Biomedical subjects

T J Brown

Publications and source records attributed to T J Brown.

At least 19 recordsLinked to original sources

PubMed2026

Age-stratified mutation patterns in early-onset colorectal cancer reveal distinct molecular features and therapeutic implications.

BACKGROUND: Colorectal cancer (CRC) is increasingly diagnosed in younger adults, with evidence that early-onset cases (age <50 years) differ in the spectrum of prevalent gene mutations compared with older individuals. To evaluate how these age-related differences may inform testing guidelines and therapeutic development, we examined mutation rates of the most prevalent gene mutations across four age-stratified cohorts. PATIENTS AND METHODS: Clinicogenomic data were obtained from Memorial Sloan Kettering Center for Harmonized Onco-genomic Research Dataset and China Pan-Cancer cohorts available in cBioPortal. A total of 6762 samples were analyzed. Mutation frequencies for a comprehensive panel of the 100 most prevalent CRC genes were compared across four age groups: 18-29 (n = 79), 30-39 (n = 402), 40-49 (n = 1064), and ≥50 (n = 5217) using chi-square analysis. False discovery rate (FDR) correction for multiple comparisons was carried out using Benjamini-Hochberg procedure. RESULTS: Statistically significant variation in mutation frequency across age groups was seen in 22 key genes. APC mutations increased with age and were seen in 49.4% of patients in the 18-29 group, 69.7% in 30-39, 73.3% in 40-49, and 75.25% of patients ≥50 (P < 0.001, FDR < 0.001). The oldest cohort was more than three times more likely to have an APC mutation than the youngest [odds ratio (OR) = 3.74, 95% confidence interval (CI) 2.44-5.74, P < 0.001]. In contrast, SMAD4 mutations were twice as common in the youngest age group at 31.6% compared with those over 40, with a prevalence of 17.29% in patients 40-49, and 18.84% in patients over 50 (OR = 2.03, 95% CI 1.26-3.27, P < 0.001, FDR < 0.001). POLE mutations peaked in the 30-39 age group with a prevalence of 10.7% compared with 6.3% in patients aged 18-29, 4.9% in patients aged 40-49, and 5.9% in patients aged ≥50 (P < 0.001, FDR < 0.001). Individuals in the 30-39 group were nearly twice as likely to carry a POLE mutation compared with those over 40 (OR = 1.96, 95% CI 1.41-2.74, P < 0.001). CONCLUSIONS: Differences in mutations of key genes including a lower prevalence of APC mutations and increased SMAD4 mutations in younger individuals provides further supporting evidence that early-onset CRC may represent a distinct biological subtype of CRC. Enrichment of POLE mutations in younger patients highlights the importance of expanded molecular profiling in early-onset CRC, which could help identify patients most likely to benefit from immunotherapy and advance personalized treatment strategies in CRC. Together, these findings reinforce the need to approach early-onset CRC as a distinct biological entity and ensure that appropriate molecular assays are incorporated to guide care.

APC↗

PubMed1995

Localization and measurement of occupied androgen receptors in thaw-mounted rat and human prostate tissue sections by in vitro autoradiography.

In this paper we present an in vitro exchange binding assay procedure for measurement of androgen receptors in slide-mounted tissue sections. This method allows quantitative autoradiographic determinations with an anatomical resolution approaching the cellular level. Tissue sections are incubated with the synthetic androgen [3H]R1881 in the presence of triamcinolone acetonide to suppress possible binding of the radioligand to the progestin receptor. Adjacent tissue sections are incubated with [3H]R1881 in the presence of excess unlabeled 5 alpha- dihydrotestosterone or R1881 to assess nonspecific binding. Following incubation, the tissue sections are washed to remove unbound radioligand and either scraped for immediate determination of androgen receptor binding or placed against emulsion-coated film for the production of an autoradiographic image. In validation experiments with rat prostate sections from castrated, gonad-intact, and androgen-supplemented animals, maximum levels of androgen binding were observed with incubation at 4 degrees C or 72 h. Markedly less binding was detected with shorter incubations or with incubations at even slightly elevated temperatures. Very little androgen receptor binding was detected in castrated animals whereas receptor levels in intact and androgen-supplemented animals were 79.3 fmol/mg and 143.6 fmol/mg protein, respectively, suggesting that the method is selective for occupied receptors. Saturation binding analysis revealed binding to a single class binding site with high affinity (kd = 1.475 +/- 0.12 nM). Autoradiographic images of androgen binding in the prostate reflected the findings with the scraped sections: essentially no specific binding was present in sections from castrated animals whereas much heavier labeling was present in sections from intact animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Immunocytochemical detection of androgen receptor in human temporal cortex characterization and application of polyclonal androgen receptor antibodies in frozen and paraffin-embedded tissues.

