Respiration-dependent proton translocation and the mechanism of protonmotive force generation in Nitrobacter winogradskyi

Abstract Proton translocation associated with electron flow to oxygen has been observed with cells of Nitrobacter winogradskyi in the presence of either potassium ferrocyanide or isoascorbate plus N , N , N ', N ' tetramethyl- p -phenylenediamine. The data are consistent with a proton pumping function for the terminal oxidase, cytochrome aa ₃, in this organism as the mechanism for generating a protonmotive force. The failure of previous work with Nitrobacter [4] to detect proton translocation linked to oxidation of nitrite, the physiological substrate, is discussed.     

Differences in reactivity of confluent and nonconfluent cultures of human endothelial cells toward thrombin-stimulated platelets or heparinized salt solution

Summary This study examined whether nonconfluent endothelial cell cultures reacted differently than confluent ones toward thrombin-stimulated platelets or a heparinized salt solution. The adherence to the endothelial cell cultures of⁵¹Cr-labeled human platelets stimulated at different thrombin concentrations was studied. There was significantly higher adherence of stimulated platelets to nonconfluent cultures compared with confluent ones. This was confirmed by scanning electron microscopy, which also revealed a tendency for the platelets to adhere at the cell periphery. Electron microscopy also showed that thrombin-stimulated platelets induced endothelial cell contraction. Part of the peripheral endothelial cell surface toward the bottom of the culture dish was inverted, facing the lumen of the dish. This phenomenon was particularly seen in nonconfluent cultures. When⁵¹Cr-labeled endothelial cultures were incubated with a mildly injurious fluid as heparinized sodium acetate and 20% serum, at 20° C for 30 min, the nonconfluent cultures showed significantly more cell detachment and release of⁵¹Cr than the confluent ones. We conclude that under the conditions of the present experiments there are differences in the reactivity of confluent and nonconfluent endothelial cell cultures. These differences probably reflect biological dissimilarities. In experiments where properties of cultured endothelium are studied, care should be taken that the degree of confluency is standardized.     

Immunological comparison of glyoxalase I from yeast and mammals and quantitative determination of the enzyme in human tissues by radioimmunoassay

Antibodies to glyoxalase I from yeast, rat liver, porcine erythrocytes and human erythrocytes were raised in rabbits. Gel precipitation and immunotitration experiments demonstrated that the mammalian enzymes were immunologically related, but distinct from the yeast enzyme. Fab fragments of the antibodies to human glyoxalase I did not inhibit the catalytic activity, indicating that the antigen binding sites were not directed towards the active site of the enzyme. A radioimmunoassay for glyoxalase I was developed. Quantitative analysis of human adult as well as fetal organs demonstrated that glyoxalase I was present in a concentration of approximately 0.2 micrograms/mg protein in most human tissues.     

Analysis and detection of chlamydial DNA

Elementary bodies of lymphogranuloma venereum (LGV) strains of Chlamydia trachomatis contain, in addition to the genomic DNA, a 6.7 kb plasmid. The plasmid from serovar L2 (434-B) was cloned at the BamHI site of pBR327 into Escherichia coli and a restriction cleavage map of this pLGV125 recombinant plasmid determined. All 15 C. trachomatis serovars contained DNA sequences that hybridized with pLGV125. When total DNA from L2 elementary bodies was used as a probe in Southern blotting and spot hybridization, serovars L1, L2 and L3 exhibited significant homology. The detection level of homologous DNA was 100 pg and LGV DNA was detectable in infected cells when total L2 probe was used in the nucleic acid hybridization test. These DNA probes may be useful as investigative and diagnostic reagents for C. trachomatis.     

