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At least 199 records · Page 11Linked to original sources
PubMed2002

[Physiological significance of pituitary adenylate cyclase-activating polypeptide (PACAP) in the nervous system].

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been conserved remarkably during evolution and is widely expressed in the nervous system across phyla. PACAP has an amino acid sequence homology of 68% with that of vasoactive intestinal polypeptide (VIP) and of 37% with that of secretin, indicating that PACAP is a member of the VIP/glucagon/secretin superfamily. PACAP exerts its actions via three heptahelical G-protein-linked receptors: one PACAP-specific (PAC1) receptor and two receptors (VPAC1 and VPAC2) shared with VIP. PACAP stimulates several different signaling cascades in neurons, leading to the activation of adenylate cyclase, phospholipase C, and mitogen-activated protein kinase and mobilization of calcium. Although PACAP and VIP have no apparent homology with calcitonin and parathyroid hormone (PTH), PAC1, VPAC, secretin, glucagon, glucagon-like peptide 1, growth hormone-releasing hormone, calcitonin, and PTH/PTH-related peptide receptors are related to each other and constitute a subfamily of the G-protein-coupled receptors. Distribution analysis of PACAP and its receptors and pharmacological studies have elucidated its pleiotropic effects in the central and peripheral nervous systems. However, the relevance of the pharmacological PACAP effects to the actual physiological activities of endogenous PACAP has not been addressed, because potent and selective low-molecular-weight PACAP antagonists have not yet been developed. To assess the function of PACAP in vivo, we have recently generated PAC1 receptor- and PACAP-targeted mice, and provided evidence that PACAP plays a previously uncharacterized role in the regulation of psychomotor behaviors. In this review, we focus on the physiological and or pathophysiological roles mediated by PACAP in the nervous system.

Amino Acid Sequence↗
PubMed2004

[Prevention of delayed neuronal cell death by PACAP and its molecular mechanism].

Ischemic delayed neuronal cell death (apoptosis) in the hippocampus is prevented by PACAP. PACAP inhibits the increasing activity of the MAP kinase family, especially on JNK/SAPK and p38, thereby protecting against apoptotic cell death. After the ischemia-reperfusion, both pyramidal cells and astrocytes increased their expression of PACAP receptors (PAC1-Rs). The pyramidal cells degenerated but reactive astrocytes increased their expression of PAC1-R. PACAP does not only inhibit apoptotic cell death directly, but also affects astrocytes through PAC1-Rs. Interleukin-6 (IL-6), produced in astrocytes, has several effects on the prevention of brain ischemia and trauma and stimulating neuronal growth. IL-6 secretion into the CSF was markedly stimulated after PACAP infusion, suggesting that PACAP stimulates IL-6 secretion from astrocytes. We studied the effects of PACAP on the wild type and IL-6 KO mice after brain ischemia. In wild-type animals, PACAP significantly inhibited cell death, but in IL-6 KO animals, no cytoprotective effect of PACAP was seen. These results suggest that PACAP inhibits apoptotic cell death partly through IL-6. It is considered that PACAP itself and IL-6, stimulated secretion by PACAP, both synergistically inhibit the JNK/SAPK and p38 signaling pathway, thereby protecting against neuronal cell death.

Animals↗
PubMed2000

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuron-derived peptide regulating glial glutamate transport and metabolism.

In the brain, glutamatergic neurotransmission is terminated predominantly by the rapid uptake of synaptically released glutamate into astrocytes through the Na(+)-dependent glutamate transporters GLT-1 and GLAST and its subsequent conversion into glutamine by the enzyme glutamine synthetase (GS). To date, several factors have been identified that rapidly alter glial glutamate uptake by post-translational modification of glutamate transporters. The only condition known to affect the expression of glial glutamate transporters and GS is the coculturing of glia with neurons. We now demonstrate that neurons regulate glial glutamate turnover via pituitary adenylate cyclase-activating polypeptide (PACAP). In the cerebral cortex PACAP is synthesized by neurons and acts on the subpopulation of astroglia involved in glutamate turnover. Exposure of astroglia to PACAP increased the maximal velocity of [(3)H]glutamate uptake by promoting the expression of GLT-1, GLAST, and GS. Moreover, the stimulatory effects of neuron-conditioned medium on glial glutamate transporter expression were attenuated in the presence of PACAP-inactivating antibodies or the PACAP receptor antagonist PACAP 6-38. In contrast to PACAP, vasoactive intestinal peptide promoted glutamate transporter expression only at distinctly higher concentrations, suggesting that PACAP exerts its effects on glial glutamate turnover via PAC1 receptors. Although PAC1 receptor-dependent activation of protein kinase A (PKA) was sufficient to promote the expression of GLAST, the activation of both PKA and protein kinase C (PKC) was required to promote GLT-1 expression optimally. Given the existence of various PAC1 receptor isoforms that activate PKA and PKC to different levels, these findings point to a complex mechanism by which PACAP regulates glial glutamate transport and metabolism. Disturbances of these regulatory mechanisms could represent a major cause for glutamate-associated neurological and psychiatric disorders.

