Entamoeba histolytica: gene linkage groups and relevant features of its karyotype

We identified some gene linkage groups in Entamoeba histolytica using a 4-M urea improved transversal alternating field electrophoresis (TAFE) method. Complex rosette-structured DNA molecules were found trapped along the gel lanes, explaining the fuzziness of the patterns. Using several episomal probes, including 16 S, 5.8 S, and 25 S ribosomal (r)Dna genes, an autonomous replication sequence (ARS), and EhVR1, we identified a complete ribosomal episome linkage group (CELG) at the 1.2-Mb position. Three other incomplete groups were found: IELG-1, formed by EhVR1,16 S, 5.8 S, and 25 S genes; IELG-2 formed by EhVR1, 16 S and 25 S; and IELG-3 formed only by 5.8 S. Ehadh3, Ehpfo, and Ehredox genes migrated at the 1.8-Mb position, forming the non-ribosomal linkage group, NRLG-1.8, while the Ehenl-1 gene migrated at 1.6 Mb forming the NRLG-1.6 group. Ehhk was located at 1.2, 0.8, and 0.17 Mb in three different groups: NRLG-1.2, IELG-3-0.8, and NRLG-0.17. Putative lineal chromosomes were also identified using an heterologous telomeric probe. By in situ hybridization experiments, the rDNA and Ehhk genes were located in both nucleus and cytoplasm, while the Ehpfo and Ehredox genes were found mainly in the nucleus. We propose a model hypothezising that the 16 S and 25?S genes are in a linear molecule, duplicated in two inverted repeats, which may be looped out of the linear DNA to form an episome probably lacking or not the 5.8 S sequence, which could be added later by recombination.     

Oxidative stress and antioxidant content in Chlorella vulgaris after exposure to ultraviolet-B radiation

Growth of Chlorella vulgaris was measured in cultures irradiated with 0, 0.8, 2.0 and 4.4 kJ m² UV-B. Growth expressed as chlorophyll content, declined significantly with increased UV-B dose. Ultraviolet-B irradiated cultures in log phase of growth showed a 284% increase in oxygen radical generation and a 145% increase in lipid peroxidation compared with unirradiated cultures, whereas cultures in the stationary growth phase showed no significant changes in these parameters. The activities of superoxide dismutase and catalase increased by 40 and 500%, respectively, after exposure to a UV-B dose of 4.4 kJ m⁻². Contents of the lipophilic antioxidants α-tocopherol and β-carotene increased by 180 and 63 amol cell⁻¹ respectively, between log and stationary phases in unirradiated cultures; but in UV-B-irradiated cultures these increases were significantly depressed. Photoreducing capacities of chloroplasts were decreased following UV-B irradiation of both isolated chloroplasts and those isolated from irradiated algae. Cells exposed to UV-B exhibited increased size and starch accumulation. These results suggest that oxidative stress conditions related to UV-B exposure trigger an antioxidant response that includes an increase in the activity of the antioxidant enzymes (superoxide dismutase and catalase).     

Vanadium derivatives act as growth factor — mimetic compounds upon differentiation and proliferation of osteoblast-like UMR106 cells

The effect of different vanadium compounds on proliferation and differentiation was examined in osteoblast-like UMR106 cells. Vanadate increased the cell growth in a biphasic manner, the higher doses inhibiting cell progression. Vanadyl stimulated cell proliferation in a dose-responsive manner. Similar to vanadate, pervanadate increased osteoblast-like cell proliferation in a biphasic manner but no inhibition of growth was observed. Vanadyl and pervanadate were stronger stimulators of cell growth than vanadate. Only vanadate was able to regulate the cell differentiation as measured by cell alkaline phosphatase activity. These results suggest that vanadium derivatives behave like growth factors on osteoblast-like cells and are potential pharmacological tools in the control of cell growth.     

Structural diversity in the extracellular faces of peptidergic G-protein-coupled receptors. Molecular cloning of the mouse C5a anaphylatoxin receptor.

