Nucleolin (also called C23) is the major nucleolar protein of exponentially growing eukaryotic cells. It is found associated with intranucleolar chromatin and preribosomal particles. Through use of a polyclonal antiserum, nucleolin cDNA clones were isolated from a Chinese hamster ovary cell library constructed in the expression vector lambda gt11. The isolated cDNAs encoded a polypeptide containing 679 residues of the 713 amino acids of nucleolin. The amino acid sequence presents several unusual features: in particular, repetitive sequences are found at both ends of the molecule. A repeat, Hy-Thr-Pro-Hy-Lys-Lys-Hy-Hy, in which Hy is a nonpolar residue, is found six times in the NH2-end proximal portion, followed by three acidic stretches containing 25, 25, and 33 glutamic acid or aspartic acid residues. Four potential phosphorylation sites (serines) are also observed in this region. The COOH-terminal proximal portion of the protein carries a glycine-rich region with fairly regularly interspersed phenylalanine and dimethylarginine residues. The two terminal portions of the molecule exhibit unique potential secondary structures: alpha-helix (NH2 terminus) and extended (COOH terminus). The central region exhibits alternating hydrophobic and hydrophilic stretches. Five potential N glycosylation sites are detected. The structure of this protein may reflect two functions in preribosome biogenesis: interaction with chromatin (NH2 terminus) and with preribosomes (COOH terminus).

To get further information on bacterial surface sensing and biofilm-dependent regulation of gene expression in Escherichia coli K-12, random insertion mutagenesis with Mu dX, a mini-Mu carrying the promoterless lacZ gene, was performed with an ompR234 adherent strain, and a simple screen was developed to assess changes in gene expression in biofilm cells versus planktonic cells. This screen revealed that major changes in the pattern of gene expression occur during biofilm development: the transcription of 38% of the genes was affected within biofilms. Different cell functions were more expressed in sessile bacteria: the OmpC porin, the high-affinity transport system of glycine betaine (encoded by the proU operon), the colanic acid exopolysaccharide (wca locus, formerly called cps), tripeptidase T (pepT), and the nickel high-affinity transport system (nikA). On the other hand, the syntheses of flagellin (fliC) and of a putative protein of 92 amino acids (f92) were both reduced in biofilms. Such a genetic reprogramming of gene expression in biofilms seems to result from changes in multiple environmental physicochemical conditions. In this work, we show that bacteria within biofilms encounter higher-osmolarity conditions, greater oxygen limitation, and higher cell density than in the liquid phase.

The Martini coarse-grained force field has been successfully used for simulating a wide range of (bio)molecular systems. Recent progress in our ability to test the model against fully atomistic force fields, however, has revealed some shortcomings. Most notable, phenylalanine and proline were too hydrophobic, and dimers formed by polar residues in apolar solvents did not bind strongly enough. Here, we reparametrize these residues either through reassignment of particle types or by introducing embedded charges. The new parameters are tested with respect to partitioning across a lipid bilayer, membrane binding of Wimley-White peptides, and dimerization free energy in solvents of different polarity. In addition, we improve some of the bonded terms in the Martini protein force field that lead to a more realistic length of α-helices and to improved numerical stability for polyalanine and glycine repeats. The new parameter set is denoted Martini version 2.2.

The major type of receptor for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), called the GABAA receptor, is a member of a gene superfamily of ligand-gated ion channels. This receptor is a hetero-oligomeric protein composed of several distinct polypeptide types (alpha, beta, gamma, and delta). Molecular cloning of these polypeptides reveals that they show 20-40% identity with each other, and 10-20% identity with polypeptides of the nicotinic acetylcholine receptors and strychnine-sensitive glycine receptor. Each polypeptide type is also represented by a family of genes whose members have 60-80% amino acid sequence identity. Regions of conserved and variable amino acid sequence suggest structural and functional domains within each polypeptide. All of the polypeptides when expressed in heterologous cells produce GABA-activated chloride channels, and the different subtypes express different pharmacological properties. The distributions of mRNAs for the different GABAA receptor polypeptides and their subtypes show significant brain regional variation consistent with pharmacological and biochemical evidence for receptor heterogeneity. Subpopulations of GABAA receptors with different cellular and regional locations show differential sensitivity to GABA, to modulators like steroids, to physiological regulation, to disease processes, and to pharmacological manipulation by drugs such as benzodiazepines. The properties of the different subpopulations of GABAA receptors are determined by which one or more of the different polypeptides and their subtypes are expressed in a given cell to produce a variety of different oligomeric protein structures. Molecular cloning techniques have produced rapid advances in understanding the GABAA receptor protein family.

