Design and screening of in vivo expressed antimicrobial peptide library

The genetic library of -helical amphipathic peptides, 20 amino acid long, was designed and expressed under the T7 promoter in the E. coli JM109(DE3) and BL21 (DE3). Clones that inhibited the growth of the host cell were screened by the relative size of colonies on the plates. Clones which strongly inhibited growth of Escherichia coli JM109(DE3) were further selected. The method developed in this study is useful for the structure activity relationship study of antimicrobial peptides.     

The effect of nickel on the growth, photosynthesis, and nitrogenase activity of Anabaena inaequalis.

Anabaena inaequalis was sensitive to nickel ion in the order of decreasing sensitivity of growth, photosynthesis, and acetylene reduction. At a culture density of 9 x 10(4) cells per millilitre, growth after 12 days was completely inhibited by 0.125 ppm (microgram/mL) Ni2+. Nickel caused the increase of both the lag phase of growth and the culture doubling time, and caused the retardation phase to be sooner. Photosynthesis and acetylene reduction were completely inhibited by 10 and 20 ppm Ni2+, respectively, at a cell concentration of 1.3 x 10(6) cells per millilitre. Preincubation for 24 h in the presence of nickel ion significantly increased the sensitivity of photosynthesis and acetylene reduction. Under these conditions acetylene reduction was more sensitive than photosynthesis. Nickel ion reduced culture growth by 35% at a level of 0.05 ppm and inhibited that culture's acetylene-reducing ability by 29% while leaving photosynthesis unaffected. Nickel caused some damage to filament apical cells and induced pigment bleaching in aged cultures. Nickel toxicity was proposed to be due to poisoning of intracellular enzyme systems by nickel ions.     

Combined action of micafungin, a new echinocandin, and human phagocytes for antifungal activity against Aspergillus fumigatus

Micafungin, a new echinocandin, inhibits fungal cell wall beta-glucan synthesis. We postulated micafungin and host phagocytic cells could act together in damaging fungi. Using the metabolic XTT assay, micafungin alone (0.01 and 0.10 microg/ml) inhibited Aspergillus fumigatus germlings by 48% and 61%, respectively. Polymorphonuclear neutrophils (PMNs) inhibited germlings by 53%. Micafungin at 0.01 or 0.10 microg/ml and PMNs resulted in additive inhibition, 82% and 99%, respectively. Monocyte-derived macrophage (MDM) monolayers inhibited germling growth by 66%; micafungin (0.01 or 0.10 microg/ml) alone inhibited by 32% and 42%, respectively. MDMs and micafungin (0.01 or 0.10 microg/ml) caused an additive inhibition of growth, 85% and 95%, respectively. Hyphae were generated by incubation of conidia for 24 h with or without micafungin. PMNs alone, added to hyphae, inhibited growth by 19% in the subsequent 20 h. Hyphae generated in the presence of micafungin (0.10 microg/ml) and subsequently cultured with micafungin for 24 h inhibited growth by 64%. PMNs plus micafungin resulted in 82% inhibition. Monocytes alone inhibited hyphal growth by only 5%. Hyphae produced in the presence of micafungin (0.01 microg/ml) and incubated again with micafungin for 24 h inhibited growth by 47%; combination with monocytes resulted in 62% inhibition. These data indicate that micafungin inhibits growth of tissue forms of A. fumigatus, and phagocytes and micafungin together have an additive effect. These findings support the thesis that the greater efficacy of micafungin in vivo compared with in vitro could be due to combined effect of phagocytic cells and micafungin.     

Inhibition of growth and of thymidine incorporation into DNA in Tetrahymena by chlorpromazine, pimozide and penfluridol

The antipsychotic drugs chlorpromazine, pimozide, and penfluridol caused a 50% inhibition of growth of Tetrahymena at concentrations of 4.5, 5.5, and 1.5 microM, respectively. The degree of growth inhibition was dependent on the concentration of cells; higher drug concentrations were needed to produce inhibition of denser cell cultures. Binding studies with penfluridol showed that 50% growth inhibition resulted when approximately 50 mumoles of drug were bound per 10(6) cells. A 20-min preincubation of cells with chlorpromazine (14.7 microM) inhibited DNA synthesis by 46%, and with penfluridol (4 microM) DNA synthesis was inhibited by 27%. The incorporation of labeled thymidine into the thymidine triphosphate pool was inhibited by chlorpromazine but not by penfluridol, indicating that the drugs produce their growth inhibitory effects by different mechanisms. TDP kinase activity was demonstrated in a particle-free fraction of the cells. Its enzymatic activity was not affected by added chlorpromazine, penfluridol, or calmodulin, suggesting that inhibition of DNA synthesis by these drugs may be a consequence of growth inhibition.     