Immunocytochemical and biochemical studies have demonstrated the presence of androgen receptor protein in various regions of the rodent and non-human primate cortex. Localization of androgen receptor in the human brain has, however, not been studied as extensively, because of difficulties in obtaining suitable tissue samples. In the present study, we have localized androgen receptors in both frozen and paraffin-embedded temporal cortex from epileptic patients undergoing resection. Polyclonal antibodies were raised against fusion proteins containing fragments of the human androgen receptor protein. The antibodies were affinity-purified against the corresponding fusion protein. Immunoprecipitation and Western blotting using extracts from human cell lines demonstrated the specificity of the antibodies for the human androgen receptor and lack of cross-reactivity with other steroid hormone receptors. Immunocytochemistry was performed on frozen and paraffin sections of human temporal cortex and in paraffin-embedded benign hyperplastic prostates (BPH), as well as prostate and breast carcinomas, by the streptavidin-biotin-peroxidase method. Antigen-retrieval was performed in paraffin-embedded sections using microwave irradiation. Specific nuclear and cytoplasmic immunoreactivity for androgen receptor was detected in neurons, astrocytes, oligodendrocytes, and microglia cells of the temporal cortex. In contrast, only nuclear staining was observed in BPH, prostate and breast carcinomas. Immunoprecipitation of human temporal cortex lysate and subsequent Western blot analysis demonstrated the expression of a 98 kDa immunoreactive protein, slightly smaller than the reported molecular weight of the wild-type androgen receptor. These results provide further evidence for the expression of androgen receptor in the human temporal cortex. The use of these immunocytochemical techniques should enable the retrospective determination of possible changes in androgen receptor expression in a variety of archival paraffin-embedded tissues, including samples of the human central nervous system.

Adolescent↗

PubMed1992

Synthesis and biological activity of trans(+-)-N-methyl-2-(3-pyridyl)-2-tetrahydrothiopyrancarbothioamide 1-oxide (RP 49356) and analogues: a new class of potassium channel opener.

The synthesis and biological activity of trans-(+-)-N-methyl-2-(3-pyridyl)-2-tetrahydrothiopyrancarbothioamid+ ++ e 1-oxide (8a, RP 49356) and analogues is reported. These compounds constitute a new structural class of K(+)-channel opener. The effects of changes in pyridyl group, thioamide, and thiane ring on in vitro K(+)-channel opening reactivity are discussed. A 3-pyridyl or 3-quinolyl group, a small N-alkyl thioamide function, and a thiane oxide ring, in which the sulfoxide is in a trans relationship to the thioamide, are preferred for activity. Selected compounds were tested intravenously in the normotensive anaesthetized rat for hypotensive effects, and the activities reflect their in vitro K(+)-channel opening activity. This led to further evaluation of compound 8a and the selection of the (-)-enantiomer 8b (RP 52891) for development as an antihypertensive and antianginal agent.

Animals↗

PubMed1992

CD4 and its role in infection of rabbit cell lines by human immunodeficiency virus type 1.

Human CD4 (HuCD4) is the principal receptor for human immunodeficiency virus type 1 (HIV-1) in human cell infection. Susceptibility of rabbit cell lines to infection with HIV-1 raised questions concerning whether a CD4 homolog serves as HIV-1 receptor on rabbit cells. Sequence comparisons of rabbit CD4 (RbCD4) cloned from a rabbit thymus cDNA library showed that 6 of the 18 residues implicated in HIV-1 binding by CD4 differ between the human and rabbit proteins. No correlation between RbCD4 expression by rabbit cell lines and their ability to support HIV-1 infection was seen. Transfection of RbCD4-negative, HTLV-I-transformed cell lines with HuCD4 significantly enhanced HIV-1 infectivity, suggesting that these lines lack a receptor present on other RbCD4-negative lines that produce high levels of p24 in their native state. Inhibition of HIV-1 infection with soluble HuCD4 was demonstrated for all rabbit lines tested, but complete inhibition was obtained only with a rabbit T-cell line expressing RbCD4 and with HuCD4 transfectants. The results suggest that HIV-1 infection of the RbCD4-positive line proceeds through a receptor similar to HuCD4 but that an additional receptor or receptors may serve this purpose in RbCD4-negative lines.

