Neuraxial block anesthetic technique in a patient with SCN8A encephalopathy: case report.

Mutations in SCN8A gene lead to changes in sodium channels in the brain, which are correlated with severe epileptic syndrome. Due to the rarity, there are few studies that support anesthesia in that population. The present study aims to report alternatives to inhalation anesthesia at epileptic encephalopathy. CASE REPORT: Male, 4 years old, with SCN8A encephalopathy with surgical indication of orchidopexy. Neuroaxis block was performed and dexmedetomidine was used as a pre-anesthetic and sedation. The anestheticsurgical act was uneventful. CONCLUSION: The association of neuraxial block and dexmedetomidine proved to be a viable alternative for surgery in patients with SCN8A encephalopathy.

Constraints on Dark Matter Properties from Observations of Milky Way Satellite Galaxies.

We perform a comprehensive study of Milky Way (MW) satellite galaxies to constrain the fundamental properties of dark matter (DM). This analysis fully incorporates inhomogeneities in the spatial distribution and detectability of MW satellites and marginalizes over uncertainties in the mapping between galaxies and DM halos, the properties of the MW system, and the disruption of subhalos by the MW disk. Our results are consistent with the cold, collisionless DM paradigm and yield the strongest cosmological constraints to date on particle models of warm, interacting, and fuzzy dark matter. At 95% confidence, we report limits on (i) the mass of thermal relic warm DM, m_{WDM}>6.5 keV (free-streaming length, λ_{fs}≲10h^{-1} kpc), (ii) the velocity-independent DM-proton scattering cross section, σ_{0}<8.8×10^{-29} cm^{2} for a 100 MeV DM particle mass [DM-proton coupling, c_{p}≲(0.3 GeV)^{-2}], and (iii) the mass of fuzzy DM, m_{ϕ}>2.9×10^{-21} eV (de Broglie wavelength, λ_{dB}≲0.5 kpc). These constraints are complementary to other observational and laboratory constraints on DM properties.

Widespread methane seepage along the continental margin off Svalbard - from Bjørnøya to Kongsfjorden.

Numerous articles have recently reported on gas seepage offshore Svalbard, because the gas emission from these Arctic sediments was thought to result from gas hydrate dissociation, possibly triggered by anthropogenic ocean warming. We report on findings of a much broader seepage area, extending from 74° to 79°, where more than a thousand gas discharge sites were imaged as acoustic flares. The gas discharge occurs in water depths at and shallower than the upper edge of the gas hydrate stability zone and generates a dissolved methane plume that is hundreds of kilometer in length. Data collected in the summer of 2015 revealed that 0.02-7.7% of the dissolved methane was aerobically oxidized by microbes and a minor fraction (0.07%) was transferred to the atmosphere during periods of low wind speeds. Most flares were detected in the vicinity of the Hornsund Fracture Zone, leading us to postulate that the gas ascends along this fracture zone. The methane discharges on bathymetric highs characterized by sonic hard grounds, whereas glaciomarine and Holocene sediments in the troughs apparently limit seepage. The large scale seepage reported here is not caused by anthropogenic warming.

Methane excess in Arctic surface water-triggered by sea ice formation and melting.

Arctic amplification of global warming has led to increased summer sea ice retreat, which influences gas exchange between the Arctic Ocean and the atmosphere where sea ice previously acted as a physical barrier. Indeed, recently observed enhanced atmospheric methane concentrations in Arctic regions with fractional sea-ice cover point to unexpected feedbacks in cycling of methane. We report on methane excess in sea ice-influenced water masses in the interior Arctic Ocean and provide evidence that sea ice is a potential source. We show that methane release from sea ice into the ocean occurs via brine drainage during freezing and melting i.e. in winter and spring. In summer under a fractional sea ice cover, reduced turbulence restricts gas transfer, then seawater acts as buffer in which methane remains entrained. However, in autumn and winter surface convection initiates pronounced efflux of methane from the ice covered ocean to the atmosphere. Our results demonstrate that sea ice-sourced methane cycles seasonally between sea ice, sea-ice-influenced seawater and the atmosphere, while the deeper ocean remains decoupled. Freshening due to summer sea ice retreat will enhance this decoupling, which restricts the capacity of the deeper Arctic Ocean to act as a sink for this greenhouse gas.

