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C L Brooks

Publications and source records attributed to C L Brooks.

107 records · Page 6Linked to original sources
PubMed1983

Alloxan inhibition of a Ca2+- and calmodulin-dependent protein kinase activity in pancreatic islets.

Alloxan was found to inhibit a Ca2+- and calmodulin-dependent protein kinase recently identified in pancreatic islets. This effect of alloxan may be specifically related to the inhibitory action of alloxan on insulin secretion from islets since: 1) in islet-cell subcellular fractions, alloxan at micromolar concentrations irreversibly inhibits the Ca2+- and calmodulin-dependent protein kinase activity; 2) pretreatment of intact islets with alloxan at concentrations that inhibit insulin secretion similarly inhibits the protein kinase activity; and 3) alloxan inhibition of both insulin secretion and protein kinase activity in intact islets can be prevented by D-glucose. This inhibition by alloxan appears to be a direct effect on the enzyme since alloxan treatment of either the islet homogenate or the microsomal fraction enriched in protein kinase activity inhibited the kinase activity with similar concentration dependence. These results suggest that alloxan-induced inhibition of a Ca2+- and calmodulin-dependent protein kinase may represent a critical inhibitory site which mediates alloxan-induced inhibition of insulin secretion.

Alloxan↗
PubMed1983

Correlation of Ca2+-and calmodulin-dependent protein kinase activity with secretion of insulin from islets of Langerhans.

A Ca2+-activated and calmodulin-dependent protein kinase activity which phosphorylates predominantly two endogenous proteins of 57kDa and 54kDa was found in a microsomal fraction from islet cells. Half-maximal activation of the protein kinase occurs at approx. 1.9 microM-Ca2+ and 4 micrograms of calmodulin/ml (250 nM) for phosphorylation of both protein substrates. Similar phosphoprotein bands (57kDa and 54kDa) were identified in intact islets that had been labelled with [32P]Pi. Islets prelabelled with [32P]Pi and incubated with 28 mM-glucose secreted significantly more insulin and had greater incorporation of radioactivity into the 54 kDa protein than did islets incubated under basal conditions in the presence of 5 mM-glucose. Thus the potential importance of the phosphorylation of these proteins in the regulation of insulin secretion is indicated both by activation of the protein kinase activity by physiological concentrations of free Ca2+ and by correlation of the phosphorylation of the substrates with insulin secretion in intact islets. Experiments undertaken to identify the endogenous substrates indicated that this calmodulin-dependent protein kinase may phosphorylate the alpha- and beta-subunits of tubulin. These findings suggest that Ca2+-stimulated phosphorylation of islet-cell tubulin via a membrane-bound calmodulin-dependent protein kinase may represent a critical step in the initiation of insulin secretion from the islets of Langerhans.

Animals↗
PubMed1982

Biochemical and immunohistochemical characterization of prolactin binding in rat ventral, lateral, and dorsal prostate lobes.

A radioligand assay and the soluble peroxidase-antiperoxidase (PAP) technique were used to compare ovine and rat prolactin as ligands for the demonstration of prolactin binding sites in rat ventral, lateral, and dorsal prostate lobes. Biochemical techniques were effective in demonstrating prolactin binding sites in ventral prostate with both ovine and rat prolactin; however, binding sites could not be characterized in lateral or dorsal lobes with either ligand. Using the PAP technique, rat prolactin consistently produced a discrete, dense reaction product over binding sites in the Golgi zone of the ventral prostate; ovine prolactin produced a more dispersed pattern of staining throughout the entire supranuclear region of the cells. In ventral prostate, most epithelial cells were immunoreactive when either ligand was used. However, lateral prostate contained fewer densely immunoreactive cells than did ventral prostate, and rat prolactin was more effective in demonstrating these binding sites than was ovine prolactin. Dorsal prostate epithelium was highly immunoreactive for binding of both ligands; however, there was considerably more staining in this lobe that remained after absorption of the antibody with prolactin. These data suggest that the inability to demonstrate prolactin binding sites in the lateral lobe using radioligand assay may have been due to the fewer numbers of binding sites that were diluted below the sensitivity of the assay when a tissue homogenate preparation was used. Noncompetitive binding in the dorsal lobe, as seen in the radioligand assay, coincided with immunocytochemical findings of relatively high nonabsorbable background staining.

Animals↗
PubMed1982

Calculating specific binding for prolactin receptor assays.

