Cyclic GMP stimulates inositol phosphate production in cultured pituitary cells: possible implication to signal transduction

Addition of the stable and permeable analog 8-bromo cyclic GMP (8-BR-cGMP) to myo-[2-3H]inositol prelabeled cultured rat pituitary cells results in enhanced formation of [3H]-myo-inositol monophosphate (IP1). The stimulatory effect of the cyclic nucleotide analog is additive to the effect of Li+, which accumulates IP1 via inhibition of inositol 1-monophosphatase, and also to the effect of gonadotropin releasing hormone (GnRH) which stimulates the formation of IP1, as well as that of inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3) via enhanced hydrolysis of polyphosphoinositides. Many Ca2(+)-mobilizing hormones acting via phosphoinosite turnover also stimulate cGMP formation. The cyclic nucleotide might then serve as a modulator by further hydrolysis of phosphoinositides needed for protein kinase C activation.     

Competition for nectar resources does not affect bee foraging tactic constancy

1. Competition alters animal foraging, including promoting the use of alternative resources. It may also impact how animals feed when they are able to handle the same food with more than one tactic. Competition likely impacts both consumers and their resources through its effects on food handling, but this topic has received little attention. 2. Bees often use two tactics for extracting nectar from flowers: they can visit at the flower opening, or rob nectar from holes at the base of flowers. Exploitative competition for nectar is thought to promote nectar robbing. If so, higher competition among floral visitors should reduce constancy to a single foraging tactic as foragers will seek food using all possible tactics. To test this prediction, field observations and two experiments involving bumble bees visiting three montane Colorado plant species (Mertensia ciliata, Linaria vulgaris, Corydalis caseana) were used under various levels of inter- and intra-specific competition for nectar. 3. In general, individual bumble bees remained constant to a single foraging tactic, independent of competition levels. However, bees that visited M. ciliata in field observations decreased their constancy and increased nectar robbing rates as visitation rates by co-visitors increased. 4. While tactic constancy was high overall regardless of competition intensity, this study highlights some intriguing instances in which competition and tactic constancy may be linked. Further studies investigating the cognitive underpinnings of tactic constancy should provide insight on the ways in which animals use alternative foraging tactics to exploit resources.     

Differential expression of multiple protein kinase C subspecies in rat central nervous tissue

Protein kinase C from a number of areas of rat central nervous tissue was resolved into three distinct fractions upon hydroxyapatite column chromatography. One of the enzyme fractions, designated type II, could be further distinguished into two subspecies with polyclonal antisera, which were raised against synthetic peptides specific for the predicted amino acid sequences of two alternative cDNA clones encoding this enzyme type. Using a combination of these biochemical and immunological techniques, the relative activity of the multiple subspecies of protein kinase C was assessed for each brain area. A distinct regional pattern of expression was found, which per se may be an important factor in determining the response of different neuronal cell types to extracellular stimuli.     

Effect of phorbol ester on stimulus-secretion coupling mechanisms in gonadotropin releasing hormone-stimulated pituitary gonadotrophs

The feedback regulatory control mechanism exerted by activated Ca2+/phospholipid-dependent protein C kinase upon gonadotropin releasing hormone (GnRH) binding, stimulation of phosphoinositide turnover and gonadotropin secretion was investigated in cultured pituitary cells. Addition of the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), at concentrations which activate pituitary protein C kinase, to cultured pituitary cells resulted in up-regulation of GnRH receptors (155% at 4 h). The stimulatory effect of GnRH on [3H]inositol phosphates (Ins-P) production in myo-[2-3H]inositol prelabeled pituitary cells was not inhibited by prior treatment of the cells with TPA (10(-9)-10(-7) M). Higher concentrations of TPA (10(-6)-10(-5) M) inhibited the effect of GnRH on [3H]Ins-P production. Increasing concentrations of TPA or the permeable analog of diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) stimulated luteinizing hormone (LH) release from cultured pituitary cells with ED50 values of 5 x 10(-9) M and 10 micrograms/ml, respectively. No consistent inhibition or additivity of LH release was observed when increasing doses of TPA or OAG were added with a submaximal dose of GnRH. These results suggest that protein C kinase might mediate the known homologous up-regulation of GnRH receptors during the reproductive cycle. Protein C kinase is positively involved in mediating the process of gonadotropin secretion. Unlike many other systems, activation of protein C kinase in pituitary gonadotrophs is not involved in negative feed-back regulation of stimulus-secretion-coupling mechanisms in GnRH-stimulated gonadotrophs.     

Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the CuII(Cys112Asp) protein

Replacement of the cysteine at position 112 of Pseudomonas aeruginosa azurin with an aspartic acid residue results in a mutant (Cys112Asp) protein that retains a strong copper-binding site. Cu^II(Cys112Asp) azurin can be reduced by excess [Ru^II(NH₃)₆]²⁺, resulting in a Cu^I protein with an electronic absorption spectrum very similar to that of wild-type Cu^I azurin. Cys112Asp azurin exhibits reversible interprotein electron-transfer reactivity with P. aeruginosa cytochrome c ₅₅₁ (μ?=?0.1?M sodium phosphate (pH?7.0);E°(Cu^II/I)?=?180 mV vs NHE); this redox activity indicates that electrons can still enter and exit the protein through the partially solvent-exposed imidazole ring of His117. The structure of Cu^II(Cys112Asp) azurin at 2.4-Å resolution shows that the active-site copper is five coordinate: the pseudo-square base of the distorted square-pyramidal structure is defined by the imidazole N^δ atoms of His46 and His117 and the oxygen atoms of an asymmetrically-bound bidentate carboxylate group of Asp112; the apical position is occupied by the oxygen atom of the backbone carbonyl group of Gly45. The Cu^II–Asp112 interaction is distinguished by an approximately 1.2-Å displacement of the metal center from the plane defined by the Asp112 carboxylate group.     

Isolated soluble fractions from the murine B16 melanoma induce primary in vitro syngeneic antitumor responses

Summary This paper extends our previous studies, which documented our ability to isolate immunogenic entities from nonimmunogenic or weakly immunogenic tumors.B16 melanoma cells failed, in our in vitro experimental system, to induce anti-B16 cytotoxic responses in spleen cells derived from normal syngeneic C57BL/6 mice. The B16 melanoma cellular homogenate was fractionated on an Ultrogel AcA 34 column, and the various fractions were tested for their ability to induce anti-B16 cytotoxic responses under the same conditions as those used for intact B16, the nonimmungenic tumor cells. Certain fractions, some of them with relatively low protein concentrations, induced anti-B16 cytotoxic responses in spleen cells of normal C57BL/6 mice, whereas others, some of them with relatively high protein concentrations, failed to induce such responses. One fraction (Fr.), designated Fr. 5/6, was examined in detail. It was found that in normal syngeneic spleen cells this fraction induced effector cells that efficiently killed (at various E : T ratios) the relevant B16 target cells and RBL5 syngeneic tumor cells, but not the YAC allogeneic tumor cells or C57BL/6 lymphoblasts. Furthermore, an excess of unlabeled B16 cells most efficiently blocked the ability of these anti-B16 effector cells to kill radiolabeled B16 target cells. RBL5 tumor cells, YAC tumor cells, or C57BL/6 lymphoblasts failed to block these effector cells efficiently. A significant fraction of the effector cells induced with Fr. 5/6 was characterized as thymus-derived cells (Thy-1⁺, Thy-2⁺3⁺ cells). It was suggested that another fraction of the cellular population was natural killer cells, which cytolyzed the RBL5 target cells. Various theoretical and practical aspects of these findings are discussed.     

A possible role for cyclic GMP in mediating the effect of luteinizing hormone releasing hormone on gonadotropin release in dispersed pituitary cells of the female rat.

In continuing studies on cyclic nucleotide involvement in the regulation of gonadotropin release, we have measured the cyclic nucleotide content and rate of LH and FSH release during stimulation by LHRH of dispersed overnight cultured cells from the pituitaries of adult female rats. The minimal effective concentration of LHRH was 0.1 nM and half maximal stimulation of gonadotropin release was observed in the presence of 1.0 nM LHRH. Significant release of both LH and FSH was detectable after only 10 min in the presence of 5 nM LHRH. The presence of fetal calf serum (FCS) in the overnight culture medium increased basal cGMP levels significantly, whereas horse serum (HS) had no effect, therefore all experiments were conducted on cells cultured in the presence of HS. Treatment of the cultured cells with the phosphodiesterase inhibitors theophylline (TH) or isobutyl-methyl-xanthine (MIX) revealed a preferential stimulatory effect of TH on basal cAMP levels and of MIX on cGMP levels. Throughout these experiments, LHRH had no effect on cAMP levels. In the presence of MIX, concentrations of the releasing hormone as low as 1 nM induced a significant rise in the level of cGMP whereas in its absence, cGMP levels appeared to be unchanged by LHRH. The increase was detectable after 10 min of incubation. MIX alone slightly increased LH and FSH release and significantly potentiated the response of the cells to increasing doses of LHRH up to, but not beyond, 10 nM. The data support the possibility that cGMP may be involved in the mechanism of action of LHRH.