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Biomedical subjects

C Richardson

Publications and source records attributed to C Richardson.

At least 19 recordsLinked to original sources
PubMed1995

Preselection of transduced murine hematopoietic stem cell populations leads to increased long-term stability and expression of the human multiple drug resistance gene.

We have been using the human multiple drug resistance (MDR) gene to transduce murine hematopoietic cells via retroviruses as a model system for potential human gene therapy. In this paper, we show that transplantation of MDR-transduced midgestational fetal liver cells (FLCs) into lethally irradiated mice leads to the continued presence and expression of the human MDR gene in the short-lived granulocyte-macrophages of recipients' peripheral blood (PB) for up to 12 months. We have also shown the ability of this retroviral system to efficiently transduce several murine FLC subpopulations enriched for hematopoietic stem cells (FL-HSCs) both (1) short-term by MDR-polymerase chain reaction analysis of individual day 12 colony-forming unit-spleen and (2) long-term by in vivo maintenance of MDR and expression of its product, p-glycoprotein, up to 1 year in PB. More highly enriched FL-HSC subpopulations show the greatest number of circulating granulocyte-macrophage cells expressing MDR long-term. These studies also show that preselection by fluorescence-activated cell sorting of MDR-transduced and -expressing cells before transplant significantly increases the percentage of circulating granulocyte-macrophage cells that express MDR at all time points analyzed posttransplant as compared with unsorted cells transduced in the same manner (P < .01). These results have potentially significant implications for future human gene therapy trials.

3T3 Cells↗
PubMed1995

A redrawn Vandenberg and Kuse mental rotations test: different versions and factors that affect performance.

The available versions of the Vandenberg and Kuse (1978) Mental Rotations Test (MRT) have physically deteriorated because only copies of copies are available. We report results from a redrawn version of the MRT and for alternate versions of the test. Males perform better than females, and students drawn from the physical sciences perform better than students drawn from the social sciences and humanities, confirming other reports with the original version of the MRT. Subjects find it very hard to perform the MRT when stimuli require rotation along both the top/bottom axis and the left/right axis. The magnitude of effect sizes for sex (which account, on average, for some 20% of the variance) does not increase with increasing difficulty of the task. Minimal strategy effects were observed and females did not perform differently during the menstrual period as opposed to the days between the menstrual periods. Practice effects are dramatic, confirming other reports with the original MRT, and can also be shown to be powerful in a transfer for practice paradigm, where test and retest involve different versions of the MRT. Main effects of handedness on MRT performance were not found.

Adult↗
PubMed1995

The Ypt1 GTPase is essential for the first two steps of the yeast secretory pathway.

Small GTPases of the rab family are involved in the regulation of vesicular transport. The restricted distribution of each of these proteins in mammalian cells has led to the suggestion that different rab proteins act at different steps of transport (Pryer, N. K., L. J. Wuestehube, and R. Sheckman. 1992. Annu Rev. Biochem. 61:471-516; Zerial, M., and H. Stenmark. 1993. Curr. Opin. Cell Biol. 5:613-620). However, in this report we show that the Ypt1-GTPase, a member of the rab family, is essential for more than one step of the yeast secretory pathway. We determined the secretory defect conferred by a novel ypt1 mutation by comparing the processing of several transported glycoproteins in wild-type and mutant cells. The ypt1-A136D mutant has a change in an amino acid that is conserved among rab GTPases. This mutation leads to a rapid and tight secretory block upon a shift to the restrictive temperature, and allows for the identification of the specific steps in the secretory pathway that directly require Ypt1 protein (Ypt1p). The ypt1-A136D mutant exhibits tight blocks in two secretory steps, ER to cis-Golgi and cis- to medial-Golgi, but later steps are unaffected. Thus, it is unlikely that Ypt1p functions as the sole determinant of fusion specificity. Our results are more consistent with a role for Ypt1/rab proteins in determining the directionality or fidelity of protein sorting.

Base Sequence↗
PubMed1995

Factors affecting illicit and licit drug use among adolescents and young adults in Greece.

