ATP binding cassette transporter G1 (ABCG1) mediates the transport of cholesterol from cells to high density lipoprotein (HDL) but not to lipid-depleted apolipoprotein A-I. Here we show that human ABCG1 overexpressed in baby hamster kidney cells in the absence of lipoproteins traffics to the plasma membrane and redistributes membrane cholesterol to cell-surface domains accessible to treatment with the enzyme cholesterol oxidase. Cholesterol removed by HDL was largely derived from these domains in ABCG1 transfectants but not in cells lacking ABCG1. Overexpression of ABCG1 also increased cholesterol esterification, which was decreased by the addition of HDL, suggesting that a proportion of the cell-surface cholesterol not removed by HDL is transported to the intracellular esterifying enzyme acyl-CoA:cholesterol acyltransferase. A 638-amino acid ABCG1, which lacked the 40 N-terminal amino acids of the predicted full-length protein, was fully functional and of a similar size to ABCG1 expressed by cholesterol-loaded human monocyte-derived macrophages. Mutating an essential glycine residue in the Walker A motif abolished ABCG1-dependent cholesterol efflux and esterification and prevented localization of ABCG1 to the cell surface, indicating that the ATP binding domain in ABCG1 is essential for both lipid transport activity and protein trafficking. These studies show that ABCG1 redistributes cholesterol to cell-surface domains where it becomes accessible for removal by HDL, consistent with a direct role of ABCG1 in cellular cholesterol transport.

Mutation analysis of the KRAS oncogene is now established as a predictive biomarker in colorectal cancer (CRC). Large prospective clinical trials have shown that only CRCs with wild-type KRAS respond to anti-epidermal growth factor receptor (EGFR) treatment. Therefore, mutation analysis is mandatory before treatment, and reliable benchmarks for the frequency and types of KRAS mutations have to be established for routinely testing large numbers of metastatic CRCs. A thousand and eighteen cases (879 primary tumors and 139 metastases) of metastatic colorectal cancer were analyzed for the KRAS mutational status of codons 12 and 13 of the KRAS gene by genomic sequencing in a routine setting. Results were analyzed separately for specimens derived from primary tumors and metastases. KRAS mutations in codons 12 and 13 were present in 39.3% of all analyzed CRCs. The most frequent types of mutations were glycine to aspartate on codon 12 (p.G12D, 36.0%), glycine to valine on codon 12 (pG12V, 21.8%), and glycine to aspartate on codon 13 (p.G13D, 18.8%). They account for 76.6% of all mutations and prevail in primary tumors and distant metastases, indicating a robustness of the KRAS mutational status during neoplastic dissemination. The frequency of KRAS mutations and the preponderance of three types of mutations in codons 12 and 13 in a large, unselected cohort of metastatic CRC confirm the previous data of small and selected CRC samples. Thus, a mutation frequency of 40% and a cluster of three mutation types (p.G12D, pG12V, and p.G13D) in primaries and metastases can be defined as benchmarks for routine KRAS analyses.

Expression analysis of genes encoding components of the phosphotyrosine signaling system by cDNA array hybridization revealed elevated levels of FGFR4 transcripts in several mammary carcinoma cell lines. In the FGFR4 gene transcript from MDA-MB-453 mammary carcinoma cells, a G to A conversion was discovered that results in the substitution of glycine by arginine at position 388 in the transmembrane domain of the receptor. The Arg(388) allele was also found in cell lines derived from a variety of other tumor types as well as in the germ-line of cancer patients and healthy individuals. Analysis of three geographically separated groups indicated that it occurs in approximately 50% of the human population. Investigation of the clinical data of 84 breast cancer patients revealed that homo- or heterozygous carriers of the Arg(388) allele had a significantly reduced disease-free survival time (P = 0.01) within a median follow-up of 62 months. Moreover, the FGFR4 Arg(388) allele was associated with early lymph node metastasis and advanced tumor-node-metastasis (TNM) stage in 82 colon cancer patients. Consistent with this finding, MDA-MB-231 mammary tumor cells expressing FGFR4 Arg(388) exhibited increased motility relative to cells expressing the FGFR4 Gly(388) isotype. Our results support the conclusion that the FGFR4 Arg(388) allele represents a determinant that is innocuous in healthy individuals but predisposes cancer patients for significantly accelerated disease progression.