Light effects in yeast: inhibition by visible light of growth and transport in Saccharomyces cerevisiae grown at low temperatures.

Growth rate, sugar transport, and amino acid transport of yeast cells grown at 12 degrees C were inhibited by cool-white fluorescent light. At light intensities below 1,250 lx, growth and membrane transport were only slightly inhibited. Above 1,250 lx, there was increasing inhibition of both processes. Transport of histidine was completely inhibited after 3 to 5 days in cultures grown at 12 degrees C under 3,500-lx illumination. Cells grown at 20 degrees C were not inhibited by light intensities that caused complete loss of viability and membrane transport activity in cells grown at 12 degrees C.     

A proposed sequence of hormones controlling the induction of luteal 20alpha-hydroxy steroid dehydrogenase and progesterone withdrawal in the late-pregnant rat.

1. The previously reported induction of luteal 20alpha-hydroxy steroid dehydrogenase by administration of aminoglutethimide to late-pregnant rats was shown to be unaffected by prior removal of the foetuses. Aminoglutethimide therefore does not act via the foetuses in this context. 2. The ability of injected oestrogen to prevent the above induction was lost by delaying the injection for 12h after aminoglutethimide, although the increase in enzyme activity begins only after 24h. 3. Induction of 20alpha-hydroxy steroid dehydrogenase by foetoplacental removal on day 18 of pregnancy was inhibited by human choriogonadotropin, lutropin (luteinizing hormone) and pregnant-mare serum gonadotropin, but not by somatotropin (growth hormone), thyrotropin or follitropin (follicle-stimulating hormone) 4. Indomethacin blocked the normal induction of 20alpha-hydroxy steroid dehydrogenase in late pregnancy and that caused by aminoglutethimide. It partially blocked that caused by human choriogonadotropin given on days 19-20 and that caused by 2-bromo-alpha-ergocryptine on days 5-6, but failed to block that caused by human choriogonadotropin on days 15-16 or by foetoplacental removal on day 18 of pregnancy. 5. These findings, and the control of progesterone synthesis in late pregnancy, are interpreted in terms of a sequence of hormonal or enzymic syntheses, each of which is inhibited by the product of the preceding synthesis.     

Assessing the pathogenic effect ofFusarium, Geosmithia andOphiostoma fungi from broad-leaved trees

Phytopathogenic effect of Geosmithia pallida, G. langdonii, Ophiostoma grandicarpum, O. querci, two isolates of O. piceae, and two isolates of Fusarium solani was compared using plant growth test (stem and root length of garden cress plants seeded on mycelium-covered potato carrot agar); Ophiostoma spp. and F. solani were isolated from oak, Geosmithia spp. from galleries of Scolytus intricatus on beech. All fungi inhibited more the root elongation than that of stems. F. solani led to plant collapse after briefly stimulating the growth of stem and in one case also root. G. langdonii inhibited stem and root growth to 20% and led to plant collapse. G. pallida inhibited root growth to 25% whereas stem growth was almost unimpaired. Ophiostoma spp. reduced stem growth to approximately 60-80% and root growth to 25-60%. O. piceae and O. querci caused plant collapse after 15-20 d.     

The effect of carbon sources and mononucleotides on the production of extracellular alkaline phosphatase by Bacillus intermedius

The biosynthesis of extracellular alkaline phosphatase in the streptomycin-resistant strains Bacillus intermedius S3-19 and S7 in the presence in the medium of 5'-nucleoside monophosphates and different sources of carbon--glucose, sodium pyruvate, sodium lactate, or glycerol--was studied. It was established that, in the presence of mononucleotides, the content of extracellular alkaline phosphatase in both strains increased; the maximal effect was caused by 5'-AMP at a concentration of 20 micrograms/ml. In medium with a low orthophosphate content, where active biosynthesis of alkaline phosphatase occurred, 1% glucose and 0.5% pyruvate stimulated this process 2.5-4 times, and 2% sodium lactate and sodium pyruvate, on the contrary, inhibited it by 20-40%. Analysis of the dynamics of growth and accumulation of extracellular phosphatase in the presence of different sources of carbon in the medium gives evidence of an interrelationship between the biosynthesis of alkaline phosphatase and carbon metabolism in Bacillus intermedius.     