Amino Acid Sequence↗

PubMed1992

Oncostatin M induces tyrosine phosphorylation in endothelial cells and activation of p62yes tyrosine kinase.

Oncostatin M is a polypeptide cytokine produced by activated and transformed T lymphocytes that has diverse biologic effects, including growth inhibition of tumor cells and induction of IL-6 expression in cultured human endothelial cells (HEC). HEC are highly responsive to oncostatin M and express high levels of oncostatin M receptors relative to other cell types. Oncostatin M has previously been found to bind a specific receptor of 150 to 160 kDa. We have found through the use of anti-phosphotyrosine immunoblotting that oncostatin M induces tyrosine phosphorylation in HEC. Anti-phosphotyrosine antibodies specifically immunoprecipitated labeled oncostatin M cross-linked to its receptor, demonstrating that the oncostatin M receptor is either directly phosphorylated on tyrosine after ligand binding or is tightly associated with a phosphotyrosyl protein in these cells. The tyrosine kinase inhibitor herbimycin A blocked the induction of IL-6 by oncostatin M in HEC. In addition, immune complex kinase assays showed that oncostatin M markedly increased the activity of the p62yes tyrosine kinase with a small increase in p59fyn but no increase in p56lyn tyrosine kinase activity in HEC. We conclude that oncostatin M utilizes a tyrosine phosphorylation signal transduction pathway in HEC involving the activation of the p62yes tyrosine kinase, and that this tyrosine phosphorylation pathway leads to the induction of IL-6 expression.

Benzoquinones↗

PubMed1992

Estrogen receptors colocalize with low-affinity nerve growth factor receptors in cholinergic neurons of the basal forebrain.

The rodent and primate basal forebrain is a target of a family of endogenous peptide signaling molecules, the neurotrophins--nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3--and of the gonadal steroid hormone estrogen, both of which have been implicated in cholinergic function. To investigate whether or not these ligands may act on the same neurons in the developing and adult rodent basal forebrain, we combined autoradiography with 125I-labeled estrogen and either nonisotopic in situ hybridization histochemistry or immunohistochemistry. We now report colocalization of intranuclear estrogen binding sites with the mRNA and immunoreactive protein for the low-affinity nerve growth factor receptor, which binds all three neurotrophins, and for the cholinergic marker enzyme choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). Colocalization of estrogen and low-affinity nerve growth factor receptors implies that their ligands may act on the same neuron, perhaps synergistically, to regulate the expression of specific genes or gene networks that may influence neuronal survival, differentiation, regeneration, and plasticity. That cholinergic neurons in brain regions subserving cognitive functions may be regulated not only by the neurotrophins but also by estrogen may have considerable relevance for the development and maintenance of neural substrates of cognition. If estrogen-neurotrophin interactions are important for survival of target neurons, then clinical conditions associated with estrogen deficiency could contribute to the atrophy or death of these neurons. These findings have implications for the subsequent decline in those differentiated neural functions associated with aging and Alzheimer disease.

Animals↗

PubMed1992

Sex differences in estrogen receptor binding in the rat hypothalamus: effects of subsaturating pulses of estradiol.

The effects of subsaturating pulses of estradiol on cell nuclear retention of estrogen receptors in brain regions of male and female rats were determined. In the first experiment, age-matched adrenalectomized/gonadectomized (ADX/GDX) rats were killed 1 h after i.v. injection of a subsaturating dose of free estradiol (1.0 microgram/kg b. wt.) and cell nuclear estrogen receptor binding was measured in microdissected brain areas by an in vitro exchange assay. As we have previously reported with saturating doses of estradiol (3.6-36.0 micrograms/kg b. wt.), a greater concentration of receptors was measured in the ventromedial nucleus (VMN), periventricular preoptic area (PVPOA), and medial preoptic area (mPOA) of the female than in the same regions of the male brain. Binding levels did not differ between the sexes in the bed nucleus of the stria terminalis, arcuate nucleus, or the corticomedial amygdala. In the second experiment, GDX/ADX male and female rats received either a single or double pulse of 0.5 microgram estradiol/kg b. wt. administered 6 h apart. Rats were killed 1 h after the second pulse. In animals treated with a single pulse of estradiol 1 h before sacrifice, higher concentrations of estrogen binding were measured in the female VMN and PVPOA than in the male. Except in the mPOA, the concentration of estrogen receptor binding measured was not different from that measured in animals that had received an additional, earlier pulse of estradiol. In this area, receptor concentrations were lower in male rats treated with two pulses than in males treated with one pulse.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗

PubMed1992

Recombinant oncostatin M stimulates the production of acute phase proteins in HepG2 cells and rat primary hepatocytes in vitro.

Acute inflammation is characterized by increased liver output of acute phase proteins (APP). Several cytokines including IL-6, leukemia inhibitory factor, and IL-11 are capable of stimulating APP synthesis by hepatocytes and hepatoma cells. We have tested the activity of a separate and unique cytokine oncostatin M (OM) and have found potent APP-inducing activity of human recombinant OM on hepatocytes. OM acted in a dose-dependent fashion (ED50 5 to 10 ng/ml) in stimulating APP synthesis in human HepG2 cells, rat H35 cells, and primary rat hepatocyte cultures, but not human Hep3B cells. Human OM induced equivalent to or greater responses than IL-6 in HepG2 cells, however, it was less effective than human IL-6 in stimulating rat cells. Northern analysis showed that OM stimulated mRNA levels of haptoglobin and alpha 1-antichymotrypsin in HepG2 cells. OM induced CAT activity in HepG2 cells transfected with CAT constructs containing IL-6-responsive elements, suggesting that OM induces transcription of native proteins through mechanisms involving IL-6-responsive element-like sequences in gene promoters. OM was also shown to act additively with IL-6 or leukemia inhibitory factor and synergistically with glucocorticoid or IL-1 in the induction of specific APP. These results suggest that OM plays a role as a mediator of APP synthesis in inflammatory responses.

Acute-Phase Proteins↗

PubMed1992

Hemodynamic and liver function predictors of serum hyaluronan in alcoholic liver disease.

To define hepatic predictors of serum hyaluronan in patients with chronic liver disease, 62 patients with alcoholic liver disease were evaluated. In group 1, 30 patients had concurrent assessment of serum hyaluronan, liver function tests, Pugh grade and hemodynamic indices. A second, overlapping group of 42 patients (group 2) also had antipyrine clearance measured but without hemodynamic assessment. All but six patients had elevated serum hyaluronan levels. In both groups, serum hyaluronan levels differed between Pugh grades and, in each group, was significantly greater in Pugh grade C compared with those in Pugh grade A (p less than 0.05, Kruskal-Wallis test). When analyzed by correlation, serum hyaluronan was significantly associated with several indices in group 1, but on multivariate linear regression only three statistically independent predictors of serum hyaluronan were identified: serum albumin (p = 0.008), indocyanine green clearance (p = 0.024) and indocyanine green extraction (p = 0.036). The overall R2 for these correlates was 65%. In the second group, antipyrine clearance was not significantly associated with serum hyaluronan (r = 0.29, p = 0.06), but other associations were similar to the first group. On multivariate analysis, only serum albumin predicted serum hyaluronan (p less than 0.001; R2 = 43%). In conclusion, indices of hepatocyte synthetic function, sinusoidal blood flow and degree of intrahepatic shunting are independent predictors of serum hyaluronan in alcoholic liver disease. These data show the unique nature of serum hyaluronan and suggest its potential application to the assessment of acute hemodynamic changes in patients with liver disease.

Adult↗

PubMed1992

Binding of antibodies in sera from Type 1 (insulin-dependent) diabetic patients to glutamate decarboxylase from rat tissues. Evidence for antigenic and non-antigenic forms of the enzyme.

