We report on ten individuals with a fatal infantile encephalopathy and/or pulmonary hypertension, leading to death before the age of 15 months. Hyperglycinemia and lactic acidosis were common findings. Glycine cleavage system and pyruvate dehydrogenase complex (PDHC) activities were low. Homozygosity mapping revealed a perfectly overlapping homozygous region of 1.24 Mb corresponding to chromosome 2 and led to the identification of a homozygous missense mutation (c.622G>> T) in NFU1, which encodes a conserved protein suggested to participate in Fe-S cluster biogenesis. Nine individuals were homozygous for this mutation, whereas one was compound heterozygous for this and a splice-site (c.545 + 5G>> A) mutation. The biochemical phenotype suggested an impaired activity of the Fe-S enzyme lipoic acid synthase (LAS). Direct measurement of protein-bound lipoic acid in individual tissues indeed showed marked decreases. Upon depletion of NFU1 by RNA interference in human cell culture, LAS and, in turn, PDHC activities were largely diminished. In addition, the amount of succinate dehydrogenase, but no other Fe-S proteins, was decreased. In contrast, depletion of the general Fe-S scaffold protein ISCU severely affected assembly of all tested Fe-S proteins, suggesting that NFU1 performs a specific function in mitochondrial Fe-S cluster maturation. Similar biochemical effects were observed in Saccharomyces cerevisiae upon deletion of NFU1, resulting in lower lipoylation and SDH activity. Importantly, yeast Nfu1 protein carrying the individuals' missense mutation was functionally impaired. We conclude that NFU1 functions as a late-acting maturation factor for a subset of mitochondrial Fe-S proteins.

BACKGROUND

[corrected] The Staphylococcus aureus collagen-binding protein Cna mediates bacterial adherence to collagen. The primary sequence of Cna has a non-repetitive collagen-binding A region, followed by the repetitive B region. The B region has one to four 23 kDa repeat units (B(1)-B(4)), depending on the strain of origin. The affinity of the A region for collagen is independent of the B region. However, the B repeat units have been suggested to serve as a 'stalk' that projects the A region from the bacterial surface and thus facilitate bacterial adherence to collagen. To understand the biological role of these B-region repeats we determined their three-dimensional structure.

RESULTS

B(1) has two domains (D(1) and D(2)) placed side-by-side. D(1) and D(2) have similar secondary structure and exhibit a unique fold that resembles but is the inverse of the immunoglobulin-like (IgG-like) domains. Comparison with similar immunoglobulin superfamily (IgSF) structures shows novel packing arrangements between the D(1) and D(2) domains. In the B(1)B(2) crystal structure, an omission of a single glycine residue in the D(2)-D(3) linker loop, compared to the D(1)-D(2) and D(3)-D(4) linker loops, resulted in projection of the D(3) and D(4) in a spatially new orientation. We also present a model for B(1)B(2)B(3)B(4).

CONCLUSIONS

The B region of the Cna collagen adhesin has a novel fold that is reminiscent of but is inverse in nature to the IgG fold. This B region assembly could effectively provide the needed flexibility and stability for presenting the ligand binding A region away from the bacterial cell surface.

The critical problem of oxygen toxicity for nitrogen-fixing organisms may be related to damage caused by oxygen radicals and peroxides. An enzymatic mechanism is described for removal of peroxides in root nodules of soybean (Glycine max). The system utilizes ascorbate as an antioxidant and glutathione as a reductant to regenerate ascorbate. The enzymes involved are ascorbate peroxidase (ascorbate:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1), and glutathione reductase (NADPH:oxidized-glutathione oxidoreductase, EC 1.6.4.2). The reactions are essentially the same as those involving scavenging of H(2)O(2) in chloroplasts. Glutathione peroxidase (glutathione:hydrogenperoxide oxidoreductase, EC 1.11.1.9) was not detected. During the course of early nodule development, ascorbate peroxidase and dehydroascorbate reductase activities and total glutathione contents of nodule extracts increased strikingly and were positively correlated with acetylene reduction rates and nodule hemoglobin contents. The evidence indicates an important role of glutathione, ascorbate, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase as components of a peroxide-scavenging mechanism in soybean root nodules.

