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Biomedical subjects

J J Roberts

Publications and source records attributed to J J Roberts.

At least 19 recordsLinked to original sources
PubMed2001

Effect of 6 weeks of vitamin E administration on renal haemodynamic alterations following a single dose of neoral in healthy volunteers.

BACKGROUND: A single oral dose of cyclosporin-A (CsA) transiently reduces renal plasma flow (RPF) and glomerular filtration rate (GFR) in transplant patients and, in some patients, chronic administration of CsA leads to renal impairment and fibrosis. Based on experimental studies, several mediators including free radicals have been proposed to account for CsA-nephrotoxicity. We have previously reported that administration of the antioxidant vitamin E in a rat model of chronic CsA-nephrotoxicity reduces renal fibrosis and maintains renal function. METHODS: In the present study, the effect on renal haemodynamics of a single dose of the new oral formulation of CsA (neoral) was assessed before and after 6 weeks of vitamin E (800 IU/day, 2-fold increase in serum vitamin E). GFR (inulin clearance) and RPF (para-amino hippuric acid clearance) were measured before and after a single dose of 5 mg/kg of neoral in 12 healthy subjects under standardised conditions. RESULTS: Although the mean area under the curve of the CsA levels was 21% lower after the vitamin E period, the peak CsA level at 120 min after neoral was similar both before and after vitamin E administration. At 120 min after neoral, a transient reduction in RPF and GFR was noted both before and after vitamin E administration. The nadir of the reductions in RPF (-81 +/-27 ml/min) and GFR (-14 +/- 6 ml/min) at 120 min compared with baseline tended to be lower before than after the treatment with vitamin E (-51 +/- 33 ml/min of RPF and -12 +/- 8, ml/min of GFR, respectively). Plasma and urine levels of F2-isoprostanes (free radical-catalysed vasoconstrictive prostanoids (F2-iso) at 120 min after the administration of neoral were not different from the pre-neoral levels. CONCLUSION: The findings demonstrate that a single oral dose of neoral causes transient, yet significant, reductions in RPF and GFR, and suggest that F2-iso might not be involved in the CsA-induced acute renal vasoconstriction. The tendency for a lower reduction in RPF and GFR following CsA during the vitamin E period in healthy humans warrants additional studies in transplant patients.

Adult↗
PubMed1995

Platelet activation induced by a murine monoclonal antibody directed against a novel tetra-span antigen.

MAb 14A2.H1 identifies a novel low-abundance platelet surface antigen, PETA-3, which is a member of the tetra-span (TM4) family. This MAb brings about platelet aggregation and mediator release, which is completely inhibitable by prostaglandin E1, and partially inhibitable by aspirin and ketanserin. Platelet activation by MAb 14A2.H1 is dependent on interaction with both the platelet Fc receptor, Fc gamma RII, and the specific antigen as it was prevented by either a blocking MAb to Fc gamma RII (IV.3) or F(ab')2 fragments of 14A2.H1. The extent of platelet activation by the antibody varied considerably between donors, and is believed to reflect the polymorphism of Fc gamma RII. Subaggregating concentrations of 14A2.H1 synergized with other platelet agonists, ADP, adrenaline, collagen and serotonin, indicating signalling via a pathway distinct from these activators. Synergy was also blocked by MAb IV.3, or F(ab')2 fragments of 14A2.H1. The similar low copy number of PETA-3 and Fc gamma RII in the platelet membrane (approximately 1000/platelet), together with the dependence on Fc gamma RII for activation by MAb 14A2.H1, suggests that PETA-3 may be a component of the Fc gamma RII signal transducing complex in platelets.

Adenosine Diphosphate↗
PubMed1992

Establishment and characterization of an in vitro model of acquired resistance to cisplatin in a human testicular nonseminomatous germ cell line.

