Maximum Number Of Cells Research Articles

Influence of temperature rise on kinetic parameters during batch propagation of Kluyveromyces fragilis in cheese whey under ambient conditions

Three 5 l working volume fermenters were used to investigate the growth of the yeast Kluyveromyces fragilis in acid cheese whey under ambient temperature in order to assess the specific growth rate and yield, the lactose and oxygen uptake rates during the various phases of batch culture, the effect of increasing temperature on the various kinetic parameters, and the need for a cooling unit for single cell production batch systems. The initial dissolved oxygen in the medium was 5.5 mg l−1 and the pH was maintained at 4.5. The observed lag phase, specific growth rate and maximum cell number were 4 h, 0.2 h−1 and 8.4 × 108 cells ml−1, respectively. About 99% of the lactose in cheese whey was utilized within 20 h, 85% during the exponential growth phase. The specific lactose utilization rates by K. fragilis were 0.20 × 10−12, 1.457 × 10−12, 0.286 × 10−12 and 0.00 g lactose cell−1 h−1, for the lag, exponential, stationary and death phases, respectively. The dissolved oxygen concentration in the medium decreased as the cell number increased. The lowest oxygen concentration of 1.2 mg l−1 was observed during the stationary phase. The volumetric oxygen transfer coefficient was 0.41 h−1 and the specific oxygen uptake rates were 0.32 × 10−12, 2.14 × 10−12, 0.51 × 10−12 and 0.003 × 10−12 mg O2 cell−1 h−1, for the lag, exponential, stationary and death phases, respectively. The maximum temperature recorded for the medium was 33 °C, indicating that a cooling unit for batch production of single cell protein at ambient temperature is not needed for this type of bioreactor. The increase in medium temperature affected the cell growth and the lactose and oxygen uptake rates.

Stable transfection of CHO cells with the c-myc gene results in increased proliferation rates, reduces serum dependency, and induces anchorage independence.

Induction of the transcription factor Myc promotes cell proliferation and transformation by activating growth-promoting genes and/or by transcriptionally repressing the expression of growth arrest genes. However, a number of studies have shown that c-Myc is a potent inducer of apoptosis in the absence of serum or growth factors. To further examine the role of Myc in cell growth and proliferation, and the advantages of this positive regulator in cell culture we transfected the CHO-K1 cell line with a human c-myc gene driven by MMLV 5'-LTR promoter. Over-expression of ectopic c-Myc resulted in a significant increase in growth rate and maximum cell number, in both suspension and attached batch culture accompanied by a similar decrease in specific glucose consumption rate. Interestingly, there was no manifestation of the widely reported apoptotic death by c-myc in the absence of serum. Additionally, over-expression of c-Myc appeared to induce morphological transformation and partial anchorage-independence.

Bacterial growth rate and growth pouch nodulation profile differences as possible ways of Bradyrhizobium japonicum strain screening for low P soils

This work studied the effects of P fertilization on nodulation of field-grown soybean by two Bradyrhizobium strains (SMGS1 and THA7), and checked if differences between strains were consistent with bacterial growth and growth pouch nodulation ability in response to P availability. In the field, nodule dry weight and nitrogen fixation activity of inoculated soybean were studied on typical acid soils of Thailand at the flowering (R1) stage and at the end of grain filling. Grain yield, growth and phosphorus content were recorded. The bradyrhizobial strains were cultivated in culture medium, and growth parameters recorded. Nodulation patterns were observed during growth pouch experiments: infective root cells were inoculated with strains cultivated at two P concentrations in their culture media, namely 1 μM and 1 mM. Ten days after inoculation, the position of each nodule was measured relative to the root tip (RT) mark, expressed relative to the smallest emerging root hairs-RT distance in the nodulation frequency profile, and the consistency of responses was tested. In the field, on P deficient soils, dry weight of nodules was higher with Bradyrhizobium japonicum strain SMGS1 than with strain THA7. P supply increased the number and dry weight of nodules for both strains, with a higher dry weight response for THA7 than for SMGS1. It also had a positive effect on tissue phosphorus status and grain yield at R8 stage. In growth media, significant differences were recorded between strains under P-limiting conditions: The growth rate was higher for strain SMGS1, as well as the maximal number of bacterial cells supported. With growth pouch, inoculating plants with bacteria grown in P-deficient medium resulted in a less intense nodulation of roots by THA7, and with nodules appearing earlier on roots than in the case of SMGS1. At 1 mM P, there was no significant difference between strains. Thus, strain THA7 is more affected by P deficiency than strain SMGS1. Although P was not supplied in the same way in the soil and in the growth pouch experiments, this consistency of behaviour between work scales indicates that phosphorus availability is a key component for a successful inoculation. Furthermore, the study of bacterial growth rates and nodulation profile represents an interesting step for bacterial screening for low P soils. [-11pt]

