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Biomedical subjects

J A Nelson

Publications and source records attributed to J A Nelson.

At least 127 records · Page 7Linked to original sources
PubMed1995

Targeting 6-thioguanine to the kidney with S-(guanin-6-yl)-L-cysteine.

Recently, S-(purin-6-yl)-L-cysteine (GC) was shown to be a kidney-selective prodrug of 6-mercaptopurine. In the present study, for further development of kidney-selective chemotherapeutic agents, GC was synthesized, and its metabolism was examined in the rat by cysteine conjugate beta-lyase (beta-lyase) to yield the antitumor and immunosuppressant drug, 6-thioguanine (6-TG). The apparent Km values obtained with renal mitochondrial and cytosolic beta-lyases were similar, but the Vmax value obtained with renal mitochondrial beta-lyase was approximately 45-fold higher than the Vmax value obtained with renal cytosolic beta-lyase. After rats were administered GC (400 mumol/kg), the concentrations of GC in the kidney, liver and plasma at 30 min were higher than the corresponding values at 15 or 60 min. GC concentrations in plasma and kidney were, however, 3- and 5-fold higher than that in liver, respectively. Although GC metabolites were not detected in plasma, they were detectable in liver and kidney; metabolite concentrations at 30 min were higher than those at 15 or 60 min. Renal 6-TG concentration at 30 min was nearly 4-fold higher than hepatic 6-TG concentration; hepatic and renal 6-thioxanthine and 6-thiouric acid concentrations were similar. The amount of GC metabolites excreted in urine within 24 hr was linearly proportional to the administered GC dose. Rats administered GC (400 mumol/kg) excreted nearly 5-fold the amount of metabolites as rats given an equimolar dose of 6-chloroguanine, a GC precursor. These results and the finding that renal 6-TG concentrations after GC treatments were in excess of the ED50 of 6-TG (0.5-1.0 microM) in two human renal carcinoma cell lines (A-498 and CAKI-1) suggest that GC may have clinical usefulness as a prodrug of 6-TG.

Animals↗
PubMed1995

Cysteine conjugate beta-lyase activity in human renal carcinomas.

Cysteine conjugate beta-lyase, an enzyme that converts cysteine S-conjugates to free thiols, pyruvate and ammonia, is normally expressed primarily in the liver and kidney. In theory, this selective distribution affords the opportunity to target thiol-containing drugs to these organs and, perhaps, to tumors derived from them. To assess the potential for delivery of such drugs to kidney-derived tissue, we have used a typical beta-lyase substrate, S-(2-benzothiazolyl)-L-cysteine, to measure the beta-lyase activity in normal and tumor tissue of kidneys removed from patients with renal carcinoma. Although considerable heterogeneity in enzyme activity levels was observed in normal and tumor-derived samples, a high proportion of tumor samples had enzyme activity that was at least 50% of that observed in adjacent normal tissue. Frequently, hypoxanthine-guanine phosphoribosyltransferase activity was observed to be greater in the tumor than in normal tissue. These results may aid in the development of therapy for renal carcinomas.

Adult↗
PubMed1995

Assessment of bystander effect potency produced by intratumoral implantation of HSVtk-expressing cells using surrogate marker secretion to monitor tumor growth kinetics.

A molecular marker, human alpha-1-antitrypsin (hAAT) was transduced into tumor cells and its secretion was found to correlate with tumor growth or regression, allowing for an accurate and continuous measurement of tumor growth kinetics. Using this system, we investigated the therapeutic potential produced purely from the bystander effect of HSVtk+ CT26 cells to eradicate established CT26 colon carcinomas in mice by direct intratumoral implantation and subsequent ganciclovir administration. With lower ratios (0.1% and 1% of initial tumor burden), tumor growth kinetics went into a static (remission) phase of approximately 2 weeks duration before relapse and resumption of progressive tumor growth. When the number of CT26tk+ modified cells injected into the tumor equaled 10% to 100% of the initial tumor cell number the bystander effect was sufficient to completely eradicate established tumors indicating that a potent bystander killing effect is produced in this system, and that a cellular therapy based on this approach may have applications.

