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Biomedical subjects

M E Harper

Publications and source records attributed to M E Harper.

At least 55 records · Page 3Linked to original sources
PubMed1996

Comparison of the subcellular distribution of G-proteins in hepatocytes in situ and in primary cultures.

The subcellular localization of the heterotrimeric G-proteins in hepatocytes in situ was compared to that in hepatocytes in primary culture. The ability of various ligands to activate adenylyl cyclase (AC) in membrane preparations was also investigated. In hepatocytes in situ the G proteins were mainly localized at the plasma membrane while in hepatocytes in culture they were predominantly cytoplasmic. The localization of the G-proteins in hepatocytes in situ correlates with their role in signal transduction. In homogenates prepared from the cultured cells, ligands which stimulate AC via Gs alpha were without effect, which was consistent with the localization of Gs alpha in the cytoplasmic and nuclear compartments. The "relocalization" of the G proteins to the cytoplasm when cells are cultured suggests that transmembrane signalling may be regulated by cell differentiation and cell-cell and cell-extracellular matrix interactions.

Actins↗
PubMed1996

Low incidence of androgen receptor gene mutations in human prostatic tumors using single strand conformation polymorphism analysis.

It is possible that structural changes of the androgen receptor (AR) contribute to the insensitivity of prostatic carcinomas to endocrine therapy. We have isolated DNA from 58 human prostate tumor specimens (31 carcinomas pretreatment, 13 carcinomas after relapse to hormonal therapy, and 14 benign prostatic hyperplasia), three established human prostate carcinoma cell lines and two transplantable human prostatic carcinoma xenografts. Twelve pairs of oligonucleotide primers were used to amplify the majority of the coding region of the AR gene and the products screened for mutations using single-strand conformation polymorphism (SSCP) techniques. In one tumor sample a cytosine to guanine transition in exon F which leads to substitution of glutamic acid for the wild type glutamine at position 798 of the ligand binding domain was detected. The same mutation was also found in the patient's genomic DNA and as been described in a patient with partial androgen insensitivity syndrome. Intronic mutations were detected in two of the benign prostatic hyperplasia samples, and a silent mutation at nucleotide 995 was found to be present in eight poorly differentiated carcinomas, one BPH specimen, as well as in the cell line DU145 (18% of the samples studied). In agreement with most of the literature, these studies indicate that AR mutations are rare both prior to therapy and even in androgen relapsed tumors.

Humans↗
PubMed1996

Comparative analysis of mRNA and protein expression for epidermal growth factor receptor and ligands relative to the proliferative index in human prostate tissue.

The expression of epidermal growth factor receptor (EGF-R), transforming growth factor alpha (TGFalpha), and epidermal growth factor (EGF) was evaluated in a series of prostate cancer (CaP; n = 55) and benign prostate hyperplasia (BPH; n = 44) specimens using immunocytochemistry (ICC) and Northern blotting. In situ hybridization (ISH), performed on a subgroup of these specimens, proved to be a more informative technique for the assessment of messenger RNA (mRNA) in this heterogeneous tissue. A comparative analysis was made in relation to the proliferative index, assessed using the MIB-1 antibody. Elevated levels of EGF-R and TGFalpha, mRNA, and protein were observed in carcinoma cells compared with benign, secretory epithelium using in situ hybridization and immunocytochemistry. In carcinoma specimens evidence of an autocrine growth loop is provided by a correlation between EGF-R and TGFalpha, mRNA (P < .0001), and protein expression (P < .01). A trend toward increased expression of EGF-R and TGFalpha protein with dedifferentiation and a similar trend in the growth fraction suggest a role in tumor progression. Although there was a correlation between EGF-R and the proliferative index (P < .01), no relationship was found between this latter parameter and TGFalpha immunoreactivity (P > .05), indicating that this growth factor may be linked with other aspects of malignant activity rather than directly stimulating proliferation.

Cell Division↗
PubMed1996

Vascular endothelial growth factor (VEGF) expression in prostatic tumours and its relationship to neuroendocrine cells.