Inhibition of Growth and of Thymidine Incorporation into DNA in Tetrahymena by Chlorpromazine,Pimozide and Penfluridol1

The antipsychotic drugs chlorpromazine, pimozide, and penfluridol caused a 50% inhibition of growth of Tetrahymena at concentrations of 4.5, 5.5, and 1.5 μM, respectively. The degree of growth inhibition was dependent on the concentration of cells; higher drug concentrations were needed to produce inhibition of denser cell cultures. Binding studies with penfluridol showed that 50% growth inhibition resulted when approximately 50 μmoles of drug were bound per 10⁶ cells. A 20-min preincubation of cells with chlorpromazine (14.7 μM) inhibited DNA synthesis by 46%, and with penfluridol (4 μM) DNA synthesis was inhibited by 27%. The incorporation of labeled thymidine into the thymidine triphosphate pool was inhibited by chlorpromazine but not by penfluridol, indicating that the drugs produce their growth inhibitory effects by different mechanisms. TDP kinase activity was demonstrated in a particle-free fraction of the cells. Its enzymatic activity was not affected by added chlorpromazine, penfluridol, or calmodulin, suggesting that inhibition of DNA synthesis by these drugs may be a consequence of growth inhibition.     

Effect of sulphite on superoxide dismutase activity in the mycorrhizal fungus Rhizopogon roseolus

 Sulphite at a concentration of 1 mM did not strongly affect the growth of mycelium. Higher concentrations of 5–20 mM almost completely inhibited the growth of mycelium and superoxide dismutase (SOD) [EC.1.15.1.1.] activity. The activity of this enzyme was not detectable on polyacrylamide gels. The lack of induction of SOD and the resulting oxidative stress may in part be responsible for the growth inhibition caused by high concentrations of sulphite. Accepted: 20 July 1995     

Antagonistic effect of fluorescent pseudomonads against Macrophomina phaseolina that causes charcoal rot of groundnut

Maximum colony growth inhibition was observed due to Pseudomonas PS2 (74%) as compared to PS1 (71%) on trypticase soy agar (TSM) plates after 5 days of incubation. Light and scanning electron microscopic examination showed hyphal coiling, vacuolation, coagulation and granulation of cytoplasm resulting in lysis of hyphae of M. phaseolina by pseudomonads. Cell free culture filtrates of strains PS1 and PS2 restricted the growth of mycelium of M. phaseolina. PS1 and PS2 caused maximum colony growth inhibition by 57 and 61% respectively at 20% concentration of culture filtrate after 4 days of incubation. Volatile substances produced by PS1 and PS2 also inhibited the colony growth of M. phaseolina by 25 and 32%, respectively. Inhibitory effect of volatile substances, however, decreased with advancing in incubation period. Colony growth of M. phaseolina was significantly decreased by PS1 and PS2 as compared to control both in iron- sufficient and iron-deficient conditions. PS2 showed higher antagonistic activity than PS1, as evidenced by pronounced colony growth inhibition.     

Redistribution of a rat sperm epididymal glycoprotein after in vitro and in vivo capacitation.

Rat epididymal glycoprotein DE (37 kDa) associates with the sperm surface during maturation and is localized over the dorsal region of the acrosome. In the present study we examine, by indirect immunofluorescence, the localization of DE after in vitro and in vivo capacitation. While 49% of sperm capacitated in vitro for 5 hr still presented fluorescence over the dorsal region, 51% showed labeling distributed over a domain that corresponds to the equatorial segment of the sperm head. This change in the localization of fluorescence was not associated with sperm deterioration or death and increased gradually as a function of capacitation time, reaching the maximum at 5 hr. The presence of labeling over the equatorial segment results from protein migration and cannot be induced by permeabilization, proteinase, or high ionic strength treatments. The omission of Ca2+ from the standard capacitation medium inhibited the relocalization of DE, and incubation with Ca2+ ionophore A23187 for induction of the acrosome reaction (AR) significantly raised the percentage of cells with DE localized over the equatorial region. Finally, while free and cumulus-associated spermatozoa recovered from the oviducts of in vivo inseminated females presented 15% and 21% of cells with redistribution respectively, all perivitelline (acrosome reacted) spermatozoa showed DE over the equatorial segment. These results indicate that epididymal protein DE migrates to the equatorial segment under in vitro and in vivo capacitating conditions and suggest a possible association between the redistribution of DE and the occurrence of the AR.     