ATP-Binding Cassette Transporters↗
PubMed2000

Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor subtypes in human tumors and their tissues of origin.

The evaluation of peptide receptors in man is needed not only to discover the physiological target tissues of a given peptide but also to identify diseases with a sufficient receptor overexpression for diagnostic or therapeutic interventions. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) receptors have been evaluated in human tumors and in their tissues of origin using in vitro receptor autoradiography with 125I-VIP or 125I-acetyl-PACAP-27 in tissue sections. The VIP/PACAP receptor subtypes VPAC1, VPAC2, and PAC1 were evaluated in these tissues by determining the rank order of potencies of VIP and PACAP as well as VPAC1- and VPAC2-selective analogues. The VIP/PACAP receptors expressed in the great majority of the most frequently occurring human tumors, including breast (100% receptor incidence), prostate (100%), pancreas (65%), lung (58%), colon (96%), stomach (54%), liver (49%), and urinary bladder (100%) carcinomas as well as lymphomas (58%) and meningiomas (100%), are predominantly of the VPAC1 type. Their cells or tissues of origin, i.e., hepatocytes, breast lobules and ducts, urothelium, prostate glands, pancreatic ducts, lung acini, gastrointestinal mucosa, and lymphocytes, also predominantly express VPAC1. Leiomyomas predominantly express VPAC2 receptors, whereas paragangliomas, pheochromocytomas, and endometrial carcinomas preferentially express PAC1 receptors. Conversely, VPAC2 receptors are found mainly in smooth muscle (i.e., stomach), in vessels, and in stroma (e.g., of the prostate), whereas PAC1 receptors are present in the adrenal medulla and in some uterine glands. Whereas the very wide distribution of VIP/PACAP receptors in the normal human body is indicative of a key role of these peptides in human physiology, the high VIP/PACAP receptor expression in tumors may represent the molecular basis for clinical applications of VIP/PACAP such as in vivo scintigraphy and radiotherapy of tumors as well as VIP/PACAP analogue treatment for tumor growth inhibition.

Adrenal Gland Neoplasms↗
PubMed2002

Platelet activation in patients with obstructive sleep apnea syndrome and effects of nasal-continuous positive airway pressure.

OBJECTIVE: Our study was undertaken to determine whether increased platelet activation occurs in patients with obstructive sleep apnea syndrome (OSAS) and whether a therapy with nasal-continuous positive airway pressure (N-CPAP) alters this activation. METHODS: We measured the positive rate of activated platelets using activation-dependent monoclonal antibodies (MoAb) and flow cytometry in whole blood from 94 patients with OSAS, and from 31 age-matched controls. Thrombotic vascular diseases were surveyed as a background of alternative of platelet activation. RESULTS: The positive rate for activated platelets was significantly higher in patients with OSAS ( PAC1 52.6 +/- 22.9 %, CD62P 6.8 +/- 7.1%, mean +/- SD), as compared with healthy control subjects ( PAC1 16.7 +/- 8.6 %, CD62P 0.7 +/- 0.5 %, p < 0.001). The activation indexes were significantly reduced after 1 month with N-CPAP treatment as a whole (PAC1; from 52.6 +/- 22.9 to 44.2 +/- 22.4, p < 0.05, CD62P; from 6.8 +/- 7.1 to 5.3 +/- 5.5, p < 0.05). In nearly 60 % of patients, platelets activation remained high despite significant improvement of sleep apnea-episodes after N-CPAP. These patients had significantly higher incidence of previous myocardial infarction and/or cerebral infarction and abnormalities of head MRI and carotid sonograpy; indicating that the platelet activation appears to be induced by existing atheroma plaque and not by sympathetic activity in OSAS. CONCLUSION: In conclusion, patients with OSAS have increased percentages of activated platelets as assessed by flow cytometrical analysis of activation dependent surface markers, and were divided into two groups, one group with response to N-CPAP treatment in the reduction of platelet activation and the other without. One possible reason of no response to N-CPAP treatment in the reduction of platelet activation was suggested to be thrombotic diseases.

