Xanthone and flavonol constituents of Swertia hookeri

The whole plant of Swertia hookeri, collected at flowering has been shown to contain two tri- and nine tetraoxygenated free, glucosyloxy, and stearyl ester xanthones and one flavonol stearyl ester. Among these, three are previously unreported in nature and one was known previously only as a synthetic compound. The xanthones are based on 1,3,5,-, 1,3,5,8- and 1,3,7,8-oxygenated systems with the middle oxygenation pattern predominating. The two ester compounds appeared only at the flowering stage. Plants collected at the pre-flowering stage gave the corresponding free compounds. The biochemical and biological significance of these findings are appraised.     

Association of cyclic nucleotide phosphodiesterase of buffalo spermatozoa with sperm chromatin

Buffalo sperm heads contain more than 50% of the total cyclic AMP-phosphodiesterase activity (EC 3.1.4.17) present in spermatozoa. Its distribution in sperm heads revealed no activity in acrosome and other membrane structures present in the head. All the cyclic AMP-phosphodiesterase activity was found firmly bound to sperm chromatin which could not be solubilized. In addition to cyclic AMP, cyclic GMP was also hydrolysed by chromatin preparation. The rate of hydrolysis was 2.5-times more rapid with cyclic AMP than with cyclic GMP at their optimum pH of 7.5 and 8.0, respectively. The pH and heat stability profiles, inhibition studies and the effect of divalent metal ions indicated that the two activities are not associated with the same protein. Mixed substrate analysis showed two sites at which the hydrolysis of cyclic AMP and cyclic GMP is catalysed. Chromatin cyclic nucleotide phosphodiesterases exhibited kinetics typical of one enzyme species both for cyclic AMP (K m = 100 microM; V = 1.0 nmol/min per mg protein) and cyclic GMP (Km = 23 microM; V = 0.4 nmol/min per mg protein). Each cyclic nucleotide was found to be a competitive inhibitor of the hydrolysis of the other with a Ki value of 30.18 microM for cyclic AMP hydrolysis and 256 microM for cyclic GMP hydrolysis. Hill coefficients of 1.0 obtained in the presence of cyclic AMP for cyclic GMP hydrolysis and vice-versa indicated no allosteric interactions. It is suggested that chromatin cyclic nucleotide phosphodiesterase may have a role post fertilization in cell growth and differentiation with no role in sperm motility which is regulated by similar enzymes present in sperm flagella.     

Superoxide release by zymosan-stimulated rat Kupffer cells in vitro

Kupffer cells were isolated from pronase-perfused rat livers and were maintained as a monolayer culture in a state of high purity and viability. Immediately after contact with zymosan particles, O2 uptake of the Kupffer cells increased fivefold; about 50% of the net oxygen consumed was accounted for as superoxide released into the medium. Concomitantly, a transient burst of luminol-dependent chemiluminescence, an increased activity of NAD(P)H oxidase and a stimulation of the flow of glucose through the hexose monophosphate shunt were observed. Chemiluminescence and O2- production were almost completely inhibited by superoxide dismutase and iodoacetate. Zymosan-induced chemiluminescence was not inhibited in the presence of the non-penetrating thiol reagents, 5,5'-dithio-bis-2-nitrobenzoate and iodoacetyl-sepharose. Iodoacetate acted on the cytosolic glucose-6-phosphate dehydrogenase rather than on NAD(P)H oxidase of the cell membrane.     

Inhibition of hydroxyproline synthesis by palladium ions.

Palladium ions, administered as PdSO4, markedly affect the incorporation of L-[3,4-3H2] proline into non-dialyzable fractions in 10-day chick embryo cartilage explants with a 55-65% reduction in the concentration range 0.06-0.6 mM. Under these conditions the synthesis of [3H]hydroxyproline was nearly completely inhibited. Experiments with prolyl hydroxylase (EC 1.14.11.2) indicated a strong irreversible inhibition of the enzyme with a competition between Fe2+ and Pd2+. The Ki for the inhibition was 0.02 mM. Pd2+-treated enzyme remained inactive after extensive dialysis. These studies suggest that Pd2+ may inhibit collagen synthesis by replacing Fe2+ in the active site of prolyl hydroxylase and forming strong complexes with the enzyme. These studies also point to a potential mechanism of Pd2+ toxicity.     

