Calcium-regulated exocytosis of dense-core vesicles requires the activation of ADP-ribosylation factor (ARF)6 by ARF nucleotide binding site opener at the plasma membrane

Vacuole fusion requires a coordinated cascade of priming, docking, and fusion. SNARE proteins have been implicated in the fusion itself, although their precise role in the cascade remains unclear. We now report that the vacuolar SNAP-23 homologue Vam7p is a mobile element of the SNARE complex, which moves from an initial association with the cis-SNARE complex via a soluble intermediate to the docking site. Soluble Vam7p is specifically recruited to vacuoles and can rescue a fusion reaction poisoned with antibodies to Vam7p. Both the recombinant Vam7p PX domain and a FYVE domain construct of human Hrs block the recruitment of Vam7p and vacuole fusion, demonstrating that phosphatidylinositol 3-phosphate is a primary receptor of Vam7p on vacuoles. We propose that the Vam7p cycle is linked to the availability of a lipid domain on yeast vacuoles, which is essential for coordinating the fusion reaction prior to and beyond docking.     

Mercury methylation independent of the acetyl-coenzyme A pathway in sulfate-reducing bacteria

Sulfate-reducing bacteria (SRB) in anoxic waters and sediments are the major producers of methylmercury in aquatic systems. Although a considerable amount of work has addressed the environmental factors that control methylmercury formation and the conditions that control bioavailability of inorganic mercury to SRB, little work has been undertaken analyzing the biochemical mechanism of methylmercury production. The acetyl-coenzyme A (CoA) pathway has been implicated as being key to mercury methylation in one SRB strain, Desulfovibrio desulfuricans LS, but this result has not been extended to other SRB species. To probe whether the acetyl-CoA pathway is the controlling biochemical process for methylmercury production in SRB, five incomplete-oxidizing SRB strains and two Desulfobacter strains that do not use the acetyl-CoA pathway for major carbon metabolism were assayed for methylmercury formation and acetyl-CoA pathway enzyme activities. Three of the SRB strains were also incubated with chloroform to inhibit the acetyl-CoA pathway. So far, all species that have been found to have acetyl-CoA activity are complete oxidizers that require the acetyl-CoA pathway for basic metabolism, as well as methylate mercury. Chloroform inhibits Hg methylation in these species either by blocking the methylating enzyme or by indirect effects on metabolism and growth. However, we have identified four incomplete-oxidizing strains that clearly do not utilize the acetyl-CoA pathway either for metabolism or mercury methylation (as confirmed by the absence of chloroform inhibition). Hg methylation is thus independent of the acetyl-CoA pathway and may not require vitamin B(12) in some and perhaps many incomplete-oxidizing SRB strains.     

Method for monitoring alginate released in biological fluids by high-performance anion-exchange chromatography with pulsed amperometric detection

The use of alginate-entrapped cells in cell therapy requires a method for monitoring possible released compound within biological fluids following either their implantation or inoculation in artificial organs. Oligomannuronic and oligoguluronic acids were prepared by enzymatic depolymerization with alginate lyase from Pseudomonas alginovora, characterized by high-performance anion-exchange chromatography with pulsed amperometric detection and quantitated in human, pig and rabbit blood, urine and tissue samples. The method was tested for linearity and detection limit, accuracy, intra- and inter-day precision. The limit of detection was 3 microgram/ml in both urine and plasma and 5 mg/g of tissues. The relative standard deviations (RSDs) of intra-day precision were 6.0-16.6% and 4.8-8.7% in plasma and urine, respectively; the RSDs of inter-day precision were 5.1-14.4% and 5.0-11.6% in plasma and urine, respectively. Thus, this method appears suitable for the measurement of released alginate from entrapped cells used in cell therapy.     

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.     

