MR Activation Research Articles

Gluco- and mineralocorticoid receptor-mediated regulation of neurotrophic factor gene expression in the dorsal hippocampus and the neocortex of the rat.

Gluco- and mineralocorticoid receptors (GR and MR) act via common promoter elements but may exert different effects on gene regulation in various regions of the forebrain. In order to separately analyse the role of GR and MR in the regulation of neurotrophic factor genes and their receptors, we used adrenalectomy and subsequent hormone injections in the rat as a model system. Twenty-four hours after adrenalectomy rats were injected with a single dose of corticosterone (2 and 10 mg/kg), aldosterone (0.5 mg/kg) or the synthetic glucocorticoid agonist RU 28362 (4 mg/kg). Gene expression of basic fibroblast growth factor (bFGF) and its high-affinity receptors [fibroblast growth factor receptor subtypes 1-3 (FGF-R1, FGF-R2, FGF-R3)], as well as brain-derived growth factor (BDNF) and neurotrophin-3 (NT-3) was analysed at 4 h after the hormone injection in CA1-CA4 (cornus of Ammon areas of the hippocampus) and dentate gyrus of the dorsal hippocampus and in neocortex by means of in situ hybridization. We found that bFGF is regulated in CA2, CA3 and dentate gyrus by GR and MR together, and in CA1, CA4 and neocortex by GR alone. FGF-R2 expression in the hippocampus seems to be regulated only by MR, while BDNF expression appears to depend on both receptors. FGF-R1, FGF-R3 and NT-3 were only moderately affected by the hormone activation of GR and MR acting in concert or alone in the various regions. Thus, the present findings suggest that the adrenal cortical system through GR and MR participate in the control of neurotrophic factor signalling in a highly subregion- and cellular-dependent manner.

The relationship between 'deep-hole' mitochondria-rich cells and salinity adaptation in the euryhaline teleost, Oreochromis mossambicus.

The present study elucidates the relationship between deep-hole MR cells (Lee et al. 1996) and salinity adaptation in tilapia (Oreochromis mossambicus). Freshwater tilapia were transferred to salt water of various salinities, i.e., 5 (hypotonic), 10 (isotonic), 20 (hypertonic), and 30‰ (hypertonic), for 2 weeks. The density of MR cells, protein content, activity, and localization of the sodium pump were examined. There was no significant difference in serum osmolarity and Na+, K+, Cl− levels in fish of the various treatment groups. The amounts of protein and activity of Na,K-ATPase were elevated in fish from SW with the highest salinity. MR cells observed by scanning electron microscope revealed small pits (0.5–1.0 μm in diameter) in groups from hypotonic and isotonic water and large crypts (2.4–3.8 μm in diameter) in fish from hypertonic water. Moreover, the density of these deep-hole MR cells increased significantly in fish adapted to hypertonic SW. Larger and more numerous deep-hole MR cells of euryhaline tilapia may account for higher protein amounts and activities of Na,K-ATPase, probably to meet the physiological demand of euryhaline teleosts engaged in hyporegulation.

Differential regulation of hippocampal glucocorticoid receptors mRNA and fast feedback: relevance to post-traumatic stress disorder.

Hippocampal glucocorticoid receptors (GR and MR) play an important role in glucocorticoid negative feedback. Abnormalities in negative feedback are found in depression and in post-traumatic stress disorder (PTSD), suggesting that GR and MR might be involved in the pathophysiology of these disorders. Enhanced negative feedback, the PTSD-specific neuroendocrine abnormality, can be induced in animals using a single prolonged stress (SPS) paradigm (a number of different stressors in one prolonged session, 'no stress' interval and a testing session one week later). In the current study, we examined hippocampal GR and MR mRNA distribution in the same animals that exhibited altered negative feedback following the SPS. Seven groups of adult Sprague-Dawley male rats (seven animals each) were used in two studies, comparing unstressed controls to acutely stressed animals (SPS: 24 h group), SPS animals (seven and 14 days), and SPS + chronic stress animals. GR and MR mRNA distribution across hippocampal subfields was studied using in-situ hybridization with 35S-labelled cRNA probes. Acute stress produced down-regulation of GR and MR mRNA across all hippocampal subfields. Seven days later (SPS-7 group), there was a differential recovery, with GR mRNA reaching higher than the prestress levels, and MR mRNA remaining down-regulated. The same differential regulation was present in the 14-day group. Chronically stressed animals that exhibited normal fast feedback also had normalization in their GR and MR mRNA levels. The MR/GR ratio was decreased only in animals that had enhanced fast feedback. These findings suggest that the increase in GR, in hippocampus is involved in the fast feedback hypersensitivity observed in the SPS animals, and might also underlie enhanced dexamethasone sensitivity found in PTSD. Since differential activation of GR and MR can modulate memory, behavioural responsivity, anxiety and fear, change in MR/GR ratio might also explain other PTSD-related phenomena.

