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PubMed1994

Selectivity of agonists for cloned alpha 1-adrenergic receptor subtypes.

The potencies and intrinsic activities of agonists in activating cloned alpha 1-adrenergic receptor (AR) subtypes were compared. The hamster alpha 1B-, bovine alpha 1C-, or rat alpha 1A/D-ARs were expressed at high levels in human embryonic kidney 293 cells. Catecholamines and phenylethylamines, but not lower efficacy agonists, were more potent in inhibiting radioligand binding to the expressed alpha 1A/D subtype than to the alpha 1B or alpha 1C subtypes; this selectivity remained in the presence of different buffers, nucleotides, and cations. Activation of all three subtypes caused substantial increases in [3H]inositol phosphate formation in cells grown in 96-well plates. Pretreatment with phenoxybenzamine decreased maximal responses to norepinephrine (NE) with only small decreases in apparent potency, suggesting similar small receptor reserves for all three subtypes. The catecholamines NE, epinephrine, and 6-fluoro-NE were full agonists with similar potencies at the three subtypes; alpha-methyl-NE was also a full agonist but was about 20-fold less potent at alpha 1B-ARs than at alpha 1C- or alpha 1A/D-ARs. Phenylephrine had similar potencies at all three subtypes but gave a submaximal response at alpha 1B-ARs. Methoxamine was a full agonist at alpha 1C- and alpha 1A/D-ARs, with about 20-fold greater potency at the alpha 1C subtype, but showed lower intrinsic activity at alpha 1B-ARs. A number of imidazolines, amidephrine, and SKF 89748 had substantial intrinsic activity at alpha 1C-ARs but little or no intrinsic activity at the other two subtypes. We conclude that the potencies of many agonists in competing for radioligand binding sites are related to their potencies in activating functional responses but that this relationship is not the same for all subtypes. NE and epinephrine activate all three cloned alpha 1-AR subtypes with similar potencies and intrinsic activities, but many widely used agonists show significant selectivity for different alpha 1-AR subtypes.

Adrenergic alpha-1 Receptor Agonists↗

PubMed1995

Identification of alpha 1-adrenergic receptor subtypes in human corpus cavernosum tissue and in cultured trabecular smooth muscle cells.

Recent pharmacological and functional studies have suggested the presence of more than one alpha-1 adrenergic receptor subtype in human corpus cavernosum (HCC). In this study, we sought to identify the alpha-1 adrenergic receptor (alpha 1-AR) subtypes expressed in HCC whole tissue and in trabecular smooth muscle subcultured from this tissue. We have utilized RNase protection assays and in situ hybridization (ISH) techniques to identify and localize these receptor subtypes. RNase protection assays of mRNA isolated from whole tissue demonstrated the presence of mRNA transcripts for three alpha 1-AR receptor subtypes (alpha 1d, alpha 1b, and alpha 1a). alpha 1d-AR and alpha 1a-AR appear to be more abundant than alpha 1b-AR. The identification and localization of mRNA for alpha 1-AR subtypes in whole tissue was demonstrated by RNA protection assays and ISH analysis. Immunocytochemical analysis of alpha 1-AR by an antipeptide antibody developed against a specific amino acid sequence derived from alpha 1d-AR subtype demonstrated specific staining of the smooth muscle cells, suggesting the expression of alpha 1d-AR subtype. In cultured HCC smooth muscle cells (HCC SMC), phenylephrine,alpha 1-AR agonist stimulated Na+/K+ ATPase activity, suggesting the presence of functional alpha 1-AR. RNase protection assay of mRNA isolated from HCC SMC grown in culture further demonstrated the presence of mRNA transcripts for alpha 1d-AR and alpha 1a-AR subtypes. ISH analysis and confocal microscopy also indicate that the SMC express the alpha 1d-AR and alpha 1a-AR subtypes. The data presented suggests that HCC and SMC derived from this tissue express at least three alpha 1-AR subtypes. Identification of these receptor subtypes should allow characterization of the functional role of these receptor subtypes in regulation of trabecular smooth muscle tone and penile detumescence.

Cells, Cultured↗

PubMed2001

HIV type 1 non-B subtype prevalence in Spain, 1997-1998.

