Oxidised glutathione reductase activity in mouse epidermis: TPA induced change and its modulation by vitamin A

Single cutaneous application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) increased epidermal oxidised glutathione reductase activity in adult mouse by almost 100%. Pretreatment of animals with vitamin A for a week resulted in 75% inhibition of TPA induced change in the enzyme activity which remained unaffected in skin treated with vitamin A alone. This biochemical change in skin induced by TPA and modulated by vitamin A has been discussed in relation to epidermal hyperplasia.     

Interrelation of active oxygen species,membrane damage and altered calcium functions

Incubation of freshly isolated rat liver mitochondria in the presence of oxygen free radical generating hypoxanthine —xanthine oxidase system led to swelling of mitochondria as measured by the change in optical density, which was reversed by the addition of superoxide dismutase. O₂ ^– in the presence of CaCl₂ enhanced the peroxidative decomposition of mitochondrial membrane lipids along with swelling of the organelle. Free radical generation led to enhancement of monoamine oxidase activity while glutathione peroxidase and cytochrome c oxidase were inhibited. Tertbutyl hydroperoxide (t-BHP) caused mitochondrial swelling through oxidative stress. Incorporation of ruthenium red, which is a Ca²⁺ transport blocker, during assay abolished peroxidative membrane damage and swelling. Dithiothreitol (DTT) accorded protection against t-BHP induced mitochondrial swelling. The above in vitro data suggest a possible interrelationship of active oxygen species, membrane damage and calcium dynamics.     

Role of calcium channel in postvagal potentiation of contraction as evident from the effects of Mn2+, La3+, D-600, and deoxycholate on isolated guinea pig atria-vagus nerve preparation

The role of the calcium channel in the first large contraction (postvagal potentiation, PVP) of the atria at the end of the inhibitory phase of its response (IPR) to vagal stimulation has been investigated by studying the effects of agents acting on the calcium channel (e.g., Ca2+, Mn2+, La3+, and D-600) or sarcoplasmic reticulum (SR) (e.g., deoxycholate (DOC)). IPR was potentiated by high [Ca2+]o (3-16 mM) and also by the calcium channel blockers, Mn2+ (1 microM-0.5 mM), La3+ (0.1 microM-0.5 mM), D-600 (1.0-10 microM), and DOC (1 microM-0.5 mM). PVP was also potentiated by enhanced [Ca2+]o, but the PVP ratio, which employs a correction for the simultaneous changes in the force of spontaneous contraction was inhibited. This indicated greater potentiation of contractility during spontaneous activity by Ca2+ than during PVP. Mn2+, La3+, and D-600 and even DOC in the above concentrations inhibited PVP but increased the PVP ratio. High concentrations of DOC (greater than 1 mM), which disrupt SR, strongly inhibited PVP. It is concluded that the calcium channel plays a more prominent role in spontaneous contractions than in PVP in guinea pig atria. PVP is suggested to be generated by excessive triggered release of Ca2+ from SR leading to a marked increase in [Ca2+]i. The calcium channel and the calcium trapped in the glycocalyx also play significant roles in PVP.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Relationship between Endopolyploidy and Cell Size in Epidermal Tissue of Arabidopsis

Relative quantities of DNA in individual nuclei of stem and leaf epidermal cells of Arabidopsis were measured microspectrofluorometrically using epidermal peels. The relative ploidy level in each nucleus was assessed by comparison to root tip mitotic nuclei. A clear pattern of regular endopolyploidy is evident in epidermal cells. Guard cell nuclei contain levels of DNA comparable to dividing root cells, the 2C level (i.e., one unreplicated copy of the nuclear DNA). Leaf trichome nuclei had elevated ploidy levels of 4C, 8C, 16C, 32C, and 64C, and their cytology suggested that the polyploidy represents a form of polyteny. The nuclei of epidermal pavement cells were 2C, 4C, and 8C in stem epidermis, and 2C, 4C, 8C, and 16C in leaf epidermis. Morphometry of epidermal pavement cells revealed a direct proportionality between nuclear DNA level and cell size. A consideration of the development process suggests that the cells of highest ploidy level are developmentally oldest; consequently, the developmental pattern of epidermal tissues can be read from the ploidy pattern of the cells. This observation is relevant to theories of stomate spacing and offers opportunities for genetic analysis of the endopolyploidy/polyteny phenomenon.     

Intraspecific diversity in the fungal speciesChaunopycnis alba: Implications for microbial screening programs

Intraspecific variation among 84 isolates of the anamorphic fungusChaunopycnis alba from 26 different geographical locations was analyzed by investigating optimal growth temperatures, differences in the production of secondary metabolites and presence or absence of the cyclosporin synthetase gene. The genetic diversity was assessed using random amplified polymorphic DNA (RAPD). Analysis of these data showed high genetic, metabolic and physiological diversity within this species. Isolates from the Antarctic represented the most homogeneous group withinC. alba and together with isolates from the Arctic these polar strains differed from alpine, temperate and tropical strains by low optimal growth temperatures and by low production of secondary metabolites. Isolates from tropical climes were characterized by high optimal growth temperatures and by the production of comparatively diverse metabolite spectra. Most of the isolates that were similar in the combination of their physiological and metabolic characters were also genetically related. Isolates from different geographical origins did not show many similarities, with the exception of the cyclosporin A-producing isolates, and large diversity could be observed even within a single habitat. This leads us to the suggestion that for pharmaceutical screening programs samples should be collected from a diversity of different geographical and climatic locations. For the selection of strains for screening the RAPD assay seems to be the most powerful tool. It reflected the highest intraspecific diversity and the results corresponded well with the other characteristics.