Plasticity of hippocampal corticosteroid receptors during aging in the rat

Aging is commonly associated with dysregulation of the hypothalamo-pituitary-adrenal axis and cognitive impairment. On the basis of suggestions that these disruptions ensue from changes in the hippocampal complement of corticosteroid (mineralocorticoid and glucocorticoid) receptors (MR and GR), we examined the availability of hippocampal MR and GR by measuring the in vivo uptake of 3H-aldosterone and 3H-dexamethasone (selective MR and GR agonists, respectively); MR and GR mRNA levels were also measured. We observed age-related declines in both the synthesis of MR and GR and the uptake of their respective ligands. Whereas MR mRNA levels and ligand uptake declined in parallel, GR binding declined more steeply than GR mRNA. This latter result, together with our finding that aged rats show impaired corticosteroid receptor mRNA and protein up-regulation after corticosteroid withdrawal, indicates decreased transcription of MR and GR genes and posttranslational modification of GR mRNA during aging. Given that corticosteroids can influence MR and GR synthesis and binding, and based on the finding that aged subjects show reduced basal secretion of corticosterone, we propose that this relative hypocorticalism may be responsible for the changes observed in MR and GR activity, which then leads to disturbances in neuroendocrine regulation and cognitive function in aged subjects.

Regulation of the Human Na/K-ATPase β1 Gene Promoter by Mineralocorticoid and Glucocorticoid Receptors

Expression of the human Na/K-ATPase beta1 subunit is regulated by a mineralocorticoid- and glucocorticoid-responsive elements. Here we identified an MR and GR responsive element, at positions -650 to -630, within the beta1 gene promoter that is required for both MR and GR activation. Independent expression of MR and GR activated by aldosterone or triamcinolone acetonide (TA) leads to significant transactivation of the beta1 promoter. Yet coexpression of both receptors activated by aldosterone plus TA or cortisol results in a much lower induction, indicating that coexpression of MR and GR is inhibitory. Gel shift mobility assay using an oligonucleotide including the 21-base pair MRE/GRE with whole cell extracts prepared from CV-1 cells overexpressing MR or GR showed specific MR and GR binding to this sequence. Additionally, antibodies to both MR and GR effectively supershifted the protein-DNA complexes, indicating that these receptors bound to the DNA sequence. Finally, the 21-base pair MRE/GRE was capable of activating transcription from a heterologous promoter in response to both aldosterone and TA. Together these data indicate that the 21-base pair sequence represents a true MRE/GRE and that optimal activation of the human Na/K-ATPase beta1 promoter is controlled by mineralocorticoid and glucocorticoid hormones. It appears that an interaction of MR with GR on the beta1 promoter effectively down-regulates transcription.

Brain corticosteroid receptor balance in health and disease.

