Diamide inhibits pulmonary vasoconstriction induced by hypoxia or prostaglandin F2 alpha

Diamide oxidizes glutathione and other cellular sulfhydryl groups. It decreases calcium ATPase activity and alters mitochondrial calcium flux, probably as a result of the sulfhydryl oxidation. We examined the effect of diamide (5 mg/kg, iv) on pulmonary vascular reactivity in 12 anesthetized dogs. Diamide reversed the pulmonary vasoconstriction caused by hypoxia in seven dogs (control delta PVR + 2.5 +/- 0.6 mm Hg/liter/min; postdiamide delta PVR - 0.1 +/- 0.4 mm Hg/liter/min; P less than 0.01). The pulmonary pressor response to prostaglandin F2 alpha (5 micrograms/kg/min, iv) was also reduced (control delta PVR + 3.8 +/- 0.5 mm Hg/liter/min; postdiamide delta PVR + 1.1 +/- 0.7 mm Hg/liter/min; P less than 0.01). However, in a further five dogs, diamide had only a small effect on the pulmonary vasoconstriction caused by angiotensin II, while the pressor response to hypoxia was again inhibited. The mechanism by which diamide reverses pulmonary vasoconstriction is not certain but the effect is rapid, consistent, and reversible. Because the intravenous infusion of diamide does not produce systemic hypotension, during its period of action on the pulmonary vasculature, unlike the drugs currently available for the clinical treatment of pulmonary hypertension, further studies of its mechanism of action are indicated.     

Mapping of B-cell epitopes of the human hepatitis B virus X protein.

The immune response to the X protein of human hepatitis B virus (HBV) was studied by epitope mapping by using a set of MS2-HBx fusion proteins and synthetic peptides. Antibodies in sera of patients with acute and chronic HBV infection showed a multispecific immune response. Each serum contained antibodies to a different set of epitopes, which taken together cover most of the HBx sequence. Some of the epitopes were detectable only by immunoblotting with fusion proteins; others were detectable only by an enzyme-linked immunosorbent assay (ELISA) with synthetic peptides. The carboxy-terminal half of the HBx protein was preferentially recognized by antibodies from patients with chronic hepatitis and contained a short immunodominant antigenic region with at least two major nonoverlapping epitopes. Anti-HBx antibody titers as revealed by peptide ELISAs were highest and most frequent in patients with chronic hepatitis and usually low in acutely infected patients and asymptomatic carriers. The data demonstrate a remarkable qualitative and quantitative heterogeneity of the humoral HBx immune response which can be monitored by HBx-specific peptide ELISAs. Such tests may become useful diagnostic tools.     

Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein.

In this study we used duck hepatitis B virus (DHBV)-infected Pekin ducks and heron hepatitis B virus (HHBV)-infected heron tissue to search for epitopes responsible for virus neutralization on pre-S proteins. Monoclonal antibodies were produced by immunizing mice with purified DHBV particles. Of 10 anti-DHBV specific hybridomas obtained, 1 was selected for this study. This monoclonal antibody recognized in both DHBV-infected livers and viremic sera a major (36-kilodalton) protein and several minor pre-S proteins in all seven virus strains used. In contrast, pre-S proteins of HHBV-infected tissue or viremic sera did not react. Thus, the monoclonal antibody recognizes a highly conserved DHBV pre-S epitope. For mapping of the epitope, polypeptides from different regions of the DHBV pre-S/S gene were expressed in Escherichia coli and used as the substrate for immunoblotting. The epitope was delimited to a sequence of approximately 23 amino acids within the pre-S region, which is highly conserved in four cloned DHBV isolates and coincides with the main antigenic domain as predicted by computer algorithms. In in vitro neutralization assays performed with primary duck hepatocyte cultures, the antibody reduced DHBV infectivity by approximately 75%. These data demonstrate a conserved epitope of the DHBV pre-S protein which is located on the surface of the viral envelope and is recognized by virus-neutralizing antibodies.     

Reserve mass and dispersal investment in relation to geographic range of plant species: phylogenetically independent contrasts

Recent studies have reported conflicting evidence about correlations between seed size and plant species geographic range sizes. Using phylogenetically independent contrasts (PICs) within genera, we found no consistent differences in reserve mass between species with similar dispersal morphology and «wide>> versus «narrow>> geographic ranges. There was also no tendency within genera for broad ranged species to be those that allocate a larger percentage of the resources invested in each diaspora to dispersal structures. PICs were also constructed between species having a tenfold difference in seed size. In these PICs, the larger seeded species often occupied a greater number of regions than species with smaller seed sizes. This result was generated primarily through the comparison of species from different genera, families or higher level taxa which differed not only in seed mass but also in dispersal modes and growth forms. Finally, comparing species within Acacia and Eucalyptus having similar seed size but different dispersal modes, we found that bird dispersal (in Acacia ) and possession of a wing for wind dispersal (in Eucalyptus ) was associated with wider geographic range compared to lower-investment dispersal modes. Taken together, these comparisons indicate that seed size is not itself important as a factor influencing breadth of geographic range. Dispersal mode and growth form may have an influence, however, and seed size differences may be associated with contrasts in dispersal mode or growth form.     

