Secondary effect of aldosterone on Na-KATPase activity in the rabbit cortical collecting tubule.

The possibility that mineralocorticoids have a direct influence on renal Na-K ATPase activity has been the focus of intense research effort and some controversy for a number of years. Early studies were hindered by an inability to differentiate between possible glucocorticoid vs. mineralocorticoid effects on this enzyme within the multitude of cells that comprise the heterogeneous mammalian nephron. This study attempts to circumvent this problem by monitoring Na-K ATPase activity in the rabbit renal cortical collecting tubule (CCT), a proposed target epithelium for mineralocorticoids. Using an ultramicro assay, Na-K ATPase activity was measured in CCT from normal, adrenalectomized (adx), and adx rabbits subjected to one of several corticosteroid treatment protocols. The results indicate that Na-K ATPase activity in the CCT decreased by 86% subsequent to adrenalectomy. Injection of physiological doses of aldosterone (10 micrograms/kg) but not dexamethasone (100 micrograms/kg) restored CCT Na-K ATPase activity in adx rabbits to normal levels within 3 h after injection. An insignificant rise in activity was observed 1.5h after aldosterone treatment. In addition, spirolactone SC 26304, a specific mineralocorticoid antagonist, blocked the action of aldosterone on Na-K ATPase.. Therefore an acute increase in Na-K ATPase activity participates in the action of aldosterone on Na transport in this segment. To differentiate between primary vs. secondary activation of this enzyme, adx animals were treated with amiloride before the injection of aldosterone with the intent of blocking luminal membrane Na entry into CCT. In these animals, pretreatment with amiloride blocked the increase in CCT Na-K ATPase act activity seen with aldosterone alone at 3 h. Thus the increase in activity with aldosterone appears to be a secondary adaptation that is dependent on an aldosterone-enhanced increase in the passive entry of Na across the luminal membrane. The subcellular mechanism by which Na modulates Na-K ATPase activity remains obscure.

Correlation between ATP synthesis and the decay of the arotenoid band shift after single flash activation of chromatophores from Rhodopseudomonas capsulata.

ATP synthesis and the acceleration of the decay of the carotenoid absorption band shift after single flash excitation of Rhodopseudomonas capsulata chromatophores were compared. The two processes behave similarly with respect to: (1) ADP and Pi concentration; (2) inhibition by efrapeptin and venturicidin, and (3) inhibition by valinomycin/K+ and by ionophores. Taken together with earlier evidence for the electrochromic nature of the carotenoid band shift the data support the contention that positive charge moves outwards across the chromatophore membrane during ATP synthesis and justify the method for determination of the H+/ATP ratio (Petty, K.M. and Jackson, J.B. (1979) FEBS Lett. 97, 367-372). The ability of nucleotide diphosphates in the presence of Pi and Mg2+ to give rise to the acceleration of the carotenoid shift decay closely correlates with the rate of phosphorylation of the nucleotides in steady-state light. Nucleotide triphosphates enhance the decay in parallel with their rate of hydrolysis. Adenylyl imidodiphosphate is itself without effect on the decay of the carotenoid shift and it does not prevent the ADP-induced acceleration. The analogue does prevent the ATP effect but only after repeated flashes.

Kinetic factors limiting the synthesis of ATP by chromatophores exposed to short flash excitation.

ATP synthesis was measured after chromatophores from Rhodopseudomonas capsulata had been subjected to illumination by single turnover flashes fired at variable frequencies. Three processes were examined, which under different conditions can limit the net yield of ATP. (1) A process with an apparent relaxation time of 10-20 ms. This reaction probably limits the rate of ATP synthesis in continuous illumination. It has similar time dependence to the stimulation of the carotenoid shift decay by ADP after a single flash. (2) An active state of the ATPase only persists when the chromatophores are excited more often than once in 10 s. This state decays with similar kinetics to the entire carotenoid shift decay. Full activation is achieved after two flashes. (1) and (2) are not significantly affected by concentrations of antimycin A sufficient to block electron flow through the cytochrome b/c2 oxidoreductase and abolish phase III in the generation of the carotenoid shift. (3) In the presence of antimycin A, after the third, fourth and subsequent flashes ATP synthesis is limited by the quantity of reducing equivalents transported through the reaction centre rather than by the level of the electrochemical proton gradient.

