Divergence in nitrogenases of Azoarcus spp., Proteobacteria of the beta subclass

Nitrogenase is a functionally constant protein catalyzing N2 reduction, which is found in many phylogenetic lineages of Archaea and Bacteria. A phylogenetic analysis of nif genes may provide insights into the evolution of the bacterial genomes. Moreover, it may be used to study diazotrophic communities, when classical isolation techniques may fail to detect all contributing populations. Among six species of the genus Azoarcus, diazotrophic Proteobacteria of the beta subclass, the deduced amino acid sequences of nifH genes of two species were unusually divergent from each other. Nitrogenases of the "authentic" Azoarcus branch formed a monophyletic unit with those of gamma Proteobacteria, thus being in accordance with 16S ribosomal DNA phylogeny. The nitrogenase proteins of the two aberrant strains clustered within the alpha proteobacterial clade with rhizobial nitrogenases. This relationship was supported by bootstrap values of 87 to 98% obtained by various distance and maximum parsimony methods. Phylogenetic distances of NifH proteins indicate a possible lateral gene transfer of nif genes to Azoarcus from a common donor of the alpha subclass at the time of species diversification or several more recent, independent transfers. Application of the phylogenetic analysis to DNA isolated from environmental samples demonstrated novel habitats for Azoarcus: in guts of termites and rice grown in Japan, nifH genes belonging to the authentic Azoarcus branch were detected. This is the first evidence suggesting the occurrence of Azoarcus spp. in a plant other than its originally described host, Kallar grass. Moreover, evidence for expression of nif genes inside grass roots was obtained by in situ hybridization studies with antisense nifH probes.     

Phylogeny of Methanopyrus kandleri based on methyl coenzyme M reductase operons

The mcrBDCGA operon that encodes methyl coenzyme M reductase (MR) in the hyperthermophile Methanopyrus kandleri was cloned and sequenced. The results of a phylogenetic analysis of the nine MR sequences now available support the position that M. kandleri is a separate methanogen lineage. As in other methanogens, the M. kandleri mcr operon is located immediately upstream of the mtrE gene, the promoter-proximal gene in an operon that encodes the N5-methyltetrahydromethanopterin:coenzyme M methyltransferase that catalyzes the step preceding the MR-catalyzed reaction in methanogenesis. In contrast to other methanogens and hyperthermophilic members of the Archaea, CG dinucleotides and CG-containing codons occur frequently in M. kandleri DNA. The MR subunit-encoding genes are preceded by sequences consistent with ribosome binding sites, indicating that mRNA-rRNA base pairing can still direct translation initiation in cells growing at temperatures above 100 degrees C.     

Production of heparin-binding endothelial mitogens by bovine uterine fibroblastic and epithelial cells

This study was conducted to determine whether early-passage cultures of bovine endometrial fibroblastic (Fb, n = 7 uteri) and epithelial (Ep, n = 3 uteri) cells produce endothelial mitogens in vitro and to begin characterization of these mitogens. Confluent cultures of Fb and Ep were incubated for 72 h in serum-free media, and the resulting conditioned media (CM) were evaluated for effects on proliferation of bovine aortic endothelial cells. CM from these Fb cultures (n = 8) and Ep cultures (n = 4) stimulated (147 +/- 10% (mean +/- SEM), p < 0.01, and 124 +/- 8%, p < 0.10, respectively) proliferation of endothelial cells compared with control (unconditioned) media. Most of the mitogenic activity of a sample of Fb CM and a sample of Ep CM from one individual uterus bound to heparin-agarose, and each exhibited two major peaks of activity that eluted at 0.9-1.0 and 1.7-1.8 M NaCl; the Fb CM also exhibited an additional heparin-binding peak eluting at 0-0.1 M NaCl. Pooled Fb CM (n = 8 cultures from 7 animals) also contained mitogenic activity for endothelial cells that bound to heparin-agarose, but exhibited three major peaks, eluting at 0.6, 1.1, and 1.8 M NaCl. Pooled Ep CM (n = 4 cultures from 3 animals) showed only one peak of mitogenic activity, which eluted at 0.9 M NaCl. Further characterization indicated that heat treatment reduced the activity of all heparin-binding Fb CM and Ep CM peaks, except the Fb CM peak eluting at 1.7 M NaCl. Trypsin reduced the activity of all peaks except one. Protein-A-purified antibody against fibroblast growth factor 1 (FGF-1) had no or only a slight effect on the mitogenic activity of the peaks. Mitogenic activity of the Fb CM peak eluting at 0.6 M NaCl was reduced by antibody against FGF-2. Activity of the Fb CM and Ep CM peaks that eluted at 1.7-1.8 M NaCl also was immunoneutralized by antibody to FGF-2. These data demonstrate that early passage cultures of endometrial Fb and Ep cells produce heparin-binding endothelial mitogens that appear to be immunologically related to FGF-2. These heparin-binding endothelial mitogens may influence endometrial vascular growth.     