Vascular endothelial growth factor (VEGF) expression was examined by immunohistochemistry in 45 prostatic carcinoma specimens and ten benign prostatic tumours (BPH). The majority of carcinoma specimens exhibited cytoplasmic staining for VEGF and showed a trend of increasing expression with dedifferentiation (2p = 0.003). Immunoreactive VEGF was also seen in the prostatic carcinoma cell lines, the order of staining intensity was PC3 > DU145 > LNCaP. Intense granular cytoplasmic staining for VEGF was observed in neuroendocrine-like cells which were seen focally in many of the prostatic specimens. Consecutive sections were incubated with a chromogranin A antibody to confirm the neuroendocrine phenotype of these cells. A significant correlation (P < 0.0001) between the total number of intensely stained VEGF-positive cells and chromogranin A-positive cells was found. A subpopulation of neuroendocrine-like cells also showed intense immunoreactivity for transforming growth factor alpha (TGF-alpha). A correlation was observed (2p = 0.0092) between the intensity of VEGF and TGF-alpha immunostaining in carcinoma cells which were not of neuroendocrine differentiation. The presence of these two angiogenic factors may aid the neovascularisation of carcinomas and their increased expression in tumour-associated neuroendocrine cells may contribute to a more aggressive phenotype.

Chromogranin A↗
PubMed1995

Targeted disruption of the beta 3-adrenergic receptor gene.

beta 3-Adrenergic receptors (beta 3-ARs) are expressed predominantly in white and brown adipose tissue, and beta 3-selective agonists are potential anti-obesity drugs. However, the role of beta 3-ARs in normal physiology is unknown. To address this issue, homologous recombination was used to generate mice that lack beta 3-ARs. This was accomplished by direct injection of a DNA-targeting construct into mouse zygotes. Twenty-three transgenic mice were generated, of which two had targeted disruption of the beta 3-AR gene. Mice that were homozygous for the disrupted allele had undetectable levels of intact beta 3-AR mRNA, as assessed by RNase protection assay and Northern blotting, and lacked functional beta 3-ARs, as demonstrated by complete loss of beta 3-agonist (CL 316,243)-induced stimulation of adenylate cyclase activity and lipolysis. beta 3-AR-deficient mice had modestly increased fat stores (females more than males), indicating that beta 3-ARs play a role in regulating energy balance. Importantly, beta 1 but not beta 2-AR mRNA levels up-regulated in white and brown adipose tissue of beta 3-AR-deficient mice (brown more than white), strongly implying that beta 3-ARs mediate physiologically relevant signaling under normal conditions and that "cross-talk" exists between beta 3-ARs and beta 1-AR gene expression. Finally, acute treatment of normal mice with CL 316,243 increased serum levels of free fatty acids (FFAs) (3.2-fold) and insulin (140-fold), increased energy expenditure (2-fold), and reduced food intake (by 45%). These effects were completely absent in beta 3-AR-deficient mice, proving that the actions of CL are mediated exclusively by beta 3-ARs. beta 3-AR-deficient mice should be useful as a means to a better understanding of the physiology and pharmacology of beta 3-ARs.

Adenylyl Cyclases↗
PubMed1995

Proportional activation coefficients during stimulation of oxidative phosphorylation by lactate and pyruvate or by vasopressin.

A 'proportional activation' approach designed to deal with the influence of external effectors within biochemical systems is described. The proportional activation coefficient, which enables the quantitative determination of the relative stimulation of different parts of a system by a given effector, is defined. The proportional activation approach was used to calculate the relative activation of delta p-producing and delta p-consuming subsystems during stimulation of the respiration rate of cells by a variety of different effectors. Oxidative phosphorylation was stimulated by the addition of either lactate and pyruvate (10 mM and 1 mM) or vasopressin. The addition of lactate and pyruvate to suspensions of resting hepatocytes increased the respiration rate by about 50%. The delta p-consuming subsystem was stimulated about 60% as much as the delta p-producing subsystem. Quinolinic acid, commonly considered to be a specific inhibitor of gluconeogenesis, was found to block the delta p-producing oxidative subsystem as well as the delta p-consuming subsystem, indicating some nonspecific effects of this inhibitor. Addition of vasopressin to hepatocytes that were incubated in the presence of lactate and pyruvate resulted in an increase of the respiratory rate by up to 35%. The relative stimulation of the delta p-producing and delta p-consuming subsystems was essentially equal. Using the 'proportional activation approach' to analyse these and previously published data, it is shown that substrates (lactate/pyruvate and fatty acids), Ca(2+)-acting hormones (vasopressin and others) and calcium in muscles (heart muscle and skeletal muscle) activate both subsystems to a similar extent (it concerns especially Ca(2+)-dependent systems).

Animals↗
PubMed1995

Epidermal growth factor receptor expression by northern analysis and immunohistochemistry in benign and malignant prostatic tumours.

