Neuroanatomical and pharmacological evidence implicates glutamate transmission in drug-environment conditioning that partly controls drug seeking and relapse. Glutamate receptors could be targets for pharmacological attenuation of the motivational properties of drug-paired cues and for relapse prevention. The purpose of the present study was therefore to investigate the involvement of ionotropic and metabotropic glutamate receptor subtypes in cue-induced reinstatement of cocaine-seeking behavior. Rats were trained to self-administer cocaine using a second-order schedule of reinforcement (FR4(FR5:S)) under which a compound stimulus (light and tone) associated with cocaine infusions was presented contingently. Following extinction, the effects of the competitive NMDA receptor antagonist CGP 39551 (0, 2.5, 5, 10 mg/kg intraperitoneally (i.p.)), two competitive AMPA/kainate antagonists, CNQX (0, 0.75, 1.5, 3 mg/kg i.p.) and NBQX (0, 1.25, 2.5, 5 mg/kg i.p.), the NMDA/glycine site antagonist L-701,324 (0, 0.63, 1.25, 2.5 mg/kg i.p.), and the mGluR5 antagonist MPEP (0, 1.25, 2.5, 5 mg/kg i.p.) on cue-induced reinstatement of cocaine seeking were examined. The AMPA/kainate receptor antagonists CNQX and NBQX, the NMDA/glycine site antagonist L-701,324, and the mGluR5 antagonist MPEP attenuated significantly cue-induced reinstatement. The NMDA antagonist CGP 39551 failed to affect reinstatement. Additional control experiments indicated that attenuation of cue-induced reinstatement by CNQX, NBQX, L-701,324, and MPEP was not accompanied by significant suppression of spontaneous locomotor activity. These results suggest that conditioned influences on cocaine seeking depend on glutamate transmission. Accordingly, drugs with antagonist properties at various glutamate receptor subtypes could be useful in prevention of relapse induced by conditioned stimuli.

A single genomic clone (14 kb) isolated from bean (Phaseolus vulgaris L.) contains two genes that encode glycine-rich proteins. These genes are present as single copies in the genome, are separated by 2.85 kb and encode transcripts of 1.8 kb and 1.0 kb respectively. The encoded proteins contain 60% glycine and have amino-terminal signal peptides. The 1.8 kb transcript is present in young hypocotyls and in ovary tissue. Excision-wounding transiently induced this transcript in old, but not in young hypocotyl tissue. Antibodies raised against regions of the glycine-rich protein 1.8, expressed as a lacZ fusion protein in bacteria, react with a protein of 53 kd in a protein fraction extracted from cell walls of bean ovaries. Tissue imprints of bean ovaries treated with anti-glycine-rich protein antibodies showed that the glycine-rich protein was distributed in a regular pattern of small, highly localized discrete sites. The immunoreactive regions correspond to the pattern of vascular tissue in the pod. In young hypocotyls, glycine-rich protein is present at four pairs of discrete sites symmetrically arranged on the inner side of the vascular ring. These results suggest a close relationship between glycine-rich proteins and development of the vascular system.

We have used stably transfected CHO cell lines to characterize the pathway of intracellular transport of the lgp120 (lgp-A) to lysosomes. Using several surface labeling and internalization assays, our results suggest that lgp120 can reach its final destination with or without prior appearance on the plasma membrane. The extent to which lgp120 was transported via the cell surface was determined by two factors: expression level and the presence of a conserved glycine-tyrosine motif in the cytoplasmic tail. In cells expressing low levels of wild-type lgp120, the majority of newly synthesized molecules reached lysosomes without becoming accessible to antibody or biotinylation reagents added extracellularly at 4 degrees C. With increased expression levels, however, an increased fraction of transfected lgp120, as well as some endogenous lgp-B, appeared on the plasma membrane. The fraction of newly synthesized lgp120 reaching the cell surface was also increased by mutations affecting the cytoplasmic domain tyrosine or glycine residues. A substantial fraction of both mutants reached the surface even at low expression levels. However, only the lgp120G----A7 mutant was rapidly internalized and delivered from the plasma membrane to lysosomes. Taken together, our results show that the majority of newly synthesized wild-type lgp120 does not appear to pass through the cell surface en route to lysosomes. Instead, it is likely that lysosomal targeting involves a saturable intracellular sorting site whose affinity for lgp's is dependent on a glycine-tyrosine motif in the lgp120 cytoplasmic tail.

