Addition of Cryoprotectant Significantly Alters the Epididymal Sperm Proteome

Although cryopreservation has been developed and optimized over the past decades, it causes various stresses, including cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. During cryopreservation, addition of cryoprotective agent (CPA) is crucial for protecting spermatozoa from freezing damage. However, the intrinsic toxicity and osmotic stress induced by CPA cause damage to spermatozoa. To identify the effects of CPA addition during cryopreservation, we assessed the motility (%), motion kinematics, capacitation status, and viability of epididymal spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, the effects of CPA addition were also demonstrated at the proteome level using two-dimensional electrophoresis. Our results demonstrated that CPA addition significantly reduced sperm motility (%), curvilinear velocity, viability (%), and non-capacitated spermatozoa, whereas straightness and acrosome-reacted spermatozoa increased significantly (p < 0.05). Ten proteins were differentially expressed (two decreased and eight increased) (>3 fold, p < 0.05) after CPA, whereas NADH dehydrogenase flavoprotein 2, f-actin-capping protein subunit beta, superoxide dismutase 2, and outer dense fiber protein 2 were associated with several important signaling pathways (p < 0.05). The present study provides a mechanistic basis for specific cryostresses and potential markers of CPA-induced stress. Therefore, these might provide information about the development of safe biomaterials for cryopreservation and basic ground for sperm cryopreservation.     

Beneficial effects of n-hexane bark extract of Onosma echioides L. on diabetic peripheral neuropathy

Onosma echioides Linn (Boraginaceae) is the most frequently used curative herb widely used for kidney obstruction, sciatic pain, and gout. The present study was designed to investigate the therapeutic effects of n-hexane bark extract of O. echioides (OE) L. root in vivo against Streptozotocin-induced diabetic neuropathy in SD rats. For in vivo activity, the experiment was categorized into five different groups (n = 5). Group-I was considered as nondiabetic/normal control (NC) treated with 0.5% carboxymethyl cellulose (CMC), Group II as diabetic control, Group-III, IV, and V served as diabetic treated with OE 50, OE 100, and pregabalin at a dose of 50, 100, and 10 mg/kg body weight, orally, respectively. Body weight, blood glucose, oral glucose tolerance test, behavioral studies (motor coordination test, thermal hyperalgesia, cold allodynia, locomotor activity, oxidative biomarkers (thio barbituric acid reactive substances [TBARS], superoxide dismutase [SOD], glutathione [GSH], and catalase), and histopathology of the sciatic nerve were performed. Treatment with OE showed a dose-dependent increase in neuroprotective activity by improving the myelination and decreasing the axonal swelling of nerve fibers. The verdicts of behavioral activities showed a remarkable effect on animals after the treatment of extract and standard drug pregabalin. In conclusion, our findings supported the traditional application of OE and explored its importance in the management of diabetic neuropathy. Additional clinical experiments may provide novel therapeutic drugs for diabetes and its complications.     

Purification of a kappa-carrageenase from marine Cytophaga species

A mixture of extracellular carrageenases was isolated from the cell-free medium of a culture of marine Cytophaga sp. 1k-C783 grown on ZoBell 2216 E broth with 0.1% commercial carrageenan. A single active peak of kappa-carrageenase was separated and purified from the mixture by ammonium sulfate precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration chromatography. Molecular weight of the purified kappa-carrageenase was estimated as 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified kappa-carrageenase had pH optimum 7.6 and temperature optimum 25 C.     

Mini-exon derived RNA gene ofLeishmania donovani: structure,organization and expression

Mini-exon derived RNA is a small nuclear RNA of trypanosomatid protozoa such asLeishmania which donates its 5′-terminal 39 nucleotides to the 5’-ends of cellular messenger RNAs by trans-splicing. We have cloned a mini-exon derived RNA gene fromLeishmania donovani and studied its organization and expression. About 200 copies of the gene per haploid genome are organized as a tandem repeat on a single chromosome. The gene is transcribed as a 95-nucleotide RNA. The first 39 nucleotides of mini-exon derived RNA is also found at the 5′-terminus of a cellular mRNA (Β-tubulin), thus confirming its identity. Sequence analysis of the gene and its flanking regions showed that while classical RNA polymerase II promoter elements such as TATA and CAAT are absent from the 5′-upstream region, intragenic sequence motifs resembling RNA polymerase III promoter elements are present. The implications of this finding for mini-exon derived RNA expression are discussed.     

Effect of carbohydrates on the growth ofRhizoctonia solani Kühn

The growth OfRhizoctonia solani in different carbohydrates was studied. The rate of growth of the fungus was traced by taking the dry weights of mycelia obtained from the carbohydrate medium at regular intervals and shifts in the pH were recorded. Different carbohydrate sources had different effects on the growth of the organism. The exoenzymes from the organism were capable of cleaving carbohydrates irrespective of whether the fungus grew in them or not.     

Characterization and expression of uterine and placental alkaline phosphatases in the mouse.

Alkaline phosphatase (ALP) is rapidly induced in the uterine subepithelial stroma after a natural or artificial decidual stimulus. During gestation ALP-specific activity peaked at Day 7 to 8 (Day 1 is day of detection of the copulation plug) followed by a rapid decline to control levels by Day 9. This elevation in enzyme activity was preceded by an 8-fold induction of a 2.6 kilobase (kb) mRNA. This mRNA was not preferentially localized to implantation sites. ALP activity was detected in the placenta at Day 9 and reached maximum specific activity at Day 19. The placental ALP was also encoded by a 2.6 kb mRNA. Uterine and placental ALPs were inhibited to the same extent by levamisole, L-tryptophan and homoarginine. The calculated Ki values for these inhibitors were not statistically different between the uterine and placental forms. Km values towards the substrate p-nitrophenylphosphate, however, were statistically different between the uterine and placental forms. Both uterine and placental ALPs were stimulated 3-4-fold by addition of 2 mM-Mg2+. Electrophoretic mobilities on SDS polyacrylamide gel, where the enzyme migrated as a single band, were the same. The uterine form, however, could be distinguished from the placental isoenzyme by separation on non-denaturing polyacrylamide gels; the uterine form had a single zone of activity which migrated with an intermediate mobility between the two zones of activity detected for the placental enzyme. These differences in mobility could be ascribed to the sialic acid content of the enzyme because treatment with neuraminidase resulted in the uterine and placental forms migrating with comparable but slower mobilities in non-denaturing gels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oil palm (Elaeis guineensis) protoplasts: isolation, culture and microcallus formation

Procedures are deseribed for the efficient isolation of protoplasts from a variety of oil palm (Elaeis guineensis Jacq.) tissues. Various factors including donor source, composition of enzyme mixture and culture medium affected the yield and viability of the protoplasts Polyembryogenic cultures of oil palm were the most suitable starting material in terms of yield, viability and metabolic competence. Pectolyase Y-23 in association with cellulase and hemicellulase was required for the efficient release of protoplasts from the oil palm tissues. Limited cell division to form microcallus was observed at very low frequency (<0.01%) when glutathione and catalase were incorporated in the culture medium.Abbreviations 2,4-d dichlorophenoxyacetic acid - DTT dithiothreitol - MES 2[N-morpholino] ethanesulphonic acid - NAA 1-naphthalene acetic acid - PVP polyvinylpyrrolidone