Epidermal growth factor receptor (EGFR) expression in 44 benign prostatic hyperplasia (BPH) and 55 prostatic carcinoma specimens has been investigated using Northern blot analysis and immunohistochemistry. The values obtained for the EGFR mRNA in the BPH and carcinoma specimens were not significantly different and in the latter there was no correlation with grade. In the immunohistochemical assays, two antibodies to the external and one to the internal domain of EGFR were used. The former ones stained the basal cell membranes intensely whilst cytoplasmic staining of secretory epithelium was seen in BPH specimens with the latter. In the carcinoma specimens, the intensity of membrane staining correlated with the two external domain antibodies, r = 0.640, P < 0.001, but neither of these correlated with the EGFR mRNA results. All three antibodies demonstrated a trend towards elevated expression of EGFR with dedifferentiation which reached significance only with the internal domain antibody results, P < 0.02. No correlation was observed with tumour EGFR mRNA values and the EGFR immunohistochemical results. The EGFR immunoreaction with the external domain antibody in 14 treated high-grade tumours was comparable to that obtained in 15 untreated anaplastic prostatic tumours. In 5 patients, both pre- and post-treatment samples were available and these exhibited little or no difference in EGFR expression with therapy.

Blotting, Northern↗
PubMed1995

Use of top-down elasticity analysis to identify sites of thyroid hormone-induced thermogenesis.

Top-down elasticity analysis is a novel extension of top-down metabolic control analysis. It has provided researchers with a theoretical and practical platform upon which quantitative analyses of the sites of action of hormones and drugs can be based. This approach is easy to apply and involves dividing up the metabolic system in question into two or three blocks of enzyme reactions around an intermediate between the blocks of reactions. The kinetic response of each block to the intermediate is measured in the steady state in situ by determining the flux through the block at different measured intermediate concentrations. The intermediate can be manipulated by titrating the other blocks with suitable inhibitors or activators. Then, to determine which blocks of reactions are quantitatively the most important in terms of any change in the flux rate of the system, a quantitative comparison of the titration curves from the experimental preparations is made with those of the control preparations. In this minireview we will examine, as an example, the use of top-down elasticity analysis for the quantitative identification of the important sites of action of thyroid hormones on oxidative phosphorylation in hepatocytes. The experimental results show that approximately 50% of the change in resting oxygen consumption in hepatocytes from hypothyroid and hyperthyroid rats (compared with euthyroid controls) is attributable to changes in the rate of the mitochondrial proton leak; the remaining 50% is accounted for by changes in nonmitochondrial- and ATP turnover-dependent oxygen consumption in hypothyroid and hyperthyroid hepatocytes, respectively.

Adenosine Triphosphate↗
PubMed1995

Cell proliferation in prostatic carcinoma: comparative analysis of Ki-67, MIB-1 and PCNA.

Antibodies to assess the proliferative index of tumours are being increasingly employed together with established markers for prognostic evaluation. This study set out to compare three cell proliferation markers, Ki-67, MIB-1 and PCNA, utilizing a semi-quantitative method of assessment, in 20 human prostatic carcinomas. The streptavidin-biotin immunostaining system was used for the monoclonal antibodies MIB-1 and PCNA and an indirect immunoperoxidase assay for the monoclonal antibody Ki-67. Significant correlations were found between the expression of Ki-67 in frozen tissues and MIB-1 in formal saline-fixed wax-embedded tissues (p = 0.0003); between Ki-67 and PCNA expression in Bouin's-fixed tissues (p < or = 0.0001); and MIB-1 (formalin-saline-fixed tissues) and PCNA (Bouin's-fixed tissues) (p < or = 0.0001). A more intense nuclear staining pattern with less heterogeneity was observed for MIB-1 compared with PCNA, suggesting the antibody of choice, on formal saline-fixed tissues, is MIB-1, which closely correlated with Ki-67, a marker we have previously shown to be of prognostic value in prostatic carcinoma.

Antibodies, Monoclonal↗
PubMed1994

Transforming growth factor beta 1 expression in benign and malignant prostatic tumors.

