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Biomedical subjects

J D Roberts

Publications and source records attributed to J D Roberts.

At least 109 records · Page 6Linked to original sources
PubMed1990

Phase I trial of intraperitoneal recombinant interleukin-2/lymphokine-activated killer cells in patients with ovarian cancer.

Ten patients with ovarian cancer refractory to conventional therapy were treated with intraperitoneal (i.p.) recombinant interleukin-2 (rIL-2) and lymphokine-activated killer cells (LAK). The 28-day protocol consisted of 6 priming i.p. rIL-2 infusions on days 0, 4, 6, 8, 10, and 12. Leukapheresis was performed for mononuclear cell collection on days 15, 16, 17, and 18 and lymphokine-activated killer cells were given i.p. with the rIL-2 on days 19 and 21. Three additional i.p. rIL-2 infusions were given on days 23, 25, and 27. Three dose levels of rIL-2 were tested: 5 X 10(5), 2 X 10(6), and 8 X 10(6) units/m2 body surface area. The dose-limiting toxicity was abdominal pain secondary to ascites accumulation with significant weight gain. Other toxic effects included decreased performance status, fever, nausea and vomiting, diarrhea, and anemia. Peripheral lymphocytosis and eosinophilia were seen at all dose levels. The maximum tolerated dose is 8 X 10(6) units/m2/dose. Peripheral and peritoneal IL-2 levels were measured with a bioassay using an IL-2-dependent cell line. At the highest dose level, serum IL-2 was greater than 10 units/ml for 18 h. After the first infusion, a 2-log dilution of the i.p. IL-2 was measured in the serum. In the postleukapheresis i.p. IL-2-dosing period less IL-2 was detected in the serum than in the earlier i.p. IL-2-priming period. The induction and persistence of LAK activity were studied. Peritoneal LAK activity was detected as early as 4 days after the first i.p. infusion, by day 11 in all evaluable patients, and persisted for the 6-day interval between priming IL-2 and LAK/IL-2 infusion. Peritoneal lytic activity persisted until day 28 in 5 tested patients. These peritoneal cells retained lytic activity 48 h in culture medium without rIL-2 present. Peritoneal LAK activity correlated with the percentage of mononuclear cells and the percentage of CD56-positive mononuclear cells in the peritoneum. The yield of peripheral lymphocytes after the six i.p. priming doses of rIL-2 correlated with the dose level of rIL-2 infused. Peripheral blood LAK activity showed a minimal, however progressive, increase during the treatment protocol. LAK activity could be enhanced if rIL-2 was present during the 4-h assay. These studies indicate that i.p. rIL-2 infusion induced durable regional LAK activity and primes peripheral blood cells for LAK activity if exposed briefly to additional IL-2.

Adult↗
PubMed1990

Fidelity of animal cell DNA polymerases alpha and delta and of a human DNA replication complex.

We are investigating the mechanisms by which mutations are produced or avoided during DNA synthesis. Using in vitro fidelity assays, we have defined the error frequency and mutational specificity of the replicative animal cell DNA polymerases (alpha and delta). With DNA polymerase alpha or the four-subunit DNA polymerase alpha-DNA primase complex, neither of which contains detectable associated exonuclease activity, the fidelity of the polymerization step is low relative to spontaneous mutation rates in vivo. DNA polymerase delta is much more accurate, partly due to proofreading by the 3'----5' exonuclease activity associated with this polymerase. These fidelity studies have been extended to the replication apparatus present in extracts of human HeLa cells. The replication complex is highly accurate, suggesting that additional fidelity components are operating in the extract during bidirectional, semiconservative replication of double-stranded DNA. Nevertheless, in highly sensitive reversion assays, base substitution errors can be readily detected at frequencies greater than the estimated rate of spontaneous mutation in vivo. This suggests that fidelity components may be missing and/or that human cells depend heavily on postreplicative repair processes to correct replication errors.

Animals↗
PubMed1990

Subcellular localization of a putative kainate receptor in Bergmann glial cells using a monoclonal antibody in the chick and fish cerebellar cortex.