In response to low ambient temperature, mammalian cells as well as microorganisms change various physiological functions, but the molecular mechanisms underlying these adaptations are just beginning to be understood. We report here the isolation of a mouse cold-inducible RNA-binding protein (cirp) cDNA and investigation of its role in cold-stress response of mammalian cells. The cirp cDNA encoded an 18-kD protein consisting of an amino-terminal RNAbinding domain and a carboxyl-terminal glycine-rich domain and exhibited structural similarity to a class of stress-induced RNA-binding proteins found in plants. Immunofluorescence microscopy showed that CIRP was localized in the nucleoplasm of BALB/3T3 mouse fibroblasts. When the culture temperature was lowered from 37 to 32 degrees C, expression of CIRP was induced and growth of BALB/3T3 cells was impaired as compared with that at 37 degrees C. By suppressing the induction of CIRP with antisense oligodeoxynucleotides, this impairment was alleviated, while overexpression of CIRP resulted in impaired growth at 37 degrees C with prolongation of G1 phase of the cell cycle. These results indicate that CIRP plays an essential role in cold-induced growth suppression of mouse fibroblasts. Identification of CIRP may provide a clue to the regulatory mechanisms of cold responses in mammalian cells.

A high-throughput proteomic approach was employed to determine the expression profile and identity of hundreds of proteins during seed filling in soybean (Glycine max) cv Maverick. Soybean seed proteins were analyzed at 2, 3, 4, 5, and 6 weeks after flowering using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This led to the establishment of high-resolution proteome reference maps, expression profiles of 679 spots, and corresponding matrix-assisted laser desorption ionization time-of-flight mass spectrometry spectra for each spot. Database searching with these spectra resulted in the identification of 422 proteins representing 216 nonredundant proteins. These proteins were classified into 14 major functional categories. Proteins involved in metabolism, protein destination and storage, metabolite transport, and disease/defense were the most abundant. For each functional category, a composite expression profile is presented to gain insight into legume seed physiology and the general regulation of proteins associated with each functional class. Using this approach, an overall decrease in metabolism-related proteins versus an increase in proteins associated with destination and storage was observed during seed filling. The accumulation of unknown proteins, sucrose transport and cleavage enzymes, cysteine and methionine biosynthesis enzymes, 14-3-3-like proteins, lipoxygenases, storage proteins, and allergenic proteins during seed filling is also discussed. A user-intuitive database (http://oilseedproteomics.missouri.edu) was developed to access these data for soybean and other oilseeds currently being investigated.

Crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK), has also recently shown efficacy in the treatment of lung cancers with ROS1 translocations. Resistance to crizotinib developed in a patient with metastatic lung adenocarcinoma harboring a CD74-ROS1 rearrangement who had initially shown a dramatic response to treatment. We performed a biopsy of a resistant tumor and identified an acquired mutation leading to a glycine-to-arginine substitution at codon 2032 in the ROS1 kinase domain. Although this mutation does not lie at the gatekeeper residue, it confers resistance to ROS1 kinase inhibition through steric interference with drug binding. The same resistance mutation was observed at all the metastatic sites that were examined at autopsy, suggesting that this mutation was an early event in the clonal evolution of resistance. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