Factors controlling the binding of two protons per electron transferred through the ubiquinone and cytochrome b/c2 segment of Rhodopseudomonas sphaeroides chromatophores.

1. On every turnover, 2.0 protons can be bound by the membrane for each single electron moving through the Q-b/c2 oxidoreductase. 2. One proton (H+II) binding reaction is, and one (H+I) is not, sensitive to antimycin. 3. The redox states of electron transfer components other than the proton binding agents can affect both the rate of proton uptake and the apparent pK values on the agents binding the protons. 4. The presence of valinomycin under certain well-defined conditions can strongly influence the value of the measured pK on the H+II binding agent.

Cytochrome c2 and reaction center of Rhodospeudomonas spheroides Ga. membranes. Extinction coefficients, content, half-reduction potentials, kinetics and electric field alterations.

The reduced minus oxidized extinction coefficients (delta epsilon-red-ox) of reaction center P605 when in the chromatophore is about 20% smaller than in the detergent-isolated state. Presumably the coupling of the reaction center protein to the antenna bacteriochlorophylls and carotenoids causes this hypochromism. The chromatophore values for P605 are 19.5 mM- minus 1 times cm- minus 1 with the spectrophotometer on single beam mode at 605 nm, and 29.8 mM- minus 1 times cm- minus 1 on dual wavelength mode set at 605--540 nm. Cytochrome c2, which is not affected by detergent, has a delta epsilon-red-ox value at 550--540 nm of 19.0 mM- minus 1 times cm- minus 1.2. The total bacteriochlorophyll to reaction center bacteriochlorophyll protein (P) ratio is about 100: 1. The cytochrome c2: reaction center protein ratio approaches 2. In current French press chromatophore preparations, about 70% of the reaction centers are each associated on a rapid kinetic basis with two cytochrome c2 molecules (intact P-c2 units). The remaining reaction center proteins are not associated with cytochrome c2 on a kinetically viable bais and may be the result of damage incurred during mechanical rupture of the cells. 3...

A TATA binding protein-associated factor functions as a coactivator for thyroid hormone receptors.

Transcriptional regulation by thyroid hormone receptors (TRs) requires the TR to interact with various proteins. The TATA binding protein-associated factors (TAFs) are cofactors for several transcription factors and, therefore, are candidate cofactors for the TR. To determine whether one or more of the TAFs are cofactors for TRs, direct protein interactions between human TR beta and several Drosophila TAFs were quantitated in vitro. The human (h) TR beta bound specifically to dTAFII110 and weakly to dTAFII60, but did not bind to dTAFII30 alpha, dTAFII30 beta, dTAFII40, dTAFII80, or dTAFII150. The dTAFII110:hTR beta interaction required the carboxyl-terminals of both proteins. The dTAFII110 also interacted with the hTR alpha 1 carboxyl-terminus in a yeast two-hybrid system. Thyroid hormone destabilized the dTAFII110:TR interaction in vitro, but had no effect on the interaction in the two-hybrid system. The dTAFII110 did not bind to human retinoid X receptor alpha in vitro, indicating that this TAF interacts differentially with nuclear receptors. The transcriptional function of hTR beta was enhanced by dTAFII110 in transfection assays, indicating that this TAF can function in the thyroid hormone signalling pathway. Thus, TAFII110 functions as a cofactor for TRs, and the interactions between specific TAFs and nuclear receptors may provide another level of selectivity for transcriptional responses to hormones.

Dominant inheritance of resistance to thyroid hormone not linked to defects in the thyroid hormone receptor alpha or beta genes may be due to a defective cofactor.

