Immunolocalization of tenascin and cellular fibronectins in diverse glomerulopathies

Frozen samples of minimal change glomerulopathy (MCG), and of membranous, segmental and diffuse lupus glomerulonephritis (MGN, SGN, DLGN) were studied to assess the distribution of tenascin (Ten), and the extradomains A and B (EDA-and EDB-) and oncofetal (Onc-) isoforms of cellular fibronectin (cFn). Cryosections were immunostained by the ABC method with specific monoclonal antibodies. In MCG, mesangial Ten and EDA-cFn reactions were increased. In MGN, mesangial Ten and EDA-cFn staining was enhanced except in segmental scars; convincing reactions were seen in cases with membranous transformation; spikes stained strongly. In SGN, variably intense staining for Ten and all cFn isoforms was seen in glomerular necrosis, proliferation and crescents; parietal epithelium EDA-cFn staining was noted. In DLGN, strong and extensive mesangial Ten and EDA-cFn staining was seen as were focal EDB-and Onc-cFn reactions. Parietal cells with and without crescents stained variably with all Mabs. Obsolete glomeruli were unreactive save for rare periglomerular Ten rims. Interstitial inflammation and fibrosis in MGN, SGN and DLGN had moderate to strong Ten and EDA-cFn staining with rare traces of EDB-and Onc-cFn. We conclude that enhanced Ten and EDA-cFn is a potentially reversible response to glomerular injury whereas the expression of EDB-and Onc-cFn apparently result from necrosis and/or cellular proliferation which lead to scarring. And, while mesangial cells are the major source of these molecules, epithelial cells might also partake in their synthesis.     

Phase and amplitude maps of the electric organ discharge of the weakly electric fish,Apteronotus leptorhynchus

Summary The electric organ discharge (EOD) potential was mapped on the skin and midplane of several Apteronotus leptorhynchus. The frequency components of the EOD on the surface of the fish have extremely stable amplitude and phase. However, the waveform varies considerably with different positions on the body surface. Peaks and zero crossings of the potential propagate along the fish's body, and there is no point where the potential is always zero. The EOD differs significantly from a sinusoid over at least one third of the body and tail. A qualitative comparison between fish showed that each individual had a unique spatiotemporal pattern of the EOD potential on its body.The potential waveforms have been assembled into high temporal and spatial resolution maps which show the dynamics of the EOD. Animation sequences and Macintosh software are available by anonymous ftp (mordor.cns.caltech.edu; cd/pub/ElectricFish).We interpret the EOD maps in terms of ramifications on electric organ control and electroreception. The electrocytes comprising the electric organ do not all fire in unison, indicating that the command pathway is not synchronized overall. The maps suggest that electroreceptors in different regions fulfill different computational roles in electroreception. Receptor mechanisms may exist to make use of the phase information or harmonic content of the EOD, so that both spatial and temporal patterns could contribute information useful for electrolocation and communication.Abbreviations EOD electric organ discharge - EO electric organ - CV coefficient of variance     

NEW ENDOLITHIC CYANOBACTERIA FROM THE ARABIAN GULF. I. HYELLA IMMANIS SP. NOV.1

Hyella immanis, a new species of endolithic cyanobacterium that penetrates carbonate ooid sand grains in the Arabian/Persian Gulf, is formally described. Natural populations of the new taxon were sampled in moving ooid shoals at four locations along the east coast of Saudi Arabia, together with seven other endolithic taxa. The new species was isolated, and its properties were studied under experimental conditions. Small reproductive cells (baeocytes) exhibited positively phototactic gliding motility following release. In culture they grew into colonies forming isodiametric packages (prevalent on agar) and distinct pseudofilaments (prevalent in liquid culture). Carbonate penetration of the cultured strain in liquid culture proceeded at a rate of up to 10 μ· d^?1. Agitation of cultures with magnetic stirrers enhanced the frequency of borings and the initial boring rates, but it had no effect on the continuing boring activity. A fossil counterpart of the new species was identified in Upper Proterozoic (700–800 million years old) silicified oolitic and pisolitic rocks of East Greenland.     

