Isolation of HIV type 1 from long-term nonprogressors in Herpesvirus saimiri-immortalized T cells.
Explore the source record for details and available documents.
Cell Line, Transformed↗
Biomedical subjects
R Daniels
Publications and source records attributed to R Daniels.
Subclinical polydipsia and polyuria in young patients with schizophrenia or obsessive-compulsive disorder vs normal controls.
1. Increased water intake and output is more common among psychiatric patients, especially those with schizophrenia, than in the general population. Animal studies suggest that polydipsia and polyuria derive, in part, from dopamine dysregulation. Stimulated by these observations this study sought to elucidate relationships among water homeostasis, monoamine metabolism, and electrolyte excretion in schizophrenic patients with and without paranoid hallucinatory symptoms (PH vs. NP), thought to reflect hyper- and hypo-dopaminergic states respectively, and to compare these with those shown by patients with obsessive compulsive disorder (OCD). 2. 24 hr-urine samples for electrolyte, monoamine and metabolite measures were taken from 14 schizophrenic patients with PH symptoms, 13 with predominantly nonparanoid (NP) symptoms, 11 OCD patients and 27 healthy controls (matched for age, weight and creatinine production). Water intake and serum electrolytes was sampled during psychological testing. 3. PH patients drank 2-3 times more than the others in a 3-4 hr test, yet 24 hr-urinary volumes were 75% larger in both PH and NP patients than in the two comparison groups. 4. Daily potassium excretion was a bit higher in PH patients, but concentrations of sodium, potassium and phosphate tended to be lower in PH and NP patients than in the others. 5. Positive associations of electrolyte with homovanillic acid excretion were consistent across groups and not directly related to medication. But associations of electrolyte excretion with noradrenergic activity in controls were absent in psychotic patients and associations with serotonin in OCD patients were absent in the other groups. 6. Increased water intake and output in PH patients along with the disturbed association with noradrenergic metabolism are consistent with altered autonomic activity in these patients. 7. The independence of measures of water homeostasis from dopaminergic medication indicates that the associations in clinically responding PH patients of polydipsia with DA function (decreased DA levels) may be pertinent to this subgroup but not to schizophrenia in general.
Adolescent↗
Workplace health concerns: a focus group study.
Addresses the workplace health concerns of employees at an Acute Hospital Trust. The research conducted utilised a focus group methodology to investigate the health concerns of multidisciplinary groups of health care workers (n = 27). The findings indicate that the concern for the majority was workplace stress. Stressors that were identified as important to the groups were for example, the nature of the work they were undertaking, staffing levels, volume of the work, management styles and their work environments. Health promotion and prevention topics such as motivation and health status, smoking, alcohol and drug misuse, nutrition and weight control and physical exercise were seen of subsidiary concern. Limitations of the study are given. Future research in the subject area is identified.
Attitude of Health Personnel↗
Plasma neuropeptide-Y levels, monoamine metabolism, electrolyte excretion and drinking behavior in children with attention-deficit hyperactivity disorder.
Against a background of (a) increased drinking behavior in children with attention-deficit hyperactivity disorder (ADHD); (b) the parallel between some behaviors associated with ADHD and hypertension; (c) the use of the spontaneously hypertensive rat as a model for ADHD; and (d) similarities in the changes of neuropeptide Y (NPY) and catecholamine in studies of hypertension and drinking, NPY, catecholamines and electrolyte balance were compared in the plasma and urine of healthy children and those with ADHD. Drinking was monitored during 3 h of neuropsychological tests over 2 days in 14 ADHD and nine healthy children. Patients drank four times as much water and showed twice the levels of NPY found in controls. In controls there were positive and in patients there were negative relationships for NPY with drinking and restless behavior. Patients' plasma levels of norepinephrine (NE) and epinephrine were slightly elevated, but urinary levels of NE and the serotonin metabolite were markedly increased. Urinary excretion rates for sodium (not potassium), phosphate and especially calcium were decreased in patients even after covarying for less urine production in the ADHD group. NPY levels were inversely related to calcium excretion and drinking was inversely related to circulating sodium. Increases of drinking and circulating NPY in ADHD children and decreased electrolyte excretion may reflect a common disturbance in metabolic homeostasis.
Adolescent↗
The construction of cDNA libraries from human single preimplantation embryos and their use in the study of gene expression during development.
