Chromatographic separation of DNA dependent RNA polymerases and molecular properties of RNA polymerase II from a Leishmania Spp

Multiple forms of DNA-dependent RNA polymerases have been isolated and characterized from Leishmania strain UR6 promastigotes. RNA polymerases from this organism fail to resolve into multiple forms by conventional chromatography on DEAE-Sephadex A25, but could be separated by a modification of the method using CM-Sephadex C25. The CM-Sephadex bound enzyme is resistant toamanitin even up to a concentration of 250g/ml. The activity which flows through CM-Sephadex further resolves into two forms upon chromatography on DEAE-Sephadex A25. These forms are sensitive to -amanitin to different extent. Enzyme activity in peak I is 50% inhibited by 3g/ml and in peak II by 50g/ml of the drug respectively. The enzyme in peak I has been further purified by heparin agarose and fast performance liquid chromatography (FPLC) on MonoQ. The enzyme has Stoke's radius of 70å, a sedimentation coefficient of 17.6S and an f/fo of 1.35. Analysis of ammonium sulfate and met n peak I, relative activities with Mn+2 versus Mg+2 and template specificities gave results similar to those reported for other type II RNA polymerases in eukaryotes. The MonoQ purified enzyme resolves into 16 polypeptides on denaturing polyacrylamide gel and densitometric analysis suggests that 9 major bands are present in the stoichiometry expected of RNA polymerase subunits having molecular weights: 154000; 104000; 77000; 64000; 52000; 48000; 46000; 45000 and 39000 respectively.     

The aPKC/Par3/Par6 Polarity Complex and Membrane Order Are Functionally Interdependent in Epithelia During Vertebrate Organogenesis

The differential distribution of lipids between apical and basolateral membranes is necessary for many epithelial cell functions, but how this characteristic membrane organization is integrated within the polarity network during ductal organ development is poorly understood. Here we quantified membrane order in the gut, kidney and liver ductal epithelia in zebrafish larvae at 3–11 days post fertilization (dpf) with Laurdan 2‐photon microscopy. We then applied a combination of Laurdan imaging, antisense knock‐down and analysis of polarity markers to understand the relationship between membrane order and apical‐basal polarity. We found a reciprocal relationship between membrane order and the cell polarity network. Reducing membrane condensation by exogenously added oxysterol or depletion of cholesterol reduced apical targeting of the polarity protein, aPKC. Conversely, using morpholino knock down in zebrafish, we found that membrane order was dependent upon the Crb3 and Par3 polarity protein expression in ductal epithelia. Hence our data suggest that the biophysical property of membrane lipid packing is a regulatory element in apical basal polarity.     

The relationship between heat-stress and photobleaching in green and blue-green algae

Two characteristic temperatures were identified from measurements of the temperature dependence of O₂ evolution by Chlorella vulgaris and Anacystis nidulans: T₁, the threshold temperature for inhibition of O₂ evolution under saturating light conditions, and T₂, the upper temperature limit for O₂ evolution. Measurement of delayed light emission from photosystem II (PSII) showed that it passed through a maximum at T₁ and was virtually eliminated on heating the samples to T₂. Related changes were observed in low-temperature (77K) fluoresence emission spectra. Heat-stress had little effect on the absorption properties of the cells at temperatures below T₁ but incubation at higher temperatures, particularly under high-light conditions, resulted in extensive absorption losses. An analysis of these measurements suggests that this increased susceptibility to photobleaching is triggered by an inhibition of the flow of reducing equivalents from PSII that normally serves to protect the light-harvesting apparatus of the cells from photo-oxidation. Adaptation to higher growth temperatures resulted in increases in the values of T₁ and T₂ for Anacystis nidulans but not for Chlorella vulgaris.Abbreviations PSI photosystem I - PSII photosystem II - Chl a chlorophyll a - Chl b chlorophyll b - DCMU 3-(3 4 dichlorophenyl)-11-dimethylurea - PC plastocyanin - APC allophycocyanin CIW-DPB Publication No. 887.     

The effect of calcium on the bilayer stability of lipids from bovine rod outer segment disk membranes

The phase behavior of bovine rod outer segment disk lipids has been investigated using freeze-fracture and ³¹P nuclear magnetic resonance (NMR) techniques. ³¹P-NMR spectra of isolated disk membranes were taken as a function of temperature between 25°C and 45°C. The ³¹P-NMR spectrum characteristic of phospholipid bilayers was observed at all temperatures both in the absence of Ca²⁺ and in the presence of 10 mM and 50 mM Ca²⁺. A similar study was performed on lipids isolated from the disk membranes. In the absence of Ca²⁺ only lamellar phase behavior was observed. In the presence of less than 10 mM Ca²⁺, however, there was a change in morphology to non-lamellar structures. Removal of the Ca²⁺ caused the system to reassume the lamellar form.     

Polyamine metabolism in Ancylostoma ceylanicum and Nippostrongylus brasiliensis

Spermidine was detected as the major polyamine of Ancylostoma ceylanicum as well as Nippostrongylus brasiliensis. Spermine was present in lower amounts whereas the level of putrescine was even less. S-Adenosylmethionine decarboxylase, a rate-limiting enzyme in the biosynthetic pathway of polyamines, was demonstrated at low levels in both parasites. Decarboxylation of lysine and arginine was absent or negligible and that of ornithine questionable, as the enzyme activity was not inhibited by alpha-difluoromethylornithine while RMI 71,645, an irreversible inhibitor of ornithine aminotransferase, strongly inhibited the liberation of CO2 from ornithine. High activity of ornithine aminotransferase was observed in both the parasites and may interfere with the assay for ornithine decarboxylase. Adults of A. ceylanicum were found to rapidly take up spermidine and spermine from incubation medium while uptake of putrescine was very low. These results indicate that hookworms depend on uptake and interconversion rather than de novo synthesis for their polyamine requirement.     

Evaluation of radical scavenging potential and total phenol content in Woodfordia fruticosa from different altitudes

Woodfordia fruticosa Kurz is a member of family Lythraceae, commonly used in the treatment of diseases like leucorrhoea, dysentery, leprosy and menorrhagia. The plant material such as leaves, bark and flowers were collected from three different (200, 402 and 600?m) altitudes of Khandala (M.S. India) and the extracts were prepared in solvents like methanol, ethanol and distilled water. The radical scavenging potential and total phenolic content of the extracts were evaluated. The methanol extract of bark collected from the location one (200?m) showed high radical scavenging activity (96.52?±?0.02) than distilled water and ethanol extracts (57.80?±?0.2 and 86.52?±?0.03). The bark of the plant showed highest (663?±?37.85) total phenolic content that of flowers and leaves. The methanol extracts of leaves, bark and flowers showed high tannic acid content, while TBARS assay of ethanol extract of flowers showed maximum protection (27.65). It was observed that there is no significant difference in percent scavenging activity in different plant parts collected from three different altitudes but showed difference in the solvent system used. The results obtained are in support of extensive use of Woodfordia fruticosa in traditional medicine and endorses the use of bark, while it needs further investigations on the plants growing in different geographic areas.