Suppressive effects of coffee on the SOS responses induced by UV and chemical mutagens

SOS-inducing activity of UV or chemical mutagens (AF-2, 4NQO and MNNG) was strongly suppressed by instant coffee in Salmonella typhimurium TA1535/pSK1002. As decaffeinated instant coffee showed a similarly strong suppressive effect, it would seem that caffeine, a known inhibitor of SOS responses, is not responsible for the effect observed. The suppression was also shown by freshly brewed coffee extracts. However, the suppression was absent in green coffee-bean extracts. These results suggest that coffee contains some substance(s) which, apart from caffeine, suppresses SOS-inducing activity of UV or chemical mutagens and that the suppressive substance(s) are produced by roasting coffee beans.     

Loss of heterocyclic amine mutagens by insoluble hemicellulose fiber and high-molecular-weight soluble polyphenolics of coffee

The presence of 2 kinds of components in brewed and instant coffee that could remove and destroy heterocyclic amine mutagens was demonstrated. The component that could remove the mutagens was insoluble fiber composed of hemicellulose. The fiber could tightly adsorb the mutagens Trp-P-1, Trp-P-2, Glu-P-1 and A alpha C, and those generated in roasted coffee beans. The component that could destroy the mutagens was high-molecular-weight soluble polyphenolics. They might be converted into quinone derivatives in the presence of molecular oxygen. The quinone derivatives might destroy the mutagens. The fibers and the polyphenolics in one cup of brewed or instant coffee had the capacity to remove and destroy a substantial amount of the mutagens in pyrolysates of foodstuffs.     

Characteristics of major mutagenicity of instant coffee

Detailed quantitative studies on the mutagenicity of methylglyoxal showed that its contribution to the total mutagenicity of instant coffee on S. typhimurium TA100 was minor although we reported previously (Kasai et al., 1982) that its contribution to the mutagenicity of freshly brewed coffee was about 50%. Cysteine suppressed the mutagenicity of methylglyoxal and of methylglyoxal when added to instant coffee, but did not affect the mutagenicity of coffee itself. Catalase suppressed most of the mutagenicity of coffee, but not that of methylglyoxal or of methylglyoxal added to coffee.     

A new gene in E. coli RNA synthesis

A novel spontaneous temperature sensitive mutant of Escherichia coli, which stops synthesizing stable RNA and some proteins immediately upon temperature shift from 30 degrees C to 42 degrees C, is described. Stable RNA species are not preferentially degraded in the mutant at the nonpermissive temperature. The guanine polyphosphate compounds, ppGpp (MS1) and pppGpp (MS2), are not produced at 42 degrees C. The mutant strain does not grow at 42 degrees C in either broth or defined minimal medium supplemented with any of a variety of carbon sources. The temperature sensitive mutation in this strain maps between dap A, E and pts I and defines a new locus affecting RNA synthesis in E. coli.     

Enterotoxigenic E. coli in humans.

E. coli strains isolated from persons having diarrhoeal disease were tested for the production of TS enterotoxin. The production of TSE was demonstrated in 2.5% in a series of 80 strains isolated from children under one year of age, having acute diarrhoea. TSE was produced by 8.4% of E. coli strains out of 59 strains isolated from patients over one year of age. Among these strains, an interesting E. coli strain was isolated from the patient T. J., which produced TSE for more than 15 months. The production of TLE was tested though not proved in all strains by experiment on an isolated intestinal loop of an adult rabbit. The test on suckling mice so far appears to be the most suitable test for the demonstration of TSE. The results were considered positive when the index (the ratio of the weight of the whole intestine to the weight of the rest of the body) was higher than 0.08 while indices up to 0.078 were considered negative. E. coli strains with indices of intermediate values and strains with temporary production of TSE, occurring particularly in very small children, deserve special attention. The height of the indices was not influenced by a 30-minute exposure at 60 degrees C, but a decrease in the values of the indices was observed after boiling for a period of 15 min. The occurrence of E. coli strains producing TSE is evidently small in humans in European countries but, without doubt, they are important in the aetiology of diarrhoeal diseases.     

Expression of polyoma early gene products in E. coli.

