The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel

Background: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels; delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. Results: The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 3(10) helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-l, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. Conclusions: delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.

Inhibition of phospholipase A(2) increases Tau phosphorylation at Ser214 in embryonic rat hippocampal neurons

Background: Arachidonic acid is released from cellular membranes by the action of phospholipase A(2) (PLA(2)) and is implicated in microtubule-associated protein Tau phosphorylation. Tau hyperphosphorylation affects its ability to stabilize microtubules. Objective: To determine the effect of PLA(2) inhibition on the phosphorylation state of Tau phosphoepitopes in primary cultures of hippocampal neurons. Methods: 4 DIC neurons were incubated at different concentrations of methyl-arachidonylfluorophosphonate (MAFP), an irreversible inhibitor of cPLA(2) and iPLA(2). Changes on Tau phosphorylation were determined by Western blotting with a panel of anti-Tau antibodies (C-terminal, Ser199/202, Ser202/205, Ser396 and Ser214). Results: The Ser214 site was hyperphosphorylated upon MAFP treatment. Significant differences were observed with 10 mu M (p = 0.01), 50 mu M (p = 0.01) and 100 mu M (p = 0.05) of MAFP. Less-intense changes were found in other phosphoepitopes. Conclusion: The present findings indicate that the phosphorylation of Ser214 is regulated by c- and/or iPLA(2), whereas other phosphoepitopes primarily regulated by GKS3b were not affected. (C) 2009 Elsevier Ltd. All rights reserved.

Mutations in NOTCH2 cause Hajdu-Cheney syndrome, a disorder of severe and progressive bone loss

We used an exome-sequencing strategy and identified an allelic series of NOTCH2 mutations in Hajdu-Cheney syndrome, an autosomal dominant multisystem disorder characterized by severe and progressive bone loss. The Hajdu-Cheney syndrome mutations are predicted to lead to the premature truncation of NOTCH2 with either disruption or loss of the C-terminal proline-glutamate-serine-threonine-rich proteolytic recognition sequence, the absence of which has previously been shown to increase Notch signaling.

Similar Intracellular Peptide Profile of TAP1/beta 2 Microglobulin Double-Knockout Mice and C57BL/6 Wild-Type Mice as Revealed by Peptidomic Analysis

Cells produce and use peptides in distinctive ways. In the present report, using isotope labeling plus semi-quantitative mass spectrometry, we evaluated the intracellular peptide profile of TAP1/beta 2m(-/-) (transporter associated with antigen-processing 1/beta 2 microglobulin) double-knockout mice and compared it with that of C57BL/6 wild-type animals. Overall, 92 distinctive peptides were identified, and most were shown to have a similar concentration in both mouse strains. However, some peptides showed a modest increase or decrease (similar to 2-fold), whereas a glycine-rich peptide derived from the C-terminal of neurogranin (KGPGPGGPGGAGGARGGAGGGPSGD) showed a substantial increase (6-fold) in TAP1/beta 2m(-/-) mice. Thus, TAP1 and beta 2microglobulin have a small influence on the peptide profile of neuronal tissue, suggesting that the presence of peptides derived from intracellular proteins in neuronal tissue is not associated with antigens of the class I major histocompatibility complex. Therefore, it is possible that these intracellular peptides play a physiological role.

Molecular Characterization of Strains of Respiratory Syncytial Virus Identified in a Hematopoietic Stem Cell Transplant Outpatient Unit Over 2 Years: Community or Nosocomial Infection?

Respiratory syncytial virus (RSV) is recognized as the leading cause of nosocomial respiratory infection among hematopoietic stem cell transplant (HSCT) recipients, causing considerable morbidity and mortality. RSV is easily transmitted by contact with contaminated surfaces, and in HSCT units, more than 50% of RSV infections have been characterized as of nosocomial origin. From April 2001 to October 2002, RSV was identified by direct immunofluorescent assay in 42 symptomatic HSCT recipients. Seven RSV strains from 2001 and 12 RSV strains from 2002 were sequenced. RNA extraction, cDNA synthesis, and seminested polymerase chain reaction (PCR) with primers complementary to RSV genes G and F were pet-formed. PCR products were analyzed by nucleotide sequencing of the C-terminal region of gene G for typing (in group A or B). Of the 7 strains analyzed in 2001, only 2 belonged to group B; the other 5 belonged to group A. Of these 7 strains, 3 were identical and were from recipients receiving outpatient care. In 2002, of the 12 strains analyzed, 3 belonged to group A and the other 9 belonged to group B. Of these 9 strains, 7 were genetically identical and were also from recipients receiving outpatient care. Therefore, multiple strains of RSV cocirculated in the hematopoietic stem cell transplant units (ward and outpatient units) between 2001 and 2002. Nosocomial transmission was more likely to occur at the HSCT outpatient unit than in the HSCT ward. Infection control practices should also be implemented in the outpatient setting.