In vitro reconstitution of mammalian U1 snRNPs active in splicing: the U1-C protein enhances the formation of early (E) spliceosomal complexes.

We have established an in vitro reconstitution/splicing complementation system which has allowed the investigation of the role of mammalian U1 snRNP components both in splicing and at the early stages of spliceosome formation. U1 snRNPs reconstituted from purified, native snRNP proteins and either authentic or in vitro transcribed U1 snRNA restored both early (E) splicing complex formation and splicing-activity to U1-depleted extracts. In vitro reconstituted U1 snRNPs possessing an m3G or ApppG cap were equally active in splicing, demonstrating that a physiological cap structure is not absolutely required for U1 function. However, the presence of an m7GpppG or GpppG cap was deleterious to splicing, most likely due to competition for the m7G cap binding proteins. No significant reduction in splicing or E complex formation was detected with U1 snRNPs reconstituted from U1 snRNA lacking the RNA binding sites of the U1-70K or U1-A protein (i.e., stem-loop I and II, respectively). Complementation studies with purified HeLa U1 snRNPs lacking subsets of the U1-specific proteins demonstrated a role for the U1-C, but not U1-A, protein in the formation and/or stabilization of early splicing complexes. Studies with recombinant U1-C protein mutants indicated that the N-terminal domain of U1-C is necessary and sufficient for the stimulation of E complex formation.     

Interrelationship of renal vascular development and nephrogenesis

Within the cortex region of the neonatal rabbit kidney the developing microvasculature was investigated by means of two endothelium-detecting antibodies (EnPo 1 and EC1). Rows of antibody-labelled cells were found within tissue regions that had previously been described as avascular. We conclude that these vessel-like structures detected by EnPo 1 and EC1 are capillary precursors without lumina. Furthermore, beneath the fibrous capsule within the morphologically homogeneous mesenchyme two cell populations can be discriminated by use of differential antigen expression. The EnPo 1 antigen, which is abundant on endothelial cells and podocytes at different developmental stages, was detected on a subpopulation of mesenchymal cells. These cells were exclusively detected surrounding the tip of the collecting duct ampulla. Due to the unique specificity of EC1 and EnPo 1 the process of microvascular development can be readily followed on serial optical sections gained by laser scan microscopy. (1) Adjacent to EnPo 1-positive mesenchymal cell islets vessel-like structures are found that are in contact with the differentiated vasculature. (2) The renal vesicle is enclosed by a network of vessel-like structures establishing contact with differentiated vessels. (3) No guidance of invading capillary sprouts toward the developing glomerulus and nephron is required, since vascular elements already accompany the earliest detectable nephron stage.     

Mouse centromere mapping using oligonucleotide probes that detect variants of the minor satellite

Cytologically, the centromere is found at the very end of most Mus musculus chromosomes, co-localizing with an array of minor satellite sequences. It is separated from the euchromatin of the long arm by a large domain of heterochromatin, composed in part of arrays of major satellite sequences. We used oligonucleotide probes that specifically detect regions of sequence variation found in certain cloned minor satellite sequences. They detect a limited subset of the minor satellite arrays in the mouse genome, based on both pulsed-field gel electrophoresis and in situ hybridization data, and provide direct molecular genetic markers for individual centromeres in some inbred mouse strains. Array size polymorphisms detected by these probes map to positions consisten with the centromeres of chromosomes 1 and 14 in the BXD recombinant inbred (RI) strains. The genetic distances between these minor satellite arrays and loci on the long arms of chromosomes 1 and 14 are consistent with repression of meiotic recombination in the heterochromatic domains separating them. The existence of chromosome-specific minor satellite sequences implies that the rate of sequence exchange between non-homologous chromosomes relative to the rate between homologous chromosomes is much lower than has previously been postulated. We suggest that the high degree of sequence homogeneity of mouse satellite sequences may instead reflect recent common ancestry.     

Ecology of Microbial Saprophytes and Pathogens in Tissue Culture and Field-Grown Plants: Reasons for Contamination Problems In Vitro

This review compares published surveys of microbial populations in plant tissue and cell cultures with the microbial saprophytes and pathogens found on field grown plants and microbial populations in the laboratory environment. From this comparison and the measured reduction in contamination after improvements in working practices in the laboratory, conclusions can be drawn about the importance of the explant and the laboratory as sources of contamination.

Mechanisms of pathogenicity in vitro are described to explain why bacteria, fungi, and yeasts that are not pathogenic to plants in the field become pathogens in plant tissue cultures. Conversely, plant metabolism and its effect on the tissue culture environment are described to explain why prokaryotes, viruses, and viroids that cause disease in the field can stay latent in vitro.

Detection methods for latent contaminants in plant tissue cultures are summarized, and the strategies and methods for prevention or treatment of contamination are discussed.     

Synthesis and antibody-mediated detection of oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups.

A series of non-nucleoside-based 2,4-dinitrophenyl (DNP) phosphoramidites have been prepared and used in the multiple labelling of oligonucleotides during solid-phase synthesis. The length of spacer arm between the DNP label and the oligonucleotide phosphate backbone, and the number of attached DNP groups have both been varied in order to determine the optimum conditions for anti-DNP antibody binding. Detection using enzyme-linked colorimetric techniques showed sensitivity equivalent to that obtainable using biotinylated oligonucleotides.