PURPOSE: The construction and application of polymerase chain reaction (PCR)-based cDNA libraries from unfertilized human oocytes and single preimplantation-stage embryos are described. The purpose of these studies is to provide a readily available resource for the study of gene expression during human preimplantation development. METHODS: Rapid, reproducible, and efficient procedures for the construction of PCR-based cDNA libraries from fewer than 10 cells were first developed in small populations of fibroblast cells. We then constructed cDNA libraries from eight unfertilized oocytes and single two-cell, -4-cell, -7-cell, and blastocyst-stage embryos. Differential display PCR using the libraries as template allows the analysis of stage-specific expression of embryonic genes. Genomic libraries are also prepared from parental samples (cumulus cells and sperm) corresponding to the individual embryo generating the cDNA library. RESULTS: The complexities (between 10(5) and 10(6) clones) of the human embryo libraries indicate that they may represent the entire active gene population at these early stages of human development. Nucleotide sequence analyses of random clones showed the presence of a variety of transcripts, such as the human transposable element. LINE-1, Alu repeat sequences, housekeeping genes, and tissue-specific genes, (e.g., alpha-globin, FMR-1, and interleukin-10). Also present at the expected frequency are the ubiquitous cytoskeletal elements, beta-actin, keratin-18, and alpha-tubulin. In addition to cDNAs corresponding to known expressed sequence tags (ESTs) in the GenBank and dbEST databases, a high proportion of novel sequences was also detected. Several cDNAs were detected only at specific stages of preimplantation development by the differential display analysis. CONCLUSIONS: PCR-based cDNA libraries from single human preimplantation embryos provide a new and important resource for the identification and study of novel genes or gene families. As such, they will increase our basic understanding of the molecular control of human development. In the clinical context, the libraries identify the time of onset of specific genes, and hence the diseases resulting from mutation of these genes, and provide information about new methods of preimplantation diagnosis. The molecular analysis of early gene transcription in human embryogenesis may be expected to lead to advances in contraception, assisted reproduction, and preimplantation genetic diagnosis.
Blastocyst↗
Imprinted expression of SNRPN in human preimplantation embryos.
Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two clinically distinct neurogenetic disorders arising from a loss of expression of imprinted genes within the human chromosome region 15q11-q13. Recent evidence suggests that the SNRPN gene, which is defective in PWS, plays a central role in the imprinting-center regulation of the PWS/AS region. To increase our understanding of the regulation of expression of this imprinted gene, we have developed single-cell-sensitive procedures for the analysis of expression of the SNRPN gene during early human development. Transcripts of SNRPN were detected in human oocytes and at all stages of preimplantation development analyzed. Using embryos heterozygous for a polymorphism within the SNRPN gene, we showed that monoallelic expression from the paternal allele occurs by the 4-cell stage. Thus, the imprinting epigenetic information inherited in the gametes is recognized already in the preimplantation embryo. The demonstration of monoallelic expression in embryos means that efficient preimplantation diagnosis of PWS may be made by analysis for the presence or absence of SNRPN mRNA.
Angelman Syndrome↗
Detection of a novel splice variant of the hypoxanthine-guanine phosphoribosyl transferase gene in human oocytes and preimplantation embryos: implications for a RT-PCR-based preimplantation diagnosis of Lesch-Nyhan syndrome.
We have detected a novel splice variant of the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in two human oocytes and four preimplantation embryos from the 4-cell to the 8-cell stage of development. The novel HPRT transcript lacks exons 4, 5 and 6 of the normal HPRT gene. The same parental origin for the two oocytes and two of the preimplantation embryos, in which the alternatively spliced transcript was detected, might suggest that the alternative splicing is influenced by genetic background. Mutations in the HPRT gene which cause alternative mRNA splicing are implicated in Lesch-Nyhan syndrome. However, the relatively high frequency of detection of this novel HPRT transcript described here (6/109 oocytes and preimplantation embryos) suggests that it is not involved in Lesch-Nyhan syndrome. It is probable that the alternative HPRT transcript is derived from the aberrant splicing of a small percentage of the total mRNA produced from normal HPRT alleles. The presence of this alternative transcript in human preimplantation embryos may complicate an reverse transcription-polymerase chain reaction-based preimplantation diagnosis of Lesch-Nyhan syndrome.
Base Sequence↗
cDNA libraries from single human preimplantation embryos.
