Assessing the tumorigenic phenotype of VERO cells in adult and newborn nude mice.

VERO cell lines are important substrates for viral vaccine manufacture. The mechanism by which these cells became neoplastically transformed is unknown. During tissue-culture passage, VERO cells can develop the capacity to form tumors. Although at the passage levels (around p140) currently used for vaccine manufacture, VERO cells are non-tumorigenic, questions have been raised about safety issues that might be associated with this capacity to acquire a tumorigenic phenotype. To begin to address these issues, the tumorigenicity of VERO cell lines, derived at different passage levels under different growth conditions, were evaluated in 365-day assays in adult and newborn nude mice. High passage (p>200) VERO cell lines established by random passaging in tissue culture produced tumors in adult (10 out of 27) mice and newborn (21 out of 30) mice, respectively. In contrast, a high passage (p>250) cell line established by passage at sub-confluence produced tumors only in newborn mice (16 out of 30). Progressively growing tumors began forming at 36 days in newborns and at 69 days in adults. Higher tumor incidences and shorter tumor latencies suggest that newborn nude mice may be more sensitive than adults in detecting the expression of a tumorigenic phenotype by some VERO cell lines.     

Isolation and characterization of a novel banana rhizosphere bacterium as fungal antagonist and microbial adjuvant in micropropagation of banana

AIM: Isolation and characterization of a bacterial isolate (strain FP10) from banana rhizosphere with innate potential as fungal antagonist and microbial adjuvant in micropropagation of banana. METHODS AND RESULTS: Bacterium FP10 was isolated from the banana rhizosphere and identified as Pseudomonas aeruginosa based on phenotypic, biochemical traits and sequence homology of partial 622-bp fragment of 16S ribosomal DNA (rDNA) amplicon, with the ribosomal database sequences. Strain FP10 displayed antibiosis towards fungi causing wilt and root necrosis diseases of banana. Production of plant growth hormone, indole-3-acetic acid (IAA), siderophores and phosphate-solubilizing enzyme in FP10 was determined. Strain FP10 tested negative for hydrogen cyanide, cellulase and pectinase, the deleterious traits for plant growth. Screening of antibiotic genes was carried out by polymerase chain reaction using gene-specific primers. Amplification of a 745-bp DNA fragment confirmed the presence of phlD, which is a key gene involved in the biosynthesis of 2,4-diacetylphloroglucinol (DAPG) in FP10. The antibiotic produced by FP10 was confirmed as DAPG using thin layer chromatography, high performance liquid chromatography and Fourier transform infrared and tested for fungal antibiosis towards banana pathogens. Procedures for encapsulation of banana shoot tips with FP10 are described. CONCLUSIONS: Strain FP10 exhibited broad-spectrum antibiosis towards banana fungi causing wilt and root necrosis. DAPG by FP10 induced bulb formation and lysis of fungal mycelia. Encapsulation of banana shoot tips with FP10 induced higher frequency of germination (plantlet development) than nontreated controls on Murashige and Skoog basal medium. Treatment of banana plants with FP10 enhanced plant height and reduced the vascular discolouration as a result of Fusarium oxysporum f. sp. cubense FOC. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the innate potential of fungal antibiosis by DAPG antibiotic and production of siderophore, plant-growth-promoting IAA and phosphatase, the strain FP10 can be used as biofertilizer as well as a biocontrol agent.     

L-NAME does not affect exercise-induced pulmonary hypertension in Thoroughbred horses

The present study was carried out to examine theeffects of nitric oxide synthase inhibition withN-nitro-L-arginine methyl ester(L-NAME) on the right atrial as well as on the pulmonary arterial, capillary, and venous blood pressures of horses during rest and exercise performed at maximal heartrate (HR_max). Experiments werecarried out on seven healthy, sound, exercise-trained Thoroughbredhorses. Using catheter-tip manometers, with signals referenced at thepoint of the shoulder, we determined phasic and mean right atrial andpulmonary vascular pressures in two sets of experiments [control(no medications) and L-NAME (20 mg/kg iv given 10 min before exercise studies)]. The studies werecarried out in random order 7 days apart. Measurements were made atrest and during treadmill exercise performed on a 5% uphill grade at6, 8, and 14.2 m/s. Exercise on a 5% uphill grade at 14.2 m/s elicitedHR_max and could not be sustainedfor >90 s. In quietly standing horses,L-NAME administration caused asignificant rise in right atrial, as well as pulmonary arterial, capillary, and venous pressures. This indicates that nitric oxide synthase inhibition modifies the basal pulmonary vasomotor tone. Inboth treatments, exercise caused progressive significant increments inright atrial and pulmonary vascular pressures, but the values recordedin the L-NAME study were notdifferent from those in the control study. The extent ofexercise-induced tachycardia was significantly decreased in theL-NAME study at 6 and 8 m/s butnot at 14.2 m/s. Thus, L-NAMEadministration may not modify the equine pulmonary vascular tone duringexercise at HR_max. However, asindicated by a significant reduction in heart rate,L-NAME seems to modify thesympathoneurohumoral response to submaximal exercise.

     

Differential regulation of antioxidant enzymes in response to oxidants.

