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Biomedical subjects

R A Bennett

Publications and source records attributed to R A Bennett.

At least 55 records · Page 3Linked to original sources
PubMed1993

Spontaneous cleavage of bleomycin-induced abasic sites in chromatin and their mutagenicity in mammalian shuttle vectors.

The stability of oxidized abasic sites induced by bleomycin and neocarzinostatin was examined in chromatin reconstituted from a supercoiled plasmid and core histones. Most of the drug-induced abasic sites were found to undergo spontaneous cleavage in chromatin, probably by reaction with histone amine groups. However, there was considerable heterogeneity in the rate of spontaneous cleavage, with some sites being cleaved almost immediately and some remaining intact even after 7 h. Bleomycin-induced abasic sites with closely opposed strand breaks were more unstable than lone abasic sites. Neocarzinostatin-induced abasic sites, which have a different chemical structure, were cleaved somewhat more slowly than those induced by bleomycin. To assess the mutagenic potential of bleomycin-induced abasic sites, bleomycin-treated shuttle vectors were transfected into mammalian cells, and mutations in progeny plasmids were sequenced. Bleomycin treatment resulted primarily in deletions of various sizes in the shuttle vectors, including a number of one-base deletions occurring at potential bleomycin damage sites. However, under certain conditions, substitutions occurring at expected sites of bleomycin attack were also observed. The results suggest that bleomycin-induced abasic sites have only a slight potential to produce base substitutions in mammalian cells and that a substantial fraction of the double-strand breaks induced by bleomycin and most of the double-strand breaks induced by neocarzinostatin are the result of spontaneous cleavage of abasic sites with closely opposed strand breaks. Inaccurate repair of these double-strand breaks may account for the large deletions, and perhaps the one-base deletions, induced by bleomycin.

Apurinic Acid↗
PubMed1992

Interstitial pulmonary macrophages produce platelet-derived growth factor that stimulates rat lung fibroblast proliferation in vitro.

Alveolar macrophages from humans and several animal species produce factors in vitro that modulate fibroblast growth and have been proposed as mediators of interstitial pulmonary fibrosis. Pulmonary interstitial macrophages (IMs) have not been studied previously in this regard. Pulmonary IMs were isolated from prelavaged rat lungs by enzymatic digestion of tissue and subsequent differential adherence of cells to culture dishes. The ability of IMs to release modulators of fibroblast growth into the culture medium was assessed by measuring [3H]thymidine incorporation into DNA and/or nuclear labeling of early-passage rat lung fibroblasts exposed to medium conditioned by IMs. The percentages of nuclei labeled in fibroblast cultures exposed to interstitial macrophage-conditioned medium (IMCM) alone did not significantly differ from that observed in controls, but fibroblasts exposed to IMCM supplemented with 2% platelet-poor plasma showed a 2.6-fold increase in labeling, indicating that IMCM contains predominantly "competence" growth factor activity. Similar results were obtained using purified human platelet-derived growth factor (PDGF). The level of growth factor activity released by IMs increased in cells that had phagocytized iron spheres during the culture period. In addition, fractionation of IMCM by high-performance liquid chromatography demonstrated most of the growth factor activity at a relative molecular mass of about 35 kd. Subsequent quantitative analysis of the fractions by an enzyme immunoassay for PDGF demonstrated that IMCM contains a homologue of human PDGF. These results show that IMs are capable of producing a PDGF-like growth factor for autologous fibroblasts and that release of this factor is enhanced by exposure to an insoluble inorganic particle. Because PDGF is a potent growth factor for fibroblasts and is released by IMs, it is essential to ask in future studies whether this or similar macrophage products play a significant role in mediating fibroblast proliferation in vivo.

Animals↗
PubMed1991

Thermolabile adenine adducts and A.T base pair substitutions induced by nitrogen mustard analogues in an SV40-based shuttle plasmid.