Fused in sarcoma (FUS) is a nuclear protein that carries a proline-tyrosine nuclear localization signal (PY-NLS) and is imported into the nucleus via Transportin (TRN). Defects in nuclear import of FUS have been implicated in neurodegeneration, since mutations in the PY-NLS of FUS cause amyotrophic lateral sclerosis (ALS). Moreover, FUS is deposited in the cytosol in a subset of frontotemporal lobar degeneration (FTLD) patients. Here, we show that arginine methylation modulates nuclear import of FUS via a novel TRN-binding epitope. Chemical or genetic inhibition of arginine methylation restores TRN-mediated nuclear import of ALS-associated FUS mutants. The unmethylated arginine-glycine-glycine domain preceding the PY-NLS interacts with TRN and arginine methylation in this domain reduces TRN binding. Inclusions in ALS-FUS patients contain methylated FUS, while inclusions in FTLD-FUS patients are not methylated. Together with recent findings that FUS co-aggregates with two related proteins of the FET family and TRN in FTLD-FUS but not in ALS-FUS, our study provides evidence that these two diseases may be initiated by distinct pathomechanisms and implicates alterations in arginine methylation in pathogenesis.

The collagens represent a family of trimeric extracellular matrix molecules used by cells for structural integrity and other functions. The three alpha chains that form the triple helical part of the molecule are composed of repeating peptide triplets of glycine-X-Y. X and Y can be any amino acid but are often proline and hydroxyproline, respectively. Flanking the triple helical regions (i.e., Col domains) are non-glycine-X-Y regions, termed non-collagenous domains. These frequently contain recognizable peptide modules found in other matrix molecules. Proper tissue function depends on correctly assembled molecular aggregates being incorporated into the matrix. This review highlights some of the structural characteristics of collagen types I-XXVIII.

Human embryonic stem cells (hESCs) have the potential to self-renew and generate multiple cell types, producing critical building blocks for tissue engineering and regenerative medicine applications. Here, we describe the efficient derivation and chondrogenic differentiation of mesenchymal-like cells from hESCs. These cells exhibit mesenchymal stem cell (MSC) surface markers, including CD29, CD44, CD105, and platelet-derived growth factor receptor-alpha. Under appropriate growth conditions, the hESC-derived cells proliferated without phenotypic changes and maintained MSC surface markers. The chondrogenic capacity of the cells was studied in pellet culture and after encapsulation in poly(ethylene glycol)-diacrylate (PEGDA) hydrogels with exogenous extracellular proteins or arginineglycine- aspartate (RGD)-modified PEGDA hydrogels. The hESC-derived cells exhibited growth factor- dependent matrix production in pellet culture but did not produce tissue characteristic of cartilage morphology. In PEGDA hydrogels containing exogenous hyaluronic acid or type I collagen, no significant cell growth or matrix production was observed. In contrast, when these cells were encapsulated in RGDmodified poly(ethylene glycol)hydrogels, neocartilage with basophilic extracellular matrix deposition was observed within 3 weeks of culture, producing cartilage-specific gene up-regulation and extracellular matrix production. Our results indicate that precursor cells characteristic of a MSC population can be cultured from differentiating hESCs through embryoid bodies, thus holding great promise for a potentially unlimited source of cells for cartilage tissue engineering.

The clock-regulated RNA-binding protein AtGRP7 is part of a negative feedback circuit through which the protein influences circadian oscillations of its own transcript. Constitutive overexpression of AtGRP7 in transgenic plants leads to the appearance of a low amount of an alternatively spliced Atgrp7 transcript with a premature stop codon. It is generated by the use of a 5' cryptic splice site in the middle of the intron at the expense of the fully spliced mRNA, indicating a role for AtGRP7 in splice site selection. Accelerated decay of this transcript accounts for its low steady state abundance. This implicates a mechanism for the AtGRP7 feedback loop: Atgrp7 expression is downregulated, as AtGRP7 protein accumulates over the circadian cycle, partly by the generation of an alternate transcript that due to its instability does not accumulate to high levels and does not produce a functional protein. Recombinant AtGRP7 protein specifically interacts with the 3' untranslated region and the intron of its transcript, suggesting that the shift in splice site selection and downregulation involves binding of AtGRP7 to its pre-mRNA. AtGRP7 also influences the choice of splice sites in the Atgrp8 transcript encoding a related RNA-binding protein, favoring the production of an alternatively spliced, unstable Atgrp8 transcript. This conservation points to the importance of this regulatory mechanism to control the level of the clock-regulated glycine-rich RNA-binding proteins and shows how AtGRP7 can control abundance of target transcripts.