A substance in L-929 cell extracts which replaces the ascorbate requirement for prolyl hydroxylase in a tritium release assay for reducing cofactor; correlation of its concentration with the extent of ascorbate-independent proline hydroxylation and the level of prolyl hydroxylase activity in these cells

Reductant used as cofactor for the prolyl hydroxylase reaction, was measured by a tritium release assay modified from an enzyme assay by making all components of the assay system saturating except for the reductant, but including prolyl hydroxylase. Reduced glutathione (6 mm), which had little activity as a cofactor, and thymol (0.1 mm), an antioxidant which exhibited no cofactor activity at all, were required for optimal proline hydroxylation dependent on reducing cofactor, with thymol fulfilling the previously described requirement for catalase. Ascorbate, cysteine and 6,7-dimethyltetrahydropterin were active as cofactors, in descending order of activity at equimolar concentrations, and activity was concentration dependent for all of these compounds. Sonicates of stationary phase L-929 cells which exhibit ascorbate-independent proline hydroxylation in culture contained reducing cofactor which could replace ascorbate in the cofactor assay, while sonicates of log phase cells which exhibit an ascorbate requirement in culture contained about one-third or less of that amount. NADH and NADPH, which themselves have little or no activity as cofactor, increased the cofactor activity of log phase cell sonicates but had relatively little effect on the activity of stationary cell sonicates suggesting that the cofactor is in a more reduced state in stationary phase. Within 24 h after replating dense, stationary phase cell cultures at low density, conditions where cells return to ascorbate dependence, prolyl hydroxylase activity had decreased to one-fifth the original activity while the concentration of functional reducing cofactor had decreased to less than 1% of its original concentration, largely as a result of oxidation. Ascorbate was not present in L-929 cells sonicates and the levels of tetrahydropterin and cysteine in sonicates could not account for the amount of cofactor activity exhibited by the sonicates in the assay system. Treatment of L-929 cultures with aminopterin did not decrease ascorbate independence, suggesting that tetrahydrofolate did not contribute significantly to cellular proline hydroxylation. These results suggest that an unidentified reductant present in L-929 cells can account for ascorbate-independent proline hydroxylation and also regulate prolyl hydroxylase activity in these cells and that cellular levels of reduced pyridine nucleotides may regulate the reduction state of this substance.     

Inhibitory effects of orotate on precursor incorporation into nucleic acids.

The influence of orotic acid on the incorporation of precursors into nucleic acids was studied in mice and rats and in isolated cells. In vivo, orotate levels were modified by two diets which are known to increase the rate of pyrimidine nucleotide synthesis in rat liver. Of these diets, a 1% orotate diet had greater inhibitory effects than an arginine-deficient diet on the incorporation of [3H]orotate into RNA of mouse kidney than mouse liver. This contrasted with the situation in the rat where there was a greater effect in the liver than the kidney. The situation in the rat was more readily interpreted than in the mouse in terms of previously established effects of these diets on ribonucleotide pool sizes. However, studies using [3H]adenosine as a precursor for incorporation into RNA suggested that even in the mouse the effects of orotate were on pool sizes rather than an inhibitory effect on RNA synthesis. The incorporation of [3H]thymidine into DNA was inhibited by orotate to a similar degree in cultured HTC hepatoma cells and a line of rat liver epithelial cells. An effect on DNA synthesis rather than solely on pool sizes was suggested by the observation that the pool size of dTTP was not increased by 5 mM orotate under conditions in which there was a four-fold increase in the level of UTP in HTC cells. An inhibitory effect of orotate on DNA synthesis was further supported by an observation of decreased incorporation of [3H]deoxyadenosine into DNA and a lower rate of cellular proliferation.     

Submicrosomal localization of prolyl hydroxylase from chick embryo limb bone.