It was previously shown that the predominant mutations induced by melphalan (L-phenylalanine mustard) in the supF gene of shuttle plasmid pZ189 during replication in human cells are A.T----T.A transversions. In order to determine whether adenine adducts were formed at sequence positions corresponding to these mutations, melphalan-induced thermolabile adducts were mapped in the supF gene by selective depurination followed by strand cleavage in alkali. All A.T base pairs which were frequent sites for melphalan-induced A.T----T.A transversions were also prominent sites for formation of thermolabile adenine adducts. Although no mutations were detected at some prominent adduct sites, there was a significant correlation between adduct sites and mutation sites. While runs of two or more adenines were particularly prominent adduct sites, comparison of results obtained with 3'- and 5'-end-labeled DNA gave no evidence for intrastrand cross-links between adjacent adenines. Chlorambucil, another aromatic nitrogen mustard, showed sequence specificities for both mutagenesis and adenine adduct formation nearly identical to those seen with melphalan. The nonaromatic analogues mechlorethamine and phosphoramide mustard were much less efficient in inducing thermolabile adenine adducts, and mechlorethamine induced significantly fewer transversions at A.T base pairs than chlorambucil or melphalan. Formation of thermolabile adenine adducts by the aromatic nitrogen mustards was markedly reduced by blockage of the minor groove with distamycin, or by prior heat denaturation of the DNA. These results suggest that alkylation occurs primarily at the N-3 rather than N-7 position of adenine, probably as a consequence of the affinity of the aromatic rings of melphalan and chlorambucil for the minor groove.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1991

Frequency of inflammatory bowel disease in offspring of couples both presenting with inflammatory bowel disease.

A private practice was surveyed for prevalence of inflammatory bowel disease (IBD) in husband-wife partners and in their children. Among approximately 2500 patients with IBD in our files, 19 couples were identified. They were subdivided into group A (n = 5) in which both partners had symptoms of IBD before marriage; group B (n = 7) in which only one spouse had IBD before marriage and the other partner experienced symptoms afterwards; and group C (n = 7) in which neither spouse had symptoms before marriage but IBD subsequently developed in both. In group B, the disease developed in the second spouse 2-16 years after marriage (mean, 6.4 years). In group C, IBD developed in the first spouse 0.3-41 years after marriage and in the second spouse 1-14 years after the first spouse (mean, 6.8 years). Thirty-five children have been born to 16 of the couples. Two children died before age 20. Of the surviving 33 children, IBD has already developed in 12 (36%). Among the other 21 unaffected children, 1 had uveitis at age 9 and 10 are still under the age of 21, perhaps too young to have yet developed IBD. The frequency of IBD in children was slightly higher if both parents had already developed IBD at the time of conception (67%) compared with when only 1 parent (50%) or neither parent (50%) had developed IBD when conception occurred. Although these data do not distinguish genetic from environmental factors, they show a higher risk of IBD in children when both parents have this illness.

Adult↗
PubMed1991

Capillary spatial pattern and muscle fiber geometry in three hamster striated muscles.

Since most oxygen exchange in muscle is thought to occur by diffusion across the walls of capillaries, it is important to determine the spatial relationship between capillaries and muscle fibers. We have extended the work of Kayar et al. (Microvasc. Res 24: 326-341, 1982) to include other statistical tests that allow one to make stronger statements regarding the spatial pattern. Data for hamsters were obtained from two sartorius, three retractor, and five soleus muscle sections. Distances between all pairs of capillaries, distances between a capillary and its first nearest neighbor for all capillaries, and distances between random tissue sample points and the closest capillary were used to test the spatial arrangement of capillaries. The null hypothesis tested of complete spatial randomness of capillary locations was rejected in favor of a regular alternative in one each of the sartorius and retractor fields and in all five soleus fields. We formulated a geometric model, composed of a space-filling array of identical hexagonal muscle fibers with capillaries placed randomly at the juncture of three fibers or between two fibers, according to the observed relative probability of those occurrences. The model simulations of muscle fibers and capillaries were then analyzed by the same statistical tests used on the histological sections. The findings were similar in both cases, providing confidence that the assumptions of the model were sufficient approximations. The results of this study provide a basis for the placement of capillaries around muscle fibers in mathematical models of oxygen transport in capillary networks.

Animals↗
PubMed1990

Melphalan-induced mutagenesis in an SV40-based shuttle vector: predominance of A.T----T.A transversions.

The base and sequence specificity of mutagenesis by the carcinogenic antitumor agent melphalan (L-phenylalanine mustard) was examined by in vitro treatment of the plasmid pZ189 followed by replication in human 293 cells, rescue in bacteria, and sequence analysis of mutations in the supF gene. Melphalan was strongly mutagenic in this assay. The induced mutations were predominantly base substitutions, with a minor component of small deletions (3-80 base pairs). Surprisingly, A.T----T.A transversions were by far the most frequent substitutions, suggesting that modifications of adenine may play a major role in mutagenesis. More than half the substitutions were clustered in a C-T-A-A sequence in the anticodon loop. No base substitutions were detected at G-N-C sequences, which are thought to be potential sites of DNA interstrand cross-links. The results raise the possibility that the cytotoxic and mutagenic effects of melphalan may be separable.

