Purification and partial characterization of rat liver bilirubin glucuronoside glucuronosyltransferase.

Bilirubin glucuronoside glucuronosyltransferase (EC 2.4.1.95) converts bilirubin monoglucuronide to bilirubin diglucuronide and is concentrated in plasma membrane-enriched fractions of rat liver homogenates. The enzyme was purified 2,000-fold to homogeneity from rat liver. The pI of the enzyme is 7.9 +/- 0.2. The enzyme has a molecular weight of 160,000 and is an oligomer of 28,000 dalton subunits. Km for purified enzyme was 35 microM and Vmax was 2.2 mumol of bilirubin diglucuronide formed/min/mg of protein. Freshly biosynthesized bilirubin monoglucuronide was injected intravenously into homozygous Gunn rats which had bile duct cannulation. Gunn rats lack UDP-glucuronate glucuronyltransferase activity (EC 2.4.1.17), have normal bilirubin glucuronoside glucuronosyltransferase activity, cannot form bilirubin monoglucuronide in vitro or in vivo, and do not excrete bilirubin glucuronides after intravenous injection of unconjugated bilirubin. Within 1 h, approximately 75% of the injected conjugated bilirubin was recovered in bile, of which 20% consisted of bilirubin diglucuronide. These results indicate that bilirubin glucuronide glucuronosyltransferase catalyzes conversion of bilirubin monoglucuronide to diglucuronide in vivo.     

Effects of an LH-RH analogue in male rats pretreated with oestradiol benzoate.

Treatment of adult male rats with oestradiol benzoate (OB) for 21 days significantly decreased the body, testicular and accessory sex organ weights but increased anterior pituitary weight. OB treatment also significantly suppressed circulating FSH and LH levels as well as plasma and testicular concentrations of testosterone. The seminiferous tubules and interstitial cells were partly atrophied, and there was some effect on spermatogenesis, with step 14 to 19 spermatids being fewer than normal. Rats treated with OB for 21 days were then treated daily with LH-RH analogue ((D-Leu6, des-Gly-NH2(10))-LH-RH-ethylamide), to see if testicular function could be recovered. Circulating gonadotrophins were significantly elevated, testicular histology was normal and testicular and plasma testosterone concentrations and the accessory sex organ weights remained suppressed. These results suggest possible extra-pituitary effects of the LH-RH analogue, including a direct action on the testes and/or accessory sex organs.     

The effects of testosterone propionate and estradiol benzoate on the vitro synthesis of FSH.

The effects of estradiol benzoate (EB) and testosterone propionate (TP) on pituitary and plasma concentrations of follicle stimulating hormone (FSH) and the in vitro synthesis of FSH in pituitary tissue was studied in mature male rats. By the 4th day of treatment with EB, pituitary content and concentration of FSH had declined, and content had fallen to 6% and concentration to 3% of pretreatment values. Similar results occurred during in vitro synthesis. However, serum levels of FSH did not show any decline until the 21st and 28th days of treatment. Administration of TP produced a progressive increase in pituitary content and concentration of FSH, though serum levels remained unchanged for the 1st 7 days, after which they fell slightly. The effect of TP on the in vitro synthesis of FSH showed no consistent pattern, though in no case was a decrease in the uptake of labeled leucine into immunoprecipitable FSH observed. The results suggest that EB and TP have different effects on pituitary FSH in normal adult male rats.     

Radiation and fermentation treatment of cellulosic wastes

The effect of radiation pasteurization of sugar cane bagasse and rice straw and fermentation using various strains of fungi were studied for upgrading of cellulosic wastes. The initial contamination by fungi and aerobic bacteria both in bagasse and straw was high. The doses of 30 kGy for sterilization and 8 kGy for elimination of fungi were required. Irradiation effect showed that rice straw contained comparatively radioresistant microorganisms. It was observed that all the fungi (Hericium erinacium, Pleurotus djamor, Ganoderma lucidum, Auricularia auricula, Lentinus sajor-caju, Coriolus versicolor, Polyporus arcularius, Coprinus cinereus) grow extending over the entire substrates during one month after inoculation in irradiated bagasse and rice straw with 3% rice bran and 65% moisture content incubated at 30°C. Initially, sugar cane bagasse and rice straw substrates contained 39.4% and 25.9% of cellulose, 22.9% and 26.9% of hemicellulose, and 19.6% and 13.9% of lignin + cutin, respectively. Neutral detergent fibre (NDF) values decreased significantly in sugar cane bagasse fermented byG. lucidum, A. auricula andP. arcularius, and in rice straw fermented by all the 8 strains of fungi. Acid detergent fibre (ADF) values also decreased in bagasse and rice straw fermented by all the fungi.P. arcularius, H. erinacium, G. lucidum andC. cinereus were found to be the most effective strains for delignification of sugar cane bagasse.     

