Biomedical subjects

T K Chatterjee

Publications and source records attributed to T K Chatterjee.

At least 19 recordsLinked to original sources

PubMed2006

Isolation of pure compound R/J/3 from Pluchea indica (L.) Less. and its anti-amoebic activities against Entamoeba histolytica.

The plant Pluchea indica is known for its anti-inflammatory, anti-ulcer, anti-pyretic, hypoglycemic, diuretic and anti-microbial activities besides many other pharmacological activities. We have isolated and purified seven compounds from the methanolic root extract of this plant by column chromatography. The compounds were identified by spectroscopic analyses. The anti-amoebic activities of the pure compound R/J/3 was investigated against the HM1 strain of Entamoeba histolytica. The compound, R/J/3 showed the most pronounced anti-proliferative activity at a dose of 50 microg/ml. It also showed a marked activity on cell lysis of trophozoites, 4h after administration. The cell lytic activity was compared with metronidazole (5 microg/ml) as positive control.

Animals↗

PubMed2006

Caspase-3 induced apoptosis in transgenic zebrafish.

Zebrafish is an attractive model organism for studying apoptosis development because of its genetic accessibility. Here we describe the induction of clonally derived apoptosis in transgenic zebrafish expressing mouse caspase-3 (CASP3) under control of the zebrafish beta-actin promoter (betap). Visualization of apoptotic cells, expressing a chimeric transgene encoding CASP3 fused to green fluorescent protein (GFP) gene, revealed that apoptosis arose in the thymus, spread locally into gill arches and retro-orbital soft tissue, and then disseminated into abdominal organs like testis, kidney. This transgenic model provides a platform for over-expression of caspase-3 induced extensive apoptosis in embryos and adult.

Actins↗

PubMed2000

Crystal structure of S-glutathiolated carbonic anhydrase III.

S-Glutathiolation of carbonic anhydrase III (CAIII) occurs rapidly in hepatocytes under oxidative stress. The crystal structure of the S-glutathiolated CAIII from rat liver reveals covalent adducts on cysteines 183 and 188. Electrostatic charge and steric contacts at each modification site inversely correlate with the relative rates of reactivity of these cysteines toward glutathione (GSH). Diffuse electron density associated with the GSH adducts suggests a lack of preferred bonding interactions between CAIII and the glutathionyl moieties. Hence, the GSH adducts are available for binding by a protein capable of reducing this mixed disulfide. These properties are consistent with the participation of CAIII in the protection/recovery from the damaging effects of oxidative agents.

Amino Acid Sequence↗

PubMed2000

Novel alternative splicing and nuclear localization of human RGS12 gene products.

RGS proteins are GTPase-activating proteins for certain Galpha subunits, accelerating the shutoff mechanism of G protein signaling, and also may interact with receptors and effectors to modulate G protein signaling. Here, we report identification of 12 distinct transcripts of human RGS12 that arise by unusually complex splicing of the RGS12 gene, which spans 70 kilobase pairs of genomic DNA and contains 16 exons. These transcripts arise by both cis- and trans-splicing mechanisms, are expressed in a tissue-specific manner, and encode proteins ranging in size from 356 to 1447 amino acids. Both 5'- and 3'-splicing of two primary RGS12 transcripts occur to generate RGS12 mRNAs encoding proteins with four distinct N-terminal domains, three distinct C-terminal domains, and a common internal region where the semiconserved RGS domain is located. Confocal microscopy and subcellular fractionation of COS-7 cells expressing RGS12 proteins with three different N termini (brain (B), peripheral (P), and trans-spliced (TS)) and a shared short (S) C-terminal domain demonstrated exclusive nuclear localization of these proteins and an influence of the N-terminal region on the pattern of intranuclear distribution. Both native RGS12TS-S in HEK-293T cells and ectopically expressed RGS12TS-S localized to discrete nuclear foci (dots), a characteristic of various tumor suppressor proteins. Subnuclear localization of RGS12TS-S into nuclear dots was cell cycle-dependent. Native RGS12TS-S associated with the metaphase chromosome during mitosis, and ectopically expressed RGS12TS-S induced formation of abnormally shaped and multiple nuclei in COS-7 cells. Expression of RGS12 proteins with long and intermediate C-terminal domains was not observed in COS-7 cells, suggesting that 3'-splicing of RGS12 transcripts may influence the expression or stability of the encoded proteins. These results document extraordinary structural complexity in the RGS12 family and the role of alternative splicing and cell cycle-dependent mechanisms in expression and subnuclear targeting of RGS12 proteins.

