Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.     

Somatic embryogenesis and shoot regeneration from intact seedlings of Phaseolus acutifolius A., P. aureus (L.) Wilczek,P. coccineus L., and P. wrightii L.

A rapid, one-step procedure has been developed for inducing direct organogenesis and somatic embryogenesis in cultures of Phaseolus coccineus L., P. acutifolius A., P. aureus L. [Vigna radiata L. Wilczek] and P. wrightii L. Development of somatic embryos and shoot buds occurred within 6–8 weeks of culture from intact seedlings raised on MS (Murashige and Skoog 1962) medium supplemented with N⁶-benzylaminopurine (BAP). Shoot buds or embryoids originated from subepidermal tissue of the regions adjacent to the shoot apex, hypocotyl and cotyledonary axils. While P. acutifolius and P. aureus were regenerated via shoot formation and P. wrightii by somatic embryogenesis, both embryogenesis and shoot regeneration were observed in P. coccineus. Relatively higher levels of BAP, 50–80 M, were found to be optimal for inducing regeneration while lower concentrations were ineffective. About 40–70 shoots and 70–250 somatic embryos were produced per responding seedling. Regenerated shoots and somatic embryos developed into whole plants on a basal medium or the one supplemented with 1 M naphthaleneacetic acid.     

The occurrence of long chain polyprenols in leaves of plants of Rosaceae family and their isolation by time-extended liquid chromatography.

The long chain polyprenols composed of 30 and more isoprene units from leaves of plants belonging to the genera Potentilla and Rosa have been described. They occur in the form of fatty acid esters. The composition of polyprenol mixture was species dependent and its content reached ca. 0.5% wet weight. Large scale preparation of individual polyprenols from a natural polyprenol mixture was performed using time-extended liquid chromatography on the hydrophobic gel Lipidex-5000.     

Stress studies in lentil (Lens esculenta Moench)

The response of soil exchangeable sodium percentage levels to nitrate reductase activity, nitrite reductase activity, free proline, DNA, RNA, chlorophyll a and b contents and yield components in lentil (Lens esculenta Moench)cv. PL 406 was studied in a replicated pot experiment. All the biochemical observations were recorded at four growth stages i.e. 30, 60, 90 and 120 days after sowing (DAS). Germination occurred up to exhangeable sodium percentage of 30, but plants survived only up to 25. With increasing exchangeable sodium percentage, there was a continuous decrease in chlorophyll a and b content, nitrate and nitrite reductase enzyme activities and DNA and RNA content. Increasing level of sodicity enhanced the free proline content up to 60 DAS, after which values fell.Number of pods per plant, 1000 grain weight and grain yield were significantly reduced with increasing level of sodicity, but the number of grains per pod was not affected.     

Flavonoid glycosides from Cassia alata.

Two new glycosides, chrysoeriol-7-O-(2"-O-beta-D-mannopyranosyl)-beta-D-allopyranos ide and rhamnetin-3-O-(2"-O-beta-D-mannopyranosyl)-beta-D-allopyranosid e, were isolated from the seeds of Cassia alata. The structures were established on the basis of chemical evidence and spectroscopic methods, especially NMR.     

Embryo transfer as a means of controlling the transmission of viral infections. XIII. Failure to transmit foot-and-mouth disease virus through the transfer of embryos from viremic donors

A total of 436 embryos/unfertilized ova was collected from 30 foot-and-mouth disease (FMD) viremic cattle; 106 of these embryos/ova were from eight donors that had FMD virus in their reproductive tracts. The 436 embryos/ova were washed and then either assayed in cell culture or intradermally in steer tongues or transferred to recipients. Foot-and-mouth infectivity was not found to be associated with any of the embryos/ova assayed in cell culture or intradermally. The 149 embryos transferred produced two abortions, five sets of twins born prematurely, and 15 normal calves. All of the recipients and all of the calves remained FMD-seronegative.     

Shift in product formation from acetate to ethanol using metabolic inhibitors inFusarium oxysporum

Summary Fusarium oxysporum 841 produces a mixture of ethanol and acetic acid from glucose, xylose or Avicel (microcrystalline cellulose) substrates. Some metabolic inhibitors viz. sodium azide, dinitrophenol and polyethylene glycol were used for shifting product formation from acetic acid to ethanol. Using these inhibitors 1.5- to 2- fold increase in ethanol production was achieved with significant repression (by 80 to 90%) of acetic acid. Almost theoretical yields of ethanol were obtained.     

Evidence for a role of glutamine synthetase in assimilation of amino acids as nitrogen source in the cyanobacterium Nostoc muscorum.

Methylammonium/ammonium ion, glutamine, glutamate, arginine and proline uptake, and their assimilation as nitrogen sources, was studied in Nostoc muscorum and its glutamine synthetase-deficient mutant. Glutamine served as nitrogen source independent of glutamine synthetase activity. Glutamate was not metabolised as a nitrogen source but still inhibited nitrogenase activity and diazotrophic growth. Glutamine synthetase activity was essential for the assimilation of N2, ammonia, arginine and proline as nitrogen sources but not for the control of their transport, heterocyst formation, and production of ammonia or aminoacid dependent repressor signal for N2-fixing heterocysts. These results also suggest that glutamine synthetase serves as the sole route of ammonia assimilation and glutamine synthesis, and ammonia per se as the repressor signal for N2-fixing heterocysts and methylammonium (ammonium) transport.     

Methyl mercury uptake by free and immobilized cyanobacterium

Methyl mercury uptake in free cells and different immobilizates of the cyanobacteriumNostoc calcicola has been examined. The general growth of the immobilized cyanobacterial cells could be negatively correlated with methyl mercury uptake. Alginate spheres proved most efficient in terms of uptake rate (0.48 nmol mg protein^–1 min^–1, 10 min) and total bioaccumulation (10.71 nmol mg protein^–1, 1 h) with a bioconcentration factor of 3.3×10³. Alginate biofilms showed a faster methyl mercury accumulation rate (0.83 nmol mg protein^–1 min^–1, 10 min) with a saturation of 10.28 nmol mg protein^–1 reached within only 30 min (bioconcentration factor, 3.1×10³). Foam preparations with a slow initial uptake approximated biofilms but were characterized by a lower bioconcentration factor (2.8×10³). Free cells, in comparison, maintained the initial slow rate of uptake (0.62 nmol mg protein^–1 min^–1, 10 min), saturating at 30 min (8.81 nmol mg protein^–1), and the resultant lowest bioconcentration factor (2.7×10³). Cell ageing (30 days) brought a drastic reduction (3-fold) in organomercury uptake by free cells while alginate spheres maintained the same potential. Foam preparations of the same age showed a significant improvement in methyl mercury uptake followed by only a marginal decline in alginate biofilms. Data are discussed in the light of the physiological efficiency and longevity of immobilized cells.