Neurally expressed Drosophila genes encoding homologs of the NSF and SNAP secretory proteins

Several lines of investigation have now converged to indicate that the neurotransmitter release apparatus is formed by assembly of cytosolic proteins with proteins of the synaptic vesicle and presynaptic terminal membranes. We are undertaking a genetic approach in Drosophila melanogaster to investigate the functions of two types of cytosolic proteins thought to function in this complex: N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs). We have identified Drosophila homologs of the vertebrate and yeast NSF and SNAP genes. Both Drosophila genes encode polypeptides that closely resemble their vertebrate counterparts and are expressed in the nervous system; neither appears to be in a family of closely related Drosophila genes. These results indicate that the Drosophila NSF and SNAP genes are excellent candidates for mutational analysis of neurotransmitter release.     

Nucleotide sequence of grapevine (Vitis vinifera) cDNA similar to SNAP proteins

A cDNA clone (VS1) homologous to SNAP proteins was isolated from a grapevine cDNA library. The cDNA insert was 1167 bp long and contained a single open reading frame coding for 289 amino acids. The amino acid sequence of VS1 shows similarity (35%-45%) to SNAP proteins from various sources.     

Integrins alpha(v)beta3 and alpha5beta1 mediate attachment of lyme disease spirochetes to human cells

Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin alpha(IIb)beta3 was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins alpha(v)beta3 and alpha5beta1, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified alpha(v)beta3 and alpha5beta1. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified alpha(v)beta3 and alpha5beta1 was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to alpha(v)beta3 was also shown by using a transfected cell line that expresses this receptor but not alpha(IIb)beta3. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both alpha5beta1 and alpha(v)beta3. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.     

Association of N-ethylmaleimide sensitive fusion (NSF) protein and soluble NSF attachment proteins-alpha and -gamma with glucose transporter-4-containing vesicles in primary rat adipocytes

To investigate the role of N-ethylmaleimide sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAP)-containing fusion complexes in glucose transporter-4 (GLUT4) membrane trafficking, the subcellular distributions of NSF, alpha-SNAP, and gamma-SNAP in primary rat adipocytes were determined. A large fraction of the NSF and SNAPs were associated with intracellular membranes, distributed between the low-density microsomes (LDM) and high-density microsomes. Very little of the NSF and SNAPs were associated with the plasma membrane fraction. This distribution did not change after insulin stimulation. Approximately 75% of the NSF and SNAPs in the LDM fraction were coimmunoprecipitated with 85% of the GLUT4 and 60% of the vesicle associated membrane proteins (VAMPs; synaptobrevins) VAMP-2 and cellubrevin in anti-GLUT4 immunoadsorptions. In contrast to NSF and the SNAPs, the beta-coatomer protein (beta-COP) found in the LDM fraction was excluded from GLUT4 vesicles. When LDM fractions were solubilized with Thesit (octaethylene glycol dodecyl ether) or Triton X-100, approximately 40% of the alpha-SNAP was colocalized with NSF on glycerol gradients in large (approximately 20S), ATP-sensitive complexes. VAMP-2 and cellubrevin are concentrated in the LDM fractions and in GLUT4 vesicles; both were excluded from these complexes. These data suggest that the steady state association of NSF and the SNAPs with GLUT4 vesicles and cell membranes is independent of the formation of fusion complexes.     

Venous versus arterial actions of diethylamine/nitric oxide (DEA/NO) complex and S-nitroso-N-acetylpenicillamine (SNAP) in vivo

We studied the effects of diethylamine/NO complex (DEA/NO) and S-nitroso-N-acetylpenicillamine (SNAP), relative to those of sodium nitroprusside (SNP) and nitroglycerin (NTG), on mean arterial pressure (MAP), mean circulatory filling pressure (MCFP), arterial resistance (Ra), venous resistance (Rv), heart rate (HR), cardiac output (CO) and stroke volume (SV) in groups of Inactin-anaesthetized rats pre-treated with i.v. mecamylamine (3.7 micromol kg(-1)) and noradrenaline (6.8 nmol kg(-1) min(-1)). Doses of each that reduced MAP by 30%, 80% and the lowest dose that maximally reduced MAP were examined to allow a comparison of the compounds' dilator actions at equivalent effective depressor doses. DEA/NO (4, 32 and 256 microg kg(-1) min(-1)), SNAP (4, 32 and 256 microg kg(-1) min(-1)) and SNP (8, 32 and 128 microg kg(-1) min(-1)) caused similar dose-dependent reductions in MAP and Ra, and increases in CO and SV. NTG (0.2, 0.8 and 6.4 microg kg(-1) min(-1)) dose-dependently reduced Ra, and increased CO and SV, but lowered MAP only at the highest dose. DEA/NO, SNAP and SNP but not NTG lowered MCFP with efficacy: DEA/NO > SNAP > SNP. All four drugs reduced Rv with efficacy: DEA/NO approximately equal to SNAP > SNP approximately equal to NTG. Therefore, all compounds lowered Ra and Rv. DEA/NO, SNAP and SNP but not NTG reduced MCFP. The pharmacological profiles of DEA/NO and SNAP resemble SNP more than NTG.     

Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex

In a screen for suppressors of a temperature-sensitive mutation in the yeast SNAP-25 homolog, Sec9, we have identified a gain-of-function mutation in the yeast synaptobrevin homolog, Snc2. The genetic properties of this suppression point to a specific interaction between the C-termini of Sec9 and Snc2 within the SNARE complex. Biochemical analysis of interactions between the wild-type and mutant proteins confirms this prediction, demonstrating specific effects of these mutations on interactions between the SNAREs. The location of the mutations suggests that the C-terminal H2 helical domain of Sec9 is likely to be aligned in parallel with Snc2 in the SNARE complex. To test this prediction, we examined the structure of the yeast exocytic SNARE complex by deep-etch electron microscopy. Like the neuronal SNARE complex, it is a rod approximately 14 nm long. Using epitope tags, antibodies and maltose-binding protein markers, we find that the helical domains of Sso, Snc and both halves of Sec9 are all aligned in parallel within the SNARE complex, suggesting that the yeast exocytic SNARE complex consists of a parallel four helix bundle. Finally, we find a similar arrangement for SNAP-25 in the neuronal SNARE complex. This provides strong evidence that the exocytic SNARE complex is a highly conserved structure composed of four parallel helical domains whose C-termini must converge in order to bring about membrane fusion.     

Characterization of the Golgi complex cleared of proteins in transit and examination of calcium uptake activities

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were > 99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that > 90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.     

Localization and characterization of the human ADP-ribosylation factor 5 (ARF5) gene

ADP-ribosylation factor 5 (ARF5) is a member of the ARF gene family. The ARF proteins stimulate the in vitro ADP-ribosyltransferase activity of cholera toxin and appear to play a role in vesicular trafficking in vivo. We have mapped ARF5, one of the six known mammalian ARF genes, to a well-defined yeast artificial chromosome contig on human chromosome 7q31.3. In addition, we have isolated and sequenced an approximately 3.2-kb genomic segment that contains the entire ARF5 coding region, revealing the complete intron-exon structure of the gene. With six coding exons and five introns, the genomic structure of ARF5 is unique among the mammalian ARF genes and provides insight about the evolutionary history of this ancient gene family.     

(R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors mediate a calcium-dependent inhibition of the metabotropic glutamate receptor-stimulated formation of inositol 1,4,5-trisphosphate

L-glutamate (3-1,000 microM) and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 10-1,000 microM), a selective agonist for the metabotropic glutamate receptor, stimulated the formation of inositol 1,4,5-trisphosphate in a concentration-dependent manner. L-Glutamate was half as efficacious as 1S,3R-ACPD. N-methyl-D-aspartate (NMDA; 1 nM to 1 mM) did not significantly influence the response to a maximally effective concentration of 1S,3R-ACPD (100 microM). On the other hand, coapplication of (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA; 1-300 nM) produced a concentration- and time-dependent inhibition of the 1S,3R-ACPD effect, with a maximal inhibition (97%) at 100 nM. Ten micromolar 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of the AMPA receptor, blocked the inhibitory effect of AMPA. Reduced extracellular calcium concentration, as well as 10 microM nimodipine, an L-type calcium channel antagonist, inhibited the AMPA influence on the 1S,3R-ACPD response. W-7, a calcium/calmodulin antagonist, prevented the inhibition by AMPA, whereas H-7, an inhibitor of protein kinase C, had no effect. These data suggest that activation of AMPA receptors has an inhibitory influence on inositol 1,4,5-trisphosphate formation mediated by stimulation of the metabotropic glutamate receptor. The mechanism of action involves calcium influx through L-type type calcium channels and possible activation of calcium/calmodulin-dependent enzymes.     

