Increased oxidative stress in the RAW 264.7 macrophage cell line is partially mediated via the S-nitrosothiol-induced inhibition of glutathione reductase

We investigated whether endogenously or exogenously produced nitric oxide (NO) can inhibit cellular glutathione reductase (GR) via the formation of S-nitrosothiols to decrease cellular glutathione (GSH) and increase oxidative stress in RAW 264.7 cells. The specificity of this inhibition was demonstrated by addition of a NO-synthase inhibitor, and met- or oxyhemoglobin. Using isolated GR we found that only certain NO donors inhibit this enzyme via S-nitrosothiol. Furthermore, we found that cellular GSH decrease is paralleled by an increase of superoxide anion production. Our results show that the GR enzyme is a potential target of S-nitrosothiols to decrease cellular GSH levels and to induce oxidative stress in macrophages.     

Molecular cloning and functional expression of a phospholipase D from cabbage (Brassica oleracea var. capitata)

We cloned and expressed a full-length cDNA encoding a phospholipase D of type alpha (PLDalpha) from cabbage. Analysis of the cDNA predicted an 812-amino-acid protein of 92.0 kDa. The deduced amino acid sequence of cabbage PLD has 83% and 80% identity with Arabidopsis PLDalpha and castor bean PLD, respectively. Expression of this cDNA clone in E. coli shows a functional PLD activity similar to that of the natural PLD.     

Cloning and characterization of overlapping DNA fragments of the toxin A gene of clostridium difficile

Clostridium difficile, a human pathogen, produces two very large protein toxins, A and B (250-600 kDa), which resist dissociation into subunits. To clone the toxin A gene, a genomic library of 3-8 kb chromosomal DNA fragments of C. difficile strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum. Thirty-five clones were isolated which carried 2.5-7.0 kb inserts representing a 10 kb region of the C. difficile genome. All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the lac promoter of the pUC12 vector. Western blot experiments revealed the presence of low amounts of fusion proteins of variable size (30-170 kDa) in Escherichia coli strains harbouring recombinant plasmids. As deduced from subcloning experiments, the DNA sequences encoding toxin A comprised about 4 kb, corresponding to about 140 kDa of the 300-600 kDa protein. This was either due to incomplete cloning of the gene or it might indicate a subunit composition of toxin A. No additional gene(s) with homology to the cloned toxin A gene was detected.     

The role of macrophages in B cell tolerance. I. Antigen-specific failure of Thy-1, Ly-2 negative adherent cells from tolerant mice to reconstitute immunocompetence in adherent cell deficient spleen cells

T-Cell-independent B-cell tolerance to the hapten derivatives of carboxymethyl cellulose (CMC) or methyl cellulose (MC) appears to be controlled by Thy-1-, Ly-2- adherent (A) cells contained in the spleen or peritoneal fluid. Immunocompetence in nonadherent (NA) normal spleen cells could be restored in vitro by irradiated A cells from normal mice. However, NA cells reconstituted with irradiated A cells derived from hapten specifically tolerant mice failed to respond to the same hapten, but responded normally to an immunogenic challenge with another unrelated antigen. A cells that had been preincubated at 4 degrees C with hapten derivatized MC also failed to restore immunocompetence. While preincubation of unfractionated spleen cells with the tolerogen under the same conditions resulted in B-cell unresponsiveness, such treatment of NA cells failed to render B cells tolerant. Treatment of A cells from tolerant mice with the reducing agent potassium iodide (KI) in vitro restored their capacity to render cultures of NA cells immunocompetent to the relevant hapten. Moreover, treatment with KI of spleen cells from mice injected with the tolerogen was shown to render them responsive. We suggest that B-cell tolerance induced by hapten derivatives of CMC and MC is mediated by suppressive macrophages contained among A cells. Certain subpopulations of macrophages are known to exert cytotoxic effects upon target cells by the release at close range of oxidating agents. We postulate that hapten derivatized CMC and MC, through unique properties of the carrier, bind to and possibly activate macrophages rendering them specifically suppressive for hapten binding B cells.     

The terminal deoxynucleotidyltransferase gene is located on human chromosome 10 (10q23----q24) and on mouse chromosome 19

Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase expressed in immature lymphocytes of the thymus and bone marrow, as well as certain leukemic cells. Chromosomal assignment of the gene coding for human TdT was accomplished by in situ hybridization of a 3H-labeled cDNA probe to human chromosome preparations and by Southern blot analysis of somatic cell hybrid DNAs. The human TdT gene was mapped to the region q23----q24 of chromosome 10. Breaks at this site have been reported in different translocations in human leukemias. The mouse TdT gene was assigned to chromosome 19 by Southern blot analysis of mouse X Chinese hamster somatic cell hybrids. This result adds a fourth locus to the conserved syntenic group on mouse chromosome 19 and human chromosome 10.     