Resistance to thyroid hormone (RTH) is an inherited syndrome of reduced tissue responsiveness to thyroid hormone. To date, all individuals expressing the RTH phenotype have been found to harbor mutations in the thyroid hormone receptor beta (TR beta) gene that impair T3-mediated function. We describe a unique family in which the dominantly inherited RTH is not associated with abnormalities in the TR beta or TR alpha genes, as determined by gene sequencing and linkage analysis. However, affected family members manifest a severe form of RTH, with reduced responses of thyrotrophs and peripheral tissues requiring 8- to 10-fold the normal replacement doses of L-T4 and L-T3. No other endocrine abnormalities were detected. The defect developed de novo in the proposita and was transmitted to her two children of unrelated fathers. As cultured fibroblasts from the proposita responded poorly to T3 despite a normal concentration of TR, other abnormalities in the mediation of T3 action were sought. Nucleotide sequences of the TSH beta promoter, containing thyroid hormone response elements, and TR-interacting protein 1 were normal. Nuclear extracts (NE) of cultured skin fibroblasts from affected individuals of this family were tested for their interaction with normal TR beta and thyroid hormone response elements by the electrophoretic mobility shift assay. NE from the proposita showed a strong additional band compared to NEs from normal individuals and patients with RTH caused by TR beta mutations or deletion. Far Western analysis of NE from the affected daughter hybridized with labeled TR beta demonstrated an additional band that was not seen in NEs from a normal control or patients with TR beta gene defects. It is concluded that the etiology of RTH is not confined to abnormalities in the TR beta gene. An abnormal cofactor with a specific function in the regulation of thyroid hormone action is probably involved in the expression of the RTH phenotype in this family.

Tissue- and cell-specific distribution of proteins that interact with the human thyroid hormone receptor-beta.

Variable tissue effects of thyroid hormones depend on many factors that could include tissue-specific proteins that interact with nuclear thyroid hormone receptors. To study this possibility, direct interactions between the thyroid hormone receptor beta and other nuclear proteins were examined by far western blotting assays. Purified hTR beta bound to human retinoid X receptor alpha and human general transcription factor IIB (TFIIB) immobilized on a membrane and it also bound to several different hTR beta binding proteins (TRBPs) in nuclear extracts from rat brain, kidney, liver, GH3 cells, and HeLa cells. Each tissue or cell line showed unique distributions of TRBPs and some TRBPs were regulated by thyroid hormone in vivo. These studies show that multiple proteins interact with the hTR beta and they could be important in regulating TR function.

Effects of enteric-coated, low-dose aspirin on parameters of platelet function.

BACKGROUND: Aspirin is widely used as an anti-thrombotic drug; however, it has been suggested that enteric-coated formulations of aspirin may be less bioavailable and less effective as anti-thrombotic agents. AIM: To assess the effect of a formulation of enteric-coated, low-dose (81 mg) aspirin on serum generated thromboxane B2 and platelet aggregation in healthy subjects. METHODS: Twenty-four subjects participated in a double-blind, randomized, placebo-controlled, parallel-group, multiple-dose study. Twelve subjects in each of two groups received a daily oral dose of enteric-coated aspirin (81 mg) or matching placebo for 7 days. Serum thromboxane B2 and platelet aggregation (using 1 mm arachidonic acid and 1 microg/mL collagen as agonists) were measured 1-3 days prior to day 1, on day 1 (prior to therapy) and 4 h after the last dose on day 7. RESULTS: After seven daily doses of enteric-coated aspirin, the mean percentage inhibition from baseline of ex vivo generated serum thromboxane B2 was 97.4%, compared with a 7.8% increase after placebo treatment. The mean percentage inhibition of arachidonic acid- and collagen-induced platelet aggregation was 97.9% and 70.9%, respectively, following enteric-coated aspirin, compared with - 1.0% and 2.7%, respectively, after placebo. CONCLUSIONS: The anti-platelet effects of multiple, daily, low-dose aspirin (as assessed by inhibition of serum thromboxane B2 and platelet aggregation) are not adversely affected by enteric coating.

Effect of aprepitant on the pharmacokinetics and pharmacodynamics of warfarin.

OBJECTIVE: To examine the effect of aprepitant on the pharmacokinetics and pharmacodynamics of warfarin. Aprepitant is a neurokinin-1 (NK1)-receptor antagonist developed as an antiemetic for chemotherapy-induced nausea and vomiting. METHODS: This was a double-blind, placebo-controlled, randomized, two-period, parallel-group study. During period 1, warfarin was individually titrated to a stable prothrombin time (expressed as international normalized ratio, INR) from 1.3 to 1.8. Subsequently, the daily warfarin dose remained fixed for 10-12 days. During period 2, the warfarin dose was continued for 8 days, and on days 1-3 administered concomitantly with aprepitant (125 mg on day 1, and 80 mg on days 2 and 3) or placebo. At baseline (day -1 of period 2) and on day 3, warfarin pharmacokinetics was investigated. INR was monitored daily. During period 2, warfarin trough concentrations were determined daily. RESULTS: The study was completed by 22 healthy volunteers (20 men, 2 women). On day 3, steady-state pharmacokinetics of warfarin enantiomers after aprepitant did not change, as assessed by warfarin AUC(0-24 h) and C(max). However, compared with placebo, trough S(-) warfarin concentrations decreased on days 5-8 (maximum decrease 34% on day 8, P<0.01). The INR decreased after aprepitant with a mean maximum decrease on day 8 of 11% versus placebo (P=0.011). CONCLUSION: These data are consistent with a significant induction of CYP2C9 metabolism of S(-) warfarin by aprepitant. Subsequently, in patients on chronic warfarin therapy, the clotting status should be monitored closely during the 2-week period, particularly at 7-10 days, following initiation of the 3-day regimen of aprepitant with each chemotherapy cycle.

