Resistance of selected strains of Pseudomonas aeruginosa to low-intensity ultraviolet radiation.

The resistances of 10 strains of Pseudomonas aeruginosa and other microorganisms to an ultraviolet (UV) intensity of 100 muW/cm2 were determined. Organisms were exposed in 2- or 15-ml saline suspensions contained in uncapped polyethylene bottles for increasing periods of time, and the surviving fractions were enumerated. Decimal reduction times were calculated by regression analysis, using the least-squares method. The 10 strains of P. aeruginosa, compared with Micrococcus radiodurans and Candida albicans, were very susceptible to low-intensity UV radiation. Results from this study showed that a UV intensity of 100 muW/cm2 penetrated saline suspensions up to 40 mm deep sufficiently to kill high levels of microbial cells, especially P. aeruginosa cells. These results allowed us to design a system for determining and monitoring the sterilization capability of low-intensity UV radiation. In our particular case, UV proved to be an efficient mode for sterilizing saline suspensions of P. aeruginosa in polyethylene bottles. The significance and application of these findings with regard to supporting UV as a sterilant are discussed.     

Oxidation of various substrates by host grown Mycobacteria leprae and M. lepraemurium.

Oxidation of various substrates by whole cell suspensions of M. Lepraemurium and M. leprae was investigated using manometric techniques. Yeast extract, L-cysteine, dithioerythritol, and DL-penicillamine were oxidized by both M. lepraemurium as well as by M. leprae. Although tween 80 was oxidized by M. lepraemurium cell suspensions, it was not by M. leprae. Succinate was readily oxidized by whole cells of M. leprae (without being frozen) whereas it was oxidized only by M. lepraemurium cells frozen at -40 degrees C for one minute. The results indicate that M. leprae and M. lepraemurium are capable of oxidizing some substrates without requiring any cofactor and are not dependent upon host cells for respiration.     

Tissue distribution and ultrastructure of B lymphocytes reacting with the monoclonal antibody anti-Y29/55

The reactivity of normal tonsilar cells with the monoclonal antibody anti-Y29/55 is characterized at the tissue and ultrastructural cytological level. Using an indirect immuno-alkaline phosphatase method on frozen sections the antibody labels mantle zone and germinal center lymphocytes. This staining reaction is more generalized in B-lymphocyte areas than that obtained with antibodies to IgM and IgD. By indirect immunoperoxidase staining, as well as by an indirect rosetting procedure in cell suspensions, the reactive cell population were either small resting lymphocytes or activated lymphocytes corresponding to centrocytes, centroblasts, immunoblasts and plasmoblasts; some plasma cells were also labeled. These results characterize the monoclonal antibody anti-Y29/55 as a pan-B-marker antibody, useful for labeling resting and activated peripheral B-lymphocytes in frozen tissue sections and cell suspensions.     

Studies on Rapidly Frozen Suspensions of Yeast Cells by Differential Thermal Analysis and Conductometry

Few, if any, yeast cells survived rapid cooling to -196°C and subsequent slow warming. After rapid freezing, the suspensions absorbed latent heat of fusion between -15° and 0°C during warming, and the relation between the amount of heat absorbed and the concentration of cells was the same as that in equivalent KCl solutions, indicating that frozen suspensions behave thermally like frozen solutions. The amount of heat absorbed was such that more than 80 per cent of the intracellular solution had to be frozen. The conductometric behavior of frozen suspensions showed that cell solutes were still inside the cells and surrounded by an intact cell membrane at the time heat was being absorbed. Two models are consistent with these findings. The first assumes that intracellular freezing has taken place; the second that all freezable water has left the cells and frozen externally. The latter model is ruled out because rapidly cooled cells do not shrink by an amount equal to the volume of water that would have to be withdrawn to prevent internal freezing.     

The Effect of Helium on Cryopreservation of HeLa and L929 Cells

The effect of helium on cell survival during cryopreservation was studied using the HeLa and L929 cell lines. Cell suspensions were incubated in an atmosphere of helium, nitrogen, or air and frozen in the presence or absence of glycerol as a cryoprotectant. After thawing, the cell viability was evaluated by the Trypan Blue exclusion test and culture development for 18 h. Helium was found to provide better preservation of cell suspensions compared with nitrogen and air. The positive effect of helium was the greatest in the case of freezing without cryoprotectants (the HeLa cell survival increased by a factor of 1.5–2) and somewhat lower in the case of freezing in the presence of low glycerol concentrations (the L929 cell survival increased by a factor of 1.2–1.5 in the presence of 3% glycerol). Use of helium for cell suspensions may improve cryopreservation methods by making it possible to reduce the concentrations of conventional cryoprotectants, which are generally highly toxic and undesirable to use for cryopreservation of biological material for medical applications.     

