A physical genome map of Pseudomonas aeruginosa PAO.

A complete macrorestriction map of the 5.9 Mb genome of Pseudomonas aeruginosa PAO (DSM 1707) was constructed by the combination of various one- and two-dimensional pulsed field gel electrophoresis techniques. A total of 51 restriction sites (36 SpeI sites, 15 DpnI sites) were placed on the physical map yielding an average resolution of 110 kb. Several genes encoding virulence factors and enzymes of metabolic pathways were located on the anonymous map by Southern hybridization. Distances between the gene loci were similar on the genetic and physical maps, suggesting an even distribution of genome mobility throughout the bacterial chromosome. The four rRNA operons were organized in pairs of inverted repeats. The two-dimensional macro-restriction techniques described herein are generally applicable for the genome mapping of any prokaryote and lower eukaryote which yields resolvable fragment patterns on two-dimensional pulsed field gels.     

Reactive extraction of penicillin G from mycel-containing broth in a countercurrent extraction decanter

Penicillin was recovered from mycel-containing fermentation broth by direct reactive extraction into a counter-current extraction decanter, Type CA 226-290 of the Westfalia Separator Co., at room temperature via steady state operation. Penicillin concentrations in the feed varied from 3 to 41 g L(-1), Amberlite LA-2 carrier concentrations from 7 to 20 g L(-1)and/or DITDA carrier concentrations from 7.2 to 84 g L(-1), the LA-2-to-penicillin mole concentration ratio from 4 to 6.4, and/or the DITDA-to-penicillin mole concentration ratio was maintained at 2. The throughputs of the fermentation broth (520 to 880 L h(-1)) of the solvent phase (200 to 860 L h(-1)) and the over all throughput (800 to 1750 L h(-1)) were high. Extraction degrees of 72 to 96% were achieved between pH 4.6 and 5.1. Without carriers in the same pH range, extraction degrees of only 17 to 19% were attained. By reducing the pH to 2.3 and in the absence of carriers, the degree of extraction was increased to 61%. However, during the extraction, 6.5% of the penicillin decomposed. At these high throughputs, the steady state was attained within 1 to 4 min. Through the mechanical stress, the length of the hyphae was reduced and the protein content of the broth was increased by 50 to 100%. However, this protein content had no appreciable influence on the phase separation.     

Isolation of heparin-binding growth factors from bovine, porcine and canine hearts

Fresh bovine, porcine and canine hearts were homogenized and mitogens for mesoderm-derived cells were purified in three different steps. Extraction by two different ammonium sulfate precipitations was followed by cation-exchange chromatography and by heparin-Sepharose affinity chromatography. A heparin-Sepharose fraction from heart (eluted at 1.1 M NaCl) increased mitotic activity in serum-deprived cultures of porcine aortic endothelial and smooth muscle cells, and in human fibroblasts. This mitogenic activity is potentiated by heparin and inhibited by gamma-interferon. The heart mitogenic fraction showed one double peak on HPLC at A215 and one polypeptide band on SDS/PAGE. These peaks and bands were identical to those obtained from bovine brain. The heart acidic fibroblast growth factor (aFGF) showed a positive signal in Western blots using antibodies raised against brain aFGF. Gas-phase amino acid sequencing established that the mitogens were identical to aFGF and the N-terminally truncated aFGF. Extraction in the presence of a protease inhibitor (pepstatin A) produced a higher-molecular mass form of aFGF with a blocked amino terminus. Another mitogen, eluted at 1.6 M NaCl from heparin-Sepharose, reacted with polyclonal antiserum against human recombinant basic fibroblast growth factor (bFGF) and showed a 66% (12 from 18 amino acids determined by gas-phase sequencing) similarity with bFGF. This polypeptide increased the mitotic activity of the same cell lines but was more potent than aFGF.     

A novel L-glutamate oxidase from Streptomyces endus. Purification and properties

A new flavoenzyme using molecular oxygen to oxidize L-glutamic acid has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the culture medium of Streptomyces endus. Hydrogen peroxide, 2-oxoglutaric acid and ammonia are formed as products. Among 25 amino acids tested including D-glutamic acid, L-glutamine and L-aspartic acid, only L-glutamic acid is converted. The molecular mass of the enzyme was estimated to be about 90 kDa by gel chromatography and 50 kDa by SDS/PAGE. The subunit contains 1 molecule noncovalently bound FAD. The absorption spectrum shows maxima at 273, 355 and 457 nm and the isoelectric point is at pH 6.2. The Km value for L-glutamic acid in air-saturated phosphate pH 7.0 was estimated to be 1.1 mM, the Km for oxygen was calculated to be 1.86 mM at saturating concentration of L-glutamic acid. The enzymic reaction is inhibited by Ag+ and Hg2+ ions. The enzyme described here distinctly differs from two microbial L-glutamate oxidases purified hitherto, with regard to extremely high substrate specificity and to the subunit structure.     

