Effect of incubation conditions, inhibitors, 2-mercaptoethanol and testosterone on RNA synthesis in ram spermatozoa

In vitro studies on RNA synthesis using washed ram spermatozoa were carried out by measuring the incorporation of (3)H-uridine into RNA. Penicillin-G (100 mug/ml medium) was added to prevent contamination by microorganisms. Spermatozoa were quickly separated from seminal plasma by washing twice in Tris-HCl buffer (at pH 7.2) and centrifuged at 1,000 g for 5 min. Washed spermatozoa were then diluted to 1 10 , 1 20 or 1 40 (v/v) by the same buffer system (containing 400 mg% glucose) and were incubated in air at 37 degrees C for 1, 2 and 4 h. Results indicated that the rate of RNA synthesis was maximal at 1 40 semenbuffer dilution (5-8 x 10(7) spermatozoa/ml) and increased linearly up to 4 h of incubation. The rate of RNA synthesis at 1 40 dilution also increased linearly as the dose of exogenous glucose substrate was increased up to 400 mg%. Denaturation of the ram spermatozoa by 1% HgCl(2) caused almost complete inhibition of RNA synthesis that amounted to 97% of the control samples. Incubation of spermatozoa with 50, 100 or 200 mug/ml chloramphenicol also inhibited uridine incorporation by 86 to 94%, while equivalent doses of cycloheximide did not. On the other hand, the incorporation of (3)H-uridine into the RNA of ram spermatozoa was significantly enhanced by graded doses of 2-mercaptoethanol (0.2, 0.4 and 0.8 muM) and of testosterone (15 and 30 mug/ml). The results of this study indicate RNA synthesis, mainly of mitochondrial origin, by mature ram sperm. The data also suggest a role for intracellular cyclic adenosine monophosphate in the regulation of sperm RNA synthesis.     

Synaptic effects of the synthetic pyrethroid resmethrin in rat brain in vitro

Resmethrin (30 microM) induced release of transmitters was not affected by manipulation of the Na+ current with either choline or tetrodotoxin agents which readily reversed the effects of veratridine, deltamethrin and cypermethrin. Resmethrin (I50: 2.2 microM) inhibited the ATP dependent uptake of Ca2+ but deltamethrin and cypermethrin were much less effective. Resmethrin also displaced Ca2+ from crude synaptosomal membranes. The release promoting effects of resmethrin in rat brain in vitro are better explained by its effects on Ca2+ rather than through a specific effect on the Na+ channel. In contrast, the effects of deltamethrin and cypermethrin promote transmitter release by a Na+ dependent process.     

Analysis of aminophospholipid molecular species by high performance liquid chromatography

A new method is described for the separation of individual molecular species of the aminophospholipids, phosphatidylethanolamine and phosphatidylserine. Trinitrobenzene-sulfonic acid was used to derivatize both aminophospholipids and the derivatives were purified by thin-layer chromatography. A reversed-phase high performance liquid chromatography technique was developed to separate and quantify individual molecular species based upon ultraviolet detection of the attached chromophore. The retention times of the molecular species on the C18 reversed-phase column were longer with increasing carbon chain length and decreasing degree of unsaturation of fatty acyl chain. The overall procedure allowed a quantitative recovery of the aminophospholipid species. The lower limit of detection was about 10 pmol and a linear response was observed in the range of 0.1-10 nmol of phospholipid. Using this method, we were able to separate and quantify trinitrophenyl-phosphatidylethanolamine molecular species of both subclasses (diacyl and alkenyl) from human red blood cells and rat brains. Separation of species was confirmed by gas-liquid chromatographic analysis of the fatty acid content of each peak and by thermospray liquid chromatography-mass spectrometry. This new method provides a convenient and sensitive technique for studies of aminophospholipid molecular species composition. Furthermore, it appears to be a useful tool for the analysis of asymmetric distribution of these species in biological membranes.     

Preparation and spectroscopic characterization of molecular species of brain phosphatidylserines

This study describes the first preparation and spectroscopic characterization of naturally occurring phospholipids separated according to degree of unsaturation. Phosphatidylserines (PS) have been prepared from bovine brain and shown to be pure by extensive thin layer chromatographic analysis as well as by infrared spectroscopy and fatty acid analysis. The PS has been separated according to degree of unsaturation and prepared using AgNO3-impregnated silica gel H thin-layer chromatography. Fatty acid analysis of the two principal PS subfractions indicates that they are enriched in the molecular species 1-octadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphorylserine and 1-octadecanoyl-2-octadecenoyl-sn-glycero-3-phosphorylserine. The identity of the two PS subfractions was further verified by rechromatographing on several thin layer systems and by infrared spectroscopy. With the use of a 100 MHz Fourier transform nuclear magnetic resonance (NMR) spectrometer, the spectra of bovine whole brain, white matter, gray matter, monoenoic, and hexaenoic PS were obtained. Distinct proton resonances were assigned to double bond protons, protons adjacent to a double bond, and protons between two double bonds, using fatty acid methyl ester standards. The various PS preparations gave different intensities of the various proton resonances which correlated with differences in fatty acid composition. The method provides a convenient, non-destructive spectroscopic method for distinguishing monoenoic and polyunsaturated species of intact phospholipids. Electron spin resonance studies of nitroxide-labelled cholestane in sonicated PS vesicles showed greater probe motion as the unsaturation of the acyl chains was increased. The hexaenoic PS vesicles were more fluid than monoenoic PS vesicles at all temperatures in the range 10-55 degrees C. These results suggest that neuronal membranes are more fluid than myelin membranes as neuronal membranes contain more hexaenoic phospholipids.     

