Role of Ni in coupling angiotensin receptors to inhibition of adenylate cyclase in hepatocytes

Angiotensin II can inhibit glucagon-stimulated cyclic AMP production in hepatocytes and adenylate cyclase activity in hepatic membranes. Pertussis toxin, an exotoxin produced by Bordetella pertussis, was used to investigate the role of the inhibitory guanine nucleotide-binding regulatory protein of adenylate cyclase (Ni) in coupling angiotensin receptors to the adenylate cyclase system. An assay was developed using [32P] NAD+ to quantitate the amount of Ni protein in the membrane and the extent of its ADP-ribosylation catalyzed by toxin. The ability of angiotensin to inhibit adenylate cyclase and interact with its receptor was compared with the degree of modification of Ni in membranes prepared from isolated hepatocytes. In control membranes angiotensin II inhibited basal adenylate cyclase by 35%. When all of the Ni molecules in the membrane were ADP-ribosylated, angiotensin did not inhibit adenylate cyclase. However, the attenuation of angiotensin's effect on cyclase was not linearly correlated with the degree of modification of Ni; ADP-ribosylation of greater than 80% of the Ni was required before a reduction of the angiotensin effect was observed. A possible explanation for this finding is an excess of Ni molecules in the membrane (approximately 3.4 pmol/mg of membrane protein) over angiotensin II receptors (approximately 1.2 pmol/mg of membrane protein). 125I-angiotensin bound to sites in the membrane with two affinities. Computer fitting of the binding isotherms yielded parameters of N1 = 279 fmol/mg protein, Kd1 = 0.2 nM; N2 = 904 fmol/mg protein, Kd2 = 1.4 nM. When all of the Ni molecules in the membrane were ADP-ribosylated, angiotensin bound to only one site with binding parameters of N = 349 fmol/mg protein, Kd = 0.4 nM. GTP-gamma-S caused a 7-fold increase in the Kd of this site to 2.7 nM. Overall, the data indicate that the Ni protein mediates the effect of angiotensin on adenylate cyclase. The observation that GTP-gamma-S can markedly decrease the affinity of angiotensin receptors when all Ni molecules are ADP-ribosylated suggests that angiotensin receptors may couple to other GTP-binding proteins which may mediate the effects of angiotensin in other signal transduction systems.     

Effects of guanyl nucleotides and rhodopsin on ADP-ribosylation of the inhibitory GTP-binding component of adenylate cyclase by pertussis toxin

Hormonal inhibition of adenylate cyclase is mediated by a guanyl nucleotide binding protein, Gi, which is composed of alpha, beta, and gamma subunits (Gi alpha, G beta gamma). Pertussis toxin blocks hormonal inhibition by catalyzing the ADP-ribosylation of Gi alpha. With purified Gi subunits, but without nucleotides, it was observed that toxin-catalyzed ADP-ribosylation of Gi alpha was negligible in the absence of G beta gamma; ATP, previously shown to increase ADP-ribosylation in membranes, enhanced the ADP-ribosylation of Gi alpha in the absence, more than in the presence, of G beta gamma. Prior studies (Kanaho, Y., Tsai, S.-C., Adamik, R., Hewlett, E.L., Moss, J., and Vaughan, M. (1984) J. Biol. Chem. 259, 7378-7381) had demonstrated that rhodopsin, the retinal photon receptor protein, can replace inhibitory hormone receptors, and stimulate the hydrolysis of GTP by Gi alpha in the presence of G beta gamma. Photolyzed rhodopsin, but not the inactive, dark protein, inhibited ADP-ribosylation of Gi alpha in the presence of G beta gamma. ADP-ribosylation of Gi alpha, in the presence of G beta gamma and photolyzed (but not dark) rhodopsin was increased by guanosine 5'-O-(2-thiodiphosphate) or GDP, but not by (beta, gamma-methylene)guanosine triphosphate or guanosine 5'-O-(3-thiotriphosphate). Presumably, photolyzed rhodopsin and nucleoside triphosphate analogues activate Gi, whereas with dark rhodopsin and nucleoside diphosphates Gi is in the inactive state. The latter appears to be the preferred substrate for pertussis toxin. These observations are consistent with other evidence that rhodopsin and inhibitory hormone receptors are functionally similar.     