In this paper, the construction, evaluation, and application of cDNA libraries from eight unfertilized oocytes and single four-cell-, seven-cell-, and blastocyst-stage embryos are described. Rapid, reproducible, and efficient procedures for the construction of PCR-based cDNA libraries from fewer than 10 cells were first developed in small populations of fibroblast cells. The human embryo libraries display complexities sufficient (between 10(5) and 10(6) clones) to represent the entire active gene population at these early stages of human development. The ubiquitous cytoskeletal elements, beta-actin, keratin-18, and alpha-tubulin, were detected at the expected frequency. Sequencing of consecutively picked random clones, without selection, showed the presence of a variety of sequences, such as the human transposable element, LINE-1 and Alu repeat sequences, housekeeping genes, and tissue-specific genes, such as alpha-globin and FMR-1. In addition to cDNAs corresponding to known ESTs (expressed sequence tags) in the GenBank and dbEST databases, a high proportion of novel sequences were detected. Applications of the libraries to several areas of interest, such as expression of CpG-island-containing "tissue-specific" genes, developmental genes expressed in a stage-specific manner, and a search for monoallelic expression of imprinted genes, are described. The libraries are a valuable resource for the study of gene expression during human preimplantation development and obviate the need for research on the human embryos themselves.
Cells, Cultured↗
XIST expression in human oocytes and preimplantation embryos.
During mouse preimplantation development, the exclusive expression of the Xist gene from the paternally inherited allele is thought to play a role in the inactivation of the paternally inherited X chromosome in the extra-embryonic cell lineages of the developing female embryo. Recently, inactivation of the paternally inherited X chromosome has also been shown to occur in the extraembryonic cell lineages of the human female conceptus. In this paper, we determine whether the pattern of XIST expression in human preimplantation embryos is similarly correlated with paternal X inactivation. We developed procedures sensitive to the single cell, for the simultaneous analysis of XIST and HPRT expression and of sexing, initially using human fibroblast cells. Application of these procedures to human cleavage-stage embryos derived by in vitro fertilization revealed a pattern of XIST expression different from that in the mouse. Transcripts of the XIST gene were detected as early as the 1-cell zygote and, with increasing efficiency, through to the 8-cell stage of preimplantation development. In addition, transcripts of XIST were detected in both male (hence from the maternally inherited allele) and female preimplantation embryos. This pattern of expression is not consistent with a role for the early expression of the XIST gene in the choice of paternal X inactivation in the extraembryonic cell lineages of the developing human embryo.
Animals↗
Transcription of tissue-specific genes in human preimplantation embryos.
We have recently detected de-novo transcripts of the predominantly muscle-specific myotonin protein kinase gene in human preimplantation embryos from the 1-cell to the 4-cell stages. Others have shown de-novo transcripts of the Y-linked genes, ZFY and SRY, in the 1-cell zygote. In order to assess the significance of early transcription of these predominantly tissue-specific genes in preimplantation development, we have analysed individual human oocytes and preimplantation embryos for the presence of transcripts of two further tissue-specific genes, alpha-globin and beta-globin, and two house-keeping genes, HPRT and APRT. Reverse transcriptase polymerase chain reaction assays were developed to the required single cell sensitivity, using human red blood cells and fibroblasts, prior to their application to human oocytes and embryos. As expected, transcripts of the house-keeping genes, HPRT and APRT, were detected at all stages of preimplantation development. Transcripts of 'tissue-specific' alpha-globin were readily detected in preimplantation embryos from the 1-cell stage. However, transcripts of beta-globin were detected only rarely (in only one of the 11 embryos analysed). This difference may be due to the fact that alpha-globin contains a CpG island. A survey of the data on gene expression in early human development suggests that CpG-island-containing genes may be expressed in preimplantation embryos. Expression of these genes in gametes and early embryos may be involved in the survival of CpG islands in evolution.
Adenine Phosphoribosyltransferase↗
Herpesvirus saimiri-immortalized human T-cells support long-term, high titred replication of human immunodeficiency virus types 1 and 2.
Herpesvirus saimiri strain C488 transforms human CD4+ T-lymphocytes to continuous interleukin-2-dependent growth. Unlike human T-cell lines derived from tumours or those transformed by human T-lymphotropic virus 1, herpesvirus saimiri-immortalized T-cells (HVS T-cells) retain many functions of primary activated T-lymphocytes. We have characterized the course of human immunodeficiency virus types 1 and 2 (HIV-1/-2) infection in three HVS T-cell lines. Our results confirm that HVS T-cells are highly permissive to both HIV-1/-2 prototype viruses and to poorly replicating HIV-2 strains of restricted cell tropism. However, the infection was persistently productive for up to 5 months. The down-regulation of surface CD4 molecules was delayed and virus yields significantly exceeded those obtained in T-cell lines.