Alternative Splicing↗

PubMed2000

Cytoplasmic, nuclear, and golgi localization of RGS proteins. Evidence for N-terminal and RGS domain sequences as intracellular targeting motifs.

RGS proteins comprise a family of proteins named for their ability to negatively regulate heterotrimeric G protein signaling. Biochemical studies suggest that members of this protein family act as GTPase-activating proteins for certain Galpha subunits, thereby accelerating the turn-off mechanism of Galpha and terminating signaling by both Galpha and Gbetagamma subunits. In the present study, we used confocal microscopy to examine the intracellular distribution of several RGS proteins in COS-7 cells expressing RGS-green fluorescent protein (GFP) fusion proteins and in cells expressing RGS proteins endogenously. RGS2 and RGS10 accumulated in the nucleus of COS-7 cells transfected with GFP constructs of these proteins. In contrast, RGS4 and RGS16 accumulated in the cytoplasm of COS-7 transfectants. As observed in COS-7 cells, RGS4 exhibited cytoplasmic localization in mouse neuroblastoma cells, and RGS10 exhibited nuclear localization in human glioma cells. Deletion or alanine substitution of an N-terminal leucine repeat motif present in both RGS4 and RGS16, a domain identified as a nuclear export sequence in HIV Rev and other proteins, promoted nuclear localization of these proteins in COS-7 cells. In agreement with this observation, treatment of mouse neuroblastoma cells with leptomycin B to inhibit nuclear protein export by exportin1 resulted in accumulation of RGS4 in the nucleus of these cells. GFP fusions of RGS domains of RGS proteins localized in the nucleus, suggesting that nuclear localization of RGS proteins results from nuclear targeting via RGS domain sequences. RGSZ, which shares with RGS-GAIP a cysteine-rich string in its N-terminal region, localized to the Golgi complex in COS-7 cells. Deletion of the N-terminal domain of RGSZ that includes the cysteine motif promoted nuclear localization of RGSZ. None of the RGS proteins examined were localized at the plasma membrane. These results demonstrate that RGS proteins localize in the nucleus, the cytoplasm, or shuttle between the nucleus and cytoplasm as nucleo-cytoplasmic shuttle proteins. RGS proteins localize differentially within cells as a result of structural differences among these proteins that do not appear to be important determinants for their G protein-regulating activities. These findings suggest involvement of RGS proteins in more complex cellular functions than currently envisioned.

Amino Acid Sequence↗

PubMed1998

Mutation of a putative amphipathic alpha-helix in the third intracellular domain of the platelet-activating factor receptor disrupts receptor/G protein coupling and signaling.

Platelet-activating factor (PAF) is a potent phospholipid mediator that interacts with G protein-coupled PAF receptors to elicit diverse physiological and pathophysiological actions. We recently demonstrated that the third intracellular domain of the rat PAF receptor (rPAFR) is a critical determinant in its coupling to phosphoinositide phospholipase C-activating G proteins. Here, we report identification of a putative amphipathic helix in the third intracellular domain of the rPAFR and the effects of mutational disruption of its amphipathic character on G protein coupling of and signaling by the rPAFR. Modeling of the third intracellular domain and adjacent transmembrane regions of the rPAFR identified a single amphipathic helix located in the amino-terminal region of the third intracellular domain of the receptor. Baby hamster kidney cells were transiently transfected with cDNAs encoding the rPAFR or rPAFR mutants in which nonconserved substitutions were made separately in the hydrophobic or polar face of this amphipathic helix. The number and affinity of binding sites for specific PAF receptor antagonist WEB2086 were identical in membranes prepared from rPAFR and amphipathic helix mutant PAFR transfectants. However, only membranes derived from rPAFR transfectants possessed high affinity PAF binding sites that were sensitive to the G protein-uncoupling effects of guanosine-5'-O-(3-thio)triphosphate. These results show that substitutions into either face of the amphipathic helical domain abolished the ability of the rPAFR to undergo coupling to G proteins to form a high affinity agonist/receptor/G protein ternary complex. To examine the effects of these mutations on rPAFR signaling, PAF-stimulated inositol phosphate accumulation was determined in cells transfected with cDNAs encoding the wild-type or amphipathic helix mutant PAFRs. Although PAF stimulated 10-fold increases in inositol phosphate accumulation in rPAFR transfectants, it had no effects on inositol phosphate accumulation in amphipathic helix mutant PAFR transfectants. These results suggest that an amphipathic helix located in the amino-terminal region of the third intracellular domain of the rPAFR is required for its coupling to and activation of G proteins. This study provides the first insight into the structure of the receptor interface for G protein coupling of a PAFR and suggests a conserved role of amphipathic helices in G protein coupling of receptors ranging from those for biogenic amines to the phospholipid mediator PAF.

