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PubMed1995

Identification of prostaglandin E receptor 'EP2' cloned from mastocytoma cells EP4 subtype.

We previously cloned a cDNA for a mouse PGE receptor positively coupled to adenylate cyclase from mouse mastocytoma cells, and reported it as EP2 subtype of PGE receptor [Honda, A., Sugimoto, Y., Namba, T., Watabe, A., Irie, A., Negishi, M., Narumiya, S. and Ichikawa, A. (1993) J. Biol. Chem. 268, 7759-7762]. However, it is not sensitive to one of the EP2 agonists, butaprost. Recently another subtype of PGE receptor coupled to adenylate cyclase has been identified pharmacologically and named EP4. These findings have led us to examine whether the cloned receptor is the EP4 subtype. AH23848B, a selective EP4 antagonist, not only displaced the [3H]PGE2 binding to the cloned receptor but antagonized the PGE2-stimulated cAMP formation in the receptor. In contrast, EP2 specific agonist, butaprost and 19(R)OH-PGE2 neither bound to the receptor nor stimulated the cAMP formation. These results suggest that this receptor previously reported as 'EP2' subtype is identical to the pharmacologically defined EP4 subtype and not of EP2 subtype.

Adenylyl Cyclases↗

PubMed1993

Expression of bombesin-receptor subtypes and their differential regulation of colonic smooth muscle contraction.

BACKGROUND: Bombesin-related peptides show different potencies, suggesting the existence of at least two receptor subtypes. The aim of this study was to assess the relationship and contribution of each receptor subtype on smooth muscle contraction. METHODS: The expression of bombesin-receptor subtype messenger RNA (mRNA) was examined in human and rabbit smooth muscle from the rectosigmoid colon, and the contribution of each of the receptor subtypes to smooth muscle contraction was investigated by blocking mRNA translation of either neuromedin B or gastrin-releasing peptide (GRP) receptor subtype or of both. RESULTS: Neuromedin B and GRP receptor mRNAs were detected in human and rabbit colonic smooth muscle cells. Incubation with neuromedin B receptor antisense oligonucleotides inhibited the neuromedin B-induced contraction, whereas incubation with GRP receptor antisense oligonucleotides inhibited the GRP-induced contraction. Incubation with GRP plus neuromedin B receptor antisense oligonucleotides inhibited the contractile response induced by bombesin, neuromedin B, and GRP. CONCLUSIONS: Distinct neuromedin B and GRP receptor subtypes are present on smooth muscle cells of the rectosigmoid colon, and bombesin interacts with both neuromedin B and GRP receptors, resulting in a complex contraction that is sustained in nature.

Animals↗

PubMed1979

Preparation of sera for subtyping of influenza A viruses by immunofluorescence.

The conditions for preparation of type-specific and subtype-specific influenza A virus reagents to be used in the immunofluorescence technique have been evaluated. Type A-specific antibodies were prepared by passing an antivirion hyperimmune serum through an immunoadsorbent column containing antigens from disrupted virions of a different influenza A virus subtype. The type-specific antibodies were recovered from the immunoadsorbent by desorption with 3 M NaI. For subtype determination, antisera against the various hemagglutinins were used. Such sera could be prepared by removal of irrelevant influenza A virus antibodies from sera directed against purified virions and isolated peplomers, respectively. This was performed by passing the antisera through immunoadsorbent columns containing antigens from disrupted virions of appropriate strains. However, attempts to obtain a hemagglutin-specific antiserum from a serum directed against allantoic fluid virus suspension failed with this procedure. Antisera obtained after immunization with purified hemagglutinin were also elaborated. These sera were shown to be superior for subtyping of influenza A virus infections by immunofluorescence, but could not a priori be regarded as subtype-specific. The usefulness of subtype-specific sera has been demonstrated on clinical specimens for rapid virus diagnosis.

Animals↗

PubMed1991

Angiotensin receptor subtypes in rat, rabbit and monkey tissues: relative distribution and species dependency.

The displacement of [125I]Sar1, Ile8 angiotensin II binding by the receptor subtype selective angiotensin II antagonists, DuP-753 and WL-19 (PD121981) was used to define the relative proportion of angiotensin subtype AT1 and subtype AT2 receptors, respectively in various tissues (aorta, heart, adrenal cortex, kidney cortex and brain) of the rat, rabbit and monkey. The relative abundance of these receptor subtypes varied greatly not only among different tissues of the same species but also within the same tissue of different species. The relative affinity of the DuP-753 and WL-19 for the angiotensin receptor subtypes did not vary markedly suggesting that the two angiotensin receptor subtypes in these tissues and species are similar.