Insulin-like growth factor (IGF)-binding protein 5 forms an alternative ternary complex with IGFs and the acid-labile subunit

Up to 90% of circulating insulin-like growth factors (IGF-I and IGF-II) are carried in heterotrimeric complexes with a binding protein (IGFBP) and a liver-derived glycoprotein known as the acid-labile subunit. IGFBP-3 is considered unique among the six well characterized IGFBPs in its ability to complex with the acid-labile subunit. However, a basic carboxyl-terminal domain of IGFBP-3, known to be involved in its interaction with the acid-labile subunit, is shared by IGFBP-5, suggesting the possibility of ternary complexes containing IGFBP-5. We now demonstrate using three independent methods that human IGFBP-5, when occupied by IGF-I or IGF-II, forms ternary complexes of approximately 130 kDa with the acid-labile subunit. IGFBP-3 competes with approximately twice the potency of IGFBP-5 for the formation of such complexes. No other IGFBP complexes with the acid-labile subunit itself or competes with IGFBP-5 for complex formation. As observed for IGFBP-3, ternary complexes containing IGFBP-5 form preferentially in the presence of IGF-I, even though IGFBP-5 has a preferential affinity for IGF-II over IGF-I. By size fractionation chromatography, serum IGFBP-5 co-elutes predominantly with ternary complexes. The demonstration of IGFBP-5-containing ternary complexes indicates an unrecognized form of IGF transport in the circulation and an additional mechanism for regulating IGF bioavailability.     

Growth factor regulation of insulin-like growth factor (IGF) binding proteins (IGFBP) and preadipocyte differentiation in porcine stromal-vascular cell cultures

The influence of anti-IGF-1 and anti-transforming growth factor beta (TGF-beta) neutralizing antibodies on preadipocyte differentiation and secretion of IGFBPs was examined in serum free porcine stromal-vascular cultures. Cultures were stained for morphological analysis and conditioned media were collected for: TGF-beta determination by ELISA, IGF-1 by RIA, and IGFBP analysis by ligand blotting. After 6 d of treatment, anti-TGF-beta increased fat proportions by 2.7 fold compared to controls. Anti-IGF-1 decreased fat cell proportions by 14-fold. Anti-TGF-beta increased concentrations of IGF-1 5.8-fold and IGFBP-2 and IGFBP-3 by 8- and 7-fold in conditioned media whereas IGFBP-4 decreased 5-fold. Anti-IGF-1 increased concentrations of IGFBP-2 and 3 by 9- and 35-fold, respectively. TGF-beta increased concentrations of IGFBP-1, 2 and 3 by 3-fold, 18-fold and 3-fold, respectively, after 9 d in culture (6 d of treatment). There was no change in TGF-beta levels in anti-IGF-1 treated cultures compared to controls. Control antibodies and negative controls had no effect. These results provide evidence that endogenously produced IGF-1 and TGF-beta has a major influence on preadipocyte differentiation in serum free media by modulating IGFBP production/secretion.     

Association between the serotonin transporter promoter polymorphism (5-HTTLPR) and adult unresolved attachment.

Research on antecedents of organized attachment has focused on the quality of caregiving received during childhood. In recent years, research has begun to examine the influence of genetic factors on quality of infant attachment. However, no published studies report on the association between specific genetic factors and adult attachment. This study examined the link between the 5-HTTLPR promoter polymorphism of the serotonin transporter gene and adult unresolved attachment assessed with the Adult Attachment Interview. Genetic material and information on attachment-related loss or trauma were available for 86 participants. Multivariate regression analyses showed an association between the short 5-HTTLPR allele and increased risk for unresolved attachment. Temperament traits and psychological symptoms did not affect the association between 5-HTTLPR and unresolved attachment. The authors hypothesize that the increased susceptibility to unresolved attachment among carriers of the short allele of 5-HTTLPR is consistent with the role of serotonin in modulation of frontal-amygdala circuitry. The findings challenge current thinking by demonstrating significant genetic influences on a phenomenon previously thought to be largely environmentally driven. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Serotonin (5-HT)3 receptors: antagonists and their pharmacological profiles

The dysuria syndrome consists of the persistence or accentuation following adenomectomy of the symptoms which caused the patient to seek the urologist's advice. It is frequent event whose causes are largely connected to physiopathological events which are also influenced by the developing role between the urologist and patients in view of prostate disease. The authors analyse the various causes of post-adenomectomy dysuria and emphasise the importance of a precise diagnosis and the correct indications for surgery for the prevention of this disease.     