Effects of Tropical Rotation Crops on Meloidogyne arenaria Population Densities and Vegetable Yields in Microplots

The effects of 12 summer crop rotation treatments on population densities of Meloidogyne arenaria race 1 and on yields of subsequent spring vegetable crops were determined in microplots. The crop sequence was: (i) rotation crops during summer 1991 ; (ii) cover crop of rye (Secale cereale) during winter 1991-92; (iii) squash (Cucurbita pepo) during spring 1992; (iv) rotation crops during summer 1992; (v) rye during winter 1992-93; (vi) eggplant (Solanum melongena) during spring 1993. The 12 rotation treatments were castor (Ricinus communis), cotton (Gossypium hirsutum), velvetbean (Mucuna deeringiana), crotalaria (Crotalaria spectabilis), fallow, hairy indigo (Indigofera hirsuta), American jointvetch (Aeschynomene americana), sorghum-sudangrass (Sorghum bicolor x S. sudanense), soybean (Glycine max), horsebean (Canavalia ensiformis), sesame (Sesamum indicum), and peanut (Arachis hypogaea). Compared to peanut, the first eight rotation treatments resulted in lower (P ≤ 0.05) numbers of M. arenaria juveniles on most sampling dates. Soybean, horsebean, and sesame rotations were less effective in suppressing nematodes. Yield of squash was greater (P ≤ 0.05) following castor, cotton, velvetbean, and crotalaria than following peanut. Compared to the peanut rotation, yield of eggplant was enhanced (P ≤ 0.10) following castor, crotalaria, hairy indigo, American jointvetch, and sorghum-sudangrass. Several of these rotation crops may provide a means for depressing M. arenaria population densities on a short-term basis to enhance yields in a subsequent susceptible vegetable crop.     

Adenosine and related compounds counteract tumor necrosis factor-alpha inhibition of neutrophil migration: implication of a novel cyclic AMP-independent action on the cell surface

Human rTNF-alpha (greater than or equal to U/ml) decreased PMN nondirected and directed migration to FMLP to approximately 50% of control. Adenosine (100 microM) almost completely restored hrTNF-inhibited migration (nondirected from 54 to 92% and directed migration to from 54 to 93% of control). The lowest concentration of adenosine that restored hrTNF-inhibited migration was 3 microM, and the adenosine analogue, 5'-(N-cyclopropyl)-carboxamido-adenosine (CPCA) was more potent than adenosine. Although CPCA binds to A2-receptors and stimulates adenylate cyclase, the reversal of hrTNF-inhibited chemotaxis was found to be independent of both PMN cAMP content and binding to A2-receptors, because neither 8-Br-cAMP nor pertussis adenylate cyclase restored hrTNF-inhibited PMN chemotaxis and the A2-receptor antagonist, 1,3-dipropyl-7-methylxanthine decreased CPCA stimulated cAMP but enhanced CPCA-restoration of hrTNF-inhibited chemotaxis. The effect of adenosine could be augmented by inhibition of adenosine uptake and decreased by adenosine deamination. Pentoxifylline, (3,7 dimethyl-1-[5 oxo-hexyl] xanthine), like adenosine also restored PMN chemotaxis inhibited by hrTNF. The adenosine receptor antagonist, 1,3-dipropyl-8(phenyl-p-acrylate)-xanthine (BW A1433U), decreased restoration of hrTNF-inhibited chemotaxis by CPCA or pentoxifylline. Thus, the inhibitory effect of hrTNF on PMN migration can be counteracted by adenosine, CPCA, pentoxifylline, and compounds that increase adenosine availability to the surface of the PMN. Inasmuch as an A1-selective agonist N6-cyclopentyladenosine was less active, and the action of the A2-selective agonist CPCA was enhanced by an A2-receptor antagonist, we hypothesize that neither A1 or A2 receptors are involved in adenosine restoration of hrTNF-inhibited chemotaxis. Further, increased cAMP, an A2-regulated event, does not cause the effect, and adenosine restoration of hrTNF-inhibited migration does not appear to be mediated by changes in PMN [F-actin], FMLP receptor expression, or cytosolic calcium. Hence, the restoration of hrTNF-inhibited chemotaxis is controlled by a novel cyclic AMP-independent action on the PMN surface.     

