PubMed Health⌕ Search

Biomedical subjects

R A Knazek

Publications and source records attributed to R A Knazek.

At least 19 recordsLinked to original sources
PubMed1991

Inhibitory effects of gossypol on adrenal function.

Gossypol, an antifertility agent, has inhibitory actions on many membrane-associated enzymes, suggesting that this agent might have a generalized effect on cell membranes. This hypothesis was examined in the present study using membranes and dispersed cells prepared from human and rat adrenal glands. Four parameters were determined: microviscosity as measured by fluorescence polarization of human adrenal microsomal- and mitochondrial-enriched membranes, adrenal steroidogenic enzymes; and cAMP and cortisol responses to ACTH. It was found that gossypol increased the polarization constants of microsomes and mitochondria in a dose-dependent manner. Of the three adrenal enzymes tested, both 3 beta-hydroxysteroid dehydrogenase delta 5-delta 4 isomerase and 11-hydroxylase were inhibited by gossypol, but not 21-hydroxylase. Using intact human adrenocortical cells, high doses of gossypol also inhibited the ACTH-stimulated cAMP and cortisol levels. The in vivo corticosterone response to ACTH in rats subjected to chronic gossypol treatment was also found to be reduced. These findings suggest that gossypol has multiple effects on adrenal function. Its effects on membrane microviscosity, adrenal steroidogenesis, cAMP and corticosterone responses to ACTH stimulation probably occur through a generalized membrane effect.

Adrenal Glands↗
PubMed1990

Culture of human tumor infiltrating lymphocytes in hollow fiber bioreactors.

Human tumor infiltrating lymphocytes (TIL) from metastatic melanoma of six patients were grown using a new hollow fiber bioreactor system. After inoculating 0.35-10 X 10(8) TIL into the extra-fiber space (EFS), each Cellmax bioreactor was perfused with AIM-V medium, supplemented with rIL-2. The cells subsequently expanded 124-1170-fold to yield 1.5-5.4 X 10(10) TIL over a 14-32 day period. TIL were flushed from the EFS using 200 ml medium and possessed an average viability = 91%. The phenotype and the autologous tumor cell lytic capacity of these TIL were similar to those of TIL grown in the currently used gas-permeable culture bags. Tissue culture media use averaged 4.3 liters/10(10) TIL harvested. The TIL of one patient were re-expanded twice from cells remaining within the same bioreactor after harvest suggesting that one bioreactor cartridge could be used for repetitive, periodic studies. An estimated 80% decrease in technical time expended and in incubator space requirements were realized using this methodology. Cell culture on hollow fibers appears to be a useful method for producing large quantities of primary human lymphocytes for experimental, and perhaps, therapeutic needs.

Cells, Cultured↗
PubMed1989

An in vitro and in vivo study of antitumor effects of gossypol on human SW-13 adrenocortical carcinoma.

The present study investigated the in vitro and in vivo antitumor effects of gossypol on human SW-13 adrenocortical carcinoma cells. In vitro gossypol concentrations greater than or equal to 0.5 microM reduced the growth rate of the SW-13 cells. Membrane microviscosity was determined by fluorescence polarization of diphenylhexatriene. The membranes of viable SW-13 cells exposed to gossypol became more rigid after a 1-day exposure to gossypol, the polarization constant, P, increasing from 0.229 to 0.352. Gossypol also increased the microviscosities of isolated mitochondrial and microsomal enriched membrane preparations. Tumor was also transplanted into nude mice by s.c. injection of SW-13 cells. A 1-week pretreatment period followed by daily administration of gossypol in which 30 mg gossypol/kg body weight/day was administered via orogastric tube delayed the onset of visible tumor in the subsequent weeks. Five weeks after transplantation, tumor prevalence rate was 95.8% in the control group and 54.5% in the gossypol-treated group. A second experiment, consisting of 12 weeks of gossypol treatment, reduced a preexisting 71% tumor prevalence to 54% while the tumor prevalence increased to 83% in the control group. This was accompanied by a 41.6% mortality in the control group versus 8.3% in the gossypol-treated group. These data suggest that gossypol may provide a beneficial effect in patients with adrenocortical carcinoma.

Adrenal Cortex Neoplasms↗
PubMed1988

Receptor-mediated effects of glucocorticoids on inflammation: enhancement of the inflammatory response with a glucocorticoid antagonist.