Disturbed circadian rhythms have been observed in seasonal affective disorder (SAD). The aim of this study was to further investigate this connection, and to test for potential association between polymorphisms in circadian clock-related genes and SAD, seasonality (seasonal variations in mood and behavior), or diurnal preference (morningness-eveningness tendencies). A total of 159 European SAD patients and 159 matched controls were included in the genetic analysis, and subsets were screened for seasonality (n=177) and diurnal preference (n=92). We found that diurnal preference was associated with both SAD and seasonality, supporting the hypothesis of a link between circadian rhythms and seasonal depression. The complete case-control material was genotyped for polymorphisms in the CLOCK, Period2, Period3, and NPAS2 genes. A significant difference between patients and controls was found for NPAS2 471 Leu/Ser (chi(2)=9.90, Bonferroni corrected P=0.035), indicating a recessive effect of the leucine allele on disease susceptibility (chi(2)=6.61, Bonferroni corrected P=0.050). Period3 647 Val/Gly was associated with self-reported morningness-eveningness scores (n=92, one-way ANOVA: F=4.99, Bonferroni corrected P=0.044), with higher scores found in individuals with at least one glycine allele (t=3.1, Bonferroni corrected P=0.013). A second, population-based sample of individuals selected for high (n=127) or low (n=98) degrees of seasonality, was also genotyped for NPAS2 471 Leu/Ser. There was no significant difference between these seasonality extreme groups, and none of the polymorphisms studied were associated with seasonality in the SAD case-control material (n=177). In conclusion, our results suggest involvement of circadian clock-related polymorphisms both in susceptibility to SAD and diurnal preference.

Adaptation of cells to hypertonicity often involves changes in gene expression. Since the concentration of salt in the interstitial fluid surrounding renal inner medullary cells varies with operation of the renal concentrating mechanism and generally is very high, the adaptive mechanisms of these cells are of special interest. Renal medullary cells compensate for hypertonicity by accumulating variable amounts of compatible organic osmolytes, including sorbitol, myo-inositol, glycine betaine, and taurine. In this review we consider how these solutes help relieve the stress of hypertonicity and the nature of transporters and enzymes responsible for their variable accumulation. We emphasize recent developments concerning the molecular basis for osmotic regulation of these genes, including identification and characterization of osmotic response elements. Although osmotic stresses are much smaller in other parts of the body than in the renal medulla, similar mechanisms operate throughout, yielding important physiological and pathophysiological consequences.

Members of the ERF transcription factor family play important roles in regulating gene expression in response to biotic and abiotic stresses. In soybean (Glycine max L.), however, only a few ERF genes have been studied so far. In this study, 98 unigenes that contained a complete AP2/ERF domain were identified from 63,676 unique sequences in the DFCI Soybean Gene Index database. The phylogeny, gene structures, and putative conserved motifs in soybean ERF proteins were analysed, and compared with those of Arabidopsis and rice. The members of the soybean ERF family were divided into 12 subgroups, similar to the case for Arabidopsis. AP2/ERF domains were conserved among soybean, Arabidopsis, and rice. Outside the AP2/ERF domain, many soybean-specific conserved motifs were detected. Expression analysis showed that nine unigenes belonging to six ERF family subgroups were induced by both biotic/abiotic stresses and hormone treatment, suggesting that they were involved in cross-talk between biotic and abiotic stress-responsive signalling pathways. Overexpression of two full-length genes from two different subgroups enhanced the tolerances to drought, salt stresses, and/or pathogen infection of the tobacco plants. These results will be useful for elucidating ERF gene-associated stress response signalling pathways in soybean.

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.

