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.     

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.     

Rhythmic signalling and entrainment inVespa orientalis larvae: Characterization of the underlying interactions

Rhythmic “circa-second” contrations of larvae of the hornetVespa orientalis, believed to serve as hunger signals, were studied. A considerable degree of coordination among individual larvae, both in frequency and phase of these contractions, has been observed. The oscillations of singly isolated larvae are of short duration, non-constant, with increasing intervals in between and there is a substantial variability in the patterns shown by different larvae. In contrast, the association of two or more larvae leads to enhancement of their periodic behaviour and to (partial) entrainment. Communication among larvae may perhaps be mediated by the sound pulses (“scratching” noises) which are generated by these contractions. We have subjected individual and grouped larvae to external sound pulses and were able to demonstrate: (a) enhancement of rhythmic activity; (b) phase resetting; (c) entrainment to an external oscillator within a range of frequencies; (d) the existence of a subharmonic mode of entrainment. We propose a simple phenomenologic model to account for these larvae responses. Our model assumes the existence of an “energy” variable which declines with time but is upgraded, in a phase-dependent way, by external stimuli. Based in part on work performed by V. Barenholz-Paniry in partial fulfillment of the requirements for the M.Sc. degree from the Sackler Faculty of Medicine, Tel Aviv University, 1986.     

Partial amino acid sequence of porcine elastase II. Active site and the activation peptide regions

The partial amino acid sequence of porcine elastase II, isolated from crude trypsin Type II, was determined. The enzyme consists of two polypeptide chains, a light chain composed of 11 residues, and a heavy chain (Mr = 23 500) with four intrachain disulfide bridges; the two chains are held together by one interchain disulfide bond. Elastase II was fragmented into several peptides by chemical cleavages with CNBr at the two methionine residues, 99 and 180 (chymotrypsinogen numbering), and with hydroxylamine at the peptide bond following DIP-Ser195. About 50% of the sequence was determined and the positions of 120 amino acids were located, including the light chain residues and most of the active site residues. The partial sequence shows 64% difference between porcine elastase II and elastase I and only 26% difference between porcine elastase II and bovine chymotrypsin B.     

Involvement of two differenturf-s related mitochondrial sequences in the molecular evolution of the CMS-specificS-Pcf locus in petunia

In petunia, a mitochondrial (mt) locus,S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). TheS-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF,Pcf, contains parts of theatp9 andcoxII genes and an unidentified reading frame,urf-s. The second and third ORFs contain NADH dehydrogenase subunit 3 (nad3) and ribosomal protein S12 (rps12) sequences, respectively. Thenad3 andrps12 sequences included in theS-Pcf locus are identical to the corresponding sequences on the mt genome of fertile petunia. In both CMS and fertile petunia, only a single copy ofnad3 andrps12 has been detected on the physical map of the main mt genome. The origin of theurf-s sequence and the molecular events leading to the formation of the chimericS-Pcf locus are not known. This paper presents evidence indicating that two different mt sequences, related tourf-s and found in fertile petunia lines (orf-h and Rf-1), might have been involved in the molecular evolution of theS-Pcf locus. Southern analysis of mtDNA derived from both fertile and sterile petunia plants suggests that one of theseurf-s related sequences (showing 100% homology tourf-s and termedorf-h) is located on a sublimon. An additional, low-homologyurf-s related sequence (Rf-1) is shown to be located on the main mt genome 5′ to thenad3 gene. It is, thus, suggested that the sequence of events leading to the generation of theS-Pcf locus might have involved introduction of theorf-h sequence, via homologous recombination, into the main mt genome 5′ tonad3 at the region where the Rf-1 sequence is located.     

Involvement of two differenturf-s related mitochondrial sequences in the molecular evolution of the CMS-specificS-Pcf locus in petunia

In petunia, a mitochondrial (mt) locus,S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). TheS-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF,Pcf, contains parts of theatp9 andcoxII genes and an unidentified reading frame,urf-s. The second and third ORFs contain NADH dehydrogenase subunit 3 (nad3) and ribosomal protein S12 (rps12) sequences, respectively. Thenad3 andrps12 sequences included in theS-Pcf locus are identical to the corresponding sequences on the mt genome of fertile petunia. In both CMS and fertile petunia, only a single copy ofnad3 andrps12 has been detected on the physical map of the main mt genome. The origin of theurf-s sequence and the molecular events leading to the formation of the chimericS-Pcf locus are not known. This paper presents evidence indicating that two different mt sequences, related tourf-s and found in fertile petunia lines (orf-h and Rf-1), might have been involved in the molecular evolution of theS-Pcf locus. Southern analysis of mtDNA derived from both fertile and sterile petunia plants suggests that one of theseurf-s related sequences (showing 100% homology tourf-s and termedorf-h) is located on a sublimon. An additional, low-homologyurf-s related sequence (Rf-1) is shown to be located on the main mt genome 5′ to thenad3 gene. It is, thus, suggested that the sequence of events leading to the generation of theS-Pcf locus might have involved introduction of theorf-h sequence, via homologous recombination, into the main mt genome 5′ tonad3 at the region where the Rf-1 sequence is located. Contribution [No. 1581-E (1995 series)] from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel 50 250     

Conformation and interactions of bombolitin I analogues with SDS micelles and phospholipid vesicles: CD,fluorescence, two-dimensional NMR and computer simulations

Bombolitins are five structurally related heptadecapeptides acting at the membrane level able to lyse erythrocytes and liposomes and to enhance the activity of phospholipase A ₂(PLA₂). In the presence of SDS or phospholipid vesicles bombolitins are able to form amphiphilic α-helical structures and this property seems to be the major determinant of bioactivity. In order to test the model of interaction between bombolitin I and membranes, an analogue was synthesized in which all the lysines were replaced by arginines: ([Arg^2,9,12, Ile¹⁰] bornbolitin I). The design ofthis sequence allowed the synthesis of a second analogue through a specijic postsynthetic dansylation at the ?-amino group qf a lysine residue replacing the original leucine residue at position 7. The, first analogue was, fiilly characterized by CD and two-dimensional nmr in the presence of SDS or phospholipid vesicles. The peptide, folds into an amphiphilic α-helical confbrrnation with the helical segment spanning the central part of the sequencefrom Ile³ to His¹⁶. This behavior is identical to that observed for the native sequence. The replacement of Iysine residues by arginine hus no detectable effect on the conformational prderence of the peptide chain. By CD and fluorescence spectroscopy measurements, the fluorophore-containing analogue [Arg^2,9,12, Lys⁷(?-dansyl)] bombolitin I also folded into the α-helical conformation in the presence of SDS micelles or phospholipid vesicles. In particular, the dansyl fluorophore, which is located approximately in the middle of the apolar surface ojthe amphiphilic helix, is clearly buried in a hydrophobic environment when the peptide is bound to phospholipid vesicles. These findings support the hypothesis that the peptide helices are oriented parallel to the vesicle surface. © 1995 John Wiley & Sons, Inc.     

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.