Structural analysis of cDNAs for human and rat androgen receptors (ARs) indicates that the amino-terminal regions of ARs are rich in oligo- and poly(amino acid) motifs as in some homeotic genes. The human AR has a long stretch of repeated glycines, whereas rat AR has a long stretch of glutamines. There is a considerable sequence similarity among ARs and the receptors for glucocorticoids, progestins, and mineralocorticoids within the steroid-binding domains. The cysteine-rich DNA-binding domains are well conserved. Translation of mRNA transcribed from AR cDNAs yielded 94- and 76-kDa proteins and smaller forms that bind to DNA and have high affinity toward androgens. These rat or human ARs were recognized by human autoantibodies to natural ARs. Molecular hybridization studies, using AR cDNAs as probes, indicated that the ventral prostate and other male accessory organs are rich in AR mRNA and that the production of AR mRNA in the target organs may be autoregulated by androgens.

Many bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. Surprisingly, the amides are not generated by a transamidation reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-C alpha bond. In higher organisms, this reaction is catalyzed by a single bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The PAM gene encodes one polypeptide with two enzymes that catalyze the two sequential reactions required for amidation. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyzes the stereospecific hydroxylation of the glycine alpha-carbon of all the peptidylglycine substrates. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5), generates alpha-amidated peptide product and glyoxylate. PHM contains two redox-active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. The structure of the catalytic core of rat PHM at atomic resolution provides a framework for understanding the broad substrate specificity of PHM, identifying residues critical for PHM activity, and proposing mechanisms for the chemical and electron-transfer steps in catalysis. Since PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase (DBM; EC 1.14.17.1), the enzyme that converts dopamine to norepinephrine during catecholamine biosynthesis, these structural and mechanistic insights are extended to DBM.

Reelin and glutamic acid decarboxylase (GAD)67 expressed by cortical gamma-aminobutyric acid-ergic interneurons are down-regulated in schizophrenia. Because epidemiological studies of schizophrenia fail to support candidate gene haploinsufficiency of Mendelian origin, we hypothesize that epigenetic mechanisms (i.e., cytosine hypermethylation of CpG islands present in the promoter of these genes) may be responsible for this down-regulation. Protracted l-methionine (6.6 mmolkg for 15 days, twice a day) treatment in mice elicited in brain an increase of S-adenosyl-homocysteine, the processing product of the methyl donor S-adenosyl-methionine, and a marked decrease of reelin and GAD67 mRNAs in both WT and heterozygous reeler mice. This effect of l-methionine was associated with an increase in the number of methylated cytosines in the CpG island of the reelin promoter region. This effect was not observed for GAD65 or neuronal-specific enolase and was not replicated by glycine doses 2-fold greater than those of l-methionine. Prepulse inhibition of startle declined at a faster rate as the prepulsestartle interval increased in mice receiving l-methionine. Valproic acid (2 mmolkg for 15 days, twice a day) reverted l-methionine-induced down-regulation of reelin and GAD67 in both WT and heterozygous reeler mice, suggesting an epigenetic action through the inhibition of histone deacetylases. The same dose of valproate increased acetylation of histone H3 in mouse brain nearly 4-fold. This epigenetic mouse model may be useful in evaluating drug efficacy on schizophrenia vulnerability. Hence the inhibition of histone deacetylases could represent a pharmacological intervention mitigating epigenetically induced vulnerability to schizophrenia in individuals at risk.

Wild relatives of crops are an important source of genetic diversity for agriculture, but their gene repertoire remains largely unexplored. We report the establishment and analysis of a pan-genome of Glycine soja, the wild relative of cultivated soybean Glycine max, by sequencing and de novo assembly of seven phylogenetically and geographically representative accessions. Intergenomic comparisons identified lineage-specific genes and genes with copy number variation or large-effect mutations, some of which show evidence of positive selection and may contribute to variation of agronomic traits such as biotic resistance, seed composition, flowering and maturity time, organ size and final biomass. Approximately 80% of the pan-genome was present in all seven accessions (core), whereas the rest was dispensable and exhibited greater variation than the core genome, perhaps reflecting a role in adaptation to diverse environments. This work will facilitate the harnessing of untapped genetic diversity from wild soybean for enhancement of elite cultivars.