The mouse C5a receptor gene was isolated using the human C5a receptor cDNA probe recently described (Gerard, N. P., and C. Gerard. 1991. Nature 349:614). By analogy with the human gene, the mouse homolog contains two exons with the 5' untranslated region and initiating methionine codon present in exon 1 and the remainder of the molecule in exon 2. Generation of an expressible cDNA for the mouse C5a receptor was accomplished using the polymerase chain reaction and a sense oligodeoxynucleotide primer which included an initiation codon just 5' to the sequence encoding the N-linked glycosylation site. When transfected into human 293 kidney epithelial cells the cloned cDNA directs expression of a binding site for human C5a anaphylatoxin with a binding constant of 2.5 +/- 0.3 nM; the human C5a receptor expressed under identical conditions has a Kd of 1.7 +/- 0.2 nM. Overall, the deduced amino acid sequences of the receptors are 65% identical given the analogous gene structures. Alignment of the sequences as seven transmembrane segment receptors reveals that the greatest structural diversity (approximately 70%) exists in the putative extracellular domains. In contrast, species differences among other members of this family of seven membrane-spanning receptors is generally only 10 to 20%, even for receptors whose ligands are relatively small and not expected to interact with sites on the extracellular surfaces. A high degree of structural identify is observed for the C5a receptors in the transmembrane segments and in all but one of the loops predicted to exist in the cytoplasm. Inasmuch as critical structures responsible for high affinity binding of the 74 amino acid polypeptide to both C5a receptors involve features conserved between species, these data provide the starting point for mutagenesis studies to determine the nature of the binding and activation sites for the chemotactic receptors. Additionally, these data provide a reagent for immunologic and molecular genetic studies on the role of C5a receptors in inflammatory models.     

Immunocytolocalization of glutamine synthetase in mesophyll and phloem of leaves ofSolanum tuberosum L.

Summary Localization of glutamine synthetase inSolanum tuberosum leaves was investigated by techniques of Western tissue printing and immunogold electron microscopy. Anti-GS antibodies used in immunolocalization recognize two peptides (45 kDa and 42 kDa) on Western blots. Antibody stained tissue prints on nitrocellulose membranes allowed low resolution localization of GS. Immunostaining was most evident in the adaxial phloem of the leaf midribs and petiole veins. High-resolution localization of glutamine synthetase by immunogold electron microscopy revealed that this enzyme occurs in both the chloroplasts and the cytosol ofS. tuberosum leaf cells. However, GS was specifically associated with the chloroplasts of mesophyll cells and with the cytoplasm of phloem companion cells. The evidence for cell-specific localization of chloroplast and cytosolic GS presented here agrees with the recently reported cell-specific pattern of expression of GUS reporter gene, directed by promoters for chloroplast and cytosolic GS form in tobacco transgenic plants. These data provide additional clues to the interpretation of the functional role of these different isoenzymes and its relationship with their specific localization.Abbreviations BSA bovine serum albumin - EM electron microscope - GOGAT glutamate synthase - GS glutamine synthetase - GUS -glucuronidase - IgG immunoglobulin - PBS phosphate buffer saline - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Aluminium Stress Affects Nitrogen Fixation and Assimilation in Soybean (Glycine max L.)

Nitrogen fixation and assimilation in nodules and roots were studied in soybean (Glycine max L.) exposed to different levels of aluminium (Al) stress (0, 50, 200 and 500 μM). Al at 500 μM induced oxidative stress, which became evident from an increase in lipid peroxidation accompanied by a concomitant decline in antioxidant enzyme activities and leghaemoglobin breakdown. Consequently, there was also a reduction in nitrogenase activity. However, the leghaemoglobin levels and nitrogenase activity were unexpectedly found to be higher in nodules when the plants were treated with 200 μM Al. Of the enzymes involved in nitrogen assimilation, the activity of glutamate dehydrogenase-NADH was reduced in nodules under Al stress, but it was significantly higher in roots at 500 μM Al as compared to that in the control. In nodules, the glutamine synthetase/glutamate synthase-NADH pathway, assayed in terms of activity and expression of both the enzymes, was inhibited at >50 μM Al; but in roots this inhibitory effect was apparent only at 500 μM Al. No significant changes in ammonium and protein contents were recorded in the nodules or roots when the plants were treated with 50 μM Al. However, Al at ≥200 μM significantly increased the ammonium levels and decreased the protein content in the nodules. But these contrasting effects on ammonium and protein contents due to Al stress were observed in the roots only at 500 μM Al. The results suggest that the effect of Al stress on nitrogen assimilation is more conspicuous in nodules than that in the roots of soybean plants.     

Hybrid Cluster Proteins and Flavodiiron Proteins Afford Protection to Desulfovibrio vulgaris upon Macrophage Infection

Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection.