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Reactions of Ugandan antisera with peptides encoded by V3 loop epitopes of human immunodeficiency virus type 1.

The specificities of antibodies reacting with peptides encoded by V3 loop apical epitopes were determined for sera from 230 seropositive Ugandans, including asymptomatic persons and AIDS patients, sampled between 1986 and 1992. Most (71%) of the sera reacted with the peptide encoded by HIV-MN, 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the HIV-1 global subtype A (referred to as the Uganda A consensus), 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the global subtype D (the Uganda D consensus); 19% of the sera also reacted with peptides encoded by the divergent Ugandan variant U31. There was no obvious correlation between the specificities of antibody binding and the V3 loop sequence of the corresponding virus isolate or provirus. Competitive inhibition and antibody adsorption experiments indicated that the MN peptide, the Uganda A consensus peptide, the Uganda D consensus peptide, and the U31 peptide were recognized by different sets of antibodies. Eighteen percent of the sera from AIDS patients and 26% of the sera from asymptomatic persons were monospecific for one of the MN, Uganda A, or Uganda D peptides. Whereas all except one of the singly reactive AIDS sera were specific for MN, 39% of the singly reactive asymptomatic sera were specific for MN, 39% for the Uganda A peptide, and 21% for the Uganda D peptides. We conclude that analysis of the specificities of antibodies against the V3 loop epitopes in sera from asymptomatic persons could provide useful epidemiological data about the prevalence of viral subtypes within a population.

Adult↗

Molecular epidemiological typing of Enterobacter cloacae isolates from a neonatal intensive care unit: three-year prospective study.

Since 1992, there has been an increase in the incidence of Enterobacter sepsis in the neonatal intensive care unit (NICU) of the authors' hospital. From 1995 to 1997, a prospective molecular epidemiological survey of the colonizing and infecting strains isolated from neonates was conducted. Enterobacter cloacae was the most frequent cause of neonatal sepsis, accounting for 19.2% of all neonatal infections, reaching a peak incidence of 2.2/1000 during 1996. Fifty isolates from the NICU and four epidemiologically unrelated strains were characterized by pulse-field gel electrophoresis (PFGE), ribotyping, enterobacterial repetitive intergenic consensus (ERIC)-PCR and plasmid profiling. PFGE was the most discriminatory technique and identified 13 types (two of them classified into two and three subtypes) compared with ERIC-PCR, plasmid profiling and ribotyping that identified 11, 11 and seven types, respectively. A good correlation was found between all techniques. Five different clones caused 15 cases of sepsis. Clones A and B were prevalent in 1995 and 1996, but they were not isolated in 1997. An outbreak caused by clone G in 1997 was controlled by cohort nursing and hygienic measures, without changing the antibiotic policy. Strains were characterized by their antibiotic resistance pattern and divided into three groups. Group I correlated with PFGE types A, B1 and B2, which hyperproduced Bush type 1 chromosomal beta-lactamase and expressed extended-spectrum ?-lactamases (ESBLs). Group II only hyperproduced Bush type 1 chromosomal beta-lactamase and correlated with PFGE-types D1, D2, D3 and I. Finally, Group III, with inducible beta-lactamases, correlated with the rest of PFGE types. The sudden disappearance of E. cloacae after reinforcement of hygienic measures confirms the importance of patient-to-patient transmission.

Bacterial Typing Techniques↗

Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions.

There are two estrogen receptor (ER) subtypes in fish, ERalpha and ERbeta, and increasing evidence that the ERbeta subtype has more than one form. However, there is little information on the characteristics and functional significance of these ERs in adults and during development. Here, we report the cloning and characterization of three functional ER forms, zfERalpha, zfERbeta1, and zfERbeta2, in the zebrafish. The percentages of identity between these receptors suggest the existence of three distinct genes. Each cDNA encoded a protein that specifically bound estradiol with a dissociation constant ranging from 0.4 nM (zfERbeta2) to 0.75 nM (zfERalpha and zfERbeta1). In transiently transfected cells, all three forms were able to induce, in a dose-dependent manner, the expression of a reporter gene driven by a consensus estrogen responsive element; zfERbeta2 was slightly more sensitive than zfERalpha and zfERbeta1. Tissue distribution pattern, analyzed by reverse transcription polymerase chain reaction, showed that the three zfER mRNAs largely overlap and are predominantly expressed in brain, pituitary, liver, and gonads. In situ hybridization was performed to study in more detail the distribution of the three zfER mRNAs in the brain of adult females. The zfER mRNAs exhibit distinct but partially overlapping patterns of expression in two neuroendocrine regions, the preoptic area and the mediobasal hypothalamus. The characterization of these zfERs provides a new perspective for understanding the mechanisms underlying estradiol actions in a vertebrate species commonly used for developmental studies.

Amino Acid Sequence↗

Therapeutic significance of peroxisome proliferator-activated receptor modulators in diabetes.

Peroxisome proliferator-activated receptors (PPARs) are a group of three nuclear receptor isoforms, identified and encoded by different genes: PPARalpha, PPARdelta and PPARgamma. Each subtype of PPAR appears to be differently expressed in a tissue-specific manner due to its binding to a specific consensus DNA sequence of peroxisome proliferator response elements (PPREs). PPARalpha plays a significant role in the regulation of nutrient metabolism, including fatty acid oxidation, gluconeogenesis and amino acid metabolism. PPARdelta is expressed ubiquitously and has been found to be effective in controlling dyslipidemia and cardiovascular diseases, while PPARgamma isotype is mainly expressed in adipose tissue where it stimulates adipogenesis and lipogenesis. Thus PPARs have emerged as potential molecular targets for the design and synthesis of a different class of compounds, considering the conformation of receptors for the treatment of human metabolic disorders. This review concerns the therapeutic importance of PPARs in diabetes drug development.

Animals↗

Molecular epidemiology of HIV type 1 infection in Portugal: high prevalence of non-B subtypes.

In this study, we have investigated the diversity of current HIV-1 strains circulating in the metropolitan area of Lisbon, Portugal. A total of 217 HIV-1-positive blood samples, collected between October 1998 and December 2000, was genetically characterized in the gp120 C2V3C3 region (n = 205) or part of the gp41 N-terminal segment (n = 12) by heteroduplex mobility assay (HMA) and/or DNA sequencing. The HMA subtyping efficiency (number of samples unambiguously subtyped by HMA divided by the total number of samples subtyped) was 65.9% (143 of 217), with indeterminate migration patterns of subtype A and G strains contributing significantly to this value. On the overall, subtype B was the most prevalent (50.2%), followed by subtypes G (21.7%), A (17.5%), and F (5.5%), whereas subtypes C, D, H, and J accounted altogether for 5.1% of the infections. Non-B subtypes were responsible for 77.4 and 33.1% of the infections among African immigrants and Portuguese subjects, respectively. Angolan individuals (n = 25) were the only ones infected with all the HIV-1 subtypes documented, probably reflecting a high degree of viral genetic diversification in their country of origin. Phylogenetic analysis showed a predominance of IbNG-like viruses among subtype A sequences and two new major subclusters within subtype G (G(P) and G(P)'). The majority of the Portuguese G sequences described formed a well-defined subcluster (G(P)), supported by bootstrap values >90%, phylogenetically distant from clade G sequences in databases. gag (p24/p7) sequence analysis of these variants confirmed the maintenance of the subtype G subclusters. The multiple subclustering observed for the major clades A, B, D, and G, as well as the variety of subtypes found, indicate a high diversity of HIV-1 variants circulating in Portugal and suggest a need for continuous epidemiologic surveillance.

Adolescent↗

Molecular characterization of the hemagglutinin gene and oral immunization with a waterfowl-origin avian influenza virus.