Adult↗
PubMed1992

Regulation of glycoprotein IIb-IIIa receptor function studied with platelets permeabilized by the pore-forming complement proteins C5b-9.

Recent evidence suggests that the cytoplasmic domains of platelet glycoprotein (GP) IIb-IIIa are involved in the agonist-initiated transformation of this integrin into a receptor for fibrinogen. To identify intracellular reactions that regulate the receptor function of GP IIb-IIIa, membrane-impermeable agonists and antagonists were introduced into the platelet by permeabilizing the plasma membrane with the pore-forming complement proteins C5b-9. Platelet responses were then analyzed by flow cytometry. Non-lytic concentrations of C5b-9 caused permeabilization of the platelet plasma membrane, as determined by uptake of a water-soluble fluorescent tracer dye. The complement pores were large enough to permit the entry of fluorescein isothiocyanate (FITC)-labeled oligopeptides in a size-dependent manner. Under conditions of low external Ca2+, C5b-9 treatment per se did not activate GP IIb-IIIa, as measured by binding of the activation-dependent antibody FITC-PAC1. However, FITC-PAC1 binding to C5b-9-permeabilized platelets was stimulated by a thrombin receptor agonist acting at the cell surface and by guanosine 5'-O-(thiotriphosphate), a membrane-impermeable activator of G proteins. Permeabilization also permitted the entry of cyclic AMP and the peptide, RFARKGALRQKNV, a pseudo-substrate inhibitor of protein kinase C. Each of these inhibited agonist-induced FITC-PAC1 binding to permeabilized platelets but not to intact platelets. Agonist-induced GP IIb-IIIa activation in permeabilized platelets was also inhibited by tyrphostin-23, a protein tyrosine kinase inhibitor. Thus, C5b-9 can be used to permeabilize the plasma membrane to permit the selective entry of small peptides and other bioactive compounds into permeabilized platelets. Results obtained with these platelets indicate that GP IIb-IIIa receptor function is regulated by a network of signaling reactions involving G proteins, serine/threonine kinases, and tyrosine kinases.

Amino Acid Sequence↗
PubMed1990

Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein IIb-IIIa with conformation-specific monoclonal antibodies.

Normal primary platelet aggregation requires agonist-mediated activation of membrane GPIIb-IIIa, binding of fibrinogen to GPIIb-IIIa, and cellular events after ligand binding. PAC1 monoclonal antibody distinguishes between resting and activated states of GPIIb-IIIa, and other antibodies preferentially recognize GPIIb (PMI-1) or IIIa (anti-LIBS1) after the binding of fibrinogen or fibrinogen-mimetic peptides, such as GRGDSP. Using these antibodies and platelet flow cytometry, we studied two distinct persistent platelet aggregation abnormalities. Platelets from a thrombasthenic variant, which contained near-normal amounts of GPIIb-IIIa, failed to aggregate or bind PAC1 in response to agonists. In addition, GRGDSP, which binds to normal GPIIb-IIIa without prior cell activation, failed to increase the binding of PMI-1 or anti-LIBS1 to the thrombasthenic platelets, suggesting a primary defect in ligand binding. Chromatography of detergent-solubilized platelets on a KYGRGDS affinity column confirmed that the patient's GPIIb-IIIa lacked the fibrinogen binding site. In another patient with myelofibrosis and defective aggregation, PAC1 failed to bind to adenosine diphosphate-stimulated platelets, but did bind when protein kinase C was directly activated with phorbol myristate acetate. Furthermore, the binding of PMI-1 and anti-LIBS1 increased in response to GRGDSP, confirming a defect in agonist-mediated fibrinogen receptor activation rather than in fibrinogen binding or events distal to binding. These studies indicate that this immunochemical approach is useful in classification of clinical abnormalities of platelet aggregation as defects in either (a) fibrinogen receptor activation, (b) fibrinogen binding, or (c) postoccupancy events.

Antibodies, Monoclonal↗
PubMed1996

Phorbol ester enhances integrin alpha IIb beta 3-dependent adhesion of human erythroleukemic cells to activation-dependent monoclonal antibodies.