A Biocultural Investigation of Gender Differences in Tobacco Use in an Egalitarian Hunter-Gatherer Population

In the developing world, the dramatic male bias in tobacco use is usually ascribed to pronounced gender disparities in social, political, or economic power. This bias might also reflect under-reporting by woman and/or over-reporting by men. To test the role of gender inequality on gender differences in tobacco use we investigated tobacco use among the Aka, a Congo Basin foraging population noted for its exceptionally high degree of gender equality. We also tested a sexual selection hypothesis—that Aka men’s tobacco use is related to risk taking. Tobacco use, income, tobacco purchases, tobacco sharing, reasons for using tobacco, risk taking, and other variables were measured using structured surveys and peer reports. Tobacco use was verified by testing for salivary cotinine, a nicotine metabolite. Contrary to expectations, we found a very large male bias in tobacco use. Low levels of use among females appeared to be explained by aversions to tobacco, concerns over its negative effects on fetal health, and a desire to attract husbands, who prefer nonsmoking wives. High male use appeared to be related to a desire to enhance hunting abilities and attract and/or retain wives, who prefer husbands that smoke. We conclude that low levels of smoking by Aka women are better explained by the hypothesis that women evolved to avoid plant toxins to protect their fetuses and nursing infants. High male use might be better explained by sexual selection. We also highlight the important role that recreational drugs appear to play in hunter-gatherer sharing relationships.     

Effects of Monoclonal Antibodies,Cationized Ferritin,and Other Organic Molecules on Adhesion of Pasteuria penetrans Endospores to Meloidogyne incognita

The incidence of adhesion of Pasteuria penetrans endospores to Meloidogyne incognita second-stage juveniles (J2) was studied after pretreatment of the latter with monoclonal antibodies (MAb), cationized ferritin, and other organic molecules in replicated trials. Monoclonal antibodies developed to a cuticular epitope of M. incognita second-stage juveniles gave significant reductions in attachment of P. penetrans endospores to treated nematodes. MAb bound to the entire length of J2 except for the area of the lateral field, where binding was restricted to the incisures. Since reductions in attachment with MAb treatment were modest, it is uncertain if these results implicated a specific surface protein as a factor that interacted in binding of the endospore to the nematode cuticle. Endospore attachment was decreased following treatment of the nematode with the detergents sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB). Endospore attachment to live nematodes was significantly greater than attachment to dead nematodes. Attachment rates of three P. penetrans isolates to M. incognita race 3 varied between isolates. The effects of neuraminidase, pronase, pepsin, trypsin, lipase, and Na periodate on endospore attachment were inconsistent. The cationic dye alcian blue, which binds sulfate and carboxyl groups on acidic glycans, had no consistent effect on endospore attachment. The incidence of endospore attachment was significantly lower but modest, at best, for nematodes that were treated with cationized ferritin alone or cationized ferritin following monoclonal antibody. The lack of consistency or extreme reduction in most experiments suggests that attachment of P. penetrans spores to M. incognita is not specified by only one physico-chemical factor, but may involve a combination of at least two physico-chemical factors (including surface charge and movement of the J2). This points to a need for analysis of combined or factorial treatment effects.     

Breast Reconstruction for Malignant or Premalignant Disease

In ten patients breast reconstruction was done after surgical treatment for a premalignant or malignant breast disease. In six of these, prophylactic subcutaneous mastectomy and implant reconstruction were carried out, and in the remaining four reconstruction was done after simple or modified radical mastectomy. It is suggested that these procedures should be considered by those physicians and surgeons who undertake evaluation and treatment of breast disease in women. Breast reconstruction should be considered and offered to patients who suffer from the severe personal and emotional trauma attendant to surgical operation for breast disease.     

