Phosphorylation of membrane-bound guanylate cyclase of sea urchin spermatozoa

When Arbacia punctulata spermatozoa are incubated in seawater containing ammonium hydroxide (pH 8.8), the sperm plasma membrane-bound guanylate cyclase is dephosphorylated, its electrophoretic mobility increases (from an apparent molecular mass of 160 to 150 kD), and its enzymatic activity decreases 3.5-fold. Transfer of these cells into ammonium-free seawater (pH 7.4) results in the rephosphorylation of the cyclase, its reconversion to 160 kD, and recovery of the enzymatic activity lost upon dephosphorylation. This is the first direct demonstration that the activity of membrane-bound guanylate cyclase can be regulated by phosphorylation. A plasma membrane preparation is described that specifically supports the in vitro phosphorylation of the guanylate cyclase. This preparation will be useful in more detailed studies on the relationship between phosphorylation state and enzymatic activity of membrane-bound guanylate cyclase.     

Mechanisms of dimethylbenzanthracene-induced immunotoxicity

Traditional methods for toxicological assessment have implicated the immune system as a frequent target organ of toxic insult following chronic exposure to certain environmental chemicals, radiation or therapeutic drugs (xenobiotics). Immunotoxicity is expressed as autoimmunity, chemical hypersensitivity or immunosuppression. A tiered approach for characterizing chemical and drug-induced immunomodulation has been developed and validated in laboratory animals. Polycyclic aromatic hydrocarbons (PAH) have been studied because of their ubiquitous presence in the environment and carcinogenic potential. Since immunosuppression induced by PAH carcinogens has been implicated as an epigenetic mechanism in the outgrowth of initiated cells, this tiered approach was used to characterize the mechanism of PAH immunosuppressive capacity. Previously, studies in this laboratory have demonstrated that subchronic exposure of B6C3F1 mice to PAH carcinogens suppresses both humoral immunity (HI) and cell-mediated immunity (CMI), concurrently with decreased resistance to tumor challenge. The potent carcinogenic PAH, 7,12-dimethylbenz[a]anthracene (DMBA) was subchronically administered subcutaneously at 5, 50, or 100 micrograms/g of body weight. Natural killer (NK) cell tumor cytolysis, generation of cytotoxic T-cells (CTL), and lymphoproliferation to mitogens and allogeneic splenocytes in mixed leukocyte cultures (MLC) were quantitated 3-5 days after exposure to assess CMI. Mitogen and alloantigen-induced proliferation (MLC) of splenocytes was suppressed up to 90%. CTL and NK tumor cytolysis of radiolabelled target cells were similarly depressed up to 88 and 82%, respectively. Impairment of MLC or CTL responses correlated with increased susceptibility to challenge with PYB6 sarcoma cells. HI was measured by quantitating the number of antibody (IgM) plaque-forming cells (PFC) produced in response to T-cell dependent antigen challenge (sheep erythrocytes) and was similarly suppressed up to 95%. To understand the mechanism of PAH-induced immunotoxicity, splenocytes from DMBA-exposed mice were sensitized to alloantigens in the presence of interleukin-2 (IL-2) because there were indications that T-helper cell function was suppressed. In these preliminary studies, CTL suppression could be completely restored by the addition of the T-cell growth supporting lymphokine (IL-2) during the inductive phase of CTL generation, suggesting that DMBA exposure directly or indirectly induced deficits in T-helper cell function.     

Study of the effect of β-1,3-glucanase fromBasidiomycete QM 806 on yeast extract production

Summary Cell free supernatants, containing -1,3-glucanase fromBasidiomycete QM 806, dramatically augmented the effect of papain on yeast autolysis. This enables the process time to be significantly reduced and/or the yield of extract to be substantially increased.     

Heme regulation of HeLa cell transferrin receptor number

The number of diferic transferrin receptors on HeLa cells decreases when cells are grown in iron-supplemented media. The experiments reported here suggest that heme is the iron-containing compound which serves as the signal for receptor number regulation. When HeLa cells were grown in the presence of hemin, transferrin receptor number decreased to a greater degree than when cells were grown in equivalent amounts of iron supplied as ferric ammonium citrate. Incubation of cells in conditions which increased cellular heme content resulted in a decrease in cellular transferrin receptors. Incubating cells with 5-aminolevulinic acid (thus bypassing the rate-limiting step in heme biosynthesis, 5-aminolevulinic acid synthase) led to a decrease in transferrin receptor number. Incubation of cells with an inhibitor of heme oxygenase, Sn-protoporphyrin IX, also led to a decrease in transferrin receptor number. When cellular heme content was decreased by inhibiting heme synthesis with succinylacetone (an inhibitor of 5-aminolevulinic acid dehydratase), or by depriving cells of iron with deferoxamine, an increase in HeLa cell transferrin receptor number was seen. When HeLa cells were incubated with inducers of heme oxygenase (CoCl2, SnCl2, Co-protoporphyrin IX), transferrin receptor number also increased. The effects of all compounds which alter transferrin receptor number were dependent on the concentration of the supplement, as well as the duration of the supplementation. These experiments suggest that intracellular heme content may be an important signal controlling transferrin receptor number.     

The chemistry of vitamin B12. The coordination of biologically important molecules

The following equilibrium constants (given as logK in units of m⁻¹) were determined for the substitution of co-ordinated H₂O in aquocobalamin by glycine (bound through N) 5.8, cysteine (bound through S) 6.0 or 8.3, depending on the value chosen for the pK of the thiol group, and phenolate 2.9. The spectrum of the phenolate cobalamin shows an additional intense absorption band at 468nm with a molar extinction coefficient of 1.1×10⁴, which is assigned to a charge transfer from the phenolate to the cobalt ion. Equilibrium constants have also been determined for the equilibria between adenylcobamide cyanide and CN⁻, HO⁻ and H⁺, which show that the adenine is more easily displaced by CN⁻ and HO⁻ than is 5,6-dimethylbenziminazole in vitamin B₁₂, but can be protonated by acid while still remaining co-ordinated to the cobalt. It is shown that in the binding of corrinoids to proteins and polypeptides the formation of hydrogen bonds is far more important than co-ordination by the metal.