An islet protein of M(r) 64000, identified as the gamma-amino butyric acid (GABA)-synthesizing enzyme, glutamate decarboxylase, is a major target for antibodies in Type 1 (insulin-dependent) diabetes mellitus. This enzyme is also expressed in brain and in some other tissues and may exist in multiple forms. The aim of this study was to determine the ability of antibodies from diabetic patients to recognize glutamate decarboxylase from rat islets, brain and other normal rat tissues. Glutamate decarboxylase was detected at high activity levels in brain and at lower levels in islets, kidney, liver, pituitary gland, thyroid gland, adrenal gland, testis and ovary. The ability of antibodies in sera of diabetic patients to immunoprecipitate enzyme activity from detergent extracts of tissues was determined. Antibodies in sera from diabetic patients were found to bind the enzyme from islet and brain extracts, but bound less than 20% of the activity from other tissues. The ability of antibodies to immunoprecipitate the brain enzyme was significantly correlated with the presence of antibodies to the islet 64 kilodalton antigen. These studies show that the glutamate decarboxylase activity expressed in brain shares antigenic determinants with the islet 64 kilodalton antigen. Isoforms of the enzyme expressed in other non-neuronal tissues may be antigenically distinct and may lack determinants recognized by diabetes-associated antibodies.

Adolescent↗

PubMed1992

Transmitter content and afferent connections of estrogen-sensitive progestin receptor-containing neurons in the primate hypothalamus.

Progestin receptor-containing cells in the hypothalamus of the adult female green monkey (Cercopithecus aethiops) were examined by double-label immunocytochemical methods to determine their anatomical location, neurotransmitter content and afferent connections. Animals were ovariectomized and administered either estradiol valerate or the oil injection vehicle, and were sacrificed after 10 days of treatment. Using a monoclonal antibody raised against rabbit uterine progestin receptor (PR), the distribution of PR-immunoreactive cells in the mediobasal hypothalamus and the effect of estrogen treatment on this distribution was determined. PR-immunoreactive cells were found throughout the ventromedial nucleus (VMN), in the area between the VMN and fornix, and in the medial portion of the infundibular nucleus. Estrogen treatment dramatically increased both the number of labeled cells and the intensity of immunoreaction product in these regions. In double-immunostained sections, boutons immunoreactive for antigens indicative of serotonin, pro-opiomelanocortin derived peptides, GABA, catecholamine, neuropeptide Y, substance P, cholecystokinin, and somatostatin were demonstrated to establish synaptic contact with the soma of PR-immunoreactive hypothalamic neurons. In colchicine-pretreated animals, all PR-containing neurons in the mediobasal hypothalamus were found to contain immunoreactivity for glutamic acid decarboxylase, the enzyme required for synthesis of GABA. No evidence of colocalization with other antigens, including LHRH, was observed. Because LHRH neurons are known to receive a rich GABAergic innervation PR-containing GABAergic cells may represent steroid-sensitive sites of integration for inputs from other neural systems involved in the control of gonadotropin secretion.

Afferent Pathways↗

PubMed1992

Antibodies to GAD and tryptic fragments of islet 64K antigen as distinct markers for development of IDDM. Studies with identical twins.

Insulin-dependent diabetes mellitus (IDDM) is associated with antibodies to a 64,000-M(r) islet cell protein, at least part of which is identified as glutamic acid decarboxylase (GAD). These antibodies are detected as two distinct antibody specificities to 50,000-M(r) and 37,000/40,000-M(r) tryptic fragments of the autoantigen (50K and 37K antibodies, respectively). We determined the frequencies of antibodies to intact GAD, tryptic fragments of islet 64,000-M(r) antigen, islet cell antibodies (ICAs), and insulin autoantibodies (IAAs) in sera from 58 nondiabetic identical twins of patients with IDDM, of whom 12 subsequently developed diabetes. ICA, antibodies to intact GAD, and those to tryptic fragments were detected at similar frequencies in prediabetic twins (67-75%), but only 25% had IAA. Of 46 twins who remain nondiabetic, GAD antibodies, 50K antibodies, and ICA were detected in 6 (13%), 7 (15%), and 5 (11%), respectively, whereas only 1 (2%) possessed 37K antibodies and 2 (4%) had IAA. Eight of 9 twins with 37K antibodies and all 6 twins with ICA greater than 20 Juvenile Diabetes Foundation U have developed diabetes. Antibodies to GAD are sensitive markers for diabetes development but may also be present in genetically susceptible individuals who are unlikely to develop disease. Antibodies to 37,000/40,000-M(r) fragments of the 64,000-M(r) antigen or high-titer ICA were the best markers for diabetes development in these twins.