Base Sequence↗
PubMed1990

Secretion of a platelet-derived growth factor homologue by rat alveolar macrophages exposed to particulates in vitro.

Lung macrophages secrete a homologue of platelet-derived growth factor (PDGF) which induces the proliferation of fibroblasts in vitro. In previous studies, we showed that such a PDGF homologue is produced by rat alveolar macrophages and that rat lung fibroblasts have specific receptors for the macrophage-derived PDGF. In this study, we demonstrate the biological and physicochemical properties of the growth factor, as well as the time-related production of this factor following macrophage activation in vitro by organic and inorganic particles. Alveolar macrophages (AMs) collected by saline lavage from the lungs of rats were cultured in serum-free Dulbecco's modified Eagle's medium (SF-DMEM) for varying periods of time up to 72 h. The SF-DMEM "conditioned" by the AMs was used to treat early passage rat lung fibroblasts (RLFs), which were rendered quiescent by culturing in 2% platelet-poor plasma (PPP). Alveolar macrophage conditioned media (AMCM) in the presence of PPP caused increases in the number of fibroblasts, the percent of labeled fibroblast nuclei and tritiated [3H]thymidine incorporation. AMCM alone caused no detectable changes in fibroblast growth rate. These results indicate that AMs release a "competence-like" growth factor. The AMs were left untreated or were exposed to opsonized zymosan, carbonyl iron spheres or chrysotile asbestos fibers. Macrophages attached to a plastic substrate spontaneously produced the factor, and subsequent addition of the organic and inorganic particles to the macrophage cultures significantly increased the fibroblast-stimulating activity of the AMCM. The growth factor was stable after concentration (100-fold), lyophilization and reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Capillary spatial pattern and muscle fiber geometry in three hamster striated muscles.

The primary goal of this project was to elucidate the spatial pattern of capillaries in three hamster striated muscles according to statistical techniques of pattern analysis. The spatial pattern of capillaries and traditional measures of capillarity are important to understanding the supply and distribution of oxygen and nutrients in a tissue. Statistical tests based on the distance between nearest neighbor capillaries are the most sensitive for detecting regularity in a pattern. A mathematical model was created to simulate the observed muscle fields. The same statistical tests that were performed on the empirical data were performed on the modelled data. The results of the analysis of the modelled data agree sufficiently with those of the empirical data to justify overall confidence in the assumptions. Conclusions that may be drawn from this investigation are (1) the spatial pattern of capillaries tends to be more regular than random and never was there evidence for aggregation using the test statistics; (2) as many as 60-75% of capillaries are located at the corners of muscle fibers indicating that there is some preferential placement for capillaries, and (3) the model developed is a good first approximation to the real situation.

Animals↗
PubMed1989

Cardiopulmonary changes in conscious dogs with induced progressive pneumothorax.

Cardiopulmonary function was measured in 6 conscious dogs with progressive degrees of induced pneumothorax. Minute volume, respiratory rate, central venous pressure, systemic arterial pressure, pulmonary arterial pressure, pulmonary arterial occlusion pressure, heart rate, cardiac output, and arterial and mixed venous blood gases were determined before pneumothorax and at progressive volumes of pneumothorax equivalent to 50, 100, and 150% of the calculated lung volume. Tidal volume, pulmonary vascular resistance, alveolar to arterial O2 tension difference, physiologic dead space fraction, and pulmonary venous admixture also were calculated. Linear increases in respiratory rate, central venous pressure, alveolar to arterial O2 tension difference, and pulmonary venous admixture differed significantly (P less than 0.05). Linear decreases in tidal volume, pHv, pHa, PvO2, and PaO2 were also significantly different. Quadratic increases were significantly different for pulmonary arterial pressure and pulmonary vascular resistance. Trends were not significantly different for other values.

Animals↗
PubMed1988

A homologue of platelet-derived growth factor produced by rat alveolar macrophages.