Specific binding of [125I]iodo-prolactin and receptor is usually calculated by subtracting the amount of bound radioactivity of assays containing excess unlabeled hormone (competed) from that of noncompeted assays. A proportional method to calculate specific binding of prolactin-receptor or other hormone-receptor interactions is introduced as a replacement for the currently used subtraction method. The proportional method calculates specific binding by multiplying the ratio of free radioactivities of noncompeted and competed assays by the radioactivity retained in the competed assay and subtracting this value from the radioactivity retained by the noncompeted assay. In prolactin-receptor assays greater specific binding is seen when data is calculated by this proportional method rather than the subtraction method. The amount of this increase relates directly to the levels of specific and nonspecific binding. Additionally, the binding characteristics of prolactin and receptor in both equilibrium and kinetic studies are significantly different when specific binding is calculated by the proportional method rather than the subtraction method.

Animals↗
PubMed1982

A survey of calmodulin-activated protein kinase activity in several tissues of Rattus rattus.

1. In each of five tissues (brain, heart, spleen, mammary acini and pancreatic acini) the prominent endogenous protein substrate possessed a molecular weight between 51,900 and 56,800 on SDS gel electrophoresis. 2. Evidence was obtained for two species of protein substrate, differing slightly in molecular weight, which appear to be distributed in a tissue-specific manner. 3. One species, with a molecular weight of 52,900, was found in spleen, mammary acini and brain; the other species, with a molecular weight of 56,200, was found in heart and pancreatic acini. 4. The specific activity of calmodulin-activated protein kinase in homogenates varied from a high of 44 pmol/min/mg in brain to 4.2 pmol/min/mg in mammary acini. 5. Subcellular fractionation of these tissues demonstrated that most of the activity was found in the cytosolic fraction and a "light-particle" fraction obtained by ultracentrifugation, but the kinase was not associated with endoplasmic reticulum. 6. High concentrations of calmodulin were required to activate the protein kinase activity from each tissue. 7. Calmodulin concentrations producing half-maximal activation were 94 nM for brain, 377 nM for spleen, 132 nM for pancreatic acini, 350 nM for heart, and 117 nM for mammary acini. 8. The calmodulin-activated protein kinase activity in these tissues were similar, but the few differences in properties from tissue to tissue left open the possibility that multiple, calmodulin-activated kinase activities exist in these tissues.

Animals↗
PubMed1982

Characterization of [125I]-iodo-ovine prolactin and evaluation of prolactin receptor assay methods.

In an attempt to establish more rigorous conditions for assay of prolactin receptor, a study of the preparation, recovery and specific binding of iodoprolactin was conducted. Averaging the results of 20 preparations starting with 5 micrograms prolactin preparation and iodinated by a lactoperoxidase method, an average of 0.62 +/- 0.12 micrograms of the iodinated prolactin was recovered in a form capable of binding to excess rat liver membrane preparations, corresponding to between 24 and 51% of the total recovered iodinated prolactin. Based on preliminary amino acid analysis and N-terminus determination, 53% of a weighed amount of ovine prolactin was determined to be protein with the N-terminal amino acid being consistent with homogeneity. The percent specific binding assay (single point assay) and Scatchard analysis were tested for their ability to give assay results in a direct relationship to sample receptor. Scatchard analyses were determined to be a superior method to the percent specific binding assay. Using commonly employed conditions for the percent specific binding assay, the relative amounts of receptor found in increasing amounts of sample were significantly less than that found by Scatchard analysis in all likelihood due to the use of subsaturating concentrations of iodohormone. As more hormone was added per assay tube, the relationship of sample receptor to the results of the single point assay improved, but the results never equaled those of Scatchard analyses. Thus, many workers who have used the percent specific binding assay under nonsaturating conditions have underestimated the relative receptor contents of samples.

Animals↗
PubMed1979

Inhibition of mammary tumorigenesis in carcinogen-treated Lewis rats by suppression of prolactin secretion.

One hundred sixty-eight female Lewis rats were treated intragastrically with 10 mg 7,12-dimethylbenz[a]anthracene at 56 and 63 days of age. Pituitary prolactin secretion was suppressed in one-half of these rats by daily sc administrations of 2-bromoergocryptine mesylate (CB-154; 0.4 mg/100 g body wt) from 29 to 90 days of age (series 1) and from 90 to 140 days of age (series 2). Treatment with CB-154 was initiated prior to the onset of palpable mammary carcinomas. Control rats were given injections of saline. Inguinal mammary glands were excised from 10 control and 10 CB-154-treated rats at the cessation of saline and CB-154 treatments and examined for hyperplastic nodules (HN). The remaining rats were palpated weekly for mammary carcinomas (MC) and killed at 200 days of age. Mean number of HN per rat, mean number of MC per rat, and percent of rats with MC were, respectively: series 1--controls, 0.6, 1.5, and 68; CB-154 treatment, 0.5, 1.1, and 62; series 2--controls, 10.4, 2.0, and 94; CB-154 treatment, 5.1, 1.1, and 56. The number of HN and MC was only slightly reduced in rats when prolactin was suppressed during carcinogen treatment (series 1) but markedly reduced when prolactin was suppressed after carcinogen treatment (series 2). These results provide evidence that prolactin is involved in the early development of mammary dysplasias in the carcinogen-treated female Lewis rat.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1977

