Modulation of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) acetyltransferase in human polymorphonuclears. The role of cyclic AMP-dependent and phospholipid sensitive, calcium-dependent protein kinases

In order to characterize the mechanism of activation of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (EC 2.3.1.67) which is the limiting step in the regulation of the synthesis of the potent inflammatory mediator 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; homogenates from human polymorphonuclear leukocytes were incubated in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase and in the presence of a partially purified phospholipid sensitive, calcium-dependent protein kinase (PrKC). The first kinase was found to enhance up to 3-fold acetyltransferase activity in a dose- and time-dependent manner. In homogenates from PMN previously stimulated with complement-coated zymosan particles, the decay of acetyltransferase activity was partially prevented by the addition of soybean trypsin inhibitor and almost completely inhibited when the homogenates were supplemented with inhibitors of alkaline phosphatase such as 50 mM KF and 100 microM paranitrophenylphosphate. Under these conditions it was possible to initiate the decay of acetyltransferase activity by adding an excess of alkaline phosphatase. Preincubation of PMN with 12-O-tetradecanoylphorbol-13-acetate previous or simultaneously to the addition of ionophore A23187 reduced the increase in acetyltransferase produced by ionophore A23187, whereas the generation of superoxide anions was enhanced. Addition of partially purified PrKC to homogenates from ionophore A23187-stimulated PMN, reduced acetyltransferase activity by 63%, whereas only a 16% inhibition was observed on homogenates from resting PMN. These data indicate the modulation of acetyltransferase activity in human polymorphonuclear leukocytes by a phosphorylation-dephosphorylation mechanism linked to cyclic AMP-dependent protein kinase. Phospholipid sensitive, calcium-dependent protein kinase seems not to be involved in the mechanism of activation, but, most probably, in the generation of negative activation signals.     

Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor

Digestion of the rat liver glucocorticoid receptor with chymotrypsin results in the generation of a 42-kDa fragment which contains the steroid-binding and DNA-binding domains and the antigenic site for the BuGR anti-glucocorticoid receptor monoclonal antibody, while digestion with trypsin generates a 15-kDa receptor fragment containing only the DNA-binding function and the BuGR epitope (Eisen, L.P., Reichman, M.E., Thompson, E.B., Gametchu, B., Harrison, R. W., and Eisen, H.J. (1985) J. Biol. Chem. 260, 11805-11810). In this paper, glucocorticoid receptor of mouse L cells that were grown in the presence of [32P]orthophosphate was digested with trypsin or chymotrypsin (either before or after immune purification with BuGR antibody) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and Western blotting. The receptor is endogenously phosphorylated only on serine residues. Chymotrypsin digestion results in a 32P-labeled 42-kDa receptor fragment which contains steroid-binding, DNA-binding, and BuGR-reactive sites. Trypsin digestion generates a 27-kDa steroid-bound fragment (meroreceptor) which is not labeled with 32P and a 32P-labeled 15-kDa fragment which contains both the DNA-binding domain and the BuGR epitope. We have calculated that there are 4 times as many phosphate residues in the intact receptor than in the 42-kDa chymotrypsin fragment. From examination of 32P-labeled receptor fragments, we have deduced that one phosphate is located between amino acids 398 and 447, a region containing the BuGR epitope and about one-third of the DNA-binding domain, and the remaining three phosphates appear to be clustered just to the amino-terminal side of the BuGR epitope in a region defined by amino acids 313 to 369. Treatment of intact 32P-labeled receptor in cytosol with alkaline phosphatase removes these three phosphates, but it does not remove the phosphate from the DNA-binding-BuGR-reactive fragment and it does not affect the ability of the transformed receptor to bind to DNA-cellulose.     

Single-channel activity of bilayers derived from sea urchin sperm plasma membranes at the tip of a patch-clamp electrode

Changes in the plasma membrane permeability of echinoderm sperm play a fundamental role in the acrosome reaction. During the reaction there is an increase in intracellular Ca2+ and Na+ and an efflux of H+ and K+. We have formed bilayers at the tip of patch pipets from a mixture of lipid vesicles and sea urchin sperm plasma membranes (12-50 microgram protein/ml). We observed three types of K+ channels (conductances: 22, 46, and 82 pS), two of which are partially blocked by TEA, and one Cl- channel (148 pS). The presence of K+ channels in sperm plasma membranes is consistent with the inhibition by TEA of the acrosome reaction in whole sperm and the membrane potential change that occurs during the reaction.     