In this review, we have described the function of MR and GR in hippocampal neurons. The balance in actions mediated by the two corticosteroid receptor types in these neurons appears critical for neuronal excitability, stress responsiveness, and behavioral adaptation. Dysregulation of this MR/GR balance brings neurons in a vulnerable state with consequences for regulation of the stress response and enhanced vulnerability to disease in genetically predisposed individuals. The following specific inferences can be made on the basis of the currently available facts. 1. Corticosterone binds with high affinity to MRs predominantly localized in limbic brain (hippocampus) and with a 10-fold lower affinity to GRs that are widely distributed in brain. MRs are close to saturated with low basal concentrations of corticosterone, while high corticosterone concentrations during stress occupy both MRs and GRs. 2. The neuronal effects of corticosterone, mediated by MRs and GRs, are long-lasting, site-specific, and conditional. The action depends on cellular context, which is in part determined by other signals that can activate their own transcription factors interacting with MR and GR. These interactions provide an impressive diversity and complexity to corticosteroid modulation of gene expression. 3. Conditions of predominant MR activation, i.e., at the circadian trough at rest, are associated with the maintenance of excitability so that steady excitatory inputs to the hippocampal CA1 area result in considerable excitatory hippocampal output. By contrast, additional GR activation, e.g., after acute stress, generally depresses the CA1 hippocampal output. A similar effect is seen after adrenalectomy, indicating a U-shaped dose-response dependency of these cellular responses after the exposure to corticosterone. 4. Corticosterone through GR blocks the stress-induced HPA activation in hypothalamic CRH neurons and modulates the activity of the excitatory and inhibitory neural inputs to these neurons. Limbic (e.g., hippocampal) MRs mediate the effect of corticosterone on the maintenance of basal HPA activity and are of relevance for the sensitivity or threshold of the central stress response system. How this control occurs is not known, but it probably involves a steady excitatory hippocampal output, which regulates a GABA-ergic inhibitory tone on PVN neurons. Colocalized hippocampal GRs mediate a counteracting (i.e., disinhibitory) influence. Through GRs in ascending aminergic pathways, corticosterone potentiates the effect of stressors and arousal on HPA activation. The functional interaction between these corticosteroid-responsive inputs at the level of the PVN is probably the key to understanding HPA dysregulation associated with stress-related brain disorders. 5. Fine-tuning of HPA regulation occurs through MR- and GR-mediated effects on the processing of information in higher brain structures. Under healthy conditions, hippocampal MRs are involved in processes underlying integration of sensory information, interpretation of environmental information, and execution of appropriate behavioral reactions. Activation of hippocampal GRs facilitates storage of information and promotes elimination of inadequate behavioral responses. These behavioral effects mediated by MR and GR are linked, but how they influence endocrine regulation is not well understood. 6. Dexamethasone preferentially targets the pituitary in the blockade of stress-induced HPA activation. The brain penetration of this synthetic glucocorticoid is hampered by the mdr1a P-glycoprotein in the blood-brain barrier. Administration of moderate amounts of dexamethasone partially depletes the brain of corticosterone, and this has destabilizing consequences for excitability and information processing. 7. The set points of HPA regulation and MR/GR balance are genetically programmed, but can be reset by early life experiences involving mother-infant interaction. 8. (ABSTRACT TRUNCATED)

Corticosteroid Regulation of Ion Channel Conductances and mRNA Levels in Individual Hippocampal CA1 Neurons

Overexposure to corticosteroid hormones is harmful to hippocampal neuronal integrity, likely by perturbation of calcium homeostasis. To identify molecular mechanisms at the single-cell level, we characterized mRNA expression corresponding to voltage- and ligand-gated Ca channels in individual dissociated CA1 neurons in response to long-term corticosterone (CORT) exposure. Predominant mineralocorticoid receptor occupation (ADC-LO group) resulted in low levels of P/Q- and L-type Ca channel mRNAs, high levels of GluR-2 versus GluR-1, and a high ratio of NMDAR-2A to NMDAR-2B mRNA. Corresponding alterations in protein expression were consistent with the restriction of Ca influx. In contrast, additional glucocorticoid receptor occupation (ADC-HI group) altered the expression of these mRNAs in a manner consistent with enhanced Ca influx; interestingly, qualitatively similar alterations were seen in control ADX neurons. Electrophysiological data from the same neurons indicate that Ca current amplitudes also are modulated by CORT, although on a shorter time scale. Finally, principal components analysis (PCA) suggests that neuronal AMPA and NMDA receptor composition may be regulated by MR and GR activation in a complex manner. Therefore, our data implicate molecular events by which CORT may regulate Ca influx into CA1 hippocampal neurons.

Role of inosine in prevention of methaemoglobinaemia in the pig: in vitro studies.

Methaemoglobin reductase activity was studied in pig, human and cattle erythrocytes incubated in a medium containing glucose or inosine as NADH generator. With glucose, methaemoglobin reduction was very fast in human, less so in cattle and slowest in pig erythrocytes. With inosine, pig red cells reduced methaemoglobin more rapidly than human red cells and in bovine erythrocytes the enzyme activity was undetectable. In intact red cells the ability to reduce methaemoglobin depends on the amount of NADH the cell can produce with glucose or inosine utilization. The results show that pig red blood cells utilize inosine as the NADH generator for enzymatic reduction of methaemoglobin. The greater efficiency of pig red cells could be due to several factors: better inosine transport, more active metabolic pathway for inosine utilization (so that more NADH is produced), or greater MR activity when NADH is in excess. In any case, the high rate of methaemoglobin reduction could explain the lower prevalence of methaemoglobinaemia in pigs than in cattle.