A cross-sectional survey on psychosocial issues, drug use, alcohol and health was carried out in Greece, with a nationwide probability sample of 2448 respondents aged 12-17 and 18-24. Factors potentially associated with illicit and unprescribed licit drug use were tested by logistic regression analysis. Several predictors were revealed. Sex and age were related to a higher lifetime use of illicit drugs, the male young adults reported a higher lifetime use of illicit drugs than the females. Positive attitudes toward hashish use, systematic smoking and use of drugs by close friends were more closely related to illicit than to unprescribed licit drug use. In addition "low self-esteem, family members using tobacco and alcohol" and "problematic drinking" were found only to predict illicit drug use. Other variables, "family members using drugs with or without a doctor's prescription", "being dissatisfied with social life" and "suffering from anxiety, depression and depersonalization symptoms" were found to be significantly associated with licit but unprescribed drug use.

Adolescent↗
PubMed1994

Transfer and expression of the human multiple drug resistance gene in human CD34+ cells.

The human multiple-drug resistance (MDR1) gene has been transferred into human hematopoietic progenitors using retroviral gene transfer. Human bone marrow cells and isolated CD34+ cells isolated from marrow were exposed to growth factors interleukin-3 (IL-3), IL-6, and stem cell factor for 48 hours and then to two changes of MDR retroviral supernatants over the next 24 hours. Progenitor assays in methylcellulose at this time showed that 18% to 70% of BFU-E and 30% to 60% of CFU-GM contain the transferred MDR gene by polymerase chain reaction analysis. Up to 11.2% of the progeny of these cells express increased amounts of MDR glycoprotein on their surface by fluorescence-activated cell sorter (FACS) analysis. In addition, transduced cells are enriched in high MDR-expressing cells after exposure to taxol as assessed by FACS analysis, and by resistance of BFU-E to taxol (Bristol-Myers Squibb, Princeton, NJ). These studies indicate the feasibility of using MDR gene transfer as a means of enriching marrow for MDR-transduced cells. They also provide the basis of a phase 1 clinical protocol in patients with advanced cancers not involving the bone marrow for the use of MDR gene transfer as a means of protecting marrow cells, which normally express low levels of MDR, from the myelosuppressive effects of drugs like taxol.

Antigens, CD↗
PubMed1994

Mouse fetal liver cells lack functional amphotropic retroviral receptors.

We have been transducing mouse hematopoietic cells with the human MDR1 (MDR) gene in retroviral vectors to determine the optimal conditions for retroviral gene transfer as a model system for potential human gene therapy. In these studies, we have demonstrated transduction and expression of the human MDR gene using ecotropic and amphotropic MDR-retroviral producer lines. To obtain more mouse hematopoietic cells for detailed study, mouse fetal liver cells (FLC) have been used for MDR transduction and expression, and to reconstitute the ablated marrows of live adult mice. FLC contain hematopoietic cells that have a reconstituting capacity comparable to that of adult mouse bone marrow cells. However, to our surprise, FLC can only be transduced with ecotropic retrovirus and not with amphotropic virus. This restriction of transduction of FLC cannot be overcome by higher titer virus. The resistance to amphotropic transduction by FLC may be part of a changing developmental program that results in a different antigen repertoire on FLC as compared with adult bone marrow cells.

Animals↗
PubMed1994

Changing cognitive functioning in rehabilitation of schizophrenia.

A three-factor model is proposed for clinical assessment of cognitive and neuropsychological impairments in schizophrenia. The first factor is stable, pervasive and vulnerability-linked. The second and third include executive, memory and conceptual abilities and are episode-linked. The third factor may be amendable to psychosocial treatment.

Cognition Disorders↗
PubMed1994

Characterization of a cleavage mutant of the measles virus fusion protein defective in syncytium formation.