Adrenal Cortex↗

PubMed1993

Visualization of muscarinic m4 mRNA and M4 receptor subtype in rabbit lung.

We have used in situ hybridization to study the localization of muscarinic receptor subtype mRNAs and compared this with the distribution of muscarinic receptor subtypes using receptor autoradiography in tissue sections of the rabbit lung. To localize muscarinic receptor subtype mRNAs, subtype-specific synthetic oligonucleotide probes or riboprobes were generated. Two muscarinic receptor mRNAs (m3 and m4) were detected. The m3 mRNA was expressed in high concentration in tracheal smooth muscle, epithelium and blood vessels and to a lesser extent in bronchiolar smooth muscle while the m4 mRNA was abundantly expressed in bronchiolar smooth muscle and alveolar walls. No expression of m1, m2 or m5 mRNAs was found in any lung structures. This was confirmed by Northern blot analysis which revealed only a single transcript of 3.6 kb for m4 mRNA in rabbit lung. With the exception of the airway epithelium and blood vessels, there was a correspondence between mRNA localization and the distribution of muscarinic receptor subtypes visualized by [3H](-)-quinuclidinyl benzilate ([3H]QNB) autoradiographically in the presence of relatively selective muscarinic subtype antagonists (M1-M3) and direct labelling with selective [3H]AF-DX 384. The significance and the reasons for the presence of m4 mRNA and M4 receptors in the rabbit lung are uncertain and warrant further investigation.

Animals↗

PubMed1992

Genetic variation in rotavirus serotype 4 subtypes.

Serotype 4 rotavirus strains have been classified into two antigenic "subtypes" by a solid phase immune electron microscopy technique in which cross-absorbed animal polyclonal immune sera are used as the source of antibodies. The sequences of the gene encoding the outer capsid glycoprotein VP7 from a single serotype 4 rotavirus field strain identified as subtype A ("ST3-like") and from three field strains identified as subtype B ("VA70-like") were determined. A comparison of the deduced amino acid sequences indicated that 15 amino acid residues were divergent between subtypes but were conserved within a subtype. Three of these 15 amino acid residues (at positions 96, 212, and 217) were located in regions of the VP7 that have been defined as serotype-specific antigenic sites. These data suggest that VP7 subtype differences may result from critical amino acid substitutions within an immunodominant serotype 4-specific antigenic site. Whether these differences are an important mechanism in the epidemiology of rotaviruses requires further investigation.

Amino Acid Sequence↗

PubMed1989

The loss of subtypic determinants in alleles, d/y or w/r, on hepatitis B surface antigen.

Hepatitis B surface antigen possesses the group-specific determinant called a and one or another member from each of two pairs of allelic determinants, d and y as well as w and r, thereby creating the four major subtypes, adw, adr, ayw and ayr. In the sequence of major surface antigen polypeptides made of 226 amino acid residues, lysine or arginine at amino acid position 122 specifies d or y determinant, and lysine or arginine at position 160 specifies w or r determinant, respectively. By means of site-directed mutagenesis and expression of mutant genes in cultured cells, the mechanism for the loss of subtypic determinants on surface antigens was investigated at the molecular level. A rare sample of surface antigen of subtype ad, devoid of w or r determinant, had asparagine at position 160. When it was converted to lysine, the surface antigen of subtype adw was obtained. Two samples of surface antigen were subtyped as ar. They lacked d determinant, despite having lysine at position 122 which usually specified it. They differed from all reported sequences of surface antigen in amino acid 144 or 145. They displayed d determinant when amino acid 144 was converted from glutamic acid to aspartic acid, or when amino acid 145 was changed from alanine to glycine. These results indicate that the key amino acid residue at position 122 or 160 is indispensable for the expression of subtypic determinants and that some distant residues are also crucially involved in conforming them.

Alleles↗

PubMed1990

Subtyping familial schizophrenia: reliability, concordance, and stability.

This report examines the reliability, concordance, and long-term stability of the subtypes of schizophrenia defined by four major diagnostic systems (DSM-III, DSM-III-R, ICD-10, and Tsuang-Winokur criteria) and rated both for the first hospitalization and for a best estimate diagnosis reflecting lifetime evolution of symptomatology. Schizophrenics studied belonged to two samples of multiply affected families, namely a sample selected in France and a sample of non-metropolitan French identified in the island of La Réunion. ICD-10 and DSM-III-R show opposite stringency regarding subtyping of schizophrenia, with DSM-III-R having a narrow and ICD-10 a broader definition of specific subtypes. Long-term stability of each subtype was fairly good, stability being the highest for hebephrenics and only intermediate for paranoid and undifferentiated subtypes. Comparison of two different cultural and geographical regions reveals an overall similarity of subtype frequencies in familial schizophrenia. The implications of the results for the choice of diagnostic procedures in family studies of schizophrenia are discussed.