Ternary complex between elongation factor Tu.GTP and Phe-tRNA(Phe)

The effect of aminoacylation and ternary complex formation with elongation factor Tu.GTP on the tertiary structure of yeast tRNA(Phe) was examined by 1H-NMR spectroscopy. Esterification of phenylalanine to tRNA(Phe) does not lead to changes with respect to the secondary and tertiary base pair interactions of tRNA. Complex formation of Phe-tRNA(Phe) with elongation factor Tu.GTP results in a broadening of all imino proton resonances of the tRNA. The chemical shifts of several NH proton resonances are slightly changed as compared to free tRNA, indicating a minor conformational rearrangement of Phe-tRNA(Phe) upon binding to elongation factor Tu.GTP. All NH proton resonances corresponding to the secondary and tertiary base pairs of tRNA, except those arising from the first three base pairs in the aminoacyl stem, are detectable in the Phe-tRNA(Phe)-elongation factor Tu-GTP ternary complex. Thus, although the interactions between elongation factor Tu and tRNA accelerate the rate of NH proton exchange in the aminoacyl stem-region, the Phe-tRNA(Phe) preserves its typical L-shaped tertiary structure in the complex. At high (> 10(-4) M) ligand concentrations a complex between tRNA(Phe) and elongation factor Tu-GDP can be detected on the NMR time-scale. Formation of this complex is inhibited by the presence of any RNA not related to the tRNA structure. Using the known tertiary structures of yeast tRNA(Phe) and Thermus thermophilus elongation factor Tu in its active, GTP form, a model of the ternary complex was constructed.     

Early career achievements of National Science Foundation (NSF) graduate applicants: Looking for Pygmalion and Galatea effects on NSF winners.

NSF Graduate Fellowships are awarded to approximately half of a homogeneous group of applicants in a procedure that approximates random assignment to the conditions of either fellowship or honorable mention. This natural experiment permits assessment of the effect on early career accomplishments of being named an NSF fellow. The authors found a consistent effect for PhD completion—overall, fellows were 7% more likely to complete the PhD than were nonawardees—but found no reliable fellowship effect on achieving faculty status, achieving top faculty status, or submitting or receiving an NSF or a National Institutes of Health research grant. The authors conclude that the positive expectancies associated with this prestigious fellowship have only a small influence (Pygmalion or Galatea effect) in graduate school and no effect thereafter. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Expression of interleukin (IL)-4 and IL-5 proteins in asthma induced by toluene diisocyanate (TDI)

BACKGROUND: TDI-induced asthma exhibits clinical, functional and morphological similarities with allergen-induced asthma, suggesting that an immunological mechanism is involved in the sensitization to TDI. In vitro studies using the technique of cloning lymphocytes demonstrated that a great proportion of T-cell clones derived from bronchial mucosa of subjects with TDI-induced asthma produced IL-5 and interferon-gamma, but not IL-4, upon in vitro stimulation. OBJECTIVES: To investigate in vivo the role of IL-4 and IL-5 on the inflammatory response of the bronchial mucosa to TDI in sensitized subjects, we performed a quantitative analysis of bronchial biopsies. METHODS: We obtained bronchial biopsies from six subjects with TDI asthma 48 h after an asthmatic reaction induced by TDI challenge (challenged group), in six subjects with TDI asthma 1-4 weeks after the last exposure to TDI (chronic group), and in six non-asthmatic controls. The number of eosinophils, mast cells, T-lymphocytes, and IL-4 and IL-5 protein positive cells was determined by immunohistochemistry in the area 100 microm beneath the epithelial basement membrane. RESULTS: The characteristic increase of submucosal eosinophils, but not of mast cells and T-lymphocytes, was observed in the subjects with TDI-induced asthma when compared with controls. No differences were detected between the two groups of asthmatics. In the subjects with TDI-induced asthma, cell immunoreactivity for IL-5 was increased when compared with normal controls. There was no difference in the expression of IL-5 protein between challenged and chronic asthmatics. In contrast, the expression of IL-4 protein was increased only in the asthmatic subjects tested after recent exposure to TDI. CONCLUSIONS: We demonstrated that TDI asthma 48 h after specific bronchial challenge was associated with increased numbers of cells expressing IL-4 and IL-5, whereas chronic TDI asthma was associated with increased expression of IL-5, but not of IL-4. The results suggest that subjects who developed TDI asthma exhibit increased production of IL-5 even in the absence of a recent trigger by the exogenous sensitizer and that production of TH2-like cytokines in TDI-induced asthma may not always be co-ordinately regulated in vivo.     

Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that interacts with eIF5

Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a complex of approximately 600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNAiMet binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.     

LF(VD)精炼工艺对控制28MnCr5齿轮钢中硫和硫化物的影响

试验了45 t LF精炼渣的碱度、喂硫线和脱氧工艺对28MnCr5钢(％:0.25～0.30C、0.60～0.80Mn、0.020～0.035S、0.80～1.00Cr)硫含量的控制、氧化物含量和钢中硫化物的影响。结果表明,LF精炼渣碱度控制在2.8～5.1喂硫线,VD后硫的回收率达80％～90％;钢中氧化物级别≤1.5级;精炼结束喂适量CaSi线可改善钢中硫化物的形貌。