Membrane fusion caused by measles virus (MV) is a function of the fusion (F) protein. This process is essential for penetration into the host cell and subsequent initiation of the virus replicative cycle. The biological activity of the MV F protein is generated by endoproteolytic cleavage of a precursor protein (F0) into a large F1 subunit and a smaller F2 subunit held together by disulfide bonds. The cleavage site consists of a cluster of five basic amino acids (amino acids 108 to 112) within the predicted primary structure of the F protein. To investigate the role of the arginine residue at the carboxy terminus of the F2 subunit (arginine 112), site-directed mutagenesis was used to construct a cleavage mutant of the MV F protein in which this arginine residue was changed to a leucine residue. The mutated F gene, encoding four out of the five basic amino acids at the cleavage site, was inserted into the genome of vaccinia virus. The resulting recombinant virus was used to study expression of the mutant F protein in infected cells. Analysis of the Leu-112 mutant protein made in infected cells demonstrated that this single-amino-acid substitution resulted in a reduced rate of transport of the mutant protein to the cell surface, despite its efficient cleavage to yield F1 and F2 subunits. However, the electrophoretic mobilities of the Leu-112 polypeptides suggested that the protein was cleaved incorrectly. This aberrant cleavage appears to have abolished the ability of the F protein to cause syncytium formation. The data indicate that the arginine 112 residue is critical for the correct proteolytic cleavage that is required for the membrane fusion activity of the MV F protein.

Amino Acid Sequence↗
PubMed1994

Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors.

The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes.

Animals↗
PubMed1993

Mutation of glutamate 309 to glutamine alters one Ca(2+)-binding site in the Ca(2+)-ATPase of sarcoplasmic reticulum expressed in Sf9 cells.

Sf9 cells infected with a baculovirus vector containing SERCA1 cDNA expressed immunoreactive rabbit fast-twitch muscle Ca(2+)-ATPase at levels up to 3 mg/liter. The microsomal fraction isolated from infected Sf9 cells catalyzed Ca2+ transport at rates 6-fold above control values. To obtain direct evidence for the postulate (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H., et al. (1989) Nature 339, 476-478) that Glu309 contributes to a Ca(2+)-binding site in the transmembrane sector of the Ca(2+)-ATPase, Ca2+ binding to wild type and mutant (Glu309 to Gln) Ca(2+)-ATPases was measured. The wild type Ca(2+)-ATPase, expressed in Sf9 cells and purified using a monoclonal antibody bound to Sepharose beads, bound approximately 1.6-1.7 mol Ca2+/mol of enzyme at 2 microM Ca2+ in a buffer favoring the E1 conformation of the enzyme and at 10 microM Ca2+ in a buffer favoring the E2 conformation. Under identical conditions, the mutant Ca(2+)-ATPase bound less than 0.1 mol of Ca2+/mol of enzyme in E1 buffer, but 0.8 mol Ca2+/mol in the E2 buffer. In spite of the ability of the Glu309 to Gln mutant enzyme to bind about 1 mol of Ca2+/mol of enzyme, E2P formation was not inhibited by up to 100 microM Ca2+, and E1P formation from ATP and Ca2+ was not observed with up to 100 microM Ca2+ in intact microsomal vesicles from Sf9 cells. Nevertheless, with detergent-solubilized and purified mutant Ca(2+)-ATPases, E2P formation was inhibited with a K0.5 of 2 microM Ca2+. These observations are consistent with the view that a single intact Ca(2+)-binding site is present in the mutant Ca(2+)-ATPase, which is accessible to Ca2+ only from the lumenal side and only in the E2 conformation. Transition from E2 to E1-Ca2+ may occur during or following Ca2+ binding, accounting for the relatively high Ca2+ affinity and inhibition by Ca2+ of phosphorylation from Pi.

Animals↗
PubMed1993

Factors associated with HIV seropositivity in European injecting drug users. The European Community Study Group on HIV in Injecting Drug Users.