Treatment of acute paraquat intoxication using recommended megadose of vitamin C: a reappraisal.

Megadose of vitamin C (MVC) has been proposed for an emergent treatment of acute paraquat (PQ) poisoning. However, the safety issue of this treatment protocol has not been evaluated. Here, we present the first evidence that vitamin C can promote aggravated production of hydroxyl radical (OH(•)) via interacting with preexisting PQ(+•)/H2O2 system in a nonmetal-catalyzed manner. This enhanced oxidative stress would therefore expect to cause more deleterious effect during acute PQ intoxication. To lend support to this possibility, we set out to attest the effects of MVC on a simulated, PQ-intoxicated, Madin-Darby canine kidney (MDCK) cell model. First, PQ alone could trigger oxidative-nitrosative stress (ONS) through robust generation of reactive oxygen species and nitric oxide (NO) that could induce apoptotic killing via promoting effective release of mitochondrial cytochrome c, an apoptogenic factor. The percentage of apoptosis for MDCK cells treated with 1.0 mM PQ for 24 h was 16.3 ± 13.0%. However, when MDCK cells were treated with a combination of PQ (1.0 mM) and MVC (20 mM) for 24 h, the severity of apoptotic killing was further exacerbated as reflected by a nearly 7-fold increase in the release of mitochondrial cytochrome c and the percentage of apoptotic cell population rose sharply to 90.7 ± 5.1%. These data indicate that MVC apparently exacerbates further killing rather than cytoprotection on this simulated, PQ-intoxicated MDCK cell model and suggest that the treatment of PQ poisoning using MVC protocol should be cautious.

The arabinogalactan-proteins from pressed juice of Echinacea purpurea belong to the hybrid class of hydroxyproline-rich glycoproteins.

Pressed juices from Echinacea purpurea are used as non-specific immunostimulants, and arabinogalactan-proteins (AGPs) are part of the active principle. An AGP fraction was isolated from pressed juice of E. purpurea by precipitation with ss-glucosyl Yariv reagent, followed by gel-permeation chromatography. Polyclonal antibodies directed against the carbohydrate moiety of this AGP fraction showed a preferential specificity for E. purpurea AGPs from pressed juice over those extracted from E. purpurea suspension culture and other plant species. Native AGPs purified from this AGP fraction by RP-HPLC were then deglycosylated for N-terminal protein sequencing resulting in the identification of three major polypeptides. They show characteristic motifs of classical AGPs but also some features of extensins, suggesting these may be "hybrid" hydroxyproline-rich glycoproteins (HRGPs).

ICP is lower during propofol anaesthesia compared to isoflurane and sevoflurane.

OBJECTIVES: Propofol is a cerebral vasoconstrictor while inhalation anaesthetics like isoflurane and sevoflurane act as cerebral vasodilators in both animal and human studies. This difference of action upon cerebral vessels might implicate a lower ICP during propofol anaesthesia. Cerebral metabolism is decreased by all three anaesthetics. In a prospective, randomised multicenter study ICP was compared during anaesthesia with propofol, isoflurane and sevoflurane. METHODS: 117 patients subjected to elective craniotomy for supratentorial tumour. Propofol: N = 41; isoflurane: N = 38; sevoflurane: N = 38. Nitrous oxide was omitted and all anaesthetics were supplemented with a continuous infusion of fentanyl. ICP was measured subdurally after removal of the bone flap. MABP, CPP, PCO2, AVDO2, rectal temperature, tumour size and midline shift were registered too. STATISTICS: Kruskal-Wallis Variance on Ranks. All values in medians with range. P < 0.05 was considered significant. RESULTS: ICP (mmHg): propofol 7 (-1-20), isoflurane 12 (1-29), sevoflurane 11 (2-32). ICP was significantly lower in the propofol group compared to the isofluane and sevoflurane groups. CPP (mmHg): propofol 80 (45-104), isoflurane 60 (32-84), sevoflurane 63 (44-77). CPP was significantly higher in the propofol group compared to the isoflurane and sevoflurane groups. AVDO2 (mmol/l): propofol 3.1 (0.9-5.1), isoflurane 2.5 (1.1-4.5), sevoflurane 2.6 (0.8-4.1). AVDO2 was significantly higher in the propofol group compared to the isoflurane and sevoflurane groups. No significant differences in PCO2, rectal temperature, tumour size and midline shift were found. CONCLUSIONS: Subdural ICP is significantly lower during propofol anaesthesia compared to isoflurane and sevoflurane anaesthesia. CPP and AVDO2 are significantly higher during propofol anaesthesia compared to isoflurane and sevoflurane anaesthesia.