We have evaluated the prevalence of HIV-1 non-B subtypes in Spain by means of an enzyme immunoassay (EIA) for discrimination between B and non-B subtypes. Samples were obtained from newly diagnosed patients attended at internal medicine outpatient clinics between October 1997 and October 1998. Discrimination between HIV-1 B and non-B subtypes was carried out by means of the EIA, with V3 synthetic peptides specific to the different subtypes. Non-B-serotyped samples were genetically analyzed in the gp41 region from the original sera. During the study period, 909 samples were collected from 21 medical units located in various Spanish geographical regions. Serotyping was possible in 885 cases, of which 791 were assigned as B serotype (89.38%), 70 showed no reactivity to any of the peptides (7.91%), and the remaining samples displayed other reaction patterns (2.72%). Of the 94 non-B-assigned samples, 65 were genetically characterized in the gp41 region of the env gene: 55 were B subtype, 5 were A subtype (4 clustered with CRF02AG reference strains), 3 were C subtype, and 2 were G subtype. The prevalence rate for non-B subtypes in Spain was established at 1.13% (95% CI, 0.59-2.21). Although the B subtype is predominant in the Spanish population, other subtypes have been detected.

Enzyme-Linked Immunosorbent Assay↗

PubMed2003

Introduction of HIV type 1 non-B subtypes into Eastern Andalusia through immigration.

A study of the distribution of HIV-1 subtypes in the native and immigrant populations of Eastern Andalusia (Southern Spain) was conducted to determine any changes between 1983 and 2001 and to identify antiretroviral resistance mutations in non-B subtype strains among the immigrant population. The study included 111 native patients from Eastern Andalusia: 94 infected with HIV before 1996 and 17 infected since 1996. A parallel study was conducted on 26 HIV-positive immigrants from Africa. Subtyping was done with the heteroduplex mobility assay. Resistance mutations were determined by line probe assay. A total of 137 patients were studied: 9.2% had subtype A (n = 12), 80.8% subtype B (n = 105), and 1.5% subtype C (n = 2). Among the Eastern Andalusia population infected before 1996, 10.9% had non-B subtypes, compared with 23.5% of those infected after that year. The greatest percentage of non-B subtypes (52.4%) was found among the immigrant population. Resistance mutation K70R was detected in one of the six immigrants with non-B subtype and M41L in another. There has been a slight increase in the diversity of HIV-1 subtypes in Eastern Andalusia over the past few years, possibly influenced by non-B subtypes introduced by immigrants from sub-Saharan Africa.

Adult↗

PubMed1995

Biological phenotypes of HIV-1 subtypes A and B strains of diverse origins.

The identification of specific biologic phenotypic traits that can be correlated with different HIV-1 genetic subtypes was sought. The genetic subtypes were determined by either sequencing (Cameroonian strains, n = 18) or by the heteroduplex mobility assay (HMA) (Belgian strains, n = 21 and Ivorian strains, n = 25). Seventeen (81%) of the 21 Belgian isolates belonged to subtype B and 4 (17%) were subtype A strains. Subtype A variants were predominant in the two African countries studied; 11 (61%) of 18 strains from Cameroon and 23 (92%) of 25 strains from the Ivory Coast. Of the 64 isolates, 38 (58%) and 19 (29%) belonged to subtypes A and B, respectively. No significant difference was observed for biological phenotypes (slow/low and rapid/high) of both genetic subtypes. In symptomatic individuals, however, a significantly higher number of subtype B isolates were of rapid/high phenotype, compared with subtype A (5 of 10; 50%) vs. 2 of 22; 9%), respectively; X2 = 6.7, P = 0.02). The findings suggest that overall HIV-1 isolates belonging to genetic subtype B are not distinguishable from subtype A variants on the basis of their biological phenotypes. Syncytium-inducing variants were less prevalent regardless of the geographic origin of the isolates.

Base Sequence↗

PubMed2004

Effects of potent antiretroviral therapy on the immune activation marker soluble CD27 in patients infected with HIV-1 subtypes A-D.