A family of octamer binding proteins is expressed during mouse development. Oct-4 and Oct-6 have been identified as two octamer binding proteins present in embryonal stem cells. Here we report the complementary DNA cloning and characterization of the mouse Oct-6 gene. The protein of 448 amino acids contains a glycine/alanine-rich amino terminal region, a histidine-rich sequence with homology to a region of kininogen associated with clotting, a POU domain and a short proline/histidine-rich carboxy terminal region. Expression of Oct-6 in HeLa cells is sufficient for transcriptional activation from the octamer motif, identifying Oct-6 as a transcription factor. The Oct-6 expression is downregulated upon embryonic stem cell differentiation increasing again during brain development. Expression in brain is present in certain areas of telencephalon, mesencephalon and brain stem with abundant expression in the cortex anlagen and in the developing colliculi. Thus Oct-6 is a new octamer binding transcription factor specifically regulated during mouse development.

It is well established that long-term potentiation (LTP), a paradigm for learning and memory, results in a stable enlargement of potentiated spines associated with recruitment of additional GluA1-containing AMPA receptors (AMPARs). Although regulation of the actin cytoskeleton is involved, the detailed signaling mechanisms responsible for this spine expansion are unclear. Here, we used cultured mature hippocampal neurons stimulated with a glycine-induced, synapse-specific form of chemical LTP (GI-LTP). We report that the stable structural plasticity (i.e., spine head enlargement and spine length shortening) that accompanies GI-LTP was blocked by inhibitors of NMDA receptors (NMDARs; APV) or CaM-kinase kinase (STO-609), the upstream activator of CaM-kinase I (CaMKI), as well as by transfection with dominant-negative (dn) CaMKI but not dnCaMKIV. Recruitment of GluA1 to the spine surface occurred after GI-LTP and was mimicked by transfection with constitutively active CaMKI. Spine enlargement induced by transfection of GluA1 was associated with synaptic recruitment of Ca(2+)-permeable AMPARs (CP-AMPARs) as assessed by an increase in the rectification index of miniature EPSCs (mEPSCs) and their sensitivity to IEM-1460, a selective antagonist of CP-AMPARs. Furthermore, the increase in spine size and mEPSC amplitude resulting from GI-LTP itself was blocked by IEM-1460, demonstrating involvement of CP-AMPARs. Downstream signaling effectors of CP-AMPARs, identified by suppression of their activation by IEM-1460, included the Rac/PAK/LIM-kinase pathway that regulates spine actin dynamics. Together, our results suggest that synaptic recruitment of CP-AMPARs via CaMKI may provide a mechanistic link between NMDAR activation in LTP and regulation of a signaling pathway that drives spine enlargement via actin polymerization.

In addition to photoreceptors and neurons, glial cells (in particular Müller cells) contribute to the removal and metabolization of neurotransmitters in the neural retina. This review summarizes the present knowledge regarding the role of retinal glial cells in the uptake of glutamate, N-acetylaspartylglutamate, gamma-aminobutyric acid, glycine, and d-serine, as well as the degradation and removal of purinergic receptor agonists. Some major pathways of glutamate metabolism in Müller cells are described; these pathways are involved in the glutamate-glutamine cycle of the retina, in the defense against oxidative and nitrosative stress via the production of glutathione, and in the production of substrates for the neuronal energy metabolism. In addition, the developmental regulation of the major glial glutamate transporter, GLAST, and of the glia-specific enzyme glutamine synthetase is described, as well as the importance of a malfunction and even reversal of glial glutamate transporters, and a downregulation of the glutamine synthetase, as pathogenic factors in different retinopathies.

Lactobacillus plantarum C11 secretes a small cationic peptide, plantaricin A, that serves as induction signal for bacteriocin production as well as transcription of plnABCD. The plnABCD operon encodes the plantaricin A precursor (PlnA) itself and determinants (PlnBCD) for a signal transducing pathway. By Northern (RNA) and sequencing analyses, four new plantaricin A-induced operons were identified. All were highly activated in concert with plnABCD upon bacteriocin induction. Two of these operons (termed plnEFI and plnJKLR) each encompass a gene pair (plnEF and plnJK, respectively) encoding two small cationic bacteriocin-like peptides with double-glycine-type leaders. The open reading frames (ORFs) encoding the bacteriocin-like peptides are followed by ORFs (plnI and -L, respectively) encoding cationic hydrophobic proteins resembling bacteriocin immunity proteins. On the third operon (termed plnMNOP), a similar bacteriocin-like ORF (plnN) and a putative immunity ORF (either plnM or -P) were identified as well. These findings suggest that two bacteriocins of two-peptide type (mature PlnEF and PlnJK) and a bacteriocin of one-peptide type (mature PlnN) could be responsible for the observed bacteriocin activity. The last operon (termed plnGHSTUV) contains two ORFs (plnGH) apparently encoding an ABC transporter and its accessory protein, respectively, known to be involved in processing and export of peptides with precursor double-glycine-type leaders. Promoter structure was established. A conserved regulatory-like box encompassing two direct repeats was identified in the promoter regions of all five plantaricin A-induced operons. These repeats may serve as regulatory elements for gene expression.