Clinically, human testicular nonseminomatous germ cell tumors exhibit remarkable sensitivity to platinum-based chemotherapy. To define better the mechanistic basis for this unusual sensitivity, the biochemical determinants of platinum-induced cytotoxicity have been investigated in a human testicular tumor cell line (GCT27) established from a previously untreated patient and in an in vitro derived 5.6-fold cisplatin-resistant stable variant (GCT27cisR). Compared to 12 ovarian and 5 cervical human tumor cell lines, the parent GCT27 line was among the most sensitive to the cytotoxic effects of both cisplatin (dosage producing 50% inhibition, 0.2 microM) and carboplatin (dosage producing 50% inhibition, 2.9 microM), thus reflecting clinical data. A 4-day exposure sulforhodamine B-staining assay was used to determine that GCT27cisR was cross-resistant to carboplatin and iproplatin and the classical bifunctional alkylating agents melphalan and chlorambucil. Partial cross-resistance was observed to tetraplatin, methotrexate, and mitomycin C. No cross-resistance was observed to Adriamycin, etoposide, vinblastine, bleomycin, 1-beta-D-arabinofuranosylcytosine, and 5-fluorouracil. Intracellular cisplatin accumulation across the dose range 2.5-100 microM (for 2 h) was 1.6 +/- 0.39-fold (mean +/- SD) greater for the parent line. There was no significant difference in glutathione levels between the two lines. The acquired resistance line was 1.9-fold more resistant than the parent line to the cytotoxic effects of cadmium chloride. There was no significant difference between the two lines, however, in the total amounts of platinum bound to DNA after cisplatin exposure (25, 50, or 100 microM for 2 h). The removal of total platinum adducts from DNA was significantly faster for GCT27cisR compared to the parent line (half-times of removal, 32 and 67 h, respectively). These data suggest that the abnormal sensitivity of the parent testicular tumor cell line to platinum-containing anticancer drugs may be due predominantly to an inherent defect in the ability of these cells to remove platinum from their DNA. This defect is apparently lost in the acquired resistance counterpart. Reduced intracellular accumulation and increased cytoplasmic concentrations of metallothionein may also contribute, in part, to the acquisition of cisplatin resistance in this model.

Antineoplastic Agents↗
PubMed1992

The properties of total adducts and interstrand crosslinks in the DNA of cells treated with CB 1954. Exceptional frequency and stability of the crosslink.

CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) becomes, upon bioactivation, a difunctional alkylating agent. It can be up to a 100,000-fold more cytotoxic in cells that are able to bioactivate it than in those that cannot. This increase in cytotoxicity is much greater than would be predicted from the conversion of a monofunctional alkylating agent to a difunctional one. We now show that the interstrand crosslink formed in the DNA of CB 1954-sensitive cells has some unusual properties. In Walker cells, which are able to activate CB 1954, the interstrand crosslink is the major adduct and can constitute up to 70% of the total adducts. These crosslinks are only poorly excised, as are those produced in V79 cells (which are themselves unable to activate CB 1954) by co-culturing them with Walker cells. Also, CB 1954 is approximately 10-fold more reactive toward the DNA of Walker cells than V79 cells. These observations may explain the extent of the increase in cytotoxicity accompanying the bioactivation of CB 1954.

Animals↗
PubMed1992

Risk-taking behaviour on the decline in intravenous drug users.

In order to assess behaviour changes among intravenous drug users we interviewed 51 IVDUs attending a general practice in Edinburgh. The data obtained was compared with equivalent data gathered in 1986. We were able to show that significant changes in injecting behaviour have occurred and have been maintained over time. A decrease in HIV seroprevalence in this group was also found. The primary drugs of abuse are benzodiazepines and pharmacological opiates, a fact which has important implications for the medical authorities.

Attitude to Health↗
PubMed1992

Problems of drug abuse, HIV and AIDS: the burden of care in one general practice.

Responsibility for many of the problems of intravenous drug abuse and human immunodeficiency virus (HIV) infection lies with community care agencies, such as general practitioners, community psychiatric and district nurses and drug agencies. It is in general practice that this burden is most clearly observed, given that general practitioners are in charge of the day-to-day care of patients. In an attempt to quantify this workload in an inner city practice with 11,200 patients, data were gathered from several sources relating to drug use and HIV infection. The study identified 432 patients who had consulted with problems of drug abuse and/or HIV infection over the period 1981-90. Among this group of patients 161 (37%) were HIV antibody positive. Among 191 drug abusers who were still registered with the practice in 1990 dihydrocodeine was the most commonly prescribed substitute treatment (130 patients) and only nine patients were prescribed methadone. Forty seven per cent of drug users continued to inject drugs occasionally. However, analysis of urine samples revealed that there was a shift away from injecting mainly heroin to multiple drug use, including benzodiazepines, usually originating from prescribed sources. Drug abusers who were HIV positive consulted their general practitioner significantly more often over one year than those who were not (mean 24.9 versus 15.8 consultations, P < 0.01). However, there was no significant difference between these two groups in terms of days spent in hospital. A total of 61 patients were referred to a community psychiatric nurse over an eight month period.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1991

Bioactivation of CB 1954: reaction of the active 4-hydroxylamino derivative with thioesters to form the ultimate DNA-DNA interstrand crosslinking species.

