Denitrification of PVA-immobilized denitrifying photosynthetic bacterium,Rhodobacter sphaeroides

A newly isolated denitrifying strain, Rhodobacter sphaeroides NII2 was immobilized in polyvinyl alcohol (PVA) gel, and the properties of the cells in the gel were examined. The immobilized cells had low or almost no denitrification activity, but the cells were activated by incubation in light with culture medium for denitrification containing 0.5% nitrate and no other nitrogen source. Cells grown in the dark were activated by incubation at an earlier stage and to a higher rate than the light-grown cells. The activation was markedly enhanced in the PVA gel with a low cell concentration. The immobilized cells consumed nitrate with a temporary accumulation of NO₂ and evolved nitrogen gas. The immobilized cells could use various organic compounds as electron donors for denitrification. Thus, the immobilized cells were applied to a continuous treatment of synthetic wastewater using an aparatus devised by this laboratory. The results showed an efficient removal of NO₃-N from the test water.     

Light dependence of sexual agglutinability in Chlamydomonas eugametos

The mating activity of mating-type plus gametes of Chlamydomonas eugametos depends on light. Cells lost their ability to agglutinate with mating-type minus gametes after a dark period of 30 min. They regained their agglutinability after 10 min exposure to light. Other mating reactions, such as tipping and flagellar tip activation, were not dependent upon light. Since cycloheximide and tunicamycin did not affect the light-induced activation of flagellar agglutinability, no protein synthesis or glycosylation is involved in this process. Equal amounts of biologically active agglutination factor could be extracted from cells placed either in light or in darkness. A minor portion of the active material was found to be located on the flagellar surface of illuminated cells. No active material was found on the flagellar surface of dark-exposed cells, whereas their cell bodies contained the same amount of active material as the cell bodies of illuminated cells. Since a light-induced flow of agglutination factors from the cell body to the flagella could not be detected and dark-exposed cells could be slightly activated by amputation or fixation by glutaraldehyde, we propose that light affects flagellar agglutinability by an in-situ modification of the agglutination factor on the flagella. When mt ⁺ and mt ^- strains were crossed and the progeny examined for light-sensitivity, it was apparent that this phenomenon is not mating type-linked.Abbreviations and symbols FTA flagellar tip activation - mt ^+/- mating type plus or minus - WGA wheat-germ agglutinin     

Steroid transformation by living cells immobilized in calcium alginate

Summary Whole cells of Arthrobacter simplex were immobilized in a living state in calcium alginate gel. The bacteria showed steroid-¹-dehydrogenase activity and the production of prednisolone from cortisol was investigated. The ¹-dehydrogenase activity of the immobilized cells could be increased about ten-fold by incubation in nutrient media (e.g., containing 0.5% peptone abd 0.2% glucose). The reason for this activation was examined and it was found that the immobilized cells were capable of multiplying when supplied with nutrients. Furthermore, provided that an inducer, cortisol, was present, the steroid-¹-dehydrogenase activity increased in proportion to the increase in the number of cells and it was thus concluded that microbial growth was the cause of activation.Experiments on repeated, batch-wise pseudocrystallofermentation with immobilized A. simplex cells also showed that immobilized cells could be advantageously used for pseudocrystallofermentation of steroids.     

Biodegradation of dichloromethane by the polyvinyl alcohol-immobilized methylotrophic bacterium Ralstonia metallidurans PD11

A dichloromethane (DCM)-degrading bacterium, Ralstonia metallidurans PD11 NBRC 101272, was immobilized in a polyvinyl alcohol (PVA) gel to use in a bioreactor for DCM treatment. After 4-month incubation of PVA gel beads with R. metallidurans PD11 and DCM in a mineral salt medium, the cells were tightly packed in the mesh of the gel. Forty beads of the gel in 10 ml of a batch system model showed effective activity degrading 500 and 1,000 mg l⁻¹ DCM within 2 and 3 h, respectively. Although reduction of pH due to accumulation of chloride ion liberated from DCM decreased the activity, it was recovered by adjustment to neutral pH. The activity of the immobilized cells was not affected by addition of nutrients which were preferentially utilized by R. metallidurans PD11, unlike the activity of the free-living cells. A continuous flow system with a column was more effective for rapid degradation of DCM. Thus, the PVA gel–immobilized cell of R. metallidurans PD11 is thought to be a prospective candidate to develop the bioreactor.     