Amino Acid Sequence↗

PubMed1997

Genomic organization, 5'-flanking region, and chromosomal localization of the human RGS3 gene.

RGS3 is the largest member of a recently discovered family of proteins (RGS proteins) that appear to function as negative regulators of heterotrimeric G-protein signaling. Seventeen mammalian RGS proteins have been identified by cloning or by comparison to expressed sequence tags, and several of these proteins have been shown recently to function as GTPase-activating proteins for G-protein alpha subunits. Despite the intense interest in RGS proteins as physiological regulators of G-protein signaling, there is little understanding of the structure and regulation of mammalian RGS genes. Using long-distance PCR, we amplified and characterized the entire coding and 5'-untranslated region of the human RGS3 gene. The coding region of the human RGS3 gene spans 14.7 kb and contains six exons, and the 5'-untranslated region spans 3.2 kb and contains two exons. Mapping of the exons revealed that the RGS domain, conserved among all RGS proteins, was encoded by three exons, while the unique amino-terminal domain of RGS3 was encoded by a single exon. Comparison of the location of the intron-exon boundaries of the human RGS3 gene to that of the human RGS2 gene, the only mammalian RGS gene described previously, revealed a remarkable similarity, providing the first conceptual support for a common ancestral mammalian RGS gene. 5'-RACE analysis was used to map the transcription start site 517 bp upstream of the translation start site, and anchored PCR was performed to amplify 1.0 kb of genomic DNA upstream of the transcription start site. Analysis of the 5'-flanking region revealed the presence of many potential regulatory elements, the presence of an initiator (Inr) element overlapping the transcription start site, and the absence of a TATA or a CCAAT box at the usual positions. By radiation hybrid mapping, the RGS3 gene was assigned to human chromosome 9q31-q33. This study is the first to elucidate the structure, chromosomal location, and regulatory sequences of the RGS3 gene, and it establishes the genetic basis for RGS3 gene research in humans.

Amino Acid Sequence↗

PubMed1997

A truncated form of RGS3 negatively regulates G protein-coupled receptor stimulation of adenylyl cyclase and phosphoinositide phospholipase C.

Identification of a new family of proteins (RGS proteins) that function as negative regulators of G protein signaling has sparked new understanding of desensitization of this signaling process. Recent studies with several mammalian RGS proteins has delineated their ability to interact with and function as GTPase-activating proteins specifically for G proteins in the Gi family. Here, we investigated the functional activity of RGS3 and a truncated form of RGS3 on G protein-coupled receptor-mediated activation of adenylyl cyclase, phosphoinositide phospholipase C, and mitogen-activated protein kinase in intact cells. Polymerase chain reaction and 5'-rapid amplification of cDNA ends analyses revealed the tissue-specific expression of a short form of the RGS3 transcript that encodes the approximate carboxyl-terminal half of RGS3. This truncated form of RGS3 (RGS3T) was shown recently to function as a negative regulator of pheromone signaling in yeast (Druey, K. M., Blumer, K. J., Kang, V. R., and Kehrl, J. H. (1996) Nature 379, 742-746). Baby hamster kidney cells transiently transfected with RGS3T cDNA exhibited a pronounced impairment in platelet-activating factor receptor-stimulated inositol phosphate production, a pertussis toxin-insensitive response. Similarly, calcitonin gene-related peptide receptor-stimulated increases in intracellular cAMP and pituitary adenylate-cyclase activating polypeptide receptor-stimulated increases in both cAMP and inositol phosphates were reduced significantly in RGS3T transfectants compared with vector-transfected control cells. In contrast, baby hamster kidney cells transfected with the full-length RGS3 cDNA showed no impairment in cAMP and inositol phosphate production mediated by these G protein-coupled receptors. However, lysophosphatidic acid receptor-stimulated phosphorylation of endogenous ERK1 and ERK2 was impaired markedly in both RGS3 and RGS3T transfectants, demonstrating the functional ability of both RGS forms to modulate Gi-mediated signaling. These results provide the first evidence for regulatory effects of an RGS protein on Gs- and Gq-mediated signaling in intact cells and document that the carboxyl-terminal region of RGS3 comprises the structural domain for this activity.