Cross talk between substance P and melittin-activated cellular signaling pathways in rat lactotroph-enriched cell cultures.

We have investigated the possible interaction (cross talk) between the phospholipase A2 (PLA2) and inositol 1,4,5-trisphosphate/protein kinase C (PKC) signaling pathways in rat lactotroph-enriched cell cultures. Melittin, a bee venom peptide, stimulated release of [3H]arachidonic acid ([3H]AA) from [3H]AA-labeled enriched lactotrophs in a dose-dependent manner. Moreover, melittin and exogenous AA induced a redistribution of PKC catalytic activity and PKC alpha and beta immunoreactivity from the soluble to the particulate fraction in resting and substance P (SP)-stimulated cells. Melittin had no effect on phospholipase C (PLC) activity. Pretreatment of cell cultures with the PLA2 inhibitors quinacrine and aristolochic acid resulted in a dose-dependent inhibition of melittin-stimulated PKC isozyme translocation as did the inhibitor of lipoxygenase, nordihydroguaiaretic acid, whereas the cyclooxygenase inhibitor indomethacin had no effect. SP and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) dose-dependently increased levels of [3H]AA released from cells. Pretreatment of cell cultures with quinacrine reduced the effect of SP on [3H]AA formation. After long-term treatment (24 h) of cells with TPA, the effect of TPA on [3H]AA production was not different from control, whereas SP still displayed [3H]AA-releasing abilities although not at full scale. Pretreatment of cells with thapsigargin, U 73122, methoxyverapamil, and RHC 80267, an inhibitor of diacylglycerol lipase, all resulted in reduced SP-stimulated [3H]AA liberation. Treatment of cell cultures with pertussis toxin (PTX) reduced the release of [3H]AA induced by SP, whereas PTX had no effect on SP-stimulated generation of 3H-inositol phosphates. On the basis of these results, it is concluded that (1) the PLA2 pathways interfere with the phosphoinositide-PLC signaling system at the level of PKC isozymes alpha and beta, the product responsible for this interaction being either AA or a metabolite produced by the action of lipoxygenase; (2) SP and TPA are able to activate the PLA2 pathway at a level at or beyond PLA2, and this effect is mediated, in part, through PKC alpha and beta species and (for SP) intracellular Ca2+ recruited from internal stores as well as from external sources; and (3) SP also activates PLA2 through a PTX-sensitive pathway distinct from the one coupled to phosphoinositide-PLC, which is PTX insensitive.

Effects of withdrawal of dopamine on translocation of protein kinase C isozymes and prolactin secretion in rat lactotroph-enriched pituitary cells. Modulation of substance P-mediated responses.