Glucocorticoids suppress the inflammatory response by altering leukocyte traffic and function, cytokine secretion and action, and phospholipid metabolism. We employed the glucocorticoid receptor antagonist RU 486, to examine whether glucocorticoids suppress the inflammatory response through a receptor-mediated mechanism and whether basal glucocorticoid secretion exerts antiinflammatory effects in the resting (non-stress) state. To test these hypotheses we evaluated the effects of increasing doses of dexamethasone, RU 486, or dexamethasone plus RU 486 on the exudate volume and concentrations of leukocytes, prostaglandin E2, (PGE2) and leukotriene B4 (LTB4) in intact rats that received subcutaneous carrageenin. Exudate volume, leukocyte concentration and LTB4 and PGE2 levels were all suppressed by dexamethasone in a dose-dependent fashion (P less than 0.005). RU 486 was able to antagonize fully the suppressive effects of dexamethasone on the inflammatory response (P less than 0.001) and to cause increases of exudate volume and leukocyte, PGE2 and LTB4 concentrations when given alone (P less than 0.05). These increases ranged between 30 and 100% above the basal inflammatory response. We conclude that glucocorticoids most likely suppress the inflammatory response by a glucocorticoid receptor-mediated mechanism and under basal conditions exert tonic antiinflammatory effects.

Alprostadil↗
PubMed1988

Effects of long-chain, saturated fatty acids on membrane microviscosity and adrenocorticotropin responsiveness of human adrenocortical cells in vitro.

Adrenoleukodystrophy (ALD) and adrenomyeloneuropathy are inherited disorders in which long-chain, saturated fatty acids (LCFA) accumulate in various tissues. A mechanism by which LCFA cause the endocrine and neurological dysfunction characteristic of these diseases is proposed based on in vitro response of human adrenocortical cells to ACTH in the presence of various fatty acids. Human adrenocortical cells cultured in the presence of 5 microM hexacosanoic (C26:0) or lignoceric (C24:0) acids showed decreased basal and ACTH-stimulated cortisol release compared with cells cultured without exogenous fatty acids or in the presence of linoleic acid (C18:2). Measurement of fluorescence polarization demonstrates a significant increase in the membrane microviscosity of cells cultured in the presence of LCFA. It is hypothesized that cells exposed to LCFA have increased membrane microviscosity with a consequent decrease in their ability to respond to ACTH. This decrease in trophic support may contribute to the adrenal insufficiency and atrophy in patients with ALD.

Adrenal Cortex↗
PubMed1988

Monocytes stimulate cortisol production by cultured human adrenocortical cells.

Studies using cultured human adrenocortical cells were performed to determine if the immune modulation of glucocorticoid production previously described in animal studies also occurs in humans. Human monocytes significantly increased (P less than 0.001) cortisol production from adrenocortical cells after 24, 48, and 72 h of culture. This stimulation was not CRH dependent, and the presence of CRH alone did not augment cortisol production. The active factor was soluble, since supernatants of monocyte cultures stimulated cortisol production by adrenocortical cells; ACTH levels in these supernatants were undetectable by RIA. While interleukin-1 also stimulated cortisol production by adrenal cells, the degree of stimulation was only 30% of that in the monocyte experiments. In contrast to previous animal studies, we found that human monocytes stimulate cortisol production by human adrenocortical cells by a factor that is neither CRH dependent nor mediated by immunoreactive ACTH-(1-24) or ACTH-(1-39).

Adrenal Cortex↗
PubMed1985

Prostaglandin and hydroxyeicosatetraenoic acid synthesis by human mesenchymal tumors.

The metabolism of arachidonic acid was investigated by radioimmunoassay and chromatographic techniques in 5 sarcomas and one embryonal carcinoma of human origin maintained as transplantable tumors in nude mice. The results obtained indicate that: the absolute quantities of arachidonic acid metabolites produced by a given tumor varied between experiments but the overall distribution pattern of these products, in general, remained constant from passage to passage; each tumor showed a different arachidonic acid metabolite profile in quality and quantity; 2 sarcomas of the same histological type could be clearly distinguished by their arachidonic acid metabolites; the predominant product in all tumors was 12-HETE or 15-HETE, whereas thromboxane A2 was synthesized in low quantities by all tumors; PGF2 alpha was synthesized at the highest rate by an alveolar rhabdomyosarcoma; PGE2 synthesis was highest in a malignant fibrous histiocytoma; and total prostaglandin synthesis was low in the chondrosarcoma and synovial-cell sarcomas. All results reported in this study are for the complete tumor which includes both neoplastic and stromal cells. The role that these products play in the biological behavior of mesenchymal tumor cells and normal tissues of the host remains to be determined.

Animals↗
PubMed1983

The effect of copper on the binding of prostaglandin E1 to NMU-induced rat mammary carcinoma membranes.