We show that the non-DNA-binding precursor for the p50 subunit (p110), like NF-kappa B, is subject to control of nuclear uptake. In contrast to p50, p110 was excluded from nuclei and unable to associate detectably with p50 or p65 NF-kappa B subunits. The nuclear location signal in the N-terminal half of p110 was not accessible for monospecific antibodies. Removal of only 191 amino acids from the C-terminus of p110 restored antibody accessibility as well as nuclear uptake. The C-terminal half of p110, which is linked to the p50 portion via a glycine-rich hinge, could also noncovalently bind to p50. This helps to explain why p50, after cleavage of the precursor in intact cells, was still retained in an inactive form in the cytoplasm. Our study describes a novel mechanism of nuclear uptake control by masking of a nuclear location signal through a remote domain within a precursor molecule.

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.

Infection of metazoan cells with some viruses alters the balance of cellular mRNA export to favor viral RNA export and to retain cellular transcripts in the nucleus. Here, evidence is presented to show that the herpes simplex virus 1 (HSV-1) essential regulatory protein ICP27, which inhibits host cell-splicing, resulting in the accumulation of unspliced transcripts in the nucleus, mediates RNA export of viral intronless mRNAs. ICP27 was shown to shuttle between the nucleus and cytoplasm through a leucine-rich nuclear export signal, which alone was able to direct the export of the heterologous green fluorescent protein. In vivo UV irradiation studies demonstrated that ICP27 could be crosslinked to poly(A)+ RNA in the nucleus and the cytoplasm, supporting a role in export. Furthermore, the amount of hnRNP A1, which has been implicated in the export of cellular spliced mRNAs, that was bound to poly(A)+ RNA in HSV-1-infected cells was reduced compared with uninfected cells. In addition, it was demonstrated that ICP27 bound seven intronless HSV-1 transcripts in both the nucleus and the cytoplasm, and export of these transcripts was diminished substantially during infection with an ICP27 null mutant virus. In contrast, ICP27 did not bind to two HSV-1 mRNAs that undergo splicing. Finally, binding of ICP27 to RNA in vivo required an arginine-glycine region that resembles an RGG box. These results indicate that ICP27 is an important viral export factor that promotes the transport of HSV-1 intronless RNAs.

Repression of transcription from the silent mating loci (HML alpha and HMRa) is essential for mating ability in Saccharomyces cerevisiae. This silencing is known to require at least five proteins (SIR1, SIR2, SIR3, SIR4, and histone H4) and is accompanied by a change in chromatin structure. We show here that four positions of histone H4 (N-terminal residues 16, 17, 18, and 19) are crucial to silencing. HML alpha and HMRa are efficiently repressed when these positions are occupied by basic amino acids but are derepressed when substituted with glycine. These results suggest that acetylation of Lys-16 would lead to derepression of the silent mating loci. Three strong extragenic suppressors of the latter H4 mutations were isolated and determined to be located in SIR3. These suppressors allow high mating efficiencies in cells expressing either wild-type H4 or H4 containing single amino acid substitutions. They did not allow efficient mating in a strain that contained an H4 N-terminal deletion. These results indicate that the SIR3 mutations do not bypass the requirement for the H4 N terminus but, rather, allow repression in the presence of a less than optimal H4 N terminus. This provides a link between one of the SIR proteins and a component of chromatin.

Many surface proteins of Gram-positive bacteria are anchored to the cell wall envelope by a transpeptidation mechanism, requiring a C-terminal sorting signal with a conserved LPXTG motif. Sortase, a membrane protein of Staphylococcus aureus, cleaves polypeptides between the threonine and the glycine of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl-group of threonine and the amino-group of peptidoglycan cross-bridges. S. aureus mutants lacking the srtA gene fail to anchor and display some surface proteins and are impaired in the ability to cause animal infections. Sortase acts on surface proteins that are initiated into the secretion (Sec) pathway and have their signal peptide removed by signal peptidase. The S. aureus genome encodes two sets of sortase and secretion genes. It is conceivable that S. aureus has evolved more than one pathway for the transport of 20 surface proteins to the cell wall envelope.