Preparation and characterization of two major isotypes of parvalbumins from skeletal muscle of bullfrog (Rana catesbeiana)

Two major isotypes of parvalbumins (PA1 and PA2) have been isolated from the skeletal muscle of bullfrog, Rana catesbeiana. The Mr values were estimated to be 10,100 (PA1) and 11,800 (PA2) by SDS/polyacrylamide gel electrophoresis, and the isoelectric points were determined to be 4.78 (PA1) and 4.97 (PA2) by polyacrylamide gel isoelectric focusing. The amino acid compositions and isoelectric points indicate that PA1 corresponds to Rana esculenta pI 4.50 and Rana temporaria pI 4.75 parvalbumins and PA2 to Rana esculenta pI 4.88 and Rana temporaria pI 4.97 parvalbumins, showing that PA1 is genetically a beta-parvalbumin and PA2 an alpha-parvalbumin. However, in terms of the amino acid compositions, PA1 and PA2 are distinctly different from the corresponding parvalbumins of Rana esculenta or Rana temporaria. The ultraviolet spectra of PA1 and PA2 are consistent with their amino acid compositions. An ultraviolet difference spectrum of the Ca2+-loaded form vs. metal-free form indicates that a Tyr and some Phe residues in PA1 are affected by a conformational change associated with the binding of Ca2+. On electrophoresis in polyacrylamide gel in 14 mM Tris and 90 mM glycine, the Ca2+-loaded form of PA1 migrated twice as fast as the Mg2+-loaded form. Both PA1 and PA2 show increased mobility in the Ca2+-loaded forms, like troponin C but different from calmodulin.     

Physical map of the chromosome of the apple proliferation phytoplasma

A physical map of the apple proliferation phytoplasma strain AT chromosome was constructed from genomic DNA extracted from diseased tobacco plants. The map was generated with single and double digestions of the chromosome with BssHII, SmaI, MluI, and ApaI restriction endonucleases and resolving the fragments by pulsed-field gel electrophoresis. Partial digestion and Southern blot analysis were used to assist in the arrangement of the 14 contiguous restriction fragments obtained. From the restriction fragments generated by double digestions, the size of the circular chromosome was calculated to be approximately 645 kb. Locations of the two rRNA operons, the operon including the fus and tuf genes, and three other genes were placed on the map. Genome sizes and BssHII restriction profiles of apple proliferation strain AP15 and the pear decline and European stone fruit yellows phytoplasmas were different from that of strain AT.     

Ultrastructure of Tricornia muhezae N. G., n. sp. (Microspora, Thelohaniidae), a parasite of Mansonia africana (Diptera: Culicidae) from Tanzania.

Collections of Mansonia africana mosquito larvae were made at one site in N.E. Tanzania in 1985 and 1987 and from two additional sites, both within about 2 mi of the original one in 1987. An octosporous microsporidian, present at all three sites, was found in both years infecting from 7 to 22% of larvae. Spores (stained in Giemsa) measured 3.0 microns +/- 0.25 microns x 2.25 microns +/- 0.26 microns. Ultrastructurally, spores were seen to have an anterior rim surrounding a depressed area where the endospore was at its thinnest. In transmission electron microscopy section, the rim appeared as two processes into which all layers of the wall extended. At the posterior end all layers of the wall extended into a simple knob-like structure which could be interpreted as a section through a crest running longitudinally around the spore. The polar filament was anisofilar, with two anterior coils of greater diameter than the three posterior coils. Although most closely resembling the genera Amblyspora and Parathelohania in the family Thelohaniidae, the species in M. africana differs from the former, which has oval spores, broadly rounded at the ends, and from the latter, which has a prominent, ridged posterior extension to the spores. The new species has been placed in a new genus and the name Tricornia muhezae proposed.     

Nutritional and hormonal regulation of the translatable levels of malic enzyme and albumin mRNAs in avian liver cells in vivo and in culture

Relative synthesis of malic enzyme is stimulated 25-to 100-fold by feeding neonatal ducklings or by incubating embryonic chick hepatocytes in culture with triiodothyronine. Synthesis of the enzyme is almost completely blocked when fed birds are starved or when triiodothyronine-treated hepatocytes in culture are also treated with glucagon. Cytoplasmic poly(A)+ RNA was isolated from livers of intact ducklings or hepatocytes in culture treated as described above and translated in an mRNA-dependent rabbit reticulocyte lysate. The identity of malic enzyme synthesized in the cell-free system was confirmed by virtue of its antigenicity, subunit molecular weight, and proteolytic peptide pattern. Translatable levels of malic enzyme mRNA paralleled changes in relative synthesis of malic enzyme in vivo and in hepatocytes in culture. Translatable levels od albumin mRNA were either unaffected or changed in a direction opposite to that of malic enzyme mRNA. Thus, both nutritional and hormonal regulation of malic enzyme synthesis involves regulation of cytoplasmic translatable malic enzyme mRNA levels. The hepatocyte culture system is ideally suited for future studies on the regulation of malic enzyme mRNA synthesis and/or degradation by thyroid hormone and glucagon.