Functional association of human Ki-1/57 with pre-mRNA splicing events

The cytoplasmic and nuclear protein Ki- 1 / 57 was first identified in malignant cells from Hodgkin`s lymphoma. Despite studies showing its phosphorylation, arginine methylation, and interaction with several regulatory proteins, the functional role of Ki- 1 / 57 in human cells remains to be determined. Here, we investigated the relationship of Ki- 1 / 57 with RNA functions. Through immunoprecipitation assays, we verified the association of Ki- 1 / 57 with the endogenous splicing proteins hnRNPQ and SFRS9 in HeLa cell extracts. We also found that recombinant Ki- 1 / 57 was able to bind to a poly- U RNA probe in electrophoretic mobility shift assays. In a classic splicing test, we showed that Ki- 1 / 57 can modify the splicing site selection of the adenoviral E1A minigene in a dose- dependent manner. Further confocal and. uorescence microscopy analysis revealed the localization of enhanced green. uorescent protein - Ki- 1 / 57 to nuclear bodies involved in RNA processing and or small nuclear ribonucleoprotein assembly, depending on the cellular methylation status and its N- terminal region. In summary, our findings suggest that Ki- 1 / 57 is probably involved in cellular events related to RNA functions, such as pre- mRNA splicing.

New transposons to generate GFP protein fusions in Candida albicans

Transposon elements are important tools for gene function analysis, for example they can be used to easily create genome-wide collections of insertion mutants. Transposons may also carry sequences coding for an epitope or fluorescent marker useful for protein expression and localization analysis. We have developed three new Tn5-based transposons that incorporate a GFP (green fluorescent protein) coding sequence to generate fusion proteins in the important fungal pathogen Candida albicans. Each transposon also contains the URA3 and Kan(R) genes for yeast and bacterial selection, respectively. After in vitro transposition, the insertional allele is transferred to the chromosomal locus by homologous recombination. Transposons Tn5-CaGFP and Tn5-CaGFP-URA3:FLIP can generate C-terminal truncated GFP fusions. A URA3 flipper recycling cassette was incorporated into the transposon Th5-CaGFP-UFRA3:FLIP. After the induction of Flip recombinase to excise the marker, the heterozygous strain is transformed again in order to obtain a GFP-tagged homozygous strains. In the Tn5-CaGFP-FL transposon the markers are flanked by a rare-cutting enzyme. After in vitro transposition into a plasmid-borne target gene, the markers are eliminated by restriction digestion and religation, resulting in a construct coding for full-length GFP-fusion proteins. This transposon can generate plasmid libraries of GFP insertions in proteins where N- or C-terminal tagging may alter localization. We tested our transposon system by mutagenizing the essential septin CDC3 gene. The results indicate that the Cdc3 C-terminal extension is important for correct septin filament assembly. The transposons described here provide a new system to obtain global gene expression and protein localization data in C. albicans. (c) 2008 Elsevier B.V. All rights reserved.

Consequences of lifetime isolated growth hormone (GH) deficiency and effects of short-term GH treatment on bone in adults with a mutation in the GHRH-receptor gene

Growth hormone (GH) influences bone mass maintenance. However, the consequences of lifetime isolated GH deficiency (IGHD) on bone are not well established. We assessed the bone status and the effect of 6 months of GH replacement in GH-naive adults with IGHD due to a homozygous mutation of the GH-releasing hormone (GHRH)-receptor gene (GHRHR). We studied 20 individuals (10 men) with IGHD at baseline, after 6 months of depot GH treatment, and 6 and 12 months after discontinuation of GH. Quantitative ultrasound (QUS) of the heel was performed and serum osteocalcin (OC) and C-terminal cross-linking telopeptide of type I collagen (ICTP) were measured. QUS was also performed at baseline and 12 months later in a group of 20 normal control individuals (CO), who did not receive GH treatment. At baseline, the IGHD group had a lower T-score on QUS than CO (-1.15 +/- 0.9 vs. -0.07 +/- 0.9, P < 0.001). GH treatment improved this parameter, with improvement persisting for 12 months post-treatment (T-score for IGHD = -0.59 +/- 0.9, P < 0.05). GH also caused an increase in serum OC (baseline vs. pGH, P < 0.001) and ICTP (baseline vs. pGH, P < 0.01). The increase in OC was more marked during treatment and its reduction was slower after GH discontinuation than in ICTP. These data suggest that lifetime severe IGHD is associated with significant reduction in QUS parameters, which are partially reversed by short-term depot GH treatment. The treatment induces a biochemical pattern of bone anabolism that persists for at least 6 months after treatment discontinuation.