The structure of a ternary complex of the catalytic subunit of cAMP-dependent protein kinase, MgATP, and a 20-residue inhibitor peptide was determined at a resolution of 2.7 A using the difference Fourier technique starting from the model of the binary complex (Knighton et al., 1991a). The model of the ternary complex was refined using both X-PLOR and TNT to an R factor of 0.212 and 0.224, respectively. The orientation of the nucleotide and the interactions of MgATP with numerous conserved residues at the active site of the enzyme are clearly defined. The unique protein kinase nucleotide binding site consists of a five-stranded antiparallel beta-sheet with the base buried in a hydrophobic site along beta-strands 1 and 2 and fixed by hydrogen bonds to the N6 amino and N7 nitrogens. The small lobe secures the nucleotide via a glycine-rich loop and by ion pairing with Lys72 and Glu91. While the small lobe fixes the nontransferable alpha- and beta-phosphates in this inhibitor complex, the gamma-phosphate is secured by two Mg2+ ions and interacts both directly and indirectly with several residues in the large lobe--Asp184, Asn171, Lys168. Asp166 is positioned to serve as a catalytic base. The structure is correlated with previous chemical evidence, and the features that distinguish this nucleotide binding motif from other nucleotide binding proteins are delineated.

Glycine is one of the most important inhibitory neurotransmitters in the spinal cord and the brainstem, and glycinergic synapses have a well-established role in the regulation of locomotor behavior. Research over the last 15 years has yielded new insights on glycine neurotransmission. Glycinergic synapses are now known not to be restricted to the spinal cord and the brainstem. Presynaptic machinery for glycine release and uptake, the structure and function of postsynaptic receptors and the factors (both pre- and postsynaptic) which control the strength of glycinergic inhibition have been extensively studied. It is now established that glycinergic synapses can be excitatory in the immature brain and that some inhibitory synapses can corelease gamma-aminobutyric acid (GABA) and glycine. Moreover, the presence of glycine transporters on glial cells and the capacity of these cells to release glycine suggest that glycine may also act as a neuromodulator. Extensive molecular studies have revealed the presence of distinct subtypes of postsynaptic glycine receptors with different functional properties. Mechanisms of glycine receptors aggregation at postsynaptic sites during development are better understood and functional implications of variation in receptor number between postsynaptic sites are partly elucidated. Mutations of glycine receptor subunits have been shown to underly some human locomotor disorders, including the startle disease. Clearly, recent work on glycine receptor channels and the synapses at which they mediate inhibitory signalling in both young and adult animals necessitates an update of our vision of glycinergic inhibitory transmission.

Kynurenic acid is an endogenous glutamate antagonist with a preferential action at the glycine-site of the N-methyl D-aspartate-receptor. Mounting evidence indicate that the compound is significantly involved in basal neurophysiological processes in the brain. In the present investigation, cerebrospinal fluid (CSF) level of kynurenic acid was analyzed in 28 male schizophrenic patients and 17 male healthy controls by means of high pressure liquid chromatography and fluorescence detection. Schizophrenic patients showed elevated CSF levels of kynurenic acid (1.67+/-0.27 nM) compared to the control group (0.97+/-0.07 nM). Furthermore, CSF levels of kynurenic acid in schizophrenic patients were also found to correlate with age. The present finding is indicative of a contribution of kynurenic acid in the pathogenesis of schizophrenia.

Hemolytic uremic syndrome (HUS) in adults carries a high morbidity and mortality, and its cause remains unknown despite many theories. Although familial HUS is rare, it affords a unique opportunity to elucidate underlying mechanisms that may have relevance to acquired HUS. We have undertaken a genetic linkage study based on a candidate gene approach. A common area bounded by the markers D1S212 and D1S306, a distance of 26 cM located at 1q32 segregated with the disease (Z max 3.94). We demonstrate that the gene for factor H lies within the region. Subsequent mutation analysis of the factor H gene has revealed two mutations in patients with HUS. In an individual with the sporadic/relapsing form of the disease we have found a mutation comprising a deletion, subsequent frame shift and premature stop codon leading to half normal levels of serum factor H. In one of the three families there is a point mutation in exon 20 causing an arginine to glycine change, which is likely to alter structure and hence function of the factor H protein. Factor H is a major plasma protein that plays a critical regulatory role in the alternative pathway of complement activation. In light of these findings and previous reports of HUS in patients with factor H deficiency, we postulate that abnormalities of factor H may be involved in the etiology of HUS.

