Reference ranges of retinol, tocopherols, lycopene and alpha- and beta-carotene in plasma by simultaneous high-performance liquid chromatographic analysis

Using a high-performance liquid chromatographic procedure, age- and gender-based reference ranges for plasma retinol, alpha- and gamma-tocopherol, lycopene, and alpha- and beta-carotene have been established. In addition to confirming higher retinol levels in men than in women (p less than 0.001), this study demonstrates significantly higher plasma levels of both carotenes in women than in men (p less than 0.001). The plasma levels of retinol and beta-carotene showed a positive relationship with age after adjusting for plasma lipids (p less than 0.001 and p less than 0.02, respectively). In contrast to beta-carotene, there was no gender difference for plasma lycopene and a strong (p less than 0.001) inverse relationship of lycopene with age. These results suggest that differences may exist between beta-carotene and lycopene in their intestinal absorption, plasma transport, or tissue metabolism.     

Requirement for BSF-1 in the induction of antigen-specific B cell proliferation by a thymus-dependent antigen and carrier-reactive T cell line

We report here a role of B cell stimulatory factor 1 (BSF-1) in the induction of antigen-specific proliferation of affinity-purified small B lymphocytes by a thymus-dependent antigen and a carrier-reactive T cell line. By using an ovalbumin-reactive T cell line (designated Hen-1), which does not produce BSF-1 following activation, it was possible to demonstrate that the antigen-specific proliferative response of trinitrophenyl (TNP)-binding B cells to TNP-ovalbumin required exogenous BSF-1 in addition to direct interaction with irradiated Hen-1 T cells. The activation obtained under these conditions was highly efficient, being sensitive to antigen doses as low as 0.001 microgram/ml. The addition of saturating amounts of BSF-1 did not alter the antigen-specificity or the requirements for hapten-carrier linkage or major histocompatibility complex-restricted T-B interaction in this system. The involvement of BSF-1 was confirmed by the ability of 11B11 anti-BSF-1 antibody to specifically suppress the response of TNP-binding B cells to TNP-ovalbumin, BSF-1, and irradiated Hen-1 T cells. Finally, this response was augmented by addition of the monokine interleukin 1. These data indicate that the proliferative response of small B cells to the thymus-dependent antigen and carrier-reactive T cell line used in our experiments can be regulated by the same factors that govern B cell proliferation induced by thymus-independent type 2 antigens or anti-IgM antibodies.     

Active site of human liver aldehyde dehydrogenase

Bromoacetophenone (2-bromo-1-phenylethanone) functions as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) and has been found specifically to label a unique tryptic peptide in the enzyme. Amino-terminal sequence analysis of the labeled peptide after purification by two different procedures revealed the following sequence: Val-Thr-Leu-Glu-Leu-Gly-Gly-Lys. Radioactivity was found to be associated with the glutamate residue, which was identified as Glu-268 by reference to the known amino acid sequence. This paper constitutes the first identification of an active site of aldehyde dehydrogenase.     

Enhanced release and synthesis of lipoprotein lipase in rat heart cell cultures exposed to high concentrations of Hepes

While attempting to optimize conditions for synthesis of lipoprotein lipase by cultured heart cells, we encountered an unexpected rise in enzyme activity when media were supplemented inadvertently with 100 mM Hepes buffer (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid). This finding was further investigated and optimal results were obtained at pH 7.0-7.2. The increase in lipoprotein lipase activity was time dependent; after 3-6 h there was a rise in medium activity but cellular activity increased only after 24 h. The increased enzyme activity was defined as lipoprotein lipase by inhibition with antiserum to rat adipose tissue lipoprotein lipase. A 72-h exposure to Hepes resulted in a 30% increase in the incorporation of [35S]methionine into cellular proteins and a 2-fold increase into heparin-releasable proteins. Using heparin Sepharose chromatography and stepwise elution, a lipoprotein lipase enriched fraction was recovered with 2 M NaCl. The amount of [35S]methionine and [3H]galactose incorporated into protein of this fraction derived from Hepes-treated cells was 2-6-fold that of controls. A 4-fold increase in cellular lipoprotein lipase mass in Hepes-treated cells was shown by immunoblotting. Results obtained with Hepes-conditioned medium suggest the presence of cell-derived compounds that enhance release and subsequent synthesis of lipoprotein lipase. The effect of Hepes-conditioned medium on lipoprotein lipase resembled to some extent that of the addition of heparin. Therefore, it appears that when Hepes is first added to the culture medium, it might promote a release of heparan sulfate or related compounds, possibly by virtue of its negatively charged sulfonic acid residue. The accumulated heparan sulfate could then promote a sustained release of lipoprotein lipase into the culture medium which in turn leads to increased enzyme synthesis.     