A monoclonal antibody, IX-50, that was raised against a kainate binding protein (Mr = 49,000) from chicken cerebellum, was used in light and electron microscopic immunocytochemical studies to localize putative kainate receptors. Pre- and postembedding immunoperoxidase and immunogold methods were used in the cerebellar cortices of one to 26-day old chickens and adult rainbow trout. Immunoreactivity was detected only in association with Golgi epithelial/Bergmann glial cells. Intracellular immunoreactivity was present in the granular and agranular endoplasmic reticulum, Golgi apparatus and in lysosomes, representing the sites of synthesis, glycosylation and degradation of the protein. In the fish the granular endoplasmic reticulum was not immunoreactive. Extracellular immunoreactivity was associated with the plasma membrane. In the fish it was established that the epitope is on the outer surface of the membrane. The protein seems to be uniformly distributed along the membrane including the somata, the radial stem processes and the leafy lamellae surrounding Purkinje cell dendrites. Areas of the glial membrane in contact with other glial cells were also immunopositive. High-resolution light microscopy demonstrated all the Bergmann glial plasma membrane in the cortex, providing a "negative" image of Purkinje cell dendrites. It is apparent that Bergmann glial processes selectively outline the dendrites of the Purkinje cells by surrounding the parallel fibre terminal/Purkinje cell spine synaptic complexes. The parallel fiber terminals were highly immunoreactive for glutamate, as shown by an immunogold procedure. The association of Bergmann glial processes, carrying the Mr = 49,000 kainate binding protein, with the Purkinje cell dendrites and spine synapses could provide a basis for neuronal signalling to the Bergmann glia, possibly by glutamate.

Animals↗
PubMed1990

Synaptic and Nonsynaptic Localization of Benzodiazepine/GABAA Receptor/Cl- Channel Complex Using Monoclonal Antibodies in the Dorsal Lateral Geniculate Nucleus of the Cat.

The two monoclonal antibodies, bd-17 and bd-24, are specific for beta- and alpha-subunits of the GABAA/benzodiazepine receptor/chloride channel complex respectively. An abundance of both subunits has been revealed in the visual thalamus of the cat by light microscopic immunocytochemistry using these antibodies. The alpha-subunit specific antibody and electron microscopy were used to determine the subcellular distribution of immunoreactivity with respect to specific cell classes in the dorsal lateral geniculate nucleus. Immunoreactivity was always associated with membranes and the degree of immunoreactivity varied greatly between different types of cell as defined by: (i) immunoreactivity for GABA; (ii) soma area; (iii) presence or absence of cytoplasmic laminated bodies (CLB). GABA negative neurons with the smallest soma area showed the strongest immunoreactivity, mainly in the endoplasmic reticulum and also on the somatic plasma membrane. Cytoplasmic laminated bodies could be found in the majority of these neurons. Large GABA negative cells without CLBs were strongly immunoreactive on the plasma membrane of the soma and dendrites, but showed scant if any intracellular immunoreactivity. GABA-positive cells showed weak intracellular immunoreactivity but negligible if any immunoreactivity at the somatic and proximal dendritic plasma membrane. A similar reaction pattern was found in GABA negative cells which contained no CLBs and which constituted a medium sized cell population. It is suggested that the degree of intracellular receptor immunoreactivity is positively correlated with receptor turnover. The dendrites of projection cells, particularly outside the glomeruli, showed strong immunoreactivity on the plasma membrane. The synaptic junctions formed by many boutons (F terminals) establishing symmetrical synapses with dendrites of relay cells were immunopositive, but no immunoreactivity could be detected at the synapses established by the presynaptic dendrites of the local interneurons. Many axo-somatic F1 junctions were also immunoreactive. However, immunoreactivity for the receptor/channel complex was also widely distribution on nonsynaptic plasma membranes of somata and dendrites. Thus GABA may act at both synaptic and non-synaptic sites. Furthermore, the correlation of immunoreactivity for the GABAA receptor complex with previously published properties of physiologically identified cells suggests that the strongly immunoreactive, small, GABA negative cells with CLBs might correspond to the 'lagged' X-type cells, and the large GABA negative receptor outlined cells without CLBs might correspond to some of the Y-type neurons.

