Purification of nuclear cAMP-independent protein kinases from rat ventral prostate

Two nuclear cAMP-independent protein kinases (designated PK-N1 and PK-N2) were purified from rat ventral-prostate and liver. The yield of enzyme units was 4-5% and 7-9% for each enzyme from the prostatic nuclei and liver nuclei, respectively. The average fold purification for prostatic nuclear protein kinase N1 and N2 was 1360 and 1833, respectively. The respective average specific activity of the two enzymes towards casein was 81,585 and 110,000 nmol 32P incorporated/hr/mg of enzyme. Protein kinase N1 comprised one polypeptide of Mr 35,000 which underwent phosphorylation in the presence of Mg2+ + ATP. Protein kinase N2 comprised two polypeptides Mr 40,000 and 30,000 of which only the Mr 30,000 polypeptide was autophosphorylated. Both enzymes were active towards casein, phosvitin, dephosphophosvitin, spermine-binding protein, and non-histone proteins in vitro. Little activity was detected towards histones. Both enzymes were stimulated by 150-200 mM NaCl. MgCl2 requirement varied with the protein substrate but was between 2-4 mM for both enzymes. With dephosphophosvitin as substrate, the apparent Km for ATP for N1 protein kinase was 0.01 mM. GTP did not replace ATP in this reaction. Protein kinase N2 was active in the presence of ATP or GTP. The apparent Km was 0.01 mM for ATP, but 0.1 mM for GTP.     

Identification of N epsilon-carboxymethyllysine as a degradation product of fructoselysine in glycated protein

The chemistry of Maillard or browning reactions of glycated proteins was studied using the model compound, N alpha-formyl-N epsilon-fructoselysine (fFL), an analog of glycated lysine residues in protein. Incubation of fFL (15 mM) at physiological pH and temperature in 0.2 M phosphate buffer resulted in formation of N epsilon-carboxymethyllysine (CML) in about 40% yield after 15 days. CML was formed by oxidative cleavage of fFL between C-2 and C-3 of the carbohydrate chain and erythronic acid (EA) was identified as the split product formed in the reaction. Neither CML nor EA was formed from fFL under a nitrogen atmosphere. The rate of formation of CML was dependent on phosphate concentration in the incubation mixture and the reaction was shown to occur by a free radical mechanism. CML was also identified by amino acid analysis in hydrolysates of both poly-L-lysine and bovine pancreatic ribonuclease glycated in phosphate buffer under air. CML was also detected in human lens proteins and tissue collagens by HPLC and the identification was confirmed by gas chromatography/mass spectroscopy. The presence of both CML and EA in human urine suggests that they are formed by degradation of glycated proteins in vivo. The browning of fFL incubation mixtures proceeded to a greater extent under a nitrogen versus an air atmosphere, suggesting that oxidative degradation of Amadori adducts to form CML may limit the browning reactions of glycated proteins. Since the reaction products, CML and EA, are relatively inert, both chemically and metabolically, oxidative cleavage of Amadori adducts may have a role in limiting the consequences of protein glycation in the body.     

Evidence for a pool of non-recycling transferrin receptors in peripheral sheep reticulocytes

Sheep reticulocytes from phlebotomized animals have a total transferrin binding potential that may exceed by an order of magnitude the surface binding capacity. Steady state uptake of transferrin at 37 degrees C is generally less than 50% of the total transferrin binding capacity. During long-term incubation of the reticulocytes, all transferrin binding ability is lost, the ability to internalize being lost most rapidly. The loss in ability to bind transferrin during long-term incubation is independent of the number of surface transferrin binding sites, since removal of surface receptors with pronase does not affect the rate of loss of the internal pool of receptors during long-term incubation. Moreover, after removing surface receptors with pronase, only a fraction of the original number of receptors is restored to the surface, despite the presence of a large pool of internal receptors. These data suggest that only a fraction of the internal pool of receptors is capable of recycling to the cell surface in sheep reticulocytes.     