A fraction greatly enriched in microsomes was prepared from chick embryo limb bone tissue homogenates by differential centrifugation in a high density solution of Metrizamide. This fraction was used to determine the submicrosomal localization of prolyl hydroxylase. At a low concentration (0.05%) of the non-ionic detergents Triton X-100 and Brij-35, 90 to 93% of prolyl hydroxylase activity was released from microsomes. Concentrations of Triton X-100 greater than 0.1% were required to solubilize the intrinsic membrane enzyme NADH-ferricyanide reductase and to release membrane-bound ribosomes, while Brij-35 did not extensively solubilize membrane components even at concentrations up to 0.4%. In addition, prolyl hydroxylase activity which could subsequently be released from microsomes by Brij-35 was relatively resistant to trypsin proteolysis at concentrations which removed more than 50% of the ribosomes and approximately 40% of the protein from microsomes. These results suggest that 90 to 93% of prolyl hydroxylase activity in connective tissue is located within the cisternae of the endoplasmic reticulum. Gel filtration of prolyl hydroxylase released from microsomes or found in the soluble fraction of limb bone homogenates revealed two peaks of activity corresponding to molecular weights of 230,000 and 450,000 to 500,000. The latter is twice the value reported for purified chick embryo prolyl hydroxylase. A fraction of the total prolyl hydroxylase activity (generally 20 to 35%) in microsome preparations could be measured in the absence of detergent, although the microsomal membrane should be impermeable to the large unhydroxylated collagen chains used as substrate. On the basis of experimental data, it was concluded that detergent-independent activity was most likely due to damaged microsomal membranes and that this damage was sufficient to allow substrate and trypsin to enter the cisternae but not to allow prolyl hydroxylase to be released.     

Effect of oil pollution on euendolithic cyanobacteria of the Arabian Gulf

Microbial euendoliths (true borer) cyanobacteria are carbonate-boring microorganisms found in modern and ancient marine environments. Modern euendoliths include a wide range of prokaryotes as well as eukaryotes, which have been reported world-wide. The importance of euendolithic cyanobacteria concerns their role in bio-erosion of calcium carbonate substrates and as ecological indicators of shallow, tropical and subtropical marine environments. Arabian Gulf ooids from four sites along the east coast of Saudi Arabia have been bored and inhabited by several species of euendolithic cyanobacteria. This assemblage of different species exists simultaneously within the same ooid grain. Comparisons of 1989 and 1992 data reveal a drastic reduction in active euendoliths, and the average numbers of colonies in these ooids. This study reveals the harmful effect of the 1991 oil spill on these unique microorganisms residing in these ooids.     

An improved protocol for electroporation of Oenococcus oeni ATCC BAA‐1163 using ethanol as immediate membrane fluidizing agent

Aims: To finalize an effective and reproducible electroporation procedure to transform Oenococcus oeni ATCC BAA‐1163 strain. Methods and Results: The vector pGID052 was selected to optimize the electroporation procedure. Transformation efficiency was 5·8 × 10³ per μg of DNA. Transformation was improved when competent cells were prepared with exponential phase cultures; optimum electroporation parameters were an electric pulse of 12·5 kV cm^?1, under a resistance of 200 Ω and the presence of 10% (v/v) ethanol in the electroporation buffer (EPB). Conclusions: An effective protocol to transform O. oeni ATCC BAA‐1163 strain by electroporation has been obtained by addition of ethanol to the EPB. A heterologous expression was obtained in O. oeni ATCC BAA‐1163 by introducing a recombinant vector encoding a truncated form of ClpL2 protein. Significance and Impact of the Study: This is the first report of a successful electroporation of O. oeni ATCC BAA‐1163. The major improvement was the addition of ethanol to the EPB, which has never been reported before as means of enhancing the incorporation of foreign DNA molecules into prokaryote cells by electroporation. This method constitutes a useful tool for the genetic study of this lactic bacterium.     

Sphingosine kinase: biochemical and cellular regulation and role in disease

Sphingolipids have emerged as molecules whose metabolism is regulated leading to generation of bioactive products including ceramide, sphingosine, and sphingosine-1-phosphate. The balance between cellular levels of these bioactive products is increasingly recognized to be critical to cell regulation; whereby, ceramide and sphingosine cause apoptosis and growth arrest phenotypes, and sphingosine-1-phosphate mediates proliferative and angiogenic responses. Sphingosine kinase is a key enzyme in modulating the levels of these lipids and is emerging as an important and regulated enzyme. This review is geared at mechanisms of regulation of sphingosine kinase and the coming to light of its role in disease.