Four mutants defective in endocytosis were isolated by screening a collection of temperature-sensitive yeast mutants. Three mutations define new END genes: end5-1, end6-1, and end7-1. The fourth mutation is in END4, a gene identified previously. The end5-1, end6-1, and end7-1 mutations do not affect vacuolar protein localization, indicating that the defect in each mutant is specific for internalization at the plasma membrane. Interestingly, localization of actin patches on the plasma membrane is affected in each of the mutants. end5-1, end6-1, and end7-1 are allelic to VRP1, RVS161, and ACT1, respectively. VRP1 and RVS161 are required for correct actin localization and ACT1 encodes actin. To our surprise, the end6-1 mutation fails to complement the act1-1 mutation. Disruption of the RVS167 gene, which is homologous to END6/RVS161 and which is also required for correct actin localization, also blocks endocytosis. The end7-1 mutant allele has a glycine 48 to aspartic acid substitution in the DNase I-binding loop of actin. We propose that Vrp1p, Rvs161p, and Rvs167p are components of a cytoskeletal structure that contains actin and fimbrin and that is required for formation of endocytic vesicles at the plasma membrane.

Osteopontin is a secreted glycoprotein with a multidomain structure and functions characteristic of a matricellular protein. Osteopontin interacts with cell surface receptors via arginine-glycine-aspartate (RGD)- and non-RGD containing adhesive domains, in addition to binding to components of the structural extracellular matrix. While normally expressed in bone and kidney, osteopontin levels are elevated during wound healing and inflammation in most tissues studied to date. Since 1986, over one thousand studies have been published on osteopontin, including recent experiments in osteopontin-deficient mice. These studies reveal osteopontin as a cell adhesive, signaling, migratory, and survival stimulus for various mesenchymal, epithelial, and inflammatory cells, in addition to being a potent regulator of osseous and ectopic calcification. Based on these reports, a general picture of osteopontin as an important regulator of inflammation and biomineralization is emerging. A common denominator in osteopontin function in these situations is its ability to regulate the function of macrophage and macrophage-derived cells (i.e. osteoclasts). While we have learned much about osteopontin and the processes it appears to regulate over the past decade, many questions regarding this important multifunctional protein remain unanswered and provide important directions for future studies.

Osmoregulated transporters sense intracellular osmotic pressure and respond to hyperosmotic stress by accumulation of osmolytes to restore normal hydration levels. Here we report the determination of the X-ray structure of a member of the family of betaine/choline/carnitine transporters, the Na(+)-coupled symporter BetP from Corynebacterium glutamicum, which is a highly effective osmoregulated uptake system for glycine betaine. Glycine betaine is bound in a tryptophan box occluded from both sides of the membrane with aromatic side chains lining the transport pathway. BetP has the same overall fold as three unrelated Na(+)-coupled symporters. Whereas these are crystallized in either the outward-facing or the inward-facing conformation, the BetP structure reveals a unique intermediate conformation in the Na(+)-coupled transport cycle. The trimeric architecture of BetP and the break in three-fold symmetry by the osmosensing C-terminal helices suggest a regulatory mechanism of Na(+)-coupled osmolyte transport to counteract osmotic stress.

Hyperammonemia resulting from inherited urea cycle enzyme deficiencies or liver failure results in severe central nervous system dysfunction including brain edema, convulsions and coma. Neuropathologic evaluation in these disorders reveals characteristic alterations of astrocyte morphology ranging from cell swelling (acute hyperammonemia) to Alzheimer Type II astrocytosis (chronic hyperammonemia). Having no effective urea cycle, brain relies on glutamine synthesis for the removal of excess ammonia and the enzyme responsible, glutamine synthetase, has a predominantly astrocytic localization. Accumulation of ammonia in brain results in a redistribution of cerebral blood flow and metabolism from cortical to sub-cortical structures. In addition to changes in astrocyte morphology, increased brain ammonia concentrations result in alterations in expression of key astrocyte proteins including glial fibrillary acidic protein, glutamate and glycine transporters and "peripheral-type" (mitochondrial) benzodiazepine receptors. Such changes result in alterations of astrocytic volume and increased extracellular concentrations of excitatory and inhibitory substances. In addition, the ammonium ion has direct effects on excitatory-inhibitory transmission via distinct mechanisms involving cellular chloride extrusion and postsynaptic receptor function. Acute ammonia exposure leads to activation of NMDA receptors and their signal transduction pathways. Chronic hyperammonemia also results in increased concentrations of neuroactive L-tryptophan metabolites including serotonin and quinolinic acid. Therapy in hyperammonemic syndromes continues to rely on ammonia-lowering strategies via peripheral mechanisms (reduction of ammonia production in the gastrointestinal tract, increased ammonia removal by muscle).