A permanent fish cell line (EPC) for genotoxicity testing of marine sediments with the comet assay

Genotoxicity and cytotoxicity were evaluated in an in vitro system with a permanent cell line Epithelioma papulosum cyprini (EPC) derived from a skin tumour of carp (Cyprinus carpio L.). EPC cells were exposed to different concentrations of organic sediment extracts from the North Sea for 24 h. After incubation the cells were analysed for viability and DNA strand breaks with the comet assay or single cell gel electrophoresis (SCGE). The results confirm the sensitivity of this assay. Out of 10 marine sediment samples from the North Sea, 9 showed a dose-dependent genotoxic effect. The EC₅₀ of sediment extracts ranged from 7 to 307 mg sediment dry weight/ml assay volume. Hepatic microsomal enzymes from dab (Limanda limanda L.) was proposed for enzymatic activation of benzo[a]pyrene (BAP) or sediment extracts, respectively. The suitability of this in vitro test system for assessing genotoxic and cytotoxic effects of marine sediment extracts on EPC cells could be demonstrated.     

Renouvellement des protéines et effet spécifique de la kinétine sur des cultures de cellules de Tabac

Determination and fractionation of proteins of tobacco cell suspensions requireing kinetin for cell division. — Cell suspensions either in stationary phase without kinetin in the medium or dividing in the presence of this factor have been compared. It was found a) that the specific rates of protein synthesis and protein degradation were not changed by the addition of kinetin during the early period of growth. A quantitative change ocurred only after the first generation period, b) Soluble proteins of these cells were mapped by polyacrylamide gel electrophoresis. The observed protein patterns were very similar as well as the patterns or radioactivity incorporated into the same proteins during the incubation period of the cells. However, a small number of specific discrepancies appeared in the pattern of cells growing in the presence of kinetin matched to the patterns of stationary cells. At least one specific difference in these patterns could be observed before the first cell division occurred.     

Steroid transformation by activated living immobilized Arthrobacter simplex cells

A preparation of living Arthrobacter simplex cells immobilized in polyacrylamide gel, which showed steroid-Δ¹-dehydrogenase activity, was studied. The entrapped microorganisms catalyzed the transformation of cortisol to prednisolone and this reaction was followed spectrophotometrically or with the aid of thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC). About 40% of the original activity found with free bacteria was retained after immobilization. The steroid dehydrogenase activity of polyacrylamide-entrapped A. simplex could be raised to a minor extent in alcoholic solvents or by addition of a cofactor such as menadione. On incubation in various nutrient media, on the other hand, the activity could be increased considerablyl, usually 7–10 times. Possible causes for the observed increase in activity have been investigated, and microbial growth of the original entrapped microorganisms appears to be the major reason. Frozen activated preparations of immobilized A. simplex showed only a small loss of activity on storage for at least four months. A semicontinuous batch wise operation with immobilized A. simplex in different nutrient media was carried out. At the end of the experiment the steroid transformation capacity was 0.5 g steroid per day per g gel (wet weight).     

枯草芽孢杆菌刺激小鼠肠上皮细胞分泌的IL-33在活化树突状细胞中发挥重要作用

【目的】枯草芽孢杆菌能有效诱导肠道黏膜免疫应答,但活化黏膜下树突状细胞(DC)的具体机制不完全清楚。【方法】本研究首先用不同浓度枯草芽孢杆菌刺激小鼠肠上皮CMT93细胞,用荧光定量PCR和ELISA检测细胞因子表达水平,然后将枯草芽孢杆菌刺激细胞的培养上清与小鼠骨髓源树突状细胞(BMDC)进行共孵育,用流式细胞术检测BMDC活化标志,最后用RNA干扰技术证明IL-33在活化BMDC中的作用。【结果】枯草芽孢杆菌能显著刺激CMT93细胞分泌IL-6、IL-33和IFN-γ等细胞因子,对刺激IL-33表达呈现剂量依赖性;枯草芽孢杆菌刺激CMT93细胞产生的细胞因子能活化BMDC,在RNA干扰IL-33基因表达和枯草芽孢杆菌刺激后,CMT93细胞培养上清活化BMDC的能力显著降低。【结论】本研究结果表明枯草芽孢杆菌刺激肠上皮细胞产生的IL-33在BMDC活化中具有重要作用。     

Production of β-galactosidase by Kluyveromyces marxianus MTCC 1388 using whey and effect of four different methods of enzyme extraction on β-galactosidase activity