Characterization of 5-aminolevulinate synthase from Agrobacterium radiobacter,screening new inhibitors for 5-aminolevulinate dehydratase from Escherichia coli and their potential use for high 5-aminolevulinate production

The hemA gene encoding 5-aminolevulinate synthase (ALAS) from Agrobacterium radiobacter zju-0121 showed 92.6% homology with that from A. radiobacter ATCC4718 and contained several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was used as the host to construct an efficient recombinant strain. And the encoded protein was over-expressed as fusion protein and was purified by affinity purification on Ni-NTA agarose and by gel filtration chromatography on Sephadex G-25 Medium resin. The recombinant protein was partly characterized, and d-glucose, d-fructose, d-xylose, d-mannose, l-arabinose, d-galactose, lactose, sucrose and maltose were detected to have no distinct inhibition on this recombinant ALAS. Meanwhile, 20 mM d-glucose or d-xylose inhibited about 20% activity of ALA dehydratase (ALAD) from Escherichia coli Rosetta(DE3). Combining d-xylose as a new inhibitor for ALAD with d-glucose in fed-batch culture and based on the optimal culture system using Rosetta(DE3)/pET28a-hemA, the yield of ALA achieved was 7.3 g/l (56 mM) under the appropriate conditions in the fermenter.     

Isozymes of alpha-amylase in the porcine pancreas: population distribution

1. Three isozymes of pancreatic alpha-amylase, PPA 1, PPA 2, and PPA 3, were observed in a porcine population of 50 animals. 2. Isozyme PPA 2 was common to each pancreas. 3. Three phenotypic patterns were described as: (A) consisting of PPA 2 alone (20%); (B) consisting of PPA 1 and PPA 2 (78%); and (C) consisting of all three forms (2%). 4. Amylase isozymes were separated by anion exchange chromatography using DE53. 5. Individual isozymes corresponded to one of the three isozymes found in pancreatin. 6. Individual isozymes were inhibited equally by an amylase inhibitor from wheat. 7. Differences in amylase isozymes were attributed to genetically controlled mechanisms and not to artifacts of isolation.     

Benefits and Risks of the Hormetic Effects of Dietary Isothiocyanates on Cancer Prevention

The isothiocyanate (ITC) sulforaphane (SFN) was shown at low levels (1–5 µM) to promote cell proliferation to 120–143% of the controls in a number of human cell lines, whilst at high levels (10–40 µM) it inhibited such cell proliferation. Similar dose responses were observed for cell migration, i.e. SFN at 2.5 µM increased cell migration in bladder cancer T24 cells to 128% whilst high levels inhibited cell migration. This hormetic action was also found in an angiogenesis assay where SFN at 2.5 µM promoted endothelial tube formation (118% of the control), whereas at 10–20 µM it caused significant inhibition. The precise mechanism by which SFN influences promotion of cell growth and migration is not known, but probably involves activation of autophagy since an autophagy inhibitor, 3-methyladenine, abolished the effect of SFN on cell migration. Moreover, low doses of SFN offered a protective effect against free-radical mediated cell death, an effect that was enhanced by co-treatment with selenium. These results suggest that SFN may either prevent or promote tumour cell growth depending on the dose and the nature of the target cells. In normal cells, the promotion of cell growth may be of benefit, but in transformed or cancer cells it may be an undesirable risk factor. In summary, ITCs have a biphasic effect on cell growth and migration. The benefits and risks of ITCs are not only determined by the doses, but are affected by interactions with Se and the measured endpoint.     

Growth inhibition of Monodus subterraneus by free fatty acids

Monodus subterraneus is a microalga, which is known for its high eicosapentaenoic acid (EPA; 20:5omega3) content. To produce EPA commercially, high volumetric productivities of microalgae are required. These high productivities can be reached in flat panel photobioreactors with small optical paths that have to be operated at high cell densities (>10 g/L). However, at these cell densities a reduction of productivity is observed. This growth inhibition is probably caused by growth inhibitors released by the microalgae, which have been suggested to be fatty acids. Our aim was to investigate if free fatty acids produced by M. subterraneus inhibited growth of this species. Therefore a bioassay was developed and saturated, unsaturated and poly-unsaturated fatty acids occurring in Monodus were tested on their growth inhibiting properties. Growth of M. subterraneus was completely inhibited at a saturated concentration (96 microM) of palmitoleic acid (16:1omega7). But, the saturated fatty acid palmitic acid (16:0) and the mono-saturated oleic acid (18:1omega9) were much stronger inhibitors. Growth was inhibited for 50% already at concentrations of 0.4 microM 16:0 and 3 microM 18:1omega9, respectively. These fatty acids probably cause the growth inhibition in high cell density cultures of M. subterraneus.     