Senescence-induced alteration in cell surface carbohydrates correlated using proton NMR spectroscopy and a lectin-based affinity-binding assay

Changes in the cell surface oligosaccharides in human fetal lung fibroblasts (IMR-90) are studied as the cells progress to senescence using nuclear magnetic resonance spectroscopy (NMR) and a biochemical assay. A lectin-based affinity-binding technique is used which measures the organization of carbohydrates on the cell surface. Proton NMR studies of the water in samples of frozen cell suspensions of young and old cells provide information on the local dynamics of the cell surface by monitoring the motion of bound water. Changes in the lectin binding density and affinity class distribution correlate with a decrease in the water proton linewidth in frozen cells. These observations reflect alterations in the conformation or structure of the cell surface oligosaccharides and local constituent water.     

Influence of the container material on the hemolysis of glycerolized red cells after freezing and thawing

Postthaw hemolysis of glycerolized human red cells is influenced by the container material. The effect does not appear to be the result of toxic materials extracted from the plastic or of differences in thermal conductivity. In materials other than UCAR there is a correlation between post-thaw hemolysis and the area of container surface exposed to the cells. Rough handling of frozen cell suspensions increases the postthaw hemolysis of cells frozen in PVC bags but not those frozen in UCAR bags. Hemolysis at glass surfaces is particularly high. The mechanism of these effects remains a mystery.     

Serotyping of Pseudomonas aeruginosa strains by slide coagglutination

Serological typing of Pseudomonas aeruginosa strains (228 strains) by slide coagglutination, using our own reagents (5 polyvalent and 22 monovalent ones, corresponding to the 22 serotypes in Meitert-Meitert scheme), led to identical results obtained by conventional slide agglutination. Utilization of live Ps. aeruginosa cells suspensions, killed by boiling or autoclaving, showed a 100% concordance of results, when using the second and the third suspension types and a 97.37% one between them and the live cells suspension. We noticed that reactions intensity was higher when using bacterial suspensions, boiled for 2.5 hours, in comparison with autoclaved cells suspensions, 30 minutes at 120 C. Compared to conventional slide agglutination, the slide coagglutination presents more advantages, being simple, rapid, specific and economical.     

Determination of maximal bactericidal activity in human granulocytes

A conventional in vitro test assay was used to determine maximal bactericidal capabilities of human granulocytes. By means of a mathematical model the maximal phagocytosis and killing activity could be calculated for S. aureus and P. aeruginosa serving as test organisms. The evaluation allowed moreover the determination of the optimal bacterial load and also of critical bacterial concentrations leading to a complete depression of observable granulocyte killing functions. In contrast to other studies frozen suspensions of bacteria were used allowing the employment of identical microorganisms within a complete series of experiments. On average one granulocyte was found to ingest a maximum of 17 CFU of S. aureus with 9 CFU killed under optimal ratios of bacteria per granulocyte. For P. aeruginosa the granulocyte function reached peak values of 96 CFU ingested and 62 CFU killed per one granulocyte. The new assay might provide a highly reproducible method for clinical assessment of granulocyte dysfunctions in various diseases.     

Human monoclonal antibodies against surface antigens of Langerhans cells from lymphocytes of diabetics transformed by Epstein-Barr virus

Human monoclonal antibody against islet cell surface antigens was generated from a pre-diabetic patient's peripheral blood lymphocytes transformed with Epstein-Barr virus. Reactivity of these transformed lymphocytes was evaluated using indirect immunofluorescence on rat islet cell suspensions and frozen sections of human pancreas. Several lymphoblastoid cell lines that react with islet cell surface were obtained. Preliminary immunoblots with enriched rat islet cell membrane antigens suggest a reactivity toward a 64 kdalton antigen.     

Death of Lactobacillus bulgaricus Resulting from Liquid Nitrogen Freezing

Concentrated cell suspensions of Lactobacillus bulgaricus prepared from cells grown in semisynthetic media were frozen in liquid nitrogen. After storage for 24 hr, the cell suspensions were found to have decreased colony counts and acid-producing capacity in milk. The amount of loss varied among the different strains tested. The addition of known cryoprotective agents to cell suspensions of the most labile strain before freezing provided little or no protection to the cells. However, storage stability of all strains investigated was improved by supplementing the growth medium with Tween 80 (polyoxyethylene sorbitan monooleate). The concentration of Tween 80 necessary for maximal storage stability varied among strains.     