Whey containing 4.4% (w/v) lactose was inoculated with Kluyveromyces marxianus MTCC 1389 for carrying out studies related to β-galactosidase production. β-galactosidase activity was found to be maximum after 30 h and further incubation resulted in decline in activity. The maximum cell biomass of 2.54 mg mL⁻¹ was observed after 36 h of incubation. Lactose concentration dropped drastically to 0.04 % from 4.40% after 36 h of incubation. Out of the four methods tested for extraction of enzyme, SDS — Chlorofom method was found to be best followed by Toluene — Acetone, sonication and homogenization with glass beads in that order. It could be concluded through this study that SDS — Chloroform is cheap and simple method for enzyme extraction from Kluyveromyces cells, which resulted in higher enzyme activity as compared to the activity observed using the remaining extraction methods. The study could also establish that whey could effectively be utilized for β-galactosidase production thus alleviating water pollution problems caused due to its disposal into the water streams.     

Generation of cell surface-bound plasmin by cell-associated urokinase-type or secreted tissue-type plasminogen activator: A key event in melanoma cell invasiveness in vitro

Recently, we have shown that plasminogen activators (PAs) of both types, urokinase-type (uPA) as well as tissue-type (tPA), are involved in the in vitro invasiveness of human melanoma cells. The present study is focused on the generation and importance of cell surface-bound plasmin in this process. The human melanoma cell lines MelJuso and MeWo expressed plasminogen binding sites on the cell surface. Plasminogen binding was saturable and not species-specific, since human and bovine plasminogen bound to the cells with comparable efficiency. The activation of the proenzyme plasminogen bound on MelJuso cells, which expressed surface-associated uPA activity, occurred almost synchronously with binding to the cell surface. Removal of cell-associated uPA considerably reduced plasmin generation on these cells. In contrast, plasminogen activation on Me Wo cells, which secreted tPA into the culture supernatant and which were devoid of surface-associated PA activity, was by far less effective. The efficiency of the activation process could be increased by addition of exogenous tPA. With both cell lines, plasmin generation on the cell surface was suppressed by inhibitory monoclonal antibodies specific for the respective PA type. Selective inhibition of cell surface-associated plasmin by preincubating the cells with an inhibitory monoclonal antibody or with aprotinin, as well as removal of plasmin from the cell surface, led to a significant decrease in cellular invasiveness of both cell lines into various biological substrates such as fibrin gel, the basement membrane extract Matrigel, or intact extracellular matrix. Both cell lines were able to penetrate an intact cell layer of the human keratinocyte line HaCaT, a process, which also proved to be dependent on cell-associated plasmin. In conclusion, these data provide evidence that plasminogen activation associated with the surface of human melanoma cells is catalyzed much more efficiently by cell-associated uPA (MelJuso) than by secreted tPA (MeWo). Cell-associated plasmin, which is protected from inactivation by serum inhibitors, represents the essential component of the proteolytic cascade of plasminogen activation during in vitro invasiveness of human melanoma cells.     

Monitoring protein expression by proteomics: human plasma exposed to benzene

Low levels and long term exposure to benzene is associated with hematotoxicity including aplastic anemia, acute myelogenous leukemia, and lymphoma. Current biomonitoring methods such as urinary phenol, S-phenylmercapturic acid, and trans-trans muconic acid were found to be unreliable as analytical methods to detect benzene exposure. Therefore, to search for a specific protein for biomonitoring benzene exposure, we investigated plasma proteins from workers (n = 50) at a printing company who were exposed to benzene, by two-dimensional gel electrophoresis. The protein profiles are significantly different (p < 0.05) between benzene exposed and unexposed groups, as identified by matrix-assisted laser desorption ionization/time of flight mass spectrometry and confirmed by Western blot analyses. T cell receptor beta chain (TCR beta), FK506-binding protein, and matrix metalloproteinase-13 were expressed only in benzene exposed workers. In addition, interleukin-4 receptor alpha chain and T cell surface glycoprotein CD1b precursor were found to be up-regulated in the plasma of benzene exposed workers. When we treated Jurkat cells with benzene (10 microM-10 mM), TCR beta expression was increased in the membrane more than 6-9-fold compared to untreated cells. In addition, the amount of TCR beta released into the culture media, at benzene concentrations greater than 50 microM, increased up to 10 mM. Therefore, TCR beta levels in plasma could be used as a biomarker and a possible therapeutic target for benzene exposure.     