The Tudor domain is an approximately 60-amino acid structure motif in search of a function. Herein we show that the Tudor domains of the spinal muscular atrophy gene product SMN, the splicing factor 30 kDa (SPF30), and the Tudor domain-containing 3 (TDRD3) proteins interacted with arginine-glycine-rich motifs in a methylarginine-dependent manner. The Tudor domains also associated with methylarginine-containing cellular proteins, providing evidence that methylated arginines represent physiological ligands for this protein module. In addition, we report that spliceosomal small nuclear ribonucleoprotein particles core Sm proteins accumulated in the cytoplasm when arginine methylation was inhibited with adenosine dialdehyde or in the presence of an excessive amount of unmethylated arginine-glycine-rich peptides. These data provide in vivo evidence in support of a role for arginine methylation in the proper assembly and localization of spliceosomal Sm proteins.

BACKGROUND

Archaeobotany, the study of plant remains from sites of ancient human activity, provides data for studying the initial evolution of domesticated plants. An important background to this is defining the domestication syndrome, those traits by which domesticated plants differ from wild relatives. These traits include features that have been selected under the conditions of cultivation. From archaeological remains the easiest traits to study are seed size and in cereal crops the loss of natural seed dispersal.

METHODS

The rate at which these features evolved and the ordering in which they evolved can now be documented for a few crops of Asia and Africa. This paper explores this in einkorn wheat (Triticum monococcum) and barley (Hordeum vulgare) from the Near East, rice (Oryza sativa) from China, mung (Vigna radiata) and urd (Vigna mungo) beans from India, and pearl millet (Pennisetum glaucum) from west Africa. Brief reference is made to similar data on lentils (Lens culinaris), peas (Pisum sativum), soybean (Glycine max) and adzuki bean (Vigna angularis). Available quantitative data from archaeological finds are compiled to explore changes with domestication. The disjunction in cereals between seed size increase and dispersal is explored, and rates at which these features evolved are estimated from archaeobotanical data. Contrasts between crops, especially between cereals and pulses, are examined.

CONCLUSIONS

These data suggest that in domesticated grasses, changes in grain size and shape evolved prior to non-shattering ears or panicles. Initial grain size increases may have evolved during the first centuries of cultivation, within perhaps 500-1000 years. Non-shattering infructescences were much slower, becoming fixed about 1000-2000 years later. This suggests a need to reconsider the role of sickle harvesting in domestication. Pulses, by contrast, do not show evidence for seed size increase in relation to the earliest cultivation, and seed size increase may be delayed by 2000-4000 years. This implies that conditions that were sufficient to select for larger seed size in Poaceae were not sufficient in Fabaceae. It is proposed that animal-drawn ploughs (or ards) provided the selection pressure for larger seeds in legumes. This implies different thresholds of selective pressure, for example in relation to differing seed ontogenetics and underlying genetic architecture in these families. Pearl millet (Pennisetum glaucum) may show some similarities to the pulses in terms of a lag-time before truly larger-grained forms evolved.

The elastic properties of many tissues such as the lung, dermis, and large blood vessels are due to the presence of elastic fibers in the extracellular space. These fibers have been shown by biochemical and ultrastructural analysis to be composed of two distinct components, a more abundant amorphous component and a 10-12 nm microfibrillar component, which is located primarily around the periphery of the amorphous component. The protein elastin makes up the highly insoluble amorphous component and is responsible for the elastic properties. Elastin is found throughout the vertebrate kingdom and possesses an unusual chemical composition rich in glycine, proline, and hydrophobic amino acids, consonant with its characteristic physical properties. The 72-kDa biosynthetic precursor, tropoelastin, is secreted into the extracellular space where it becomes highly cross-linked into a rubber-like network through the activity of the copper-requiring enzyme lysyl oxidase. Analysis of the elastin gene has demonstrated that hydrophobic and cross-linking domains are encoded in separate exons and that there is significant alternative splicing, resulting in multiple isoforms of tropoelastin. The elastin gene promoter contains many potential binding sites for various modulating factors indicative of a complex pattern of transcriptional regulation. The microfibrils contain several proteins, including fibrillin, and probably act as an organizing scaffold in the formation of the elastin network. There appears to be a fibrillin gene family in which each protein contains multiple repeats of a motif previously found in epidermal growth factor and a second motif observed in transforming growth factor beta 1-binding protein. Mutations in the fibrillin gene located on human chromosome 15 have been strongly implicated as the cause of the Marfan syndrome.