RNA binding proteins often contain multiple arginine glycine repeats, a sequence that is frequently methylated by protein arginine methyltransferases. The role of this posttranslational modification in the life cycle of RNA binding proteins is not well understood. Herein, we report that Sam68, a heteronuclear ribonucleoprotein K homology domain containing RNA binding protein, associates with and is methylated in vivo by the protein arginine N-methyltransferase 1 (PRMT1). Sam68 contains asymmetrical dimethylarginines near its proline motif P3 as assessed by using a novel asymmetrical dimethylarginine-specific antibody and mass spectrometry. Deletion of the methylation sites and the use of methylase inhibitors resulted in Sam68 accumulation in the cytoplasm. Sam68 was also detected in the cytoplasm of PRMT1-deficient embryonic stem cells. Although the cellular function of Sam68 is unknown, it has been shown to export unspliced human immunodeficiency virus RNAs. Cells treated with methylase inhibitors prevented the ability of Sam68 to export unspliced human immunodeficiency virus RNAs. Other K homology domain RNA binding proteins, including SLM-1, SLM-2, QKI-5, GRP33, and heteronuclear ribonucleoprotein K were also methylated in vivo. These findings demonstrate that RNA binding proteins are in vivo substrates for PRMT1, and their methylation is essential for their proper localization and function.

The third most common stroke complication is infection. We studied the rates of aspiration pneumonia and urinary tract infection (UTI), their risk factors and their effect on outcome in the 1455 Glycine Antagonist (Gavestinel) in Neuroprotection (GAIN) International patients with ischaemic stroke. Forward stepwise logistic regression and Cox proportional hazards modelling identified baseline factors that predicted events and the independent effect of events up to day 7 on poor stroke outcome at 3 months in patients alive at day 7, after correcting for prognostic factors. Higher baseline National Institute of Health Stroke Scale (NIHSS) and age, male gender, history of diabetes and stroke subtype predicted pneumonia, which occurred in 13.6% of patients. Female gender and higher baseline NIHSS and age predicted UTI, which occurred in 17.2% of patients. Pneumonia was associated with poor outcome by mortality (hazard ratio, 2.2; 95% confidence interval, 1.5-3.3), Barthel index (<60) (odds ratio, 3.8; 2.2-6.7), NIHSS (4.9; 1.7-14) and Rankin scale >>/=2) (3.4; 1.4-8.3). UTI was associated with Barthel index (1.9; 1.2-2.9), NIHSS (2.2; 1.2-4.0) and Rankin scale (3.1; 1.6-4.9). Pneumonia and UTI are independently associated with stroke poor outcome. Patients with identified risk factors must be closely monitored for infection.

Partial agonists produce submaximal activation of ligand-gated ion channels. To address the question of partial agonist action at the NR1 subunit of the NMDA receptor, we performed crystallographic and electrophysiological studies with 1-aminocyclopropane-1-carboxylic acid (ACPC), 1-aminocyclobutane-1-carboxylic acid (ACBC), and 1-aminocyclopentane-1-carboxylic acid (cycloleucine), three compounds with incrementally larger carbocyclic rings. Whereas ACPC and ACBC partially activate the NMDA receptor by 80% and 42%, respectively, their cocrystal structures of the NR1 ligand binding core show the same degree of domain closure as found in the complex with glycine, a full agonist, illustrating that the NR1 subunit provides a new paradigm for partial agonist action that is distinct from that of the evolutionarily related GluR2, AMPA-sensitive receptor. Cycloleucine behaves as an antagonist and stabilizes an open-cleft conformation. The NR1-cycloleucine complex forms a dimer that is similar to the GluR2 dimer, thereby suggesting a conserved mode of subunit-subunit interaction in AMPA and NMDA receptors.

The neurotransmitters GABA and glycine mediate fast synaptic inhibition by activating ligand-gated chloride channels--namely, type A GABA (GABA(A)) and glycine receptors. Both types of receptors are anchored postsynaptically by gephyrin, which self-assembles into a scaffold and interacts with the cytoskeleton. Current research indicates that postsynaptic gephyrin clusters are dynamic assemblies that are held together and regulated by multiple protein-protein interactions. Moreover, post-translational modifications of gephyrin regulate the formation and plasticity of GABAergic synapses by altering the clustering properties of postsynaptic scaffolds and thereby the availability and function of receptors and other signalling molecules. Here, we discuss the formation and regulation of the gephyrin scaffold, its role in GABAergic and glycinergic synaptic function and the implications for the pathophysiology of brain disorders caused by abnormal inhibitory neurotransmission.