Vaccination against highly pathogenic (HP) subtypes of avian influenza (AI) virus in poultry has been prohibited in the United States. Recently, policy has been changed to potentially allow use of inactivated vaccines in emergency programs to control HP H5 and H7 AI. Vaccination with inactivated virus against non-highly pathogenic AI viruses has been allowed in the U.S. turkey industry since 1979 (1) but requires expensive handling of individual birds for parenteral inoculation. Oral immunization would provide a less expensive method to protect commercial poultry from AI. Prime candidates for oral vaccines are waterfowl-origin (WFO) isolates, which have a tropism for the alimentary tract. One WFO isolate, A/mallard/Ohio/556/1987 (H5N9) (MOh87), was characterized by determining the complete nucleotide sequence of its hemagglutinin (HA) gene. The HA protein of this isolate possessed a deduced amino acid sequence nearly identical to the consensus amino acid sequence for all published H5 genes, indicating that it has potential as a broadly effective vaccine. Experimental results demonstrated measurable serum antibody responses to orally delivered live and inactivated preparations of MOh87. Oral vaccination also protected chickens from diverse, lethal H5 AI virus challenge strains and blocked cloacal shedding of challenge virus.

Administration, Oral↗

A consensus structure for omega-conotoxins with different selectivities for voltage-sensitive calcium channel subtypes: comparison of MVIIA, SVIB and SNX-202.

The omega-conotoxins are a set of structurally related peptides that have a wide range of specificities for different subtypes of the voltage-sensitive calcium channel (VSCC). To understand their VSCC subtype differentiation we studied the structure of two naturally occurring omega-conotoxins, MVIIA (specific to N-type) and SVIB (specific to P/Q-type) and a synthetic hybrid, SNX-202, which has altered specificities to both VSCC subtypes. The secondary structures of the three peptides are almost identical, consisting of a triple-stranded beta-sheet and several turns. A comparison of NMR data emphasizes the structural similarities between the peptides and highlights some minor structural differences. In the three-dimensional structures of SVIB and MVIIA these are manifested as orientational differences between two key loops. The structural rigidity of MVIIA was also examined. H alpha shifts are similar in a range of solvents, indicating that there are no solvent-induced changes in structure. The omega-conotoxins form a consensus structure despite differences in sequence and VSCC subtype specificity. This indicates that the omega-conotoxin macrosites for the N/P/Q-subfamily of VSCCs are related, with specificity for receptor targets being conferred by the positions of functional side-chains on the surface of the peptides.

Amino Acid Sequence↗

Molecular cytogenetic analysis of adult testicular germ cell tumours and identification of regions of consensus copy number change.

A series of adult testicular germ cell tumours consisting of eight seminomas, 14 non-seminomas (including two cell lines) and two combined tumours was analysed by comparative genomic hybridization and, in some cases, by interphase fluorescence in situ hybridization. The gain of 12p was identified in all cases and additional material from chromosomes 7 and 8 was found in over 70% of cases, in keeping with previous analyses. Other consistent regions of gain included 1q24-q31 (50%), 2p16-pter (41%), 2q22-q32 (45%) and Xq11-q21 (50%). The loss of 1p32-p36 (36%), 9q31-qter (36%), 11q14-qter (50%), 16p (36%) and 18p (45%) and the loss of material from chromosomes 4 and 5 (50% and 36% respectively) were also found in all histological subtypes. The loss of 1p material was confirmed in four cases by interphase FISH analysis and shown, with one exception, not to involve the loss of the D1Z2 locus at 1p36.3, which is commonly deleted in paediatric germ cell tumours. An association between gain of 6q21-q24 with cases resistant to chemotherapy (P < 0.01) was observed. In addition, loss of chromosome 19 and 22 material and gain of 5q14-q23, 6q21-q24 and 13q were found at a significantly lower frequency in seminoma than non-seminoma. These regions may contain genes involved in the divergent development of seminoma and non-seminoma.

Adult↗

The dopamine D1D receptor. Cloning and characterization of three pharmacologically distinct D1-like receptors from Gallus domesticus.