THE EFFECTS OF RED BLOOD CELL EXTRACTS ON THE PROLIFERATION OF ERYTHROCYTE PRECURSOR CELLS, IN VIVO

Saline incubation extracts of mature erythrocytes were assayed in vivo by a variety of techniques in order to study their ability to modify the proliferation of maturing erythroid cells. Using comparable extracts from granulocytes and lymphocytes, the specificity of the effect of the red cell extract for erythroid cells was confirmed by measurement of autoradiographic labelling indices, radio-iron incorporation and spleen colony growth. The erythroid cells were found to be very sensitive to the effects of the extract, as little as 10 μg per mouse producing a maximum effect on iron incorporation. It was found that the extract does not block erythroid cell proliferation completely but simply lengthens the cell cycle, mainly by increasing the G₁ phase of the cycle. There was no effect on the committed erythroid precursor cells. The in vivo activity, specificity and non-toxicity to the cells, together with the cells' sensitivity to red cell extract suggest, therefore, that this inhibitor may play a physiological role in the control of red cell production.     

Enhanced benzene-induced DNA damage in PMA-stimulated cells in vitro and in LPS-treated animals

The present study investigated the interaction between inflammatory reactions and benzene in vitro and in vivo with respect to oxidative DNA damage. In the in vitro models the oxidative burst of cells was induced by the pretreatment with phorbol myristate acetate (PMA) and in the in vivo models of inflammation mice were pretreated with lipopolysaccharide (LPS). The oxidative DNA damage was indicated by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and strand breaks as assessed by alkaline single cell gel electrophoresis (SCGE, Comet assay). The results showed that combination of PMA and benzene enhanced the level of 8-oxodG in DNA from mouse bone marrow cells by 197%, from human lymphocytes by 188% and from human neutrophils by 205% (p < .05). Pretreatment of mice with LPS and benzene resulted in an enhanced Comet score formation in bone marrow cells by 98% and in lymphocytes by 39% in Comet score (p < .05) and in an enhanced 8-oxodG level in bone marrow cells by 290%. The effects of the combined treatment with PMA/LPS and benzene exceeded the sum of the effects induced by PMA/LPS or benzene alone. The production of nitrate/nitrite showed a two fold increase in the supernatant from incubation of benzene and PMA-pretreated neutrophils. The increase in the 8-oxodG level in the human neutrophil incubation system demonstrated a correlation with nitrate/nitrite production, indicating a possible relationship with the generation of reactive nitrogen species.     

In vitro augmentation of rat natural killer (NK) cell activity

In vitro augmentation of rat natural killer (NK) cell activity was produced by 2 types of treatment. Increased activity occurred "spontaneously" when spleen cells were cultured alone at 37 degrees C. This augmentation was dependent on the presence of adherent, phagocytic cells, presumably macrophages, and was independent of LPS of FCS. Normally low levels of NK activity, present in macrophage-depleted cultured cells, could also be boosted in vitro by incubation with Corynebacterium parvum. This augmentation appeared to be independent of both B cells and macrophages and may be due to stimulation of rat NK cells themselves. Both forms of augmentation were associated with the production of interferon, were found in rats of all ages and strains tested, and should provide an excellent in vitro system for detailed studies of activation of rat NK cells.     

Stabilization of chitin synthetase and purification of chitosomes from several mycelial Mucorales

Stability of chitin synthetase in cell-free extracts from mycelial fungi was markedly improved by the presence of sucrose in the homogenization media. Breakage of mycelium in sucrose-containing buffer yielded enzyme preparations from which chitosomal chitin synthetase could be purified by a procedure involving ammonium sulfate precipitation, gel filtration and centrifugation in sucrose density gradients. Purified chitosomes catalyzed the synthesis of chitin microfibrils in vitro upon incubation with substrate and activators. Chitosomal chitin synthetase from the filamentous form of M. rouxii was similar to the enzyme from yeast cells, except for the poorer stability and diminished sensitivity to GlcNAc activation of the former.     

Calcium alginate immobilized cells of clostridium acetobutylicum for solvent production

Conclusions Immobilized vegetative cells ofC. acetobutylicum has a similar product formation pattern when incubated in a simple glucose-salts solution as ordinary growing cells. If vegetative cells of the organism are immobilized in the solvent production phase, solvents are continuously produced on extended incubation.By immobi1izing spores of the organism the disturbance of the cells metabolic activity during the immobilization procedure was avoided. After the outgrowth of viable cells within the gel, the washed gel preparation retained at a high production capacity in the non-growth stage and the results indicate that continuous production might be fully possible. The butanol productivity was also found to be higher with immobilized cells than in a normal batch process.     