Journal Article↗
PubMed1990

Efficiency factors and ATP/ADP ratios in nitrogen-fixing Bacillus polymyxa and Bacillus azotofixans.

The efficiency factor, the number of moles of ATP generated per mole of glucose fermented, was determined in anaerobic, non-carbon-limited N2-fixing cultures of Bacillus polymyxa, Bacillus macerans, Bacillus azotofixans, and Clostridium butyricum through identification and quantitation of the fermentation products by 13C nuclear magnetic resonance spectroscopy and measurement of acetate kinase activities. All three Bacillus species had acetate kinase activities and produced acetate and ethanol as the major fermentation products. The maximum amounts of ATP generated per mole of glucose fermented were 2.70, 2.64, and 2.88 mol in B. polymyxa, B. macerans, and B. azotofixans, respectively, compared with 3.25 mol in C. butyricum. Thus, in the N2-fixing Bacillus species, the efficiency factors are lower than that in C. butyricum. Steady-state ATP/ADP concentration ratios were measured in non-carbon-limited N2-fixing cultures of B. polymyxa and B. azotofixans through separation and quantitation of the adenylates in cell extracts by ion-pair reversed-phase high-performance liquid chromatography. The observed ATP/ADP ratios were 4.5 and 3.8, and estimated energy charges were 0.81 to 0.86 and 0.81 to 0.83, respectively, for B. polymyxa and B. azotofixans. The results suggest that under these growth conditions, the rate of ATP regeneration is adequate to meet the energy requirement for N2 fixation in the Bacillus species, in contrast to N2-fixing Clostridium pasteurianum and Klebsiella pneumoniae, for which substantially lower steady-state ATP/ADP ratios and energy charges have been reported. Implications of the results are discussed in relation to possible differences between Bacillus and Clostridium species in energy requirements for N2 fixation and concomitant ammonia assimilation.

Acetate Kinase↗
PubMed1990

Acute hematomas: effects of deoxygenation, hematocrit, and fibrin-clot formation and retraction on T2 shortening.

Acute hematomas can appear hypointense on T2-weighted magnetic resonance (MR) images at field strengths as low as 0.35 T. Using Raman spectroscopy to measure blood oxygenation and taking T2 measurements at 2.1 and 9.4 T, the authors examined the relaxation mechanisms acting during deoxygenation, increases in hematocrit, and fibrin-clot formation and retraction. Individual contributions to overall T2 from deoxyhemoglobin and the interactions of water with protein hydration layers in hemoglobin, plasma proteins, and fibrin were measured. Overall T2 values estimated by summing individual relaxation rates were in reasonable agreement with the T2 values of clotted blood. Results suggest that deoxygenation may be most important in T2 shortening, followed by increased hematocrit. T2 shortening from fibrin polymerization was minimal at the field strengths used. Effects of deoxygenation and increasing hematocrit are more sensitive to field strength than fibrin T2 shortening. Effects of fibrin may be more significant at middle and low field strengths.

Acute Disease↗
PubMed1990

Multicenter trial of single-dose modified bovine surfactant extract (Survanta) for prevention of respiratory distress syndrome. Ross Collaborative Surfactant Prevention Study Group.

