Micropropagation of Rollinia mucosa (JACQ.) baill

Summary A protocol for in vitro propagation of Rollinia mucosa, an important medicinal plant, was developed. The presence of 500 mg l⁻¹ polyvinylpyrrolidone (PVP) during explant excision was important to avoid browning. Axillary buds, adventitious buds, and shoot cluster proliferation were achieved from epicotyl and hypocotyl explants from nursery-grown seedlings. The highest direct organogenesis percentage from hypocotyl explants was obtained upon culture of explants on Murashige and Skoog medium supplemented with 2.2 μM benzyladenine (BA) plus 2.32 μM kinetin. Epicotyl explants display highest regeneration frequency on a medium containing 8.8 μM BA and 0.54 μM naphthaleneacetic acid. Gibberellic acid was necessary for shoot elongation. Root induction was observed when shoots were pretreated with activated charcoal for 7 d in the dark before culture on Woody Plant Medium supplemented with 49.21 μM indolebutyric acid for 10 d. Root development was observed when 20 g l⁻¹ sucrose was used. Rooted plantlets were acclimatized and grown in the greenhouse.     

Depletion of taurine in the adult rat retina

Taurine is the major free amino acid of the vertebrate retina. Treatment of rats with guanidinoethyl sulfonate (GES), a taurine analogue which competes with taurine for transport sites, leads to depletion of 60% of retinal taurine with little effect on other free amino acids. Supplementation of the diet with 0.3% taurine gives partial protection against depletion, confirming that taurine-GES competition underlies part of the effects. The magnitude of the depletion suggests the importance of taurine transport across the blood-retinal barrier for the maintenance of retinal taurine levels.     

Medicinal plants of the Argentine Yungas plants of the Las Yungas biosphere reserve, Northwest of Argentina, used in health care

We have compared the species richness of medicinal plants and the differential patterns of use amongst settlements in the Andean communities of Northwest Argentina which have differing levels of isolation. About 259 ethnoespecies, belonging to 74 plant families, were included, representing between 70 and 80% of the total estimate. The results indicate that Coronopus didymus is the most relevant and important species. The method of use of medicinal plants and the ailments treated by rural doctors compared to those of the layperson is different. Native and exotic plants are used differently according to the body system treated. There are some relationships between internal and external use and body systems and recipes. The greater medicinal species richness found in the less isolated locations is due to external enriching cultural influences.     

Continuous-flow NMR culture system for mammalian cells

A continuous-flow NMR culture system for mammalian cells has been developed on which ³¹P-NMR experiments under complete and strictly physiologic conditions have been performed. Observations on the response of the cellular metabolism to stresses such as starvation, low temperature and changes in environmental pH monitored by ³¹P-NMR are reported. The response of the intracellular pH relative to the external pH of the growth medium is studied. We find that under the experimental conditions used there exists a ΔpH varying between less than 0.2 and more than 0.6 pH units. These results are compatible with those obtained using other techniques.     

Trypsin and α-amylase inhibitors are differentially induced in leaves of amaranth (Amaranthus hypochondriacus) in response to biotic and abiotic stress

Seeds of Amaranthus hypochondriacus L. are known to accumulate a trypsin-inhibitor (ATI) member of the potato-I inhibitor family and an α -amylase inhibitor (AAI), possessing a knottin-like fold. They are believed to have a defensive role due to their inhibition of trypsin-like enzymes and α -amylases of insect pests. In this work, both inhibitory activities were found in leaves of young A. hypochondriacus plants. High constitutive levels of foliar inhibitory activity against bovine trypsin and insect α -amylases were detected in in vitro assays. Trypsin inhibitory activity was further increased by exposure to diverse treatments, particularly water stress. Salt stress, insect herbivory and treatment with exogenous methyl jasmonate (MeJA) or abscisic acid (ABA) also induced trypsin inhibitor activity accumulation, although to a lesser degree. In gel and immunoblot analyses showed that foliar trypsin inhibitor activity was constituted by at least three different inhibitors of approximately 29, 8 (including ATI) and 3 kDa, respectively. These inhibitors showed differing patterns of accumulation in response to diverse treatments. On the other hand, significant increases in α -amylase inhibitor activity and AAI levels were detected in leaves of insect-damaged, MeJA- and ABA-treated A. hypochodriacus plantlets, but not in those subjected to water- or salt-stress. A differential induction of trypsin inhibitor activity and α -amylase inhibitor accumulation in response to insect herbivory by two related species of lepidopterous larvae was observed, whereas mechanical wounding failed to induce either inhibitor. The overall results suggest that trypsin and α -amylase inhibitors could protect A. hypochondriacus against multiple types of stress.     