OBJECTIVE: To study factors associated with HIV seropositivity among European injecting drug users (IDU). METHODS: Data on behavioural and other factors potentially associated with HIV status were collected retrospectively by personal interview with IDU (n = 2330) in a multicentre cross-sectional survey conducted in 12 European countries from 1989 to 1990. HIV status tested at the time of the survey was examined in relation to demographic data, history of imprisonment, travel to foreign countries, high-risk drug-using practices in the previous 6 months (re-using other IDU's injecting equipment without effective disinfection) and high-risk sexual behaviour in the last 6 months (multiple partners, unless none was IDU and condoms were always used). Analysis excluded IDU who claimed to be seropositive from previous testing. RESULTS: Imprisonment was significantly associated with current HIV status [odds ratio (OR), 1.70; 95% confidence interval (CI), 1.2-2.87]. High-risk drug-use practices were significantly associated with current HIV status in respondents not previously tested (OR, 2.86; 95% CI, 1.09-7.35) but not in respondents claiming to be seronegative. No significant association was found for high-risk sexual behaviour. CONCLUSIONS: Our experience supports the validity of retrospective investigation of behaviour in a population that is notoriously difficult to study. However, any interpretation of the apparently different associations of HIV status with high-risk sexual behaviour and high-risk drug-use practices must consider that factors tending to attenuate the correlations may operate differently in these two areas.

Adolescent↗
PubMed1993

Measurement of hemoglobin flow and blood flow by near-infrared spectroscopy.

A noninvasive method of measuring hemoglobin flow through an organ by near-infrared spectroscopy (NIRS) is described that allows blood flow to be calculated. The method is derived from the Fick principle and uses a small change in arterial oxyhemoglobin concentration (brought about by a change in the fractional inspired O2 concentration) as an intravascular tracer. Changes in deoxyhemoglobin and oxyhemoglobin concentrations are quantified by monitoring variations in the absorption of near-infrared light in the organ, thus providing a measure of tracer accumulation. The tracer input function is calculated from the change in arterial O2 saturation, measured by pulse oximetry. The method was used to determine hemoglobin flow in the forearms of six healthy young adults on 10 occasions. Forearm hemoglobin flow ranged from 22.5 to 82.6 mumol.l-1.min-1. Calculated forearm blood flow ranged from 1.01 to 4.01 ml.100 g-1.min-1. For comparison, forearm blood flow was measured by venous occlusion plethysmography, and the relation between flow calculated by NIRS (y) and plethysmography (x) was y = 0.93x + 0.30 (r2 = 0.95). The mean difference between the methods was 0.14 ml.100 g-1.min-1. The technique may be widely applicable.

Adult↗
PubMed1993

Acid secretion and serum gastrin in normal subjects and patients with duodenal ulcer: the role of Helicobacter pylori.

OBJECTIVES: To compare gastric secretory function in patients with duodenal ulcer and in healthy volunteers with and without Helicobacter pylori infection. METHODS: Basal acid output, peak acid output, meal-stimulated acid output, fasting and meal-stimulated serum gastrin concentrations were measured in 136 healthy volunteers (63 H. pylori positive, 73 H. pylori negative) and 52 duodenal ulcer patients, all but one of whom were H. pylori positive. RESULTS: By multivariate linear regression analysis, H. pylori infection was a significant negative predictor of basal acid output and a positive predictor of fasting and meal-stimulated gastrin concentrations. When compared to truly normal (i.e., H. pylori-negative) control subjects, duodenal ulcer patients had elevated basal acid output, peak acid output, fasting and meal-stimulated gastrin concentrations. CONCLUSIONS: Our results show that in patients with duodenal ulcer disease, hypergastrinemia is largely related to gastric H. pylori infection, whereas acid hypersecretion is due to factors other than H. pylori.

Adult↗
PubMed1993

Use of gene therapy to induce human-mouse xenogeneic chimerism.

BACKGROUND: Bone marrow transplantation (BMT) has been used in the laboratory to overcome the immunologic barriers to xenotransplantation and results in chimerism and specific tolerance to donor antigens in lethally irradiated mice. Clinically, BMT carries the considerable risks of graft-versus-host disease and graft failure. Retrovirus-mediated gene transfer could provide a means of introducing foreign major histocompatibility (MHC) genes into host bone marrow cells (BMC) and thus accomplish the immunologic goals of BMT, without the associated risks. METHODS: Using a Moloney virus-based vector, a replication defective retrovirus was constructed that contained a complementary DNA encoding the human MHC antigen HLA-A2. Three million C57BL/6 mouse BMC were cocultured for 48 hours with 1 x 10(6) HLA-A2 virus "producer" cells in the presence of 15% WEHI supernatant (interleukin-3) and 200 units/ml interleukin-6. Putatively infected BMC were then used at 2 to 3 x 10(6) BMC/animal to reconstitute lethally irradiated syngeneic mice. RESULTS: Twelve days after reconstitution, spleen colonies were found to have integrated the full-length retroviral sequences. Thirty days after BMT, the introduced DNA could be found in the bone marrow, thymus, and spleen, and approximately 5% of T cells in the spleen expressed the HLA-A2 surface antigen. CONCLUSIONS: These data show that xenogeneic MHC genes can be introduced and expressed in mouse hematopoietic cells in vivo and indicate that gene therapy potentially may be used in the future to manipulate the immune system to induce transplantation tolerance.