Comparative analysis of rabies virus isolates from Brazilian canids and bats based on the G gene and G-L intergenic region

Rabies virus (RABV) isolates from two species of canids and three species of bats were analyzed by comparing the C-terminal region of the G gene and the G-L intergenic region of the virus genome. Intercluster identities for the genetic sequences of the isolates showed both regions to be poorly conserved. Phylogenetic trees were generated by the neighbor-joining and maximum parsimony methods, and the results were found to agree between the two methods for both regions. Putative amino acid sequences obtained from the G gene were also analyzed, and genetic markers were identified. Our results suggest that different genetic lineages of RABV are adapted to different animal species in Brazil.

Lp95, a novel leptospiral protein that binds extracellular matrix components and activates e-selectin on endothelial cells

Objectives: The study of a predicted outer membrane leptospiral protein encoded by the gene LIC12690 in mediating the adhesion process. Methods: The gene was cloned and expressed in Escherichia coli BL21 (SI) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and used to assess its ability to activate human umbilical vein endothelial cells (HUVECs). Results: The recombinant leptospiral protein of 95 kDa, named Lp95, activated E-selectin in a dose-dependent fashion but not the intercellular adhesion molecule 1 (ICAM-1). In addition, we show that pathogenic and non-pathogenic Leptospira are both capable to stimulate endothelium E-selectin and ICAM-1, but the pathogenic L. interrogans serovar Copenhageni strain promotes a statistically significant higher activation than the non-pathogenic L. biflexa serovar Patoc (P < 0.01). The Lp95 was identified in vivo in the renal tubules of animal during experimental infection with L. interrogans. The whole Lp95 as well as its fragments, the C-terminal containing the domain of unknown function (DUF), the N-terminal and the central overlap regions bind laminin and fibronectin ECM molecules, being the binding stronger with the DUF containing fragment. Conclusion: This is the first leptospiral protein capable to mediate the adhesion to ECM components and the activation of HUVECS, thus suggesting its participation in the pathogenesis of Leptospira. (C) 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Solid-state NMR structure determination of melittin in a lipid environment

Solid-state C-13 NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-C-13]Gly3-[2-C-13]Ala4, [1-C-13]Gly3-[2-C-13]Leu6, [1-C-13]Leu13-[2-C-13]Ala15, [2-C-13]Leu13-[1-C-13]Ala15, and [1-C-13]Leu13-[2-C-13]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-C-13]Gly3-[2-C-13]Ala4 and [1-C-13]Gly3-[2-C-13]Leu6 were consistent with alpha -helical structure in the N-terminus irrespective of environment. The Internuclear distances measured in [1-C-13]Leu13-[2-C-13]Ala15, [2-C-13]Leu13-[1-C-13]Ala15, and [1-C-13]Leu13-[2-C-13]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139 degrees -145 degrees) than in lyophilized powder (121 degrees -139 degrees) or crystals (129 degrees). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (similar to 160 degrees) (R. Smith, F. Separovic, T. J. Milne, A. Whittaker, F. M. Bennett, B. A. Cornell, and A. Makriyannis, 1994, J. Mol, Biol 241:456-466). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.

Discovery and structure of a potent and highly specific blockerof insect calcium channels

We have isolated a novel family of insect-selective neurotoxins that appear to be the most potent blockers of insect voltage-gated calcium channels reported to date. These toxins display exceptional phylogenetic specificity, with at least a 10,000-fold preference for insect versus vertebrate calcium channels. The structure of one of the toxins reveals a highly structured, disulfide-rich core and a structurally disordered C-terminal extension that is essential for channel blocking activity. Weak structural/functional homology with omega -agatoxin-IVA/B, the prototypic inhibitor of vertebrate P-type calcium channels, suggests that these two toxin families might share a similar mechanism of action despite their vastly different phylogenetic specificities.