Effects of short-term administration of sex hormones on normal and autoimmune mice

The effects of short-term administration (2 to 4 wk) of sex hormones on the immune system of normal (C57BL/6) and autoimmune (C57BL/6-lpr, C3H/lpr, B/W) strains of mice were investigated. Both estrogen (E2) and testosterone (Te) had significant effects on the numbers of T and B cells as well as on the density of cell surface antigens as demonstrated by flow cytometry. For example, Te depleted Thy-1.2+ thymocytes in normal mice and brought about a shift to lower density cells. Lyt-2+ cells appeared to be the main target cells of hormonal modulation in normal and autoimmune mice. Both sex hormones significantly depleted these cells in the thymus but had differential effects in the peripheral lymphoid organs, particularly in the spleen. In general, E2 depleted Lyt-2+ cells, whereas Te increased or maintained this subpopulation of cells in spleen and lymph nodes. Similarly, the suppressor cell activity and IL 2 production on a per cell basis in E2-treated animals was diminished, whereas Te-treated animals had normal or enhanced activity. The relevance of these findings to differential sex susceptibility in autoimmune diseases is discussed.     

Inhibition of plasma prolactin in the rat by amantadine

A single iv injection of 15 or 30 but not 7.5 mg/kg BW of an antiviral drug, amantadine, significantly (P less than 0.05) decreased plasma prolactin (PRL) concentrations in male rats. This effect was dose-dependent, with the highest dose producing a longer-lasting decrease in plasma PRL. The amantadine-induced decrease was unaffected by a simultaneous injection of 5-hydroxytryptophan (30 mg/kg BW) but was completely blocked by a simultaneous injection of haloperidol (0.05 mg/kg BW). It is concluded that this novel effect of amantadine on PRL is produced by an interaction with the dopaminergic system.     

3α, 11α-Dihydroxy-23-oxo-lup-20(29)-en-28-oic acid from Acanthopanax trifoliatus

The new triterpene 3α,11α-dihydroxy-23-oxo-lup-20(29)-en-28-oic acid was isolated from Acanthopanax trifoliatus. Its structure has been determined on the basis of spectroscopic data and chemical transformations.     

Asplenetin,a flavone and its glycoside from Launaea asplenifolia

A new flavone, asplenetin, has been isolated from Launea asplenifolia and characterized as 5,7,3′,4′,5′-pentahydroxy-3-(3-methylbutyl)flavone. Its glycoside, asplenetin 5-O-neohesperidoside, is also reported.     

Solvent influence on rate and mechanism of oxidation of ethylenediaminetetraacetatocobaltate(II) by periodate

The kinetics of the oxidation of Co^II(EDTA)²⁻ by IO₄⁻ were studied in various ethanol + water mixtures covering the range 7.9 to 58.0 wt% ethanol, at five different temperatures in the range 15–35 °C. The effect of solvent on the rate and mechanism of the reaction was investigated. An inner-sphere mechanism for the reaction was proposed and supported by the calculated activation parameters.     

Prekallikrein and kininogens in blister fluids in burn patients

Blister fluids removed from benign cutaneous burns contain the components of the prekallikrein-kininogens system in a non activated state. Their occurrence in the fluids is explained by a diffusion from the blood plasma.     

A rapid procedure for DNA sequencing using transposon-promoted deletions in Escherichia coli

A simple procedure has been developed for sequencing long fragments of DNA. The fragment (which can be several kb in length) is cloned in pAA3.7X, and subdivided into many overlapping segments by Tn9-promoted deletions. The deletions are isolated by positive selection for galactose resistance. A rapid plasmid preparation from several hundred galactose-resistant colonies is fractionated by agarose gel electrophoresis to pick a series of deletions terminating at approx. 200-bp intervals across the entire length of the fragment. Selected plasmids are purified by rapid alkaline extraction, and used directly for supercoil sequencing with a primer derived from IS1. Sequences of adjacent deletions contain overlaps which are used to connect individual sequences to give the complete sequence.