18-substituted steroids--Part 17. 2 alpha-hydroxylated liver metabolites of aldosterone identified by high-field [1H]NMR spectroscopy

11 beta,18-Epoxy-2 alpha,3 alpha,18,21-tetrahydroxy-5 alpha,17 alpha- pregnan-20-one (2 alpha-hydroxy-3 alpha,5 alpha-tetrahydro-17-isoaldosterone) and its apo isomer have been identified by high-field NMR studies, supported by thermospray HPLC/MS, to be among the major polar metabolites formed from incubation of aldosterone with rat liver microsomal fraction. Indications that unreduced 2 alpha-hydroxy-aldosterone is also present among the metabolites have still to be confirmed.     

Primary Anorexia Nervosa or Weight Phobia in the Male: Report on 13 Cases

The cases of 13 men with anorexia nervosa are reported. While the disorder as seen in the clinic is much less common in males than females this may not be a true reflection of the differential sex or overall prevalence rates. The disorder is found to have the same basic characteristics in the male as in the female: namely, a phobic avoidance of normal weight associated with elective carbohydrate starvation and emaciation. As in the female the mechanism appears to develop out of normal adolescent dieting behaviour and to arise as a means of avoiding overwhelming psychosocial maturational demands of adolescence. Premorbid and family features include a state of overnutrition and a high degree of family psychopathology reflected in high rates of parental psychiatric morbidity and marital difficulty.     

The properties of subunits of avidin coupled to Sepharose

Avidin that had been coupled to Sepharose 4B activated with CNBr retained over 90% of its biotin-binding capacity. When low concentrations of CNBr were used about 75% of the protein could be removed from the Sepharose by washing with guanidinium chloride (6 m). The remaining 25%, the covalently bound subunits, had an almost undiminished capacity for biotin but a decreased affinity. Addition of avidin subunits in guanidinium chloride to the coupled subunits followed by dilution or dialysis restored the original biotin-binding capacity and affinity. Three classes of binding sites were present in preparations of the subunits. About 25% were weak (K=5x10(-8)m), about one third exchanged their biotin in a few minutes (K approximately 10(-10)m) and the remainder were indistinguishable from the native tetramer. The last-named exchanged their bound biotin at a similar rate at pH5 and at pH2, they did not lose their biotin in 6 m-guanidinium chloride and they were resistant to tryptic digestion in the absence of biotin. The proportion of these stable sites could be increased to 65% when the subunits coupled to Sepharose were incubated at 37 degrees C. This increase was reversed by guanidinium chloride, which suggested that it was caused by a temperature-dependent association of covalently linked subunits. This in turn implies a temperature-dependent mobility of the agarose matrix of the Sepharose. Analysis of the spatial distribution of subunits within the Sepharose beads led to the conclusion that the association of subunits implied that they could move through distances greater than 20nm (several hundred A). This mobility and consequent formation of tetramer was greatly decreased when avidin subunits were coupled to Sepharose that had been cross-linked with divinyl sulphone.     

Crystal structure of a coiled-coil domain from human ROCK I

The small GTPase Rho and one of its targets, Rho-associated kinase (ROCK), participate in a variety of actin-based cellular processes including smooth muscle contraction, cell migration, and stress fiber formation. The ROCK protein consists of an N-terminal kinase domain, a central coiled-coil domain containing a Rho binding site, and a C-terminal pleckstrin homology domain. Here we present the crystal structure of a large section of the central coiled-coil domain of human ROCK I (amino acids 535-700). The structure forms a parallel α-helical coiled-coil dimer that is structurally similar to tropomyosin, an actin filament binding protein. There is an unusual discontinuity in the coiled-coil; three charged residues (E613, R617 and D620) are positioned at what is normally the hydrophobic core of coiled-coil packing. We speculate that this conserved irregularity could function as a hinge that allows ROCK to adopt its autoinhibited conformation.     

Functional Characterization of Trehalose Biosynthesis Genes from <Emphasis Type="Italic">E</Emphasis>. <Emphasis Type="Italic">coli</Emphasis>: An Osmolyte Involved in Stress Tolerance

Trehalose (1-α-d-glucopyranosyl-1-α-d-glucopyranoside), a non-reducing disaccharide is a major compatible solute, which maintains fluidity of membranes and protects the biological structure of organisms under stress. In this study, trehalose-6-phosphate synthase (otsA) and trehalose-6-phosphate phosphatase (otsB) genes encoding for trehalose biosynthesis from Escherichia coli was cloned as an operon and expressed in E. coli M15(pREP4). The recombinant E. coli strain showed a threefold increase in the activity of otsBA pathway enzymes, compared to the control strain. The transgenic E. coli accumulated up to 0.86 mg/l of trehalose. The sequence of otsA and otsB genes reported in this study contains several base substitutions with that of reported sequences in GenBank, resulting in the altered amino acid sequences of the translated proteins.     

The emergence and spread of finch trichomonosis in the British Isles

Finch trichomonosis, caused by the protozoal parasite Trichomonas gallinae, was first recognized as an emerging infectious disease of British passerines in 2005. The first year of seasonal epidemic mortality occurred in 2006 with significant declines of greenfinch Carduelis chloris and chaffinch Fringilla coelebs populations. Here, we demonstrate that large-scale mortality, principally of greenfinch, continued in subsequent years, 2007-2009, with a shifting geographical distribution across the British Isles over time. Consequent to the emergence of finch trichomonosis, the breeding greenfinch population in Great Britain has declined from ca 4.3 million to ca 2.8 million birds and the maximum mean number of greenfinches (a proxy for flock size) visiting gardens has declined by 50 per cent. The annual rate of decline of the breeding greenfinch population within England has exceeded 7 per cent since the initial epidemic. Although initially chaffinch populations were regionally diminished by the disease, this has not continued. Retrospective analyses of disease surveillance data showed a rapid, widespread emergence of finch trichomonosis across Great Britain in 2005 and we hypothesize that the disease emerged by T. gallinae jumping from columbiforms to passeriforms. Further investigation is required to determine the continuing impact of finch trichomonosis and to develop our understanding of how protozoal diseases jump host species.     

Protein kinase A-dependent step(s) in hepatitis C virus entry and infectivity

Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.     

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.