A dynamic role for HDAC7 in MEF2-mediated muscle differentiation

The overlapping expression profile of MEF2 and the class-II histone deacetylase, HDAC7, led us to investigate the functional interaction and relationship between these regulatory proteins. HDAC7 expression inhibits the activity of MEF2 (-A, -C, and -D), and in contrast MyoD and Myogenin activities are not affected. Glutathione S-transferase pulldown and immunoprecipitation demonstrate that the repression mechanism involves direct interactions between MEF2 proteins and HDAC7 and is associated with the ability of MEF2 to interact with the N-terminal 121 amino acids of HDAC7 that encode repression domain 1. The MADS domain of MEF2 mediates the direct interaction of MEF2 with HDAC7, MEF2 inhibition by HDAC7 is dependent on the N-terminal repression domain and surprisingly does not involve the C-terminal deacetylase domain. HDAC7 interacts with CtBP and other class-I and -II HDACs suggesting that silencing of MEF2 activity involves corepressor recruitment. Furthermore, we show that induction of muscle differentiation by serum withdrawal leads to the translocation of HDAC7 from the nucleus into the cytoplasm. This work demonstrates that HDAC7 regulates the function of MEF2 proteins and suggests that this class-II HDAC regulates this important transcriptional (and pathophysiological) target in heart and muscle tissue. The nucleocytoplasmic trafficking of HDAC7 and other class-II HDACs during myogenesis provides an ideal mechanism for the regulation of HDAC targets during mammalian development and differentiation.

Isolation and identification of the toxic peptides from Lophyrotoma zonalis (Pergidae) sawfly larvae

The broad-leaved paper bark tree Melaleuca quinquenervia (Cav) (Myrtaceae) was introduced into Florida (USA) early in this century it has proliferated to such an extent that urgent measures are now required to control it. The sawfly Lophyrotoma zonalis (Pergidae) has been introduced as a possible biological control agent due to its ability to defoliate M. quinquenervia. Because toxic D-amino acid- containing peptides have been isolated from some sawfly species, L. zonalis larvae were processed using the previously reported method for the recovery of these compounds. The toxins lophyrotomin (as the free C-terminal acid) and a mixture of pergidin and Val(4)-pergidin were isolated at 0.36 and 0.43% yield of the dried larvae, respectively. Both compounds when dosed intraperitoneally to C57/B16 male mice were hepatotoxic with lowest lethal doses of 8 and 32 mg/kg, respectively. The pathology of the liver was different for each compound, with the lophyrotomin free acid causing a periportal haemorrhagic necrosis and the pergidin causing a periacinar coagulative necrosis. (C) 2001 Elsevier Science Ltd. All rights reserved.

The solution structure of C1-T1, a two-domain proteinase inhibitor derived from a circular precursor protein from Nicotiana alata

A two-domain portion of the proteinase inhibitor precursor from Nicotiana alata (NaProPI) has been expressed and its structure determined by NMR spectroscopy. NaProPI contains six almost identical 53 amino acid repeats that fold into six highly similar domains; however, the sequence repeats do nut coincide with the structural domains. Five of the structural domains comprise the C-terminal portion of one repeat and the N-terminal portion of the next. The sixth domain contains the C-terminal portion of the sixth repeat and the N-terminal portion of the first repeat. Disulphide bonds link these C and N-terminal fragments to generate the clasped-bracelet fold of NaProPI. The three-dimensional structure of NaProPI is not known, but it is conceivable that adjacent domains in NaProPI interact to generate the circular bracelet with the N and C termini in close enough proximity to facilitate formation of the disulphide bonds that form the clasp The expressed protein, examined in the current study, comprises residues 25-135 of NaProPI and encompasses the first two contiguous structural domains, namely the chymotrypsin inhibitor C1 and the trypsin inhibitor T1, joined by a five-residue linker, and is referred to as C1-T1. The tertiary structure of each domain in C1-T1 is identical to that found in the isolated inhibitors. However, no nuclear Overhauser effect contacts are observed between the two domains and the five-residue linker adopts an extended conformation. The absence of interactions between the domains indicates that adjacent domains do not specifically interact to drive the circularisation of NaProPI. These results are in agreement with recent data which describe similar PI precursors from other members of the Solanaceae having two, three, or four repeats. The lack of strong interdomain association is likely to be important for the function of individual inhibitors by ensuring that there is no masking of reactive sites upon release from the precursor. (C) 2001 Academic Press.