All living cells must conduct protein synthesis with a high degree of accuracy maintained in the transmission and flow of information from gene to finished protein product. One crucial "quality control" point in maintaining a high level of accuracy is the selectivity by which aminoacyl-tRNA synthetases furnish correctly activated amino acids, attached to tRNA species, as the building blocks for growing protein chains. During selection of amino acids, synthetases very often have to distinguish the cognate substrate from a homolog having just one fewer methyl group in its structure. The binding energy of a methyl group is estimated to contribute only a factor of 100 to the specificity of binding, yet synthetases distinguish such closely related amino acids with a discrimination factor of 10,000 to 100,000. Examples of this include methionine versus homocysteine, isoleucine versus valine, alanine versus glycine, and threonine versus serine. Many investigators have demonstrated in vitro the ability of certain aminoacyl-tRNA synthetases to edit, that is, correct or prevent incorrect attachment of amino acids to tRNA molecules. Several major editing pathways are now established from in vitro data. Further, at least some aminoacyl-tRNA synthetases have recently been shown to carry out the editing function in vivo. Editing has been demonstrated to occur in both Escherichia coli and Saccharomyces cerevisiae. Significant energy is expended by the cell for editing of misactivated amino acids, which can be reflected in the growth rate. Because of this, cellular levels of aminoacyl-tRNA synthetases, as well as amino acid biosynthetic pathways which yield competing substrates for protein synthesis, must be carefully regulated to prevent excessive editing. High-level expression of recombinant proteins imposes a strain on the biosynthetic capacity of the cell which frequently results in misincorporation of abnormal or wrong amino acids owing in part to limited editing by synthetases. Unbalanced amino acid pools associated with some genetic disorders in humans may also lead to errors in tRNA aminoacylation. The availability of X-ray crystallographic structures of some synthetases, combined with site-directed mutagenesis, allows insights into molecular details of the extraordinary selectivity of synthetases, including the editing function.

New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to BKIs. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable.

Sulfur is an essential macro-element in plant and animal nutrition. Plants assimilate inorganic sulfate into two sulfur-containing amino acids, cysteine and methionine. Low supply of sulfate leads to decreased sulfur pools within plant tissues. As sulfur-related metabolites represent an integral part of plant metabolism with multiple interactions, sulfur deficiency stress induces a number of adaptive responses, which must be coordinated. To reveal the coordinating network of adaptations to sulfur deficiency, metabolite profiling of Arabidopsis has been undertaken. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry techniques revealed the response patterns of 6,023 peaks of nonredundant ion traces and relative concentration levels of 134 nonredundant compounds of known chemical structure. Here, we provide a catalogue of the detected metabolic changes and reconstruct the coordinating network of their mutual influences. The observed decrease in biomass, as well as in levels of proteins, chlorophylls, and total RNA, gives evidence for a general reduction of metabolic activity under conditions of depleted sulfur supply. This is achieved by a systemic adjustment of metabolism involving the major metabolic pathways. Sulfur/carbon/nitrogen are partitioned by accumulation of metabolites along the pathway O-acetylserine to serine to glycine, and are further channeled together with the nitrogen-rich compound glutamine into allantoin. Mutual influences between sulfur assimilation, nitrogen imbalance, lipid breakdown, purine metabolism, and enhanced photorespiration associated with sulfur-deficiency stress are revealed in this study. These responses may be assembled into a global scheme of metabolic regulation induced by sulfur nutritional stress, which optimizes resources for seed production.