Evaluation of assimilable organic carbon in the Yodo River and the effect of coagulation on its control

Biodegradable organic compounds in Yodo River water, the coagulated water and those ozonated waters were characterized on the concentration of assimilable organic carbon (AOC), the apparent molecular size and the sensitivity to E260 (UV absorbance at the wavelength of 260 nm). High molecular (>5,000 Da) organic compounds which were insensitive to E260 were contained with relatively high concentration in Yodo River, whereas they were not detectable as AOC. The AOC concentration in Yodo River was reduced by coagulation because the phosphorus concentration sharply decreased. The AOC concentration rapidly increased with the reduction of E260 and slowly with the depolymerization of high molecular organic compounds during the ozonation. However, 40-50% of AOC increasing with the ozonation was reduced by the coagulation. Furthermore, batch incubations of native bacteria living in the Yodo River were conducted in order to examine the validity of the apparent AOC concentration as an indicator of the regrowth potential. The AOC concentration calculated as acetate carbon equivalent would not exactly represent the concentration of the available carbon for the assimilation. However, maximum cell numbers for the incubation period were generally proportional to apparent AOC concentrations even if phosphorus limitation. In conclusion, it is suggested that coagulation is an effective process in controlling the concentration of AOC and is a useful indicator to evaluate the regrowth potential of heterotrophic bacteria in Yodo River.

The expression of p53, p21, Bax and induction of apoptosis in normal volunteers in response to different doses of ultraviolet radiation.

Ultraviolet radiation (UVR) damages keratinocytes. Direct DNA damage may undergo enzymatic repair followed by resumption of the normal cell cycle. Cells may also be eliminated without inflammation by the error-free process of programmed cell death or apoptosis. Necrosis of cells can occur after overwhelming damage. Failure of apoptosis leads to retention of cells with persistent mutations. This study investigates p53-dependent apoptotic responses in normal skin following solar-simulated radiation (SSR). Sun-protected buttock skin from normal volunteers with no history or clinical evidence of skin cancer was exposed to graded doses of SSR, 0.5, 1, 2 and 3 times the minimal erythema dose (MED). Biopsies taken at a range of time points (4.5, 9, 24, 33, 48 and 72 h) after UVR, quantified the time course and dose-response of apoptosis and the expression of the relevant proteins, p53, p21waf1/Cip1 and Bax, by single and double labelling techniques. Apoptosis was upregulated in a dose-dependent manner as was the expression of p53, p21waf1/Cip1 and Bax in response to SSR. Following exposure to 3 MEDs it was found that: (i) the maximum number of apoptotic cells occurred at 48 h; (ii) p53 protein expression was upregulated from 4 to 72 h preceding peak p21waf1/Cip1 protein expression (9-48 h) and peak Bax protein expression (33 h). These results suggest that, following SSR, normal human skin induces apoptosis by the p53, p21waf1/Cip1, Bax pathway in vivo. In addition, induction of apoptosis and expression of p53, p21waf1/Cip1 and Bax occurs in a dose-dependent manner.