Following platelet stimulation by agonists, integrin-alpha IIb beta 3 (or glycoprotein IIb-IIIa) is converted to an activated state that can bind soluble fibrinogen and mediate platelet aggregation. However, little is known about modulation of alpha IIb beta 3 in cell lines. In the present study, we show that agonist stimulation modulates alpha IIb beta 3-dependent adhesive properties of a human erythroleukemic (HEL) cell line. Brief treatment with phorbol 12-myristate 13-acetate (PMA) caused a significant increase in HEL cell adhesion to monoclonal antibodies (MoAbs) specific for activated alpha IIb beta 3 (PAC1 or pl-55). This adhesion was inhibited by blocking MoAbs or peptides specific for alpha IIb beta 3, but not by anti-Fc gamma receptor-specific MoAb. Similarly, PMA enhanced HEL cell adhesion to immobilized fibrinogen by 10-fold. However, the activation-dependent ligands in solution (ie, PAC1, pl-55, or fibrinogen) did not inhibit the enhanced HEL cell adhesion to immobilized MoAbs PAC1 or pl-55 after PMA treatment. Thus, PMA may increase alpha IIb beta 3-dependent adhesion to immobilized activation-dependent antibodies and fibrinogen by increasing the local concentration of alpha IIb beta 3 to participate in low-affinity interactions, resulting in an increased avidity, changing the affinity state of alpha IIb beta 3, or both.

Amino Acid Sequence↗
PubMed1997

Platelet hyporeactivity in very low birth weight neonates.

Very few studies have examined platelet function in very low birth weight (VLBW) preterm neonates, because of the relatively large volumes of blood required. In this study, platelet function in clinically stable VLBW neonates was examined by whole blood flow cytometry, which requires only 5 microliters of whole blood per assay. The following monoclonal antibodies were used: S12 (P-selectin-specific, reflecting alpha granule secretion), PAC1 (directed against the fibrinogen binding site exposed on the GPIIb-IIIa complex of activated platelets), F26 (directed against a conformational change in fibrinogen bound to the GPIIb-IIIa complex), and 6D1 (directed against the von Willebrand factor binding site on the GPIb-IX-V complex). VLBW neonates, like normal adults, did not have circulating activated platelets, as determined by the lack of binding of S12, PAC1, and F26 in the absence of an added agonist. VLBW neonatal platelets were markedly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619 (a stable thromboxane A2 analogue), as determined by the extent of increase in the platelet binding of S12, PAC1, and F26, and the extent of decrease in the platelet binding of 6D1. In summary, compared to adults, the platelets of VLBW neonates are markedly hyporeactive to thrombin, ADP/epinephrine and a thromboxane A2 analogue in the physiologic milieu of whole blood, as determined by: 1) the increase in platelet surface P-selectin; 2) the exposure of the fibrinogen binding site on the GPIIb-IIIa complex; 3) fibrinogen binding; and 4) the decrease in platelet surface GPIb. This platelet hyporeactivity may be a factor in the propensity of VLBW neonates to intraventricular hemorrhage. In addition to its previously defined use as a test of platelet hyperreactivity, the present study suggests that whole blood flow cytometry may be useful in the clinical assessment of platelet hyporeactivity.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1998

Tissue-specific expression of functional platelet factor XI is independent of plasma factor XI expression.

Platelet factor XI is an alternatively spliced product of the factor XI gene expressed specifically within megakaryocytes and platelets as an approximately 1.9-kb mRNA transcript (compared with approximately 2.1 kb in liver cells) lacking exon V. Flow cytometry with an affinity-purified factor XI antibody, with PAC1 antibody (to the GPIIb/IIIa complex on activated platelets), and with S12 antibody (to P-selectin, an alpha-granule membrane protein expressed on the platelet surface during secretion) on platelets activated with ADP, thrombin, thrombin receptor peptide (SFLLRN amide), or collagen at various concentrations exposed platelet factor XI and PAC1 antibody binding in parallel. Unactivated platelets expressed approximately 40% of total platelet factor XI but no PAC1 binding sites. Enhanced membrane exposure of platelet factor XI is independent of alpha-granule secretion, because ADP and collagen exposed platelet factor XI but no S12 binding sites. Platelets from four patients with plasma factor XI deficiency (<0.04 U/mL) had normal constitutive and activation-dependent expression of platelet factor XI. Well-washed platelets from normal and from factor XI-deficient donors incubated with low concentrations of thrombin (0. 05 to 0.1 U/mL) corrected the clotting defect observed with factor XI-deficient plasma. Thus, functionally active platelet factor XI is differentially expressed on platelet membranes in a tissue-specific manner both constitutively and in a concentration-dependent fashion by various agonists in the absence of detectable plasma factor XI.