Three genomic clones encoding dopamine D1-like receptors were isolated from the avian species Gallus domesticus. Two of these genes encode proteins of 451 and 488 amino acids, which, based on deduced amino acid sequence identity and homology of exhibited pharmacological profiles, appear to be species homologs of mammalian and vertebrate D1/D1A and D5/D1B receptors, respectively. The third genomic clone, termed D1D, encodes a protein of 445 amino acids displaying a deduced amino acid sequence identity within putative transmembrane domains of 75% to mammalian D1/D1A and 77% to D5/D1B receptors with overall sequence homologies of only 49% and 46%, respectively. Membranes from COS-7 cells transfected with D1D DNA bound [3H]SCH-23390 in a saturable manner with high affinity (approximately 300 pM) and with a pharmacological profile clearly indicative of a dopamine D1-like receptor. The D1D receptor exhibited affinities for 6,7-dihydroxy-2-aminotetralin and dopamine 10-fold higher than D1/D1A receptors, characteristic of the D5/D1B receptor subfamily. In contrast, the D1D receptor bound dopaminergic agents, such as SKF-38393, apomorphine, pergolide, and lisuride, with affinities 10-fold higher than other cloned mammalian or vertebrate D1A/D1B receptor subtypes, while both clozapine and haloperidol displayed considerably lower affinity for the D1D receptor. Based on the low overall amino acid sequence identity (54%) and unique pharmacological profile, the avian dopamine D1D receptor does not appear to be a species homolog of the recently cloned vertebrate D1C receptor (Sugamori, K.S., Demchyshyn, L. L., Chung, M., and Niznik, H. B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10536-10540). As with all cloned mammalian and vertebrate D1-like receptors, the D1D receptor stimulates adenylate cyclase activity in the presence of dopamine or SKF-82526. Northern blot analysis reveals the selective expression of both avian D1D and D1A receptor mRNAs only in brain with the D1B receptor more widely distributed and localized in tissues such as brain, kidney, and spleen. The isolation of four distinct vertebrate dopamine D1 receptor subtypes suggests the existence of additional mammalian D1 like receptor genes that may account for the observed pharmacological and biochemical multiplicity of dopamine D1-like receptor mediated events.

Amino Acid Sequence↗

Simple subtyping assay for human immunodeficiency virus type 1 subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC.

After the initial development of a human immunodeficiency virus type 1 (HIV-1) subtype-screening tool by nested multiplex PCR, we further improve it through the redesign of subtype-specific primers based on subtype signature pattern (SSP) analyses and optimization of the PCR conditions. Extracted RNA from plasma samples was used in reverse transcription and the cDNA products were added to the first round PCR, in which universal primers in the gag region were used to detect HIV-1 M group isolates. In the second round of PCR, three pairs of subtype-specific primers, detecting subtypes B, C, and CRF01-AE, were added in one tube. Subtype determination was based on the different size of PCR products on the agarose gel electrophoresis. An additional set of primers detecting only the prevalent recombinant strains CRF07-BC and CRF08-BC was used to discriminate CRF07- and CRF08-BC from pure subtype C. Testing for all kinds of HIV subtype reference strains indicated that this assay was applicable. A panel of 252 HIV-positive samples and 30 HIV-negative samples was further used to evaluate and validate this assay. Compared to the assay of sequence-based phylogenetic analysis, the newly developed assay has an adequate designated subtype sensitivity, 93.2% (69 of 74) for subtype B, 95.1% (117 of 123) for subtype C, 94.0% (47 of 50) for CRF01-AE, and 95.0% (115 of 121) for CRF07-BC and CRF08-BC. Most importantly, the intersubtype specificity of the assay was found to be 100%. The assay specificity was also found to be 100% when used to test 30 HIV-negative samples. The average reproducibility was 96.0% for subtype B, 96.7% for subtype C, and 95.0% for CRF01-AE. We have developed a simple, rapid, and low cost assay for screening subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC in China.

Acquired Immunodeficiency Syndrome↗

An HIV type 1 subtype A outbreak among injecting drug users in Kazakhstan.

Kazakhstan experienced the start of the HIV-1 outbreak among intravenous drug users (IDUs) in 1997. To characterize genetically HIV-1 strains circulating in this country, peripheral blood mononuclear cells (PBMCs) DNA samples (1999-2002) derived from HIV-infected IDUs and their sexual partners in Pavlodar (n = 19), Shymkent (n = 6), and Qaraghandy (n = 18) regions were analyzed by the gag/env heteroduplex mobility assay (HMA). The 366-bp proviral env gene fragments encoding the gp120 C2-V3 region obtained from 16 individuals were sequenced. The results of HMA revealed that all 43 HIV-1 strains studied belonged to gag/env subtype A. The nucleotide sequence analysis showed a marked genetic homogeneity with the mean genetic distance being 3.63 +/- 2.39 (range 0.00-12.13). The mean genetic distance between each sequence within the Kazakhstan set and the East-European IDU subtype A consensus was 2.94 +/- 1.92 (range 0.79-8.48). The data presented thus confirm the spreading of the same IDU subtype A virus in the former Soviet Union.