The three products of the early region of polyoma virus have been cloned for expression in E. coli using the Tac promoter. Although the identical promoter and ribosome binding site are used in each final construction, the observed level of protein expression is different for each protein. While plasmids expressing wild type T antigens as well as a plasmid expressing the truncated Py-1387T middle T antigen lacking the membrane-anchoring sequence give rise to synthesis of proteins readily detectible by 35S-methionine labeling and immunoprecipitation, only small T and the middle T of Py-1387T are made in amounts sufficient for ready detection in total cell protein. Unlike middle T expressed in animal cells, middle T produced in E. coli is not detectibly phosphorylated. Further, the E. coli protein lacks tyrosine kinase activity.     

Human uracil-DNA glycosylase complements E. coli ung mutants.

We have previously isolated a cDNA encoding a human uracil-DNA glycosylase which is closely related to the bacterial and yeast enzymes. In vitro expression of this cDNA produced a protein with an apparent molecular weight of 34 K in agreement with the size predicted from the sequence data. The in vitro expressed protein exhibited uracil-DNA glycosylase activity. The close resemblance between the human and the bacterial enzyme raised the possibility that the human enzyme may be able to complement E. coli ung mutants. In order to test this hypothesis, the human uracil-DNA glycosylase cDNA was established in a bacterial expression vector. Expression of the human enzyme as a LacZ alpha-humUNG fusion protein was then studied in E. coli ung mutants. E. coli cells lacking uracil-DNA glycosylase activity exhibit a weak mutator phenotype and they are permissive for growth of phages with uracil-containing DNA. Here we show that the expression of human uracil-DNA glycosylase in E. coli can restore the wild type phenotype of ung mutants. These results demonstrate that the evolutionary conservation of the uracil-DNA glycosylase structure is also reflected in the conservation of the mechanism for removal of uracil from DNA.     

Reconstitution of denatured E. coli alkaline phosphatase with E. coli ribosome.

E. coli alkaline phosphatase was denatured by physical/chemical means. In vitro reconstitution of this denatured enzyme was assisted by 70S E. coli ribosome, as shown by the recovery of its catalytic competence. Almost total recovery of activity of the totally inactivated enzyme was obtained in presence of equimolar concentration of 70S ribosome at 50 degrees C.     

E. coli proteolytic activity in milk and casein breakdown

Previous studies have focused on both LPS and E. coli experimental mastitis and underlined the respective roles of endogenous proteolysis (including plasmin from the blood stream and other proteases from milk leukocytes), as well as the presence of E. coli in a more intricate system. The aim of this study was to assess the role of E. coli in milk proteolysis and especially that of its proteases in casein breakdown. The first part consisted in the incubation of 104 cfu.mL(-1) of the E. coli strain in raw milk at 37 degrees C for 24 h; the same milk was also incubated with 0.04% sodium azide. Several parameters were evaluated: CFU, plasmin activity, gelatinase activity and pH 4.6 insoluble peptides, including the proportion of gamma-CN. The profile of gelatinase activity was determined by zymography and identified by immunoblotting. In the second part of the study, we examined the profile of CN (alphas-, beta- and kappa-CN) breakdown by E. coli lysate. The results suggest that E. coli proteases have a direct effect on CN, and the increase of gamma-CN in inoculated milk may be generated by both plasmin and the gelatinase. Moreover, the gelatinase activity in the inoculated milk was higher after 24 h of incubation.     

Antioxidative activity of a zinc-chelating substance in coffee

Coffee brew contains a brownish zinc-chelating polymer designated ApV. ApV was prepared from the precipitate formed in a solution of instant coffee by adding ZnCl(2) and purified using ion-exchange and cellulose column chromatographies. The antioxidative activities of ApV and related compounds were evaluated in this study. The free-radical scavenging activity of ApV estimated by ABTS assay was at a similar level to that of instant coffee, while the O(2)(-) scavenging activity of ApV, which is superoxide dismutase-like activity, was lower than that of instant coffee. The hydroxyl-radical scavenging activity of ApV was higher than that of instant coffee, and the auto-oxidation of linoleic acid was more strongly inhibited by ApV than by caffeic acid.     