Nucleosomal organization of a BPV minichromosome containing a human H4 histone gene

Summary When the body temperature of rats is elevated to 42°C, four heat shock proteins, with the molecular weights of 70000, 71000, 85000, and 100000 (hsp 70, hsp 71, hsp 85, and hsp 100, respectively), are induced in various tissues of rats (Fujio et al., J Biochem 101, 181–187, 1987). Heat shock proteins are induced by various stresses other than heat in varieties of cultured cells, so we studied whether heat shock proteins are induced in intact rats by different treatments. Analysis of the translation products of poly(A) + RNA isolated from the livers of rats recovering from ischemia of the liver showed that mRNAs for hsp 70, hsp 71, and hsp 85 were induced. These hsp-mRNAs were also induced in the livers of rats 6 h after a partial hepatectomy, and had returned to control levels 24 h after the surgery. These results suggested that heat shock proteins have not only the function of protection against various stresses but also physiological functions in the normal growth and development of animals.     

Avian adipose lipoprotein lipase: cDNA sequence and reciprocal regulation of mRNA levels in adipose and heart

cDNA clones for chicken adipose lipoprotein lipase were isolated from an expression library in lambda gt11 by antibody screening and characterized by hybridization selection and nucleotide sequencing. Based on the cDNA sequence and on N-terminal sequence analysis of the purified enzyme, chicken adipose lipoprotein lipase is a mature protein of 465 amino acids with a signal peptide of 19 or 25 amino acids, depending on which of two methionine residues is used for translation initiation. The predicted amino-acid sequence was found to be 73-77% identical to the four known mammalian adipose lipoprotein lipase sequences, with conservation of position of cysteine residues and putative functional domains, and number of potential N-glycosylation sites. Chicken lipoprotein lipase differs from mammalian lipoprotein lipases with respect to the position of one N-glycosylation site and the presence of an additional 15-17 C-terminal amino acids. 32P-labeled cDNA clones hybridized to mRNA species of 3.7 and 4.0 kb in Northern blots of heart and adipose, but not of liver RNA. In chickens that were fasted for 48 h and then refed, lipoprotein lipase mRNA levels in adipose increased to a maximal level of 350% that of controls at 10 h, whereas heart lipoprotein lipase mRNA levels fell to 40% of controls at 14 h. Concomitantly, no changes in total RNA were observed. Thus, avian lipoprotein lipase is subject to reciprocal pretranslational regulation in adipose and heart.     

Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide

The incubation of chloroplast fructose-1,6-bisphosphatase with both dithiothreitol and protein denaturants made sulfhydryl groups available for reaction with [1-14C]iodoacetamide (10-12 mol iodoacetamide incorporated/mol enzyme). Digestion of S-carboxyamidomethylated enzyme with trypsin and polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate, yielded two 14C-labeled fragments whose apparent molecular mass were 10 kDa and 16 kDa. In the absence of either dithiothreitol or protein denaturants the incorporation of iodoacetamide to the enzyme was lower than 4 mol. When chloroplast fructose-1,6-bisphosphatase was initially incubated with dithiothreitol (2.5 mM) and (a) high concentrations of both fructose 1,6-bisphosphate (4 mM) and Ca2+ (0.3 mM) or (b) low concentrations of both fructose 1,6-bisphosphate (0.8 mM) and Ca2+ (0.05 mM) in the presence of either 2-propanol (15%, by vol.), trichloroacetate (0.15 M) or chloroplast thioredoxin-f (0.5 microM) and subsequently subjected to proteolysis and electrophoresis, S-carboxyamidomethylated tryptic fragments had similar molecular masses. Thus, conditions that stimulated the specific activity of chloroplast fructose-1,6-bisphosphatase caused conformational changes which favoured both the reduction of disulfide bridges and the exposure of sulfhydryl groups. In this aspect, thioredoxin exerted structural and kinetic effects similar to compounds not involved in redox reactions (organic solvents, chaotropic anions). These results indicated that the modification of hydrophobic (intramolecular) interactions in chloroplast fructose-1,6-bisphosphatase constituted the underlying mechanism in light-activation by the ferredoxin-thioredoxin system.