HIV-1 genetic subtypes might have a different impact on disease progression and response to antiretroviral therapy (ART). Few data are available on the immune activation profile in patients with different HIV-1 subtypes. We have tested by ELISA the plasma levels of an immune activation marker, soluble CD27 (sCD27), in a cohort of 64 patients infected with HIV-1 subtypes A-D, at baseline and after 1 year of virologically successful ART. Plasma sCD27 was significantly higher in the whole HIV-1-infected population as compared to healthy subjects [522 U/ml (188-1,307) vs. 285 U/ml (174-397), P < 0.001]. Among the four different HIV-1 subtypes, patients with subtype C virus had significantly higher plasma sCD27 [684 U/ml, (188-1228)] as compared to patients with subtype A [428 U/ml (247-1307), P < 0.05] and B [454 (211-925), P < 0.05]. After 1 year of ART, plasma sCD27 significantly decreased in all groups but patients with subtype C viruses had the largest reduction of sCD27 from baseline. The data indicate that a similar immune activation profile is present in patients infected with HIV-1 subtypes A, B, and D and that in presence of successful ART these subtypes respond similarly in terms of immune activation. Intriguingly, subtype C infection seems to be associated with higher levels of plasma sCD27, suggesting that HIV-1 genetic subtype C may have a different impact on disease outcome and response to therapy.

Adolescent↗

PubMed2006

Changes in motor subtype and risk for incident dementia in Parkinson's disease.

The objective of this study was to assess the temporal relationship between changes in predominant motor symptoms and incident dementia in Parkinson's disease (PD). A community-based sample of 171 nondemented patients with PD was followed prospectively and examined at baseline and after 4 and 8 years. The motor subtype of Parkinsonism was classified into tremor-dominant (TD), indeterminate, or postural instability gait difficulty (PIGD) subtype at each visit, based on defined items in the Unified Parkinson's Disease Rating Scale, subscales II and III. Dementia was diagnosed according to DSM-III-R criteria, based on clinical interview, cognitive rating scales, and neuropsychological examination. Logistic regression was used to analyze the relationship between subtype of Parkinsonism and dementia. Transition from TD to PIGD subtype was associated with a more than threefold increase in the rate of Mini-Mental State Examination decline. Compared to patients with persistent TD or indeterminate subtype, the odds ratio for dementia was 56.7 (95% CI: 4.0-808.4; P = 0.003) for patients changing from TD or indeterminate subtype to PIGD subtype, and 80.0 (95% CI: 4.6-1400.1; P = 0.003) for patients with persistent PIGD subtype. Patients with TD subtype at baseline did not become demented until they developed PIGD subtype, and dementia did not occur among patients with persistent TD subtype of Parkinsonism. In a substantial proportion of PD patients who develop postural instability and gait disorder during the course of the disease, this transition is associated with accelerated cognitive decline and highly increased risk for subsequent dementia. These findings raise the question whether PIGD and dementia share common or parallel neuropathology.

Aged↗

PubMed1991

Occurrence of respiratory syncytial virus subtypes in hospitalized children in Cleveland, Ohio from 1985 to 1988.

In order to determine the frequency of occurrence of the two respiratory syncytial virus (RSV) subtypes in hospitalized children in Cleveland, Ohio, we analyzed clinical isolates obtained during three consecutive winter epidemic seasons between 1985 and 1988. RSV was recovered from the frozen clinical specimens of 197 patients: 176 subtype A, and 21 subtype B. Subtype A predominated during all three epidemic seasons, ranging from 83 to 94% of isolates. We surveyed the clinical records of 16 children with subtype B, and 101 children with subtype A infections, hospitalized at the University Hospitals of Cleveland during these winter epidemics and found no differences in age, sex, race, or clinical spectrum of severity of disease caused by the two subtypes. In contrast to previously reported data, subtype A predominated in each of the winter seasons studied within this community. We conclude that both subtypes circulate concurrently within the community during the winter. In hospitalized children both subtypes appear to cause a similar spectrum of disease. Both the concurrent circulation of RSV subtypes and the similar spectrum of illness pose for important considerations in the development of effective vaccines against this common respiratory agent in children.

Antigens, Viral↗

PubMed1995

Biological and molecular characterization of subtype D, G, and A/D recombinant HIV-1 transmissions in Sweden.