Adolescent↗

PubMed1994

Toxins from mamba venoms: small proteins with selectivities for different subtypes of muscarinic acetylcholine receptors.

Muscarinic acetylcholine receptors exist as five subtypes that are widely distributed throughout the body. Conventional pharmacological agents are not highly selective for particular subtypes, making investigations on the functional significance of the subtypes difficult. Recent findings indicate that mamba snake venoms contain several small proteins ('muscarinic toxins') that are highly specific for muscarinic receptors, and are discussed in this review by Diana Jerusalinsky and Alan Harvey. Some of these toxins act selectively and irreversibly on individual subtypes of receptor, and some are antagonists, while others activate muscarinic receptors. The toxins should be useful tools in studies of the functions of individual receptor subtypes, and comparisons of their three-dimensional structures should give clues about how selective binding to muscarinic receptor subtypes can be obtained.

Amino Acid Sequence↗

PubMed1991

Discrimination of angiotensin II receptor subtype distribution in the rat brain using non-peptidic receptor antagonists.

The non-peptidic angiotensin II receptor subtype selective antagonists, DuP 753 and PD123177, were used to characterize angiotensin II receptor binding sites in the rat brain. Competitive receptor autoradiography with 125I-Sar1-Ile8 angiotensin II defined a regional distribution of binding sites that were sensitive to either DuP 753 (designated AII alpha subtype) or PD123177 (designated AII beta subtype). Whereas most brain nuclei could be assigned to a category containing a predominant subtype, a multiple receptor subtype analysis indicated that some regions are homogeneous, while others contain a mixture of both AII alpha and AII beta subtypes.

Angiotensin II↗

PubMed1995

Recombinant human alpha 2-adrenoceptor subtypes: comparison of [3H]rauwolscine, [3H]atipamezole and [3H]RX821002 as radioligands.

Kinetic, saturation and competition binding assays were employed to optimize and validate radioligand binding methods for characterization of recombinant human alpha 2-adrenoceptor subtypes and for screening of new subtype-selective ligands. Stable transfected lines of Shionogi 115 mouse mammary tumour cells (S115) and three structurally different antagonist radioligands, [3H]rauwolscine, [3H]atipamezole and [3H]RX821002, were used. Specificity of alpha 2-adrenergic binding was defined with 100 microM (-)-adrenaline. Steady-state was reached with all three radioligands within 15-30 min at 25 degrees C, and the binding was rapidly reversible. The receptor affinities (alpha 2-C10) were highest in glycylglycine, almost equally high in K(+)-phosphate, and lowest in Tris buffer for all three [3H]-ligands. This was mainly caused by different association rates. [3H]RX821002 was bound with high affinity and similar kinetic properties to all three alpha 2-adrenoceptor subtypes in K(+)-phosphate buffer, and had the highest proportion of specific binding (96-98%). [3H]RX821002 and K(+)-phosphate buffer were subsequently used in competition assays. The rank order of affinity of compounds selective for alpha 2-adrenoceptor subtypes was alpha 2-C10 > alpha 2-C4 > alpha 2-C2 for oxymetazoline, alpha 2-C4 > alpha 2-C2 > alpha 2-C10 for prazosin and alpha 2-C2 > alpha 2-C4 > alpha 2-C10 for chlorpromazine. The drug affinities (Ki values) determined in this system were in close agreement with earlier results with [3H]rauwolscine in Tris buffer (r = 0.94). Agonist competition for [3H]RX821002 binding was biphasic in K(+)-phosphate buffer supplemented with 10 mM MgCl2, indicating functional coupling of receptors to G-proteins. Accordingly high-affinity binding of the agonists (-)-noradrenaline and UK14,304 was eliminated by 10 microM Gpp(NH)p in the assays. Our results confirm that [3H]RX821002 is a suitable radioligand for the characterization of all three human alpha 2-adrenoceptor subtypes and for the determination of the subtype-selectivity of new alpha 2-adrenoceptor agonists and antagonists.

Adrenergic alpha-2 Receptor Agonists↗

PubMed1993

Molecular cloning of rat cDNAs for beta and gamma subtypes of 14-3-3 protein and developmental changes in expression of their mRNAs in the nervous system.