17beta-oestradiol and Enovid mammary tumorigenesis in C3H/HeJ female mice: counteraction by concurrent 2-bromo-alpha-ergocryptine.

Chronic administration of 17beta-oestradiol (via drinking water) or the oral contraceptive Enovid (norethynodrel and mestranol) (0-1 mg injected s.c. twice weekly) to nulliparous C3H/HeJ female mice, beginning at one month of age and terminating at 20 months (17beta-oestradiol) or 22 months (Enovid), significantly increased the incidence of mammary tumours over solvent-treated controls. Concurrent treatment of the steroid-treated mice with 2-bromo-alpha-ergocryptine (CB-154) (0-1 mg s.c. injected daily) significantly reduced mammary tumour incidence and mammary hyperplastic nodule development to the control level. CB-154 is an efficacious inhibitor of pituitary prolactin secretion. These results demonstrate that steroid-induced mammary gland dysplasias can be sharply reduced by chronic CB-154 treatment, and suggest that some of the mammary tumorigenic activities of oestrogenic steroids in C3H mice are mediated via an increased secretion of pituitary prolactin.

Animals↗
PubMedDate not supplied

Effect of the association time of in vivo bound prolactin on the [125I]prolactin receptor assays of female rat livers.

Significantly (P less than 0.01) reduced 125I-labeled ovine prolactin binding (mean, --22%), as a result of an ether anesthesia-reduced rise in serum prolactin, was observed in plasma membrane preparations of liver samples of female rats taken after 5 min of etherization when compared to samples taken from the same animals during the first minute of etherization. This reduction in assayable receptors occurred after 1 h but not 2 h of assay incubation time. Significantly (P less than 0.05) reduced 125I-labeled ovine prolactin binding (mean, --16%) was also observed in liver samples exposed to a 30-min ether-induced rise in serum prolactin when compared to liver samples taken during the first minute of etherization. In contrast, this reduction was apparent at assay incubation times of 1, 2 and 4 h but not at 10 h. These results suggest that serum prolactin can bind to prolactin receptors in vivo and partially block subsequent 125I-labeled ovine prolactin receptor assay. In addition, these data provide evidence that a complex time-dependent binding of prolactin may occur in the plasma of the female rat liver.

Animals↗
PubMedDate not supplied

Pathologic effects of butylated triphenyl phosphate-based hydraulic fluid and tricresyl phosphate on the adrenal gland, ovary, and testis in the Fischer-344 rat.

Triaryl phosphates including tricresyl phosphate (TCP) and butylated triphenyl phosphates (BTPs) are used in the commercial manufacture of plastics, lubricants, and hydraulic fluids. Recent reports implicate these compounds as endocrine and reproductive toxicants in rodents. The objectives of this study were to develop and characterize a rat model to investigate the mechanism(s) of toxicity of triaryl phosphate-based hydraulic fluids and to elucidate potential mechanistic pathways of toxicity through studies of structural/functional relationships. Groups of male and female rats received daily oral doses of either sesame oil alone or 1.7 g/kg of BTP or 0.4 g/kg TCP in sesame oil vehicle or 2.8 g/kg neat BTP for 20, 40, and 60 days. Light microscopic, morphometric, ultrastructural, and histochemical studies revealed hypertrophy and cholesteryl lipidosis of adrenocortical (both sexes) and ovarian interstitial cells that were progressive with duration of exposure. Minimal degeneration was observed in the adrenal cortex and ovary. TCP caused the most severe lesions in both the adrenal gland and ovary, but the morphologic and histochemical changes produced were similar for both compounds, suggesting a common mechanism of toxicity. Decreased testicular weight and degeneration of seminiferous tubules were detected only in TCP-treated rats. The Fischer-344 rat model using TCP and BTP administered by gavage is a valuable system to study mechanisms of endocrine and reproductive toxicity induced by triaryl phosphate-based hydraulic fluids.

Adrenal Cortex↗