HIV-1 can be subdivided into at least nine genetic subtypes (A through H and O), but in Europe and the United States there is an almost complete dominance of subtype B. In this study three Swedish HIV-1 transmission chains of subtypes other than subtype B have been biologically and molecularly characterized. The three index cases were African men. The p17 gag and env V3 regions of the HIV-1 genome were directly sequenced from uncultured lymphocytes. Phylogenetic analyses showed that the HIV-1 variants with each transmission group were genetically closely related, supporting the epidemiological information. The individuals in transmission groups I (n = 3) and II (n = 2) carried subtype G and D virus, respectively. Interestingly, all four individuals in transmission group III displayed a recombinant genotype with subtype D p17 gag sequence and subtype A V3 sequence. The biological phenotype of virus isolates (rapid/high, syncytium-inducing; or slow/low, non-syncytium-inducing) correlated with the clinical stage of the infected individual. The study also suggested that the correlation between biological phenotype and V3 genotype that has been established for subtype B HIV-1 variants may be valid also for other subtypes. This study demonstrates that HIV-1 variants of subtypes other than B, including a subtype A/D recombinant, are being transmitted in Europe.

Adult↗

PubMed1998

Full genome sequences of human immunodeficiency virus type 1 subtypes G and A/G intersubtype recombinants.

Multiple genetic subtypes and intersubtype recombinant strains have been identified among isolates of HIV-1. The greatest diversity of strains has been recovered from Central Africa, where mixtures of subtypes and recombinant forms have been recovered. However, many of the HIV-1 subtypes and recombinants have been characterized by partial rather than full-length genome sequencing. Here we report the first two virtually full-length genome sequences from HIV-1 subtype G, isolated in Sweden and Finland but originating in Congo and Kenya, and from two Djibouti isolates sharing the A/G recombinant structure of Nigerian isolate, IbNG. By comparison with reference sequences of other subtypes, it appears that the subtype G strains are largely nonrecombinant, while the Djibouti strains show alternating segments from subtypes A and G. In the cytoplasmic domain of the gp41 protein of the Djibouti viruses the E, G, and IbNG strains form a single cluster, separate from subtype A, clouding the subtype origin of these particular segments. Within the resolution of current technology, the structure of the Djibouti strains is identical to that of IbNG, establishing for the first time the geographic spread of this recombinant in Africa. The geographic spread of the IbNG-like strains suggests that, like the subtype E recombinants, these should be given a specific name to facilitate future identification and tracking; the name "IbNG subtype" is proposed.

Base Sequence↗

PubMed2001

Somatostatin receptor sst1-sst5 expression in normal and neoplastic human tissues using receptor autoradiography with subtype-selective ligands.

Somatostatin receptors are known to be expressed in a large number of human tumours and represent the basis for in vivo tumour targeting. Stable somatostatin derivatives such as octreotide or lanreotide are the most frequently used radiopharmaceuticals acting through specific binding to somatostatin receptors; however, they do not bind with high affinity to all five receptor subtypes. Whereas the mRNAs for most receptor subtypes have been detected in tumours, it is in most cases unclear which of the receptor subtype proteins are expressed. Since in vitro receptor binding methods are close correlates and predictors of in vivo peptide receptor targeting, we took advantage of the recently developed subtype-selective analogues and evaluated approximately 200 tumours for their receptor subtype protein expression in specific binding assays using autoradiography with 125I-[Leu8, D-Trp22, Tyr25]-somatostatin-28 and displacement by subtype-selective analogues. The majority of the tested neuroblastomas, meningiomas, medulloblastomas, breast carcinomas, lymphomas, renal cell carcinomas, paragangliomas, small cell lung carcinomas and hepatocellular carcinomas predominantly expressed sst2. The prostate carcinomas and sarcomas preferentially expressed sstl, while a majority of inactive pituitary adenomas displayed sst3 and, to a lesser extent, sst2. Growth hormone-secreting pituitary adenomas preferentially expressed sst2 and sst5; gastroenteropancreatic tumours and phaeochromocytomas frequently displayed sst2 and/or sstl. Non-neoplastic human tissues such as vessels, nerve plexus, pancreatic islets, prostatic stroma, adrenal medulla, spleen and germinal centres of the lymphoid tissues preferentially expressed sst2. However, the human gastric mucosa predominantly expressed sst1 while colonic mucosa displayed sst2. Interestingly, a minority of tumours showed a strong 125I-[Leu8, D-Trp22, Tyr25]-somatostatin-28 binding, of which less than 50% could be displaced by the sum of the five subtype-selective analogues. This observation suggests the existence of an as yet unknown subtype in selected tumours. This study is the first report to analyse the somatostatin receptor subtype expression in tumours with binding methods. We conclude that sst2, with high affinity for current radiopharmaceuticals such as Octreoscan, is predominantly expressed in a majority of tumours. Fewer tumour types (sarcomas, prostate cancers, inactive pituitary adenomas) preferentially express another subtype. This information is of importance with regard to the clinical applications and development of somatostatin analogues with distinct receptor subtype selectivities.

