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PubMed2002

Forty-one near full-length HIV-1 sequences from Kenya reveal an epidemic of subtype A and A-containing recombinants.

OBJECTIVE: To further define the genetic diversity of HIV-1 in Kenya using approaches that clearly distinguish subtypes from inter-subtype recombinants. DESIGN: Near full genome sequencing and analysis were used, including sensitive new tools for detection and mapping of recombinants. METHODS: Purified peripheral blood mononuclear cell DNA from 41 HIV-1 positive blood donations collected from six hospitals across southern Kenya was used to amplify near full-length genomes by nested PCR. These were sequenced on an ABI 3100 automated sequencer and analyzed phylogenetically. RESULTS: Among 41 near full-length genomes, 25 were non-recombinant (61%) and 16 were recombinant (39%). Of the 25 pure subtypes, 23 were subtype A, one was subtype C and one was subtype D. Most recombinants consisted of subtype A and either subtype C or subtype D; a few contained A2, a recently identified sub-subtype. Two A2/D recombinants had identical breakpoints and may represent a circulating recombinant form. A third A2/D recombinant had the same structure as a previously described Korean isolate, and these may constitute a second A2-containing circulating recombinant form. CONCLUSIONS: In Kenya, 93% of HIV-1 genomes were subtype A or A-containing recombinant strains. Almost 40% of all strains were recombinant. Vaccine candidates tested in Kenya should be based on subtype A strains, but the methods used for evaluation of breakthrough infections during future vaccine trials should be capable of identifying non-A subtypes, the A2 sub-subtype, and recombinants.

Disease Outbreaks↗
PubMed2000

Distribution of HIV-1 subtypes among HIV-seropositive patients in the interior of Côte d'Ivoire.

Limited data exist on the distribution of HIV-1 subtypes in Côte d'Ivoire. The aim of this study is to describe the distribution of genetic subtypes of HIV-1 strains in six regions of Côte d'Ivoire. In 1997, we consecutively collected blood from 172 HIV-1-infected patients from six regional tuberculosis treatment centers. Peripheral blood mononuclear cells (PBMCs) from these people were analyzed by a restriction fragment-length polymorphism (RFLP) assay that involves a sequential endonuclease digestion of a 297-base pair polymerase chain reaction (PCR) fragment; plasma samples were tested by a V3-loop peptide enzyme immunoassay (PEIA). DNA sequencing of the protease or env genes was performed on all samples discordant in the two assays as well as a random sample of the concordant subtyped samples. Of 172 specimens, 3 were PCR-negative, and 169 were putatively classified as subtype A by RFLP. The 3 PCR-negative samples were unequivocally subtyped A by PEIA. Of the 169 RFLP subtype A samples, 159 (94%) were subtyped A by PEIA. Of the 10 discordant samples, PEIA testing classified 3 as subtype C, 2 as D, and 5 as F. Sequencing of the env gene classified these samples as 1 subtype A, 4 Ds, and 5 Gs. Thus, 163 (95%) of the specimens were subtype A, 3 subtype D, 4 subtype G, 1 A/D, and 1 A/G (IbNG) circulating recombinant forms (CRF). In conclusion, most HIV-1-infected tuberculosis patients throughout the interior of Côte d'Ivoire are infected with HIV-1 subtype A, which are very likely the A/G (IbNG) CRF. The uniform distribution of this subtype makes Côte d'Ivoire a potential site for vaccine trials.

AIDS-Related Opportunistic Infections↗
PubMed2004

Characterization of nevirapine resistance mutations in women with subtype A vs. D HIV-1 6-8 weeks after single-dose nevirapine (HIVNET 012).

OBJECTIVE: To compare the number and type of nevirapine (NVP) resistance mutations detected in Ugandan women with subtype A vs. D HIV-1 infection after single-dose NVP prophylaxis. DESIGN: In the HIVNET 012 trial, a higher rate of NVP resistance (NVPR) was seen in women with subtype D than A after single-dose NVP. In this study, the number and type of NVPR mutations detected 6-8 weeks after NVP were compared in women with subtypes A vs. D. METHODS: Plasma samples were available for 282 (92%) of 306 women who received NVP in HIVNET 012. Samples were analyzed with the ViroSeq HIV-1 Genotyping System (Applied Biosystems, Foster City, CA). Subtyping was performed by phylogenetic analysis of pol region sequences. RESULTS: Results were obtained for 279 women, including 147 with subtype A, 98 with subtype D, 6 with subtype C, and 28 with recombinant HIV-1. NVPR mutations were detected in 70 (25%) of 279 women. NVPR was more common in women with subtype D vs. A (35.7 vs. 19%, P = 0.0035). Complex patterns of NVPR mutations were detected in both subtypes. Among women with NVPR, 43% of women with subtype A and 46% of women with subtype D had >/=2 NVPR mutations. The mean number and pattern of NVPR mutations detected in women with subtypes A and D were similar. CONCLUSIONS: This study confirms a higher rate of NVPR in women with subtype D than A and further defines the pattern of NVPR mutations that emerge 6-8 weeks after single-dose NVP prophylaxis in these subtypes.

Acquired Immunodeficiency Syndrome↗
PubMed1986

Structural differences between subtype A and B strains of respiratory syncytial virus.

Differences in the properties of homologous intracellular structural components of eight strains of subtype A and eight strains of subtype B of human respiratory syncytial (RS) virus were examined. The size of the fusion (F) protein cleavage products and the phosphoprotein (P) showed systematic differences between virus strains representing the two subtypes. The apparent mol. wt. in SDS-polyacrylamide gels under reducing conditions was 48,000 (48K) and 46K to 47K for the cleavage product F1 in subtype A and B strains, respectively. The size of the F2 protein was 18K to 20K. The subtype B strains showed a slightly higher mol. wt. of this protein compared to the subtype A strains. The size of the P protein was 36K in subtype A strains, but only 34K in subtype B strains. Variations also occurred in the size of the glycoprotein (G) and the 22K to 24K structural protein. These variations did not correlate with the virus subtypes, but were strain-specific. The size of non-glycosylated forms of the F protein cleavage products was determined by use of material from tunicamycin-treated cells. A 44K to 45K non-glycosylated form of the F1 protein was detected with subtype A virus strains, but the corresponding protein of subtype B strains was not reproducibly identified, presumably due to instability in the absence of glycosylation or altered antigenicity. Monoclonal antibody immunosorbent-bound viral glycoproteins were partially digested with proteases. The pattern of breakdown products of the F1 protein was distinctly different between subtype A and B strains, but it was similar among strains of the same subtype. No subtype-specific pattern was seen in proteolytic digests of monoclonal antibody-bound G protein.

Electrophoresis, Polyacrylamide Gel↗
PubMed2005

Subtyping of Clostridium difficile PCR ribotype 001 by REP-PCR and PFGE.

The REP-PCR (repetitive sequence-based PCR using repetitive extragenic palindromic primers) typing method and a modified PFGE method were applied to isolates of Clostridium difficile PCR ribotype 001 with the aim of comparing their performance as methods of subtyping this organism. Of 200 isolates from 60 hospitals tested by REP-PCR, eight subtypes were identified and labelled as REP-PCR subtypes 001-008. The predominant subtype, REP-PCR subtype 003, accounted for 47% of the total. Fifty-two of the 200 isolates were analysed by a modified PFGE method and seven subtypes were identified, labelled as PF-A-PF-G. There was excellent correlation between REP-PCR subtypes and PFGE subtypes with both methods displaying broadly similar discriminatory powers. However, REP-PCR subtyping proved to be a much easier, cheaper and more rapid method suitable for application for routine subtyping of C. difficile ribotype 001. Application of REP-PCR subtyping to UK isolates of C. difficile PCR ribotype 001 from 60 different centres revealed a wide distribution of REP-PCR subtype 003 throughout England and Wales, with a regional clustering of REP-PCR subtype 001 around Northwest England and North Wales. Analysis of isolates from a single hospital over a 4-year period revealed a change in predominant subtype over time.

Clostridioides difficile↗
PubMed2025

Alzheimer's subtypes A supervised, unsupervised, multimodal, multilayered embedded recursive (SUMMER) AI study.

Since Alzheimer's disease (AD) is a heterogeneous disease, different subtypes may have distinct biological, genetic, and clinical characteristics, requiring tailored interventions. While several proposed subtypes of AD exist, there is still no clear consensus on a definitive classification. By leveraging complementary AI approaches, including supervised and unsupervised learning, within a recursive pipeline (SUMMER) that integrates multimodal datasets encompassing MRI measurements, phenotypes, and genetic data, our goal was to generate robust scientific evidence for identifying AD subtypes. Data was downloaded from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database and included neuroimaging data (MRI), genetics (SNPs), clinical diagnosis, and demographics. 1133 European American participants' images, aged 55-95, were included in this study. The analysis was multi-fold, where the first step involved applying an unsupervised application to a subset of the MRI sample (AD + cognitively normal (CN) aged matched groups, 100 men aged 68-85 years, and 76 women aged 68-85 years). The MRI brain gray matter was segmented into 44 regions of interest (ROIs) according to a standard atlas, and 618 features were extracted, including ROI voxel intensity measurements such as minimum, maximum, and histogram variables. Results identified a cluster of subtype AD men and a cluster of subtype AD women that were distinct from the rest of their respective samples. In the next step, the integrity of the identified subtype AD clusters was investigated using the XGBoost supervised machine learning application with genetic features (SNPs, N=36,724) and labels: the identified subtype AD cluster vs. the rest of the sample, stratified by sex. A significant AD subtype men model (accuracy=0.85, F1=0.72, AUC=0.83) and a significant women AD subtype model (accuracy=0.81, F1=0.81, AUC=0.81) were built, confirming the homogeneity of the isolated AD subtype clusters. Discriminative biomarkers were extracted from the significant models, including selected ROIs and SNPs. Finally, the subtype models were tested on an unseen subset of ADNI data. The genetic-based models identified clusters of AD subtype participants consisting of 34% of the men AD group and 47% of the women AD group. Phenotypic analysis indicates that lower body weight was associated with the women's AD subtype. Complex diseases like AD demand a sophisticated, multimodal approach for precise diagnosis. Effectively identifying disease subtypes enhances the potential for personalized treatment, ultimately improving patient outcomes.

Journal Article↗
PubMed2006

Somatostatin receptor subtypes mediate contractility on human colonic smooth muscle cells.

This study demonstrates the expression of functional somatostatin receptor (sstr) subtypes in human circular and longitudinal colonic smooth muscle cells (SMC). Native somatostatin (SS) and sstr subtype-specific analogues were used to characterize the sstr subtypes present in both cell types by contraction/relaxation studies. Qualitative and quantitative mRNA analysis and immunohistochemistry of sstr subtypes were also carried out. sstr subtype 2 mRNA was expressed in circular SMC, and various levels of subtypes 1, 2 and 3 mRNA were expressed in longitudinal colonic SMC. Native SS and each subtype-specific analogue exerted a modest, but significant, contraction, although inhibition of carbachol-induced contraction (relaxation) was the main effect on SMC from both layers. CH-288, a sstr subtype 1-specific analogue, and octreotide, a sstr subtype 2-specific analogue, were the most effective relaxant analogues on longitudinal and circular SMC, respectively. sstr subtypes display a distinct expression pattern on human colonic SMC; on circular SMC, subtype 2 is the only sstr, whereas sstr subtypes 1, 2 and 3 are expressed on human SMC isolated from the longitudinal layer. The contractile effects of SS are mediated through sstr subtype 2 and sstr subtype 1 on circular and longitudinal human colonic SMC, respectively.

Cells, Cultured↗
PubMed1999

Epidemiology of constipation (EPOC) study in the United States: relation of clinical subtypes to sociodemographic features.

OBJECTIVE: Constipation is a common heterogeneous condition, possibly encompassing different clinical subtypes. Little is known about the comparative epidemiology of constipation subtypes. This study was conducted to estimate the prevalence of constipation subtypes and determine whether subtypes differ by sociodemographic factors. METHODS: Between June and September 1997, a telephone interview was conducted with individuals about their bowel habits in the preceding 3 months. Survey data on 15 constipation-related symptoms were used to identify individuals who met prespecified symptom criteria for the following mutually exclusive subgroups: functional constipation, irritable bowel syndrome (IBS), outlet obstruction or delay (outlet), both IBS and outlet (IBS-outlet), and frequent laxative users (i.e., at least every other day). A total of 10,018 eligible individuals in the United States 18 yr of age or older completed the interview. Test-retest reliability of reporting symptoms was assessed in a separate national survey. The Spearman's correlation coefficient for reporting symptoms ranged from 0.54 to 0.83; all but three symptoms had correlations above 0.68. RESULTS: The overall prevalence of constipation was 14.7%. By subtype, prevalence was 4.6% for functional, 2.1% for IBS, 4.6% for outlet, and 3.4% for IBS-outlet. An additional 1.8% of respondents reported laxative use at least every other day. Outlet was the most common subtype among women, whereas functional constipation was the most common subtype among men. The gender ratio varied by subtype, with elevated ratios for outlet (F/M = 1.65) and IBS-outlet (F/M = 2.27) subtypes. The age pattern differed among each of the four subtypes. Prevalence of functional subtype decreased with increasing age. In contrast, outlet subtype did not seem to vary by age, and IBS (both men and women) and IBS-outlet (women only) subtypes increased to age 35 yr and declined thereafter. Prevalence of functional constipation increased with increasing education. Outlet type was more common in nonwhites compared to whites. Finally, 45% of individuals with constipation reported having the condition for 5 yr or more. CONCLUSIONS: Constipation is a heterogeneous condition. Differences in epidemiological profile by age, sex ratio, and relation to other sociodemographical factors support the distinction of two and possibly more symptom-based subtypes.

Adolescent↗
PubMed2003

Comparing the ex vivo fitness of CCR5-tropic human immunodeficiency virus type 1 isolates of subtypes B and C.

Continual human immunodeficiency virus type 1 (HIV-1) evolution and expansion within the human population have led to unequal distribution of HIV-1 group M subtypes. In particular, recent outgrowth of subtype C in southern Africa, India, and China has fueled speculation that subtype C isolates may be more fit in vivo. In this study, nine subtype B and six subtype C HIV-1 isolates were added to peripheral blood mononuclear cell cultures for a complete pairwise competition experiment. All subtype C HIV-1 isolates were less fit than subtype B isolates (P < 0.0001), but intrasubtype variations in HIV-1 fitness were not significant. Increased fitness of subtype B over subtype C was also observed in primary CD4(+) T cells and macrophages from different human donors but not in skin-derived human Langerhans cells. Detailed analysis of the retroviral life cycle during several B and C virus competitions indicated that the efficiency of host cell entry may have a significant impact on relative fitness. Furthermore, phyletic analyses of fitness differences suggested that, for a recombined subtype B/C HIV-1 isolate, higher fitness mapped to the subtype B env gene rather than the subtype C gag and pol genes. These results suggest that subtype B and C HIV-1 may be transmitted with equal efficiency (Langerhans cell data) but that subtype C isolates may be less fit following initial infection (T-cell and macrophage data) and may lead to slower disease progression.