BACKGROUND

Microglia provide continuous immune surveillance of the CNS and upon activation rapidly change phenotype to express receptors that respond to chemoattractants during CNS damage or infection. These activated microglia undergo directed migration towards affected tissue. Importantly, the molecular species of chemoattractant encountered determines if microglia respond with pro- or anti-inflammatory behaviour, yet the signaling molecules that trigger migration remain poorly understood. The endogenous cannabinoid system regulates microglial migration via CB2 receptors and an as yet unidentified GPCR termed the 'abnormal cannabidiol' (Abn-CBD) receptor. Abn-CBD is a synthetic isomer of the phytocannabinoid cannabidiol (CBD) and is inactive at CB1 or CB2 receptors, but functions as a selective agonist at this Gi/o-coupled GPCR. N-arachidonoyl glycine (NAGly) is an endogenous metabolite of the endocannabinoid anandamide and acts as an efficacious agonist at GPR18. Here, we investigate the relationship between NAGly, Abn-CBD, the unidentified 'Abn-CBD' receptor, GPR18, and BV-2 microglial migration.

RESULTS

Using Boyden chamber migration experiments, yellow tetrazolium (MTT) conversion, In-cell Western, qPCR and immunocytochemistry we show that NAGly, at sub-nanomolar concentrations, and Abn-CBD potently drive cellular migration in both BV-2 microglia and HEK293-GPR18 transfected cells, but neither induce migration in HEK-GPR55 or non-transfected HEK293 wildtype cells. Migration effects are blocked or attenuated in both systems by the 'Abn-CBD' receptor antagonist O-1918, and low efficacy agonists N-arachidonoyl-serine and cannabidiol. NAGly promotes proliferation and activation of MAP kinases in BV-2 microglia and HEK293-GPR18 cells at low nanomolar concentrations - cellular responses correlated with microglial migration. Additionally, BV-2 cells show GPR18 immunocytochemical staining and abundant GPR18 mRNA. qPCR demonstrates that primary microglia, likewise, express abundant amounts of GPR18 mRNA.

CONCLUSIONS

NAGly is the most effective lipid recruiter of BV-2 microglia currently reported and its effects mimic those of Abn-CBD. The data generated from this study supports the hypothesis that GPR18 is the previously unidentified 'Abn-CBD' receptor. The marked potency of NAGly acting on GPR18 to elicit directed migration, proliferation and perhaps other MAPK-dependent phenomena advances our understanding of the lipid-based signaling mechanisms employed by the CNS to actively recruit microglia to sites of interest. It offers a novel research avenue for developing therapeutics to elicit a self-renewing population of neuroregenerative microglia, or alternatively, to prevent the accumulation of misdirected, pro-inflammatory microglia which contribute to and exacerbate neurodegenerative disease.

Genomic DNA from 154 unrelated individuals with achondroplasia was evaluated for mutations in the fibroblast growth factor receptor 3 (FGFR3) transmembrane domain. All but one, an atypical case, were found to have a glycine-to-arginine substitution at codon 380. Of these, 150 had a G-to-A transition at nt 1138, and 3 had a G-to-C transversion at this same position. On the basis of estimates of the prevalence of achondroplasia, the mutation rate at the FGFR3 1138 guanosine nucleotide is two to three orders of magnitude higher than that previously reported for tranversions and transitions in CpG dinucleotides. To date, this represents the most mutable single nucleotide reported in the human genome. The homogeneity of mutations in achondroplasia is unprecedented for an autosomal dominant disorder and may explain the relative lack of heterogeneity in the achondroplasia phenotype.

A disparity was noted in the transport of rhodamine 123 among nine MXR/BCRP/ABCP-overexpressing cells studied; all demonstrated mitoxantrone transport, whereas only two effluxed rhodamine 123. When the MXR/BCRP/ABCP gene was sequenced in the cell lines studied, differences were noted at amino acid 482, predicted to be at the start of the third transmembrane domain. Sequencing genomic DNA revealed wild-type MXR/BCRP/ABCP to have an arginine at position 482. Cells having a threonine or glycine at position 482 were able to efflux rhodamine 123, whereas cells having an arginine were not. A vaccinia virus expression system confirmed that rhodamine as well as doxorubicin efflux is observed with R482T or R482G but not with the wild-type R482; all three MXR/BCRP/ABCP forms transported mitoxantrone. Cross-resistance studies suggest that, compared with wild-type MXR/BCRP/ABCP, cells having an R482T mutation have higher anthracycline resistance, whereas an R482G mutation seems to confer relatively less resistance to SN-38 and topotecan. These results suggest that amino acid 482 has a crucial role in MXR/BCRP/ABCP function and that mutation of a single amino acid residue significantly changes substrate specificity, thus altering the drug resistance phenotype.