Phylogeny and evolution of calcareous sponges: monophyly of calcinea and calcaronea,high level of morphological homoplasy,and the primitive nature of axial symmetry

Because calcareous sponges are triggering renewed interest with respect to basal metazoan evolution, a phylogenetic framework of their internal relationships is needed to clarify the evolutionary history of key morphological characters. Morphological variation was scored at the suprageneric level within Calcispongia, but little phylogenetic information could be retrieved from morphological characters. For the main subdivision of Calcispongia, the analysis of morphological data weakly supports a classification based upon cytological and embryological characters (Calcinea/Calcaronea) rather than the older classification scheme based upon the aquiferous system (Homocoela/Heterocoela). The 18S ribosomal RNA data were then analyzed, both alone and in combination with morphological characters. The monophyly of Calcispongia is highly supported, but the position of this group with respect to other sponge lineages and to eumetazoan taxa is not resolved. The monophyly of both Calcinea and Calcaronea is retrieved, and the data strongly rejected the competing Homocoela/Heterocoela hypothesis. The phylogeny implies that characters of the skeleton architecture are highly homoplastic, as are characters of the aquiferous system. However, axial symmetry seems to be primitive for all Calcispongia, a conclusion that has potentially far-reaching implications for hypotheses of early body plan evolution in Metazoa.     

Hyperplasia of adrenal rest tissue causing a retroperitoneal mass in a child with 11 beta-hydroxylase deficiency

We report the case of a child with congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency, with hyperplasia of adrenal rest tissue presenting as a retroperitoneal mass. Complete resolution of the mass was noted after 18 months of hydrocortisone replacement therapy.     

Lurcher GRID2-induced death and depolarization can be dissociated in cerebellar Purkinje cells

The Lurcher mutation transforms the GRID2 receptor into a constitutively opened channel. In Lurcher heterozygous mice, cerebellar Purkinje cells are permanently depolarized, a characteristic that has been thought to be the primary cause of their death, which occurs from the second postnatal week onward. The more dramatic phenotype of Lurcher homozygotes is thought to be due to a simple gene dosage effect of the mutant allele. We have analyzed the phenotype of Lurcher/hotfoot heteroallelic mutants bearing only one copy of the Lurcher allele and no wild-type Grid2. Our results show that the absence of wild-type GRID2 receptors in these heteroallelic mutants induces an early and massive Purkinje cell death that is correlated with early signs of autophagy. This neuronal death is independent of depolarization and can be explained by the direct activation of autophagy by Lurcher GRID2 receptors through the recently discovered signaling pathway formed by GRID2, n-PIST, and Beclin1.     

<Emphasis Type="Italic">Hox</Emphasis> gene survey in the chaetognath<Emphasis Type="Italic"> Spadella cephaloptera</Emphasis>: evolutionary implications

We present the isolation of six Hox genes in the chaetognath Spadella cephaloptera. We identified one member of the paralogy group 3, four median genes and a mosaic gene that shares features of both median and posterior classes ( SceMedPost). Several hypotheses may account for the presence of a mosaic Hox gene in this animal. Here we propose that SceMedPost may represent an ancestral gene, which has not diverged totally into a posterior or a median one. This hypothesis has interesting implications for the reconstruction of the evolutionary history of Hox genes and suggests that Chaetognatha lineage divergence could predate the deuterostome/protostome split. Such a phylogenetic position is considered in the light of their embryological and morphological characters.     

Microcin E492 antibacterial activity: evidence for a TonB-dependent inner membrane permeabilization on Escherichia coli

The mechanism of action of microcin E492 (MccE492) was investigated for the first time in live bacteria. MccE492 was expressed and purified to homogeneity through an optimized large-scale procedure. Highly purified MccE492 showed potent antibacterial activity at minimal inhibitory concentrations in the range of 0.02-1.2 microM. The microcin bactericidal spectrum of activity was found to be restricted to Enterobacteriaceae and specifically directed against Escherichia and Salmonella species. Isogenic bacteria that possessed mutations in membrane proteins, particularly of the TonB-ExbB-ExbD complex, were assayed. The microcin bactericidal activity was shown to be TonB- and energy-dependent, supporting the hypothesis that the mechanism of action is receptor mediated. In addition, MccE492 depolarized and permeabilized the E. coli cytoplasmic membrane. The membrane depolarization was TonB dependent. From this study, we propose that MccE492 is recognized by iron-siderophore receptors, including FepA, which promote its import across the outer membrane via a TonB- and energy-dependent pathway. MccE492 then inserts into the inner membrane, whereupon the potential becomes destabilized by pore formation. Because cytoplasmic membrane permeabilization of MccE492 occurs beneath the threshold of the bactericidal concentration and does not result in cell lysis, the cytoplasmic membrane is not hypothesized to be the sole target of MccE492.