Parkinson's disease (PD), a progressive neurodegenerative disease characterized by bradykinesia, rigidity, and resting tremor, is the most common neurodegenerative movement disorder. Although the majority of PD cases are sporadic, some are inherited, including those caused by leucine-rich repeat kinase 2 (LRRK2) mutations. The substitution of serine for glycine at position 2019 (G2019S) in the kinase domain of LRRK2 represents the most prevalent genetic mutation in both familial and apparently sporadic cases of PD. Because mutations in LRRK2 are likely associated with a toxic gain of function, destabilization of LRRK2 may be a novel way to limit its detrimental effects. Here we show that LRRK2 forms a complex with heat shock protein 90 (Hsp90) in vivo and that inhibition of Hsp90 disrupts the association of Hsp90 with LRRK2 and leads to proteasomal degradation of LRRK2. Hsp90 inhibitors may therefore limit the mutant LRRK2-elicited toxicity to neurons. As a proof of principle, we show that Hsp90 inhibitors rescue the axon growth retardation caused by overexpression of the LRRK2 G2019S mutation in neurons. Therefore, inhibition of LRRK2 kinase activity can be achieved by blocking Hsp90-mediated chaperone activity and Hsp90 inhibitors may serve as potential anti-PD drugs.

Molecular cloning has introduced an unexpected diversity of neurotransmitter receptors. In this study we review the types, the localization and possible synaptic function of the inhibitory neurotransmitter receptors in the mammalian retina. Glycine receptors (GlyRs) and their localization in the mammalian retina were analyzed immunocytochemically. Specific antibodies against the alpha 1 subunit of the GlyR (mAb2b) and against all subunits of the GlyR (mAb4a) were used. Both antibodies produced a punctate immunofluorescence, which was shown by electron microscopy to represent clustering of GlyRs at synaptic sites. Synapses expressing the alpha 1 subunit of the GlyR were found on ganglion cell dendrites and on bipolar cell axons. GlyRs were also investigated in the oscillator mutant mouse. The complete loss of the alpha 1 subunit was compensated for by an apparent upregulation of the other subunits of the GlyR. GABAA receptors (GABAARs) and their retinal distribution were studied with specific antibodies that recognize the alpha 1, alpha 2, alpha 3, beta 1, beta 2, beta 3, gamma 2 and delta subunits. Most antibodies produced a punctate immunofluorescence in the inner plexiform layer (IPL) which was shown by electron microscopy to represent synaptic clustering of GABAARs. The density of puncta varied across the IPL and different subunits were found in characteristic strata. This stratification pattern was analyzed with respect to the ramification of cholinergic amacrine cells. Using intracellular injection with Lucifer yellow followed by immunofluorescence, we found that GABAARs composed of different subunits were expressed by the same ganglion cell, however, they were clustered at different synaptic sites. The distribution of GABAC receptors was studied in the mouse and in the rabbit retina using an antiserum that recognizes the rho 1, rho 2 and rho 3 subunits. GABAC receptors were found to be clustered at postsynaptic sites. Most, if not all of the synapses were found on rod and cone bipolar axon terminals. In conclusion we find a great diversity of glycine and GABA receptors in the mammalian retina, which might match the plethora of morphological types of amacrine cells. This may also point to subtle differences in synaptic function still to be elucidated.