Islet transplantation is associated with a high rate of early graft failure, a problem that remains poorly understood. It is probable that the destruction of the islet microenvironment and loss of tropic support that occur during isolation lead to compromised survival. The purpose of this study was to determine the role of matrix-integrin interactions on beta-cell survival and function following islet isolation. Canine islets were obtained by conventional methods. Immediately after isolation, the peri-insular basement membrane (BM) was absent. The ability of islets maintained in suspension culture to attach to a collagen matrix declined progressively over 6 days. Attachment could be blocked by an arginine-glycine-aspartate (RGD) motif-presenting synthetic peptide, thereby implicating an integrin-mediated process. Characterization of cell surface integrins by immunocytochemistry (ICC) demonstrated that the expression of integrins alpha3, alpha5 and alphaV diminished during the culture period. This change was coincident with both a decrease in beta-cell function (proinsulin gene expression, islet insulin content and stimulated insulin release) and a rise in beta-cell death from apoptosis, as determined by in situ cell death detection (TUNEL) assay. These adverse events were prevented or delayed by exposure of islets to matrix proteins. In conclusion, routine islet isolation disrupts the cell-matrix relationship leading to a variety of structural and functional abnormalities, including apoptotic cell death. These alterations can be diminished by restoration of a culture microenvironment that includes matrix proteins.

This review summarizes the data on 278 different mutations found to date in the genes for types I, II, III, IX, X, and XI collagens from 317 apparently unrelated patients. A majority (217 mutations; 78% of the total) of the mutations are single-base and either change the codon of a critical amino acid (63%), or lead to abnormal RNA splicing (13%). Most of the amino acid substitutions are those of a bulkier amino acid for the obligatory glycine of the repeating-Gly-X-Y-sequence of the collagen triple helix (155; 56%). Altogether, 26 different mutations (9.4% of the mutations) occur in more than one unrelated individual. The 65 patients in whom the 26 mutations were characterized constitute almost one-fifth (20.5%) of the 317 patients analyzed. The mutations in types I, II, III, IX, X, and XI collagens cause a wide spectrum of diseases of bone, cartilage, and blood vessels, including osteogenesis imperfecta, a variety of chondrodysplasias, types IV and VII of the Ehlers-Danlos syndrome, and, rarely, some forms of osteoporosis, osteoarthritis, and familial aneurysms.

"Soft" and "hard" are the two main market classes of wheat (Triticum aestivum L.) and are distinguished by expression of the Hardness gene. Friabilin, a marker protein for grain softness (Ha), consists of two proteins, puroindoline a and b (pinA and pinB, respectively). We previously demonstrated that a glycine to serine mutation in pinB is linked inseparably to grain hardness. Here, we report that the pinB serine mutation is present in 9 of 13 additional randomly selected hard wheats and in none of 10 soft wheats. The four exceptional hard wheats not containing the serine mutation in pinB express no pinA, the remaining component of the marker protein friabilin. The absence of pinA protein was linked inseparably to grain hardness among 44 near-isogenic lines created between the soft variety Heron and the hard variety Falcon. Both pinA and pinB apparently are required for the expression of grain softness. The absence of pinA protein and transcript and a glycine-to-serine mutation in pinB are two highly conserved mutations associated with grain hardness, and these friabilin genes are the suggested tightly linked components of the Hardness gene. A previously described grain hardness related gene termed "GSP-1" (grain softness protein) is not controlled by chromosome 5D and is apparently not involved in grain hardness. The association of grain hardness with mutations in both pinA or pinB indicates that these two proteins alone may function together to effect grain softness. Elucidation of the molecular basis for grain hardness opens the way to understanding and eventually manipulating this wheat endosperm property.

