Changes in the levels of pituitary and steroid hormones in ovine and human ovarian follicular fluid

Changes in the protein and steroid hormones of follicular fluid, aspirated from different follicles of sheep and human ovaries, have been measured and correlated with the size of the follicles. As the fluid contains a number of proteins, steroids have been measured directly and after ether extraction. The follicular fluid concentrations of progesterone and 17 beta-oestradiol measured directly in the fluid increased with the size of the follicles. The levels of free testosterone remained constant in all sizes of follicles, while those of bound hormone showed a 10- to 15-fold increase over the free testosterone concentrations in both the sheep and human follicular fluid. A decrease in the levels of bound testosterone in the fluid of large follicles (LFFL) coincided with the increase in bound 17 beta-oestradiol, suggesting the possible conversion of bound testosterone to oestrogen as the follicle attained maturity. The ratio of follicle-stimulating hormone (FSH) to luteinizing hormone (LH) varied in the fluid obtained from different size follicles, being 1:7 in small (SFFL), 1.3.5 in medium (MFFL) and 1:2.3 in large (LFFL) follicles of sheep ovaries. The LH content of follicular fluid of different size follicles appeared to be the same, with LFFL showing a minor increase over SFFL. In the human, the fluid from medium follicles contained very little LH compared to LFFL. These differences in the pattern of LH levels present in the fluid from different size follicles between human and sheep ovaries presumably reflect species variations in the entry of LH into the follicles.     

Microbial transformation of rifamycin B: A new extracellular oxidase fromCurvularia lunata

Summary A highly active extracellular rifamycin oxidase was isolated fromCurvularia lunata var.aeri. The enzyme has a pH optimum of 6.5 and temperature optimum of 50°C.     

Novel assay for screening rifamycin B-producing mutants.

A simple method that allows easy identification of rifamycin B-producing strains is described. This method involves the use of an enzyme, rifamycin oxidase, which converts inactive rifamycin B to active rifamycin S. In this method, colonies to be tested are grown in pairs. The two colonies are then transferred to two plates seeded with a sensitive strain of Staphylococcus aureus, one plate of which contains the enzyme rifamycin oxidase. All paired colonies which show a larger inhibition zone diameter on the enzyme-containing plate are identified as rifamycin B producers.     

Variability of milk fat globule membrane protein gene between cattle and riverine buffalo.

A study on butyrophilin (BTN) gene was conducted to detect variability at nucleotide level between cattle and buffalo. Hae III PCR-RFLP was carried out in crossbred cattle and it revealed polymorphism at this locus. Three genotypes namely, AA, BB and AB and two alleles were observed with frequencies 0.78, 0.17, 0.04 and 0.87, 0.13, respectively. The sequences of different cattle, buffalo and sheep breeds have been reported in the EMBL gene bank with accession numbers: AY491468 to AY491475. The nucleotides, which have been substituted from allele A to B, were found to be C to G (71st nucleotide), C to T (86th nucleotide), A to T (217th nucleotide), G to A (258th nucleotide), A to C (371st nucleotide) and C to T (377th nucleotide). The nucleotide substitution at 71st, 86th and 377th position of the fragment were expected to be a silent mutation where as nucleotide changes at 217th, 258th and 371st positions were expected to be substituted by lysine with arginine, valine with isoleucine and leucine with proline in allele B. The differences of nucleotides and amino acids between cattle, buffalo and sheep breeds have been revealed and on the basis of nucleotide as well as protein variability the phylogenetic diagram have been developed indicating closeness between cattle and buffalo.     

Chick embryo fibroblasts produce two forms of hyaluronidase

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.     

Purification and physicochemical properties of polygalacturonase from Aspergillus niger MTCC 3323

Polygalacturonases are the pectinolytic enzymes that catalyze the hydrolytic cleavage of the polygalacturonic acid chain. In the present study, polygalacturonase from Aspergillus niger (MTCC 3323) was purified. The enzyme precipitated with 60% ethanol resulted in 1.68-fold purification. The enzyme was purified to 6.52-fold by Sephacryl S-200 gel-filtration chromatography. On SDS–PAGE analysis, enzyme was found to be a heterodimer of 34 and 69 kDa subunit. Homogeneity of the enzyme was checked by NATIVE-PAGE and its molecular weight was found to be 106 kDa. The purified enzyme showed maximum activity in the presence of polygalacturonic acid at temperature of 45 °C, pH of 4.8, reaction time of 15 min. The enzyme was stable within the pH range of 4.0–5.5 for 1 h. At 4 °C it retained 50% activity after 108 h but at room temperature it lost its 50% activity after 3 h. The addition of Mn²⁺, K⁺, Zn²⁺, Ca²⁺ and Al³⁺ inhibited the enzyme activity; it increased in the presence of Mg²⁺ and Cu²⁺ ions. Enzyme activity was increased on increasing the substrate concentration from 0.1% to 0.5%. The K_m and V_max values of the enzyme were found to be 0.083 mg/ml and 18.21 μmol/ml/min. The enzyme was used for guava juice extraction and clarification. The recovery of juice of enzymatically treated pulp increased from 6% to 23%. Addition of purified enzyme increased the %T₆₅₀ from 2.5 to 20.4 and °Brix from 1.9 to 4.8. The pH of the enzyme treated juice decreased from 4.5 to 3.02.     

Intense photon emission induced by fracture of γ–irradiated Dy‐, Tm‐, Sm‐ and Mn‐doped CaSO4 single crystals

When an γ‐irradiated Dy‐, Tm‐, Sm‐ or Mn‐doped CaSO₄ crystal is impulsively deformed, two peaks appear in the ML intensity versus time curve, whereby the first ML peak is found in the deformation region and the second in the post‐deformation region of the crystals. In this study, intensities I_m1 and I_m2 corresponding to first and second ML peaks, respectively, increased linearly with an impact velocity v₀ of the piston used to deform the crystals, and times t_m1 and t_m2 corresponding to the first and second ML peaks, respectively, decreased with impact velocity. Total ML intensity initially increased with impact velocity and then reached a saturation value for higher values of impact velocity. ML intensity increased with increasing γ‐doses and size of crystals. Results showed that the electric field produced as a result of charging of newly‐created surfaces caused tunneling of electrons to the valence band of the hole‐trapping centres. The free holes generated moved in the valence band and their subsequent recombination with electron trapping centres released energy, thereby resulting in excitation of luminescent centres. Copyright © 2012 John Wiley & Sons, Ltd.     

TRPC3 regulates release of brain-derived neurotrophic factor from human airway smooth muscle

Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca^2 + signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca^2 + entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP–BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20 ng/ml, 48 h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10 μM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca^2 + (EGTA; 1 mM) or intracellular Ca^2 + (BAPTA; 5 μM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca^2 + influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca^2 + and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.