嗜麦芽假单胞菌热休克基因dnaK启动子的亚克隆及其在大肠杆菌中功能的研究

通过聚合酶链式反应（PCR）得到了嗜麦芽假单胞菌（Pseudomonas maltophilia)热休克基因dnaK启动子的DNA片段dnaKp,将dnaKp的PCR的产物亚克隆在报道载体质粒pUCD615的LuxCDABE基因上游,成重组的具有启动子的质粒融合体pUCD615p。转化到大肠杆菌的pUCD615p启动子对热、有机化合物和重金属的诱导响应快速灵敏,表现出强的启动子功能,能启动LuxCDABE基因表达,使荧光酶活性提高。借助荧光敏活性的变化,可检测系统中一些有机物和重金属的存在,作为一种灵敏的环境污染生物检测工具。     

Cadaverine induces closing of E. coli porins.

We have used the electrophysiological technique of patch-clamp to study the modulation of Escherichia coli porins by cadaverine. Porin channels typically have a very high probability to be open, and were not known to be inhibited by specific compounds until the present study. Experiments performed on patches of outer membrane reconstituted in liposomes reveal that cadaverine applied to the periplasmic side increases the frequency of channel closures in a concentration-dependent fashion, and thereby decreases the total amount of ion flux through a porin-containing membrane. The positive charge on cadaverine is important for inhibition, because the effect is relieved at higher pH where fewer polyamine molecules are charged. Modulation is observed only at negative pipet voltages, and therefore confers voltage dependence to porin activity. Cadaverine increases the number and duration of cooperative closures of more than one channel, suggesting that it does not merely block the pore but exerts its kinetic effect allosterically. As a biological assay of porin inhibition, E. coli behavior in chemotaxis swarm plates was tested and found to be impaired in the presence of cadaverine. Polyamines are naturally found associated with the outer membrane of E.coli, but are lost upon fractionation. We postulate that cadaverine might be a natural regulator of porin activity.     

Sulfite suppresses the mutagenic property of coffee

The mutagenicity of instant and freshly brewed coffee on Salmonella typhimurium TA100 and TA98 without S9 mix was inactivated by sodium sulfite. Sulfite ion at a dose of 200 ppm almost completely inactivated the mutagenicity of coffee made in the ordinary way (5-15 mg dry weight/ml). Sodium bisulfite and potassium metabisulfite had similar effects. On the contrary, L-ascorbic acid enhanced the mutagenicity of coffee. Sodium sulfite also inactivated the phage-inducing activity of coffee in inductest III. Sodium sulfite completely suppressed the mutagenicities of 1,2-dicarbonyls, namely diacetyl and glyoxal. Diacetyl is present in coffee, beer, butter and other foods and drinks. Because sodium sulfite, sodium bisulfite and potassium metabisulfite are widely used as food additives, they should be useful in reducing the levels of mutagens in foods.     

Heterologous expression of barley and wheat oxalate oxidase in an E. coli trxB gor double mutant

Oxalate oxidase catalyses the degradation of oxalic acid to carbon dioxide and hydrogen peroxide and is of commercial importance for clinical analyses of oxalate in biological samples. Novel potential applications for oxalate oxidase include the prevention of the formation of calcium oxalate incrusts in pulp and paper manufacture and rapid determination of oxalic acid in process waters. The potential in using oxalate-degrading enzymes in industrial processes increases the interest in finding systems for heterologous expression. Oxalate oxidase from barley is a secreted multimeric glycosylated manganese-containing enzyme with several disulfide bridges, which have been found to be essential for the catalytic activity. Attempts to achieve expression of active heterologous oxalate oxidase in bacteria have up to now met little success. In this study, one oxalate-oxidase-encoding cDNA from barley and two from wheat were cloned and tested with regard to expression in Escherichia coli. The results suggest that the selection of a novel commercially available E. coli host strain, which has the ability to form disulfide bridges in heterologous proteins expressed in its cytoplasm, was important for successful expression. Although a considerable part of the heterologous protein was produced in an insoluble and inactive form, this strain, E. coli Origami B(DE3), in addition yielded soluble and active barley and wheat oxalate oxidase. One of the wheat cDNAs, Ta(M)OXO1, gave three-fold higher activity than the barley cDNA, Hv(H)OXO1, while the other wheat cDNA, Ta(M)OXO2, gave no detectable activity. This indicates that the choice of cDNA was also critical despite the high identity between the cDNAs and the encoded polypeptides (88-89% on the nucleotide level and 88-92% on the amino-acid level). Gel filtration of cell extracts containing heterologous barley and wheat oxalate oxidase resulted in an increase in the activity. This indicates that low molecular weight inhibitory compounds were present in the E. coli lysates but could be removed by the introduction of a purification step.     

