Adenovirus-mediated silencing of synaptotagmin 9 inhibits Ca2+-dependent insulin secretion in islets

Synaptotagmins (Syts) are involved in Ca(2+)-dependent insulin release. However, which Syt isoform is functional in primary beta-cells remains unknown. We demonstrate by electron microscopy of pancreatic islets, the association of Syt 9 with insulin granules. Silencing of Syt 9 by RNA interference adenovirus in islet cells had no effect on the expression of Syt 5, Syt 7 and Syt 3 isoforms. The latter was localized at the plasma membrane of pancreatic polypeptide cells. Insulin release in response to glucose or tolbutamide was strongly inhibited in Syt 9 deficient islets, whereas exocytosis potentiated by raising cAMP levels, was unaltered. Thus, Syt 9 may act as Ca(2+) sensor for beta-cell secretion.     

Differential scanning calorimetric studies of a Bacillus halodurans alpha-amylase

The thermal unfolding of Amy 34, a recombinant alpha-amylase from Bacillus halodurans, has been investigated using differential scanning calorimetry (DSC). The denaturation of Amy 34 involves irreversible processes with an apparent denaturation temperature (T(m)) of 70.8 degrees C at pH 9.0, with four transitions, as determined using multiple Gaussian curves. The T(m) increased by 5 degrees C in the presence of 100-fold molar excess of CaCl2 while the aggregation of Amy 34 was observed in the presence of 1000-fold molar excess of CaCl2. Increase in the calcium ion concentration from 1- to 5-fold molar excess resulted in an increase in calorimetric enthalpy (DeltaH(cal)), however, at higher concentrations of CaCl2 (up to 100-fold), DeltaH(cal) was found to decrease, accompanied by a decrease in entropy change (DeltaS), while the T(m) steadily increased. The presence of 100-fold excess of metal chelator, EDTA, resulted in a decrease in T(m) by 10.4 degrees C. T(m) was also decreased to 61.1 degrees C and 65.9 degrees C at pH 6.0 and pH 11.0, respectively.     

Conversion of acetaminophen to the bioactive N-acylphenolamine AM404 via fatty acid amide hydrolase-dependent arachidonic acid conjugation in the nervous system

Acetaminophen (paracetamol) is a popular domestic analgesic and antipyretic agent with a weak anti-inflammatory action and a low incidence of adverse effects as compared with aspirin and other non-steroidal anti-inflammatory drugs. Here we show that acetaminophen, following deacetylation to its primary amine, is conjugated with arachidonic acid in the brain and the spinal cord to form the potent TRPV1 agonist N-arachidonoylphenolamine (AM404). This conjugation is absent in mice lacking the enzyme fatty acid amide hydrolase. AM404 also inhibits purified cyclooxygenase (COX)-1 and COX-2 and prostaglandin synthesis in lipopolysaccharide-stimulated RAW264.7 macrophages. This novel metabolite of acetaminophen also acts on the endogenous cannabinoid system, which, together with TRPV1 and COX, is present in the pain and thermoregulatory pathways. These findings identify fatty acid conjugation as a novel pathway for drug metabolism and provide a molecular mechanism for the occurrence of the analgesic N-acylphenolamine AM404 in the nervous system following treatment with acetaminophen.     

Streptococcal Opacity Factor: A Family of Bifunctional Proteins with Lipoproteinase and Fibronectin-Binding Activities

The serum opacity factor (SOF) of Streptococcus pyogenes is a type-specific lipoproteinase of unknown biological significance. We have sequenced the sof gene and characterized the corresponding SOF protein from a strain of type M63. It was found that sof63 is related to sof22 and that, similar to SOF22 [25], SOF63 binds fibronectin. Moreover, we demonstrate opacity factor activity in a Streptococcus dysgalactiae fibronectin-binding protein FnBA that is structurally related to the SOF proteins of S. pyogenes. Sequence analysis of these three SOF proteins showed a unique periodical pattern of conserved and variable regions. The enzymatically active part of SOF63 was localized to the fragment corresponding to the entire set of conserved and variable sequences, while for fibronectin-binding a single repeat in the C terminal part of the protein was sufficient. The results show that streptococcal SOF proteins form a novel family of bifunctional proteins with lipoproteinase and fibronectin-binding activities. Received: 7 June 1999 / Accepted: 5 October 1999     

Determinative Value of a Portion of the nifH Sequence for the Genera Nostoc and Anabaena (Cyanobacteria)