Mammary carcinomata were induced by administration of nitrosomethyl urea to female Buffalo rats. Specific binding of PGE1 to the 15,000 xg membranes obtained from these tumors was maximal after approximately 60 minutes of incubation at 15 degrees C. An excess of unlabeled PGE1 dissociated 50% of the [3H]PGE1 from the membrane within 15 minutes. The binding of [3H]PGE1 was inhibited by various prostaglandins in a concentration-dependent manner, the order of potency being PGE1 greater than PGE2 greater than PGA2 greater than PGF2 alpha greater than PGB2. High affinity receptor sites were not definable by Scatchard analysis until 5mM CuCl2 was added. This resulted in the detection of both high and low affinity receptors (Kd = 1.01 nM and 21 microM) having binding capacities of 29 and 357 fmole/mg protein, respectively. Addition of CaCl2 or MgCl2 did not result in a significant increase in the specific binding of PGE1 to the receptors. These studies provide a means by which high affinity prostaglandin E1 receptors can be detected in neoplastic tissue and suggest a mechanism by which copper ions can increase the effect of PGE1 in tissues.

Alprostadil↗
PubMed1983

Membrane microviscosity is increased in the erythrocytes of patients with adrenoleukodystrophy and adrenomyeloneuropathy.

Adrenoleukodystrophy (ALD) and adrenomyeloneuropathy (AMN) are related X-linked disorders characterized by adrenal, gonadal, and nervous system dysfunction. While the pathologic finding common to these tissues appears to be the accumulation of excessive amounts of very long chain fatty acids, the mechanism leading to functional impairment in these tissues is unclear. Measurements of fluorescence polarization (P), using the lipid probe diphenylhexatriene, demonstrate a highly significant increase in the microviscosity of erythrocyte membranes in affected patients (P = 0.286 +/- 0.012) vs. normals (P = 0.239 +/- 0.020). Analyses of these membranes by gas-liquid chromatography revealed 1.9-, 1.6-, and 1.3-fold increases above normal values in the C25:0, C26:0, and C27:0 fatty acids, respectively. These observations are compatible with previously obtained data in animals that correlate membrane microviscosity with the number of hormone receptors in target tissues. The present data support the thesis that a decrease in responsiveness to trophic hormones in ALD and AMN is secondary to changes in the membrane microviscosity of the target tissues and suggest a mechanism by which adrenal and gonadal failure occur in such patients.

Adrenal Insufficiency↗
PubMed1983

Prolactin-binding capacity, prostaglandin synthesis and fluidity of murine hepatic membranes are modified during pregnancy and lactation.

The objective of these studies was to determine whether the changes observed in prolactin-binding capacity of mouse liver microsomal membranes during pregnancy and lactation correlated with those observed in the fluidity and prostaglandin (PG)-synthesizing activity of these membrane preparations. Prolactin-binding capacity increased with the advance of pregnancy to reach a peak at 16 days of gestation and declined thereafter to non-pregnant, non-lactating (NPNL) levels. Membrane microviscosity, studied by fluorescence polarization, was significantly decreased throughout early gestation but returned to NPNL levels by day 20 of gestation and remained unchanged thereafter. The amount of PGE synthesized in vitro by these membranes increased during gestation to reach a peak at 12 days of gestation but declined thereafter to below NPNL levels on the day of parturition and returned to NPNL levels by day 20 of lactation. Synthesis of PGF2 alpha remained at a higher level from day 12 to day 18 followed by a decline in activity at parturition to NPNL levels. These changes and other data suggest an interrelationship of receptor levels, fluidity and PG synthesis during pregnancy. Such modifications in local PG synthesis may influence the fluidity of these membranes, which ultimately play a significant role in maximal exposure of membrane receptors during pregnancy.

Animals↗
PubMed1982

Prostaglandin synthesis and binding is increased in regressing NMU mammary carcinoma.

Mammary tumors induced in Buffalo rats by treatment with nitrosomethyl urea will regress after oophorectomy. Their ability to synthesize and bind prostaglandins E and F2 alpha was studied in the growing and regressing states. Prostaglandins present in suspension of 100,000 xg tumor membranes after 2 hr incubation at 37 degrees C +/- 5x10(-4)M indomethacin were partially purified by silica gel column chromatography before assay by specific PG RIA. The amounts of PGE and F2 alpha synthesized rose from 0.13 and 10.5 ng/mg protein in the growing tumors to a maximum of 1.2 and 26.5 ng/mg protein 5 days after oophorectomy. Specific binding of 3H-PGE2 ad 3H-PGF2 alpha to 15,000 xg tumor membranes was achieved during a 45 min incubation at 23 degrees C +/- excess unlabelled PG. Free and bound prostaglandins were separated by filtration. Binding reached equilibrium after 30 min, was saturable and reversible. Scatchard analysis revealed high affinity binding of PGF2 alpha but only low affinity PGE2 binding in membranes obtained from growing tumors. A 2-3-fold increase in specific binding of PGE2 and PGF2 alpha was noted at 4 days after oophorectomy which represented an increase in the number of PGF2alpha receptors. PGE2 binding retained a low affinity character. The elevated PGF2 alpha synthesis rates observed in the regressing tumors coupled with a regression-associated increase in receptor number suggests that PGF2 alpha plays a significant role in hormone-dependent mammary tumor regression.