Animals↗
PubMed1995

Cytomegalovirus persistence in macrophages and endothelial cells.

Although human cytomegalovirus (HCMV) rarely causes overt disease in healthy individuals, the virus can be a serious, even life-threatening problem in immunosuppressed or immune-deficient patients and in the setting of maternofetal primary infection. In recent years knowledge about HCMV pathogenesis has increased significantly. Identification of the cell types infected by HCMV in vivo has demonstrated that monocytes/macrophages and endothelial cells are key elements both in latent and acute infection with HCMV. Both cell types can be involved in systemic spread of virus and specific organ disease. While it has been demonstrated that differentiation of monocytes into macrophages renders these cells permissive to productive HCMV infection, there is presently limited knowledge about the pathogenesis of HCMV in endothelial cells (ECs). These cells represent the physiological interface between blood and tissues, display heterologous phenotypes, and are functionally variable, depending on their respective microanatomic environment. Microvascular ECs are the site of monocyte transmigration into organ tissues and therefore are likely to regulate the activation state of infected monocytes. Recently, macrovascular endothelium is receiving increasing attention due to its possible involvement in atherogenesis. In this overview we present recent findings on the role of monocytes/macrophages and endothelial cells in HCMV infection.

Cytomegalovirus↗
PubMed1994

Systemic gene therapy of murine melanoma using tissue specific expression of the HSVtk gene involves an immune component.

Previously we have demonstrated safe and effective transfer of the HSVtk cytotoxic gene to primary murine melanoma tumors by direct injection of plasmid and retroviral vectors in which the HSVtk gene is driven by the tissue-specific tyrosinase promoter. However, for general clinical application such forms of therapy should, ideally, be effective against disseminated metastases. We report here that the number of recently established lung metastases of B16 melanoma in C57BL mice treated with ganciclovir is reduced compared to controls after multiple i.v. administrations of high titer retroviral supernatant encoding the HSVtk gene, but not after administration of liposome-complexed plasmid DNA. Using polymerase chain reaction analysis, integration of the provirus was observed in metastasis-bearing lungs (4 of 6 mice) and in the spleens of some ganciclovir-treated animals (2 of 6 mice) but not in the testes, brain, heart, liver, or kidney. The reduction in the number of experimental metastases in C57BL mice exceeded the anticipated extent of transduction of tumor cells, which is indicative of a marked bystander effect. This magnitude of reduction was not observed in immunodeficient athymic mice, suggesting that the immune system plays some part in the bystander effect. In support of these data, we show that, whereas the parental tumor cells are only poorly immunogenic, an effective antitumor immune response is generated following the killing of neoplastic cells in vivo as a result of treatment with ganciclovir. These effects may be responsible for augmenting the efficacy of retroviral infection. The combination of local cell killing by the HSVtk/ganciclovir system and the induction of antitumor immunity suggests new opportunities for the design of vectors for the gene therapy of cancer.

Animals↗
PubMed1994

HIV infection of human brain capillary endothelial cells--implications for AIDS dementia.

We have demonstrated that human brain capillary endothelial (HBCE) cells, unlike umbilical or aortic endothelial cells are permissively infected by HIV. HIV infection of HBCE cells is noncytolytic and is mediated by a CD4- and GalCer-independent mechanism, implying that HBCE cell tropic strains utilize a unique receptor. The V3 loop of gp120 appears to be important in this reaction. T-cell tropic but not brain-derived macrophage tropic HIV strains selectively infect brain endothelium suggesting that T-cell tropism is important for HIV entry through the blood-brain barrier (BBB). The ability of HIV to infect cells that compose the BBB implies that the virus may be directly involved in the BBB dysfunction observed in AIDS patients. HIV infection of HBCE cells may allow the flow of cytokines or toxic metabolites from the circulating blood into the brain parenchyma either by disrupting tight junctions or by altering the ability of the cells to regulate transport of substances across the BBB by transcytosis. HIV infection may also result in endothelial cell-induced astrocytosis by release of cytotoxic substances or modulation of abluminal surface antigens which contact astrocytic foot processes. Finally, HIV infection of the brain endothelium could facilitate virus entry to the CNS either by infection of HBCE cells or via entry of HIV-infected leucocytes. The establishment of our in vitro HIV-HBCE cell system will allow us to explore the potential mechanisms which mediate AIDS dementia.

