Purification and crystallization of avidin

An improved purification, including crystallization, of avidin from hen's-egg white is described. The product bound 15.1mug of biotin/mg, corresponding to an equivalent weight of 16200. The amino acid composition showed an integral number of amino acid residues per 16200g, which suggested that each molecule of avidin (mol. wt. approx. 66000) contained four identical subunits.     

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.     

The use of flow microfluorimetry in the analysis of the phenotype expression of mouse histocompatibility antigens

Quantitation of the expression of cell surface antigens has hitherto been limited to analysis by either cytotoxicity tests or radioimmune assays (5, 15). We report here the use of a new methodology to analyze and quantitate the expression of mouse histocompabililty antigens (H-2 locus) in hybrid clones and parental cell types. The binding of fluorescein-tagged antibody is measured on a cell-to-cell basis in large viable cell populations using flow microfluorimetric techniques. These techniques have been used to measure hapten and immunoglobulin binding to lymphocyte populations (8, 9, 14). However, this is the first report in which these techniques have been used to examine the expression of the H-2 locus. The advantage of this approach is twofold: first, a large and statistically significant sample population may be analyzed one cell at a time, thus revealing the fine detail of heterogeneity in the expression of the cell surface markers within a population. Second, as has been demonstrated for analysis of specific components of the immune system, this method does permit fluorescence-activated sorting of cell types according to their different surface populations (8, 9, 14).     

Contrasting evolutionary histories of chloroplast thioredoxins f and m

Fourteen thioredoxin sequences were used to construct a minimal phylogenetic tree by using parsimony. The bacterial thioredoxins clustered into three groups: one containing the photosynthetic purple bacteria, Escherichia and Corynebacterium; a second containing the photosynthetic green bacterium, Chlorobium; and a third containing cyanobacteria. These groupings are similar to those generated from earlier 16s RNA analyses. Animal thioredoxins formed a fourth group. The two thioredoxins of chloroplasts (f and m) showed contrasting phylogenetic patterns. As predicted from prior studies, spinach chloroplast thioredoxin m grouped with its counterparts from cyanobacteria and eukaryotic algae, but, unexpectedly, thioredoxin f grouped with the animal thioredoxins. The results indicate that, during evolution, thioredoxin m of contemporary photosynthetic eukaryotic cells was derived from a prokaryotic symbiont, whereas thioredoxin f descended from an ancestral eukaryote common to plants and animals. The findings illustrate the potential of thioredoxin as a phylogenetic marker and suggest a relationship between the animal and f-type thioredoxins.      

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.     

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.     

Multicomponent analysis of encapsulated marine oil supplements using high-resolution 1H and 13C NMR techniques

Multicomponent high-resolution 1H and 13C NMR analysis has been employed for the purpose of detecting and quantifying a wide range of fatty acids (as triacylglycerols or otherwise) in encapsulated marine cod liver oil supplements. The 1H NMR technique provided quantitative data regarding the docosahexaenoic acid content of these products, which serves as a valuable index of fish oil quality, and a combination of both 1H and 13C spectroscopies permitted the analysis of many further components therein, including sn-1 monoacylglycerols, sn-1,2 and -1,3 diacylglycerol adducts, together with a range of minor components, such as trans-fatty acids, free glycerol and cholesterol, and added vitamins A and E. The identities of each of the above agents were confirmed by the application of two-dimensional 1H-1H spectroscopies. The NMR techniques employed also uniquely permitted determinations of the content of nonacylglycerol forms of highly unsaturated (or other) fatty acids in these products (i.e., ethyl esters), and therefore served as a means of distinguishing "natural" sources of cod liver oils from those subjected to chemical modification to and/or supplementation with synthetic derivatives such as ethyl docosahexaenoate or eicosopentaenoate. The analytical significance and putative health effects of the results acquired are discussed.     

Deletion of Dad1 in mice induces an apoptosis-associated embryonic death

Dad1 is a putative anti-apoptosis gene identified in several distantly related organisms. Expression of Dad1 in transfected cells inhibits apoptosis in vitro. To determine whether Dad1 has a similar function in vivo, we used gene targeting to delete Dad1. Heterozygous adult mice (+/-) show no obvious phenotype or abnormalities, but genotype analysis of over 100 offspring from heterozygous matings detected no weanling, homozygous Dad1 null (-/-) mice. Subsequent analysis of embryos from heterozygous matings detected Dad1 null (-/-) embryos at E3.5 but no later, suggesting Dad1 is required for development beyond the late blastocyst stage. Increased levels of apoptosis were observed in cultured embryos lacking a functional copy of the gene, consistent with an anti-apoptotic role for Dad1.     

Modification of carboplatin by Jurkat cells

Using [(1)H,(15)N] heteronuclear single quantum coherance (HSQC) NMR and (15)N-labeled carboplatin, 1, we show that Jurkat cells affect the rate of disappearance of the HSQC NMR peak in culture medium for this Pt(2+) anticancer drug. The decay or disappearance rate constant for 1 in culture medium containing cells is k(1)=k(c)[CO(3)(2-)]+k(m)+k(u)N, where k(c) is the rate constant for reaction of 1 with carbonate in the medium, k(m) is the rate constant for reaction of 1 with all other components of the medium, and k(u) is the rate constant for reaction of 1 with cells having a number density N in the medium. Since Jurkat cells only take up a small amount of the platinum present in the medium (<1%), the observed disappearance of the HSQC NMR peak for 1 cannot be due to uptake of carboplatin by the cells.     

Variation in palaeo-shorelines explains contemporary population genetic patterns of rocky shore species

Processes driving and maintaining disjunct genetic populations in marine systems are poorly understood, owing to a lack of evidence of hard barriers that could have shaped patterns of extant population structure. Here, we map two genetically divergent lineages of an obligate rocky shore fish, Clinus cottoides, and model sea-level change during the last 110 000 years to provide the first evidence of a vicariant event along the southern coastline of Africa. Results reveal that lowered sea levels during glacial periods drastically reduced rocky intertidal habitat, which may have isolated populations in two refugia for at least 40 000 years. Contemporary coastal dynamics and oceanography explain secondary contact between lineages. This scenario provides an explanation for the origin of population genetic breaks despite a lack of obvious present-day geographical barriers and highlights the need for including palaeo-oceanography in unravelling extant population patterns.