CD4 Antigens↗
Single-cell analysis of unstable genes.
PURPOSE: We have developed sensitive diagnostic procedures for studies on the normal and mutant alleles of the triplet repeat genes associated with myotonic dystrophy and fragile X in single human somatic cells, gametes and embryos. METHODS: Polymerase chain reaction (PCR) assays for the normal alleles of the myotonic dystrophy and fragile X loci have been refined to the sensitivity of the single cell. In addition, we have developed a simple PCR-based technique, termed ¿Repeat Primer PCR', which can detect the full fragile X expansion in small samples of buccal cells. CONCLUSIONS: The assay for the triplet repeat sequence in the myotonic dystrophy locus could not be used to study stability since we observed additional PCR products derived from in vitro expansion of the triplet repeat sequence during the PCR reaction itself. The implications of in vitro expansion and allele drop-out for studies on the timing of the expansion in development and preimplantation diagnosis of triplet repeat diseases are discussed. The development of a new PCR procedure to identify the expanded alleles of the fragile X locus could prove invaluable for monitoring the timing of repeat expansion in early embryonic development. Triplet repeat polymorphisms provide a means of identifying the maternally and paternally-derived alleles of the myotonic dystrophy gene. Using single cell reverse transcriptase PCR analysis, we have monitored the onset of the myotonic dystrophy gene transcription in early preimplantation embryos. Transcripts from the paternally-inherited allele of the myotonic dystrophy gene are already detectable in the 1-cell stage human embryo.
Alleles↗
Expression of the myotonin protein kinase gene in preimplantation human embryos.
We have examined the expression of the myotonin protein kinase gene (MPK) in individual human preimplantation embryos. An allele-specific, reverse transcriptase polymerase chain reaction assay was developed to the required sensitivity using human buccal cells. Human cleavage stage embryos, derived by in vitro fertilisation, were assayed for MPK transcription directly, or after biopsy of a single blastomere used to determine the genotype of the embryo. The maternal and paternal genotypes were determined from cumulus cells and sperm, respectively. MPK gene transcripts were detected in embryos at the 1-cell--4-cell stage of development. Detection of transcripts from the paternally-inherited allele confirmed onset of embryonic MPK gene expression at this very early preimplantation stage.
Alleles↗
Refined three-dimensional structure of the Fab fragment of a murine IgGl,lambda antibody.
We report the cDNA sequence determination and the crystal structure of the Fab fragment of a murine IgG1,lambda antibody (HC19), specific for an influenza virus hemagglutinin. The HC19 Fab-fragment structure has been refined; the crystallographic R-factor is 19.5% at 2.3 A resolution. We have compared the conformation of HC19 complementarity determining regions (CDRs) with those of CDR loops of Fab structures available from the Protein Data Bank. These loops were chosen based on the identity of key residues, following the canonical-structure approach; four CDRs have a main-chain conformation very similar to the canonical structure that had been identified. HC19 L1 CDR adopts a conformation clearly distinct from all L1 CDRs that belong to a chain of a different class or origin; this is determined by the nature of a few residues at positions in the sequence different from those of key residues in other light chains. This canonical structure should be representative of most murine lambda-class light chains, as inferred from the very high sequence homologies of these polypeptides.
Journal Article↗
Visual evoked potentials to flash and pattern reversal stimulation after administration of systemic or topical scopolamine.
It has previously been shown that 0.6 mg of scopolamine produces a delay in the flash visual evoked potential of young normal volunteers, while the pattern-reversal response does not change in latency. Recent work has shown that this drug differentially affects parvocellular and magnocellular systems. To investigate this effect, two studies were performed. In the first study, 0.4 mg of scopolamine was injected intramuscularly into 11 young, healthy male volunteers who had fasted overnight. The visual evoked potential was recorded to both binocular flash stimulation and monocular pattern-reversal stimulation by means of a checker-board consisting of 56' checks in a 28 degrees field. Responses were recorded before administration of the drug and then 1, 2, 4 and 6 hours after administration. The scopolamine produced a slowing of the flash P2 latency of approximately 6 ms (p < 0.05) two hours after drug administration. There was no effect on the latency of the flash N2 or pattern-reversal N75 or P100. There was an increase in amplitude of the flash N2-P2 component 6 hours after drug administration and an increase in the amplitude of the N75 and P100 2, 4 and 6 hours after the drug. Further subjects were investigated with the use of topical administration of 0.125% scopolamine applied monocularly. In all studies the other eye acted as a control. The subjects were again young healthy volunteers. The visual evoked potential was recorded to both flash and pattern-reversal stimulation with a checkerboard consisting of 60' checks counterphasing at 2 Hz within a 5 degrees field. Results suggest that systemic scopolamine affects the tectal pathway but has no peripheral effect.