The present study deals with the effects of withdrawal of dopamine (DA) on the translocation of protein kinase C (PKC) isozymes and release of prolactin (Prl) in resting- and substance P (SP)-stimulated cultures of enriched rat pituitary lactotrophs. Following a brief tonic input (10 min), DA withdrawal induced a redistribution of PKC alpha- and beta-immunoreactivity (IR) to the particulate fraction with maximal levels, attained after 5 min, remaining translocated for 20 min. DA withdrawal prolonged the effect of SP-induced translocation of PKC alpha- and beta-IR. Similar effects were detected when the catalytic activity of PKC in response to DA withdrawal was evaluated. Thus, DA washout redistributed PKC catalytic activity and prolonged the effect of SP on catalytical PKC translocation to the particulate fraction. Pretreatment of cells with the protein kinase A inhibitor, rp-adenosine-3',5'-cyclic monophosphothionate (rp-cAMP), reduced the amount of PKC alpha- and beta-IR redistributed after DA withdrawal. Furthermore, this treatment also reduced the DA withdrawal effect on SP-mediated translocation of PKC alpha- and beta-IR. Methoxyverapamil, a blocker of voltage-gated Ca2+ channels, completely inhibited the redistribution of PKC isozymes after DA withdrawal, but also reduced the potentiating effect of DA withdrawal on SP-induced redistribution of PKC isozyme-IR. In perifused enriched lactotrophs, DA withdrawal induced a release of Prl that lasted 45-55 min and prolonged the effect of SP on Prl secretion. rp-cAMP did not significantly affect Prl release due to DA removal, but the prolonging effect of DA withdrawal on SP-induced Prl secretion was abolished. Methoxyverapamil completely abolished the rebound release of Prl after DA withdrawal, and the potentiating effect of DA removal on SP-mediated Prl release was also diminished. Readdition of DA after DA withdrawal was able to suppress the translocation of PKC isozyme-IR and catalytic activity and to reduce the release of Prl to baseline levels. Moreover, readdition of DA reduced the potentiating effects of DA withdrawal on the same parameters after SP-stimulation of cells. On the basis of these results it is concluded that in resting cells following DA withdrawal prolactin is released and specific PKC isozymes and concomitant catalytic activity are translocated to the particulate fraction in enriched lactotrophs. While cAMP/PKA and influx of Ca2+ seem to work in concert in translocating PKC, influx of Ca2+ is the primary mechanism responsible for the rebound release of Prl after DA withdrawal. DA withdrawal exerts a potentiating effect on SP-induced PKC translocation and Prl release. It is suggested that the biochemical events involved in these processes are cAMP/PKA and Ca2+ influx.

Cross-talk between cellular signaling pathways activated by substance P and vasoactive intestinal peptide in rat lactotroph-enriched pituitary cell cultures.

We have investigated cross-talk between the cAMP/protein kinase A (PKA) and protein kinase C (PKC)/inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) messenger systems probed by vasoactive intestinal peptide (VIP) and substance P (SP), respectively, in rat pituitary cell cultures enriched in lactotrophs. VIP and forskolin had no effect on the basal distribution pattern of the four PKC isozymes (alpha, beta, delta, and zeta) detectable in lactotroph-enriched cell cultures derived from peripubertal male rats, whereas both compounds significantly increased translocation of PKC alpha and beta from the cytosol to the plasma membrane induced by SP. The delta and zeta subspecies were not affected by VIP and forskolin. Moreover, VIP and forskolin also stimulated SP-induced formation of Ins(1,4,5)P3 while having no effect on basal inositol phosphate turnover. The effects of VIP and forskolin on PKC isozyme distribution could be blocked by pretreating cells with the PKA inhibitor rp-cAMP. On the other hand, SP potentiated the effect of VIP and forskolin on cAMP formation while having no effect on the cAMP pathway when it was not triggered by an appropriate agonist. Down-regulation of PKC activity by long term 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment (24 h) diminished, but did not abolish, the effect of SP on VIP-stimulated cAMP production. Staurosporine and dopamine inhibited the potentiating effect of SP on cAMP accumulation. TPA, which translocates PKC alpha, beta, and delta in lactotrophs, had a synergistic effect on cAMP formation induced by VIP, but did also, unlike SP, display cAMP rising abilities when cells were not exposed to VIP and forskolin. Discharging intracellular Ca2+ by thapsigargin pretreatment had no effect on the basal cAMP concentration or the VIP-induced cAMP response, whereas exposure of cells to SP, thapsigargin, and VIP resulted in a decrease of the cAMP response compared with SP + VIP. The potentiating effect of SP on the VIP response could also be inhibited, but not blocked, by staurosporine. On the basis of these results, it is concluded that there exists substantial cross-talk between the cAMP/PKA and PKC/Ins(1,4,5)P3 messenger systems in lactotroph-enriched cell cultures. Key effectors seem to be PKA, one or more of PKC alpha, beta, deleta and Ins(1,4,5)P3-sensitive Ca2+ stores.

Substance P increases intracellular Ca2+ in individual rat pituitary lactotrophs, somatotrophs, and gonadotrophs.