Cells, Cultured↗
PubMed2006

Distinct molecular mechanisms underlying clinically relevant subtypes of breast cancer: gene expression analyses across three different platforms.

BACKGROUND: Gene expression profiling has been used to define molecular phenotypes of complex diseases such as breast cancer. The luminal A and basal-like subtypes have been repeatedly identified and validated as the two main subtypes out of a total of five molecular subtypes of breast cancer. These two are associated with distinctly different gene expression patterns and more importantly, a significant difference in clinical outcome. To further validate and more thoroughly characterize these two subtypes at the molecular level in tumors at an early stage, we report a gene expression profiling study using three different DNA microarray platforms. RESULTS: Expression data from 20 tumor biopsies of early stage breast carcinomas were generated on three different DNA microarray platforms; Applied Biosystems Human Genome Survey Microarrays, Stanford cDNA Microarrays and Agilent's Whole Human Genome Oligo Microarrays, and the resulting gene expression patterns were analyzed. Both unsupervised and supervised analyses identified the different clinically relevant subtypes of breast tumours, and the results were consistent across all three platforms. Gene classification and biological pathway analyses of the genes differentially expressed between the two main subtypes revealed different molecular mechanisms descriptive of the two expression-based subtypes: Signature genes of the luminal A subtype were over-represented by genes involved in fatty acid metabolism and steroid hormone-mediated signaling pathways, in particular estrogen receptor signaling, while signature genes of the basal-like subtype were over-represented by genes involved in cell proliferation and differentiation, p21-mediated pathway, and G1-S checkpoint of cell cycle-signaling pathways. A minimal set of 54 genes that best discriminated the two subtypes was identified using the combined data sets generated from the three different array platforms. These predictor genes were further verified by TaqMan Gene Expression assays. CONCLUSION: We have identified and validated the two main previously defined clinically relevant subtypes, luminal A and basal-like, in a small set of early stage breast carcinomas. Signature genes characterizing these two subtypes revealed that distinct molecular mechanisms might have been pre-programmed at an early stage in different subtypes of the disease. Our results provide further evidence that these breast tumor subtypes represent biologically distinct disease entities and may require different therapeutic strategies. Finally, validated by multiple gene expression platforms, including quantitative PCR, the set of 54 predictor genes identified in this study may define potential prognostic molecular markers for breast cancer.

Biomarkers, Tumor↗
PubMed1995

Accuracy of initial stroke subtype diagnosis in the TOAST study. Trial of ORG 10172 in Acute Stroke Treatment.

Rapid identification of stroke subtype is valuable for both practicing clinicians and the optimal design of clinical stroke trials. Mechanisms of ischemic injury might differ among different stroke subtypes. Certain subtypes might be clinically identified as suboptimal for future therapeutic or prophylactic stroke trials. Some subtypes might be so clinically distinct that extensive laboratory investigation is unwarranted. Investigators in the ongoing Trial of ORG 10172 in Acute Stroke Treatment are using criteria to categorize stroke etiology among enrolled patients into one of five subtypes: large-artery atherothromboembolic, cardioembolic, small-vessel thrombotic, other etiology, or undetermined etiology. As part of the study, physicians initially predict the most likely subtype of stroke based on clinical features and baseline CT. Three months after stroke, investigators use the criteria, which also incorporate results of diagnostic testing, to reclassify stroke subtype. Initial clinical impression of subtype agreed with final determination in 62% of patients, and the rate was similar for all stroke subtypes. No stroke subtype was more accurately diagnosed than others by initial assessment. No subtype was more commonly identified by diagnostic studies. Fifteen percent of patients remained without a clear etiologic subtype diagnosis at 3 months. We conclude that clinical trials in stroke should not attempt to restrict entry into trials based on presumed stroke subtype. A careful evaluation for etiology is justified in all patients presenting with stroke, regardless of presumed subtype.