Adenylyl Cyclases↗

PubMed1997

Genomic organization of the rat pituitary adenylate cyclase-activating polypeptide receptor gene. Alternative splicing within the 5'-untranslated region.

Pituitary adenylate cyclase-activating polypeptide (PACAP) elicits its diverse biological actions by interacting with both PACAP-selective type I PACAP receptors (PACAPRs) and type II PACAPRs that do not distinguish between PACAP and vasoactive intestinal polypeptide. Using long distance polymerase chain reaction, we amplified and characterized the entire coding region of the rat type I PACAPR (rPACAPR) gene, which spans 40 kilobases and contains 15 exons. Mapping of the exons and sequencing of all intron-exon boundaries revealed a structural organization of the rPACAPR gene that is very similar to those encoding other members of the calcitonin/secretin/parathyroid hormone receptor family. Southern blot analysis demonstrated a single copy of the rPACAPR gene. A combination of rapid amplification of cDNA ends and reverse transcriptase polymerase chain reaction revealed an unexpected diversity in the rPACAPR mRNA in the 5'-untranslated (5'-UTR) region. Four rPACAPR cDNAs were identified with 5'-UTR sequences that all diverged from the genomic sequence at a site 76 bp upstream of the ATG start codon, where a consensus 3' slice acceptor sequence was located. Sequence analysis of these amplified transcripts demonstrated that they arise by tissue-specific differential usage of four exons in the 5' noncoding region of the rPACAPR gene. This study is the first to elucidate the structural organization of a PACAPR gene and to demonstrate that alternative splicing generates rPACAPR transcripts with unique 5'-UTRs.

Alternative Splicing↗

PubMed1996

Molecular cloning of a novel variant of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor that stimulates calcium influx by activation of L-type calcium channels.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel neuropeptide that produces its biological effects by interacting with G protein-coupled receptors. Molecular cloning of the PACAP receptor revealed the existence of five splice variant receptor forms differing in the third intracellular loop region, with four variants activating both adenylyl cyclase and phosphoinositide phospholipase C and one variant activating only adenylyl cyclase (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170-175). Here, we report cloning of a novel PACAP receptor variant, designated PACAPR TM4 (transmembrane domain IV), that differs from the previously cloned short form of the PACAP receptor (PACAPR) primarily by discrete sequences located in transmembrane domains II and IV. Reverse transcriptase-polymerase chain reaction and primer extension analyses demonstrated tissue-specific differential expression of mRNAs encoding PACAPR TM4 and splice variant forms of the PACAP receptor. PACAPR TM4 and PACAPR possess identical intracellular domains, implicated as primary determinants of G protein recognition by rhodopsin-like receptors. However, unlike the PACAPR, PACAPR TM4 does not activate either adenylyl cyclase or phosphoinositide phospholipase C in response to PACAP in either transient or stable expression systems. However, PACAP stimulates increases in [Ca2+]i in cells expressing PACAPR TM4 by activating L-type Ca2+ channels, a response not elicited by stimulation with vasoactive intestinal polypeptide. The signaling phenotype of PACAPR TM4 is characteristic of the PACAP receptor involved in regulation of insulin secretion from pancreatic beta islets, a tissue expressing transcripts for PACAPR TM4 but not for PACAPR or its longer splice variant forms. These findings are consistent with a role of PACAPR TM4 in the physiological control of insulin release by PACAP in beta-islet cells. The finding that PACAPR TM4 has a unique signaling phenotype, although it possesses intracellular domains identical to those of the PACAPR, suggests that receptor-G protein recognition by rhodopsin-like receptors can be determined by sequences other than those located in intracellular receptor domains.