Adult↗

Purification of the human histone H4 gene-specific transcription factors H4TF-1 and H4TF-2.

Transcription of the cell-cycle-regulated human histone genes increases approximately fivefold during S phase. One step toward the elucidation of the biochemical mechanisms that govern cell-cycle-regulated expression of these genes is to purify and characterize the transcription factors that regulate these promoters. Here, we describe the purification of two previously identified factors, H4TF-1 and H4TF-2, which bind the human histone H4 promoter. Purification was achieved through a combination of ion-exchange and oligonucleotide affinity chromatography. On the basis of analysis of purified fractions by SDS-polyacrylamide gels and UV cross-linking, we believe that H4TF-1 is two polypeptides of 105 and 110 kD. This factor binds to a GC-rich DNA sequence required for maximal expression of the H4 gene but does not bind to any Sp1 consensus elements tested. H4TF-2 is a 65-kD protein that binds specifically to sequences within that highly conserved H4 subtype-specific consensus promoter element. Both highly purified factors activated transcription in vitro only from H4 promoters that contained their binding sequences, demonstrating that H4TF-1 and H4TF-2 are H4-specific transcription factors that potentiate expression of this gene.

Base Sequence↗

Circulation of subtype A and gagA/envB recombinant HIV type 1 strains among injecting drug users in St. Petersburg, Russia, correlates with geographical origin of infections.

Countries of the former Soviet Union are experiencing an emerging HIV-1 epidemic due to a rapid expansion of HIV-1 among injecting drug users (IDUs). To study the molecular epidemiology of HIV-1 among IDUs in St. Petersburg, Russia, virus sequences were obtained from 22 individuals. Phylogenetic analysis of the env and gag regions revealed circulation of two major HIV-1 populations, one belonging to HIV-1 subtype A, and another being a recombinant of subtype A and B viruses (gagA/envB). Both virus populations were highly homogeneous, with a mean pairwise genetic distance of <2%, and similar to viruses obtained earlier from IDUs in other regions of the former Soviet Union. Distribution of the two major HIV-1 genotypes in St. Petersburg correlated with geographical origin of infections. In one individual, a virus type previously unseen among IDUs was found, which demonstrates the possibility that new viruses are entering this risk group.

Adult↗

Genetic subtypes of HIV type 1 in Hungary.

We examined the diversity of HIV-1 subtypes in 11 adults from Hungary, using the heteroduplex mobility assay (HMA) and DNA sequencing. HMA results showed that HIV-1 gp120 sequences from 10 patients were of subtype B, whereas 1 patient, infected in Africa, carried a subtype C strain. DNA sequencing confirmed the HMA results and revealed a high intrasubtype diversity in the C2V3 region of env in different clade B isolates, which suggests multiple introduction of subtype B to Hungary. This study shows that subtype B is the predominant HIV-1 clade in Hungary.

Acquired Immunodeficiency Syndrome↗

Characterization of sera from subjects infected with HIV-1 subtypes B and E in Thailand by antibody binding and neutralization.

The range and specificity of the humoral immune response to HIV-1 subtypes B and E was investigated in Thai samples. Sera from HIV-1-positive subjects, consisting of subtypes B (n = 24) and E (n = 138), were characterized in relation to the neutralization of primary isolates and T-cell line-adapted (TCLA) strains and binding to monomeric gp120, the CD4/gp120 binding site (BS), and V3 peptides. A subtype-specific pattern of antibody binding was observed with the exception of the CD4/gp 120MN BS. Neutralization of TCLA strains (n = 4) was strongly type-specific (p = .002); however, neutralization of primary isolates (n = 8) was weak and group specific. Thus, the subtype specificity of B and E sera in the neutralization of TCLA strains, but not primary isolates, supports the dominance of the V3 region in TCLA virus neutralization but does not support the distinction of subtypes B and E as discrete neutralization serotypes in Thailand.