Neuroprotective properties of topiramate in organotypic hippocampal cultures: implications for treatment of vascular and other dementias

AbstractMood stabilizers are often used in the treatment of psychosis and agitation associated with dementia. It has been shown that the antiepileptic drug topiramate (TPM) reduced brain tissue damage in a rat model of middle cerebral artery occlusion, suggesting that it may have a neuroprotective effect. We tested this possibility using confocal microscopy to measure cell death in organotypic hippocampal tissue cultures from 5–7‐day‐old Fischer 344 rats. To induce death, cultures were treated with N‐methyl‐D‐aspartate (NMDA, 50µM, 30min), kainate (KA, 1–100µm, 60min), or transferred to a fresh culture medium. Cell death was measured as propidium iodide fluorescence 24 and 48h after the insult and normalized to the maximal number of dead cells in the culture; maximal killing was achieved by exposing cultures to 4°C ambient temperature for 48h. Treatment of cultures with TPM at low concentrations (0.01–1µm) for 48 but not for 24h prior to the insult significantly increased survival. Concentrations above 1µm were ineffective. TPM (0.1µm) protected against kainate toxicity in the CA1 area, but not the CA3–4 area. TPM did not reduce cell death in the culture medium exchange model. TPM was ineffective in cultures treated with Na+ channel blocker tetrodotoxin. Pretreatment with both 0.1µm TPM and 1µm Ca2+/diacylglycerol‐dependent PKC inhibitor Gö6976 blocked the protective effect of topiramate against NMDA. These data suggest that protective action of TPM against NMDA toxicity may involve PKC activation and Na+ channel blockade and that at low doses TPM may be a clinically useful neuroprotective agent. Drug Dev. Res. 56:393–400, 2002. © 2002 Wiley‐Liss, Inc.

Limnology of an equatorial high mountain lake — Lago San Pablo, Ecuador: The significance of deep diurnal mixing for lake productivity

Equatorial high mountain lakes are a special type of lake occurring mainly in the South American Andes as well as in Central Africa and Asia. They occur at altitudes of a few thousand meters above sea level and are cold-water lakes (<20 °C). Relatively little is known about them. A long-term limnological study was therefore undertaken at Lake Lago San Pablo, Ecuador to analyze the basic limnological processes of this lake, which has a tendency for eutrophication. Lago San Pablo is spread over an area of 668 hectares, has a maximum depth of 35 m, and is located 2660 m above sea level. Its thermal stratification is a monomictic one, with only 1–2 °C difference between the epi- and hypolimnion; overturn is achieved by strong winds during the dry summer period. The stratification phase is characterized by an oxygen deficit in the lower part of the hypolimnion. Besides, strong convective currents occur due to nocturnal cooling, and partial lake mixing was observed during the nocturnal period. This type of lake mixing is called atelomixis, which is characterized by the partial mixing of isolated layers (difference in temperature or ionic content) during stratification. The nutrient level of the lake is quite high: mean P total concentration = 0.22 mg/l, mean N total = 1.05 mg/l, soluble reactive phosphorus (SRP) > 0.01 mg/l, and soluble inorganic nitrogen > 0.2 mg/l. Nitrogen and phosphorus are available in the epilimnion all year round (N sol. inorg·. = 0.3 to 1.7 mg/l N, SRP = 0.04 to 0.63 mg/l P). The N/P ratio is sometimes > 14, sometimes < 10, indicating a variability of the limiting nutrient factor. Considering the nutrient level, the phytoplankton biomass is quite low (about 4,000 cells per ml on average; maximum cell number: 13,000 in 1998 and 10,000 in 1999). The mean epilimnic chlorophyll content (Chl a was 10 mg/l in 1998 and 11 g/l in 1999, and the maximum Chl a content was 16 and 22 g/l in 1998 and 1999, respectively. Phytoplankton production can be limited by nutrients, mainly nitrogen, but convective currents can also cause a significant loss of biomass. The lake's euphotic zone is smaller than its epilimnic zone, indicating that light radiation is limiting in the deeper water body, this is caused by a weak thermocline due to destratification by nocturnal cooling, the atelomixis.

The effect of 835.62 MHz FDMA or 847.74 MHz CDMA modulated radiofrequency radiation on the induction of micronuclei in C3H 10T(1/2) cells.