Molecules from apoptotic pathways modulate HIV-1 replication in Jurkat cells

The replication of viruses involves control of some aspects of host cell homeostasis by modification of target cell metabolism and regulation of the apoptotic machinery. It is not well known whether molecules involved in apoptotic pathways affect human immunodeficiency virus type 1 (HIV-1) replication and regulate viral yields. Using the susceptible Jurkat cell line, we studied the relationship of apoptosis-associated molecules with HIV-1 virus production using a sensitive real-time RT-PCR assay. Here, we found that expression of proapoptotic proteins, including Fas ligand (FasL), FADD, or p53 significantly increased HIV-1 virus production. In contrast, the expression of antiapoptotic molecules, such as FLIP, Bcl-X_L, and XIAP, decreased HIV-1 virus production. Knockdown of Bax with siRNA and FADD with expression of its antisense mRNA also inhibited viral replication and the caspase-3 inhibitor, Z-DEVD, and decreased virus production. These data indicate that HIV-1 infection regulates the apoptosis process to facilitate viral replication and inhibition of apoptosis may inhibit HIV-1 replication and cytopathogenesis. We also discuss the effects of MAPK signaling pathways and apoptosis on HIV-1 replication.     

Damage Functions for Meloidogyne arenaria on Peanut

Microplot experiments were conducted in 1989 and 1990 to determine the relationship between yield of peanut (Arachis hypogaea) and inoculum density ofMeloidogyne arenaria race 1. Nine inoculum densities were used, ranging from 0-200 eggs/100 cm³ soil (1989) or from 0-100 eggs/100 cm³ (1990), and each density was replicated 10 times. In 1989, higher final densities (mean of 1,171 juveniles [J2]/100 cm³ soil) were obtained in plots inoculated with 0.5 to 50 eggs/100 cm³ soil than in plots inoculated with 100 to 200 eggs/100 cm³ (313 J2/100 cm³ soil). In 1990, final densities of M. arenaria reached high levels (≥ 1,111 J2/100 cm³ soil) in all inoculated plots. Pod yield and dry weight of foliage at harvest were negatively correlated (P ≤ 0.05) with inoculum density in both seasons. In 1989, the relationship between pod weight (y) and initial density (x) was described by Seinhorst''s equation, with y = 0.088 + 0.91(0.90)⁽x⁻¹⁾ and r² = 0.826. In 1990, the relationship was y = 0.22 + 0.78(0.97)⁽x⁻¹⁾ and r² = 0.794. These equations suggest tolerance limits of approximately 1 egg/100 cm³ soil, which may require specialized methods, such as bioassay, for detection.     

Genetic diversity and relationships in populations of Bordetella spp

Genetic diversity in 60 strains of three nominal Bordetella species recovered from humans and other mammalian hosts was assessed by analyzing electrophoretically demonstrable allelic variation at structural genes encoding 15 enzymes. Eleven of the loci were polymorphic, and 14 distinctive electrophoretic types, representing multilocus genotypes, were identified. The population structure of Bordetella spp. is clonal, and genetic diversity is relatively limited compared with most other pathogenic bacteria and is insufficient to justify recognition of three species. All isolates of Bordetella parapertussis were of one electrophoretic type, which was closely similar to 9 of the 10 electrophoretic types represented by isolates of Bordetella bronchiseptica. Bordetella pertussis 18-323, which is used in mouse potency tests of vaccines, is more similar genetically to isolates of B. bronchiseptica and B. parapertussis than to other isolates currently assigned to the species B. pertussis. Apart from strain 18-323, the isolates of B. pertussis represented only two closely related clones, and all isolates of B. pertussis from North America (except strain 18-323) were genotypically identical. Strain Dejong, which has been classified as B. bronchiseptica, was strongly differentiated from all of the other Bordetella isolates examined.