The clinical associations of glycine receptor antibodies have not yet been described fully. We identified prospectively 52 antibody-positive patients and collated their clinical features, investigations and immunotherapy responses. Serum glycine receptor antibody endpoint titres ranged from 1:20 to 1:60 000. In 11 paired samples, serum levels were higher than (n = 10) or equal to (n = 1) cerebrospinal fluid levels; there was intrathecal synthesis of glycine receptor antibodies in each of the six pairs available for detailed study. Four patients also had high glutamic acid decarboxylase antibodies (>1000 U/ml), and one had high voltage-gated potassium channel-complex antibody (2442 pM). Seven patients with very low titres (<1:50) and unknown or alternative diagnoses were excluded from further study. Three of the remaining 45 patients had newly-identified thymomas and one had a lymphoma. Thirty-three patients were classified as progressive encephalomyelitis with rigidity and myoclonus, and two as stiff person syndrome; five had a limbic encephalitis or epileptic encephalopathy, two had brainstem features mainly, two had demyelinating optic neuropathies and one had an unclear diagnosis. Four patients (9%) died during the acute disease, but most showed marked improvement with immunotherapies. At most recent follow-up, (2-7 years, median 3 years, since first antibody detection), the median modified Rankin scale scores (excluding the four deaths) decreased from 5 at maximal severity to 1 (P < 0.0001), but relapses have occurred in five patients and a proportion are on reducing steroids or other maintenance immunotherapies as well as symptomatic treatments. The glycine receptor antibodies activated complement on glycine receptor-transfected human embryonic kidney cells at room temperature, and caused internalization and lysosomal degradation of the glycine receptors at 37°C. Immunoglobulin G antibodies bound to rodent spinal cord and brainstem co-localizing with monoclonal antibodies to glycine receptor-α1. Ten glycine receptor antibody positive samples were also identified in a retrospective cohort of 56 patients with stiff person syndrome and related syndromes. Glycine receptor antibodies are strongly associated with spinal and brainstem disorders, and the majority of patients have progressive encephalomyelitis with rigidity and myoclonus. The antibodies demonstrate in vitro evidence of pathogenicity and the patients respond well to immunotherapies, contrasting with earlier studies of this syndrome, which indicated a poor prognosis. The presence of glycine receptor antibodies should help to identify a disease that responds to immunotherapies, but these treatments may need to be sustained, relapses can occur and maintenance immunosuppression may be required.

The composition of the human microbiota is recognized as an important factor in human health and disease. Many of our cohabitating microbes belong to phylum-level divisions for which there are no cultivated representatives and are only represented by small subunit rRNA sequences. For one such taxon (SR1), which includes bacteria with elevated abundance in periodontitis, we provide a single-cell genome sequence from a healthy oral sample. SR1 bacteria use a unique genetic code. In-frame TGA (opal) codons are found in most genes (85%), often at loci normally encoding conserved glycine residues. UGA appears not to function as a stop codon and is in equilibrium with the canonical GGN glycine codons, displaying strain-specific variation across the human population. SR1 encodes a divergent tRNA(Gly)UCA with an opal-decoding anticodon. SR1 glycyl-tRNA synthetase acylates tRNA(Gly)UCA with glycine in vitro with similar activity compared with normal tRNA(Gly)UCC. Coexpression of SR1 glycyl-tRNA synthetase and tRNA(Gly)UCA in Escherichia coli yields significant β-galactosidase activity in vivo from a lacZ gene containing an in-frame TGA codon. Comparative genomic analysis with Human Microbiome Project data revealed that the human body harbors a striking diversity of SR1 bacteria. This is a surprising finding because SR1 is most closely related to bacteria that live in anoxic and thermal environments. Some of these bacteria share common genetic and metabolic features with SR1, including UGA to glycine reassignment and an archaeal-type ribulose-1,5-bisphosphate carboxylase (RubisCO) involved in AMP recycling. UGA codon reassignment renders SR1 genes untranslatable by other bacteria, which impacts horizontal gene transfer within the human microbiota.

Activated microglia may be detrimental to neuronal survival in a number of neurodegenerative diseases. Thus, strategies that reduce microglial neurotoxicity may have therapeutic benefit. Stimulation of group II metabotropic glutamate (mGlu) receptors on rat primary microglia with the specific group II agonist 2S,2'R,3'R-2-(2',3'-dicarboxy-cyclopropyl)glycine for 24 h induced microglial activation and resulted in a neurotoxic microglial phenotype. These effects were attributable to preferential mGlu2 stimulation, because N-acetyl-L-aspartyl-L-glutamate, a specific mGlu3 agonist, did not induce microglial activation or neurotoxicity. Stimulation of microglial mGlu2 but not mGlu3 induced caspase-3 activation in cerebellar granule neurons in culture, using microglial-conditioned media as well as cocultures. Stimulation of microglial mGlu2 induced tumor necrosis factor-alpha (TNFalpha) release, which contributed to microglial neurotoxicity mediated via neuronal TNF receptor 1 and caspase-3 activation. Stimulation of microglial group I or III mGlu receptors did not induce TNFalpha release. TNFalpha was only neurotoxic in the presence of microglia or microglial-conditioned medium. The toxicity of TNFalpha could be prevented by coexposure of neurons to conditioned medium from microglia stimulated by the specific group III agonist L-2-amino-4-phosphono-butyric acid. The neurotoxicity of TNFalpha derived from mGlu2-stimulated microglia was potentiated by microglial-derived Fas ligand (FasL), the death receptor ligand. FasL was constitutively expressed in microglia and shed after mGlu2 stimulation. Our data suggest that selective and inverse modulation of microglial mGlu2 and mGlu3 may prove a therapeutic target in neuroinflammatory diseases such as Alzheimer's disease and multiple sclerosis.