Deafness is genetically very heterogeneous and forms part of several syndromes. So far, delayed rectifier potassium channels have been linked to human deafness associated with prolongation of the QT interval on electrocardiograms and ventricular arrhythmia in Jervell and Lange-Nielsen syndrome. Ca(v)1.3 voltage-gated L-type calcium channels (LTCCs) translate sound-induced depolarization into neurotransmitter release in auditory hair cells and control diastolic depolarization in the mouse sinoatrial node (SAN). Human deafness has not previously been linked to defects in LTCCs. We used positional cloning to identify a mutation in CACNA1D, which encodes the pore-forming α1 subunit of Ca(v)1.3 LTCCs, in two consanguineous families with deafness. All deaf subjects showed pronounced SAN dysfunction at rest. The insertion of a glycine residue in a highly conserved, alternatively spliced region near the channel pore resulted in nonconducting calcium channels that had abnormal voltage-dependent gating. We describe a human channelopathy (termed SANDD syndrome, sinoatrial node dysfunction and deafness) with a cardiac and auditory phenotype that closely resembles that of Cacna1d(-/-) mice.

FLOWERING LOCUS T (FT) is a key flowering integrator in Arabidopsis (Arabidopsis thaliana), with homologs that encode florigens in many plant species regardless of the type of photoperiodic response. We identified 10 FT homologs, which were arranged as five pairs of linked genes in different homoeologous chromosomal regions, in soybean (Glycine max), a paleopolyploid species. Two of the FT homologs, GmFT2a and GmFT5a, were highly up-regulated under short-day (SD) conditions (inductive for flowering in soybean) and had diurnal expression patterns with the highest expression 4 h after dawn. Under long-day (LD) conditions, expression of GmFT2a and GmFT5a was down-regulated and did not follow a diurnal pattern. Flowering took much longer to initiate under LD than under SD, and only the GmFT5a transcript accumulated late in development under LD. Ectopic expression analysis in Arabidopsis confirmed that both GmFT2a and GmFT5a had the same function as Arabidopsis FT, but the effect of GmFT5a was more prominent. A double-mutant soybean line for two PHYTOCHROME A (PHYA) genes expressed high levels of GmFT2a and GmFT5a under LD, and it flowered slightly earlier under LD than the wild type grown under SD. The expression levels of GmFT2a and GmFT5a were regulated by the PHYA-mediated photoperiodic regulation system, and the GmFT5a expression was also regulated by a photoperiod-independent system in LD. Taken together, our results suggest that GmFT2a and GmFT5a coordinately control flowering and enable the adaptation of soybean to a wide range of photoperiodic environments.

The antibiotic gramicidin, when incorporated into lipid membranes, forms pores that are exclusively permeable to monovalent cations and small unchanged molecules. We report the use of gramicidin for perforated patch-clamp recordings in the whole-cell mode. Recordings were performed in cultured rat spinal cord dorsal horn neurons. Cells had stable resting potentials and series resistances for times routinely exceeding 60 min. To test if intracellular chloride concentration ([Cl]i) remains stable with this technique, we measured responses to agonists of glycine and GABAA receptors, both of which gate chloride conductances. The driving force for these responses remained stable at values that differed significantly from values that would be expected if [Cl-]i were biased towards pipette [Cl-]. We conclude that gramicidin perforated-patch recording, in addition to other properties of the perforated-patch recording technique, has the advantage of not altering [Cl-]i. It is, therefore, an electrophysiological method particularly suitable for studies of anionic channels when [Cl-]i is a variable of interest, as well as for studies of homeostatic [Cl-]i regulation.