A purification procedure for the soluble cytochrome oxidase and some other respiratory proteins from Pseudomonas aeruginosa.

The production of the soluble cytochrome oxidase/nitrite reductase in the bacterium Pseudomonas aeruginosa is favoured by anaerobic conditions and the presence of KNO3(20g/l) in the culture medium. Of three methods commonly used for the disruption of bacterial suspensions (ultrasonication, liquid-shear homogenization and glass-bead grinding), sonication proved the most efficient in releasing the Pseudomonas cytochrome oxidase. A polarographic assay of Pseudomonas cytochrome oxidase activity with sodium ascorbate as substrate and NNN'N'-tetramethyl-p-phenylenediamine dihydrochloride as electron mediator is described. A purification procedure was developed which can be used on the small scale (40-litre cultures) or the large scale (400-litre cultures) and provides high yields of three respiratory-chain proteins, Pseudomonas cytochrome oxidase, cytochrome c551 and azurin, in a pure state. A typical preparation of 250g of Ps.aeruginosa cell paste yielded 180mg of Pseudomonas cytochrome oxidase, 81 mg of Pseudomonas cytochrome c551 and 275mg of Pseudomonas azurin.     

大鼠黄体细胞酪氨酸含量及hCG,cAMP和孕酮对含量的影响

经PMSG-hCG处理的未成年雌性大鼠卵巢,用胶原酶-DNA酶溶液消化,制成黄体细胞悬浮液。预培育1h后,加入不同浓度的hCG、cAMP及孕酮;并在加入hCG(10mIU/ml)、cAMP(2.5mmol/L)或孕酮(1nmol/L)的同时分别加入苯丙氨酸或放线菌酮,再培育1.5h,取细胞悬浮液400μl,用薄层层析扫描技术测其酪氨酸含量。结果:黄体细胞内有一定量内源酪氨酸,hCG,cAMP和孕酮均可明显促进酪氨酸的释放(P<0.05).苯丙氨酸对酪氨酸的含量无影响。酪氨酸释放也不依赖蛋白质合成过程。     

Efficient regeneration and transformation of plantain cv. “Gonja manjaya” (Musa spp. AAB) using embryogenic cell suspensions

In banana and plantain research, it is essential to establish embryogenic cell suspensions together with a highly efficient regeneration and transformation system. This article describes the development of an embryogenic cell suspension (ECS), regeneration, and transformation for plantain cv. “Gonja manjaya”. ECS was established using highly proliferative multiple buds. The frequency of embryogenic friable callus formation was 56.8% of the cultured explants. Friable embryogenic calli with many translucent proembryos were transferred to liquid medium and homogenous cell suspensions were established within 3–4 mo. Approximately 25,000 to 30,000 plants per 1.0 ml of settled cell volume were regenerated in approximately 13–14 mo. ECSs were transformed using Agrobacterium strain EHA 105 harboring the binary vector pBI121. About 50–60 transgenic plants per 0.5 ml settled cell volume were regenerated on selective medium containing 100 mg l⁻¹ kanamycin. Histochemical GUS assays using different tissues of putatively transformed plants demonstrated stable expression of uidA gene. The presence and integration of the uidA gene were confirmed by PCR and Southern blot analysis, respectively. This is the first report showing establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation of an important plantain cultivar, “Gonja manjaya”. This study shows the huge potential for genetic transformation of plantains for disease or pest resistance, as well as tolerance to abiotic factors such as drought stress using this robust regeneration and transformation protocol.     

The integration of a major hepatitis B virus gene into cell-cycle synchronized carrot cell suspension cultures and its expression in regenerated carrot plants

The percentage of cells successfully transformed by Agrobacteria is usually very low (not more than 10% and usually much less; Thomas et al., 1989). In this report we show that in carrot (Daucus carota L., ssp. sativus) cell suspensions transformation efficiency was strongly improved by using cell cycle synchronized cells. Fluorodesoxyuridine (FDU) was added for 24 h to inhibit thymidine synthesis. This blocked the cell cycle at the transition from G1- to S-phase. Then the block was released by applying thymidine. A high rate of transformation was obtained when Agrobacterium tumefaciens was added concurrently with thymidine. As examples of efficient and long term foreign gene expression in transgenic cells, the reporter enzyme -glucuronidase (GUS) as a model as well as the major hepatitis B virus surface protein were used. Both genes were linked to the MAS promoter. In carrot cell suspensions containing the viral gene, the corresponding viral protein was produced. In roots of mature transgenic carrot plants generated through somatic embryogenesis and raised in soil as well as in callus cultures derived thereof, the viral protein was also produced.     