5-(Aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is the active form of CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). This hydroxylamine is formed by the bioreduction of CB 1954 by the enzyme DT diaphorase and accounts for the highly selective cytotoxicity of this compound. The reason why the hydroxylamine derivative is so cytotoxic is that, in contrast to CB 1954, it can react difunctionally as characterized by the formation of DNA-DNA interstrand crosslinks in cells treated by this agent. However, although the 4-hydroxylamine compound can produce these crosslinks in cells it cannot crosslink naked DNA (Knox et al., Biochem Pharmacol 37: 4661-4669, 1988). We show here that 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide can become a species capable of binding to DNA and producing interstrand crosslinks, by a direct, non-enzymatic reaction with either acetyl coenzyme A, butyl and propyl coenzyme A or S-acetylthiocholine. Coenzyme A itself cannot produce these effects. The major product of the reaction between the 4-hydroxylamine and thioesters was identified as 4-amino-5-(aziridin-1-yl)-2-nitrobenzamide. However, this compound is not capable of producing the above effects and the major DNA reactive species was a minor product of the reaction. It is proposed that the ultimate, DNA reactive, derivative of CB 1954 is 4-(N-acetoxy)-5-(aziridin-1-yl)-2-nitrobenzamide.

Acetyl Coenzyme A↗
PubMed1991

Sensitive detection of DNA modifications induced by cisplatin and carboplatin in vitro and in vivo using a monoclonal antibody.

An assay that is based upon a monoclonal antibody (ICR4) is described that enables the quantitation of cisplatin-induced adducts on DNA down to 3 nmol Pt/g DNA (i.e., 1 Pt adduct/10(6) bases), the level necessary to produce toxic effects in cells in vitro and in vivo, using just a few micrograms of DNA. Detection is possible below this level (although probably not necessary for in vivo studies) but the cross-reactivity of unmodified DNA sequences complicates absolute quantitation of adducts. Therefore, it will be possible to investigate the distribution of clinically useful platinum drugs in patients undergoing chemotherapy. Rats of strain F344 appeared to be the best, among several tested, for the production of antibodies to modified DNA, and they were used for the production of hybridomas. Fifteen hybridomas which secreted antibodies that bound to DNA that was highly modified with cisplatin but not to normal DNA were obtained. One (ICR4) was chosen for further characterization because of its relatively strong binding to DNA modified to a moderate level with cisplatin. The characterization included the development of a sensitive competitive enzyme-linked immunoabsorbent assay and the use of DNA that had been reacted with cisplatin both in vitro and in vivo. The levels of platination of both types of DNA samples were determined by atomic absorbance spectroscopy. For DNA that had been exposed to cisplatin in vitro, 50% inhibition of antibody binding was caused by about 15 fmol of total DNA-bound Pt/assay well. At moderate levels of platination, heating of the DNA solution at 100 degrees C for 5 min increased its immunoreactivity such that 50% inhibition was caused by 2.5 fmol Pt adducts/well. Pt adducts on DNA extracted from cells that had been treated with cisplatin were less immunoreactive than DNA treated with cisplatin in vitro, but after heating the immunoreactivity increased such that 50% inhibition in the assay was caused by 2 fmol Pt adduct/well. This sensitivity was invariant over a wide range of levels of platinum adduct frequency. DNA adducts formed by the second generation anticancer drug carboplatin were recognized similarly to the adducts formed by cisplatin, but those formed by the clinically inactive trans-diamminedichloroplatinum(II) or chloro(diethylenetriamine)-platinum(II)-chloride were not significantly immunoreactive. Control DNA cross-reacted in the competitive assay but the immunoreactivity per mol base was 10(7) times lower than the immunoreactivity of cisplatin adducts.

Animals↗
PubMed1991

The Walker 256 carcinoma: a cell type inherently sensitive only to those difunctional agents that can form DNA interstrand crosslinks.