Materials research activities in Korea

Research and development on materials has emerged globally from the urgency for advanced materials to meet the challenges of the 21st century. Research activities have been continuously intensified in both academic and research institutes around the world and Korea has been no exception. MRS-Korea has been recently formed with the support of MRS-Japan and -USA. The global efforts in MR activities have been intensive, and paved the way for the MRS-international campaign. Current materials research in Korea encompasses mainly advanced materials (bulk, single crystals and thin film oxides, metals, and composites) as opposed to traditional materials (clay, metals, less refined materials). This article outlines the MR activities in Korea, for the benefit of MRS-international, and gives a brief history with emphasis on scientific programs. In the research community, the recent trend of MR activities has been moving towards cooperative research such as through industry-university, industry-institute, university-institute, and triads, industry-institute-university. Such types of activities are often referred to as consortium approaches, and are currently very popular throughout the world. Examples of such activities are given and the pros and cons of each system are discussed.

Injections of muscimol into the median raphe nucleus produce hippocampal theta rhythm in the urethane anesthetized rat

It has previously been shown that serotonergic [5-hydroxytryptamine (5-HT)] neurons of the median raphe nucleus (MR) are critically involved in the control of the hippocampal electroencephalogram (EEG). Activation of MR 5-HT neurons desynchronizes the hippocampal EEG, whereas inhibition of MR 5-HT activity produces hippocampal theta rhythm. The MR contains an intrinsic population of gamma-aminobutyric acid (GABA) containing neurons that synapse on 5-HT cells of the MR. The present study examined the effects on the hippocampal EEG of injections of the GABAA agonist muscimol hydrobromide into the MR. Low doses of muscimol (0.5 microgram) produced hippocampal theta rhythm at a mean latency of 6.81 min and for a mean duration of 23.6 min. Higher doses (1.0 microgram and 3.0 micrograms, respectively) produced theta at mean latencies of 2.24 min and 3.2 min and for mean durations of 31.84 min and 24.88 min. Injections of muscimol into regions adjacent to the MR generated theta at significantly longer latencies or were without effect. The present results indicate that MR injections of muscimol produce theta by inhibiting the activity of MR 5-HT neurons. It is concluded that MR GABAergic systems, via their influence on MR 5-HT cells, serve an important role in the control of the hippocampal EEG.

Quaternary naltrexone: its immunomodulatory activity and interaction with brain delta and kappa opioid receptors

To investigate the role of brain opioid receptors in immune reactions, quaternary naltrexone (QNtx), a nonselective opioid antagonist which does not cross the brain-blood barrier, was tested for its immunomodulatory activity and ability to antagonize immunological changes produced by centrally applied delta-receptor agonist methionine-enkephalin (Met-Enk) and kappa-opioid receptor agonist MR 2034. Plaque-forming cell (PFC) response served as an immunological model. For this purpose, different groups of Wistar rats were centrally (intracerebroventricularly, i.c.v.) and peripherally (intraperitoneally, i.p., or subcutaneously, s.c.) treated with different doses of Met-Enk, MR 2034 and QNtx. Centrally injected Met-Enk and MR 2034 induced a dose-dependent potentiation and suppression of PFC response, respectively. Small amounts of i.c.v. given QNtx produced a dose-dependent increase in the number of PFC, whereas peripherally administered antagonist potentiated immune response in a dose-independent manner. A dose of 10 μg/kg of QNtx given i.c.v. completely abolished the immunopotentiation by Met-Enk. However, a large dose of 5 mg/kg of QNtx given s.c., did not affect the Met-Enk-induced immunoenhancement. Immunosuppression produced by i.c.v. injected MR 2034 was totally abrogated by prior i.c.v. application of QNtx, but partially blocked by s.c. administration of the antagonist. These results suggest that: (a) QNtx itself potentiates the humoral immune response when applied centrally in small doses (1 to 10 μg/kg) or peripherally in relativelly large doses (0.2 to 5 mg/kg), (b) immunopotentiation and immunosuppression induced by i.c.v. given Met-Enk and MR 2034, respectively, appears to be preferentially mediated through brain opioid receptors, (c) QNtx antagonizes the immunomodulating activities of Met-Enk and MR 2034, and (d) peripheral opioid systems may also contribute to the immunomodulation evoked by centrally administered kappa-receptor agonist MR 2034. As a whole, this study provides evidence for the involvement of the central opioid system in mechanisms underlying the immune responsiveness.