Ca2+ dependent activation of rat uterine glycerylphosphorylcholine diesterase: Presence of a positive modulator protein in uterine secretion

Molecular cloning of human, mouse and rat brain CaM-stimulated phosphatase has suggested the existence of two genes for the subunit of the enzymes. A and A fragments of A and A from rat brain library have been expressed in bacteria to produce specific anti-calcineurin A and anti-calcineurin A antibodies (Kunoet al., J Neurochem 58: 1643–1651, 1992). Alternative mRNA splicing gives rise to additional calcineurin isozymes with some containing an insertion sequence of ATVEAIEADE. Antibody against synthetic peptide of this insertion sequence has been raised in this study. Three CaM-stimulated phosphatase isozymes previously purified from bovine brain (BPI, BPII, BPIII) (Yokoyama & Wang, J Biol Chem 266: 14822–14829, 1991), along with the bacterially expressed rat A and A fragments, were analyzed by two calcineurin subunit monoclonal antibodies VJ6 and VD3, the rat anti-calcineurin A and anti-calcineurin A specific polyclonal antibodies, and the insertion peptide antibody. The bovine brain CaM-stimulated phosphatase isozymes BPI and BPIII reacted with both anti-calcineurin A and anti-calcineurin A antibodies. While BPII reacted with anti-calcineurin A but not anti-calcineurin A antibody, it differed from the expressed A fragment in immunoreactivity towards the monoclonal antibodies. The results show that the bovine brain CaM-stimulated phosphatase isozymes cannot be simply categorized as derived from A or A genes products. On the other hand, on the basis of immunoreactivity toward the insertion antibody, the isozymes can be readily classified into those containing the insertion sequence (BPI, LPI) and those without the inserting (BPII, BPIII, LPII, LPIII).Abbreviations CaM calmodulin - SDS sodium dodecyl sulfate - BP brain peak - LP lung peak - PAGE polyacrylamide gel electrophoresis - EGTA [ethylenebis(oxyethylenenitrilo)]-tetra acetic acid - cDNA complementary DNA - mAb monoclonal antibody - NBT nitro blue tetrazolium chloride - BCIP 5-bromo 4-chloro-3-indolyl phosphate     

Nanostructured biocomposite scaffolds based on collagen coelectrospun with nanohydroxyapatite

Nanofibrous biocomposite scaffolds of type I collagen and nanohydroxyapatite (nanoHA) of varying compositions (wt %) were prepared by electrostatic cospinning. The scaffolds were characterized for structure and morphology by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), atomic force microscopy (AFM) and X-ray diffraction (XRD) techniques. The scaffolds have a porous nanofibrous morphology with random fibers in the range of 500-700 nm diameters, depending on the composition. FT-IR and XRD showed the presence of nanoHA in the fibers. The surface roughness and diameter of the fibers increased with the presence of nanoHA in biocomposite fiber as evident from AFM images. Tensile testing and nanoindendation were used for the mechanical characterization. The pure collagen fibrous matrix (without nanoHA) showed a tensile strength of 1.68 +/- 0.10 MPa and a modulus of 6.21 +/- 0.8 MPa with a strain to failure value of 55 +/- 10%. As the nanoHA content in the randomly oriented collagen nanofibers increased to 10%, the ultimate strength increased to 5 +/- 0.5 MPa and the modulus increased to 230 +/- 30 MPa. The increase in tensile modulus may be attributed to an increase in rigidity over the pure polymer when the hydroxyapatite is added and/or the resulting strong adhesion between the two materials. The vapor phase chemical crosslinking of collagens using glutaraldehyde further increased the mechanical properties as evident from nanoindentation results. A combination of nanofibrous collagen and nanohydroxyapatite that mimics the nanoscale features of the extra cellular matrix could be promising for application as scaffolds for hard tissue regeneration, especially in low or nonload bearing areas.