Adult↗
PubMed1998

Pituitary adenylate cyclase-activating polypeptide expression and modulation of neuronal excitability in guinea pig cardiac ganglia.

Cardiac output is regulated by the coordinate interactions of stimulatory sympathetic and inhibitory parasympathetic signals. Intracardiac parasympathetic ganglia are integrative centers of cardiac regulation, and modulation of the parasympathetic drive on the heart is accomplished by altering intrinsic cardiac ganglion neuron excitability. The pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) family of peptides modulates cardiac function, and in guinea pig heart, PACAP appears to act directly on intrinsic parasympathetic cardiac ganglia neurons through PACAP-selective receptors. A multidisciplinary project tested whether cardiac PACAP peptides act through PACAP-selective receptors as excitatory neuromodulators amplifying the parasympathetic inhibition from guinea pig cardiac ganglia. The in vivo sources of regulatory PACAP peptides were localized immunocytochemically to neuronal fibers and a subpopulation of intrinsic postganglionic cardiac neurons. RT-PCR confirmed that cardiac ganglia expressed proPACAP transcripts and have PACAP peptide biosynthetic capabilities. Messenger RNA encoding PACAP-selective PAC1 receptor isoforms were also present in cardiac ganglia. Alternative splicing of PAC1 receptor transcripts produced predominant expression of the very short variant with neither HIP nor HOP cassettes; lower levels of the PAC1HOP2 receptor mRNA were present. Almost all of the parasympathetic neurons expressed membrane-associated PAC1 receptor proteins, localized immunocytochemically, which correlated with the population of cells that responded physiologically to PACAP peptides. PACAP depolarized cardiac ganglia neurons and increased neuronal membrane excitability. The rank order of peptide potency on membrane excitability in response to depolarizing currents was PACAP27>PACAP38>VIP. The PACAP-induced increase in excitability was not a function of membrane depolarization nor was it caused by alterations in action potential configuration. These results support roles for PACAP peptides as integrative modulators amplifying, through PACAP-selective receptors, the parasympathetic cardiac ganglia inhibition of cardiac output.

Adenylyl Cyclases↗
PubMed2001

Pituitary adenylate cyclase activating peptide (PACAP) in the enteropancreatic innervation.

Pancreatic ganglia are innervated by neurons in the gut and are formed by precursor cells that migrate into the pancreas from the bowel. The innervation of the pancreas, therefore, may be considered an extension of the enteric nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in a subset of enteric neurons. We investigated the presence of PACAP in the enteropancreatic innervation in guinea pigs, and the response of pancreatic neurons to PACAP-related peptides. PACAP immunoreactivity was found in nerve fibers in both enteric and pancreatic ganglia and in nerve bundles that travelled between the duodenum and pancreas. PACAP-immunoreactive nerve fibers were densely distributed in the pancreatic ganglia, where they surrounded a subset of cholinergic cell bodies. Pancreatic ganglia did not contain PACAP-immunoreactive cell bodies; however, neuronal perikarya with PACAP immunoreactivity were found in the myenteric plexus of the duodenum. These cells co-stored vasoactive intestinal peptide (VIP). PACAP depolarized pancreatic neurons. Pancreatic neurons were also depolarized by VIP; however, PACAP was more efficacious at depolarizing pancreatic cells than VIP. These findings are consistent with the view that the PACAP effects were mediated through PACAP-selective (PAC1) receptors. PACAP-responsive neurons displayed PAC1 receptor immunoreactivity, which was also found in islet cells and enteric neurons. These results provide support for the hypothesis that PACAP modulates reflex activity between the gut and pancreas. The excitatory effect of PACAP would be expected to potentiate pancreatic secretion.

Animals↗
PubMed2007

Fractionated and acute irradiation induced signaling in a murine tumor.