To determine if radiofrequency (RF) radiation induces the formation of micronuclei, C3H 10T(1/2) cells were exposed to 835.62 MHz frequency division multiple access (FDMA) or 847.74 MHz code division multiple access (CDMA) modulated RF radiation. After the exposure to RF radiation, the micronucleus assay was performed by the cytokinesis block method using cytochalasin B treatment. The micronuclei appearing after mitosis were scored in binucleated cells using acridine orange staining. The frequency of micronuclei was scored both as the percentage of binucleated cells with micronuclei and as the number of micronuclei per 100 binucleated cells. Treatment of cells with cytochalasin B at a concentration of 2 microg/ml for 22 h was found to yield the maximum number of binucleated cells in C3H 10T(1/2) cells. The method used for the micronucleus assay in the present study detected a highly significant dose response for both indices of micronucleus production in the dose range of 0.1-1.2 Gy and it was sensitive enough to detect a significant (P > 0.05) increase in micronuclei after doses of 0.3 Gy in exponentially growing cells and after 0.9 Gy in plateau-phase cells. Exponentially growing cells or plateau-phase cells were exposed to CDMA (3.2 or 4.8 W/kg) or FDMA (3.2 or 5.1 W/kg) RF radiation for 3, 8, 16 or 24 h. In three repeat experiments, no exposure condition was found by analysis of variance to result in a significant increase relative to sham-exposed cells either in the percentage of binucleated cells with micronuclei or in the number of micronuclei per 100 binucleated cells. In this study, data from cells exposed to different RF signals at two SARs were compared to a common sham-exposed sample. We used the Dunnett's test, which is specifically designed for this purpose, and found no significant exposure-related differences for either plateau-phase cells or exponentially growing cells. Thus the results of this study are not consistent with the possibility that these RF radiations induce micronuclei.

Production Systems Influence Source Limitations to Growth in `Sharpblue' Southern Highbush Blueberry

`Sharpblue' southern highbush blueberry (Vaccinium corymbosum L. interspecific hybrid) was grown in either a dormant or nondormant production system to determine the effect of production system on source limitations to fruit and vegetative growth. Source limited stages were evaluated in the two production systems by reducing reproductive sink load during either the fruit cell division or fruit cell enlargement stage. Source limitation during cell division was evaluated by removing 80% of the flower buds in late fall, since the majority of cell division in blueberry ovaries occurs before bloom. Source limitation during cell enlargement was evaluated by removing 80% of the fruit after fruit set the following spring. In the dormant production (DP) system, mean fruit dry weight (DW) was greatest in the flower bud removal treatment and least in the control (nonthinned) treatment, suggesting that cell number, rather than size, is more important in determining blueberry fruit weight in the DP system. Fruit in the dormant flower bud removal treatment may have approached maximum cell number and therefore fruit size; this was supported by the observation that significant depletion of root carbohydrate concentration did not occur in this treatment, as it did in the control treatment. Mean fruit DW in the nondormant production (NDP) system was greatest in the fruit removal treatment compared with the other two treatments, suggesting that cell enlargement played a larger role in determining fruit size in this production system. However, the effect of the flower bud removal treatment (and therefore the effect of cell division) on fruit DW in the NDP system was apparently masked by continued flower bud initiation in this system after flower bud removal in late fall. Continued floral initiation was apparently an alternative sink to increasing cell division in previously formed flower buds. In both systems, fruit removal increased vegetative growth compared with the control and flower bud removal treatments. Thus, both systems exhibited source limitations to fruit and vegetative growth, although the timing and extent of the limitation to fruit growth differed between the production systems.