Differences in hydrophobic properties for human alpha 2-macroglobulin and pregnancy zone protein as studied by affinity phase partitioning

Human alpha 2-macroglobulin and pregnancy zone protein are related with regard to primary structure, physicochemical properties, and quarternary structure. Both proteins undergo conformational changes when they form complexes with proteinases or react with primary amines. The surface properties of the native, chymotrypsin-treated and methylamine-treated forms of alpha 2-macroglobulin and pregnancy zone protein were studied by partitioning in aqueous two-phase systems composed of 7.5% dextran T70 and 5% poly(ethylene glycol) 8000. All proteins and their derivatives had a high potential for hydrophobic interaction as analyzed in terms of affinity for poly(ethylene glycol) esters of fatty acids included in the phase systems. Treatment of alpha 2-macroglobulin with methylamine or chymotrypsin increased the surface hydrophobicity significantly compared to that of the native protein. No difference in hydrophobic interaction was found for native and methylamine-treated pregnancy zone protein, but the chymotrypsin-treated protein showed a marked increase in binding to the hydrophobic ligand. The changes in surface hydrophobicity parallel changes in receptor binding properties of the derivatized forms of alpha 2-macroglobulin and could be a signal for binding to cell-surface receptors, followed by internalization.     

Interruption of TPA-induced signals by an antiviral and antitumoral xanthate compound: inhibition of a phospholipase C-type reaction

The effect of tricyclodecan-9-yl-xanthogenate on the phorbolester TPA induced changes in phosphatidylcholine metabolism was investigated. In the simultaneous presence of the xanthate TPA failed to stimulate the metabolic [32P] turnover of the major phospholipids. The precursor molecule [3H] choline was incorporated into phosphatidylcholine after pulse labeling in TPA/D609-treated cells. Thus, the reduction of the [32P] phosphatidylcholine turnover did not appear to result from an inhibition of the TPA-stimulated phosphatidylcholine biosynthesis. However, the xanthate exerted an inhibitory effect on the TPA-stimulated liberation of [3H] phosphorylcholine from [3H] phosphatidylcholine in cells prelabeled with [3H] choline. Furthermore, the TPA-induced rise in the diacylglycerol level was reduced in the presence of the compound. Thus, these results provide evidence that the xanthate inhibits a TPA-induced phospholipase C activity in the intact cell.     

Generation of nitric oxide by human neutrophils

Human neutrophils were evaluated for their ability to generate nitric oxide. Neutrophils incubated with superoxide dismutase at 37 degrees C produce nitrite anion at a rate of 1.8 nmols/2 x 10(6) cells/30 min, providing indirect evidence of nitric oxide production. Incubation of the neutrophils with concentrations of serum-opsonized zymosan, N-formyl-methionyl-leucyl-phenylalanine, or phorbol myristate acetate sufficient to stimulate the respiratory burst and lysosomal enzyme release caused no additional nitrite anion production. Glass wool-adherent neutrophils exhibited a similar dissociation of nitrite anion production from the respiratory burst and lysosomal enzyme release. Direct evidence for nitric oxide production was also obtained using nitric oxide-specific chemiluminescence. These results demonstrate that human neutrophils are capable of generating nitric oxide.     

Non-C-G recognition sequences of DNA cytosine-5-methyltransferase from rat liver

The eukaryotic DNA cytosine-5-methyltransferase (E.C.2.1.1.37) is known to methylate cytosine in DNA mainly, but not exclusively in C-G. In the present study the minor, non-C-G recognition sequences of a rat DNA methyltransferase were analyzed by Maxam-Gilbert sequencing of in vitro methylated SV40 DNA. The enzyme methylates C-A and C-T at a 50-fold lower initial rate than C-G. Methylation of C-C at the 5'C was not observed in the piece of DNA sequenced. The methylation of C-A is very low in the trinucleotides ACA and CAC, the other C-A containing trinucleotides in DNA are much better methylacceptors. C-T was found methylated predominantly in the sequences CCTAA, ACTAA, and ACTGT. A comparison of the activity with different substrates is in favour of the enzyme making its recognition in the major groove of the DNA.     

Association of Tat protein and viral mRNA with nuclear matrix from HIV-1-infected H9 cells

The transactivating protein from human immunodeficiency virus type 1 (HIV-1), Tat, was found to bind to the nuclear matrix from uninfected and HIV-1-infected H9 cells. Addition of the Zn2+, Cd2+ and Cu2+ chelator o-phenanthroline destroyed the matrix fibrils and the binding affinity of Tat to the matrix. A sequential treatment of the matrix, first with o-phenanthroline and then with ZnCl2, partially restored the fibrillar-like matrix structure. Infection of H9 cells with HIV-1 resulted in a displacement of cellular mRNA by viral mRNA from the nuclear matrix. Both the matrix-bound host cell and HIV-1 mRNA were found to dissociate from the matrix in the presence of o-phenanthroline. This could be prevented by coincubation with Zn2+ or Cu2+ (but not Mg2+), which stabilize the mRNA containing nuclear matrix structure.     

Absence of the Epstein-Barr virus genome in the normal thymus, thymic epithelial tumors, thymic lymphoid hyperplasia in a European population

It has previously been shown that the Epstein-Barr virus (EBV) genome may be detected in some thymic tumors. We have investigated specimens of normal thymus, thymitis with lymphoid hyperplasia and a large spectrum of thymic epithelial tumors obtained from european patients for the presence of EBV genome by in situ hybridization and DNA-blotting methods. Cell lines established from seven of the thymic tumors were also tested for EBV. No EBV genome was demonstrated in any of the tumors examined, which included various types of thymoma and thymic carcinomas, nor in the non-neoplastic thymic specimens. However, unlike previous reports, no examples of lymphoepithelial-like thymic carcinoma, nor specimen from Asian patients were included in this study. We suggest that EBV is linked to a specific epithelial tumor type, namely the lymphoepithelial-like carcinoma, regardless of its site, and not to thymic tumors in general.