Metal-chelate affinity chromatography.

Recombinant proteins engineered to have six consecutive histidine residues on either the amino or carboxy terminus can be purified using a resin containing nickel ions (Ni(2+)) that have been immobilized by covalently attached nitrilotriacetic acid (NTA). This technique is know as metal-chelate affinity chromatography and can be performed using either native or denatured protein. This unit presents protocols for expression of histidine-tail fusion proteins and their purification in either native or denatured form (along with procedures for renaturation by either dialysis or solid-phase renaturation). Also provided are procedures for analysis of the purified produce and regeneration of the NTA resin.

Metal-chelate affinity chromatography.

Recombinant proteins engineered to have six consecutive histidine residues on either the amino or carboxyl terminus can be purified using a resin containing nickel ions (Ni(2+)) that have been immobilized by covalently attached nitrilotriacetic acid (NTA). This technique, known as metal-chelate affinity chromatography (MCAC), can readily be performed with either native or denatured protein. This unit discusses techniques for creating a fusion protein consisting of the protein of interest with a histidine tail attached. A procedure for expression of histidine-tail fusion proteins and their purification in native form by MCAC is described, and two alternate protocols describe purification of histidine-tail fusion proteins by MCAC under denaturing conditions and their renaturation by either dialysis or solid-phase renaturation. Support protocols are provided for analysis of the purified product and regeneration of the NTA resin. All of these protocols are easily adaptable to any protein expression system.

Metal-chelate affinity chromatography.

Recombinant proteins engineered to have six consecutive histidine residues on either the amino or carboxyl terminus can be purified using a resin containing nickel ions (Ni2+) that have been immobilized by covalently attached nitrilotriacetic acid (NTA). This technique, known as metal-chelate affinity chromatography (MCAC), can readily be performed with either native or denatured protein. Techniques are discussed for creating a fusion protein consisting of the protein of interest with a histidine tail attached (for purification by MCAC). The basic protocol describes expression of histidine-tail fusion proteins and their purification in native form by MCAC. Two alternate protocols describe purification of histidine-tail fusion proteins by MCAC under denaturing conditions and their renaturation by either dialysis or solid-phase renaturation. Support protocols are provided for analysis of the purified product and regeneration of the NTA resin. All of these protocols are easily adaptable to any protein expression system.

Functional characterization and receptor binding studies of the malic enzyme thyroid hormone response element.

We previously showed that the 5'-flanking region of the malic enzyme (ME) gene contains a cis-regulatory element (-281 to -261) that binds thyroid hormone receptors and confers triiodothyronine (T3) inducibility of transcription to the ME promoter (Petty, K.J., Desvergne, B., Mitsuhashi, T., and Nikodem, V. M. (1990) J. Biol. Chem. 265, 7395-7400). In this report, we have used deletion and mutation analyses of the ME thyroid hormone response element (TRE) to evaluate the roles of several subregions of TRE in T3 binding and transactivation. ME TRE was shown to act as an enhancer conferring T3 responsiveness to a heterologous promoter thymidine kinase. Although T3 treatment induced the promoter activity, the absence of hormone resulted in repression as measured by the level of chloramphenicol acetyltransferase expression in the NIH 3T3 transient expression system in the presence of overexpressed receptor. The degree of repression was similar to the degree of T3 induction observed for the same TRE mutants. Mutation and deletion analyses indicated that the functional TRE is comprised of discrete regions that are not contiguous, with a dominant role of a cluster of G residues and an AGGACA sequence. Both functions, induction and repression of transcription, correlated with receptor binding to the ME TRE as determined by competition binding assays using wild type and mutated TRE as competitors.