The present study has investigated transients in the intracellular calcium concentration [Ca2+]i in response to substance P (SP) in single pituitary cells. SP raised [Ca2+]i in three subtypes of pituitary cells: lactotrophs, somatotrophs, and gonadotrophs. In all three cell subtypes the [Ca2+]i response to SP was amplitude-modulated and a concentration of 100 nM was necessary to elicit well pronounced two phased [Ca2+]i transients. The first phase was associated with increased generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in all three cell types. In lactotrophs, the second phase, but not the first, was blunted by depletion of extracellular Ca2+ (Ca2+ free EGTA incubation buffer) and by addition of dopamine (1 microM). In somatotrophs, the second phase of the SP-induced [Ca2+]i response was inhibited by depletion of extracellular Ca2+ and by addition of somatostatin (100 nM), while the first phase was unaffected by this treatment. In gonadotrophs, the second phase, but not the first, was inhibited by the Ca2+ channel blocker methoxyverapamil and depletion of extracellular Ca2+. SP was compared with other agonists having an action on lactotrophs, somatotrophs or gonadotrophs. These experiments demonstrated that SP was a weaker agonist in terms of maximal [Ca2+]i response than thyrotropin-releasing hormone (TRH) (in lactotrophs), growth hormone-releasing hexapeptide (in somatotrophs) and GnRH (in gonadotrophs). On the basis of these results it is concluded that SP exerts direct Ca2+ mobilizing effects in single lactotrophs, somatotrophs, and gonadotrophs derived from male peripubertal rats. The first phase in SP-induced [Ca2+]i transients is likely to be brought about by inositol 1,4,5-trisphosphate-mediated Ca2+ release from internal stores while the second phase reflects an influx of calcium through voltage-gated calcium channels.

Molecular cloning of cDNAs encoding the protein backbones of arabinogalactan-proteins from the filtrate of suspension-cultured cells of Pyrus communis and Nicotiana alata.

This paper reports the isolation of cDNAs encoding the protein backbone of two arabinogalactan-proteins (AGPs), one from pear cell suspension cultures (AGPPc2) and the other from suspension cultures of Nicotiana alata (AGPNa2). The proteins encoded by these cDNAs are quite different from the 'classical' AGP backbones described previously for AGPs isolated from pear suspension cultures and extracts of N. alata styles. The cDNA for AGPPc2 encodes a 294 amino acid protein, of which a relatively short stretch (35 amino acids) is Hyp/Pro rich; this stretch is flanked by sequences which are dominated by Asn residues. Asn residues are not a feature of the 'classical' AGP backbones in which Hyp/Pro, Ser, Ala and Thr account for most of the amino acids. The cDNA for AGPNa2 encodes a 437 amino acid protein, which contains two distinct domains: one rich in Hyp/Pro, Ser, Ala, Thr and the other rich in Asn, Tyr and Ser. The composition and sequence of the Pro-rich domain resembles that of the 'classical' AGP backbone. The Asn-rich domains of the two cDNAs described have no sequence similarity; in both cases they are predicted to be processed to give a mature backbone with a composition similar to that of the 'classical' AGPs. The study shows that different AGPs can differ in the amino acid sequence in the protein backbone, as well as the composition and sequence of the arabinogalactan side-chains. It also shows that differential expression of genes encoding AGP protein backbones, as well as differential glycosylation, can contribute to the tissue specificity of AGPs.

The S-locus of Nicotiana alata: genomic organization and sequence analysis of two S-RNase alleles.

Genomic clones encoding the S2- and S6-RNases of Nicotiana alata Link and Otto, which are the allelic stylar products of the self-incompatibility (S) locus, were isolated and sequenced. Analysis of genomic DNA by pulsed-field gel electrophoresis and Southern blotting indicates the presence of only a single S-RNase gene in the N. alata genome. The sequences of the open-reading frames in the genomic and corresponding cDNA clones were identical. The organization of the genes was similar to that of other S-RNase genes from solanaceous plants. No sequence similarity was found between the DNA flanking the S2- and S6-RNase genes, despite extensive similarities between the coding regions. The DNA flanking the S6-RNase gene contained sequences that were moderately abundant in the genome. These repeat sequences are also present in other members of the Nicotianae.

Translocation of protein kinase C isozymes in rat pituitary lactotroph-enriched cell cultures by substance P: effects of sex and age.