Amino Acid Sequence↗

PubMed1996

The third intracellular domain of the platelet-activating factor receptor is a critical determinant in receptor coupling to phosphoinositide phospholipase C-activating G proteins. Studies using intracellular domain minigenes and receptor chimeras.

Platelet activating factor (PAF) is a potent phospholipid mediator which elicits a diverse array of biological actions by interacting with G protein-coupled PAF receptors (PAFR). Binding of PAF to PAFRs leads to activation of G protein(s) that stimulate phosphoinositide phospholipase C and subsequent intracellular signaling responses. To identify the potential role of intracellular domains of the rat PAFR (rPAFR) in signaling, we examined effects of transfecting minigenes encompassing rPAFR intracellular domains 1 (1i), 2 (2i), and 3 (3i) on inositol phosphate (IP) production mediated by the co-transfected rPAFR cDNA. Although transfection of the rPAFR1i and rPAFR2i minigenes had no effects on PAF-stimulated signaling, transfection of the rPAFR3i minigene inhibited PAF-stimulated IP production by approximately 50% compared to controls. The rPAFR3i domain did not inhibit IP production mediated by the multifunctional rat pituitary adenylate cyclase-activating polypeptide receptor (rPACAPR), demonstrating the specificity of the competition by the rPAFR3i domain. In further experiments, the rPAFR3i domain was engineered onto the homologous domain of a monofunctional transmembrane variant of the rPACAPR (rPACAPR2) that activates only adenylyl cyclase. The rPACAPR2/rPAFR3i chimera responded to PACAP with increases in IP production which were attenuated nearly completely in cells cotransfected with the rPAFR3i domain. In contrast, PACAP had no effects on IP production in a receptor chimera expressing a mutated form of the rPAFR3i domain (rPACAPR2/rPAFR3imut). These results demonstrate the ability of the rPAFR3i domain to confer a phospholipase C-signaling phenotype to a receptor deficient in this activity and show that this activity is specific for the engineered rPAFR3i domain. These results suggest that the third intracellular loop of the rPAFR is a primary determinant in its coupling to phosphoinositide phospholipase C-activating G proteins, providing the first insight into the molecular basis of interaction of PAFRs with signal-transducing G proteins.

Amino Acid Sequence↗

PubMed1996

Lack of constitutive activation or inactivation of the platelet-activating factor receptor by glutamate substitution of alanine 230.

The platelet-activating factor (PAF) receptor (PAFR) is a G protein-coupled receptor (GPCR) that mediates a diverse array of biological responses to PAF. Recently, we provided evidence that the third intracellular domain (3i) of the rat PAFR (rPAFR) is a critical determinant in its coupling to phosphoinositide phospholipase C (PI PLC)-activating G proteins. In the present study, we assessed the potential role of a conserved alanine in the carboxyl-terminal region of 3i of the rPAFR in rPAFR signaling activity. Previous studies with the m5 muscarinic acetylcholine and human PAF receptors revealed that substitution of a carboxyl-terminal alanine was found to impair receptor-mediated PI PLC activation. Here we report the effects of the analogous nonconservative substitution of glutamate for alanine 230 of the rPAFR (rPAFR A230E) on receptor-mediated agonist binding and PI PLC activation following transient expression of the receptor cDNA. BHK cells transfected with a cDNA encoding the rPAFR A230E exhibited PAF-stimulated increases in inositol phosphate (IP) accumulation with no increase in basal levels of IPs. PAF-stimulated IP production in rPAFR transfectants was dependent on the amount of DNA transfected, although PAF provoked a larger increase in IPs in rPAFR transfectants than in rPAFRA230E transfectants in cells transfected with equal amounts of receptor cDNA. This latter finding apparently reflects differences in the transfection efficiency or expression of the wild-type and rPAFR A230E cDNAs because PAF produced indistinguishable effects on IP accumulation in rPAFR and rPAFR A230E transfectants expressing equivalent numbers of receptors. These results provide evidence for a nonconserved role of this conserved alanine in coupling of group I GPCRs to PI PLC-activating G proteins and also suggest that this residue has differential roles in regulating expression and signaling by rat and human PAFRs.