Effect of glucose, maltose, soluble starch, and CO2 on the growth of the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermophilic archaeon Pyrococcus furiosus was cultivated in batch and continuous fermentations on different carbon substrates. The cultivation of P furiosus on soluble starch as the only carbon source resulted in cell densities three times higher than in cultivations on maltose, 1.06 x 10(10) cells/ml compared to 3.4 x 10(9) cells/ml. The yield coefficient, Y(x/y) = 0.12 g/g, and the growth rate, mu = 0.33 h(-1), were almost equal on soluble starch and on maltose, but on glucose no growth could be detected. An inhibitory effect of glucose, when added to other carbon substrates, also could not be found. Isobutyric and isovaleric acid were detected as novel metabolites produced by P. furiosus. Inhibitory effects of these acids, as well as of the well-known products acetic acid, propionic acid, and alanine, could be precluded. Concentrations of 10% CO2 in the gas supply respective to the exhaust gas enhanced the growth of P furiosus significantly. The maximum cell number was two orders of magnitude higher than was observed with pure nitrogen. Further increase of the CO2 concentration up to 100% had no significant effect on the growth of P. furiosus.

Cytokine regulation of E-selectin in rat CNS microvascular endothelial cells: differential response of CNS and non-CNS vessels

We have compared the induced expression of E-selectin in primary cultures of rat brain microvascular endothelial cells (EC), pericytes and in non-CNS microvascular endothelium stimulated with the cytokines, IL-1β (20 ng/ml), and tumor necrosis factor (TNF)-α (75 ng/ml). Expression was studied at both the protein and mRNA levels. Fluorescence in-situ hybridization (FISH) was used to examine de novo synthesis of E-selectin mRNA. Laser cytometric analysis was used as a novel approach to the quantitaion of FISH. In-situ hybridization was performed using two PCR-generated probes. The first probe (517 bp) spanned the lectin and epidermal growth factor (EGF)-like domain. The second probe (562 bp) spanned the CR3, 4, and 6 domains. E-selectin-specific mRNA was localized to the perinuclear regions of the EC. Both cytokines, IL-1β and TNF-α significantly increased E-selectin gene expression in CNS EC but not pericytes. IL-1β induced higher E-selectin mRNA levels than TNF-α. The maximum number of mRNA-positive cells was observed after stimulation for 4–6 h. Surface protein expression was sustained for up to 48 h following addition of cytokine. This was in contrast to the transient expression in non-CNS EC indicating that pure primary CNS EC display slightly different kinetics of E-selectin expression than non-CNS EC.

The Development of a Bioluminescence Assay to Compare the Efficacy of Biocides Incorporated into Plasticised PVC

A bioluminescence assay was developed using the expression of the luxAB genes in Pseudomonas veronii to allow the efficacy of biocides incorporated into plasticised polyvinylchloride (pPVC) to be determined in situ. A maximum number of cells was found to adhere to the surface after 18 h as measured by bioluminescence, radiolabelling and viable cell counts. A positive correlation was found between the level of bioluminescence and numbers of viable cells attached to the pPVC. When the biocide 10, 10-oxybisphenoxyarsine (OBPA) was incorporated into the pPVC, both bioluminescence and viable cell number were reduced by ca 60% at a concentration of 750 ppm and by >99% at 2250 ppm. When the biocide 2,3,5,6-tetrachloro-4-(methylsulphonyl)pyridine (TCMP) was incorporated into the pPVC, no reduction in viability or bioluminescence was seen after 18 h. However, over a period of 72 h at a concentration of 2250 ppm TCMP, both viable cell number and bioluminescence decreased steadily after 36 h until after 72 h, both bioluminescence and viable cell counts were less than 1% of the initial values. The viability of attached cells can therefore be measured in situ in a sensitive real-time assay by measuring bioluminescence allowing the efficacy of biocides incorporated into plastics to be compared.

Disturbance of Chromatium Population at Mid-depth of Lake Kaiike.

Two large-celled bacterial species, Chromatium sp. and Macromonas sp., densely populate the mid-depth of Lake Kaiike, a small lagoon on Kamikoshiki Island, turning the water a purplish red (bacterial plate). In recent years, the Chromatium population has showed large seasonal fluctuations; maximum cell numbers in the order of 106 cells ml-1 have dropped to 103 cells ml-1. To examine the factor(s) disturbing the population, the profiles of two bacterial species as well as environmental factors were examined during the period June 1998 to Oct. 2001. Solar heating to raising the water temperature above 30°C as well as a proliferation of Macromonas sp. were found to have disturbed the Chromatium population.