The effect of phenazine methosulfate-ascorbate on bacterial active transport and adenosine triphosphate formation: inhibition of Pseudomonas aeruginosa and stimulation of Escherichia coli.

The artificial electron-donor system, phenazine methosulfate (PMS) ascorbate, inhibited active transport of glucose by Pseudomonas aeruginosa irrespective of whether the incubation systems were in air, flushed with oxygen, or gassed with nitrogen under anaerobic denitrifying conditions. Active transport of glucose by P. aeruginosa was also inhibited by reduced 5-N-methyl-phenazonium-3-sulfonate, a membrane-impermeable electron donor. PMS-ascorbate caused rapid depletion of intracellular adenosine triphosphate (ATP) when added to respiring cell suspensions of P. aeruginosa either in the presence or absence of glucose or succinate as oxidizable energy sources. In contrast, under identical conditions, Escherichia coli formed ATP with PMS-ascorbate as the sole oxidizable energy source and ATP formation continued when glucose or succinate was present in addition to PMS-ascorbate in the incubation system.     

Changes in surface relief of suspended cells are morphological signs of the initial stage of neoplastic transformation in fibroblastic monolayer cultures.

The percentages of cells with different types of cell surface relief were determined in cell suspensions derived from monolayer cultures. Primary cultures of rat embryo fibroblasts (REF) and cell lines REF (LT) and REF-1, immortalized cells of which preserved normal phenotypic characteristics of the initial primary culture REF, as well as morphologically transformed tumorigenic lines REF (LT) ras and REF-2EJ were studied. In REF suspensions the cells with the blebbed type of surface relief were shown to be predominant as compared with those with microvillus relief whereas cell suspensions derived from both immortalized and fully transformed cultures display the reverse ratio of cells with those types of surface relief. Therefore, the pattern of cell surface relief in cell suspensions derived from fibroblastic monolayer cultures may serve as a morphological marker of the initial stage of neoplastic transformation-immortalization when typical morphological signs of cell transformation are not yet manifested in monolayer cultures.     

Evidence for small pores in the outer membrane of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa NCTC6750 and Escherichia coli K12 were used to study permeability of whole, intact cells to a series of labelled oligosaccharides. Stationary phase, oxygen depleted simple salts batch cultures were used. An efflux method was used to compare diffusion from cells of various ³H-labelled sugars (an homologous series based on isomaltitol) with diffusion of [¹⁴C]sucrose. Both plasmolysed and unplasmolysed cell suspensions were used. The data are consistent with an E. coli pore exclusion limit of approx. 833 Da for unplasmolysed cells and of about 670 Da for plasmolysed cells. For P. aeruginosa the data indicated a relatively small pore exclusion limit about the same size as sucrose with plasmolysis having little effect. These findings were confirmed with P. aeruginosa PAO1 grown in nutrient broth.     

Liquid Nitrogen Freezing in Microbiological Assay Systems: I. Preservation of Lactobacillus leichmannii for Direct Use in the Vitamin B12 Assay

Suspensions of Lactobacillus leichmannii were stored in liquid nitrogen and were used as direct inocula in vitamin B₁₂ assays. Complete recovery of viable cells was obtained when the suspensions in basal B₁₂ medium were rapidly frozen by direct immersion into liquid nitrogen and rapidly thawed by agitating the suspensions in a water bath at 40 C. Greater than 90% destruction occurred when the suspensions were in saline. However, both suspensions were usable in the B₁₂ assay system. Assay results on a number of test materials indicated good correlation between freshly prepared suspensions and frozen suspensions in basal medium stored 3 months. Suspensions in saline stored for 1 year in liquid nitrogen showed no detectable difference from the first day after freezing. Suspensions frozen slowly at the rate of 1 degree per min from 4 to -40 C and subsequently immersed in liquid nitrogen had a longer lag period of growth and were not usable in the 18-hr assay incubation system. A major advantage of a stored inoculum for direct use in a microbiological assay is the reduced day-to-day variation in the inoculum.