Amino Acid Sequence↗

Expression of recombination activating genes-1 and-2 immunoglobulin heavy chain gene rearrangements in acute myeloid leukemia: evaluation of biological and clinical significance in a series of 76 uniformly treated patients and review of the literature.

BACKGROUND AND OBJECTIVES: Early lymphoid differentiation is characterized by antigen receptor gene rearrangements; the rearrangement process is governed by two lymphoid-specific genes, RAG (recombinase activating gene)-1 and -2. The available data on the incidence and prognostic significance of clonal immunoglobulin heavy chain (IgH) gene rearrangements in acute myeloid leukemia (AML) are rather contradictory. The aim of this study was to evaluate the incidence and prognostic significance of RAG-1 and -2 mRNA transcripts and clonal IgH gene rearrangements in a cohort of uniformly treated AML patients; the available literature is also reviewed. DESIGN AND METHODS: The study was performed on 76 AML patients, newly diagnosed between August 1996 and November 1999. RAG-1/-2 gene expression was analyzed by a reverse transcriptase polymerase chain reaction technique and IgH gene rearrangements were detected with variable region (VH) family-specific and consensus framework region (FWR)-2 and/or-3 primers. Statistical associations were explored between IgH monoclonality/ RAG mRNA expression and: (i) age, gender, FAB subtype, immunophenotype, cytogenetic risk groups; (ii) response variables (response/relapse incidence, survival). RESULTS: In total, 38/75 samples (50.6%) were RAG-1 and/or -2 positive; 30/76 samples (39.5%) carried clonal IgH genes, whereas 13/30 IgH-positive samples (43.3%) were RAG-1/2-negative. Significant associations were detected only for RAG-2 positivity and unfavorable karyotype and IgH monoclonality and FAB subtypes M4/ M5; no association was identified with response outcome and survival. INTERPRETATION AND CONCLUSIONS: Lymphoid-specific molecular markers are detected in a significant proportion of AML patients, regardless of differentiation status (assessed morphologically/ immunophenotypically); however, in our experience, they do not seem to constitute an adverse prognostic factor.

Acute Disease↗

Identification of a new subgroup of SIVagm in tantalus monkeys.

Simian immunodeficiency viruses from African green monkeys (SIVagm) can be classified into three subgroups based upon the species from which they were isolated. The most extensively studied subgroup are composed of SIVagm isolated from vervet monkeys (Cercopithicus pygerythrus). Fewer isolates have been characterized from either grivets (Cercopithicus aethiops) or green monkeys (Cercopithicus sabeus). An additional distinct species of African green monkeys, tantalus monkeys (Cercopithicus tantalus), has not been characterized in terms of SIV infection. A high seroprevalence of SIV-specific antibodies was identified in sera collected from Ugandan tantalus monkeys. SIV was isolated from PBMC (SIVagm/tan), the gag region amplified by polymerase chain reaction, cloned, and sequenced. Based upon gag, SIVagm/tan isolates cluster genetically with other previously recognized SIVagm strains. However, SIVagm from tantalus monkeys forms a distinct genetic subgroup. These data confirm earlier observations of species-specific subtypes of SIVagm viruses and support the hypothesis that these viruses may have coevolved with their host during geographic dispersion throughout Africa.

Amino Acid Sequence↗

Diversity of the vif gene of human immunodeficiency virus type 1 in Uganda.

Little sequence information is available for human immunodeficiency virus type 1 (HIV-1) vif genes of African origin. Here we describe 37 new complete vif genes of 18 AIDS patients from Uganda and show that vif has a high in vivo genetic variability. vif proviral DNA sequences of peripheral blood cells were determined by direct sequencing of PCR products. Only 52% of the deduced Vif amino acids were absolutely conserved; when Vif sequences previously analysed were considered, only 32% of the Vif consensus sequence comprised conserved and as such possibly functionally important motifs. The high inter-individual vif variability was in contrast to a very low intra-individual variability. One patient carried a vif gene with a stable C-terminal deletion, but N-terminal truncations were not found in patients' predominant vif sequences. The vif genes analysed comprised subtypes A, D and an A/D mosaic. Phylogenetic analyses additionally showed that HIV- 1 in Uganda has spread across the boundaries of ethnic groups.

Acquired Immunodeficiency Syndrome↗