The effect of fractionated doses of Co(60) gamma-irradiation (2 Gy per fraction over 5 days), as is delivered in cancer radiotherapy, was compared with acute doses of 10 and 2 Gy, in a serially transplanted mouse fibrosarcoma grown in Swiss mice. The aspects that were studied included the three major mitogen-activated protein (MAP) kinases, namely p44 MAP kinase, p38 MAP kinase, and stress-activated protein (SAP) kinase, which are known to be involved in determining the cell fate following exposure to ionizing radiation. The response of dual specificity phosphatase PAC1 which is involved in the dephosphorylation of MAP kinases was also looked at. There were significant differences in the response to different dose regimens for all the factors studied. Fractionated irradiation elicited an adaptive response with a sustained activation over 7 days of prosurvival p44 MAP kinase which was balanced by the increased activation of proapoptotic p54 SAP kinase up to 1 day post-irradiation, whereas, phosphorylated p38 MAP kinase showed a decrease at most time points. PAC1 was induced following fractionated irradiation and may be acting as a feed back regulator of p44 MAP kinase. The activation of SAP kinase after fractionated irradiation may be a stress response, whereas, constitutively activated p44 MAP kinase may play an important role in the induction of radioresistance during fractionated radiotherapy of cancer and may serve as a promising target for specific inhibitors to enhance the efficacy of radiotherapy.

Animals↗
PubMed1996

Immortalization of human adult prostatic adenocarcinoma cells by human papilloma virus HPV16 and -18 DNA.

Primary prostate epithelial and prostate adenocarcinoma cells cultured in serum-free medium grew for up to 10 passages before senescence. Cells from prostate adenocarcinoma of a 55-year-old patient without lymph node involvement were transfected with plasmids containing recombinant human papilloma virus HPV16 or HPV18 DNA and the selectable neomycin-resistance gene. After G-418 selection, cells underwent crisis, and surviving cells infected with retroviruses encoding the HPV18 E6/E7 genes (HPV-PAC1), transfected with a head-to-tail dimer of the complete HPV16 genome (HPV-PAC2), or transfected with HPV18 E6/E7 early genes (HPV-PAC3) were established. HPV-PAC1 and HPV-PAC2 cultures appeared morphologically similar to primary cultures even after 40 passages. However, HPV-PAC2 cultures had a clonal morphology. All lines were positive for cytokeratin 18, had acquired vimentin expression, and contained either HPV16 or HPV18 sequences integrated into host DNA. None was tumorigenic in nude mice or formed colonies in soft agar. These cells did not secrete prostate specific antigen nor respond to androgen although tamoxifen inhibited the growth of the cells. Immunohistochemistry showed no evidence of p53 overexpression. Further characterization of these cell lines and examination of their response to chemotherapeutic agents may provide relevant information for the study of hormone-independent PC.

Adenocarcinoma↗
PubMed1989

Cloning and expression of a collagen-analog-encoding synthetic gene in Escherichia coli.

A family of totally synthetic genes coding for multiple tandem repeats of the amino acid sequence (Gly-Pro-Pro) has been prepared and inserted into the ClaI cloning site of the expression vector pJL6. A representative recombinant plasmid, pAC1, with an insert of about 340 bp was established in an Escherichia coli strain bearing a defective lambda prophage, to study expression of the CII-collagen analog fusion protein produced from pAC1 upon heat induction. Authentic fusion protein production was demonstrated by nucleotide sequencing, Northern-blot analysis, and in vivo synthesis. Conversion of a wild-type rpoH allele to the rpoH165 mutation was shown to suppress proteolysis of the unstable fusion protein.

Amino Acid Sequence↗
PubMed2005

VIP enhances synaptic transmission to hippocampal CA1 pyramidal cells through activation of both VPAC1 and VPAC2 receptors.