Cerebrovascular Disorders↗
PubMed2005

Impact of HIV-1 subtype and antiretroviral therapy on protease and reverse transcriptase genotype: results of a global collaboration.

BACKGROUND: The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate. METHODS AND FINDINGS: To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates. CONCLUSION: Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations.

Amino Acid Sequence↗
PubMed2000

Changes in the epidemiology of hepatitis C infection in Germany: shift in the predominance of hepatitis C subtypes.

Hepatitis C virus (HCV) subtype distribution was studied in 395 chronically infected patients from Germany. HCV genotype 1 was most frequent (80.5%). One hundred forty-three individuals (36.2%) were infected with subtype 1a and 175 (44.3%) were suffering from subtype 1b infection, respectively. HCV subtype 3a was found in 53 (13.42%) persons. Subtypes 2a, 2b, and 2c have been detected in 5 (1.27%), 10 (2.53%), and 4 (1.01%) individuals. Genotypes 4 and 5a accounted for HCV infections in 4 (1.01%) and 1 (0.25%) subjects. There was a notable variation in the distribution of the prevalent subtypes 1a and 1b in different age groups. Subtype 1a was detected in 53.3% and 68.0% of patients aged 1-10 and 11-20 years, whereas subtype 1b in the same groups was present only in 33.3% and 28.0% of patients, respectively. In contrast, in individuals older than 50 years subtype 1b was most frequent. Thus, subtype 1b has been gradually substituted for subtype 1a during the last 20 years. Logistic regression analysis with adjustment for sex and different modes of HCV acquisition demonstrated that age of the infected subjects was a direct explanatory variable for subtype 1a and 1b distribution. Therefore, the observed shift in HCV subtype prevalence could not be attributed to changes in the epidemiological relevance of different known risk factors of HCV transmission, as had been assumed in previous studies. The altered subtype pattern reported here may have a profound influence on the future epidemiology of HCV infection.

Adolescent↗
PubMed2005

Impact of human immunodeficiency virus type 1 subtype on first-line antiretroviral therapy effectiveness.

OBJECTIVE: The effectiveness of antiretroviral treatment (ART) was compared in 416 naive patients from a French clinical cohort infected with B and non-B HIV-1 subtypes. METHODS: Time to HIV viral load (VL) undetectability was calculated for each subtype group. Three other parameters were estimated 3, 6 and 12 months after enrolment: clinical progression (that is, AIDS-defining events or death), changes in CD4 cell counts from baseline and proportion of patients achieving an undetectable VL (<400 HIV-RNA copies/ml). RESULTS: In this cohort, 317 patients (76%) were infected with a B subtype and 99 (24%) with a non-B subtype. Median time to VL undetectability was similar in the B subtype group [147 days, 95% confidence interval (CI) 119-165] and non-B subtype group (168 days, 95% CI: 105-234; P=0.16). After adjusting for AIDS-defining events at enrolment, baseline CD4 cell counts and VL, and for the treatment on which patients were initiated, no association was found between HIV subtypes and time to VL undetectability (B subtype vs non-B subtype: hazard ratio=0.80, 95% CI: 0.62-1.02, P=0.07). In the 3, 6 and 12 months after enrolment, subtype had no impact on clinical progression, CD4 cell count or VL responses to ART. This suggests that B and non-B subtypes do not affect first-line therapy efficacy, which is encouraging in view of the worldwide spread of non-B HIV-1 subtypes and the increasing availability of ART in developing countries. However, in this study we did not take into account individual non-B subtype species, therefore further studies should be designed to evaluate the efficacy of these regimens in patients with particular non-B subtypes.

Anti-HIV Agents↗
PubMed1991

Subtype-specific antibodies for muscarinic cholinergic receptors. I. Characterization using transfected cells and avian and mammalian cardiac membranes.