Cross-reactive cellular and humoral immunity to carcinogen-induced chicken fibrosarcomas. I. Protective cross-immunity between transplantable tumor lines

Dimethylbenz[a]anthracene (DMBA)-induced transplantable fibrosarcomas in B2 homozygous chickens (SC line) grow progressively in normal chickens, but are rejected by chickens immunized previously with irradiated tumor cells and Corynebacterium parvum. Tumor-immune chickens resist challenge by the immunizing tumor lines as well as by some, but not all, fibrosarcoma lines. The pattern of cross-reactivity between four DMBA-induced transplantable tumor lines was examined in detail. Ability to reject a tumor challenge correlated very well (p less than 0.001) with the presence of delayed-type hypersensitivity (DTH) to that tumor. Immunization with one of two of the DMBA-induced lines tested also caused rejection of transplantable tumors developed from methylcholanthrene-induced and benzo(a)pyrene-induced primary fibrosarcomas. Although immunization with tumor caused DTH to chicken embryo fibroblasts (CEF), immunization with CEF failed to cause protective immunity or DTH to tumors. Presence of protective immunity, where tested, also correlated with the ability of spleen cells from immune donors to inhibit tumor growth in Winn tests. Humoral immunity exhibited even greater cross-reactivity than did cellular immunity. Distinct patterns of cross-reactivity were nevertheless observed with respect to the serum antibodies as detected in ELISA. Two of these patterns were also observed in several sera from primary tumor-bearing chickens, both including reactivity with CEF. Such reactivity was absent from normal chicken sera.     

Management of a harem breeding colony of rhesus monkeys to reduce trauma-related morbidity and mortality

A management procedure was developed for a harem breeding colony of rhesus monkeys to reduce trauma-related injuries and deaths resulting from the periodic removal of pregnant monkeys for research and their subsequent return to the population. Lower morbidity and mortality rates, a reduced mean conception interval, and a higher mean conception rate occurred when monkeys were maintained in permanent harems to which returning females were reintroduced compared to new social groups formed from aggregates of unfamiliar animals.     

Isolation and characterization of Pediococcus halophilus from salted anchovies (Engraulis anchoita)

The presence of bacteria in salted anchovies during and at the end of the curing process was investigated. Attempts to isolate bacteria under aerobic or anaerobic conditions led to the isolation of only bacteria of the genus Pediococcus which were identified as Pediococcus halophilus. The isolates correspond to a rather heterogeneous group in which some of the members differ in some biochemical tests from the types described in the literature.     

Reactions of sugar nitro-alkenes with acetoacetic esters

3,4,5,6,7-Penta-O-acetyl-1,2-dideoxy-1-nitro-d-gluco-hept-1-enitol reacts with methyl acetoacetate in an unusual Michael reaction, giving the normal adduct (6), and a bicyclic derivative (9) that arises from quasi-dimerization of the former when a high concentration of the base is used. Acetylation of compound 9 gives the hydroxylamine O-acetate (10).From the reactions of 3,4,5,6,7-penta-O-acetyl-1,2-dideoxy-1-nitro-d-galacto-hept-1-enitol with ethyl and tert-butyl acetoacetate, the normal adducts (7 and 8) were isolated. The structures of compounds 6 to 10 were established on the basis of their spectral properties (u.v., i.r., mass, and ¹H- and ¹³C-n.m.r.)     

Trifluoperazine and chlorpromazine block secretion from human platelets evoked at basal cytoplasmic free calcium by activators of C-kinase

Trifluoperazine, chlorpromazine and other drugs to inhibit calmodulin-dependent processes are also known to inhibit protein kinase C. The effect of these agents on secretion evoked by known activators of C-kinase has been studied in human platelets loaded with the fluorescent Ca indicator, quin2 and preincubated with aspirin. The secretory response stimulated by phorbol ester and exogenous diacyglycerol, at basal levels of cytoplasmic free Ca²⁺, [Ca²⁺]_i, was suppressed by trifluoperazine, chlorpromazine and W-7, as was the secretion evoked by collagen that occurs without a change in [Ca²⁺]_i, The response to thrombin, which is accompanied by elevated [Ca²⁺]_i was barely affected. Modest elevation of [Ca²⁺]_i by Ca ionophore was able to overcome the inhibitory effect of these drugs on the response to phorbol ester, diacylglycerol, and collagen.