Alanine↗

PubMed1995

Multiple affinity and guanine nucleotide sensitive forms of the calcitonin gene related peptide (CGRP) receptor.

Calcitonin gene related peptide (CGRP) is a novel neuropeptide with an impressive array of biological actions consistent with its diverse tissue distribution and suggested role as neurotransmitter or neuromodulator. Binding sites for CGRP with properties consistent with those of receptors are present in both central and peripheral tissues. Radioligand binding studies were performed to investigate the fundamental processes underlying CGRP receptor activation and signaling following agonist occupancy of the receptor. These studies documented the existence of a selective, high affinity, and homogeneous population of binding sites for CGRP in membranes prepared prepared from central and various peripheral tissues. The affinity of [125I]CGRP for these sites was regulated by GTP or its stable analog GTP--gamma S, indicating coupling of CGRP receptors to G-protein(s). Kinetic studies documented the existence of the CGRP receptor in multiple affinity states when both coupled to and uncoupled from G-protein(s). These findings suggest that CGRP occupancy of its receptor induces conformational changes in the receptor that may be involved in its coupling to G-proteins and that the resulting ligand--receptor---protein ternary complex exists in multiple affinity conformational states. It seems likely that the multiple affinity states of the CGRP receptor ternary complex are involved differentially in signaling by and desensitization of the receptor. This evidence for agonist-induced conformational changes in a G-protein-coupled receptor prior to its coupling with G-protein(s) and for the existence of the ligand--receptor--G-protein ternary complex in multiple affinity conformational states is novel and extends our current understanding of the nature of the processes involved in agonist-dependent activation of G-protein-coupled receptors.

Animals↗

PubMed1993

Fewer PGE2 and PGF2 alpha receptors in brain synaptosomes of newborn than of adult pigs.

PGE2 and PGF2 alpha decrease cerebral metabolism in the adult but increase it in the newborn. The present study was done using synaptosomes from newborn and adult pigs to find out whether these diverse cerebral effects of prostanoids are a consequence of newborn synaptosomes containing fewer or no EP2 receptors for PGE2 (mediating cAMP increase and in turn cerebral metabolism decrease) and PGF2 alpha receptors [mediating IP3 (inositol 1,4,5-triphosphate) increase and in turn cerebral metabolism decrease]. It was found that maximum binding values (fmol/mg protein) of [3H]PGE2 and [3H]PGF2 alpha were, respectively, only 11.6 +/- 0.8 and 8.6 +/- 1.4 on newborn synaptosomes compared with 41.8 +/- 1.8 and 31.2 +/- 4.8 on adult tissues. EP1 receptors were virtually absent in newborn and adult preparations. Practically all the PGE2 receptors on newborn synaptosomes were EP3 subtype, whereas adult brain synaptosomes contained both EP2 and EP3 subtypes; this was indicated by the ability of M&B 28,767, a specific EP3 receptor agonist, to cause 100% displacement of [3H]PGE2 binding to synaptosomes of the newborn but only 51% in the case of the adult. Also, PGE2, 11-deoxy PGE1 (a nonselective EP2 and EP3 agonist) and M&B 28,767 (an EP3 agonist) caused comparable decreases in cAMP formation in synaptosomes from the newborn, whereas in those from the adult, PGE2 and 11-deoxy PGE1 increased and M&B 28,767 decreased cAMP production. PGF2 alpha markedly increased IP3 synthesis in synaptosomes of the adult but not of the newborn.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1993

Solubilization and characterization of a guanine nucleotide-sensitive form of the calcitonin gene-related peptide receptor.