Polyclonal antibodies to a number of synthetic peptides corresponding to sequences of human muscarinic acetylcholine receptor (mAChR) subtypes m1, m2, and m4 have been prepared, in order to obtain specific tools with which to study the properties of the different receptors. Hydrophilic peptides from each subtype were chosen from the large intracellular loop between the fifth and the sixth transmembrane domains, because these loops are distinct in each of the mAChR subtypes and contain determinants for coupling to different GTP-binding proteins, as well as potential sites for phosphorylation. Five different antibodies were prepared and tested for their reactivity and specificity toward different mAChR subtypes by immunoprecipitation of ligand-binding activity and receptor protein and by immunoblot analysis. Each of the antisera immunoprecipitated its respective mAChR subtype, as evidenced by precipitation of 50-70% of the ligand-binding activity from stably transfected cells expressing the respective mAChR subtype. Very little cross-reactivity toward other subtypes was observed. We used these subtype-specific antibodies to probe the nature of the mAChR subtypes naturally expressed in avian and mammalian cardiac membranes. In tests using the antibodies with cardiac mAChRs, differences in the avian and mammalian mAChR subtypes were detected. Only the anti-m2 antibodies reacted with mAChR from porcine heart, confirming previous pharmacological and molecular biological studies that suggested that this tissue expressed only the m2 mAChR subtype. In contrast, both the anti-m2 and anti-m4 mAChR antibodies immunoprecipitated approximately 50% of the mAChRs solubilized from chick heart membranes. The combined or successive use of anti-m2 and anti-m4 antisera in immunoprecipitation and/or immunoblotting studies with chick heart mAChRs suggested that both antibodies appeared to recognize the same population of mAChRs from that tissue. Taken together, the results show that the subtype-specific antibodies developed in this work are useful in recognizing mAChR subtypes in cells and tissues. These antibodies should be valuable tools for further study of the properties and regulation of mAChR subtypes in various preparations. Evidence that the antibodies can modify mAChR/GTP-binding protein interactions is presented in the accompanying paper.

Amino Acid Sequence↗
PubMed1994

Selective labeling and localization of the M4 (m4) muscarinic receptor subtype.

We report here a novel strategy for the selective labeling and localization of the M4 (m4) muscarinic receptor subtype, based on the distinct kinetics of the muscarinic antagonists dexetimide and N-methylscopolamine (NMS) and on the selectivity profile of guanylpirenzepine and AF-DX 116 for the m1-m5 muscarinic receptor subtypes expressed in CHO-K1 cells. Incubation with 10 nM dexetimide, a nonselective antagonist, resulted in > 90% occupancy of each of the m1-m5 receptor subtypes. The relatively rapid rates of dexetimide dissociation from the m1, m2, and m4 receptor subtypes (t1/2 values of < 12.5 min) and the slower rates of dexetimide dissociation from the m3 and m5 receptor subtypes (t1/2 values of 65 and 75 min, respectively) favored the labeling of the m1, m2, and m4 receptor subtypes with short incubations with [3H]NMS. Inclusion of 200 nM guanylpirenzepine and 250 nM AF-DX 116 prevented the binding of [3H]NMS to the majority of the m1 and m2 receptor subtypes, respectively, resulting in primary labeling of the m4 receptor subtype. Brief dissociation of the radioligand in the presence of 1 microM atropine improved the ratio of m4 to m2 labeling by selectively removing [3H]NMS from the m2 subtype. Under these conditions, the ratio of [3H]NMS binding to the m4 versus m1, m2, m3, and m5 receptor subtypes was 4:1. In vitro autoradiography combined with these m4-selective labeling conditions demonstrated that the M4 (m4) receptor subtype was localized to the primary visual area (V1, area 17, occipital cortex) and the basal ganglia, a distribution distinct from that demonstrated for the M1 (m1), M2 (m2), and M3 (m3) receptor subtypes. These results demonstrate that a combination of the distinct kinetics of dexetimide and NMS and the receptor subtype selectivity of guanylpirenzepine and AF-DX 116 provides a valuable labeling strategy to examine the distribution and localization of the M4 (m4) muscarinic receptor subtype in brain, peripheral tissues, and cell lines.