Sulphated heteropolysaccharides from Padina pavonia

Extraction with hydrochloric acid (pH 2.5) of the brown alga Padina pavonia afforded water-soluble and water-insoluble polysaccharides comprising D-glucuronic acid, L-fucose, D-xylose, D-mannose, D-glucose and D- galactose residues. The water-soluble polysaccharide was fractionated by using ethanol, and cetylpyridinium chloride and by chromatography on DEAE-cellulose. A neutral laminaran-like glucan, a sulphated heteropolysaccharide composed of the aforementioned sugars and a protein moiety were obtained. The isolated heteropolysaccharide showed high anticoagulation activity.     

ECHOCARDIOGRAPHIC ABNORMALITIES OF THE MITRAL VALVE ASSOCIATED WITH A LARGE PERICARDIAL EFFUSION

Features of mitral stenosis and systolic anterior motion of the mitral valve were observed on the echocardiogram of a patient with a large posterior pericardial effusion. The abnormal findings disappeared with resolution of the fluid. Because pericardial effusion may result in the echocardiographic alteration of cardiac structures, repeat study is necessary after the effusion has cleared.     

Biogas manufacture from co-digestion of untreated primary sludge with raw chicken manure under anaerobic mesophilic environmental conditions

This work aimed to co-digest various wastes to assess the best combination of all mixing ratio, also at choosing the best ratio between untreated primary sludge (UPS) singly from two sources, (South valley University (SUPS) and Abu tesht wastewater station (AUPS) and raw chicken manure (RCM) and comparing the results in either case. The co-digestions of untreated primary sludge from Abu tesht wastewater treatment stations with different levels of raw chicken manure (0:100, 10:90, 30:70, 50:50, 90:10, and 100:0) to obtain the best mixtures. Also, co-digestion of untreated primary sludge from south valley university with different levels of raw chicken manure at the same ratios, to obtain the best mixtures. Batch digestion tests were applied in 2.5 L digester with a working volume of 2.0 L. The samples in triplicates were separately loaded into the digesters locally fabricated and kept for 20 days as a retention period and diluted with the same amount of water. Mesophilic under 35 °C was adopted for untreated primary sludge as well as mixtures with raw chicken manure based on total solids (TS) and volatile solid (VS) proportions. The average biogas yields from AUPS/RCM mixture obtained ranged from 8570 to 5600 ml, by the following descending order, 10: 90 > 90:10 and so on >100:0, and the average biogas yields from SUPS/RCM obtained ranged from 6330 to 5635 ml, in the order of 90: 10 > 10:90 and so on >100:0. The results showed highest biogas yield from AUPS/RCM and SUPS/RCM mixtures with mixing ratio of 10:90 and 90:10, respectively, however, the lowest biogas production detected in separate digestion of AUPS and SUPS. The results indicated that co-digestion between the sludge and raw chicken manure could increase total biogas production volume, enhance sludge treatment process, and produce eco-friendly sludge because of co-digestion process than separate processing of each feedstock.     

Preliminary note on anti-insulin antibody formation in diabetics treated with porcine insulin

In the present study, we have tested the sera of sixty five diabetic patients treated with insulin, researching the action of some physiologic and therapeutic factors (sex, age, insulin dose and time of treatment), to production of anti-insulin antibodies. Our results have shown that an important percentage of diabetic patients treated by porcine insulin produce antibodies: 72% of studied patients, concerning chiefly all the women and patients under-fourty years old. However our results have not pointed out any relation between the administered insulin dose versus the anti-insulin antibodies production, in spite of the early production of these antibodies in a great part of the patients.     

Spectrofluorimetric determination of certain biologically active phenothiazines in commercial dosage forms and human plasma

A validated simple and sensitive spectrofluorimetric method was developed for the determination of chlorpromazine hydrochloride, promethazine hydrochloride, trifluperazine hydrochloride, thioridazine hydrochloride, perazine maleate and oxomemazine. The method was based on condensation of malonic acid/acetic anhydride (MAA) under the catalytic effect of the tertiary amine moiety of the studied phenothiazines to provide a deep yellow to brown colour with green florescence. Relative fluorescence intensity of the products was measured at λ_exc 398 nm and λ_em 432 nm. Different variables affecting the reaction were studied and optimized. The method was successfully applied for the determination of the studied drugs in commercial dosage forms. The lower detection limits allowed the application of this method for the determination of the compounds in plasma as an example of a biological fluid. In addition, the method was considered specific for the determination of tertiary amines in the presence of primary and secondary amines; as a result, it was deemed suitable for the determination of the cited drugs in the presence of their degradation products resulting from N‐dealkylation or oxidation of the corresponding sulphoxides or sulphones. Copyright © 2012 John Wiley & Sons, Ltd.