Activity-dependent synapse refinement is crucial for the formation of precise excitatory and inhibitory neuronal circuits. Whereas the mechanisms that guide refinement of excitatory circuits are becoming increasingly clear, the mechanisms guiding inhibitory circuits have remained obscure. In the lateral superior olive (LSO), a nucleus in the mammalian sound localization system that receives inhibitory input from the medial nucleus of the trapezoid body (MNTB), specific elimination and strengthening of synapses that are both GABAergic and glycinergic (GABA/glycinergic synapses) is essential for the formation of a precise tonotopic map. We provide evidence that immature GABA/glycinergic synapses in the rat LSO also release the excitatory neurotransmitter glutamate, which activates postsynaptic NMDA receptors (NMDARs). Immunohistochemical studies demonstrate synaptic colocalization of the vesicular glutamate transporter 3 with the vesicular GABA transporter, indicating that GABA, glycine and glutamate are released from single MNTB terminals. Glutamatergic transmission at MNTB-LSO synapses is most prominent during the period of synapse elimination. Synapse-specific activation of NMDARs by glutamate release at GABAergic and glycinergic synapses could be important in activity-dependent refinement of inhibitory circuits.

The envelope glycoproteins of the human immunodeficiency virus (HIV) type 1 are synthesized as a precursor molecule, gp160, which is cleaved to generate the two mature envelope glycoproteins, gp120 and gp41. The cleavage reaction, which is mediated by a host protease, occurs at a sequence highly conserved in retroviral envelope glycoprotein precursors. We have investigated the sequence requirements for this cleavage reaction by introducing four single-amino-acid changes into the glutamic acid-lysine-arginine sequence immediately amino terminal to the site of cleavage. We have also examined the effects of these mutations on the syncytium formation induced by HIV envelope glycoproteins. Our results indicate that a glutamic acid to glycine change at gp120 amino acid 516, a lysine to isoleucine change at amino acid 517, and an arginine to lysine change at amino acid 518 affect neither gp160 cleavage nor syncytium formation. The results obtained with the arginine to lysine change at amino acid 518 differ significantly from the results obtained with the same mutation at the envelope precursor cleavage site of a murine leukemia virus (E. O. Freed, and R. Risser, J. Virol. 61:2852-2856, 1987). An arginine to threonine mutation at gp120 amino acid 518, the terminal residue of gp120, abolishes both gp160 cleavage and syncytium formation. These findings demonstrate that despite its highly conserved nature, the basic pair of amino acids at the site of gp160 cleavage is not absolutely required for proper envelope glycoprotein processing. This report also supports the idea that cleavage of gp160 is required for activation of the HIV envelope fusion function.

The glycine transporter subtype 1 (GlyT1) is widely expressed in astroglial cells throughout the mammalian central nervous system and has been implicated in the regulation of N-methyl-D-aspartate (NMDA) receptor activity. Newborn mice deficient in GlyT1 are anatomically normal but show severe motor and respiratory deficits and die during the first postnatal day. In brainstem slices from GlyT1-deficient mice, in vitro respiratory activity is strikingly reduced but normalized by the glycine receptor (GlyR) antagonist strychnine. Conversely, glycine or the GlyT1 inhibitor sarcosine suppress respiratory activity in slices from wild-type mice. Thus, during early postnatal life, GlyT1 is essential for regulating glycine concentrations at inhibitory GlyRs, and GlyT1 deletion generates symptoms found in human glycine encephalopathy.

The abundant nuclear RNA binding protein FUS binds the C-terminal domain (CTD) of RNA polymerase II in an RNA-dependent manner, affecting Ser2 phosphorylation and transcription. Here, we examine the mechanism of this process and find that RNA binding nucleates the formation of higher-order FUS ribonucleoprotein assemblies that bind the CTD. Both the low-complexity domain and the arginine-glycine rich domain of FUS contribute to assembly. The assemblies appear fibrous by electron microscopy and have characteristics of β zipper structures. These results support the emerging view that the pathologic protein aggregation seen in neurodegenerative diseases such as amyotrophic lateral sclerosis may occur via the exaggeration of functionally important assemblies of RNA binding proteins.