Kinetic properties of soybean net photosynthetic CO(2) fixation and of the carboxylase and oxygenase activities of purified soybean (Glycine max [L.] Merr.) ribulose 1, 5-diphosphate carboxylase (EC 4.1.1.39) were examined as functions of temperature, CO(2) concentration, and O(2) concentration. With leaves, O(2) inhibition of net photosynthetic CO(2) fixation increased when the ambient leaf temperature was increased. The increased inhibition of CO(2) fixation at higher temperatures was caused by a reduced affinity of the leaf for CO(2) and an increased affinity of the leaf for O(2). With purified ribulose 1,5-diphosphate carboxylase, O(2) inhibition of CO(2) incorporation and the ratio of oxygenase activity to carboxylase activity increased with increased temperature. The increased O(2) sensitivity of the enzyme at higher temperature was caused by a reduced affinity of the enzyme for CO(2) and a slightly increased affinity of the enzyme for O(2). The similarity of the effect of temperature on the affinity of intact leaves and of ribulose 1,5-diphosphate carboxylase for CO(2) and O(2) provides further evidence that the carboxylase regulates the O(2) response of photosynthetic CO(2) fixation in soybean leaves. Based on results reported here and in the literature, a scheme outlining the stoichiometry between CO(2) and O(2) fixation in vivo is proposed.Oxygen competitively inhibited carboxylase activity with respect to CO(2), and CO(2) competitively inhibited oxygenase activity with respect to O(2). Within the limits of experimental error, the Michaelis constant (CO(2)) in the carboxylase reaction was identical with the inhibition constant (CO(2)) in the oxygenase reaction, and the Michaelis constant (O(2)) in the oxygenase reaction was identical with the inhibition constant (O(2)) in the carboxylase reaction. The Michaelis constant, (ribulose 1,5-diphosphate) was the same in both the carboxylase and oxygenase reactions. This equality of kinetic constants is consistent with the notion that the same enzyme catalyzes both reactions.

Mice harboring the waved-1 (wa-1) and waved-2 (wa-2) mutations exhibit skin and eye abnormalities that are strikingly similar to those of TGF-alpha-deficient mice, and wa-1 and TGF-alpha were recently shown to be allelic. Because the wa-2 mutation was mapped previously to the vicinity of the EGF/TGF-alpha receptor (EGFR) gene on mouse chromosome 11, we hypothesized that the wa-2 phenotype might result from a defect in either the expression or activity of EGFR, or both. In the present report, we show that EGFR mRNA and protein of normal size are expressed in wa-2 liver and skin at levels that are comparable to those in the corresponding normal tissues, and that the ability of wa-2 EGFR to bind ligand is unaltered. However, ligand-dependent autophosphorylation of wa-2 EGFR is diminished 5- to 10-fold in vitro, and the ability of wa-2 EGFR to phosphorylate an exogenous substrate is reduced by>> 90% compared with that of the control receptor. EGF-induced tyrosine phosphorylation, including that of EGFR itself, is also diminished in skin, particularly at lower dose of exogenous EGF. To establish the nature of the wa-2 mutation, we determined the nucleotide sequence of the coding region of normal and wa-2 murine EGFR cDNAs. A comparison of these sequences revealed a single-nucleotide transversion resulting in the substitution of a glycine for a conserved valine residue near the amino terminus of the tyrosine kinase domain. The importance of this mutation was confirmed by showing that its introduction into an otherwise normal EGFR markedly reduced the receptor's tyrosine kinase activity in transfected Chinese hamster ovary cells. Finally, in situ hybridization analysis demonstrated expression of EGFR predominantly in the outer root sheath of active hair follicles in neonatal mice. As we previously localized TGF-alpha mRNA to the inner root sheath, this pattern of EGFR expression is consistent with the effect of the wa-2 mutation on hair structure, and together with our previous characterization of TGF-alpha-deficient mice, reveals a critical role for signaling by this ligand/receptor system in skin.

Using plant EST collections, we obtained 1392 potential gene duplicates across 8 plant species: Zea mays, Oryza sativa, Sorghum bicolor, Hordeum vulgare, Solanum tuberosum, Lycopersicon esculentum, Medicago truncatula, and Glycine max. We estimated the synonymous and nonsynonymous distances between each gene pair and identified two to three mixtures of normal distributions corresponding to one to three rounds of genome duplication in each species. Within the Poaceae, we found a conserved duplication event among all four species that occurred approximately 50-60 million years ago (Mya); an event that probably occurred before the major radiation of the grasses. In the Solanaceae, we found evidence for a conserved duplication event approximately 50-52 Mya. A duplication in soybean occurred approximately 44 Mya and a duplication in Medicago about 58 Mya. Comparing synonymous and nonsynonymous distances allowed us to determine that most duplicate gene pairs are under purifying, negative selection. We calculated Pearson's correlation coefficients to provide us with a measure of how gene expression patterns have changed between duplicate pairs, and compared this across evolutionary distances. This analysis showed that some duplicates seemed to retain expression patterns between pairs, whereas others showed uncorrelated expression.