A multicenter, prospective randomized controlled trial was performed comparing the efficacy of a single intratracheal dose of modified bovine surfactant extract (Survanta, 100 mg/kg, Abbott Laboratory, North Chicago, IL) with air placebo in preventing respiratory distress syndrome. Infants were enrolled if they were estimated to be between 24 and 30 weeks' gestation, weighed between 750 and 1250 g, and were intubated and stabilized within 15 minutes after birth. A total of 160 infants were treated (79 with surfactant, 81 with air placebo) between 4 and 37 minutes after birth (median time 12 minutes). Of these, 5 infants were excluded from the final analysis. The 72-hour average values for the arterial-alveolar oxygen ratio, fraction of inspired oxygen, and mean airway pressure were calculated from the area under the curve of scheduled values measured throughout 72 hours. Clinical status was classified using five ordered categories (no supplemental oxygen or assisted ventilation, supplemental oxygen only, continuous positive airway pressure or assisted ventilation with intermittent mandatory ventilation less than or equal to 6 breaths/min, assisted ventilation with intermittent mandatory ventilation greater than 6 breaths/min, death). Chest radiographs at 24 hours were graded for severity of respiratory distress syndrome. Infants receiving Survanta had less severe radiographic changes at 24 hours of age and decreased average fraction of inspired oxygen (31% vs 42%, P = .002) compared with control infants. No differences were noted in the average arterial-alveolar oxygen ratio, mean airway pressure, or clinical status on days 7 and 28. A beneficial effect was noted in the incidence of pneumothorax (P = .057) and an increase was noted in the incidence of necrotizing enterocolitis (P = .052). No differences in incidence of patent ductus arteriosus, intraventricular hemorrhage, sepsis, or bronchopulmonary dysplasia were seen. According to results of a secondary analysis, there was improvement in the fraction of inspired oxygen and a greater number of survivors without bronchopulmonary dysplasia in the subgroup of infants weighing less than 1000 g who were treated with surfactant. It was concluded that a single dose of Survanta given shortly after birth resulted in decreased severity of chest radiographic findings 24 hours after treatment and improved oxygenation during 72 hours after treatment, but did not improve other acute measures of disease severity or clinical status later in the neonatal period. The group at highest risk for respiratory distress syndrome (infants with birth weights between 750 and 999 g) may benefit the most from preventive therapy.

Animals↗
PubMed1989

Interaction of novel bis(platinum) complexes with DNA.

Bis(platinum) complexes [[cis-PtCl2(NH3)]2H2N(CH2)nNH2] are a novel series of potential anticancer agents in which two cis-diamine(platinum) groups are linked by an alkyldiamine of variable length. These complexes are potentially tetrafunctional, a unique feature in comparison with known anticancer agents. Studies of DNA interactions of bis(platinum) complexes in comparison with cisplatin demonstrate significant differences. Investigations of interstrand crosslink formation in which crosslinking of a short DNA fragment is detected by gel electrophoresis under denaturing conditions demonstrate that interstrand crosslinks are 250 fold more frequent among bis(platinum) adducts than among cisplatin-derived adducts under the conditions examined. These investigations indicate that bis(platinum) adducts contain a high frequency of structurally novel interstrand crosslinks formed through binding of the two platinum centers to opposite DNA strands. Unlike cisplatin, bis(platinum) complex binding does not unwind supercoiled DNA. Studies with the E. coli UvrABC nuclease complex demonstrate that both linear and supercoiled DNA containing bis(platinum) adducts are subject to incision by the repair enzyme complex. Initial studies using UvrABC nuclease as a probe to define the base and sequence specificity for bis(platinum) complex binding suggest that the specificity of the bis(platinum)s is similar, but not identical, to that of cisplatin.

Base Sequence↗
PubMed1989

Specificity and mechanism of error-prone replication by human immunodeficiency virus-1 reverse transcriptase.

DNA-dependent DNA synthesis in vitro by human immunodeficiency virus-1 reverse transcriptase is relatively error-prone. The enzyme, whether recombinant or from virus particles, produces errors while replicating M13mp2 DNA at a rate that, if operative in vivo, would produce about five mutations per genome per round of replication. Sequence analysis of mutants resulting from in vitro synthesis demonstrates that errors are nonrandomly distributed. Base substitution and one-base frameshift mutational hot spots are observed. The specificity and position of errors suggest that most of the frameshifts and many of the base substitutions are initiated by template-primer slippage. Processivity analysis for the enzyme on the M13mp2 DNA template reveals strong termination at specific sites. Termination sites within homopolymer sequences correlate with frameshift mutational hot spots. The results suggest that the formation and/or utilization of misaligned template-primers is increased during the dissociation-reinitiation phase of the reaction.

Base Sequence↗
PubMed1989

Effect of tamoxifen on plasma insulin-like growth factor I in patients with breast cancer.