Variazioni di attività della clorogenico ossidasi e di altri enzimi interessati all'ossidoriduzione del TPN durante l'attivazione di fettine di tubero di patata

Abstract

CHANGES IN THE ACTIVITY OF CHLOROGENIC ACID OXIDASE AND OTHER ENZYMES INVOLVED IN OXIDATION AND REDUCTION OF TPN IN AGEING POTATO TUBER SLICES. — The activation of respiration, and in particular of the pentose phosphate pathway, during incubation of potato tuber slices could depend on the increase of activity of oxidative enzymes mediating electron transfer from Gl. 6-P to oxygen.

The present report deals with the activity changes, in the first period of incubation, of the following enzymes: Gl. 6-P-dehydrogenase, TPNH-glutathione reductase, gluta-thione-dehydroascorbate reductase, chlorogenic acid oxidase and a TPNH diaphorase utilizing tetrazolium salts as electron acceptors.

The activity of all of these enzymes, with the exception of TPNH diaphorase, was found to bs, at all stages of incubation, in large excess respect that required to account for the estimated contribution of the pentose phosphate pathway to respiration.

Gl. 6-P dehydrogenase, glutathione reductase and chlorogenic oxidase activities markedly incresed during incubation; but their increase appeared to be clearly delayed (of some hours) respect that of oxygen uptake. This seems to indicate that the increase in activity of these anzymes is rather a consequence than a cause of the respiratory activation.

TPNH diaphorase showed a very low activity in the fresh slices, and it increased quite significantly already in the very first period (5 hours) of incubation. This behaviour suggests the possibility that this enzyme could limit TPNH oxidation, and thus the pentose phosphate pathway activity, and that its activation could be correlated with that of oxidative metabolism in the ageing slices. Further investigation of this hypothesis requires the identification of the natural electron acceptor of this enzyme.     

The role of red blood cell adenylyl cyclase activation in changes of erythrocyte membrane microrheological properties

The ability of red blood cells (RBCs) for the reversible change of their shape under passing through capillaries in microcirculation mainly depends on membrane elasticity of these cells. Phosphorylation of some membrane proteins can result in the changes of microrheological red blood cell properties. Here we show a significant increase in RBC deformability (RBCD) after incubation with isoproterenol (10⁻⁶ M). Red blood cell aggregation (RBCA) decreased under these conditions only slightly. When forskolin (10 μM), an adenylyl cyclase (AC) stimulator, was added to the RBC suspension, RBCD increased significantly (p < 0.05). Some more changes of deformability were found after incubation of RBC with stable penetrating analog of cyclic adenosine phosphate (cAMP), dibutyryl-cAMP, (dB-cAMP, 50 μM) and after phosphodiesterase (PDE) activity inhibition with Vinpocetine, Rolipram, or IBMX. It was found that Gs-proteins inhibitor Clonidine and specific Gi-protein stimulator Mastaparan 7 increased both RBCD and RBCA. On the whole, the data clearly show that the RBC aggregation and deformation changes are related with activation of the different intracellular signaling pathways. We suppose that RBCD increase was mainly associated with activation of the adenylyl-cyclase-cAMP system.     

A comparison of the killer character in different yeasts and its classification

The interactions between 20 killer yeasts of various genera and species were examined. Ten distinct groups were recognised with respect to killer activity and 10 distinct groups with respect to resistance to killer action. Using both killing and resistance phenotypes, 13 classes of killer yeast were found. With the exception of Torulopsis glabrata NCYC 388, non-Saccharomyces strains of yeast were not killed by a member of the genus Saccharomyces.The killer character of the 3 killing groups of Saccharomyces identified could be cured by treatment with cycloheximide or incubation at elevated temperature and the effectiveness of these procedures was indicative of the category of killer yeast examined. Killer yeasts not belonging to the genus Saccharomyces could not be cured of their activity. Double-stranded ribonucleic acids were extracted only from Saccharomyces spp. and the molecular weights of the species present were a function of the killer class to which a strain belonged.By an analysis of the effects of proteolytic enzymes, temperature and pH on killer activity and by gel chromatography of crude preparations of killer factors, the toxins of different killer classes were shown to be biochemically distinct. However all toxins had certain properties in common consistent with there being a protein component essential to killer action.