Subcellular localization of cholesterol ester hydrolase in the human intestine

Immunocytochemistry and subcellular fractionation were used to localize the cholesterol ester hydrolase in the human small intestine. A positive immunoreaction, when using antibodies directed against pancreatic cholesterol ester hydrolase, was mainly found in endocytotic vesicles. Moreover, a label by gold particles was observed in intercellular spaces where lymphatic tissue merges. No specific immunoreactivity was obtained with the mucosa when sera directed against human pancreatic chymotrypsinogen and human pancreatic lipase were used. Conventional subcellular fractionation was performed after extensive washing of enterocytes to rule out any possible contamination by pancreatic enzymes. In these conditions a bile salt-dependent cholesterol ester hydrolase activity was detected in the soluble fraction of cells. Data agree with the concept that the intestinal cholesterol ester hydrolase may have a pancreatic origin. The absorption, if any, of this enzyme by enterocytes seems specific since other pancreatic (pro)enzymes tested (lipase, chymotrypsinogen) are not detected in these cells.     

RNA synthesis in neuronal and glial cell nuclei from rat cerebral hemispheres during early postnatal development

RNA synthesis in rat cerebral hemispheres at 1, 5, and 10 days of age and the relative contribution brought by neuronal and glial nuclei to RNA synthesis was investigated. The experiments were carried out both in vivo (by i.p. injection of [³H]uridine) and in vitro (either by incubation of tissue slices with [³H]uridine or by determination of RNA polymerase activities). The labeling of RNA decreases from 1 to 10 days of age both in vivo and in vitro; the decrease is of the same extent in neuronal and glial nuclei. RNA polymerase activity Mg²⁺-dependent does not change significantly from 1 to 10 days of age either in total, in neuronal, or in glial nuclei, whereas the Mn²⁺-dependent activity increases significantly over the same developmental period studied. The significance of RNA polymerase assay as an index of in vivo RNA synthesis is discussed.     

Local breeding experience and the reproductive performance of Tree Swallows

ABSTRACT The potential advantages of repeated breeding at a particular location should improve reproductive performance in long‐lived species of birds. However, for short‐lived species, natural selection should favor individuals that most quickly develop competency in reproduction. Therefore, we hypothesized that local breeding experience beyond the first breeding attempt at a particular location would have little effect on subsequent reproductive performance of Tree Swallows (Tachycineta bicolor), a species where about 50% of adults breed only once in their lives. We tested this hypothesis using data collected from Tree Swallows in Michigan from 1993 to 2002. Because we were specifically interested in examining the effects of local breeding experience on reproductive performance, we restricted our analyses to after‐second‐year (ASY) females and their mates that we first encountered as breeders. Consistent with our hypothesis, we found no relationship between repeated local breeding experience and the reproductive performance of ASY female Tree Swallows and their mates as measured by clutch size and number of fledged young. However, pairs with more combined total local experience tended to lay eggs earlier in the season. These results suggest that Tree Swallows may benefit from breeding site fidelity, not because repeated local experience improves reproductive performance as measured by the production of fledglings, but because returning individuals acquire nest cavities earlier and are able to begin breeding earlier, providing time to renest in case of early nest failure.     

Human sperm motility: a molecular study of mitochondrial DNA,mitochondrial transcription factor A gene and DNA fragmentation

Molecular Biology Reports - Alterations affecting the mitochondrial genome and chromatin integrity of spermatozoa impair male reproductive potential. This study aimed to evaluate the impact of...     

Cobra venom phospholipase A2 inhibition by manoalide. A novel type of phospholipase inhibitor

Manoalide, an unusual nonsteroidal sesterterpenoid recently isolated from sponge, antagonizes phorbol-induced inflammation but not that induced by arachidonic acid, suggesting that manoalide acts prior to the cyclooxygenase step in prostaglandin synthesis, possibly by inhibiting phospholipase A2. We have now studied the inhibitory effect of manoalide on a homogeneous preparation of phospholipase A2 from cobra venom. For a given concentration of manoalide, the inhibition of phospholipase A2 activity toward dipalmitoylphosphatidylcholine/Triton X-100 mixed micelles is time-dependent and plateaus at about 85% inhibition of the initial velocity even after extensive preincubation. Metal ions (Ca2+, Ba2+, Mn2+) increase the inhibition, while lysophosphatidylcholine and substrate micelles protect. Increasing manoalide concentration shows increasing inhibition of the initial velocity until a plateau is reached, giving a typical saturation curve with a linear double-reciprocal plot. Under typical conditions (20-min preincubation, 40 degrees C, pH 7.1), 50% inhibition is achieved at a manoalide concentration of about 2 X 10(-6) M. The data indicate that manoalide is a potent inhibitor of the cobra venom phospholipase A2. Manoalide is now shown to react irreversibly with lysine residues in the enzyme. Surprisingly, the cobra venom phospholipase normally acts poorly on phosphatidylethanolamine as substrate, but after reaction with manoalide, the enzyme is somewhat more active toward this substrate rather than being inhibited. This suggests that a lysine residue may be important in understanding the substrate specificity of phospholipase A2.     

Esterification of cholesterol and lipid-soluble vitamins by human pancreatic carboxyl ester hydrolase

Human pancreatic carboxyl ester hydrolase is shown to catalyse the esterification of cholesterol and lipid-soluble vitamins A, E and D3 with oleic acid. The acitivity requires the presence of bile salts, and the trihydroxylated or the 3 alpha, 7 alpha dihydroxylated bile salts are better activators than the 3 alpha, 12 alpha dihydroxylated bile salts. The hydrolyzing and synthetizing activities of human pancreatic carboxyl ester hydrolase are separated by a large pH range since the synthesis of cholesterol esters is optimal at pH 5.25 and the hydrolysis of cholesterol and vitamin E esters is optimal at pH 8.0. From the comparison of the catalytic constants determined for the hydrolyzing and synthetizing activities and from the pH dependence of the two activities, it appears that human carboxyl ester hydrolase plays an important part in the intestinal lumen. The role of the enzyme in the esterification of cholesterol and lipid-soluble vitamins is questionable.