Antibody Specificity↗

PubMed1991

Regulation of IL-6 expression by oncostatin M.

Endothelial cells produce immunomodulatory cytokines in response to soluble mediators of inflammatory/immune reactions. We have previously demonstrated that the leukocyte-derived cytokine, oncostatin M (Onco M) can alter endothelial cell morphology, regeneration, and fibrinolytic activity in vitro. Here we demonstrate that Onco M stimulates the production of the pleiotropic cytokine, IL-6, in cultured human endothelial cells (HEC) in a time- and dose-dependent manner. Specific antibodies to IL-6 neutralize the growth-inhibitory activity for human breast carcinoma cells that is secreted by HEC in response to Onco M treatment. Specific immunoassays indicate greater than 10-fold increases in the IL-6 content of culture supernatants as early as 6 h post-treatment with Onco M (ED50 = 17 pM). This stimulation of IL-6 production by Onco M is associated with a sevenfold increase in intracellular levels of IL-6 mRNA. IL-1 alpha and TNF-alpha are also potent inducers of IL-6 production in these cells, the order of potency being IL-1 alpha greater than Onco M greater than TNF-alpha. TNF-alpha, but not IL-1 alpha, synergizes with Onco M to augment IL-6 production in HEC. HEC are 10 to 20 times more responsive to Onco M than are other nonendothelial cell types. In addition, HEC express 10 to 20 times greater numbers of high affinity cell-surface receptors for Onco M than do other nonendothelial cell types. Based on these findings, we propose that Onco M may represent a new immunomodulator regulating cytokine-induced gene products in endothelial cells.

Antineoplastic Agents↗

PubMed1991

Modulation of rat hepatic cytochromes P450 by chronic methapyrilene treatment.

The antihistaminic compound methapyrilene (MP) when chronically administered has been shown to be a rat-specific hepatocarcinogen. To examine the effects of chronic MP treatment on the hepatic microsomal cytochromes P450. Fischer 344 rats were gavaged for 10 weeks (5 days on, 2 days off) with either vehicle or 50, 100, or 150 mg MP/kg body weight. Chronic MP treatment was found to have a significant effect on several microsomal enzymatic activities. Small (17-28%) but significant (P less than 0.05) decreases were observed for total P450 levels and the activities of erythromycin N-demethylase (catalyzed by P450IIIA), N-nitrosodimethylamine demethylase (catalyzed by P450IIE1) and pentoxyresorufin O-dealkylase (catalyzed by P450IIB1). In addition, a relatively large decrease (approximately 80%) was observed for the activity of benzphetamine N-demethylase (representative of P450IIC11) and an induction of about 40% was observed for ethoxyresorufin O-dealkylase (catalyzed by P450IA). The metabolism of testosterone by microsomes isolated from the rats chronically treated with MP indicated that several reactions were compromized. Specifically, testosterone 2 alpha-hydroxylase, indicative of P450IIC11, was reduced greatly (86%), whereas testosterone 6 beta-hydroxylase, reflecting P450IIIA, and testosterone 7 alpha-hydroxylase, indicative of P450IIIA1, were affected only slightly by MP treatment (approximately 25%). Immunoblot analyses of the various microsomal samples were performed to determine if chronic MP treatment had direct effects on the level of expression of the cytochromes P450. Decreases in the levels of P450IIIA, IIE1, and IIC11, determined by immunoblot analyses, closely paralleled those observed for their marker catalytic activities. Further studies will be required to determine the mechanism by which MP affects the levels of the cytochromes P450 (i.e. increased degradation or decreased synthesis).

Animals↗

PubMed1991

Estrogen receptor binding in regions of the rat hypothalamus and preoptic area after inhibition of dopamine-beta-hydroxylase.