Animals↗
PubMed1997

Molecular subtyping of human T-lymphotropic virus type I (HTLV-I) by a nested polymerase chain reaction-restriction fragment length polymorphism analysis of the envelope gene: two distinct lineages of HTLV-I in Taiwan.

The major type of human T-lymphotropic virus type I (HTLV-I), generally referred to as the cosmopolitan type, has been grouped into three subtypes (A, B, and C) by phylogenetic analysis of the long terminal repeat sequences of the viral genome. Twelve subtype-specific nucleotide variations have been deduced by comparison between the envelope (env) sequences of 16 HTLV-I strains with defined subtypes and 9 Taiwanese HTLV-I strains. To gain further insights into the molecular epidemiology of HTLV-I and the origin of this virus in Taiwan, a rapid method of identification for the cosmopolitan subtypes was developed by using a nested polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) studies using the two subtype B-specific and four subtype C-specific nucleotides located within the positions 5708 to 6320 of the envgene. The nested PCR-RFLP method was used to subtype HTLV-I from four virus-positive cell lines derived from 1 Japanese and 3 North American patients, as well as 41 blood-unrelated Taiwan Chinese. The sequences of PCR products were determined and the six positions of subtype-specific nucleotide variations were examined. The sequence data completely supported the subtyping data via the nested PCR-RFLP method. The results demonstrated that, as is the case in Japan, at least two distinct cosmopolitan subtypes (A and B) of HTLV-I were present in Taiwan, but the more prevalent subtype in Taiwan is A in contrast to subtype B in Japan. Furthermore, rapid subtyping could facilitate molecular epidemiological studies of HTLV-I infection and linkage between HTLV-I subtypes and virus-associated diseases.

Base Sequence↗
PubMed2005

Instability of the DSM-IV Subtypes of ADHD from preschool through elementary school.

CONTEXT: The DSM-IV definition of attention-deficit/hyperactivity disorder (ADHD) distinguished 3 subtypes that had not been extensively studied. OBJECTIVE: To determine whether the ADHD subtypes are stable enough over time to be valid. DESIGN: Longitudinal study with a greater-than 89% retention rate in 7 assessments over 8 years. SETTING: Outpatient clinics. PARTICIPANTS: Volunteer sample of 118 4- to 6-year-olds who met DSM-IV criteria for ADHD, including impairment in 2 settings in at least 1 assessment. MAIN OUTCOME MEASURE: Meeting DSM-IV criteria for the subtypes of ADHD during years 2 through 8. RESULTS: The number of children who met criteria for ADHD declined over time, but most persisted. Children who met criteria for the combined subtype (CT, n = 83) met criteria for ADHD in more subsequent assessments than children in the predominantly hyperactive-impulsive subtype (HT, n = 23). Thirty-one (37%) of 83 CT children and 6 (50%) of 12 children in the predominantly inattentive subtype (IT) met criteria for a different subtype at least twice in the next 6 assessments. Children of the HT subtype were even more likely to shift to a different subtype over time, with HT children who persisted in ADHD mostly shifting to CT in later assessments. The subtypes exhibited consistently different mean levels of hyperactive-impulsive symptoms during years 2 through 8 that corresponded with their initial subtype classifications, but initial subtype differences in inattention symptoms diminished in later years. CONCLUSIONS: In younger children, the CT and IT may be stable enough to segregate groups for research, but they seem too unstable for use in the clinical assessment of individual children. Children rarely remain in the HT classification over time; rather, they sometimes desist from ADHD but mostly shift to CT in later years. Using continuous ratings of hyperactivity-impulsivity symptoms as a diagnostic qualifier should be considered as an alternative to classifying nominal subtypes of ADHD in DSM-V.

Attention Deficit Disorder with Hyperactivity↗