Increased susceptibility of peripheral blood mononuclear cells to equine herpes virus type 1 infection upon mitogen stimulation: a role of the cell cycle and of cell-to-cell transmission of the virus

Equine herpesvirus-1 (EHV-1) is an important pathogen of horses, causing abortion and nervous system disorders, even in vaccinated animals. During the cell-associated viremia, EHV-1 is carried by peripheral blood mononuclear cells (PBMC), mainly lymphocytes. In vitro, monocytes are the most important fraction of PBMC in which EHV-1 replicates, however, mitogen stimulation prior to EHV-1 infection increases the percentage of infected lymphocytes. The role of the cell cycle in viral replication and the role of cluster formation in cell-to-cell transmission of the virus were examined in mitogen-stimulated PBMC. Involvement of the cell cycle was examined by stimulating PBMC with ionomycin/phorbol dibutyrate (IONO/PDB) during 0, 12, 24 and 36 h prior to inoculation. Cell cycle distribution at the moment of inoculation and the percentage of EHV-1 antigen-positive PBMC at 0, 12 and 24 hours post inoculation (hpi) were determined by flow cytometry and immunofluorescence microscopy, respectively. The role of clusters was examined by immunofluorescence staining within clusters of stimulated PBMC using antibodies against EHV-1. Significant correlations were found between the increase of cells in the S- or G2/M-phase after a certain time interval of prestimulation and the increase of EHV-1 antigen-positive cells. The percentage of clusters with adjacent infected cells significantly increased from 3.3% at 8 hpi to 23.7% at 24 hpi and the maximal number of adjacent infected cells increased from 2 to 7. Addition of anti-EHV-1 hyperimmune serum did not significantly alter these percentages. Mitogen stimulation favours EHV-1 infection in PBMC by: (i) initiating cell proliferation and (ii) inducing formation of clusters, thereby facilitating direct cell-associated transmission of virus.

Fabrication of wool keratin sponge scaffolds for long-term cell cultivation

Wool keratin sponge scaffolds were fabricated by lyophilization of an aqueous wool keratin solution after controlled freezing. Freezing at −20 °C for 3 days was needed for the preparation of stable sponges, which did not show significant changes against heat treatment at 60 °C for several hours. Scanning electron microscopic observation revealed that the wool keratin sponges had a homogeneously porous microstructure, the pore size was ≈100 μm. At 1 h from seeding, the adhesion of cells was observed and at 1 day, cells spread on the sponge surface. Rapid cell growth on the sponge (doubling time: 29.0 h) was observed for at least 7 days, as well as on a commercially available plastic culture dish (doubling time: 27.4 h). At long-term (23–43 days) cultivation, cells were constantly counted to be ≈4.2–7.4 million per sponge (1 cm in diameter). The maximum cell number was 7.4 million, ≈37 times higher than on the same area dish. Living cells on the sponge were observed at 23–43 days by SEM observation and no abnormal morphology of the cells was observed. These results show that wool keratin sponges are useful scaffolds for long-term and high-density cell cultivation.

Factors influencing the detection and enumeration of Escherichia coli O157:H7 on alfalfa seeds