The expression of transforming growth factor beta 1 (TGF-beta 1) in prostate specimens obtained from patients with benign prostatic hyperplasia (BPH, n = 32) and prostate carcinoma (n = 66) was investigated using Northern blot analysis and immunohistochemistry. Northern blot analysis revealed TGF-beta 1 message (2.5 kb) in virtually all of the samples examined, reflecting the ubiquitous nature of this growth factor. No statistical difference was found between the levels of mRNA detected in benign and malignant tissues due, in part, to the inherent heterogeneity of prostate tissue. Immunohistochemical methods using an antibody to native TGF-beta 1 revealed a novel pattern of immunoreactivity. Staining observed only in certain epithelial cells of benign glands was associated with areas of infection rather than tumorigenesis. Interestingly, intense staining was also seen in polymorphonuclear leukocytes. No correlation was found with the mRNA results, suggesting that this antibody is binding to TGF-beta 1 activated in response to infection rather than detecting sites of synthesis of latent TGF-beta 1.

Blotting, Northern↗
PubMed1994

Hyperthyroidism stimulates mitochondrial proton leak and ATP turnover in rat hepatocytes but does not change the overall kinetics of substrate oxidation reactions.

Thyroid hormones have well-known effects on oxidative phosphorylation, but there is little quantitative information on their important sites of action. We have used top-down elasticity analysis, an extension of metabolic control analysis, to identify the sites of action of thyroid hormones on oxidative phosphorylation in rat hepatocytes. We divided the oxidative phosphorylation system into three blocks of reactions: the substrate oxidation subsystem, the phosphorylating subsystem, and the mitochondrial proton leak subsystem and have identified those blocks of reactions whose kinetics are significantly changed by hyperthyroidism. Our results show significant effects on the kinetics of the proton leak and the phosphorylating subsystems. Quantitative analyses revealed that 43% of the increase in resting respiration rate in hyperthyroid hepatocytes compared with euthyroid hepatocytes was due to differences in the proton leak and 59% was due to differences in the activity of the phosphorylating subsystem. There were no significant effects on the substrate oxidation subsystem. Changes in nonmitochondrial oxygen consumption accounted for -2% of the change in respiration rate. Top-down control analysis revealed that the distribution of control over the rates of mitochondrial oxygen consumption, ATP synthesis and consumption, and proton leak and over mitochondrial membrane potential (delta psi m) was similar in hepatocytes from hyperthyroid and littermate-paired euthyroid controls. The results of this study include the first complete top-down elasticity and control analyses of oxidative phosphorylation in hepatocytes from hyperthyroid rats.

Adenosine Triphosphate↗
PubMed1993

The quantitative contributions of mitochondrial proton leak and ATP turnover reactions to the changed respiration rates of hepatocytes from rats of different thyroid status.

Hepatocytes from hypothyroid rats respire more slowly than those from control animals; cells from hyperthyroid rats respire faster. We have identified the blocks of reactions whose kinetics are significantly affected by thyroid hormones and have quantified the contribution of flux through different blocks of energy-dissipating reactions to the altered oxygen consumption. In cells from hypothyroid rats compared with littermate euthyroid controls, there were significant kinetic effects on non-mitochondrial oxygen consumption reactions and on mitochondrial proton leak but not on ATP turnover. Approximately 50% of the decrease in cellular oxygen consumption of hypothyroid cells was accounted for by a decrease in mitochondrial proton leak rate and approximately 50% by decreased non-mitochondrial oxygen consumption. Metabolic control analysis showed that the distribution of control over oxidative phosphorylation and mitochondrial potential in hepatocytes from hypothyroid animals was broadly similar to its distribution in euthyroid controls. In cells from hyperthyroid rats compared with littermate euthyroid controls, there were significant kinetic effects on mitochondrial proton leak and on the reactions involved in ATP synthesis and consumption but not on non-mitochondrial oxygen consumption. Approximately 50% of the increase in cellular oxygen consumption in hyperthyroid cells was accounted for by an increased mitochondrial proton leak rate and the remainder by increased ATP turnover; there were no changes in non-mitochondrial oxygen consumption.

Adenosine Triphosphate↗
PubMed1993

Control of the effective P/O ratio of oxidative phosphorylation in liver mitochondria and hepatocytes.