AIDS Dementia Complex↗
PubMed1994

Detection of cytomegalovirus in cerebrospinal fluid autopsy specimens from AIDS patients.

Cytomegalovirus (CMV) is a common opportunistic pathogen infecting AIDS patients. Polymerase chain reaction (PCR) and antigen capture ELISA were used to detect CMV in 40 cerebrospinal fluid autopsy specimens from patients with AIDS. CMV DNA was detected by PCR in 70% of samples. Of the 21 samples from patients with systemic CMV infection, 57% had CMV encephalitis, while 81% had virus in cerebrospinal fluid detectable by PCR. Of the 24 samples from patients with no histologic evidence of CMV encephalitis, 58% had CMV DNA in cerebrospinal fluid detected by PCR. These results suggest that PCR of cerebrospinal fluid sensitively detects systemic CMV infection but is not specific for brain infection in autopsy specimens of AIDS patients.

AIDS-Related Opportunistic Infections↗
PubMed1994

Differential role of long terminal repeat control elements for the regulation of basal and Tat-mediated transcription of the human immunodeficiency virus in stimulated and unstimulated primary human macrophages.

Primary human macrophages induced to differentiate through contact with autologous activated nonadherent cells were used to investigate the transcriptional mechanisms involved in reactivation of human immunodeficiency virus (HIV) replication. Through transient transfection experiments with an HIV long terminal repeat (LTR)-chloramphenicol acetyltransferase reporter construct, we show that macrophage differentiation results in a 20-fold upregulation of basal LTR activity. To identify sequence elements responsive to the differentiation process, point mutations introduced into the LTR were tested in differentiated and undifferentiated macrophages. Several elements were identified as positive regulators of basal transcription. TATA, Sp1, and NF-kappa B binding sites were the most influential. The low-affinity site for LBP-1 (UBP-1) functioned as a negative regulator of LTR activity in undifferentiated macrophages, but this influence was lost upon differentiation. When tat was cotransfected into the expression system, the requirement for LTR elements identified as important for positive regulation of basal transcription remained in undifferentiated macrophages. Interestingly, however, the mutations in positive control elements which debilitated activity in undifferentiated macrophages had no effect on LTR activity in differentiated macrophages. Thus, it appears that while HIV-LTR activity is highly dependent on cellular transcription factors in undifferentiated cells, in differentiated macrophages the viral protein Tat confers pliability on the LTR and facilitates autonomy from absolute cellular control mechanisms. In vivo, release from either positive or negative regulation via cellular proteins may facilitate reactivation of HIV in macrophages.

Adult↗
PubMed1994

The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation.

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

Alleles↗
PubMed1994

Inhaled furosemide attenuates hyperpnea-induced obstruction and intra-airway thermal gradients.

Inhaled furosemide attenuates exercise- and isocapnic hyperventilation-induced asthma; however, the mechanism for this phenomenon is unknown. Because the magnitude of the intra-airway thermal gradient that develops between the cooling of hyperpnea and the rewarming that occurs once hyperventilation ceases is directly related to the severity of thermally induced obstruction in humans, we wondered if furosemide blunted these temperature changes. To explore this issue, eight asthmatic subjects had tracheobronchial airstream temperature measures as they performed isocapnic hyperventilation with frigid air alone (HV) or with pretreatment with inhaled saline (S + HV) or 45 +/- 3 (SE) mg of furosemide (F + HV). HV and S + HV resulted in similar degrees of obstruction, whereas the mechanical decrements after F + HV were significantly less. In concert with this protective effect, F + HV resulted in less airstream cooling during hyperventilation and slower rewarming in the recovery period. Because the major source of heat to the airways is provided by its microcirculation, inhaled furosemide may be acting as a topical vasodilator serving to enhance heat availability and thus reducing the effective thermal burden of hyperpnea.