右旋糖酐蔗糖酶工程菌株的构建及其培养条件的研究

[目的]右旋糖酐蔗糖酶是一种以蔗糖为底物,催化转移D-葡萄糖基生成α-葡聚糖或低聚糖的葡萄糖基转移酶.[方法]利用PCR扩增技术,将已获得的右旋糖酐蔗糖酶基因dexYG亚克隆到表达载体PET28a( )上,转化E.coli BL21(DE3),经过卡那霉素抗性筛选和酶切验证后,得到右旋糖酐蔗糖酶工程菌株BL21(DE3)/pET28-dexYG.[结果]经IPTG诱导该基因在E.coli BL21(DE3)中能有效表达,在诱导过程中菌体生长受到抑制.通过对培养时间、IPTG浓度、培养温度、菌浓(OD600)和pH值等产酶因素的优化考察,得到最佳培养条件为:培养时间5h、IPTG浓度0.5mmol/L、25℃、OD600值1.0和pH6.0.酶活力由最初的5.39U/mL提高到35.62U/mL,其中pH值对产酶活力影响最大,在pH6.0时的最高产酶活力是LB原始pH条件下最高酶活的3.5倍,并且pH值也是导致在诱导后期酶活迅速下降的主要原因之一.[结论]酶的表达和酶活的研究结果表明,构建的工程菌株能够异源高效表达右旋糖酐蔗糖酶,并且表现出较高的酶活力.     

Inhibition of T-2 toxin production on high-moisture corn kernels by modified atmospheres

The fungus Fusarium sporotrichioides, capable of producing T-2 toxin (T-2), was grown on irradiated corn kernels remoistened to 22% and kept in atmospheres of different CO2-O2 combinations. The production of T-2 was totally inhibited under 60% CO2-20% O2, whereas only trace amounts were detected when the gas combination was 40% CO2-5% O2. Under all other combinations tested, the amount of T-2 produced was reduced by 25 to 50% as compared with the control. Fungal growth was not inhibited by any of the gas mixtures examined, and the growth rate (measured by direct plating, dilution method, and CO2 production) was almost identical to that in grains kept under air. It is concluded that although F. sporotrichioides is tolerant to high CO2 levels, T-2 formation on corn can be inhibited by CO2 concentrations less than that required to inhibit fungal growth.     

Ochratoxin A production by Aspergillus ochraceus Wilhelm grown under controlled atmospheres.

When Aspergillus ochraceus NRRL 3174 was grown under controlled atmospheres with 1 and 5% O2 and without CO2, the amount of ochratoxin produced was the same as that produced by the control colonies. Increasing the O2 level up to 40% reduced ochratoxin production by 75%, whereas at 60% O2, ochratoxin production was enhanced. In atmospheres enriched with 10 or 20% CO2, ochratoxin production was reduced when O2 concentrations were below 20% and enhanced when the O2 concentration was 40 or 60% O2. Ochratoxin production was completely inhibited at 30% CO2 and above, regardless of the O2 level. Colony growth was partially inhibited at 60% CO2, and no growth occurred at 80% CO2 or above. However, when colonies inhibited by 60% CO2 or above were subsequently exposed to air, radial growth, number of sclerotia formed, and the amount of ochratoxin produced were the same as in the control colonies. The results indicate that A. ochraceus is tolerant to CO2 concentrations higher than those required to control storage insects.     

Inhibition of T-2 toxin production on high-moisture corn kernels by modified atmospheres.

The fungus Fusarium sporotrichioides, capable of producing T-2 toxin (T-2), was grown on irradiated corn kernels remoistened to 22% and kept in atmospheres of different CO2-O2 combinations. The production of T-2 was totally inhibited under 60% CO2-20% O2, whereas only trace amounts were detected when the gas combination was 40% CO2-5% O2. Under all other combinations tested, the amount of T-2 produced was reduced by 25 to 50% as compared with the control. Fungal growth was not inhibited by any of the gas mixtures examined, and the growth rate (measured by direct plating, dilution method, and CO2 production) was almost identical to that in grains kept under air. It is concluded that although F. sporotrichioides is tolerant to high CO2 levels, T-2 formation on corn can be inhibited by CO2 concentrations less than that required to inhibit fungal growth.