Isolating Escherichia coli O157:H7 from batches of alfalfa seeds used to produce sprouts implicated in human illness has been difficult, perhaps due to nonhomogenous and very low-level contamination and inaccessibility of the pathogen entrapped in protected areas of the seed coat. We evaluated the effectiveness of various treatments in releasing E. coli O157:H7 from seeds. The influence of homogenization (blending or stomaching for 1 or 2 min), rinsing method (shaking for 5 min), soaking time (0, 1, 3, 6, or 15 h), soaking temperature (4 or 21 °C), and the addition of surfactants (0.1%, 0.5%, or 1.0% Tween 80 or Span 20) to rinse water was determined. Blending or stomaching for 1 or 2 min, and soaking for 1 h or longer at 4 or 21 °C, respectively, resulted in maximum release of E. coli O157:H7 from seeds. Soaking seeds at 37 °C for 15 h increased cell populations of E. coli O157:H7 by approximately 3.6 log 10 CFU/g, likely due to bacterial growth. The maximum number of cells released from seeds by rinse water containing 1.0% Span 20 was at 21 °C, whereas at 37 °C, 0.1% or 0.5% Tween 80 was more effective. Detection of E. coli O157:H7 on seeds stored at 37 °C for up to 13 weeks and on sprouts derived from these seeds was compared. E. coli O157:H7 inoculated on seeds at 2.0 log 10 CFU/g was detected after storage of seeds for up to 8 weeks at 37 °C and in sprouts produced from the seeds. The pathogen was not detected on seeds stored for 13 weeks at 37 °C and was not isolated from sprouts produced from these seeds. Identifying seed treatment methods that enhance removal of E. coli O157:H7 from alfalfa seeds can aid the isolation and enumeration of the pathogen on seeds. With a combination of optimal conditions for detecting the pathogen, i.e. soaking seeds for 1 h and pummeling seeds for 1 min, an enrichment step in modified tryptic soy broth (TSB), and the use of immunomagnetic beads for separation of E. coli O157:H7 cells, E. coli O157:H7 was detected in alfalfa seeds incubated at 37 °C for up to 8 weeks as effectively as in sprouts produced from the seeds.

Mitogen stimulation favours replication of equine herpesvirus-1 in equine blood mononuclear cells by inducing cell proliferation and formation of close intercellular contacts.

In the present study, equine herpesvirus-1 (EHV-1)-infected cells were identified in ionomycin/phorbol dibutyrate (IONO/PDB)-stimulated peripheral blood mononuclear cells (PBMC) and the mechanism by which stimulation increases the percentage of infected cells was examined. In the population of viral antigen-positive PBMC, 38.4+/-4.5% were CD5(+) T-lymphocytes (18.1+/-3.2% CD4(+) 13.6+/-1.8% CD8(+)), 18.1+/-5.4% were B-lymphocytes, 8.5+/-3.9% were monocytes and 35% remained unidentified. The role of the cell cycle in the increased susceptibility to EHV-1 upon stimulation was examined by stimulating PBMC for 0, 12, 24 or 36 h prior to inoculation. A high correlation was found between the increase of cells in the S- (r=0.974) and G(2)/M-phase (r=0.927) at the moment of inoculation and the increase of infected cells at 12 h post-inoculation (p.i.). This suggests that a specific stage of the S-phase or S- and G(2)/M-phase facilitates virus replication. At 24 h p.i. lower correlations were found, suggesting that other effects are involved. From 12 h after addition of IONO/PDB, formation of clusters of PBMC became manifest. We examined whether close intercellular contacts in these clusters facilitated cell-to-cell transmission of EHV-1. Between 8 and 17 h p.i., the percentage of clusters containing adjacent infected cells increased from 1.6 to 13.4% and the maximal number of adjacent infected cells increased from two to four. Confocal microscopy visualized close intercellular contacts between adjacent infected cells. It can be concluded that mitogen stimulation favours EHV-1 infection of PBMC (i) by initiating specific cell cycle events and (ii) by inducing formation of clusters, thereby facilitating transmission of virus between cells.

Pyruvate inhibits hepatic ischemia-reperfusion injury in rats.