The control exerted by substrate oxidation reactions, by ATP turnover and by the proton leak over the oxygen consumption rate, the phosphorylation rate, the proton leak rate and the protonmotive force (delta p) in isolated rat liver mitochondria under a range of conditions between non-phosphorylating (State 4) and maximum phosphorylation (State 3) was investigated by using the top-down approach of metabolic control analysis. The experiments were carried out with saturating concentrations of the substrates succinate, glutamate with malate, or pyruvate with malate. The distribution of control was very similar with each of the three substrates. The effective P/O ratio (i.e. not corrected for leak reactions) was also measured; it varied from zero in State 4 to 80-90% of the maximum theoretical P/O ratio in State 3. Under most conditions control over the effective P/O ratio was shared between proton leak (which had negative control) and the phosphorylating subsystem (which had roughly equal positive control); near State 4, substrate oxidation reactions also acquired some control over this ratio. In resting hepatocytes the effective P/O ratio was only 50% of its maximum theoretical value, corresponding to an effective P/O ratio of only 1.3 for complete oxidation of glucose. The effective P/O ratio for intracellular mitochondrial oxygen consumption was 64% of the maximum value. The control coefficient of the mitochondrial proton leak over the effective P/O ratio in cells was -0.34; the control coefficient of phosphorylation reactions over this ratio was 0.31 and the control coefficient of substrate oxidation reactions over the ratio was 0.03, showing how the coupling efficiency in cells can respond sensitively to agents that change the proton leak or the ATP demand, but not to those that change substrate oxidation.

Adenosine Triphosphate↗
PubMed1993

Altered brown adipose tissue and Na,K pump activities during diet-induced obesity and weight loss in rats.

Brown adipose tissue (BAT) thermogenesis is an uncoupled ATPase-independent thermogenic mechanism. Ion transport by the Na,K pump is an ATPase-dependent thermogenic mechanism. Both have been proposed as mechanisms of altered energy expenditure during states of dietary energy surfeit and deficit. Our aim was to study these mechanisms during diet-induced obesity and weight loss. Over 36 weeks rats were fed lard- or tallow-based diets (63% energy as fat), or a control diet (12% energy as fat). During periods of restriction rats were fed 50% of the energy intake of controls in the form of a control diet. Several components of thermogenic response increased in rats eating high fat diets and decreased following dietary restriction. BAT activation occurred, particularly with a lard-based diet, as indicated by increased GDP binding and uncoupling protein (UCP) content. Na,K pump activity in thymocytes increased with the feeding of both high fat diets at some time points. Plasma T3 level increased in rats eating the lard-based diet and decreased with dietary restriction regardless of previous diet. Resting metabolic rate (RMR) of the animals was unchanged despite increases in these thermogenic components and was decreased in all groups following dietary restriction. Our results indicate a lack of any major role for activated BAT thermogenesis in mitigating the extent of the obesity induced by the high fat diets. The reasons for the differences in response to the two different sources of saturated fat, lard, and tallow, are not clear.

Adenosine Triphosphatases↗
PubMed1993

An immunocytochemical analysis of TGF alpha expression in benign and malignant prostatic tumors.

Transforming growth factor alpha (TGF alpha) expression was analyzed immunocytochemically on formalin-fixed wax-embedded sections obtained from 24 benign prostatic hyperplasia (BPH) specimens and 76 prostatic carcinoma tissues, 3 human prostatic tumor xenografts, normal kidney, and salivary gland. Low amounts of TGF alpha immunopositivity were encountered in the epithelium of BPH glandular tissues, whereas in the prostatic adenocarcinoma samples, a greater heterogeneity and intensity of TGF alpha immunostaining was observed. The most intense staining was exhibited by the least differentiated tumors, although a few of these were weakly stained. Statistical analysis of the relationship of histopathological grade of tumor with TGF alpha expression in the carcinomas showed a significant correlation of these parameters, 0.01 > P > 0.001. The expression of the proliferation markers Ki-67 and PCNA was also analyzed in the carcinoma specimens, and the relationship of these to TGF alpha expression indicated that there was no significant correlation in this series of tumors between increased growth activity and TGF alpha expression (p approximately 0.25 with both markers). The prostatic carcinoma xenografts TEN12 and TEN15 contained low levels of immunoreactive TGF alpha, which was uniformly distributed, whilst heterogeneous immunostaining was observed in the uroepithelial xenograft TEN16. In the normal human kidney, TGF alpha was concentrated in the epithelium of the distal convoluted tubules (DCT) and the collecting tubules (CT), and lower amounts were identified in the proximal convoluted tubules (PCT). As in the prostatic carcinomas, the immunostaining was eliminated by prior absorption of the antibody with pure TGF alpha and not with human or mouse EGF. No crossreactivity of the TGF alpha antibody with salivary EGF was demonstrated. This study concludes that, in prostate carcinoma, the least differentiated tumors more often expressed greater amounts immunoreactive TGF alpha; however, no relationship between TGF alpha expression and cellular proliferation markers was found.

Adenocarcinoma↗