Rat alveolar macrophages secrete a growth factor that renders rat lung fibroblasts competent to initiate DNA synthesis in vitro in the presence of platelet-poor plasma. This biological activity resembles that of platelet-derived growth factor (PDGF). After separation from putative associated binding proteins by chromatography under acidic conditions, the macrophage-derived factor exhibited a relative molecular weight similar to that of highly purified human PDGF. The factor bound to a monospecific antibody to human PDGF and thus could be quantitated in an enzyme immunoassay for PDGF. It competed with radiolabeled human PDGF for receptor sites for PDGF on rat lung fibroblasts, and binding to these receptor sites could be specifically inhibited by anti-PDGF. These data strongly support the view that the factor derived from rat alveolar macrophages is homologous to human PDGF and is similar to human macrophage-derived PDGF-like growth factor. Furthermore, we have demonstrated that the lung contains both an effector cell (pulmonary macrophage) and a potential target cell (interstitial fibroblast) for this cytokine. Therefore the rat appears to be an appropriate animal model in which to study macrophage-derived PDGF-like growth factors as mediators of proliferation in pulmonary fibrogenesis.

Animals↗
PubMed1987

Static mechanical properties of lungs from adrenalectomized pneumonectomized rats.

The pressure-volume characteristics of saline-filled lungs of adult rats were studied after left pneumonectomy (PNX), with or without prior bilateral adrenalectomy (ADX). Male Sprague-Dawley rats (200-240 g) remained unoperated (UNOP) or were divided into three surgical groups: left PNX, ADX alone, or PNX preceded 5 days earlier by ADX (ADX/PNX). Two weeks post-PNX, maximum lung volumes [MLV; measured at transpulmonary pressure (Ptp) of 10 cmH2O] of right lungs from PNX and ADX/PNX rats remained 20 and 37% below UNOP values, respectively, although right lung mass was equal to or exceeded that of both lungs in UNOP controls. Four weeks postoperative, MLVs approached but remained below normal values. In the ADX/PNX group, MLV was 35% below that predicted for lung mass. The effects of ADX on both lung mass and MLV were blocked by daily injections of ADX/PNX animals with hydrocortisone acetate (5 mg/kg). Neither PNX nor ADX/PNX altered lung volume at zero Ptp or changed the concentration of lung collagen or elastin. When corrected for differences in MLV, relative tissue compliance was unaffected by PNX but was reduced by ADX/PNX. These results indicate that MLV is not restored in parallel with lung mass during the rapid phase of compensatory tissue growth, which follows partial pneumonectomy, and that this discrepancy is accentuated by adrenalectomy.

Adrenalectomy↗
PubMed1987

Control of compensatory lung growth by adrenal hormones.

The effects of adrenalectomy and/or in vivo treatment with hydrocortisone acetate (HCA;5 mg X kg-1 X day-1) on lung growth were investigated in control and pneumonectomized rats of 250 g body wt. Left pneumonectomy (day 0) initiated rapid hyperplastic growth of the right lung, which was unaffected by HCA. Similarly, HCA had no effect on lung growth in unoperated control animals. Two weeks after pneumonectomy, right lung dry mass, protein, RNA, and DNA were equal to that in both lungs of unoperated rats. Adrenalectomy 5 days before (day -5) left pneumonectomy increased the rate and extent of right lung growth, but did not change its hyperplastic character. Continuous HCA treatment (days -5 to 14) prevented the adrenalectomy-mediated increase in postpneumonectomy lung growth. "Early" HCA dosing (days -5 to 6) of adrenalectomized-pneumonectomized animals suppressed lung growth to the pneumonectomy level, but from days 7 to 14 growth accelerated to the adrenalectomized-pneumonectomized rate. Conversely, "late" HCA, initiated when adrenalectomized-pneumonectomized animals had restored normal total lung mass (days 6 to 14), quickly reduced right lung growth to rates typical of unoperated controls. The latter effects were not observed unless continuous steroid treatment was provided throughout this interval. The data support a role for glucocorticosteroids in modulation of the accelerated compensatory lung growth initiated by partial resection of the tissue.

Adrenal Cortex Hormones↗
PubMed1986

Increased pulmonary uptake of exogenous polyamines after unilateral pneumonectomy.