Animals↗
PubMed1982

A rapid method for the measurement of prostaglandin receptors.

A method is presented which provides for a simple and rapid determination of prostaglandin receptors in large numbers of tissue samples. Binding of prostaglandin F2 alpha, to preparations of microsomal membranes obtained from the ovaries of PMSG/hCG-treated rats served as the model system for this study. After incubating the membranes with [3H]-PGF2 alpha +/- graded concentrations of PGF2 alpha for 90 min at 22 degrees C, dextran-coated charcoal was added to adsorb the free PGF2 alpha during a 10 minute period. Centrifugation of the mixture for 3 minutes atop a 2.2M sucrose cushion resulted in the charcoal pelleting to the bottom while the [3H]PGF2 alpha-membrane complex remained at the interface. There was no further significant diffusion of PGF2 alpha after centrifugation, enabling large numbers of samples to be handled within a short period of time. The binding data revealed both high and low affinity receptors: Kd = 5.8 nM and 77 nM with binding capacities of 360 and 4000 fmole/mg protein. Both PGE1 and PGF1 alpha partially displaced [3H]PGF2 alpha from the binding sites whereas PGA2 was without effect. Binding equilibrium was reached within 90--140 min. The results are compatible with those of investigators using other techniques.

Animals↗
PubMed1981

Virus-induced diabetes mellitus. XX. Polyendocrinopathy and autoimmunity.

Mice infected with reovirus type 1 developed transient diabetes and a runting syndrome. The diabetes was characterized by hyperglycemia, abnormal glucose tolerance tests, and hypoinsulinemia. Inflammatory cells and viral antigens were found in the islets of Langerhans, and virus particles were seen in alpha, beta, and delta cells. The runting syndrome consisted of retarded growth, oily hair, alopecia, and steatorrhea. Inflammatory cells and viral antigens were found in the anterior, but not posterior pituitary. Electron microscopy revealed virus particles in growth hormone (GH)-producing cells and radioimmunoassay showed that the concentration of GH in the blood was decreased. Examination of sera from infected mice revealed autoantibodies that, by immunofluorescence, reacted with cytoplasmic antigens in the islets of Langerhans, anterior pituitary, and gastric mucosa of uninfected mice. Absorption studies and enzyme-linked immunosorbent assays designed to identify the reactive antigens showed that some of the autoantibodies were directed against insulin and others against GH. Reovirus type 3, in contrast to reovirus type 1, did not induce autoantibodies to GH. By use of recombinant viruses, the segment of the reovirus genome responsible for the induction of autoantibodies to GH was identified. Virus containing the S1 gene segment from reovirus type 1, which codes for the sigma 1 polypeptide (i.e., hemagglutinin), infected cells in the anterior pituitary and induced autoantibodies to GH, whereas virus containing the S1 gene segment from reovirus type 3 failed to infect cells in the anterior pituitary and did not induce autoantibodies to GH. We conclude that reovirus type 1 infection can lead to polyendocrinopathy and autoimmunity and that the S1 gene segment is required for the induction of autoantibodies to GH.

Animals↗
PubMed1981

Indomethacin causes a simultaneous decrease of both prolactin binding and fluidity of mouse liver membranes.

Indomethacin suppressed the numbers of prolactin receptors detectable in the liver membranes of both male and female C3H mice. This occurred in a dose-dependent fashion with 7.5 mug/gm body weight injected every 4 hours exerting a maximal effect within 20 hours. While injection of 50 mug prolactin every 4 hr increased the number of prolactin receptors in control animals it could not in the indomethacin-treated animals. Membrane fluidity was estimated by fluorescence polarization techniques using the lipid probe 1, 6-diphenylhexatriene. Indomethacin caused a decrease in membrane fluidity, whereas, exogenous prolactin increased the fluidity of the recipients' liver membranes but again could not overcome these suppressive effects of indomethacin. The data suggest that prolactin induces its own membrane-associated receptor by means of the prostaglandin cascade, perhaps by altering the fluidity of the supporting lipid bilayer.

Animals↗