The IsTaR 1 serodeme of Trypanosoma brucei: development of a new serodeme

An extensive serodeme of sequentially-isolated antigenic variants of African trypanosomes has been produced from both syringe-passaged and cyclically-transmitted Trypanosoma brucei of the IsTaR 1 clone derived from EATRO 164. The majority of the antigenic variants were isolated from chronically-infected deer mice (Peromyscus leucopus). The pattern of parasitemias during the course of infections initiated with syringe-passaged trypanosomes differed from those initiated with cyclically-transmitted trypanosomes. Trypanosome populations from syringe-passaged (192) and cyclically-transmitted (31) clones were each amplified by growth in lethally-irradiated mice and cryopreserved for retrospective analysis. Five clones derived from a single deer mouse during the first 44 days of infection, and 2 clones derived from an acutely-infected rat were established from these amplified populations. Homogeneous populations were grown in lethally-irradiated rats and mice for antigenic analysis purification of variant-specific glycoprotein. Six of the 7 clones were distinct variants by immunological criteria using antisera derived from whole cells or purified surface glycoproteins. Two clones, one derived from the acutely-infected rat, and the other from the first parasitemia in a chronic infection that was initiated with the former clone, were immunologically identical. Production of these clones established a well-defined serodeme that will allow detailed analysis of antigenic variation.     

Evolution of a Vκ gene family

To examine the evolution of multigene families we have selected as an example an immunoglobulin light chain variable region subgroup (V24) which has been extensively characterized in inbred mice (Mus musculus domesticus). Homologous genes have been isolated and sequenced from Mus pahari, a genetically and geographically isolated species believed to be the oldest living representative of the genus. Southern blot analysis using probes corresponding to individual genes in this subgroup reveals changes in the overall size of the family occurring at the level of individual genes but not at the level of the entire family. Nucleotide sequence analysis indicates an absence of regulatory sequences such as the CAT and TATA boxes 5 to the coding region, but a decanucleotide sequence involved in light chain expression is highly conserved. Within coding regions highly complex patterns of variation are seen which appear to reflect quite different selective pressures on various subregions of the coding sequence. Complementarity determining regions (CDR) are conserved to different extents, with the first CDR region in all family members being among the most conserved segments of the molecule. Conservation is similarly variable among framework segments, indicating complex and variable evolutionary pressures not only at the level of individual genes or their products but also at subregions within homologous molecules.     

In vivo fluorescence excitation and absorption spectra of marine phytoplankton: I. Taxonomic characteristics and responses to photoadaptation

Absorption and fluorescence excitation spectra were measuredfor batch cultures of five species of marine phytoplankton grownunder high and low light. These spectra were examined for propertiescharacteristic of taxonomic position and of photoadaptive response.While regions of absorption and excitation of chlorophyll afluorescence diagnostic of pigment composition were identifiable,photoadaptive response had greater influence on spectral variability.Although reduced growth irradiance caused changes in both theabsorption and fluorescence excitation spectra, the fluorescenceexcitation spectrum appears to be more sensitive to alterationsin the ambient light field for growth than does the absorptionspectrum. For a single species. the fluorescence excitationspectrum for a sample grown at low irradiance showed greaterstructure than that for the sample grown at a high irradiance.Under low light conditions, the excitation of chlorophyll afluorescence by accessory pigments increased relative to theexcitation by chlorophyll a itself The highest fluorescenceyields occur in the blue-green region of the spectrum, correspondingto bands of peak absorption by the accessory pigments. Changesin absorption spectra are less marked, but two features recur.First. in the blue-green region of the spectrum from -500–560nm. absorption is enhanced in the low-light cells relative tothat of the high-light cells. Second, the ratio of absorptionat 435 nm to that at 676 nm was greater for the high-light cells.Correlating changes in pigment concentrations were observed.The influence of photoadaptation on the properties of fluorescenceexcitation spectra is as great or greater than the influenceof pigment complements characteristic of specific algal taxa.     

Expression of nodule-specific uricase in soybean callus tissue is regulated by oxygen

In soybean root nodules the enzyme uricase is expressed concomitantly with nodule development. The initial expression of this protein does not depend on active nitrogen fixation, as demonstrated by analysis of uricase activity in effective and ineffective root nodules. However, the maximal level of uricase activity is determined by the infecting Rhizobium japonicum strain. Sterile root cultures and callus tissue, devoid of the microsymbiont, were incubated at varying oxygen concentrations and analyzed for uricase activity. The specific activity of uricase was increased by lowering the oxygen concentration, with the highest activity obtained around 4−5% oxygen. The increase in uricase activity was due to increased uricase synthesis, as demonstrated by in vivo labelling of callus culture followed by immunoprecipitation with antibodies raised against highly purified nodule uricase.