Two distinct forms of human mast cell chymase--differences in affinity for heparin and in distribution in skin, heart, and other tissues

Chymase, a chymotrypsin-like protease secreted by human mast cells, is generally considered to be a single enzyme. However, by heparin-agarose chromatography of high-salt extracts of human skin, we have consistently resolved three peaks of chymotryptic activity, eluting at 0.4 M NaCl (peak A), 1.0-1.2 M NaCl (peak B) and 1.8-2.0 M NaCl (peak C), with peak B containing 75-90% of the recovered activity. Each peak retained its identity upon rechromatography. The three peaks of activity were similar in substrate specificity and inhibitor profile and distinctly different from other chymotryptic enzymes, including cathepsin G and the stratum corneum chymotryptic enzyme. Examination of different tissues revealed that peak C was virtually absent from synovial tissue, was present as a minor component in skin and heart, but constituted the predominant chymotryptic activity in lung. Peaks B and C from skin tissue were further purified by chromatography on Sephacryl S-200. Both had a molecular mass of 28-29 kDa, yielded the N-terminal sequence reported for chymase, and on western blots reacted with a panel of polyclonal, monoclonal and antipeptide antibodies against chymase. Chymase C required higher concentrations of NaCl to overcome the stimulatory effects of heparin than did chymase B, but had a similar pH profile. Thus, human chymase exists in at least two distinct but similar forms, and the differences in heparin binding and tissue distribution could have important consequences for enzyme function.     

Regulation of human B19 parvovirus promoter expression by hGABP (E4TF1) transcription factor

The genetic expression of human B19 parvovirus is only dependent on one promoter in vivo and in vitro. This is the P6 promoter, which is located on the left side of the genome and is a single-stranded DNA molecule. This led us to investigate the regulation of the P6 promoter and the possible resulting variability of the nucleotide sequence. After analysis of the promoter region of 17 B19 strains, only 1.5% variability was found. More exciting was the finding of mutations that were clustered around the TATA box and defined a highly conserved region (nucleotides 113-210) in the proximal part of the P6 promoter. HeLa and UT7/Epo cell extracts were found to protect this region, which contained a core motif for Ets family proteins, with YY1 and Sp1 binding sites on either side. Gel mobility shift assays performed with nuclear proteins from HeLa and UT7/Epo cells identified DNA-binding proteins specific for these sites. By supershift analysis, we demonstrated the binding of the hGABP (also named E4TF1) protein to the Ets binding site and the fixation of Sp1 and YY1 proteins on their respective motifs. In Drosophila SL2 cells, hGABPalpha and -beta stimulated P6 promoter activity, and hGABPalpha/hGABPbeta and Sp1 exerted synergistic stimulation of this activity, an effect diminished by YY1.     

MUC2 gene suppression in human colorectal carcinomas and their metastases: in vitro evidence of the modulatory role of DNA methylation