We isolated cDNAs to beta and gamma subtypes of 14-3-3 protein, a putative regulatory protein for protein kinase C, from the brain and clarified a high homology in sequences of nucleotides and deduced amino acids between the two rat subtypes and the bovine counterparts and even reciprocally between the two rat subtypes. In Northern blot analysis, the gene expression of the two subtypes was detected weakly at E13, increased progressively after birth and reached a maximum at P7-P14. Thereafter it decreased slightly. In situ hybridization analysis allowed detection of the beta but not the gamma subtype in the matrix cells of the ventricular germinal zone of the neural wall. In post-mitotic neurons in the mantle zone and maturing brain loci, genes of the two subtypes were expressed in patterns similar to each other, and three neuron types were identified: type I neurons with high levels of expression throughout development; type II neurons showing high expression during the early developmental stages with a subsequent decrease in the expression at maturing and adult stages; and type III neurons showing consistently low levels of expression throughout development. The wider and more highly-patterned expression of the 14-3-3 protein family than expected suggests that this protein may be involved in the elaborate regulation of some fundamental cellular activities and differentiation of neurons.

14-3-3 Proteins↗

PubMed1995

A molecular analysis of the telomeric end of the major histocompatibility complex. DNA typing of HLA-A3 subtypes and -B7.

A molecular investigation of the HLA-A, -B, -C, I82, D6S128, and D6S265 loci was carried out using RFLP and PCR analyses in 331 healthy individuals, 21 HLA-A3B7 homozygous subjects, and 17 HLA homozygous cell lines. New polymorphic RFLP patterns were noted at the HLA-A and -B loci which were useful for distinguishing subtypes of HLA-A3 and -B44. Strictly specific bands were observed for HLA-A3, -A11, -B7, -B57. and -Cw4. Molecular identification of two HLA-A3 subtypes was possible by HLA-A RFLP and D6S265 PCR analyses. The two alleles of the I82 locus were associated with different HLA-A3 subtypes. It was shown that the serologically HLA-A3 homozygous cell line EHM was heterozygous for the two subtypes. The common subtype formed 91% of HLA-A3 antigens. Some serologically HLA-A3 homozygous healthy individuals were heterozygous for the subtypes. Using these new polymorphisms, a thorough molecular analysis of the haplotype HLA-A3B7DR15 at the three regions of the MHC was performed. The results have implications on HLA matching in transplantation, population genetics of the MHC, and disease association studies.

Base Sequence↗

PubMed1985

Murine helper T lymphocyte response to influenza virus: recognition of haemagglutinin by subtype-specific and cross-reactive T cell clones.

Helper T cel lines specific for influenza virus were established by continuous culture of Mem 71-Bel (H3) virus-immune spleen cells in the presence of virus and antigen-presenting cells and their specificity assessed in proliferation experiments. A line stimulated in vitro with Mem 71-Bel virus was able to proliferate in response to viruses of the same, and also of different, type A haemagglutinin (HA) subtypes as the immunizing virus but not to a type B influenza virus. A component of this cross-reactivity was due to recognition of the HA molecule. Lines stimulated in vitro with purified disrupted H3 or H2 viruses showed a higher degree of cross-reactivity. Of nine clones isolated from these lines, seven were directed against the HA molecule and recognized the HA1 chain. The HA-specific T cell clones were either subtype-specific T cell clones were either subtype-specific, recognizing only viruses of the H3 subtype, or cross-reactive, also recognizing viruses of the H2 subtype of type A (but not type B). Subtype-specific and cross-reactive T cell clones were shown to function as helper T cells in vitro. In addition to collaborating with H3 virus-primed B cells responding to H3 virus in culture, the cross-reactive T cell clone could also provide help for H2 virus-primed B cells making anti-HA antibody in response to H2 virus.

Animals↗

PubMed1992

Selective potentiation of GABA-mediated Cl- current by lanthanum ion in subtypes of cloned GABAA receptors.