The Walker 256 rat tumour has been maintained in vivo for over 60 years and until recently was used as a primary screen for new antitumour agents. This screen was particularly useful in identifying difunctional alkylating agents as potentially useful anticancer agents and it would seem that the Walker tumour is composed of cells sensitive towards this type of agent. A cell line (WS) established from the Walker tumour retained the sensitivity of the tumour towards difunctional agents and we have examined its phenotype in comparison to a derived, resistant, cell line (WR). The response of WR cells to a range of cytotoxic agents was similar to other established cell lines whilst WS cells were much more sensitive only towards difunctional reacting agents. There were no significant differences in the binding of these agents to the DNA of WS or WR cells. All the agents towards which WS cells showed sensitivity were, without exception, capable of reacting with DNA in Walker cells and forming DNA-DNA interstrand crosslinks. WS cells were not sensitive to busulphan, BCNU, CCNU or Me-CCNU but these agents did not produce interstrand crosslinks in the DNA of either WS or WR cells. Thus WS cells are intrinsically sensitive to specific DNA damage and this is probably a DNA interstrand crosslink. Hybrid cells produced by fusion of WS with WR cells lacked the inherent sensitivity of the WS cells towards cisplatin; sensitivity was therefore a recessive characteristic. Transfection of WS cells with human DNA also gave rise to 2 cisplatin-resistant clones, although it could not be ascertained if these clones were true transfectants or revertants. The survival of these resistant clones, after treatment with cisplatin, was about the same as WR cells a finding which would be consistent with complementation by a transferred gene or reversion of a single gene defect in WS cells. In their sensitivity only to difunctional compounds and lack of an apparent DNA excision repair defect the phenotype of Walker cells strongly resembles those cells from human patients suffering from Fanconi's anaemia and also of yeast snm1 mutant cells. The mechanisms giving rise to this failure to tolerate specific DNA damage (which seems to involve the inability to recover from the initial inhibition of DNA synthesis and may involve a single defect of a gene involved in the late steps of crosslink repair), do not involve drug uptake, drug binding to DNA, cell size, cell doubling time or DNA excision repair.

Animals↗
PubMed1989

Caffeine, aminoimidazolecarboxamide and dicoumarol, inhibitors of NAD(P)H dehydrogenase (quinone) (DT diaphorase), prevent both the cytotoxicity and DNA interstrand crosslinking produced by 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) in Walker cells.

A form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, menadione reductase (NMOR), phylloquinone reductase, quinone reductase, EC 1.6.99.2) has been isolated from Walker 256 rat carcinoma cells. This enzyme can convert 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to a cytotoxic DNA interstrand crosslinking agent by reduction of its 4-nitro group to the corresponding hydroxylamino species (Knox et al. Biochem Pharmacol, 37: 4661-4669 and 4671-4677, 1988). 2-Phenyl-5(4)-aminoimidazole-4(5)-carboxamide and AICA [5(4)-aminoimidazole-4(5)-carboxamide] have previously been reported to be antagonists of the anti-tumour effects of CB 1954. We have shown that both these compounds are inhibitors of the above enzyme and that AICA protects against both the cytotoxicity and the formation of DNA interstrand crosslinks, produced by CB 1954 in Walker cells. Similarly, known inhibitors of NAD(P)H dehydrogenase (quinone) such as dicoumarol, also reduced the cytotoxicity and DNA-interstrand crosslinking of CB 1954 in Walker cells. Caffeine was shown to be a novel inhibitor of NAD(P)H dehydrogenase (quinone) and also elicited the above protective effects. All of the above inhibitors were also shown to potentiate the toxic effects of menadione against the Walker cell. This quinone is known to be detoxified by NAD(P)H dehydrogenase (quinone) and thus emphasises the ability of these compounds to inhibit this enzyme within the cell.

Aminoimidazole Carboxamide↗
PubMed1989

Preliminary crystallographic data for NAD(P)H quinone reductase isolated from the Walker 256 rat carcinoma cell line.

An NAD(P)H quinone reductase isolated from Walker rat 256 carcinoma cells has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell parameters a = 168.15 A, b = 105.09 A and c = 67.38 A and contain four monomeric or two dimeric enzyme molecules per asymmetric unit. Diffraction extends beyond 2.3 A resolution.

Animals↗
PubMed1989

The independent action of radiation and cisplatin on the survival or recovery of human normal or tumour cells in vitro or in vivo.

