Inhibitors of sterol synthesis. Chromatography of acetate derivatives of oxygenated sterols

The separation of the acetate derivatives of a number of oxygenated sterols was achieved by medium pressure liquid chromatography on silica gel columns and by normal and reversed phase high performance liquid chromatography. We have explored the application of these chromatographic systems for the analysis of oxygenated sterols of plasma samples from two normal human subjects. The addition of highly purified [14C]cholesterol to plasma permitted the detection and quantitation of oxygenated sterols formed by autoxidation of cholesterol during processing of the samples. Special attempts to suppress autoxidation of cholesterol included the use of an all-glass closed system for saponification and extraction under argon followed by rapid removal of cholesterol from the polar sterols by reversed phase medium pressure liquid chromatography. Chromatographic analyses of the [3H]acetate derivatives of the polar sterols provided a sensitive approach for the detection and quantitation of the individual oxygenated sterols. Oxygenated sterols detected in plasma included cholest-5-ene-3 beta,26-diol, (24S)-cholest-5-ene-3 beta,24-diol, and cholest-5-ene-3 beta,7 alpha-diol. After correction for their formation by autoxidation of cholesterol during processing of the samples, very little or none of the following sterols were observed: cholest-5-ene-3 beta,7 beta-diol, 5 alpha,6 alpha-epoxy-cholestan-3 beta-ol, 5 beta,6 beta-epoxy-cholestan-3 beta-ol, and cholestane- 3 beta, 5 alpha,6 beta-triol, and the 25-hydroxy, 22R-hydroxy, 21-hydroxy, 20 alpha-hydroxy, and 19-hydroxy derivatives of cholesterol.     

The study of vocal communication of wild mandrills in Cameroon in relation to their social structure

The vocal repertoire of the mandrill (Mandrillus sphinx), a forest living baboon, is described, and their vocal communication analyzed quantitatively. Although the vocal repertoire of mandrills corresponds well to that of savanna living baboons,Papio, some characteristics differed, such as the development of long-distance calls and differentiation of vocalizations between age-sex classes. Vocal communication within a group was closely related to changes in the spatial distribution of group members, and the two most common vocalizations, crowing and 2PG, appear to function as contact calls. Based on the wide dispersion of food trees, a group of mandrills divided into several feeding groups (subgroups). The two types of contact call were given in different and in some senses complementary contexts, and helped to facilitate and maintain group integration. According to their acoustic structure, these calls are long distance calls. Influenced by the high-level of attenuation of vocalization on the forest floor, the mandrill has developed them as contact calls, instead of using the contact “grunt,” which is common to the savanna living baboons. Comparing the patterns of vocal exchanges of mandrills with those of gelada and hamadryas baboons which have a multi-levelled society, the social structure of the mandrill is discussed. From the analysis of the spatial distribution of vocal emission, a number of clusters of vocalizations were obtained. These clusters correspond to subgroups. The frequent female-female and female-male vocal exchange between subgroups of mandrills suggest that the relationships between subgroups are less closed than between the one-male units of gelada and hamadryas baboons. Furthermore some of these clusters include more than two vocalizing adult males, while in other clusters there are no vocalizing adult males. Thus, the social structure of mandrills is suggested to be multi-male rather than a multilevelled type. The absence of contact calls specific for short distance and the functional replacement of the grunting of all group members by persistent emission of a loud call (2PG) by usually just one adult male suggests that the social structure of mandrills is not exactly equivalent to that of the multimale troop of savanna living baboons. Usually the use of 2PG is monopolized by one adult male travelling in the rear part of the group. Such monopolization of 2PG emission and the pattern of 2PG-2PG or 2PG-roar exchanges by adult males in some cases indicate the existence of strong dominance relationships among adult males, and especially the existence of a leader male within a multi-male group of mandrills.     

Characterization of Functional Primary Cilia in Human Induced Pluripotent Stem Cell-Derived Neurons

Neurochemical Research - Recent advances in human induced pluripotent stem cells (hiPSCs) offer new possibilities for biomedical research and clinical applications. Neurons differentiated from...     

Growth-dependent changes of guanylate and adenylate cyclase activities in cilia and cell bodies of Tetrahymena pyriformis

The calmodulin-dependent guanylate cyclase of Tetrahymena pyriformis was shown previously to be localized in surface membranes (ciliary and pellicular membranes) (Kudo, S, Nakazawa, K, Nagao, S & Nozawa, Y, Japan j exp med 52 (1952) 193) [21], whereas in a recent report Schultz et al, (Schultz, J E, Schonefeld, U & Klumpp, S, Eur j biochem 137 (1983) 89) [12] demonstrated the localization of this enzyme in ciliary membrane, arguing against its presence in pellicular membrane. To examine the discrepancy, the activities of guanylate and adenylate cyclases were examined in cilia and cell bodies of Tetrahymena pyriformis during transition from early log to stationary growth phase. The guanylate cyclase activity in the cell bodies increased significantly with growth of age, while in cilia the activity was rather consistent. In contrast, adenylate cyclase did not show any growth-dependent activity changes in both cilia and cell bodies. The increase of guanylate cyclase activity was not related to the increase of its activator calmodulin, because the change in enzyme activity could not be negated by addition of a saturating amount of calmodulin. These results suggest that the content of guanylate cyclase itself would be increased in the cell bodies during growth.     