Racemization in the synthesis of polytripeptide models of a collagen.

Racemization in the synthesis of tripeptide intermediates and their polymers was investigated, using L -amino acid oxidase. Stepwise investigation of peptide intermediates showed no racemization during peptide coupling steps or deprotection of benzyl esters by hydrogenolysis. Saponification of one of the methyl esters produced some racemization. Preparation of active esters from N-protected tripeptide acids containing optically active C-terminal amino acid, with one exception, produced racemization. The fractionated polymers were found to contain less racemized amino acids than the crude products or starting monomeric tripeptides, indicating that the racemized sequences gave rise to lower molecular-weight oligomers. The sequences investigated were -Pro-Pro-Ala-, -Ala-Pro-Pro-, -Val-Pro-Pro-, -Pro-Pro-Leu-, -Pro-Gly-Leu-, -Pro-Gly-Phe-, -Pro-Gly-Val-, -Gly-Val-Pro-, -Phe-Pro-Gly-, -Leu-Pro-Gly-, and Ile-Pro-Gly-.     

Polypeptide models of collagen. Solution properties of (Gly-Pro-Sar)n and (Gly-Sar-Pro)n.

Our studies on the solution conformation of (Gly-Pro-Sar)_n and (Gly-Sar-Pro)_n synthesized as polypeptide models for collagen are reported. It is found that, while (Gly-Pro-Sar)_n exists in ordered triple-helical conformation, (Gly-Sar-Pro)_n remains as a disordered random coil in water. Addition of certain helix-promoting solvents seems to generate order in (Gly-Sar-Pro)_n.     

Pollination and nucellus inAbelmoschus esculentus

Pollination induced dissolution of nucellar cells at the micropylar end forming a passage for pollen tube entry even when the pollen tubes are in the stylar region.     

Polypeptide models of collagen: Properties of (Pro-Pro-betaAla)n.

We have synthesized (Pro-Pro-βAla)_n as a model for collagen. The synthetic polytripeptide, mol wt 6500, exhibits a large negative optical rotation with a very strong negative Cotton effect centered at 216 nm. The optical rotatory dispersion of (Pro-Pro-βAla)_n followed a single-term Drude equation and the λ_c was 195 nm. The rotation decreased markedly on heating with the midpoint of the broad transition at 55°C. Preliminary studies also showed loss of structure in guadinine HCl. The circular dichroism spectrum of the polymer exhibited a deep trough at 190 nm. The marked similarities of solution properties of (Pro-Pro-βAla)_n to (Pro-Pro-Gly)_n suggest that β-alanine can replace glycine in generating collagen-like helix in solution.     

Masking of pleomorphic glycogen sites by methanolic uranyl acetate.

Rat liver tissue was fixed in 2.5% glutaraldehyde buffered with cacodylic acid (pH 7.3) for 2 hr, washed twice in buffer, and postfixed in 2% osmium tetroxide at 4 C for 1 hr. The tissue then was dehydrated, infiltrated with and embedded in Epon by routine procedures. The ultrathin sections from this tissue, when stained with spectroscopic grade methanol saturated with uranyl acetate (SMUA) for 1 min followed by aqueous lead citrate (PbCi) (Reynolds 1963) for 5 min at room temperature, showed a uniform staining of all major cellular components except glycogen. The SMUA appeared to be specific for ribonuceloprotein granules, rendering them more prominent in the cytoplasm due to the lack of glycogen staining. The question of glycogen removal from the sections due to SMUA treatment was evulated using various extractions and staining methods. It appeared that SMUA pretreatment alters the subsequent binding ability of lead salts, resulting in lack of glycogen staining, although it does not remove the glycogen from the sections.