Recovery from radiation- or cisplatin-induced lethal damage has been studied in euoxic normal human foetal lung fibroblasts (HFL cells) that remain viable for at least 20 days in plateau-phase culture. After a 1 hour treatment with cisplatin the half-time of recovery was about 2 days. By contrast recovery after radiation was more rapid with half-times of approximately 10 h. There was no further measurable recovery after 2 days. With either agent the recovery ratios (RR) were dose-dependent but recovery (following treatment with equitoxic doses of the two agents) was appreciably greater after cisplatin (RR approximately 123 after 40 microM for 1 h) than after radiation (RR approximately 15 after 900 cGy). When radiation (900 cGy) was combined with cisplatin (40 microM for 1 h) the cell survival, measured at 5 days or later times after the treatments, was not significantly less than that predicted by the additive, independent effects of both agents (calculated as the product of their respective effects on cell survival) irrespective of whether cisplatin was given 1 h before, during or for 1 h immediately after radiation. In euoxic, exponentially growing HFL or HeLa cells there was no evidence that combinations of cisplatin and radiation gave more than additive toxic effects in the protocols tested. The combined effects of cisplatin or carboplatin and whole-body irradiation given 45 min later, on human melanoma cells (assessed by their colony-forming abilities in vitro) growing in thymectomised mice, were essentially the same as that predicted by the additive, independent effects of the two agents.

Animals↗
PubMed1989

Remission and relapse in heroin users and implications for management: treatment control or risk reduction.

A general practice based study of 117 heroin users receiving minimal intervention for their drug use examines the patterns and frequency of use from onset to last medical contact. Patterns of abstinence and relapse, and episodes of dependent and nondependent use are defined and quantified, demonstrating variations between and within individuals over time. This evidence of both controlled heroin use and remission and relapse not related to intervention is discussed and contrasted with the continued fixed belief of the public and professionals that heroin use is continuous, incurable, and permanent. Implications for therapy, especially in a "post-AIDS" era, are discussed.

Adult↗
PubMed1989

Human immunodeficiency virus in drug misusers and increased consultation in general practice.

The use of general practitioner services by a group of intravenous drug users was recorded over two two-year time periods 1984-85 and 1986-87. This was felt to represent the period of maximum change in awareness of human immunodeficiency virus (HIV) infection by patients and medical staff. Fifty patients were randomly selected: 25 who were HIV positive and 25 who were HIV negative. Between the two time periods a dramatic increase in consultation rate for both high risk and infected patients attending their general practitioner was recorded (318% and 172% increase, respectively). A small increase in attendance at the accident and emergency department (30% and 34% increase, respectively) was recorded for high risk and infected patients, and there was a large increase in attendance at the infectious diseases unit for infected patients but there was little effect on use of other hospital services. The implications for resource needs in the community are discussed.

Emergency Service, Hospital↗
PubMed1988

A new cytotoxic, DNA interstrand crosslinking agent, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, is formed from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by a nitroreductase enzyme in Walker carcinoma cells.

Walker tumour cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) (Cobb LM et al., Biochem Pharmacol 18: 1519-1527, 1969). CB 1954 forms DNA interstrand crosslinks in a time-dependent manner in Walker tumour cells but not in non-toxically affected Chinese hamster V79 cells [(Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986)]. However, co-culturing Chinese hamster V79 cells with Walker cells in the presence of CB 1954 renders the hamster cells sensitive to CB 1954 and leads to the formation of interstrand crosslinks in their DNA, findings indicative of the formation by Walker cells of a diffusible toxic metabolite of CB 1954. A flavoprotein, of molecular weight 33.5 kDa as estimated by SDS-polyacrylamide gel electrophoresis, has been isolated from Walker cells and identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, EC 1.6.99.2). This enzyme, in the presence of NADH or NADPH, catalyses the aerobic reduction of CB 1954 to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. This new compound can form interstrand crosslinks in the DNA of Chinese hamster V79 cells to which it is also highly toxic.

Animals↗
PubMed1988

The nitroreductase enzyme in Walker cells that activates 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is a form of NAD(P)H dehydrogenase (quinone) (EC 1.6.99.2).

A nitroreductase enzyme has been isolated from Walker 256 rat carcinoma cells which can convert 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to a cytotoxic DNA interstrand crosslinking agent by reduction of its 4-nitro group to the corresponding hydroxylamino species (Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986; Knox RJ et al., Biochem Pharmacol 37: 4661-4669, 1988). The enzyme has now been identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, menadione reductase (NMOR), phylloquinone reductase, quinone reductase, EC 1.6.99.2) by comparison of partial protein sequences, coenzymes, substrate and inhibitor specificities, and spectroscopic data. 2-Phenyl-5(4)-aminoimidazole-4(5)-carboxamide and 5(4)-aminoimidazole-4(5)-carboxamide were shown to be inhibitors of the isolated Walker cell enzyme. This observation could explain the reported antagonistic action of the aminoimidazole carboxamides to the antitumour effects of CB 1954.

Amino Acid Sequence↗