Nucleoporin 98 (Nup98), a glycine-leucine-phenylalanine-glycine (GLFG) amino acid repeat-containing nucleoporin, plays a critical part in nuclear trafficking. Injection of antibodies to Nup98 into the nucleus blocks the export of most RNAs. Nup98 contains binding sites for several transport factors; however, the mechanism by which this nucleoporin functions has remained unclear. Multiple subcellular localizations have been suggested for Nup98. Here we show that Nup98 is indeed found both at the nuclear pore complex and within the nucleus. Inside the nucleus, Nup98 associates with a novel nuclear structure that we term the GLFG body because the GLFG domain of Nup98 is required for targeting to this structure. Photobleaching of green fluorescent protein-Nup98 in living cells reveals that Nup98 is mobile and moves between these different localizations. The rate of recovery after photobleaching indicates that Nup98 interacts with other, less mobile, components in the nucleoplasm. Strikingly, given the previous link to nuclear export, the mobility of Nup98 within the nucleus and at the pore is dependent on ongoing transcription by RNA polymerases I and II. These data give rise to a model in which Nup98 aids in direction of RNAs to the nuclear pore and provide the first potential mechanism for the role of a mobile nucleoporin.

An elicitor of phytoalexin production in soybean (Glycine max L.) tissues was isolated from purified Phytophthora megasperma var. sojae mycelial walls by a heat treatment similar to that used to solubilize the surface antigens from the cell walls of Saccharomyces cerevisiae. The wall-released elicitor is a discrete, minor portion of the P. megasperma var. sojae mycelial walls. The elicitor released from the mycelial walls was divided by diethylaminoethylcellulose and concanavalin A-Sepharose chromatography into four fractions, each having different chemical characteristics. The four fractions were obtained from each of the three races of P. megasperma var. sojae. The corresponding fractions from each of the three races are very similar in composition and elicitor activity. The results suggest that the elicitor activity of each fraction resides in the glucan component of the fraction. Evidence is presented to demonstrate that the elicitors are not race-specific and that the accumulation of glyceollin is not sufficient to account for race-specific resistance.

Full-length cDNA clones corresponding to the transcripts of the two alpha-tubulin genes in Chlamydomonas reinhardi were isolated. DNA sequence analysis of the cDNA clones and cloned gene fragments showed that each gene contains 1,356 base pairs of coding sequence, predicting alpha-tubulin products of 451 amino acids. Of the 27 nucleotide differences between the two genes, only two result in predicted amino acid differences between the two gene products. In the more divergent alpha 2 gene, a leucine replaces an arginine at amino acid 308, and a valine replaces a glycine at amino acid 366. The results predicted that two alpha-tubulin proteins with different net charges are produced as primary gene products. The predicted amino acid sequences are 86 and 70% homologous with alpha-tubulins from rat brain and Schizosaccharomyces pombe, respectively. Each gene had two intervening sequences, located at identical positions. Portions of an intervening sequence highly conserved between the two beta-tubulin genes are also found in the second intervening sequence of each of the alpha genes. These results, together with our earlier report of the beta-tubulin sequences in C. reinhardi, present a picture of the total complement of genetic information for tubulin in this organism.

N-myristoylation is an acylation process absolutely specific to the N-terminal amino acid glycine in proteins. This maturation process concerns about a hundred proteins in lower and higher eukaryotes involved in oncogenesis, in secondary cellular signalling, in infectivity of retroviruses and, marginally, of other virus types. Thy cytosolic enzyme responsible for this activity, N-myristoyltransferase (NMT), studied since 1987, has been purified from different sources. However, the studies of the specificities of the various NMTs have not progressed in detail except for those relating to the yeast cytosolic enzyme. Still to be explained are differences in species specificity and between various putative isoenzymes, also whether the data obtained from the yeast enzyme can be transposed to other NMTs. The present review discusses data on the various addressing processes subsequent to myristoylation, a patchwork of pathways that suggests myristoylation is only the first step of the mechanisms by which a protein associates with the membrane. Concerning the enzyme itself, there are evidences that NMT is also present in the endoplasmic reticulum and that its substrate specificity is different from that of the cytosolic enzyme(s). These differences have major implications for their differential inhibition and for their respective roles in several pathologies. For instance, the NMTs from mammalians are clearly different from those found in several microorganisms, which raises the question whether the NMT may be a new targets for fungicides. Finally, since myristoylation has a central role in virus maturation and oncogenesis, specific NMT inhibitors might lead to potent antivirus and anticancer agents.