Pathogenic mycobacteria, including the agent of tuberculosis, Mycobacterium tuberculosis, must replicate in macrophages for long-term persistence within their niche during chronic infection: organized collections of macrophages and lymphocytes called granulomas. We identified several genes preferentially expressed when Mycobacterium marinum, the cause of fish and amphibian tuberculosis, resides in host granulomas and/or macrophages. Two were homologs of M. tuberculosis PE/PE-PGRS genes, a family encoding numerous repetitive glycine-rich proteins of unknown function. Mutation of two PE-PGRS genes produced M. marinum strains incapable of replication in macrophages and with decreased persistence in granulomas. Our results establish a direct role in virulence for some PE-PGRS proteins.

Newly emerged hantaviruses replicate primarily in the pulmonary endothelium, cause acute platelet loss, and result in hantavirus pulmonary syndrome (HPS). We now report that specific integrins expressed on platelets and endothelial cells permit the cellular entry of HPS-associated hantaviruses. Infection with HPS-associated hantaviruses, NY-1 and Sin Nombre virus (SNV), is inhibited by antibodies to beta3 integrins and by the beta3-integrin ligand, vitronectin. In contrast, infection with the nonpathogenic (no associated human disease) Prospect Hill virus was inhibited by fibronectin and beta1-specific antibodies but not by beta3-specific antibodies or vitronectin. Transfection with recombinant alphaIIb beta3 or alphav beta3 integrins rendered cells permissive to NY-1 and SNV but not Prospect Hill virus infection, indicating that alphaIIb beta3 and alphav beta3 integrins mediate the entry of NY-1 and SNV hantaviruses. Furthermore, entry is divalent cation independent, not blocked by arginine-glycine-aspartic acid peptides and still mediated by, ligand-binding defective, alphaIIb beta3-integrin mutants. Hence, NY-1 and SNV entry is independent of beta3 integrin binding to physiologic ligands. These findings implicate integrins as cellular receptors for hantaviruses and indicate that hantavirus pathogenicity correlates with integrin usage.

Motifs rich in arginines and glycines were recognized several decades ago to play functional roles and were termed glycine-arginine-rich (GAR) domains and/or RGG boxes. We review here the evolving functions of the RGG box along with several sequence variations that we collectively term the RGG/RG motif. Greater than 1,000 human proteins harbor the RGG/RG motif, and these proteins influence numerous physiological processes such as transcription, pre-mRNA splicing, DNA damage signaling, mRNA translation, and the regulation of apoptosis. In particular, we discuss the role of the RGG/RG motif in mediating nucleic acid and protein interactions, a function that is often regulated by arginine methylation and partner-binding proteins. The physiological relevance of the RGG/RG motif is highlighted by its association with several diseases including neurological and neuromuscular diseases and cancer. Herein, we discuss the evidence for the emerging diverse functionality of this important motif.

Tissue engineering aims to replace, repair or regenerate tissue/organ function, by delivering signalling molecules and cells on a three-dimensional (3D) biomaterials scaffold that supports cell infiltration and tissue organization. To control cell behaviour and ultimately induce structural and functional tissue formation on surfaces, planar substrates have been patterned with adhesion signals that mimic the spatial cues to guide cell attachment and function. The objective of this study is to create biochemical channels in 3D hydrogel matrices for guided axonal growth. An agarose hydrogel modified with a cysteine compound containing a sulphydryl protecting group provides a photolabile substrate that can be patterned with biochemical cues. In this transparent hydrogel we immobilized the adhesive fibronectin peptide fragment, glycine-arginine-glycine-aspartic acid-serine (GRGDS), in selected volumes of the matrix using a focused laser. We verified in vitro the guidance effects of GRGDS oligopeptide-modified channels on the 3D cell migration and neurite outgrowth. This method for immobilizing biomolecules in 3D matrices can generally be applied to any optically clear hydrogel, offering a solution to construct scaffolds with programmed spatial features for tissue engineering applications.