Detection in human platelets of phospholipase A2 activity which preferentially hydrolyzes an arachidonoyl residue

It has been generally considered that highly specific liberation of arachidonic acid is induced upon the stimulation of the platelets, although the molecular mechanism of the regulation of its action has not been well understood. An aim of the present study is to clarify the role of phospholipase A2 in the arachidonic acid metabolism within human platelets. Phosphatidylcholine or phosphatidylethanolamine with arachidonate at the sn-2 position of glycerol was cleaved efficiently by phospholipase A2 activity in homogenates as well as in the cytoplasmic fraction of human platelets, leading to the selective liberation of free arachidonate, whereas phospholipids with linoleate were hardly hydrolyzed under the same conditions. Double-reciprocal plots of kinetic data further strengthened the conclusion that human platelet phospholipase A2 showed high selectivity for arachidonoyl residue. This enzyme may play a crucial role in the intracellular metabolism of the arachidonate of phospholipids.     

Construction of a secretion vector production of peptide hormones in Escherichia coli (extracellular production of calcitonin as fused protein)

A new secretion vector, pEAP84 which contained a unique restriction site (BglII) at the 3' end of the penicillinase gene to produce a fused protein, and the Ex-kil region to make the outer membrane permeable, was constructed from pEAP82. A recombinant plasmid p84h06, which contained a synthetic gene for human calcitonin with a cyanogen bromide cleavage site at the junction site of the fused protein, was constructed and introduced into Escherichia coli. The hybrid protein produced in E. coli carrying p84h06 was secreted into the culture medium. The amino acid composition of this product was consistent with that deduced from the DNA sequence. Mature calcitonin was obtained following cyanogen bromide cleavage of the fused protein.     

The primary structure of rat platelet phospholipase A2

In our previous report (Hayakawa, M., Kudo, I., Tomita, M., & Inoue, K. (1988) J. Biochem. 103, 263-266), we have shown that phospholipases A2 purified from rat platelet membrane fractions and an extracellular medium of thrombin-stimulated rat platelets were essentially identical to each other. Both purified enzymes were digested with proteases, and the resulting peptides were subjected to primary sequence determination. The sequence analysis of the HPLC-separated peptides and the alignment of the sequences showed a tentative primary structure of rat platelet phospholipase A2, which was composed of 125 amino acid residues. It showed 47% homology with snake venom Agkistrodon halys blomhoffii phospholipase A2.     

Reproducibility,Performance, and Clinical Utility of a Genetic Risk Prediction Model for Prostate Cancer in Japanese

Prostate specific antigen (PSA) is widely used as a diagnostic biomarker for prostate cancer (PC). However, due to its low predictive performance, many patients without PC suffer from the harms of unnecessary prostate needle biopsies. The present study aims to evaluate the reproducibility and performance of a genetic risk prediction model in Japanese and estimate its utility as a diagnostic biomarker in a clinical scenario. We created a logistic regression model incorporating 16 SNPs that were significantly associated with PC in a genome-wide association study of Japanese population using 689 cases and 749 male controls. The model was validated by two independent sets of Japanese samples comprising 3,294 cases and 6,281 male controls. The areas under curve (AUC) of the model were 0.679, 0.655, and 0.661 for the samples used to create the model and those used for validation. The AUCs were not significantly altered in samples with PSA 1–10 ng/ml. 24.2% and 9.7% of the patients had odds ratio <0.5 (low risk) or >2 (high risk) in the model. Assuming the overall positive rate of prostate needle biopsies to be 20%, the positive biopsy rates were 10.7% and 42.4% for the low and high genetic risk groups respectively. Our genetic risk prediction model for PC was highly reproducible, and its predictive performance was not influenced by PSA. The model could have a potential to affect clinical decision when it is applied to patients with gray-zone PSA, which should be confirmed in future clinical studies.     

Phylogenetic Analysis of Cellulolytic Enzyme Genes from Representative Lineages of Termites and a Related Cockroach

The relationship between xylophagous termites and the protists resident in their hindguts is a textbook example of symbiosis. The essential steps of lignocellulose degradation handled by these protists allow the host termites to thrive on a wood diet. There has never been a comprehensive analysis of lignocellulose degradation by protists, however, as it has proven difficult to establish these symbionts in pure culture. The trends in lignocellulose degradation during the evolution of the host lineage are also largely unknown. To clarify these points without any cultivation technique, we performed meta-expressed sequence tag (EST) analysis of cDNA libraries originating from symbiotic protistan communities in four termite species and a wood-feeding cockroach. Our results reveal the establishment of a degradation system with multiple enzymes at the ancestral stage of termite-protistan symbiosis, especially GHF5 and 7. According to our phylogenetic analyses, the enzymes comprising the protistan lignocellulose degradation system are coded not only by genes innate to the protists, but also genes acquired by the protists via lateral transfer from bacteria. This gives us a fresh perspective from which to understand the evolutionary dynamics of symbiosis.