Administration, Topical↗
Effect of dopamine and acetylcholine on the visual evoked potential.
Visual evoked potentials were measured on patients with Parkinson's disease and Alzheimer's disease and normal controls to assess the function of dopamine and acetylcholine in the visual system. Dopamine is a neurotransmitter known to be present in the retina of primates and is found to be severely depleted in the substantia nigra of patients with Parkinson's disease. Acetylcholine is also known to be present in the retina, visual cortex, and superior colliculus and is found to be grossly reduced in patients with Alzheimer's disease. Stimuli were designed to preferentially activate functionally separate pathways in the visual system described as magnocellular and parvocellular. The four stimuli were a diffuse flash; an achromatic, 73' check counterphasing at 6 Hz at a contrast of 30%; an achromatic 10' check counterphasing at 2 Hz at a contrast of 85%; and an isoluminant red/green grating of 4 cpd presented using an on and off cosine ramp of 200 ms. The results indicate that an acetylcholine deficit produces a delay to the flash P2 component of the visual evoked potential. No change was detected when other stimuli were used.
Acetylcholine↗
Antigenic and sequence analysis of H3 influenza virus haemagglutinins from pigs in Italy.
To investigate the possible mechanism of maintenance of old human influenza A (H3N2) viruses in pigs, the haemagglutinins (HAs) of seven isolates from swine were studied by analysis of nucleotide and deduced primary amino acid sequences, as well as reactivity of the HA molecule to chicken antisera and monoclonal antibodies. The swine HAs were closely similar to the HA of the A/Victoria/3/75 human variant as regards antigenic and molecular characteristics. These findings are consistent with the hypothesis that the swine HA genes were transmitted from an early human H3 virus to pigs, where they survived with limited mutations over a period of 5 years. The sequence data were also compared with swine H3 sequences to investigate genetic relationships between the H3 genes from swine viruses isolated in different geographical areas. An evolutionary tree, constructed from the nucleotide sequences of viruses isolated from pigs in China and in Italy, illustrated that, depending on the country of their isolation, the HA genes of swine influenza A (H3N2) viruses have different origins, e.g. human and avian, and evolved independently in different lineages. The study provides direct support for the hypothesis that pigs might serve as a 'mixing vessel' for the generation of pandemic strains of human influenza viruses.
Amino Acid Sequence↗
Deletion of a single N-linked glycosylation site from the transmembrane envelope protein of human immunodeficiency virus type 1 stops cleavage and transport of gp160 preventing env-mediated fusion.
The transmembrane envelope glycoprotein (gp41) of human immunodeficiency virus type 1 possesses four consensus sites (Asn-X-Ser/Thr) for the incorporation of N-linked sugars situated on the extracellular domain of the molecule. The purpose of this investigation was to determine the significance of each of these sites in relation to the structure and function of the viral envelope glycoprotein. Each of the four sites was removed by in vitro mutagenesis of gp160 sequence in the non-infectious viral clone pEVd1443, so that amino acids 616, 621, 642 and 679 were each changed from asparagine to serine. The effects of mutagenesis were assessed by syncytium assay after wild-type or mutant envelope clones had been transfected into CD4+ HeLa cells. Removal of the glycosylation site at position 642 resulted in the synthesis of precursor gp160 that was neither cleaved, to give gp120 and gp41, nor transported to the plasma membrane of transfected cells. A consequence of these events was that envelope mutant 642 failed to induce syncytia between neighbouring cells in which it had been expressed. The results of this study indicate that N-linked glycosylation of Asn-642 in the glycoprotein produced by the pEVd1443 expression system is necessary for the correct intracellular processing of gp160 to yield surface-expressed, fusogenic gp41.
Base Sequence↗