Administration, Inhalation↗
PubMed1994

P-glycoprotein and organic cation secretion by the mammalian kidney.

On the basis of physiological localization, broad substrate specificity and energy dependence, the role of the kidney P-glycoprotein was tested in the energy-dependent renal secretion of organic cations. P-glycoprotein-enriched vesicles from Cl 1D/VCR [a multidrug-resistant (MDR) cell line] displayed enhanced transport of the MDR drug vinblastine and the organic cation cimetidine but not of the organic cation tetraethylammonium (TEA) over that shown by vesicles prepared from the drug-sensitive parental line Cl 1D. An outwardly directed proton gradient stimulated TEA and cimetidine uptake by renal brush border membrane vesicles (BBMV) but this gradient did not enhance the uptake of these organic cations into Cl 1D/VCR vesicles. Vinblastine uptake was unaffected by the proton gradient in either vesicle preparation. An outwardly directed gradient of TEA enhanced the uptake of TEA into renal BBMV but did not do so in the case of Cl 1D/VCR vesicles. These data indicate that P-glycoprotein, which is normally energized by ATP hydrolysis, is incapable of catalyzing organic cation/proton exchange or organic cation/organic cation exchange, properties of the organic cation carrier of renal proximal tubule BBMV. The MDR substrates and modulators inhibited the uptake of vinblastine and cimetidine by Cl 1D/VCR vesicles and the uptake of cimetidine and TEA by renal BBMV. Several organic cations studied inhibited TEA and cimetidine uptake by renal BBMV but did not inhibit the uptake of vinblastine and cimetidine by Cl 1D/VCR vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Enhanced transepithelial flux of cimetidine by Madin-Darby canine kidney cells overexpressing human P-glycoprotein.

Cimetidine has been used as a relatively selective inhibitor of renal organic cation secretion, analogous to the use of probenecid to inhibit organic anion secretion. Many of the substrates for the multidrug transporter P-glycoprotein, which is overexpressed in multidrug-resistant tumor cells, are organic cations. Furthermore, the protein is normally expressed on the apical membranes of proximal tubule cells, the postulated site for active organic cation secretion. To test directly whether P-glycoprotein might serve as a carrier for cimetidine, we measured cimetidine transepithelial movement across Madin-Darby canine kidney cells grown as monolayers on membrane filters. A retrovirally transduced Madin-Darby canine kidney cell line (Madin-Darby canine kidney cells transfected with the human multiple drug resistance 1 cDNA for P-glycoprotein), that expresses the human form of P-glycoprotein on its apical membrane, had an increased capacity to transport cimetidine from the basolateral to apical medium (b-->a) but not in the reverse direction (i.e., a-->b). Qualitatively similar results were observed with daunomycin, a well established substrate for P-glycoprotein. Cellular uptake and energy-dependent efflux experiments further established cimetidine to be a substrate for the human P-glycoprotein. Thus, P-glycoprotein may play a role in the renal secretion of cimetidine and perhaps other organic cations.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Probe-microphone measurements with body-worn instruments: loudspeaker and reference microphone effects.

Probe-microphone measurements are typically made with behind-the-ear (BTE) and in-the-ear (ITE) hearing aids with the loudspeaker located at 0-degrees or 45-degrees azimuth at head level and the reference microphone positioned on the head near the hearing aid microphone. With body-worn instruments, these conditions may not accurately reflect in situ hearing aid performance. This study compared the real-ear aided response (REAR) and real-ear insertion response (REIR) for a body-worn hearing aid using the substitution method and an off-line equalization modified pressure method with three different loudspeaker locations (0 degrees and 45 degrees at head level and 0 degrees at body hearing aid level) and two reference microphone positions (over-the-ear [OTE] and next to the body hearing aid microphone). Results indicated that each of the responses was affected by changes in loudspeaker and reference microphone location. If the substitution method measured from 0-degrees azimuth at head level is considered to be the most realistic representation of hearing aid performance, the closest agreement with body-worn hearing aids was obtained with the modified pressure method when the loudspeaker was located at 0-degrees azimuth at head level and the reference microphone was located over the ear. If the clinician uses the modified pressure method and desires to approximate results with the substitution method, correction values are needed for REAR measurements but not for REIR measurements.

