Androgen receptor mutation (T877A) promotes prostate cancer cell growth and cell survival.

Alteration of the AR functions due to amplification, overexpression and somatic mutation of the AR itself or altered interaction of AR with other cell growth regulatory proteins, may contribute to a significant subset of advanced prostate cancer (CaP). Very little is known about the pathways impacted by AR dysfunctions, although CaP associated AR alterations suggest the biological role of the AR dysfunction in disease progression. Comparative evaluations of wild type (wt) AR and mutant (mt) ARs in appropriate experimental models should provide a better understanding of the functional impact of AR alterations in CaP. Here, we provide direct evidence showing cell growth/cell survival promoting effects of the widely studied CaP associated AR mutation (T877A). In contrast to Ad-wtAR or Ad-control infected LNCaP or LAPC4 cells, Ad-mtAR (T877A) infected LNCaP or LAPC4 cells continued to grow in the androgen-deprived medium and exhibited an androgen independent AR-transcription factor activity. Further, Ad-mtAR (T877A) infected LNCaP or LAPC4 cells exhibited enhanced cell growth in the presence of lower concentrations of the synthetic androgen, R1881. Of note, Ad-mtAR (T877A) infected LNCaP cells showed striking resistance to cell growth inhibition/apoptosis mediated by the wt p53. Taken together, these findings provide novel insights into the AR dysfunctions resulting from the T877A mutation and functionally similar AR alterations may provide selective cell growth/survival advantage for CaP progression. These observations have important implications for developing biology-based prognostic biomarkers and therapeutic strategies for CaP showing such AR dysfunctions.

Azepanone-based inhibitors of human and rat cathepsin K.

The synthesis, in vitro activities, and pharmacokinetics of a series of azepanone-based inhibitors of the cysteine protease cathepsin K (EC 3.4.22.38) are described. These compounds show improved configurational stability of the C-4 diastereomeric center relative to the previously published five- and six-membered ring ketone-based inhibitor series. Studies in this series have led to the identification of 20, a potent, selective inhibitor of human cathepsin K (K(i) = 0.16 nM) as well as 24, a potent inhibitor of both human (K(i) = 0.0048 nM) and rat (K(i,app) = 4.8 nM) cathepsin K. Small-molecule X-ray crystallographic analysis of 20 established the C-4 S stereochemistry as being critical for potent inhibition and that unbound 20 adopted the expected equatorial conformation for the C-4 substituent. Molecular modeling studies predicted the higher energy axial orientation at C-4 of 20 when bound within the active site of cathepsin K, a feature subsequently confirmed by X-ray crystallography. Pharmacokinetic studies in the rat show 20 to be 42% orally bioavailable. Comparison of the transport of the cyclic and acyclic analogues through CaCo-2 cells suggests that oral bioavailability of the acyclic derivatives is limited by a P-glycoprotein-mediated efflux mechanism. It is concluded that the introduction of a conformational constraint has served the dual purpose of increasing inhibitor potency by locking in a bioactive conformation as well as locking out available conformations which may serve as substrates for enzyme systems that limit oral bioavailability.

High-resolution inset head-mounted display.

A novel approach to inset superimposition in a high-resolution head-mounted display (HMD) is presented. The approach is innovative in its use of optoelectronic, nonmechanical devices in place of scanning mechanical devices commonly adopted previously. A paraxial layout of the overall HMD system is presented, and the benefit of employing hybrid refractive-diffractive optics for the optical component that generates the inset is discussed. A potential overall HMD design is finally presented to show the integrated system. The practical limitations of the designed system are discussed and an alternative approach is presented to compare the advantages and the limitations of these systems.

Conditions affecting the immunohistochemical detection of HIV in fixed and embedded renal and nonrenal tissues.

A number of studies have suggested that HIV infection can be detected in a variety of routinely fixed archival tissues using antibodies to various viral proteins. In order to study this immunocytochemical approach, paraffin sections were examined with a large panel of commercially available monoclonal antibodies against the various HIV proteins (5 antibodies to p24, 1 to p17, 1 to gp41, and 1 to gp120) using a streptavidin-biotin method. A polyclonal antibody against p24 was also tested. Formalin-fixed, paraffin-embedded HIV infected CEM E5 T cells were used as positive controls. Tissues from AIDS patients included 31 kidneys, 8 lymph nodes, 2 spleens and 3 brains. Non-AIDS tissues examined were 6 renal biopsies with focal segmental glomerulosclerosis, 5 with interstitial nephritis, 6 reactive lymph nodes, and a brain with encephalitis, all from patients not known to be at high risk for HIV infection. Additional negative controls included: 1) replacement of primary antibody with a hybridoma derived mouse monoclonal IgG1 standard, 2) omission of the primary antibody, and 3) sections of formalin-fixed paraffin-embedded CEM E5 T cells cultures not infected with HIV. Competition experiments with excess recombinant p24 protein were also performed. False positive staining with the IgG1 standard or with the antibodies to HIV proteins was frequently seen in tissues with pathologic findings (inflammation, hyalin degeneration), particularly following protein digestion. Protein digestion also had a major impact on specific staining. Digestion with proteinase K abolished specific staining for the core proteins of the virus (p17, p24) on the positive control sections.(ABSTRACT TRUNCATED AT 250 WORDS)

The domestic goat: a useful model to determine effects of diet and exercise on cholesterol accumulation in the body.