We have demonstrated the selective induction of manganese superoxide dismutase (MnSOD) or catalase mRNA after exposure of tracheobronchial epithelial cells in vitro to different oxidant stresses. Addition of H2O2 caused a dose-dependent increase in catalase mRNA in both exponentially growing and confluent cells. A 3-fold induction of catalase mRNA was seen at a nontoxic dose of 250 microM H2O2. Increase in the steady-state mRNA levels of glutathione peroxidase (GPX) and MnSOD were less striking. Expression of catalase, MnSOD, and GPX mRNA was highest in confluent cells. In contrast, constitutive expression of copper and zinc SOD (CuZnSOD) mRNA was greatest in dividing cells and was unaffected by H2O2 in both exponentially growing and confluent cells. MnSOD mRNA was selectively induced in confluent epithelial cells exposed to the reactive oxygen species-generating system, xanthine/xanthine oxidase, while steady-state levels of GPX, catalase, and CuZnSOD mRNA remained unchanged. The 3-fold induction of MnSOD mRNA was dose-dependent, reaching a peak at 0.2 unit/ml xanthine oxidase. MnSOD mRNA increases were seen as early as 2 h and reached maximal induction at 24 h. Immunoreactive MnSOD protein was produced in a corresponding dose- and time-dependent manner. Induction of MnSOD gene expression was prevented by addition of actinomycin D and cycloheximide. These data indicate that epithelial cells of the respiratory tract respond to different oxidant insults by selective induction of certain antioxidant enzymes. Hence, gene expression of antioxidant enzymes does not appear to be coordinately regulated in these cell types.     

Identification of Peanut Green Mosaic Virus Strains in India*

During field surveys, three peanut green mosaic virus isolates differing in symptomatology on groundnut and a few other hosts were collected. Ultrathin sections of infected groundnut leaflets showed cytoplasmic inclusions with pin wheels and scrolls. In enzyme-linked immunosorbent assay they reacted strongly with antisera to peanut green mosaic and soybean mosaic virus antisera, and moderately with adzuki bean mosaic and peanut stripe virus antisera. All isolates also reacted positively with antisera to peanut eye spot, blackeye cowpea mosaic, pea seed-borne mosaic, potato virus Y and tobacco etch viruses, and did not react with antisera to peanut mottle, bean yellow mosaic, bean common mosaic, clover yellow vein and sugarcane mosaic viruses. SDS-PAGE analysis of purified virus preparations of the three isolates showed a single polypeptide with mol. wt. of 34,500 daltons. Based on these results, the three isolates are identified as biologically distinct strains of peanut green mosaic virus.     

Rna polymerase from opaque-2 and normal Zea mays endosperm

RNA polymerase from Opaque-2 and normal maize showed qualitative differences during endosperm development. DEAE-Sephadex column chromatography indicated the presence of one and three RNA polymerases respectively at 15 and 25 days post-pollination. The polymerases from Opaque-2 and normal endosperms at 15 days post-pollination showed considerable differences in Mn²⁺ optimum. The optimum Mn²⁺ for normal polymerase was ten times higher than for Opaque-2 polymerase. The polymerase activity from endosperms at 15 days post-pollination was due to nucleoplasmic RNA polymerase II.     

Glycosidases. Ligands for affinity chromotagraphy: II. Syntheses of p-aminophenyl 1-thio-L-fucopyranosides

Syntheses of p-aminophenyl 1-thio-α-L- and β-L-fucopyranosides are described. 1,2,3,4-Tetra-O-acetyl-α-L-fucopyranose, on heating with p-nitrothiophenol in the presence of p-toluenesulfonic acid under diminished pressure, gave a mixture of p-nitrophenyl 2,3,4-tri-O-acetyl-1-thio-α- and β-L-fucopyranosides, which was separated by chromatography on silica gel. When the reaction was carried out in the presence of zinc chloride at atmospheric pressure, the β-anomer was the exclusive product. Deacetylation of the aryl α-L- and α-L-thiofucopyranosides with sodium methoxide, followed by catalytic hydrogenation in the presence of palladium on barium sulfate, afforded the respective aminophenyl 1-thiofucopyranosides. The aryl thiofucopyranosides thus synthesized were tested for their inhibitory activity toward clam α-L-fucosidase. The p-aminophenyl 1-thio α-L-fucopyranoside showed a competitive-type inhibition, with a K_i of 0.71mM.     

Influence of dietary culture material containing aflatoxin and T<Subscript>2</Subscript> toxin on certain serum biochemical constituents in Japanese quail

Forty newly hatched unsexed Japanese quail (Coturnix coturnix japonica) chicks were fed diets containing 3 ppm aflatoxin (AF) and 4 ppm T₂ toxin either singly and in combination for 35 days. Sera samples were collected from six birds in each group at the end of the trial to study the effect of certain serum biochemical parameters. Highly significant (P < 0.01) differences were observed for serum total protein, albumin, globulin, glucose, cholesterol, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT) between control and toxin treated groups. Toxin treated groups did not reveal any significant difference for serum urea nitrogen, creatinine, uric acid, calcium, phosphorus, sodium and potassium. There was a reduction in the levels of serum total protein, albumin, globulin, glucose, cholesterol, ALT and elevation of AST and GGT and variable ALP levels observed in toxin treated groups. Forms part of M.V.Sc. thesis of first author approved by the Tamil Nadu Veterinary and Animal Sciences University, Chennai 600 051, India.