Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene

Fishes represent the stem vertebrate condition and have maintained several gene arrangements common to mammalian genomes throughout the 450 Myr of divergence from a common ancestor. One such syntenic arrangement includes the GPI-PEPD enzyme association on Xiphophorus linkage group IV and human chromosome 19. Previously we assigned the Xiphophorus homologue of the human ERCC2 gene to linkage group U5 in tight association with the CKM locus. CKM is also tightly linked to the ERCC2 locus on human chromosome 19, leading to speculation that human chromosome 19 may have arisen by fusion of two ancestral linkage groups which have been maintained in fishes. To investigate this hypothesis further, we isolated and sequenced Xiphophorus fish genomic regions exhibiting considerable sequence similarity to the human DNA ligase 1 amino acid sequence. Comparison of the fish DNA ligase sequence with those of other species suggests several modes of amino acid conservation in this gene. A 2.2-kb restriction fragment containing part of an X. maculatus DNA ligase 1 exon was used in backcross hybrid mapping with 12 enzyme or RFLP loci. Significant linkage was observed between the nucleoside phosphorylase (NP2) and the DNA ligase (LIG1) loci on Xiphophorus linkage group VI. This assignment suggests that the association of four DNA repair-related genes on human chromosome 19 may be the result of chance chromosomal rearrangements.      

The lactosylceramide binding specificity of Helicobacter pylori

The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.      

Delineation of the epitope recognized by an antibody specific for N- glycolylneuraminic acid-containing gangliosides

P3 is a mouse monoclonal antibody (mAb) that binds to several NeuGc- containing gangliosides. It also reacts with antigens expressed in human breast tumors (Vazquez et al. (1995) Hybridoma , 14, 551-556). In this work, the binding specificity of P3 has been characterized in more detail using a panel of glycolipids that included several disialylated gangliosides and several chemical derivatives of NeuGc-GM3. The carboxyl group and the nitrogen function of sialic acid were found to play important roles in the antibody binding, whereas the glycerol tail appears to be nonrelevant. Molecular modeling was used to analyze the binding data, including the finding that P3 selectively recognizes the internal NeuGc in GD3. For this purpose, conformational studies of GD3 were performed using molecular dynamics. It was concluded that sialic acid binds the P3 antibody through its upper face (the one on which the carboxyl group is exposed) and the C4-C5 side of the sugar ring, whereas none or very little contact between the galactose residue and the protein is evident. Conformational analysis of GD3 revealed that, despite the large flexibility of the NeuGcalpha8NeuGc linkage, the P3 binding epitope on the external sialic acid is not well exposed for any of the possible conformations this linkage can adopt, whereas the internal sialic acid presents the epitope in a proper way for several of these conformations. As a final result, a coherent picture of the epitope that fits the wide binding data was obtained.      

A nonlinear method for estimating nucleotide polymorphism from restriction maps

Restriction mapping is used to estimate nucleotide sequence polymorphism when the regions to be studied are too long or too numerous to be sequenced. Restriction mapping is less costly than DNA sequencing, but it does not allow direct measurement of underlying nucleotide polymorphism. It is therefore useful to be able to estimate underlying nucleotide polymorphism from observations of polymorphism in restriction maps, as this offers some of the resolution afforded by DNA sequencing at a reduced cost. Previous estimators of underlying nucleotide polymorphism have assumed that each restriction-enzyme- binding site contains, at most, a single polymorphic nucleotide position (the low-polymorphism-frequency assumption), and this assumption has placed an upper limit on the level of polymorphism that can be resolved by these estimators. The present study documents an estimator which allows relaxation of this assumption. The new estimator more accurately estimates underlying nucleotide polymorphism when the polymorphism level is high enough to falsify the low-polymorphism- frequency assumption. The new estimator therefore yields good results for data sets that are too divergent for analysis by present methods.      

Challenge of chimpanzees (Pan troglodytes) immunized with human immunodeficiency virus envelope glycoprotein gp120.

The human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, infects humans and chimpanzees. To determine the efficacy of immunization for preventing infection, chimpanzees were immunized with gp120 purified from human T-cell lymphotrophic virus type IIIB (HTLV-IIIB)-infected cell membranes and challenged with the homologous virus, HTLV-IIIB. A challenge stock of HTLV-IIIB was prepared by using unconcentrated HTLV-IIIB produced in H9 cells. The titer of the virus from this stock on human and chimpanzee peripheral blood mononuclear cells and in human lymphoid cell lines was determined; a cell culture infectivity of 10(4) was assigned. All chimpanzees inoculated intravenously with 40 cell culture infectious units or more became infected, as demonstrated by virus isolation and seroconversion. One of two chimpanzees inoculated with 4 cell culture infectious units became infected. Chimpanzees immunized with gp120 formulated in alum developed antibodies which precipitated gp120 and neutralized HTLV-IIIB. Peripheral blood mononuclear cells from gp120-vaccinated and HIV-infected animals showed a significantly greater response in proliferation assays with HIV proteins than did peripheral blood mononuclear cells from nonvaccinated and non-HIV-infected chimpanzees. Two of the gp120-alum-immunized chimpanzees were challenged with virus from the HTLV-IIIB stock. One animal received 400 cell culture infectious units, and one received 40 infectious units. Both animals became infected with HIV, indicating that the immune response elicited by immunization with gp120 formulated in alum was not effective in preventing infection with HIV-1.     