It is generally accepted that the phospholipid and calcium-dependent enzyme protein kinase C (PKC) plays a significant role in secretion of hormones from anterior pituitary cells. The present study was undertaken to study age and sex-related changes in 1. levels of immunoreactivity of PKC isozymes and 2. distribution of immunoreactivity of PKC isozymes after stimulation with substance P (SP) in rat lactotroph-enriched cell cultures. The alpha, beta, delta and zeta isozymes were present in both sexes and at all ages. There was a sex-specific differential regulation of the different PKC isozymes as a function of sexual maturation. In male rats there was an up-regulation of the alpha isozyme throughout the sexual development, while the beta subtype showed a small, but significant decrease in immunoreactivity with increasing age. In female rats, on the other hand, the beta species was up-regulated with increasing age while the other subtypes remained constant. The concentration of the delta and zeta isozymes was unaffected of sex and age. Stimulation of lactotroph-enriched cell cultures with substance P (SP) resulted in translocation of the alpha and beta isozymes from the soluble to the particulate fraction while the delta and zeta species were left unchanged independently of age and sex. However, a decrease in responsiveness was observed in adult male rats, although a significant degree of translocation of alpha and beta species was still detected. On the basis of these results it is suggested that in lactotroph-enriched cell cultures basal levels of PKC subtype immunoreactivity and distribution of immunoreactivity of PKC isozymes after SP challenge might be regulated as a function of sex and age.

Growth hormone releasing hexapeptide (GHRP-6) activates the inositol (1,4,5)-trisphosphate/diacylglycerol pathway in rat anterior pituitary cells.

Growth hormone-releasing hexapeptide (GHRP-6) is known to stimulate secretion of growth hormone (GH) in vivo and in vitro in a variety of species. However, the cellular effects of GHRP-6 remain largely unknown. We have tested the influence of GHRP-6 on the inositol phospholipid second messenger system in cultured anterior pituitary cells. Cultured pituitary cells responded upon challenge with GHRP-6 with a dose-dependent release of GH. Moreover, incubation of GHRP-6 with pituitary cell cultures labelled with myo-[3H]inositol resulted in a dose-dependent rise in [3H]inositol phosphates. Brief stimulation of pituitary cells with GHRP-6 increased phosphorylation of MBP4-14, a specific protein kinase C substrate, when incubated with the cytosol- or plasma membrane fraction from the stimulated cells. Furthermore, introduction of MBP4-14 into the cytosol in digitonin permeabilized pituitary cells caused increased phosphorylation of this substrate. GHRP-6 induced a rise in intracellular Ca2+ in individual somatotrophs loaded with the Ca2+ indicator, Fura-2. Preincubation (3 min) with somatostatin (SRIF) diminished the Ca2+ spike elicited by GHRP-6, while no effect of SRIF was observed when added simultaneously with GHRP-6. These results indicate that GHRP-6-stimulated GH-secretion involves the diacylglycerol/inositol(1,4,5)trisphosphate pathway with a resulting rise in cytosolic Ca2+.

Tachykinins induce secretion of prolactin from perifused rat anterior pituitary cells by interactions with two different binding sites.

Substance P and the two other mammalian tachykinins, neurokinin A and B, are accepted to have direct regulating effects at the anterior pituitary level. We have examined the effects of substance P (SP) and neurokinin B (NKB), alone and in combination, on prolactin release from cultured anterior pituitary cells grown on collagen-coated micro beads and placed in a perfusion system. Prolactin (Prl) secretion was observed within 25 s after exposure to either secretagogue and reached a maximum within 60-80 s. Furthermore, the prolactin response induced by SP and NKB was dose-dependent. Prl secretion remained constant for up to 4 h when SP or NKB were perifused and then fell gradually towards basal levels. Simultaneous addition of submaximal concentrations of SP and NKB resulted in an additive response compared with the responses of either secretagogue alone. Continuous (8 h) perifusion with SP did not prevent a normal prolactin response by NKB or TRH. These results indicate that the tachykinins, substance P and neurokinin B, release Prl from perifused female rat anterior pituitary cells by interaction with two different receptors, possibly the NK1 and NK3 tachykinin receptor subtypes.

Molecular cloning of a gene encoding an arabinogalactan-protein from pear (Pyrus communis) cell suspension culture.