Injections of excitatory amino acid antagonists into the median raphe nucleus produce hippocampal theta rhythm in the urethane-anesthetized rat

The median raphe nucleus (MR) exerts a pronounced desynchronizing influence on the hippocampal EEG. MR stimulation disrupts theta, while MR lesions produce constant uninterrupted theta. The MR receives pronounced excitatory amino acid (EAA)-containing afferents that have been implicated in several MR-mediated behaviors. The present study examined the effects on the hippocampal EEG of MR injections of the following EAA antagonists in the urethane-anesthetized rat: 2-amino-7-phosphonoheptanoate (AP-7), dizocilpine maleate (MK-801), and γ-glutamyl-aminomethylsulfonic acid (GAMS). MR injections of the competitive (AP-7) and non-competitive (MK-801) N-methyl- d-aspartic acid (NMDA) receptor antagonists produced theta at short latencies (2.86 min; 4.02 min, respectively) and for long durations (116.1 min; 66.8 min, respectively). It was further shown that the theta-eliciting effects of AP-7 injections could be reliably and temporarily reversed with MR injections of NMDA. MR injections of the kainate/quisqualate receptor antagonist (GAMS) also produced theta at relatively short latencies (6.5 min) and for long durations (60.5 min) indicating that EAA effects on the MR are not NMDA receptor specific. Injections of each of the foregoing EAA antagonists into regions of the brainstem adjacent to the MR including the dorsal raphe nucleus and the medullary or pontine reticular formation generated theta at very long latencies or were without effect. The present findings indicate EAA afferents to the MR normally exert an excitatory influence on the MR in its desynchronization of the hippocampal EEG, whereas the removal of EAA inputs to MR produces the opposite; a reduction of MR activity and hence the elicitation of theta. Thus, EAA afferents to the MR appear to play an important modulary role in the MR control of the hippocampal EGG.

Fractionation of Heparin, Dermatan Sulfate, and Chondroitin Sulfate by Sequential Precipitation: A Method to Purify a Single Glycosaminoglycan Species from a Mixture

Purified heparin, dermatan sulfate, and chondroitin sulfate in mixtures were fractionated by sequential precipitation with increasing volumes of acetone and analyzed by agarose-gel electrophoresis and for Mr, charge density, constituent disaccharides, and anticoagulant activity (for heparin). Purified glycosaminoglycans are generally utilized for pharmaceutical purposes and show physicochemical properties of glycosaminoglycans used as drugs. Heparin is the first glycosaminoglycan to precipitate at low percentages of acetone. The relative amount of slow moving and fast moving components, the Mr and charge density, and the disaccharide pattern of fractionated heparin depend on the percentage of solvent. The activated partial thromboplastin time activity of fractions composed of heparin decreases with the charge density and Mr. Dermatan sulfate is precipitated by acetone over a narrow range (0.6-0.7 vol, 37-41%), and one of these fractions is constituted by 100% of this polysaccharide. These species of dermatan sulfate have different percentages of constituent disaccharides compared to the native polysaccharide. Nonsulfated disaccharide and disaccharide-6-sulfate are enriched. The dermatan sulfate species precipitated by acetone are also enriched in disaccharide-4,6-disulfated. Chondroitin sulfate is the most soluble glycosaminoglycan in mixed acetone/water solvent. It begins to precipitate at 0.8 vol (44%) of acetone. Different species of chondroitin sulfate can be recovered by precipitation at different percentages of solvent, and they show a decrease in Mr and charge density depending on the percentage of acetone. The chondroitin sulfate species fractionated are also enriched in disulfated disaccharides compared to native polysaccharide. A different distribution of the three disulfated disaccharides can be pointed out in the fractionated chondroitin sulfate. Sequential precipitation performed by carefully increasing acetone percentages can help obtain purified species of glycosaminoglycan with desired properties from a mixture and tissue extracts, and achieve savings in time.