We previously described that vasoactive intestinal peptide (VIP) increases synaptic transmission to hippocampal CA1 pyramidal cells at concentrations known to activate VIP-selective receptors (VPAC1 and VPAC2) but not the PACAP-selective PAC1 receptor. We now investigated the involvement of VPAC1 and VPAC2 receptors in the effects elicited by VIP as well as the transduction pathways activated by VIP to cause enhancement of synaptic transmission. Blockade of either VPAC1 or VPAC2 receptors with PG 97-269 (100 nM) or PG 99-465 (100 nM) inhibited VIP-induced enhancement of synaptic transmission. Selective activation of VPAC1 receptors with [K15, R16, L27] VIP(1-7)/GRF(8-27) (10 nM) or of VPAC2 receptors with RO 25-1553 (10 nM) increased synaptic transmission to CA1 pyramidal cells, and this increase was larger when both agonists were applied together. Inhibition of either PKA with H-89 (1 microM) or PKC with GF109203X (1 microM) attenuated the effect of VIP (1 nM). GF109203X (1 microM) abolished the effect of the VPAC1 agonist [K15, R16, L27] VIP(1-7)/GRF(8-27) (10 nM) on hippocampal synaptic transmission but that effect was not changed by H-89 (1 microM). The effect of RO 25-1553 (100 nM) obtained in the presence of both the PAC1 and VPAC1 antagonists, M65 (30 nM) and PG 97-269 (100 nM), was strongly inhibited by H-89 (1 microM) but not GF109203X (1 microM). It is concluded that VIP enhances synaptic transmission to CA1 pyramidal cell dendrites through VPAC1 and VPAC2 receptor activation. VPAC1-mediated actions are dependent on PKC activity, and VPAC2-mediated actions are responsible for the PKA-dependent actions of VIP on CA1 hippocampal transmission.

Animals↗
PubMed2004

Molecular requirements for duplex recognition and cleavage by eukaryotic RNase III: discovery of an RNA-dependent DNA cleavage activity of yeast Rnt1p.

Members of the double-stranded RNA (dsRNA) specific RNase III family are known to use a conserved dsRNA-binding domain (dsRBD) to distinguish RNA A-form helices from DNA B-form ones, however, the basis of this selectivity and its effect on cleavage specificity remain unknown. Here, we directly examine the molecular requirements for dsRNA recognition and cleavage by the budding yeast RNase III (Rnt1p), and compare it to both bacterial RNase III and fission yeast RNase III (Pac1). We synthesized substrates with either chemically modified nucleotides near the cleavage sites, or with different DNA/RNA combinations, and investigated their binding and cleavage by Rnt1p. Substitution for the ribonucleotide vicinal to the scissile phosphodiester linkage with 2'-deoxy-2'-fluoro-beta-d-ribose (2' F-RNA), a deoxyribonucleotide, or a 2'-O-methylribonucleotide permitted cleavage by Rnt1p, while the introduction of a 2', 5'-phosphodiester linkage permitted binding, but not cleavage. This indicates that the position of the phosphodiester link with respect to the nuclease domain, and not the 2'-OH group, is critical for cleavage by Rnt1p. Surprisingly, Rnt1p bound to a DNA helix capped with an NGNN tetraribonucleotide loop indicating that the binding of at least one member of the RNase III family is not restricted to RNA. The results also suggest that the dsRBD may accommodate B-form DNA duplexes. Interestingly, Rnt1p, but not Pac1 nor bacterial RNase III, cleaved the DNA strand of a DNA/RNA hybrid, indicating that A-form RNA helix is not essential for cleavage by Rnt1p. In contrast, RNA/DNA hybrids bound to, but were not cleaved by Rnt1p, underscoring the critical role for the nucleotide located at 3' end of the tetraloop and suggesting an asymmetrical mode of substrate recognition. In cell extracts, the native enzyme effectively cleaved the DNA/RNA hybrid, indicating much broader Rnt1p substrate specificity than previously thought. The discovery of this novel RNA-dependent deoxyribonuclease activity has potential implications in devising new antiviral strategies that target actively transcribed DNA.

Bacterial Proteins↗
PubMed2006

Intein-mediated rapid purification and characterization of a novel recombinant agonist for VPAC2.

In order to obtain the recombinant VPAC2 agonist efficiently by intein-mediated single column purification, a gene encoding 32-amino acids peptide was designed, synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-ROM was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the rMROM-intein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by beta-mercaptoethanol and the rMROM with the homogeneity over 95% was released from the chitin-bound intein tag. The recombinant linear rMROM competitively displaced [125I] PACAP38 on VPAC2 with a half-maximal inhibitory concentration (IC50) of 60 +/- 5 nM, whereas the IC50 of rMROM at human VPAC1 was observed up to 10 microM and no binding was detected at PAC1. rMROM stimulated the cAMP accumulation in Chinese hamster ovary (CHO) cells expressing the human VPAC2 with a half-maximal stimulatory concentration (EC50) of 0.6 nM, which was 500-fold less potent at VPAC1and had no activity on PAC1. An efficient production procedure of a novel recombinant VPAC2-selective agonist was established.

Amino Acid Sequence↗