Thyroid hormone regulation of transcription factors involved in malic enzyme gene expression.

The mechanisms by which triiodothyronine (T3) regulates gene transcription are inadequately understood. In order to examine the effect of T3 on transcriptional mechanisms, we utilized several techniques to characterize trans-acting factors which interact with cis-regulatory elements of the promoter for the T3-responsive rat malic enzyme gene. Transcription of deletion mutants of the promoter by HeLa extracts revealed the existence of three cis-regulatory elements at -144 to -123, -70 to -50, and -30 to +5 (relative to the cap site at +1). DNase I footprinting disclosed the presence of proteins which bound to each of these regions. Gel mobility shift assays showed that specific binding of liver nuclear proteins to two of these regions (-144/-114 and -76/-47) was diminished in hypothyroid and elevated in hyperthyroid rats. Similar but less marked effects of T3 on the binding of kidney (a T3-responsive tissue) nuclear proteins to the -76/-47 and cap site regions were observed while binding of testis (a T3-nonresponsive tissue) nuclear proteins was unaffected by T3 treatment. Changes in the magnitude and time course of binding of liver proteins to this promoter correlated closely with previously documented T3-induced increases in the hepatic malic enzyme gene transcription rate. These results are consistent with a model in which the regulation of malic enzyme gene transcription by T3 in responsive tissues is mediated in part by hormone-induced increases in the binding of trans-acting factors to cis-regulatory elements.

Modification of otitis media following vaccination with the capsular polysaccharide of Streptococcus pneumoniae in chinchillas.

An animal model was used to determine whether vaccination with the capsular polysaccharide of Streptococcus pneumoniae could alter the development of experimental otitis media induced by the same type of S. pneumoniae as the vaccine. Following vaccination with the capsular polysaccharide of S. pneumoniae type 7, 36(63%) of 57 chinchillas seroconverted with at least a 100% increase in concentration of antibody in serum which remained elevated for at least six weeks. The middle ears of 23 chinchillas that were vaccinated and seroconverted, 13 that were vaccinated and did not seroconvert, and 47 that were not vaccinated were inoculated with S. pneumoniae type 7. Vaccinated animals that seroconverted developed otitis media as readily as nonseroconverting and unvaccinated animals but had fewer pneumococci in their middle ears, a lower incidence of bacteremia, and lower mortality rates during the first week after inoculation of bacteria. Animals that did not seroconvert showed no evidence of modification of their middle ear infections. These results indicate that type 7 pneumococcal capsular polysaccharide vaccine is antigenic for the chinchilla and modifies experimental otitis media due to type 7 S. pneumoniae.

Identification of a thyroid hormone response element in the malic enzyme gene.

We have studied the rat malic enzyme gene as a model for thyroid hormone (triiodothyronine (T3)) regulation of transcription. Our previous studies showed that transcription of this gene is controlled by T3 in vivo. In this study, we used COS-7 cells in culture to determine the location of a T3 response element (TRE) within this gene. Cotransfection of the rat T3 alpha-receptor cDNA with a reporter gene linked to 5'-deletion mutants of the malic enzyme gene 5'-flanking region revealed the presence of a TRE between positions -315 and -248. Using T3 receptors synthesized in vitro from their cDNAs, we have identified, through the use of gel shift assays and footprinting, a single DNA-binding site (positions -281 to -261) for the receptor within the rat malic enzyme gene TRE. The site was capable of binding either the rat alpha- or human beta-receptor with similar affinities. Competition binding studies indicated that the apparent affinity of receptor binding to the malic enzyme gene was similar to that of the rat alpha-myosin heavy chain gene TRE (positions -151 to -122) and was significantly greater than that of the rat growth hormone TRE (positions -192 to -163). These results indicate that both the alpha- and beta-forms of the nuclear T3 receptor are capable of binding directly to the malic enzyme gene 5'-flanking region at a site which functions as a hormone-inducible cis-regulatory element. In conjunction with our previous finding that T3 induces or activates essential transcription factors which bind to the promoter, it appears that the regulation of transcription of the malic enzyme gene by thyroid hormone involves at least two regulatory pathways.