Aldosterone modulates glucocorticoid receptor binding in hippocampal cell cultures via the mineralocorticoid receptor

The regulation of corticosteroid receptor expression in the rat brain by adrenal steroids remains controversial. The results of in vivo studies [ Brinton and McEwen, 1988; Luttge et al., 1989] suggest that activation of type I receptors can modulate both mineralocorticoid (MR; type I) and glucocorticoid (GR; type II) receptor binding in selected brain regions. The present study utilized primary hippocampal cell cultures from rats sacrificed at E19–20 days of gestation to examine the effects of RU 28362, corticosterone (CORT) and aldosterone (ALDO) on GR binding ([ 3H]dexamethasone±RU28362). Four days of exposure to 10 nM RU 28362, a highly selective GR agonist, resulted in a robust (∼ 70%) decrease in GR binding. Similar exposure to 10 nM of either CORT or ALDO also produced a significant (50–55%) decrease in GR binding capacity. Scatchard analyses confirmed that the diminished GR binding capacity in response to ALDO was due to a decrease in total number of binding sites ( B max forControl= 112 ± 25fmol/mg vs. ALDO= 48 ± 12fmol/mg) with no significant change in the affinity constant. The calculated EC 50 from the ALDO concentration response curve was 3.5 nM. Competition studies demonstrated that such low nM concentrations of ALDO were unable to displace specific[ 3H]dexamethasone±RU28362 binding. Spironolactone, a highly specific MR antagonist, inhibited the ALDO-induced down-regulation of GR binding. These findings support the hypothesis that MR activation can modulate GR binding in hippocampal cells.

Subcellular localization and biological activity of M(r) 18,000 basic fibroblast growth factor: site-directed mutagenesis of a putative nuclear translocation sequence.

Residues 27-31 (Lys-Asp-Pro-Lys-Arg) of the 155-amino acid form of basic fibroblast growth factor (bFGF) are in good agreement with a consensus sequence for nuclear translocation. To evaluate the role of this sequence in mediating the intracellular localization and biological activity of bFGF, basic residues Lys-27, Lys-30, and Arg-31 were changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA. The bFGF mutant (M1Q-bFGF) was expressed in eukaryotic cells and in prokaryotic cells, from which it was purified to homogeneity. Transient expression of bFGF cDNA and of M1Q-bFGF cDNA in simian COS-1 cells followed by immunolocalization and by subcellular fractionation indicated that both molecules localize in the nucleus, as well as in the cytoplasm of transfected cells, and interact with nuclear chromatin and with eukaryote DNA in a similar manner. Prokaryotic expression of M1Q-bFGF cDNA yields a polypeptide endowed with a receptor-binding capacity and mitogenic activity similar to that exerted by wild-type bFGF. However, recombinant M1Q-bFGF showed a drastically reduced capacity to induce the production of urokinase-type plasminogen activator (uPA) in endothelial cells. The uPA-inducing activity of M1Q-bFGF was fully restored by the presence of soluble heparin in the culture medium. In conclusion, the sequence bFGF(27-31) does not appear to represent a nuclear translocation and/or retention sequence for bFGF. However, neutralization of its basic residues seems to modify the tertiary structure of the growth factor, thus affecting some of its biological properties.

Purification and properties of membrane and cytosolic phosphatidylinositol-specific phospholipases C from human spleen

Two phosphatidylinositol-specific phospholipases C (PI-PLC) have been purified from human spleen. PI-PLCm represents the main activity detected in the membrane, while PI-PLCc is the main activity present in the cytoplasm. PI-PLCm can be resolved into two peaks of activity of high Mr (60,000-70,000) and low Mr (16,000-18,000). High salt concentration ((NH4)2SO4, 2M) dissociates the high Mr form yielding the low molecular form and increasing the specific activity. The same effect of dissociation and potentiation of the activity is observed when membranes solubilized by n-octyl glucoside are subjected to the high voltage conditions of an isoelectric focusing run. The purified Pi-PLCm has a Mr of about 18,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration and a basic pI (9.0-9.2). Purified PI-PLCc has a Mr of 57,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration) and a slightly acid pI (6.2). Other characteristics of both enzymes, such as cations dependence, substrate specificity, optimum pH, and kinetic parameters, are also discussed.