The ABA-induced MA12 cDNA from maize, which encodes a set of highly phosphorylated embryo proteins, was used to isolate the corresponding genomic clone. This gene, called RAB-17 (responsive to ABA), encodes a basic, glycine-rich protein (mol. wt. 17,164) containing a cluster of 8 serine residues, seven of them contiguous. It is a homologue of the rice RAB-21 gene (Mundy J, Chua NH, EMBO J 7; 2279-2286, 1988). Phosphoamino acid analysis of the isolated protein indicates that only the serine residues are phosphorylated and a putative casein-type kinase phosphorylatable sequence was identified in the protein. The pattern of expression and in vivo phosphorylation of the RAB-17 protein was studied during maize embryo germination and in calli of both meristematic or embryonic origin. ABA treatment induced the synthesis of RAB-17 mRNA and protein in calli, however, the RAB-17 proteins were found to be highly phosphorylated only in embryos.

The helix propagation and N-cap propensities of the amino acids have been measured in alanine-based peptides in 40 volume percent trifluoroethanol (40% TFE) to determine if this helix-stabilizing solvent uniformly affects all amino acids. The propensities in 40% TFE are compared with revised values of the helix parameters of alanine-based peptides in water. Revision of the propensities in water is the result of redefining the capping statistical weights and evaluating the helix nucleation constant with N-capping explicitly included in the helix-coil model. The propagation propensities of all amino acids increase in 40% TFE relative to water, but the increases are highly variable. In water, all beta-branched and beta-substituted amino acids are helix breakers. In 40% TFE, the propagation propensities of the nonpolar amino acids increase greatly, leaving charged and neutral polar, beta-substituted amino acids as helix breakers. Glycine and proline are strong helix breakers in both solvents. Free energy differences for helix propagation (delta delta G) between alanine and other nonpolar amino acids are twice as large in water as predicted from side-chain conformational entropies, but delta delta G values in 40% TFE are close to those predicted from side-chain entropies. This dependence of delta delta G on the solvent points to a specific role of water in determining the relative helix propensities of the nonpolar amino acids. The N-cap propensities converge toward a common value in 40% TFE, suggesting that differential solvation by water contributes to the diversity of N-cap values shown by the amino acids.

POM121 and gp210 were, until this point, the only known membrane-integral nucleoporins (Nups) of vertebrates and, thus, the only candidate anchors for nuclear pore complexes (NPCs) within the nuclear membrane. In an accompanying study (Stavru et al.), we provided evidence that NPCs can exist independently of POM121 and gp210, and we predicted that vertebrate NPCs contain additional membrane-integral constituents. We identify such an additional membrane protein in the NPCs of mammals, frogs, insects, and nematodes as the orthologue to yeast Ndc1p/Cut11p. Human NDC1 (hNDC1) likely possesses six transmembrane segments, and it is located at the nuclear pore wall. Depletion of hNDC1 from human HeLa cells interferes with the assembly of phenylalanine-glycine repeat Nups into NPCs. The loss of NDC1 function in Caenorhabditis elegans also causes severe NPC defects and very high larval and embryonic mortality. However, it is not ultimately lethal. Instead, homozygous NDC1-deficient worms can be propagated. This indicates that none of the membrane-integral Nups is universally essential for NPC assembly, and suggests that NPC biogenesis is an extremely fault-tolerant process.

Nucleolin, a major nucleolar protein implicated in preribosome assembly and transcriptional regulation, possesses a C-terminal domain unusually rich in glycine, arginine, and phenylalanine residues. A polypeptide (p10), corresponding to this domain, has been synthesized by means of an Escherichia coli expression system and purified to homogeneity. Nitrocellulose binding assays have clearly shown that this domain of nucleolin is capable of interacting with RNA, and indeed all nucleic acids tested, in an efficient but nonspecific manner. A combination of circular dichroism and infrared spectroscopy provide strong evidence that repeated beta-turns are a major structural component of this polypeptide, which is entirely consistent with its amino acid composition and above all the presence of repeat motifs such as RGGF. Circular dichroism technique also shows that the interaction of p10 with RNA involves an unstacking of the nucleotide bases and an unfolding of the RNA secondary structure. While the role of the C-terminal domain of nucleolin in vivo has yet to be established, our findings suggest that it may act to unfold regions of ribosomal RNA so that a second domain of nucleolin has access to its specific binding site.