Ischemia/reperfusion (I/R) injury is a limiting factor in liver transplantation. We have recently shown that pyruvate (PY) inhibits intestinal and renal I/R injury. This study aims to evaluate the protective effect of PY on hepatic I/R injury. ACI rats were treated with PY, whereas control animals received placebo. Rats were killed after 60 min of partial hepatic ischemia and after 2, 6, 24, and 48 hr of reperfusion. For each time point, serum aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were measured, and liver biopsy specimens were obtained to evaluate morphology, DNA fragmentation, and apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling). The survival rate 48 hr after I/R was 83% in the control group, and 100% in the PY-treated group (P>0.05). Increased enzymatic levels and histologic findings showed increased liver damage in the untreated group compared with PY. In control rats, apoptosis was enhanced after 1 hr of ischemia and peaked after 2 hr of reperfusion, to decrease gradually 48 hr after reperfusion; in the PY group apoptosis was delayed and reduced. After 1 hr of ischemia, the number of apoptotic nuclei was significantly increased in control livers compared with normal preischemic livers, whereas the number was significantly reduced by PY. After 2 hr of reperfusion, the maximum number of apoptotic cells was observed, whereas PY significantly reduced the amount of apoptotic cells (P<0.05). Apoptosis was delayed in PY-treated livers to 6 hr after reperfusion, peaking at a significantly lower count compared with placebo-treated controls (P<0.05). These data indicate that PY has a protective effect on I/R injury of the liver.

Chitin-supplemented formulations improve biocontrol and plant growth promoting efficiency of Bacillus subtilis AF 1.

Formulations of a chitinolytic biocontrol and a plant growth promoting Bacillus subtilis AF 1 were prepared in peat, in peat supplemented with either 0.5% chitin or Aspergillus niger mycelium, or in spent compost obtained from Agaricus bisporus cultivation and were evaluated for biocontrol of two fungal pathogens and plant growth promoting activities on pigeon pea and groundnut. A steady increase in cell numbers of introduced B. subtilis AF 1 was observed in all the formulations at 30 degrees C. The increase in cell numbers was about 5.0 log units. Peat or spent compost inoculated with physiologically active and dormant states of B. subtilis AF 1 showed different time period requirements to attain maximum cell numbers. The presence of chitin or A. niger (in peat) or A. bisporus (in spent compost) as supplement in the carrier material improved the multiplication of B. subtilis AF 1. When used as seed treatments, formulations of AF 1 in peat supplemented with chitin or chitin-containing materials showed better control of A. niger (causing crown rot of groundnut) and Fusarium udum (causing wilt of pigeon pea) than AF 1 culture alone, in both groundnut and pigeon pea. Bacillus subtilis AF 1 formulations promoted seed germination and biomass of both groundnut and pigeon pea even under pathogen pressure. Survival of AF 1 on fresh culture-treated and formulation product-treated plants was similar in pathogen-infested soil.

Fluctuations of the red tide flagellates Chattonella spp. (Raphidophyceae) and the algicidal bacterium Cytophaga sp. in the Seto Inland Sea, Japan

A marine algicidal gliding bacterium Cytophaga sp. strain J18/M01 was isolated in 1990 from a station in northern Harima-Nada, the Seto Inland Sea, Japan, using the harmful red tide alga Chattonella antiqua (Hada) Ono as a susceptible organism. The bacterium can prey upon various species of microalgae. Temporal fluctuations of this bacterium and Chattonella spp. [C. antiqua and C. marina (Subrahmanyan) Hara et Chihara] were investigated weekly at the above station in the summer of 1997 and 1998, using immunofluorescence assay employing highly specific polyclonal antibodies for the bacterium. In the summer of 1997, the cell density of Chattonella spp. showed a maximum value (70 cells ml−1) on 8 July, and decreased thereafter. The bacterium Cytophaga sp. J18/M01 was commonly detected around a few hundreds of cells per milliliter or less. The number of Cytophaga sp. J18/M01 increased after the peak of Chattonella spp., and the maximum cell number of the bacterium was 1350 ml−1. This algicidal bacterium also followed the changes of total amounts of microalgal biomass (chlorophyll a+pheophytin) when Chattonella spp. were absent. In the summer of 1998, Chattonella spp. were relatively less abundant (maximum 21 cells ml−1), and the algicidal bacterium Cytophaga sp. J18/M01 showed a close relationship with the change of total microalgal biomass. The present study suggests that the algicidal bacterium Cytophaga sp. J18/M01 preyed upon, not only harmful red tide microalgae, but also other common microalgae such as diatoms, and the bacterium presumably plays an important role in regulating microalgal biomass in natural marine environments.