1,25-Dihydroxyvitamin D3 suppresses human T helper/inducer lymphocyte activity in vitro

The active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2-D3), suppresses in vitro immunoglobulin (Ig) production by activated peripheral blood mononuclear cells (PBM) from normal human subjects by inhibiting T helper/inducer TH cell activity. Normal PBM were fractionated into B, TH and T suppressor/cytotoxic (Ts) cells by fluorescence-activated cell sorting techniques. The resultant subsets were activated with mitogens and were cultured in the presence or absence of a receptor-saturating concentration of 1,25-(OH)2-D3. The sterol reduced [3H]thymidine incorporation in TH cells by 56%, with no effect on Ts or B cells. When 1,25-(OH)2-D3-treated TH cells were co-cultured with untreated B cells and culture supernatants assayed for Ig production, 1,25-(OH)2-D3 abrogated the inducing effect of TH cells on Ig synthesis by B cells. There was no inhibitory effect of the sterol on Ts or B cell activity. In addition, 1,25-(OH)2-D3 produced a dramatic inhibition of interleukin 2 (IL 2) production by activated PBM, but did not inhibit IL 2 receptor generation by these cells. Other vitamin D metabolites tested did not produce this effect. These results suggest that the TH lymphocyte is the specific cellular target for the immunoinhibitory effects of 1,25-(OH)2-D3.     

Cladistic analysis of the Cirripedia Thoracica

We present a cladistic analysis of the Cirripedia Thoracica using morphological characters and the Acrothoracica and Ascothoracida as outgroups. The list of characters comprised 32 shell and soft body features. The operational taxonomic units (OTUs) comprised 26 well-studied fossil and extant taxa, principally genera, since uncertainty about monophyly exists for most higher ranking taxonomic units. Parsimony analyses using PAUP 3.1.1 and Hennig86 produced 189 trees of assured minimal length. We also examined character evolution in the consensus trees using MacClade and Clados. The monophyly of the Balanomorpha and the Verrucomorpha sensu stricto is confirmed, and all trees featured a sister group relationship between the ‘living fossil Neoverruca and me Brachylepadomorpha. In the consensus trees the sequential progression of ‘pedunculate‘sister groups up to a node containing Neolepas also conforms to current views, but certain well-established taxa based solely on plesiomorphies stand out as paraphyletic, such as Pedunculata (= Lepadomorpha); Eolepadinae, Scalpellomorpha and Chthamaloidea. The 189 trees differed principally in the position of shell-less pedunculates, Neoverruca, the scalpelloid Capitulum, and the interrelationships within the Balanomorpha, although the 50% majority rule consensus tree almost fully resolved the latter. A monophyletic Sessilia comprising both Verrucomorpha and Balanomorpha appeared among the shortest trees, but not in the consensus. A tree with a monophyletic Verrucomorpha including Neoverruca had a tree length two steps longer than the consensus trees. Deletion of all extinct OTUs produced a radically different tree, which highlights the importance of fossils in estimating cirripede phylogeny. Mapping of our character set onto a manually constructed cladogram reflecting die most recent scenario of cirripede evolution resulted in a tree length five steps longer than any of our shortest trees. Our analysis reveals that several key questions in cirripede phylogeny remain unsolved, notably the position of shell-less forms and the transition from ‘pedunculate‘to ‘sessile‘barnacles. The inclusion of more fossil species at this point in our understanding of cirripede phylogeny will only result in even greater levels of uncertainty. When constructing the character list we also identified numerous uncertainties in the homology of traits commonly used in discussing cirripede evolution. Our study highlights larval ultrastructure, detailed studies of early ontogeny, and molecular data as the most promising areas for future research.     

Arteriovenous malformations of the brain: a teratologic challenge

Congenital arteriovenous malformations of the brain are lesions which are well-known to neurologists and neurosurgeons but are puzzling to teratologists because they occur sporadically and are unassociated with congenital malformations outside the central nervous system. They are recommended to teratologists as a field of etiologic and pathogenetic research.     

Isolation and characterization of a Saccharomyces cerevisiae mutant disrupted for the succinate dehydrogenase flavoprotein subunit

A partial genomic clone of the flavoprotein subunit of the mitochondrial enzyme, succinate dehydrogenase (EC 1.3.99.1) from Saccharomyces cerevisiae has been isolated. The partial clone was used to construct, by targeted gene disruption, a yeast mutant with a defective flavoprotein subunit gene. Submitochondrial membranes from the mutant are defective in activities requiring a functional succinate dehydrogenase but not in other respiratory chain activities. In addition, the mutant contains significantly lower levels of covalently attached flavin adenine dinucleotide cofactor than does the wild type. Disruption of the flavoprotein subunit gene results in the simultaneous loss of both the iron-sulfur and the flavoprotein subunits from mitochondrial membranes.     

Chitin/chitosan transformation by thermo-mechano-chemical treatment including characterization by enzymatic depolymerization

Chitosan, the deacetylated derivative of chitin, was until recently produced by hydrolysis in 50% (w/v) NaOH. Application of thermo-mechano-chemical technology to chitin deacetylation was evaluated as an alternative method of chitosan production. This process consists of a cascade reactor unit operating under reduced alkaline conditions of 10% (w/v) NaOH. Prior mercerization of chitin at 4 degrees C for 24 h was required for high deacetylation yields. Sudden decompression of the aqueous alkaline suspension of mercerized chitin resulted in near complete deacetylation of chitin. Reactor residence time was 90 s at 230 degrees C prior to decompression. The chitosan produced was characterized by elemental analysis, (13)C-NMR and enzymatic depolymerization. Enzymatic determination of the degree of acetylation of chitin/chitosan mixtures was also investigated. Relative chitinase and/or chitosanase digestibilities were shown to be strongly dependent on chitin deacetylation. Based on enzymatic digestibilities, the alkaline aqueous high shear process does not appear to produce significant secondary products. Correlation of chitosanase digestibility with percentage of deacetylation provides a simple biological assay to study chitosan composition.     

Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

The Putative Thiosulfate Sulfurtransferases PspE and GlpE Contribute to Virulence of Salmonella Typhimurium in the Mouse Model of Systemic Disease

The phage-shock protein PspE and GlpE of the glycerol 3-phosphate regulon of Salmonella enterica serovar Typhimurium are predicted to belong to the class of thiosulfate sulfurtransferases, enzymes that traffic sulfur between molecules. In the present study we demonstrated that the two genes contribute to S. Typhimurium virulence, as a glpE and pspE double deletion strain showed significantly decreased virulence in a mouse model of systemic infection. However, challenge of cultured epithelial cells and macrophages did not reveal any virulence-associated phenotypes. We hypothesized that their contribution to virulence could be in sulfur metabolism or by contributing to resistance to nitric oxide, oxidative stress, or cyanide detoxification. In vitro studies demonstrated that glpE but not pspE was important for resistance to H₂O₂. Since the double mutant, which was the one affected in virulence, was not affected in this assay, we concluded that resistance to oxidative stress and the virulence phenotype was most likely not linked. The two genes did not contribute to nitric oxid stress, to synthesis of essential sulfur containing amino acids, nor to detoxification of cyanide. Currently, the precise mechanism by which they contribute to virulence remains elusive.