Pyridoxal-5'-phosphate-dependent enzymes catalyze manifold reactions in the metabolism of amino acids. A comprehensive comparison of amino acid sequences has shown that most of these enzymes can be assigned to one of three different families of homologous proteins. The sequences of the enzymes of each family were aligned and their homology confirmed by profile analysis. Scrutiny of the reactions catalyzed by the enzymes showed that their affiliation with one of the three structurally defined families correlates in most cases with their regio-specificity. In the largest family, the covalency changes of the substrate occur at the same carbon atom that carries the amino group forming the imine linkage with the coenzyme. This family was thus named alpha family. It comprises glycine hydroxymethyltransferase, glycine C-acetyltransferase, 5-aminolevulinate synthase, 8-amino-7-oxononanoate synthase, all aminotransferases (with the possible exception of subgroup III), a number of other enzymes relatively closely related with the aminotransferases and very likely a certain group of amino acid decarboxylases as well as tryptophanase and tyrosine phenol-lyase which, however, catalyze beta-elimination reactions. The beta family includes L- and D-serine dehydratase, threonine dehydratase, the beta subunit of tryptophan synthase, threonine synthase and cysteine synthase. These enzymes catalyze beta-replacement or beta-elimination reactions. The gamma family incorporates O-succinylhomoserine (thiol-lyase, O-acetylhomoserine (thiol)-lyase, and cystathionine gamma-lyase, which catalyze gamma-replacement or gamma-elimination reactions, as well as cystathionine beta-lyase. The alpha and gamma family might be distantly related with one another, but are clearly not homologous with the beta family. Apparently, the primordial pyridoxal-5'-phosphate-dependent enzymes were regio-specific catalysts, which first specialized for reaction specificity and then for substrate specificity. The following pyridoxal-5'-phosphate-dependent enzymes seem to be unrelated with the alpha, beta or gamma family by the criterion of profile analysis:alanine racemase, selenocysteine synthase, and many amino acid decarboxylases. These enzymes may represent yet other families of B6 enzymes.

Heat-stable, chemotactically active peptides have been obtained from Escherichia coli culture filtrates. They range in size between 150 and 1500 daltons and are anionic at neutral pH. Free carboxyl groups but not free amino groups appear to be required for activity. The N-terminal group may be blocked. There do not appear to be internal aromatic or basic residues in the chemotactically active fractions. A highly purified, not completely characterized, fraction was found to contain aspartic acid, serine, glutamic acid, alanine, and glycine.

We have isolated a cold-sensitive mutant of Saccharomyces cerevisiae in which the first step of mRNA splicing is inhibited. The growth and splicing defects are recessive and cosegregate, thus defining a single essential gene (PRP28). The wild-type PRP28 gene was cloned, and sequence analysis reveals extensive homology to a family of proteins that are thought to function as ATP-dependent RNA helicases. The cold sensitivity is caused by a glycine-to-glutamic acid change in a conserved sequence motif. Interestingly, double mutants containing conditional alleles of PRP28 and PRP24, which encodes a U6 snRNA-binding protein, are inviable. In addition, a suppressor of prp28-1 is a mutant allele of PRP8, which encodes a U5 protein, thus linking PRP28 with U5. These data are consistent with a scenario in which PRP28 acts to unwind the U4/U6 base-pairing interaction in the U4/U6/U5 snRNP, facilitating the first covalent step of splicing.

The strychnine-sensitive glycine receptor (GlyR) mediates inhibitory synaptic transmission in the spinal cord and brainstem and is linked to neurological disorders, including autism and hyperekplexia. Understanding of molecular mechanisms and pharmacology of glycine receptors has been hindered by a lack of high-resolution structures. Here we report electron cryo-microscopy structures of the zebrafish α1 GlyR with strychnine, glycine, or glycine and ivermectin (glycine/ivermectin). Strychnine arrests the receptor in an antagonist-bound closed ion channel state, glycine stabilizes the receptor in an agonist-bound open channel state, and the glycine/ivermectin complex adopts a potentially desensitized or partially open state. Relative to the glycine-bound state, strychnine expands the agonist-binding pocket via outward movement of the C loop, promotes rearrangement of the extracellular and transmembrane domain 'wrist' interface, and leads to rotation of the transmembrane domain towards the pore axis, occluding the ion conduction pathway. These structures illuminate the GlyR mechanism and define a rubric to interpret structures of Cys-loop receptors.