Previous studies have shown that administration of diethyldithiocarbamate (DDC), a dopamine-beta-hydroxylase inhibitor, results in a decreased concentration of estrogen receptors measured in the rodent hypothalamus and preoptic area. To determine if this modulation of receptor content is region-specific, in vitro estrogen binding assays were performed on cytosol and cell nuclear extracts of microdissected brain regions from female rats treated with DDC. For cytosol binding comparisons, ovariectomized (OVX) rats were treated with 550 mg DDC/kg b. wt. or the saline vehicle 12 h before sacrifice. For cell nuclear binding comparisons, OVX rats received a maximal dose of estradiol 12 h after DDC or saline treatment and 1 h before sacrifice. No region-specific decreases in estrogen binding were observed in either cytosol or nuclear extracts. To further examine possible regional specificity, quantitative autoradiographic analysis of the in vivo hypothalamic uptake of an iodinated analog of estradiol, 11 beta-methoxy-16 alpha-[125I]iodoestradiol (MIE2), in OVX rats treated with DDC was conducted. Animals received a saturating dose of [125I]MIE2 12 h after DDC or saline treatment and 1 h before sacrifice. DDC treatment resulted in higher background levels of radioactivity and a trend toward higher uptake levels in all brain regions, but with no evidence of marked regional specific effects in any area of the brain. In tissue uptake studies, DDC treatment resulted in higher levels of radioactivity recovered from serum and neural tissues of [125I]MIE2-injected rats, suggesting that DDC slows the clearance of MIE2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Differences between the effects of cromakalim and nifedipine on agonist-induced responses in rabbit aorta.

1. The effects of cromakalim on endothelium-denuded rabbit aortic strips were compared with those of the calcium (Ca2+) entry blocking agent, nifedipine. 2. Pre-incubation with cromakalim or nifedipine had no significant effect on the initial phasic component of noradrenaline (NA)-induced responses. 3. Cromakalim (0.3-10 microM), but not nifedipine, inhibited the maintained tonic contractions produced by NA. The effects of cromakalim were antagonized by raising extracellular [K+] or by glibenclamide. 4. Nifedipine inhibited contractions produced by KCl (40 mM) whereas cromakalim had no effect. 5. In Ca2(+)-free physiological salt solution (PSS), cromakalim produced a significant inhibition of both the refilling of and the release of Ca2+ from NA-releasable Ca2+ stores, whereas nifedipine was ineffective. 6. In tissues preloaded with 42K+ cromakalim (0.3-10 microM) produced a concentration-dependent increase in the 42K+ efflux rate coefficient. NA (0.3 microM) also produced an increase in the rate of efflux of 42K+, an effect which was not antagonized by nifedipine (0.3 microM). 7. When microelectrodes were used, cromakalim (1-10 microM) produced a maintained concentration-dependent membrane hyperpolarization. However, low concentrations of cromakalim (less than 1 microM) which relaxed the aorta had no effect on membrane potential. NA had no significant effect on membrane potential. 9. It is concluded that the ability of cromakalim to relax NA-induced contractions in rabbit aorta is not exerted by the indirect closure of nifedipine-sensitive Ca2+ channels. Instead, cromakalim may exert a direct inhibitory action on Ca2+ uptake into and release from Ca2+ stores and additionally inhibit the pathway through which Ca2+ passes from the extracellular fluid to intracellular Ca2+ stores.

Animals↗

PubMed1991

Turnover of hyaluronan in synovial joints: elimination of labelled hyaluronan from the knee joint of the rabbit.

After the injection of [3H]acetyl-labelled hyaluronan into normal rabbit knee joints, about 90% of its isotope content was ultimately accounted for as 3H2O. The rate of elimination of hyaluronan from synovial fluid was therefore estimated from changes in the level of 3H2O in plasma. The half-life of plasma 3H2O was 6.2 days (S.D. 0.7). As estimated from its metabolism to 3H2O, the mean intrasynovial half-life of [3H]hyaluronan of high molecular weight (modal relative molecular mass (Mr) greater than 6.0 x 10(6) was 13.2 h (range 11-15.5 h; n = 4); an otherwise identical preparation of low molecular weight (modal Mr 0.09 x 10(6] exhibited a mean half-life of 10.2 h (range 7.8-13.5 h; n = 4). The difference between the two groups was significant (P = 0.029). Both estimates were nevertheless close to those determined by others in the same species for injected proteoglycans (Mr 2.5 x 10(6), t1/2 = 12 h) and for endogenous hyaluronan calculated from changes in concentration during intravenous infusion of fluid under anaesthesia (t1/2 = 16 h). The similarity suggests that hyaluronan and proteoglycan are removed from synovial fluid by a common pathway with limited dependence on their molecular dimensions and concentrations.

Animals↗