Autoradiography↗

PubMed2006

Subtype IV of the BK polyomavirus is prevalent in East Asia.

BK polyomavirus (BKV) is ubiquitous in human populations, infecting children asymptomatically and then persisting in the kidney. Using either serological or genotyping methods, BKV isolates have been classified into four subtypes (I-IV), with subtype I mainly detected in all countries studied so far. To elucidate the subtype of BKV prevalent in East Asia, we examined BKV-positive urine samples collected from immunocompetent elderly patients in Mongolia, Northeast China, Northwest China, Southeast China, Southwest China, Vietnam and Japan. The 287-bp typing region of the viral genome in each of these samples was PCR-amplified and sequenced, and a phylogenetic tree was constructed. According to the tree, BKV isolates in East Asia were unambiguously classified into subtype I or IV (subtypes II and III were not detected). In Japan, subtype I was mainly detected and subtype IV was rare, whereas in the other regions subtype IV was detected frequently, at rates ranging from 24 to 100%. Thus, East Asia (excluding Japan) is a region in which subtype-IV BKV is prevalent, a finding that requires the view of the geographic distribution of BKV subtypes to be revised. Furthermore, we present evidence that the immunological states of urine donors do not affect the pattern of BKV subtypes.

BK Virus↗

PubMed2006

Genetic subtypes of human immunodeficiency virus type 1 (HIV-1) in Istanbul, Turkey.

BACKGROUND: Epidemiological surveillance of HIV-1 subtypes is an important and ongoing element of preparation for global antiviral interventions. OBJECTIVE: To assess the molecular epidemiology of HIV-1 in Istanbul, Turkey. STUDY DESIGN: 27 HIV/AIDS patients were investigated. Data on age, sex, country of birth, and HIV acquisition route were collected. Following amplification with PCR the sequences of the gp41 region of the env gene were determined using a 310 DNA sequencer (ABI prism, Foster City, USA) and phylogenetically analyzed. RESULTS: Among the 27 patients (26 adults and 1 infant), 22 were male, born in Turkey, and 20 infected through heterosexual contact. Two patients acquired the virus through blood and/or blood transfusion and one infant by vertical transmission. The distribution of the subtypes was as follows: four were subtype A, 19 subtype B, one subtype C, one subtype D, and two subtype F1. According to our results, although the B subtype is still predominant, non-B subtypes are also present, even though the number of registered HIV/AIDS patients is low. CONCLUSION: These are the first subtyped HIV-1 strains in Turkey where a low level of HIV prevalence has been observed since the first reported case in 1985. These findings and Turkey's specific geographic localization indicate the need for a nationwide surveillance to detect all subtypes including the new recombinant ones.

Adult↗

PubMed2001

Expression and postnatal changes of adrenergic receptor subtype mRNA in rat submandibular glands.