The effect of lanthanum ion (La3+) on gamma-aminobutyric acid (GABA)-mediated Cl- currents was examined in the alpha 1 beta 2 or alpha 1 beta 2 gamma 2 subtype of GABAA receptors expressed in a human kidney cell line (A293), using a whole-cell configuration of patch-clamp techniques. La3+ dose-dependently stimulated the Cl- currents in the alpha 1 beta 2 gamma 2 subtype with an EC50 of 21.3 +/- 3.5 microM with a maximal potentiation of 240 +/- 16% as normalized to the GABA response at 5 microM. In the alpha 1 beta 2 subtype, however, the ion marginally potentiated GABA response, a maximal stimulation being less than 70% with an EC50 for La3+ near 200 microM. The stimulation of GABA response by La3+ in the alpha 1 beta 2 gamma 2 subtype was due to a decrease in the half maximal concentration for GABA and was more pronounced at the negative membrane potentials. This selectivity of La3+ toward the subtypes of GABAA receptors contrasts to that of Zn2+ which inhibits the currents in the alpha 1 beta 2, but not in the alpha 1 beta 2 gamma 2 subtype (Neuron, 5: (1990) 781-788). It appears that these polyvalent cations are useful in understanding the molecular basis for the functional diversity and in characterizing the molecular organization of native GABAA receptors.

Cell Line↗

PubMed1996

5-Hydroxytryptamine receptor subtype messenger RNAs in rat peripheral sensory and sympathetic ganglia: a polymerase chain reaction study.

While serotonin has been shown to play an important role in peripheral pain mechanisms, the specific subtypes of receptors involved and their differential distribution between the sensory and sympathetic nervous system remains poorly understood. In this study, the presence of messenger RNA for rat serotonin receptor subtypes in peripheral sensory and sympathetic ganglia was detected using the method of polymerase chain reaction. Lumbar dorsal root ganglia, superior cervical sympathetic ganglia and lumbar sympathetic ganglia were excised from anesthetized Sprague-Dawley rats. Oligonucleotide primers were chosen based on unique regions of complementary DNA sequence for each of the 12 cloned rat serotonin receptor subtypes (i.e. 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT5B, 5-HT6 and 5-HT7) and high stringency conditions were used during polymerase chain reaction. Within lumbar dorsal root ganglia, the presence of the 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2C, 5-HT3 and 5-HT7 receptor subtype messenger RNAs was detected. Within superior cervical ganglia, the presence of messenger RNA for 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT3, 5-HT6, and 5-HT7 receptor subtypes was detected. Lumbar sympathetic ganglia displayed banding identical to the superior cervical ganglia with the exception of the 5-HT6 receptor which was not detected in the lumbar sympathetic ganglia. The polymerase chain reaction product from each positively-detected receptor subtype was subcloned and sequenced and found to correspond to published complementary DNA sequences. Findings from this study may direct further efforts to determine the role of 5-hydroxytryptamine receptors in the peripheral nervous system.

Animals↗

PubMedDate not supplied

Positive and negative subtypes of schizophrenia. A follow-up study from India.

98 schizophrenic patients who were initially studied to examine the concept of positive and negative subtyping, were followed up for a period ranging from 18 to 30 months. Follow-up data were available for 79 patients. The major findings of the study are significant reduction in positive symptom scores and the number of patients in the positive subtype of schizophrenia. The mixed subtype of schizophrenics increased in number and at follow-up there were significantly more mixed subtype schizophrenics who did not meet criteria for either the positive or negative subtype, i.e., who had little of either type of symptomatology. The number of schizophrenics categorized as negative subtype, as well as negative symptom scores did not change appreciably. The results of the study indicate that the negative symptoms seem to become more stable over a period of time.

Adult↗

PubMed1993

Subtype selective regulation of coupling of rat cardiac beta adrenoceptors to adenylate cyclase.

There is now evidence from human studies to suggest that cardiac beta-adrenoceptor density and coupling to adenylate cyclase may be regulated in a subtype selective fashion. An animal model was used to investigate this further. Rats were infused for 6 days with the non-selective full agonist isoprenaline (n = 6) or the beta 1-selective partial agonist xamoterol (n = 6) with sham operated rats (n = 6) for control. beta-Adrenoceptor subtype density and coupling to adenylate cyclase were determined in left ventricular membranes. Isoprenaline infusion downregulated both beta 1- (30%) and beta 2- (63%) adrenoceptor subtypes with associated reduction in adenylate cyclase stimulation through both subtypes. Xamoterol did not downregulate either subtype, but selectively uncoupled beta 1-adrenoceptors. beta 1- and total beta-adrenoceptor-mediated stimulation of adenylate cyclase was reduced less by xamoterol than isoprenaline. We conclude that coupling of rat cardiac beta-adrenoceptors can be regulated in a subtype selective fashion and that the partial agonist xamoterol does not desensitise beta-adrenoceptor mediated stimulation of adenylate cyclase to the same extent as the full agonist isoprenaline.

Adenylyl Cyclases↗