Isolation of high molecular weight DNA from wholeEuglena cells

Summary A method for isolating high quality DNA from wholeEuglena cells is described. The procedure consists in: the weakening of the cell pellicle in glycerol avoiding the mechanical disruption of cells and shearing damage in DNA molecules; the decondensation ofEuglena compact chromatin directly inside the cells; the complete dissociation of cells and nucleoproteins in sarkosyl detergent; the optional digestion of proteins and RNA with DNase-free enzymes and the final purification of DNA by isopycnic banding in CsCl gradients. Degradation of DNA is prevented all along the extraction procedure by glycerol, antioxydants, EDTA and sarkosyl detergent. Using the enzymatic digestion step, DNA containing few single-stranded nicks is obtained with a yield approaching 100%. DNA with no single-stranded nick could be obtained with a 35% yield when the enzymatic digestion step was omitted. In both cases, the double-stranded DNA has an average molecular weight equal or greater than 6×10⁷. It is free of contaminants and could be easily digested with restriction enzymes. After digestion with Eco RI and size-fractionation in agarose gel this DNA has permitted specific hybridization of the rDNA sequences with a radioactive rRNA probe.Abbreviations Kbp kilobasepairs - Kb kilobases     

Effects of growth state and amines on cytoplasmic and vocuolar pH, phosphate and polyphosphate levels in Saccharomyces cerevisiae: a P-nuclear magnetic resonance study

The vacuoles of logarithmic and stationary stage cells were compared by ³¹P-NMR with regard to pH, orthophosphate (P_i) content and average size of polyphosphate. The vacuoles of stationary cells had lower pH higher P_i content, and polyphosphates of longer average chain lenght, although total polyphosphate content was about the same as in logarithmic cells. The lower vacuolar pH in stationary cells was the major cause of a larger cytoplasmic-vacuolar pH gradient. Addition of NH₄Cl, (NH₄)₂SO₄, methylamine or amantadine at pH 8 to cells in either stage caused an icnrease in both cytoplasmic and vacuolar pH, with little or no change in the cytoplasmic-vacuolar pH gradient. However, the administration of ammonium salts to the cells at pH 8.0 resulted in rapid hydrolysis of the intravacuolar polyphosphate to tripolyphosphate and P_i, with attendant redistribution of P_i between the vacuolar and cytoplasmic compartments.     

Comparative mutagenicity testing of a drug candidate, U-48753E: mechanism of induction of gene mutations in mammalian cells and quantitation of potential hazard

U-48753E is a potential human drug which was subjected to a battery of short-term assays for genetic activity. The compound was negative in the Salmonella (Ames) test, the in vitro UDS assay, the mouse bone-marrow micronucleus test and the Drosophila sex-linked recessive lethal assay. However, it was weakly positive in the CHO/HPRT assay in the presence of metabolic activation (S9). The weak positive response might easily have been labeled artifactual since there was no dose response and the dose level producing positive findings varied from experiment to experiment. In addition, the weak positive response was not confirmed in V79 cells. However, a reproducible dose-related increase in mutants was observed in the AS52/XPRT assay in the presence of S9. Metabolism of this drug proceeds through conversion of aliphatic N-methyl groups to formaldehyde. Addition of formaldehyde dehydrogenase to the S9 resulted in elimination of the mutagenicity of the compound in AS52 cells. Thus, the mutants were probably induced by formaldehyde. From the endogenous levels of formaldehyde in human blood, and the limiting potential therapeutic dose levels, the genotoxic hazard associated with U-48753E is marginal. This assessment of risk and its quantitation depend upon an understanding metabolism and exposure limits imposed by known side effects of the drug. This study can serve as a model for quantitative genetic risk assessment when mutagenicity is due to N-demethylation and formation of formaldehyde in situ.     

Flash fluorometer made from off-the-shelf photographic equipment to measure tissue levels of fluorescein

A device to measure fluorescein in tissue has been constructed from standard photographic equipment--an electronic strobe and a flashmeter both covered with interference filters. The instrument works well in the light and need not touch the area being measured, an advantage over existing fluorometers. The instrument has been used to measure the amount of dye in flaps in rats, pigs, and three humans. The results revealed that the amount of dye in a freshly made flap was rarely as much as in normal skin, and skin with less than 20 percent of the dye of control areas usually sloughed, although there were exceptions. In the future the instrument will be improved, and its readings will be compared to those obtained from radioactive microspheres, the present "gold standard" of techniques to measure vascularity. The instrument can be used to estimate the blood supply to any tissue and seems to be as reliable as the dermofluorometers already on the market.     

The dentition of fallow deer (Dama dama): a scoring scheme to assess age from wear of the permanent molariform teeth

The sequence of tooth wear was determined from skulls of fallow deer of known age. A system for scoring molariform tooth wear has been devised so that small but readily recognizable wear changes of the individual cusps may be recorded and used to assess the age of animals of unknown birth dates. The technique can be readily adapted for other ruminant species with the appropriate database.