Adrenergic receptors (ARs) are involved in regulating saliva secretion and composition in salivary glands. Nine AR subtypes, including three alpha1-ARs (alpha1a-, alpha1b- and alpha1d-ARs), three alpha2-ARs (alpha2A-, alpha2B- and alpha2C-ARs) and three beta-ARs (beta1,beta2- and beta3-ARs), have been identified through molecular cloning. The five subtype genes, alpha1a-, alpha1b-, alpha2A-, beta1-, and beta2-ARs, were expressed in rat submandibular glands. In contrast, the other four subtype mRNAs, alpha1d-, alpha2B-, alpha2C- and beta3-ARs, were not detected by reverse transcription-polymerase chain reaction (RT-PCR). The steady-state mRNA expression for the five AR subtypes in rat submandibular glands was measured by quantitative competitive RT-PCR using synthetic DNA as internal standard at different stages of postnatal development. The relative rank order of AR subtype mRNA expression was alpha1a>beta2>beta1>alpha2A>alpha1b at all stages except that beta1- and alpha2A-subtypes were reversed at 2 weeks of age. The gene expression of alpha1a-AR subtype relative to total AR was low at 2 weeks of age and increased and reached a maximum at 6 weeks of age, whereas those patterns of alpha2A-, beta1- and beta2-AR subtypes were similar to each other and their gene expressions were high at 2 weeks of age and then decreased. On the other hand, the gene expression of alpha1b-AR subtype did not change over the different stages in relation to that of a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase, and to total AR. Although rat submandibular glands contain the five AR subtype mRNAs, distinct subtype-specific expression is evident.

Age Factors↗

PubMed1997

Activation of protein kinase C subtypes alpha, gamma, delta, epsilon, zeta, and eta by tumor-promoting and nontumor-promoting agents.

Protein kinase C (PKC) subtypes alpha, gamma, delta, epsilon, zeta, and eta have been expressed using the baculovirus expression system. The partially purified PKC subtypes have been studied for their substrate specificities and phospholipid-independent activation by various chemically different nontumor- and tumor-promoting agents, as well as their inhibition of kinase activity by staurosporine and two related compounds. An endogenous PKC-like kinase activity of Sf9 cells was detected and analyzed for cofactor requirements and inhibition. Protamine sulfate was most efficiently phosphorylated by all of the PKC subtypes tested, although this phosphorylation was independent of phosphatidylserine (PS) and diacylglycerol (DAG) or 12-O-tetradecanoylphorbol 13-acetate (TPA). Except for PKC-zeta, all subtypes tested phosphorylated myelin basic protein (MBP), histone, or a peptide derived from the pseudosubstrate region of PKC-alpha in a PS/DAG-dependent manner but to varying extents. Among the various agents tested, TPA most efficiently stimulated the kinase activities of the PKC subtypes in a phospholipid-dependent manner. Phorbol 12,13-dibutyrate (PDBu) was less effective than TPA but displayed no major difference among the subtypes. Activation of PKC-alpha by bryostatin-1 reached only half of the TPA response whereas the other subtypes were activated more effectively. The weak tumor promoter resiniferonol 9,13,14-orthophenyl acetate (ROPA) mainly stimulated PKC-alpha and PKC-gamma at 1 microM concentration, whereas PKC-epsilon and PKC-eta were much less activated. Sapintoxin D, mezerein, indolactam V, and resiniferatoxin at concentrations of 1-100 nM preferentially activated PKC-alpha in a DAG-like manner, whereas at 1 microM other subtypes were activated as well. Preferential activation of PKC-alpha was also noted for tinyatoxin and thapsigargin, but their mode of activation is unclear because these two compounds did not compete for the phorbol ester binding of the PKC subtypes as the other agents did. Of the three PKC inhibitors tested, staurosporine most efficiently inhibited kinase activity of the PKC subtypes, whereas K252a and CGP 41251 were at least 10 times less effective. However, K252a showed certain specificity for inhibition of PKC-alpha, and CGP 41251 failed to inhibit PKC-epsilon and PKC-zeta. Given the different substrate specificities and modes of activation by various tumor-promoting and nontumor-promoting agents, as well as the different sensitivities towards different inhibitors, our results indicate a divergence of individual PKC subtypes in signal transduction.

Animals↗

PubMed1997

Differential coupling of alpha1-adrenoreceptor subtypes to phospholipase C and mitogen activated protein kinase in neonatal rat cardiac myocytes.