Animals↗
PubMed1992

Transfer and expression of the human multidrug resistance gene in mouse erythroleukemia cells.

Gene therapy in humans requires the transplantation of genetically modified cells, and it is important to select only those cells capable of expressing high levels of protein from the transferred gene. Expression of the human multiple drug resistance (MDR) gene confers resistance to a variety of compounds in vitro and in vivo. To determine the feasibility of conferring recipient erythroid cells with the MDR phenotype, we have transduced mouse erythroleukemia cells (MELC) with the MDR gene in a retroviral vector. We show here that MELC clones resistant to exposure to colchicine (an MDR-responsive agent) can be isolated, and demonstrate high levels of MDR RNA and protein expression. Increasing doses of colchicine increase the level of MDR RNA and protein expression significantly. These results indicate that it is possible to transfer and express the human MDR phenotype in mouse erythroid cells by retrovirally mediated gene transfer, and that drug selection can be used to enrich or purify populations of cells containing and expressing this gene.

3T3 Cells↗
PubMed1992

Transfer and expression of the human multiple drug resistance gene into live mice.

The human multiple drug resistance (MDR) gene has been used as a selectable marker to increase the proportion of bone marrow cells that contain and express this gene by drug selection. By constructing retroviral vectors containing and expressing the MDR gene and a nonselectable gene such as the beta-globin gene, enrichment for cells containing both of these genes can be achieved. A retroviral construct containing MDR cDNA in a Harvey virus-based vector has been used to transfect our ecotropic 3T3 retroviral packaging line GP+E86. Clones have been isolated by exposure of the retrovirally transfected cells (MDR producer cells) to colchicine (60 ng/ml), a selective agent that kills MDR-negative cells. Flow cytometry analysis (fluorescence-activated cell sorting) with an antibody to MDR demonstrates expression of human MDR protein on the surface of these colchicine-resistant producer clones. Untransfected GP+E86 cells are negative. Colchicine-resistant clones were titered using clone supernatants and the highest titer clone (4 x 10(4) viral particles per ml) was cocultured with 10(6) donor mouse bone marrow cells for 24-48 hr. The donor cells were then injected into congenic irradiated mice, and the presence of the MDR gene was assayed by the polymerase chain reaction (PCR) analysis using MDR-specific primers. In one experiment eight of nine transduced mice were positive for MDR by PCR of peripheral blood 14 and 50 days posttransplantation; after 240 days three of nine transduced mice were positive. Bone marrow obtained from one of these positive animals was stained with the MDR monoclonal antibody and the granulocyte population was analyzed by FACS. Approximately 14% of the total granulocyte pool contain increased levels of MDR protein. In addition, the bone marrow cells of several mice initially positive for MDR gene by PCR, and subsequently negative, were exposed to taxol, a drug whose detoxification depends on MDR gene expression; a positive signal was obtained in all of these mice, indicating drug selection of MDR-positive marrow cells. Cell sorting studies of these mice also show an increased number of high-MDR-expressing marrow cells, selected after exposure to taxol. Thus, in this live animal model MDR transduction is effective in selecting a human MDR-expressing population of marrow cells resistant to taxol chemotherapy. This strategy may, thus, be useful in humans to prevent the marrow toxicity induced by anticancer agents such as taxol and as a selectable marker to enrich for cells simultaneously transduced with a nonselectable gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