Equipment Design↗
PubMed1993

Human immunodeficiency virus infection of human brain capillary endothelial cells occurs via a CD4/galactosylceramide-independent mechanism.

Neuropathologic studies of AIDS patients have shown that brain capillary endothelial cells are a cellular target for human immunodeficiency virus (HIV) in vivo. We have established in vitro cultures of primary human brain capillary endothelial (HBCE) cells. Using this model system, we have shown a significant HIV infection of HBCE cells that is productive yet noncytopathic. The infection is mediated by a cellular interaction with gp120 that does not involve CD4 or galactosylceramide. HIV infection of HBCE cells may contribute to AIDS-associated neuropathology by disturbing the physiology of the endothelium and directly or indirectly facilitating dissemination of virus to the central nervous system.

CD4 Antigens↗
PubMed1993

The DNA-binding subunit of human transcription factor IID can interact with the TATA box as a multimer.

Transcription initiation from eukaryotic protein-coding genes is a complex process that minimally requires RNA polymerase (pol) II (B) and at least seven general transcription factors. The 38-kDa subunit (TBP) of the human general transcription factor TFIID recognizes the TATA sequence element and initiates the assembly of the other general transcription factors and RNA pol II. It is believed, based on experiments with yeast recombinant protein, that TBP binds as a monomer to DNA. Using purified recombinant human TBP protein we find that TBP interacts with the TATA element as both a monomer and a dimer. The multimeric binding of TBP to DNA revealed by this study has important implications for the role of TBP in transcription initiation and suggests novel mechanisms whereby other transcription factors may interact with a RNA pol II preinitiation complex.

Base Sequence↗
PubMed1993

Coexpression of truncated human cytomegalovirus gH with the UL115 gene product or the truncated human fibroblast growth factor receptor results in transport of gH to the cell surface.

The gH glycoprotein of herpesviruses is located on the cell surface in viral-infected cells but is retained in the endoplasmic reticulum (ER) when expressed separately from a recombinant expression vector. These observations suggested the requirement for either a viral function or a viral-induced cellular function which facilitates surface expression of gH. gL fulfills this role in the herpes simplex virus (HSV)-infected cell (J. Virol. 66, 2240-2250, 1992). We have identified the gene product of the UL 115 open reading frame (ORF) as the functional homologue of HSV gL in the human cytomegalovirus (CMV) genome. In addition, we have demonstrated that a cellular gene, the human basic fibroblast growth factor receptor (FGFr) will also facilitate some transport of CMV gH to the cell surface. Coexpression in Chinese hamster ovary cells of the gene product of the UL115 ORF or soluble FGFr with C-terminally truncated gH enhanced levels of secreted gH. These studies suggest that the coexpressed molecules act to mask an ER retention signal(s) exposed when recombinant gH is expressed outside of the context of the viral-infected cell.

Animals↗
PubMed1993

Peroxide bleaches: effect on tensile strength of composite resins.

Research concerning bleaching agents on the mechanical properties of composite resin restorative materials has produced conflicting reports. Representative brands of composite resin restorative materials were studied, including microfil, hybrid, and posterior types. The bleaching agents selected were 30% hydrogen peroxide and two commercial brands of 10% carbamide peroxide. Ten specimens of each material, prepared according to American National Standards Institute/American Dental Association specification No. 27 for diametral tensile testing, were stored in bleaching agents for 1 hour and 168 hours at 37 degrees C. An equal number of specimens stored in deionized water with identical conditions were designated controls. Qualitative examination of bleached specimens revealed a marked change in color, especially the microfilled composite resins. There was no significant difference in tensile strength between controls and exposed samples at either time except for the diminished strength of microfilled composite resins in 30% hydrogen peroxide for one week (p < 0.01).

Carbamide Peroxide↗