Activation of cardiac alpha1-adrenoreceptors has a number of physiological effects. Ascribing these effects to a specific alpha1-adrenoreceptor subtype first requires the elucidation of the subtypes that are present in the tissue of interest. In the present study, mRNA transcripts for the alpha1A, alpha1B and alpha1D-adrenoreceptor subtypes were detected in cultured neonatal rat cardiac myocytes, using reverse transcriptase-polymerase chain reaction analysis. However, binding sites for only the alpha1A and alpha1B-adrenoreceptor subtypes were detected in cultured neonatal rat cardiac myocytes, using competition binding analysis with a variety of alpha1 selective receptor antagonists. Phenylephrine-stimulated phosphatidylinositol hydrolysis was inhibited by alpha1 selective receptor antagonists with affinities consistent with the alpha1A-adrenoreceptor subtype, whereas phenylephrine-induced activation of the mitogen activated protein kinase cascade was inhibited by these same antagonists with affinities more closely resembling the alpha1B-adrenoreceptor subtype. In the case of both signaling pathways, the alpha1D selective receptor antagonist, BMY 7378, exhibited affinities suggestive of the relative absence of a alpha1D-adrenoreceptor subtype. Thus, despite the presence of mRNA transcripts for all three alpha1-adrenoreceptor subtypes, only the alpha1A and alpha1B-adrenoreceptor subtypes were expressed and functionally coupled at detectable levels in neonatal rat cardiac myocytes. Of particular interest, phenylephrine-induced activation of the mitogen activated protein kinase cascade appears to be mediated by a subtype resembling most closely the pharmacological profile of the alpha1B-adrenoreceptor subtype.

Animals↗

PubMedDate not supplied

Functional properties of recombinant GABA(A) receptors composed of single or multiple beta subunit subtypes.

GABA(A) receptor (GABAR) isoforms in the central nervous system are composed of combinations of alpha(1-6), beta(1-4), gamma(1-4), delta(1) and epsilon(1) subunit subtypes arranged in a pentamer. Many regions of the brain express high levels of mRNA encoding several different subunits and even multiple subunit subtypes. The stoichiometry of GABAR isoforms is unclear, and the number and identity of individual subunit subtypes that are coassembled remain uncertain. To examine the role of beta subunit subtypes in the functional properties of GABARS and to determine whether multiple beta subtypes can be coassembled in functional GABARs, plasmids containing cDNAs encoding rat beta1 and/or beta3, alpha5 and gamma2L subtypes were cotransfected into L929 fibroblasts. The properties of the expressed receptor populations were determined using whole-cell and single-channel recording techniques. The alpha5beta1gamma2L isoform was less sensitive to GABA than the alpha5beta3gamma2L isoform. alpha5beta1gamma2L isoform currents were also insensitive to the allosteric modulator loreclezole, while alpha5beta3gamma2L isoform currents were strongly potentiated by loreclezole. Fibroblasts transfected with plasmids containing cDNAs for both beta1 and beta3 subtypes along with alpha5 and gamma2L subtypes produced a receptor population with an intermediate sensitivity to GABA which was insensitive to loreclezole. These results suggest that functional GABARs can be formed that contain two different beta1 subunit subtypes with properties different from receptors that contain only a single beta1 subtype and that the beta1 subunit subtypes influence the response of GABARs to GABA and to the allosteric modulator loreclezole.

Animals↗

PubMed2003

Assessment of HIV-1 subtyping for Cameroon strains using phylogenetic analysis of pol gene sequences.

Phylogenetic analysis of human immunodeficiency virus type 1 (HIV-1) pol gene is a useful method for subtyping European strains of HIV-1. The suitability of this method for genetically diverse African strains was evaluated by comparing HIV-1 subtyping of Cameroon strains using a long fragment of the pol gene sequence to the findings obtained using env gene sequences. When the pol gene could not be amplified, the reverse transcriptase (RT) or the protease (PR) genes were used. Phylogenetic analysis of the env C2/V3 gene sequences of 60 HIV-1 isolates showed 52 to be subtype A, 2 subtype G, plus one each of subtypes C, F2 and H, with 3 subtypes not determined. A long fragment of the pol gene was amplified successfully and sequenced in 23% of cases. The RT region was amplified for 42% of the samples that could not be typed by analysing the long fragment, and the PR gene was amplified for 40% of them. Thus, 63% of samples were typable. Env and pol gene subtypings were in agreement in 86% of cases. It is concluded that the phylogenetic analysis of pol gene sequences is not a practical method for HIV-1 subtyping in areas of high subtype diversity, despite the good agreement between the env and pol gene subtypings. However, it can be a useful method for HIV-1 subtyping, provided that the gene is amplifiable.

Cameroon↗