient to convert a cation-selective channel into an anion-selective channel gated by acetylcholine. A critical mutation was the insertion of an uncharged residue at the amino-terminal end of MII, stressing the importance of protein geometrical constraints on ion selectivity.

Nudix hydrolases catalyze the hydrolysis of nucleoside diphosphates linked to other moieties, X, and contain the sequence motif or Nudix box, GX(5)EX(7)REUXEEXGU. The mechanisms of Nudix hydrolases are highly diverse in the position on the substrate at which nucleophilic substitution occurs, and in the number of required divalent cations. While most proceed by associative nucleophilic substitutions by water at specific internal phosphorus atoms of a diphosphate or polyphosphate chain, members of the GDP-mannose hydrolase sub-family catalyze dissociative nucleophilic substitutions, by water, at carbon. The site of substitution is likely determined by the positions of the general base and the entering water. The rate accelerations or catalytic powers of Nudix hydrolases range from 10(9)- to 10(12)-fold. The reactions are accelerated 10(3)-10(5)-fold by general base catalysis by a glutamate residue within, or beyond the Nudix box, or by a histidine beyond the Nudix box. Lewis acid catalysis, which contributes 10(3)-10(5)-fold to the rate acceleration, is provided by one, two, or three divalent cations. One divalent cation is coordinated by two or three conserved residues of the Nudix box, the initial glycine and one or two glutamate residues, together with a remote glutamate or glutamine ligand from beyond the Nudix box. Some Nudix enzymes require one (MutT) or two additional divalent cations (Ap(4)AP), to neutralize the charge of the polyphosphate chain, to help orient the attacking hydroxide or oxide nucleophile, and/or to facilitate the departure of the anionic leaving group. Additional catalysis (10-10(3)-fold) is provided by the cationic side chains of lysine and arginine residues and by H-bond donation by tyrosine residues, to orient the general base, or to promote the departure of the leaving group. The overall rate accelerations can be explained by both independent and cooperative effects of these catalytic components.

Although glycine is a major inhibitory transmitter in the mammalian CNS, the role of glycinergic neurons in defined neuronal circuits remains ill defined. This is due in part to difficulties in identifying these cells in living slice preparations for electrophysiological recordings and visualizing their axonal projections. To facilitate the morphological and functional analysis of glycinergic neurons, we generated bacterial artificial chromosome (BAC) transgenic mice, which specifically express enhanced green fluorescent protein (EGFP) under the control of the promotor of the glycine transporter (GlyT) 2 gene, which is a reliable marker for glycinergic neurons. Neurons expressing GlyT2-EGFP were intensely fluorescent, and their dendrites and axons could be visualized in great detail. Numerous positive neurons were detected in the spinal cord, brainstem, and cerebellum. The hypothalamus, intralaminar nuclei of the thalamus, and basal forebrain also received a dense GlyT2-EGFP innervation, whereas in the olfactory bulb, striatum, neocortex, hippocampus, and amygdala positive fibers were much less abundant. No GlyT2-EGFP-positive cell bodies were seen in the forebrain. On the subcellular level, GlyT2-EGFP fluorescence was colocalized extensively with glycine immunoreactivity in somata and dendrites and with both glycine and GlyT2 immunoreactivity in axon terminals, as shown by triple staining at all levels of the neuraxis, confirming the selective expression of the transgene in glycinergic neurons. In slice preparations of the spinal cord, no difference between the functional properties of EGFP-positive and negative neurons could be detected, confirming the utility of visually identifying glycinergic neurons to investigate their functional role in electrophysiological studies.

The late embryogenesis abundant (LEA) proteins are plant proteins that are synthesized at the onset of desiccation in maturing seeds and in vegetative organs exposed to water deficit. Here, we show that most LEA proteins are comprised in a more widespread group, which we call "hydrophilins." The defining characteristics of hydrophilins are high glycine content (>6%) and a high hydrophilicity index (>1.0). By data base searching, we show that this criterion selectively differentiates most known LEA proteins as well as additional proteins from different taxons. We found that within the genomes of Escherichia coli and Saccharomyces cerevisiae, only 5 and 12 proteins, respectively, meet our criterion. Despite their deceivingly loose definition, hydrophilins usually represent <0.2% of the proteins of a genome. Additionally, we demonstrate that the criterion that defines hydrophilins seems to be an excellent predictor of responsiveness to hyperosmosis since most of the genes encoding these proteins in E. coli and S. cerevisiae are induced by osmotic stress. Evidence for the participation of one of the E. coli hydrophilins in the adaptive response to hyperosmotic conditions is presented. Apparently, hydrophilins represent analogous adaptations to a common problem in such diverse taxons as prokaryotes and eukaryotes.

We have obtained the complete coding sequence of the large aggregating chondroitin sulfate proteoglycan of human cartilage (aggrecan) from a combination of cDNA and genomic exon sequencing. We screened a human costal chondrocyte cDNA library, using rat aggrecan cDNA probes, and obtained three nonoverlapping clones totaling 6.2 kilobases in length. These clones were sequenced, and the sequence of the gaps between clones was obtained from genomic exon fragments and polymerase chain reaction-amplified cDNA. The composite sequence is 7137 nucleotides long, encoding 2316 amino acids. The human and rat aggrecan amino acid sequences are about 75% identical, with domains ranging from 100% to about 60% of conserved amino acids. The human sequence contains two regions of highly conserved repeats not found in rat aggrecan: 11 repeats of a hexameric sequence in the keratan sulfate attachment domain, E-E-P-(S,F)-P-S; and a 19-amino acid sequence reiterated 19 times, in the CS-1 portion of the serine-glycine-containing region. There are at least three forms of aggrecan transcripts, generated by alternative exon usage, and the form reported here is the shortest and also the most prevalent, lacking both the epidermal growth factor-like domain, and the complement regulatory protein-like sequence.

A set of PCR primers that should amplify all subgroups of arbuscular mycorrhizal fungi (AMF, Glomeromycota), but exclude sequences from other organisms, was designed to facilitate rapid detection and identification directly from field-grown plant roots. The small subunit rRNA gene was targeted for the new primers (AML1 and AML2) because phylogenetic relationships among the Glomeromycota are well understood for this gene. Sequence comparisons indicate that the new primers should amplify all published AMF sequences except those from Archaeospora trappei. The specificity of the new primers was tested using 23 different AMF spore morphotypes from trap cultures and Miscanthus sinensis, Glycine max and Panax ginseng roots sampled from the field. Non-AMF DNA of 14 plants, 14 Basidiomycota and 18 Ascomycota was also tested as negative controls. Sequences amplified from roots using the new primers were compared with those obtained using the established NS31 and AM1 primer combination. The new primers have much better specificity and coverage of all known AMF groups.

High levels of D-serine occur in mammalian brain, where it appears to be an endogenous ligand of the glycine site of N-methyl-D-aspartate receptors. In glial cultures of rat cerebral cortex, D-serine is enriched in type II astrocytes and is released upon stimulation with agonists of non-N-methyl-D-aspartate glutamate receptors. The high levels of D-serine in discrete areas of rat brain imply the existence of a biosynthetic pathway. We have purified from rat brain a soluble enzyme that catalyzes the direct racemization of L-serine to D-serine. Purified serine racemase has a molecular mass of 37 kDa and requires pyridoxal 5'-phosphate for its activity. The enzyme is highly selective toward L-serine, failing to racemize any other amino acid tested. Properties such as pH optimum, Km values, and the requirement for pyridoxal phosphate resemble those of bacterial racemases, suggesting that the biosynthetic pathway for D-amino acids is conserved from bacteria to mammalian brain.