The biogeochemistry of basic cations in two forest catchments with contrasting lithology in the Czech Republic

The biogeochemistry of Ca, Mg, K, and Nawere investigated in two forested catchments in theCzech Republic, one underlain by leucogranite, theother by serpentinite. High weathering rates at theserpentinite site at Pluhv Bor resultedin Mg²⁺ as the dominant cation on the soilexchange complex and in drainage water. Other basiccations (Ca²⁺, K⁺, Na⁺) showedrelatively low concentrations and outflow instreamwater. The catchment exhibited high basesaturation in mineral soils (>70%), and nearneutral soil and stream pH, despite elevated inputsof acidic deposition. Slow growth of Norway spruceat Pluhv Bor may be caused by K deficiency, Mgoversupply and/or Ni toxicity. In contrast, thegranitic site at Lysina showed low concentrations ofbasic cations on the soil exchange complex and instreamwater. Soil and drainage water at Lysina werehighly impacted by acidic deposition. Soil pH wasextremely acidic (<4.5) throughout the soilprofile, and the base saturation of the mineral soilwas very low (<5%). Supplies of basic cationsfrom atmospheric deposition and soil processes wereless than inputs of SO^2- ₄ on anequivalence basis, resulting in low pH and highconcentrations of total Al in drainage water. Needle yellowing in Norway spruce was possibly theresult of Mg deficiency at Lysina. Because of theirextremely different lithologies, these catchmentsserve as valuable end-members of ecosystemsensitivity to elevated levels of acidicdeposition.     

Comparison of the effects of prostaglandins E1 and A1 on coronary occlusion induced arrhythmia

The present study compares the effects of PGE₁ and PGA₁ on ventricular arrhythmias following coronary artery occlusion. The left anterior descending coronary artery (LAD) was occluded abruptly in 55 cats anesthetized with α-chloralose. Lead II of the ECG along with arterial blood pressure were monitored for one hour after occlusion. Either vehicle or prostaglandin was infused into the left atrium (LA) or femoral vein (IV) 15 min prior to and for 1 hour after LAD occlusion at a rate of 0.15 ml/min. Prostaglandin was infused at either a high dose (1.0 μg/kg/min) or a low dose (0.1 μg/kg/min). Infusion of either PGE₁ or PGA₁ produced a marked fall in blood pressure and heart rate which returned toward control before occlusion. Abrupt occlusion of the LAD produced ventricular arrhythmia in all cats ranging from ventricular premature beats to ventricular fibrillation (VF). The control animals had a 38% incidence of VF. VF occurred in 75% of the animals in which PGE₁ was administered into the LA at either the high or low dose while the occurrence in those administered PGA₁ was 67% and 50%, respectively. Intravenous administration of the high dose of PGE₁ or PGA₁ resulted in VF in 13% and 67% of the animals after LAD occlusion, respectively. These data indicate that the IV administration of PGE₁ may protect the heart from VF while the infusion of PGE₁ or PGA₁ into the LA may enhance VF after LAD occlusion.     

An Electron Microscope Study of Autoinfection in Neogregarines (Sporozoa, Neogregarinida)

SYNOPSIS. In an electron microscope study of the neogregarine Farinocystis tribolii Weiser 1953 in the fat body of the larvae of the flour beetle Tribolium castaneum , empty oocysts and adjacent sporozoites of the gregarine were found. The empty oocysts contained host cell cytoplasm, a residuum only slightly larger than that in mature oocysts, and some remnants of the oocyst membrane pressed tightly against the inner surface of the wall. Apparently normal sporozoites were found near the empty oocysts and no damaged ones were seen. It is assumed that the sporozoites go on developing in the host, i.e., that autoinfection takes place.     

Alterations of NADPH:protochlorophyllide oxidoreductase quantity and lipid composition in etiolated barley seedlings infected by Barley stripe mosaic virus (BSMV)

To understand the phenomenon by which infection of seed-transmitted Barley stripe mosaic virus (BSMV) alters membrane structures and inhibits protochlorophyllide biosynthesis of dark-grown barley ( Hordeum vulgare L.) plants, we analysed the presence of NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) and the galactolipid content and fatty acid composition. The amount of POR in etioplasts of infected leaves, compared with non-infected leaves, was reduced, as measured by immunoelectron microscopy and Western blot. These results are in agreement with the previously described reduction of the ratio of the photoactive 650 nm to non-photoactive 630 nm absorbing protochlorophyllide forms ( Harsányi et al. , 2002 . Physiol. Plant 114 , 149–155). The galactolipid content was lower in infected leaves. Monogalactosyl-diacylglycerol (MGDG) content was reduced to 40% and digalactosyl-diacylglycerol to 55% of control plants on a fresh weight basis. In infected plants, the proportion of linolenic acid decreased in both galactolipids. The lower amount of highly unsaturated fatty acids and the reduced abundance of MGDG correlated well with the previously detected reduction in the membrane ratio of prolamellar body (PLB) to prothylakoid ( Harsányi et al. , 2002 . Physiol. Plant 114 , 149–155). The reduced amount of POR and the above described alterations in the lipid composition resulted in a disturbed structure of PLBs. As a consequence, pigment synthesis and the greening process were inhibited in infected cells, in turn explaining the appearance of chlorotic stripes of BSMV-infected barley leaves. Our results show that BSMV infection can be detected at a very early stage of leaf development.