The taxonomic positions of Nostoc and Anabaena strains are currently disputed. We selected three Nostoc and Anabaena strains, using the classic criteria of morphology and life cycle. DNA sequences of a part of the nifH gene were determined from these strains and aligned with homologous sequences from 10 other Nostoc/Anabaena strains in the public databases. Phylogenetic reconstructions were carried out to test the consistency of the taxonomic placement of these strains. The phylogenetic trees do not separate these strains into distinct groups. Our results are in agreement with other molecular-based phylogenies that also fail to differentiate the Nostoc-Anabaena groups. The data suggest that the currently recognized genera Nostoc and Anabaena may in fact belong within a single, broadly defined genus. Received: 14 February 2000 / Accepted: 21 March 2000     

New insight into the structure and function of the alternative oxidase

The alternative oxidase is a ubiquinol oxidase found in plant mitochondria, as well as in the mitochondria of some fungi and protists. It catalyzes a cyanide-resistant reduction of oxygen to water without translocation of protons across the inner mitochondrial membrane, and thus functions as a non-energy-conserving member of the respiratory electron transfer chain. The active site of the alternative oxidase has been modelled as a diiron center within a four-helix bundle by Siedow et al. (FEBS Lett. 362 (1995) 10-14) and more recently by Andersson and Nordlund (FEBS Lett. 449 (1999) 17-22). The cloning of the Arabidopsis thaliana IMMUTANS (Im) gene, which encodes a plastid enzyme distantly related to the mitochondrial alternative oxidases (Wu et al. Plant Cell 11 (1999) 43-55; Carol et al. Plant Cell 11 (1999) 57-68), has now narrowed the range of possible ligands to the diiron center of the alternative oxidase. The Im protein sequence suggests a minor modification to the recent model of the active site of the alternative oxidase. This change moves an invariant tyrosine into a conserved hydrophobic pocket in the vicinity of the active site, in a position analogous to the long-lived tyrosine radical at the diiron center of ribonucleotide reductase, and similar to the tyrosines near the diiron center of bacterioferritin and rubrerythrin. The Im sequence and modified structural model yield a compelling picture of the alternative oxidase as a diiron carboxylate protein. The current status of the relationship of structure to function in the alternative oxidase is reviewed.     

Salmonella typhimurium mutants that downregulate phagocyte nitric oxide production

To examine the potential and strategies of the facultative intracellular pathogen Salmonella typhimurium to increase its fitness in host cells, we applied a selection that enriches for mutants with increased bacterial growth yields in murine J774-A.1 macrophage-like cells. The selection, which was based on intracellular growth competition, rapidly yielded isolates that out-competed the wild-type strain during intracellular growth. J774-A.1 cells responded to challenge with S. typhimurium by mounting an inducible nitric oxide synthase (iNOS) mRNA and protein expression and a concomitant nitric oxide (NO) production. Inhibition of NO production with the use of the competitive inhibitor N-monomethyl-L-arginine (NMMA) resulted in a 20-fold increase in bacterial growth yield, suggesting that the NO response prevented bacterial intracellular growth. In accordance with this observation, five out of the nine growth advantage mutants isolated inhibited production of NO from J774-A.1 cells, despite an induction of iNOS mRNA and iNOS protein. Accompanying bacterial phenotypes included alterations in lipopolysaccharide structure and in the profiles of proteins secreted by invasion-competent bacteria. The results indicate that S. typhimurium has the ability to mutate in several different ways to increase its host fitness and that inhibition of iNOS activity may be a major adaptation.     

Corrigendum

Corrigendum

Corrigendum     

Protection against respiratory syncytial virus (RSV) elicited in mice by plasmid DNA immunisation encoding a secreted RSV G protein-derived antigen

Plasmid vectors encoding two different variants, one cytoplasmic and one secreted version, of a candidate vaccine BBG2Na to respiratory syncytial virus (RSV), were constructed and evaluated in a nucleic acid vaccination study. The two different vectors, which employed the Semliki Forest virus gene amplification system, were found to express BBG2Na appropriately in in vitro cell cultures. Immunisation of mice with the plasmid vectors elicited significant serum anti-BBG2Na IgG responses only in the mice receiving the plasmid encoding the secreted version of BBG2Na. Consistent with antibody induction data, sterilising lung protection against RSV-A challenge was also only observed in this group. These results indicate that the targeting of antigen expression (intracellular versus secreted) would be an important factor to consider in the design of nucleic acid vaccines.     

Application of imprinted synthetic polymers in binding assay development

The first part of the review describes a method for the synthesis of molecularly imprinted polymers for use in binding assays. The method considers the many factors involved that affect the recognition properties of the materials and describes an approach to screening and optimization of these factors. The second part describes the development of binding assays using such polymers. This includes the use of different labels, the effect of solvent and buffer, the scale of the assay (amount of solid polymer), and how these influence the quality of the assay in terms of sensitivity, selectivity, and speed of analysis.