Alterations in pulmonary uptake of the naturally occurring polyamine spermidine and of an exogenous polyamine substrate analogue, methylglyoxal bis(guanylhydrazone) (MGBG), were investigated during the early phase of compensatory lung growth after partial pneumonectomy (PNX) in rats. In lungs perfused in situ 3 days after left PNX, when a small (14%) but significant (P less than 0.01) increase in right lung mass could be detected, uptake of [14C]spermidine and of [14C]MGBG from the pulmonary circulation was increased. MGBG uptake exhibited saturation kinetics (1-50 microM MGBG), both in lungs of control animals apparent Km, 11.3 microM; Vmax, 479 pmol X g-1 X min-1) and on the 1st and 3rd post-PNX days. In both PNX groups, the apparent Km of the uptake pathway was decreased somewhat (8.5 microM), while Vmax increased progressively to 584 and 678 pmol X g-1 X min-1 at days 1 and 3, respectively. The effects of PNX on MGBG uptake were detected as early as 3 h after lung resection and were no longer evident when compensatory lung growth was completed 14 days after surgery. In rats adrenalectomized 5 days before left PNX, an accelerated onset and increased rate of lung restoration were associated with a doubling of the effect of PNX alone on MGBG uptake.

Adrenalectomy↗
PubMed1985

Effects of prior adrenalectomy on postpneumonectomy lung growth in the rat.

The effects of adrenalectomy, with and without subsequent glucocorticoid replacement therapy, on postpneumonectomy compensatory lung growth in the rat were investigated. Male Sprague-Dawley rats (200-230 g) were subjected to no operation (UNOP), left pneumonectomy (PNX), or PNX preceded by bilateral adrenalectomy 5 days earlier (ADX/PNX). At 14 days post-PNX, when compensatory lung growth is normally complete in 200-g rats, right lung (RL) dry weights of PNX (263 +/- 6 mg, n = 26) and ADX/PNX (334 +/- 13 mg, n = 25) rats were increased 58 and 101%, respectively, relative to UNOP controls (166 +/- 5 mg, n = 10). Increases in total DNA, RNA, and protein in the right lungs of PNX and ADX/PNX rats occurred in proportion to RL dry mass. The increase in all parameters examined in PNX and ADX/PNX rats at 7 days post-PNX was half that at 14 days, indicating linear lung growth in both treatment groups. The stimulatory effect of ADX on lung growth was blocked by hydrocortisone acetate (HCA), administered intraperitoneally in daily doses of 5 mg/kg, beginning on the day of PNX. The RL dry weights of HCA-treated ADX/PNX rats (241 +/- 7 mg, n = 10) did not differ significantly from the corresponding value in PNX rats (270 +/- 14 mg, n = 7). The lower RL weights in the HCA-treated rats resulted from an inhibition of cell division, as evidenced by the total RL DNA content, which was similar to that in PNX animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenalectomy↗
PubMed1982

Effect of inhibition of polyamine synthesis on the content of decarboxylated S-adenosylmethionine.

1. The content of decarboxylated S-adenosylmethionine (AdoMet) in transformed mouse fibroblasts (SV-3T3 cells) was increased 500-fold to about 0.4fmol/cell when ornithine decarboxylase was inhibited by alpha-difluoromethylornithine. This increase was due to the absence of putrescine and spermidine, which serve as substrates for aminopropyltransferases with decarboxylated AdoMet as an aminopropyl donor, and to the enhanced activity of AdoMet decarboxylase brought about by depletion of spermidine. The increase in decarboxylated AdoMet content was abolished by addition of putrescine, but not by 1,3-diaminopropane. 2. 5'-Methylthiotubercidin also increased decarboxylated AdoMet content, presumably by direct inhibition of aminopropyl-transferase activities, but the increase in its content and the decline in spermidine content were much less than those produced by alpha-difluoromethylornithine. 3. Decarboxylated AdoMet content of regenerating rat liver was measured in rats treated with inhibitors of ornithine decarboxylase. The content was increased by 60% 32h after partial hepatectomy in control rats, by 90% when alpha-difluoromethylornithine was given to the partially hepatectomized rats, and by 330% when 1,3-diaminopropane was used to inhibit putrescine and spermidine synthesis. After 48h of exposure to 1,3-diaminopropane, which completely prevented the increase in spermidine after partial hepatectomy, there was a 5-fold rise in hepatic decarboxylated AdoMet concentration. These increases were prevented by treatment with putrescine or with methylglyoxal bis(guanylhydrazone), an inhibitor of AdoMet decarboxylase. 4. These results show that changes in AdoMet metabolism result from the administration of specific inhibitors of polyamine synthesis. The possible consequences of the accumulation of decarboxylated AdoMet, which could, for example, interfere with normal cellular methylation or lead to depletion of cellular adenine nucleotides, should be considered in the interpretation of results obtained with such inhibitors.

Adenosylmethionine Decarboxylase↗