Calcitonin gene-related peptide (CGRP) receptors were solubilized from rat cerebellum membranes in an active, stable, and guanine nucleotide-sensitive form by using digitonin. Nearly 90% of membrane CGRP receptors and 50% of membrane protein were solubilized by digitonin treatment of cerebellum membranes. Binding of 125ICGRP to soluble receptors was specific, saturable, of high affinity, and reversible. Scatchard analysis of the saturation binding data revealed a homogeneous population of binding sites with a Kd of 178 +/- 42 pM and a Bmax of 201 +/- 17 fmol/mg of protein. Binding of 125ICGRP to soluble receptors was inhibited nearly 60% by guanosine-5'-O-(3-thio)triphosphate (GTP gamma S) (100 microM), suggesting coupling of receptors with guanine nucleotide-binding proteins (G proteins) to form high affinity binding sites. Antiserum against the amino-terminal region of Gs alpha immunoprecipitated a significant portion of soluble CGRP receptors, indicating association of receptors with Gs alpha. In agreement with the saturation binding data, association kinetic studies with soluble receptors indicated binding of 125ICGRP to a single population of sites. Dissociation kinetic data, in contrast, demonstrated that 125ICGRP dissociated from labeled receptors with fast- and slow-dissociating components. GTP gamma S significantly accelerated dissociation of 125ICGRP from labeled receptors; however, dissociation still occurred from two distinct affinity components, with rate constants significantly different from those observed in the absence of GTP gamma S. These observations suggest that soluble CGRP receptors, like native membrane-bound receptors, exist in two distinct affinity states in both G protein-coupled and -uncoupled receptor states. Soluble receptors were retained specifically on a wheat germ lectin column, and affinity cross-linking of receptors specifically labeled with 125ICGRP demonstrated labeling of a 67-kDa protein, suggesting that the rat cerebellum CGRP receptor is a 67-kDa glycoprotein. This study is the first to report solubilization of CGRP receptors retaining the native ability of the receptor to undergo functional coupling with G proteins and to provide direct evidence for association of these receptors with Gs alpha.

Animals↗

PubMed1992

Effects of plant extract Centella asiatica (Linn.) on cold restraint stress ulcer in rats.

Extract of C. asiatica (Linn.) inhibited significantly gastric ulceration induced by cold and restraint stress (CRS) in Charles-Foster rats, Antiulcer activity of plant extract was compared with famotidine (H2-antagonist) and sodium valproate (anti-epileptic). Plant extract, formotidine and sodium valproate showed a dose dependent reduction of gastric ulceration. Plant extract increased brain GABA level which was also dose dependent. Pretreatment with bicuculline methiodide (specific GABAA-antagonist) at the dose level of 0.5 mg/kg im, reversed the antiulcerogenic activity of both plant extract and sodium valproate. Bicuculline as such did not induce gastric ulceration in normal rat.

Animals↗

PubMed1992

Artificial ventilatory management in a severe, pregnant asthmatic--a case report.

A 33-year-old pregnant atopic woman, who had never suffered from asthma or atopic-related disorder, was admitted on three occasions during her fifth pregnancy with severe asthma. On her last admission she required more intensive monitoring and artificial ventilation. We report this patient and discuss the pathogenesis of asthma during pregnancy. Asthma in pregnancy is not uncommon and it may take a severe course in some pregnancies. In one prospective study 42% of the patients required aggressive management, but none required artificial ventilation. However, there was no difference in the outcome of pregnancies between asthmatic and control subjects. Here we describe a patient with severe asthma who required artificial ventilation with a successful outcome.

Adult↗

PubMed1992

Antimicrobial activities of mycotoxin MT81 and its structural derivatives.

Antimicrobial activities of mycotoxin MT81 and its acetylated, benzoylated and acetic acid derivatives were determined by zone inhibition method. MT81, acetylated MT81 and acetic acid MT81 exhibited sensitivity (31.25, 62.50, 62.50 micrograms/ml respectively) to all the test bacteria. Benzoylated-MT81 did not exhibit any activity even up to 1000 mcg/ml. MT81, its acetylated and acetic acid derivatives exhibited antifungal activity up to 62.5 micrograms/ml and 125.0 micrograms/ml respectively against test yeasts and other fungi. Benzoylated MT81 also exhibited antifungal activity but the degree was comparatively less.

Anthraquinones↗