Some exons contain exon splicing silencers. Their activity is frequently balanced by that of splicing enhancers, and this is important to ensure correct relative levels of alternatively spliced mRNAs. Using an immunoprecipitation and UV-cross-linking assay, we show that RNA molecules containing splicing silencers from the human immunodeficiency virus type 1 tat exon 2 or the human fibroblast growth factor receptor 2 K-SAM exon bind to hnRNP A1 in HeLa cell nuclear extracts better than the corresponding RNA molecule without a silencer. Two different point mutations which abolish the K-SAM exon splicing silencer's activity reduce hnRNP A1 binding twofold. Recruitment of hnRNP A1 in the form of a fusion with bacteriophage MS2 coat protein to a K-SAM exon whose exon splicing silencer has been replaced by a coat binding site efficiently represses splicing of the exon in vivo. Recruitment of only the glycine-rich C-terminal domain of hnRNP A1, which is capable of interactions with other proteins, is sufficient to repress exon splicing. Our results show that hnRNP A1 can function to repress splicing, and they suggest that at least some exon splicing silencers could work by recruiting hnRNP A1.

The mitochondrial glycine decarboxylase multienzyme system, connected to serine hydroxymethyltransferase through a soluble pool of tetrahydrofolate, consists of four different component enzymes, the P-, H-, T- and L-proteins. In a multi-step reaction, it catalyses the rapid destruction of glycine molecules flooding out of the peroxisomes during the course of photorespiration. In green leaves, this multienzyme system is present at tremendously high concentrations within the mitochondrial matrix. The structure, mechanism and biogenesis of glycine decarboxylase are discussed. In the catalytic cycle of glycine decarboxylase, emphasis is given to the lipoate-dependent H-protein that plays a pivotal role, acting as a mobile substrate that commutes successively between the other three proteins. Plant mitochondria possess all the necessary enzymatic equipment for de novo synthesis of tetrahydrofolate and lipoic acid, serving as cofactors for glycine decarboxylase and serine hydroxymethyltransferase functioning.

The distributions of GABA-like and glycine-like immunoreactivities in the rat spinal cord were compared by using postembedding immunohistochemistry on semithin sections. In laminae I, II, and III, the proportions of GABA immunoreactive cells were 28%, 31%, and 46%, respectively, whereas for glycine immunoreactive cells the proportions were 9%, 14%, and 30%. Nearly all of the glycine immunoreactive cells in this area were also immunoreactive with the anti-GABA antiserum. In lamina II, some Golgi-stained islet cells were glycine immunoreactive, whereas others were not. Immunoreactive cell bodies were also present in the remainder of the grey matter. Some of these reacted with anti-GABA or antiglycine antiserum; others showed immunoreactivity with both antisera. Immunoreactive axons were found in the ventral and lateral funiculi of the white matter. Many large axons reacted with antiglycine antiserum, whereas GABA-immunoreactive axons were mostly of small diameter. Some large and small axons showed both types of immunoreactivity. These results suggest that the inhibitory neurotransmitters GABA and glycine coexist within cell bodies and axons in the rat spinal cord.

We report the presence of excitatory and inhibitory spontaneous and evoked synaptic currents in the dorsal motor nucleus of the vagus (DMV) in the rat upon vagal and perivagal stimulation. Whole cell current-clamp recordings from anatomically identified DMV neurons in rat brain stem slices show that these neurons are capable of sustained slow-frequency action potential firing probably because of the presence of pacemaker current. Spontaneously occurring, tetrodotoxin-resistant miniature inhibitory and excitatory synaptic potentials were observed. Stimulation of the vagus mostly induced antidromic action potentials in DMV neurons. However, careful positioning of the stimulating electrode in the tissue surrounding the recording neuron, and sometimes in the vagus itself, was capable of evoking orthodromic-evoked mixed inhibitory-excitatory postsynaptic potentials, and eventually, action potentials. Whole cell voltage-clamp recordings of the synaptic currents corresponding to these synaptic potentials in the presence of pharmacological antagonists of the neurotransmitters gamma-aminobutyric acid (GABA), glutamate, and glycine receptor subtypes indicate that the inhibitory synaptic currents are mediated by GABA-activated Cl- channels, while the excitatory synaptic currents are due to activation of ionotropic glutamate receptors of the N-methyl-D-aspartic acid (NMDA) and non-NMDA subtypes.