Human breast cancer cells secrete and have membrane receptors for insulin-like growth factor I (IGF-I), a growth hormone-dependent peptide that stimulates cell replication. However, little is known about plasma concentrations of IGF-I in breast cancer patients. Plasma IGF-I levels are decreased in malnutrition, decline with advancing age, and are influenced by estrogen. We evaluated the effect of the antiestrogen agent tamoxifen on plasma IGF-I in 32 ambulatory breast cancer patients. Treatment with tamoxifen was associated with lower concentrations of plasma IGF-I (0.48 +/- 0.3 unit/ml in treated versus 1.03 +/- 0.6 units/ml in nontreated patients, P less than 0.01). However, patients treated with tamoxifen did not differ from nontreated patients in age, menopause, duration since diagnosis, metastatic disease, recent weight loss, or measures of nutritional status. We conclude that tamoxifen therapy results in a reduction of plasma IGF-I concentration. We speculate that the antitumor action of tamoxifen in breast cancer is due in part to suppression of IGF-I.

Adult↗
PubMed1989

Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum.

Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways.

Ammonia↗
PubMed1989

Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro.

We determined the fidelity of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases (RTs) during DNA synthesis in vitro using the M13mp2 lacZ alpha gene as a mutational target. Both RTs commit an error approximately once for every 30,000 nucleotides polymerized. DNA sequence analysis of mutants generated in a forward mutation assay capable of detecting many types of errors demonstrated that avian myeloblastosis virus RT produced a variety of different mutations. The majority (58%) were single-base substitutions; all of which resulted from the misincorporation of either dAMP or dGMP. Minus-one frameshifts were also common, composing about 30% of the mutations. In addition to single-base events, eight mutants contained sequence changes involving from 2 to 59 bases. The frequency of these mutants suggests that, at least during DNA synthesis in vitro, RTs also commit errors by mechanisms other than classical base miscoding and misalignment. We examined the ability of RTs to synthesize DNA from a mismatched primer terminus at a sequence where the mismatched base was complementary to the next base in the template. Unlike cellular DNA polymerases which polymerize from the mismatched template-primer, RTs preferred to polymerize from a rearranged template-primer containing a matched terminal base pair and an unpaired base in the template strand. The unusual preference for this substrate suggests that the interactions between RTs and the template-primer are different from those of cellular DNA polymerases. The overall error rate of RT in vitro is sufficient to account for the estimated mutation rate of these viruses.

Avian Myeloblastosis Virus↗
PubMed1989

Analysis of fidelity mechanisms with eukaryotic DNA replication and repair proteins.

We are investigating the mechanisms for producing or avoiding errors during DNA synthesis catalyzed by DNA replication and repair proteins purified from eukaryotic sources. Using assays that monitor the fidelity of a single round of DNA synthesis in vitro, we have defined the error frequency and mutational specificity of the four classes of animal cell DNA polymerases (alpha, beta, delta, gamma), and the fidelity of an SV40 origin-dependent DNA replication complex in extracts of HeLa cells.

Animals↗
PubMed1989

Pulmonary oxygen toxicity in the guinea pig. Effect of indomethacin.

Deleterious pulmonary effects of hyperoxia are thought, in part, to be mediated by high-energy radicals and inflammatory reactions. It has been suggested that arachidonate metabolites, such as leukotrienes, may be responsible for the latter mechanism. We sought to demonstrate that concurrent exposure to hyperoxia and indomethacin, a potent inhibitor of cyclooxygenase, would increase pulmonary inflammation. A total of 50 female guinea pigs were treated as pairs with 95.6% oxygen or air and separately with 0.3 mg/kg of indomethacin or normal saline every 12 h for 3 days. A significant increase in leukocytes in bronchopulmonary lavage subsequent to combined oxygen and indomethacin treatment was observed when compared with either oxygen, indomethacin, or air treatments alone (p less than 0.05). Although oxygen treatment did alter pulmonary volume-pressure relations (p less than 0.05), hyperoxia alone or with concurrent indomethacin treatment did not alter elastance or histologic structures. These findings suggest that pulmonary inflammation in response to hyperoxia may be augmented by indomethacin. We speculate that increased lipoxygenase products of arachidonate metabolism may be partially responsible for these changes.

Animals↗