Arabinogalactan-proteins (AGPs) are proteoglycans containing a high proportion of carbohydrate (typically > 90%) linked to a protein backbone rich in hydroxyproline (Hyp), Ala, Ser, and Thr. They are widely distributed in plants and may play a role in development. The structure of the carbohydrate of some AGPs is known in detail but information regarding the protein backbone is restricted to a few peptide sequences. Here we report isolation and partial amino acid sequencing of the protein backbone of an AGP. This AGP is a member of one of four major groups of AGPs isolated from the filtrate of pear cell suspension culture. A cDNA encoding this protein backbone (145 amino acids) was cloned; the deduced protein is rich in Hyp, Ala, Ser, and Thr, which together account for > 75% of total residues. It has three domains, an N-terminal secretion signal, a central hydrophilic domain containing all of the Pro residues, and a hydrophobic C-terminal domain that is predicted to be a transmembrane helix. Approximately 93% of the Pro residues are hydroxylated and hence are potential sites for glycosylation.

Mastoparan, a wasp venom peptide, stimulates release of prolactin from cultured rat anterior pituitary cells.

Studies have shown that mastoparan and other amphiphilic peptides induce exocytosis of hormones from anterior pituitary cells. We have studied the effect of mastoparan on the secretion of prolactin from cultured rat anterior pituitary cells and on the concomitant functional status of signal-transducing pathways in lactotroph-enriched cell cultures. Mastoparan stimulation of prolactin secretion was dose-dependent, time-dependent, reversible and required the presence of calcium. Pretreatment of pituitary cell cultures with cholera and pertussis toxin had no effect on the secretory response, whereas encapsulation of guanosine 5-[beta-thio]diphosphate (GDP-beta-S) by reversible electropermeabilization inhibited mastoparan-stimulated secretion. Incubation of mastoparan with myo-[3H]inositol-labelled lactotroph-enriched anterior pituitary cell cultures resulted in increased formation of inositol phosphates compared with control cells, and encapsulation of GDP-beta-S blocked mastoparan-induced inositol lipid hydrolysis. Mastoparan caused translocation of protein kinase C activity from a soluble to a membrane-attached form. Mastoparan was able to increase the intracellular Ca2+ concentration in Fura-2-loaded individual lactotrophs. Omission of Ca2+ from the extracellular medium did not change the Ca2+ response in lactotrophs when stimulated with mastoparan. On the basis of these results it is concluded that mastoparan-induced release of prolactin is preceded by activation of the inositol(1,4,5)trisphosphate/diacylglycerol pathway with resulting translocation of protein kinase activity and increment in intracellular Ca2+. However, other signal-transducing pathways may be involved in the secretory process.

Translocation of protein kinase C isozymes in lactotroph-enriched rat anterior pituitary cell cultures: differential effects of substance P and phorbol ester.

Translocation of protein kinase C (PKC) from the cytosol to the plasma membranes is believed to reflect activation of the enzyme. We have studied translocation of PKC in lactotroph-enriched anterior pituitary cell cultures by measuring the incorporation of gamma-32P from [gamma-32P]ATP into a synthetic peptide substrate, MBP4-14, and by immunoblotting of PKC isozymes. Using cells permeabilized with digitonin the effects of PKC cofactors on the distribution of the enzyme were studied. Ca2+ (50 nM) and dioctanoyl-sn-glycerol had no effect when tested alone, but in combination they caused a redistribution of PKC from the soluble to the particulate fraction. Arachidonic acid needed Ca2+ to induce translocation of PKC, while being ineffective under Ca(2+)-free conditions. Western blot analysis of partly purified PKC from lactotroph-enriched pituitary cells revealed the presence of the alpha, beta, delta and zeta isozymes. 12-O-Tetradecanoylphorbol 13-acetate (TPA) and substance P displayed different patterns of redistribution of PKC isozyme immunoreactivity from soluble to membrane-attached forms. Thus, TPA induced time- and dose-dependent (mean effective concentration (EC50) = 1 nM) translocation of the alpha, beta and delta species, while substance P stimulated time- and dose-dependent (EC50 = 1 nM) redistribution of the alpha and beta isozymes. zeta subtype immunoreactivity could not be translocated by either agonist; neither could the immunoreactivity of zeta be down-regulated by long-term treatment (24 h) with TPA. The results indicate that simultaneous activation of phospholipases C and A2 induces a synergistic activation of PKC. Finally it is suggested that substance P may exert some of its effects in lactotrophs by translocation of PKC isozymes alpha and beta.