丙型肝炎病毒核心蛋白在大肠杆菌中的表达

目的：建立稳定表达丙型肝炎病毒(HCV)核心蛋白的原核表达系统,获得高产量的纯化核心蛋白。方法：应用多聚酶链反应(PCR),以HCV—H株全长cDNA序列为模板,扩增获得核心区基因片段,克隆入原核表达载体pBVIL1,构建原核表达载体pBVIL1-C,转化HB101宿主菌,通过温度诱导表达核心蛋白。结果：扩增得到目的基因长度为573bp,构建pBVIL1-C表达载体,在HB101宿主菌中通过温度诱导获得稳定表达,表达蛋白占菌体总蛋白含量的21％,Western—Blot检测证实表达产物可与HCV患者阳性血清发生特异性结合反应。结论：HCV核心蛋白可在大肠杆菌中获得高表达并具有良好的反应原性。     

Characteristics of E.coli O157 strains isolated in Poland from clinical material and food samples

E. coli belonging to the O157 serological group are among the organisms isolated most frequently out of all the so called entero-hemorrhagic E. coli strains (EHEC). Since several years they have been isolated also in Poland. The purpose of the present study was determination on selected phenotypic and genotypic properties of E. coli O157 strains isolated in our country from clinical material samples and from food. The serotype of the strains was determined, together with the following properties regarded as pathogenicity markers of verotoxic E. coli strains such as absence of beta-glucuronidase activity and sorbitol fermentation ability, as well as production of verotoxins SLT I and/or SLT II and entero-hemolysin. Besides that, by the PCR method the fragments of the genes coding for verotoxins, intimin and enterohaemolysin were amplified. The products of PCR were analysed by the restriction enzyme analysis (RFLP). All verotoxic E. coli O157 strains isolated in Poland were analysed by the pulsed field gel electrophoresis of genomic DNA (PFGE). The studied group comprised E. coli O157 strains, among them 40 strains were isolated from human faeces and 5 from food. The remaining strains were the reference E. coli O157:H7 EDL 933 and G 5244 strains and strains from NIH collection. The obtained results showed that the tested strains were a very varying population. 21 of them (all isolated from food, 11 from faeces and 5 reference strains) belonged to serotype O157:H7, five were not peritrichous O157:NM and the remaining ones had other ciliary antigen than H7. All strains isolated from food, reference strains and only 3 O157:NM strains isolated from humans were verotoxic. The strains from food and two reference strains produced only SLT II, 2 of 3 strains isolated from humans and one reference strain also produced only SLT II and the other produced both verotoxins. Apart from these 13 verotoxic strains all remaining strains caused sorbitol fermentation.     

Nonspecific resistance in germ-free and E. coli monocontaminated miniature piglets]

Phagocytic activity of leukocytes, as well as the complement, properdin, and lysozyme levels in the blood serum of miniature piglets, germfree and monocontaminated with E. coli 055 and E. coli 083, were studied. E. coli 055 phagocytosis was decreased in the presence of autologous serum and complement and increased under the effect of specific opsonins (antibodies to E. coli 055). Complement, properdin, and lysozyme levels were decreased in the germfree, in comparison with conventional animals. In the E. coli contaminated piglets properdin and complement production was stimulated most, and lysozyme formation--less. No antibodies to E. coli 055 were revealed in monocontaminated piglets. The highest lysozyme levels were found in the ex-germfree animals, this indicating the participation of factors other than E. coli contamination in lysozyme stimulation. It is concluded that microbial contamination played an important role in the development of cellular and humoral factors of the organism resistance.