On the Activity of MR 889, a New Synthetic Proteinase Inhibitor

On the Activity of MR 889, a New Synthetic Proteinase Inhibitor

Protein tyrosine kinases in human breast cancer: Kinetic properties and evidence for the presence of two forms of native enzyme

The protein tyrosine kinase (PTK) of human breast tumors classified as positive (TM+) or negative (TM-) according to their estrogen and progestin receptor levels was partially characterized with regard to its distribution, kinetic parameters, molecular size, and ability to phosphorylate endogenous mammary proteins. For both types of tumors, PTK activity depended upon the presence of Mn++ (2-5 mM) and/or Mg++ (10-20 mM). The activities, total (per g of tissue) and specific (per mg of protein), were similar for both types of tumors, and an average of 60% of activity was located in cytosolic fractions. The autoradiographic detection of alkali-resistant phosphoproteins after SDS-PAGE showed very similar patterns between corresponding fractions from both types of tumors. Upon gel filtration, two peaks of activity of apparent Mr 245 kDa (peak I) and 47 kDa (peak II) were observed. Peak II was found in both cytosols and extracts from particulate fractions, while peak I was present only in the latter fraction for both TM+ and TM- tumors. The apparent Km's for ATP ranged from 4.1 to 6.6 microM, and from 11 to 34 micrograms/ml for the synthetic substrate poly [Glu80, Tyr20], at an optimal pH of 6.5-7.5. When endogenous alkali-resistant phosphorylation of peaks I and II was determined by autoradiography after SDS-PAGE, two major mammary proteins of Mr 60 and 45 kDa were phosphorylated by peak II and three, Mr 145, 74, and 62 kDa, by peak I.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermoregulation at a high ambient temperature following the oral administration of ethanol in the rat

This study was designed to assess the thermoregulatory mechanisms responsible for the elevation in body temperature following ethanol administration when exposed to a high ambient temperature (T a). Male rats of the Fischer 344 strain were gavaged with 20% ethanol at doses of 0, 2.0, 4.0, 6.0, or 8.0 g/kg and were then placed in an environmental chamber set at a T a of 37°C. Metabolic rate normalized to body mass 0.75 (MR), evaporative water loss (EWL), and motor activity were recorded for 60 min. Ethanol elicited a significant increase in colonic temperature and decrease in MR, EWL, and motor activity. Ethanol also significantly reduced the quantity of evaporated water per milliliter of oxygen consumed (E/M). Multiple linear regression analysis indicated that the two major factors which were associated with the ethanol-induced elevation in body temperature were an increase in MR and a decrease in E/M. Visual observation of behavior indicated that the normal grooming of saliva onto the fur during heat stress was impaired in ethanol-treated animals. Thus, during exposure to a high T a, the acute ethanol-induced elevation in body temperature appears to be attributed to a suppression in both autonomic and behavioral mechanisms of heat dissipation.

Gelatinolytic proteinase activities in human seminal plasma

Proteinase activities in human seminal plasma were detected using gelatin-containing sodium dodecyl sulphate-polyacrylamide gel electrophoresis zymography. Three prominent bands of activity of Mr 60,000, 66,000 and 90,000 were observed as well as 9 other bands of less intensity (34,000-158,000). These proteinases were dependent upon calcium for optimal activity, did not hydrolyse casein, and were predominantly in the soluble portion of seminal plasma. Examination of seminal plasma of men with different sperm concentrations, split ejaculates, and prostatic secretions indicated that the prostate gland was a source of most of these activities. Proteinase activities of Mr 34,000, 37,000, 82,000 and 120,000 were expressed more frequently in seminal plasma from normozoospermic men than from seminal plasma of oligo- or azoospermic men, indicating that they may also arise from spermatozoa in the semen sample. The proteinases of Mr 60,000 and 66,000 were found in all seminal plasmas whereas there was variation in the expression of the other molecular forms of enzyme, even in the normozoospermic samples. There are multiple forms of gelatinolytic proteinase activities in human seminal plasma which appear to arise from multiple sources in the reproductive tract including the Cowper's/urethral glands, the prostate gland, seminal vesicle and/or spermatozoa. Their function(s) in semen remains to be established.