The development of the majority of colorectal carcinomas is associated with a diminished expression of the intestinal mucin MUC2 in the tumor cells. The significance and the mechanism of this alteration are not yet known. We sought to determine the molecular basis of this tumor-associated change and to investigate the extent to which it might also relate to metastases. MUC2 gene expression was compared in normal (N), carcinomatous (T), and metastatic tissues (M) from nine patients by immunohistochemistry, in situ hybridization, and Northern blotting. Immunohistochemistry and in situ hybridization showed consistently lower amounts of the expressed protein and mRNA in T and in M than in N; quantitative analysis by Northern blotting confirmed that the differences between MUC2 mRNA expression between N, T, and M were significant, the expression in metastases being less than 5% of that in the normal colonic tissue. The influence of DNA methylation as a possible regulatory mechanism of MUC2 gene expression was tested after the 5' and 3'-regions flanking the first exon of MUC2 were recovered from a genomic DNA library and used as probes in Southern blot. The DNA was isolated from colon carcinoma cell lines expressing MUC2 strongly (LS174T) or moderately (T84) and from that which was nonexpressing (Colo 205), and it was digested with the methylation-sensitive enzyme HpaII. The Southern blot patterns indicated that the increased methylation in the promoter region was concomitant with the decrease of MUC2 mRNA expression. Methylation of the promoter region ligated into a reporter vector suppressed the expression of the luciferase reporter gene in the three investigated cell lines. Furthermore, the expression of MUC2 gene was enhanced by treating the MUC2-expressing colon carcinoma cells with 5-aza-2'-deoxycytidine, a methylation-inhibiting agent. To our knowledge this is the first report to show that: (a) MUC2 gene is strongly suppressed in liver and lymph node metastases of colorectal carcinomas, and (b) suppression of MUC2 gene in colon carcinoma cells in vitro is associated with methylation of the promoter region.     

Structural studies on a sulfated polysaccharide from an Arthrobacter sp. by NMR spectroscopy and methylation analysis

Interleukin-4 (IL-4) is a pleiotropic immunomodulatory cytokine secreted by T helper 2 cells. The IL-4 promoter contains multiple sites with DNA sequences homologous to the IL-2 NF-AT binding site. One of these sites--the P2 site--located between -173 and -150 was previously found to be flanked by two octamer-like motifs. NF-ATp/c and octamer proteins were suggested to bind to this region and to cooperatively activate the promoter activity (Chuvpilo et al., 1993). To precisely analyze the P2-binding factors we used antibodies against NF-ATp, NF-ATc, Fos, Jun, Oct-1 and Oct-2 in EMSA. We show here that nuclear extracts from T-cells form two P2-binding complexes--a PMA/ionomycin-inducible and a constitutive one. The PMA/ionomycin-inducible complex contains NF-ATp/c, Fos and Jun. No octamer binding factors could be detected in either of the two complexes. Analysis of the precise DNA contact points of the two complexes showed that both complexes are formed in the center of the NF-AT consensus site. No DNA contact points could be detected in the octamer-like motif site. Furthermore, purified recombinant POU domains of Oct-1 and Oct-2 failed to bind to the P2 site, suggesting that this site is not an independent octamer-binding site. Therefore, the DNA sequence at -173 to -150 of the IL-4 promoter is a binding site for NF-ATp/c and AP-1. Octamer proteins are unlikely to cooperate with NF-ATp/c at this site.     

Purification and characterization of a nuclear DNA-binding phosphoprotein in fetal and tumor tissues

The basic nonhistone phosphoprotein 110/8.4 (M.W. X 10(-3)/pI) was found in 0.35 M NaCl nuclear extracts of four tumor tissues, i.e., fast-growing Novikoff hepatoma, Morris hepatoma 3924A, HeLa cells, and Namalwa cells; it was also found in fetal rat liver. This protein was not detected in normal or regenerating liver and thus may represent an "oncofetal" protein of potential interest as a cancer "marker." Protein 110/8.4 was purified approximately 4000- to 5000-fold under nondenaturing condition from 0.35 M NaCl nuclear extracts of Novikoff hepatoma cells or Namalwa cells by ammonium sulfate fractionation, calcium phosphate gel treatment, and phosphocellulose chromatography. Sodium dodecyl sulfate:polyacrylamide gel electrophoresis of the purified native protein revealed a single polypeptide chain with a molecular weight of approximately 110,000. The pI of the protein was estimated to be 8.4 by nonequilibrium pH gradient electrophoresis in 9 M urea; accordingly, this protein was designated 110/8.4. Amino acid analysis showed that Protein 110/8.4 had an acidic:basic amino acid ratio of 1.25 and a high lysine and serine content; approximately 20% of the serine residues were found to be phosphorylated. Hydrazinolysis indicated that the carboxyl-terminal amino acid was serine; the amino terminus appeared to be blocked. Binding of Protein 110/8.4 to DNA was studied by the nitrocellulose filter assay. High-affinity binding occurred at ionic strength equal to or below 0.15 M.