1. Young goats were used to study factors contributing to atherosclerosis. 2. Cholesterol in egg yolk affected plasma cholesterol concentration more than did a similar amount of crystalline cholesterol in the diet. 3. Goats fed high fat diets developed fatty lesions in their aortas. 4. Cholesterol concentration in low-density lipoprotein was greatest in goats fed restricted-calorie diets without exercise, but least in goats fed liberally and exercised. 5. Cholesterol concentration in liver and fat deposition in aorta were greatest in the restricted-calorie, no exercise regime.

Myeloma cast nephropathy: immunohistochemical and lectin studies.

Renal disease is a common cause of morbidity and mortality in patients with plasma cell dyscrasia (PCD). We have conducted a systematic study of the formalin-fixed, paraffin-embedded renal tissues from 53 patients with plasma cell dyscrasia, 24 of whom had Bence Jones cast nephropathy (with large casts, often associated with giant cells and polymorphonuclear leukocytes). A battery of 5 immunocytochemical and lectin markers for various segments of the nephron was used [Tetragonolobus lotus, Arachis hypogaea (AH), Tamm-Horsfall protein (THP), epithelial membrane antigen (EMA), and cytokeratin (AE1/AE3)]. In particular, we sought to determine the nature of the intratubular multinucleated giant cells in Bence Jones myeloma cast nephropathy with a variety of epithelial and hematopoietic cell markers. Although tubular epithelial cells stain with their respective markers (whether inflamed, thinned, detached, or adjacent to and lining casts), true intratubular giant cells in PCD were never positive for these tubular markers. In approximately one-third of the cases studied, intratubular and extratubular giant cells stained for several of the seven hematopoietic cell markers employed [i.e., alpha 1-antitrypsin (A1AT), alpha 1-antichymotrypsin (A1ACT), vimentin, and lysozyme], suggesting that giant cells are of hematopoietic origin. The majority of the casts are present in the distal nephron, although some casts were noted in more proximal sites of the nephron. Some larger casts did not stain for THP; smaller casts often showed lamination or stratification of THP staining. Finally, in one-half of the cases, Tamm Horsfall protein (THP) and other distal tubular markers (AH, EMA, AE1/AE3) were found in Bowman's space, almost always in association with interstitial deposits of THP; these markers were virtually never noted in Bowman's spaces of PCD patients without numerous large casts. This suggests that there are communications between distal and proximal nephron, most likely by intraluminal reflux but possibly also through breaks in the tubules and via the interstitium.

Quantitation of fat and cholesterol transport in the mesenteric lymph and bile of the preruminant calf.

Nine preruminant male calves were prepared surgically with lymphatico-venous shunts and/or re-entrant gallbladder to proximal duodenum shunts to evaluate the effects of degree of saturation of dietary fat on cholesterol transport in intestinal lymph and bile. Liquid diets were formulated to contain 12.5% dried skim milk (SM) or 10.5% SM to which 2% soybean oil (SBO), milk fat (MF), beef tallow (T) or one of these fats plus supplemental cholesterol was added. After 3-d dietary treatments, total lymph collections were made to determine flow rate, total lipid and cholesterol transport. Total bile collections were made to determine flow rate and cholesterol and bile acid transport. For SM, SBO, MF and T diets, respectively, average lipid transport in mesenteric lymph was 8.94, 32.58, 64.86 and 38.12 mg/(h X kg body weight), and cholesterol transport averaged 1.09, 1.92, 2.41 and 2.70 mg/(h X kg body weight). Lipid and cholesterol transport in lymph was less (P less than 0.05) in SM-fed calves than in fat-fed calves. Source of fat or supplemental cholesterol had no statistically significant effect on amount of cholesterol or bile acid transported in bile; however, calves fed SM transported greater quantities of cholesterol in bile than did calves fed fat or fat plus cholesterol.

Cholesterol transport in mesenteric lymph of the preruminant calf: contribution of cholesterol transposition from blood to lymph.

Eight preruminant male calves were prepared surgically with lymphatico-venous shunts and re-entrant gallbladder to proximal duodenum shunts. Liquid diets were formulated to contain 12.5% dried skim milk (SM) or 10.5% SM to which was added 2% soybean oil (SBO), milk fat (MF) or beef tallow (T). Two calves were assigned to each dietary treatment. Transposition of cholesterol from blood capillaries to intestinal lymph was determined by injection of 100 microCi [4-14C]-cholesterol into the blood of calves at feeding time. To avoid recirculation of [4-14C]-cholesterol via the enterohepatic circulation, bile was diverted and replaced with bile from a donor calf fed an identical diet. For the SM, SBO, MF and T diets, respectively, cholesterol transposed from capillaries was 44, 61, 36 and 48% of the cholesterol transported in the mesenteric lymph. When cholesterol synthesis in response to test diets was calculated, we found that intestinal cholesterol synthesis is less when calves are fed SM or SBO than when fed T or MF.

Combined effects of ouabain and hypoxia on the transmembrane potential of of canine cardiac Purkinje fibers.

The effects of hypoxia on the transmembrane potential of dog Purkinje cells in isolated, superfused fiber bundles pretreated with ouabain were studied. Cells stimulated electrically at 93/min were exposed to ouabain, 2.1 X 10(-7) M, until the magnitude of phase 4 depolarization increased to 7-12 mV. Arrhythmias did not occur. Following a 10-min washout period, hypoxic solution (PO2 = 15-50 mm Hg) was applied for 2-5 min. This caused decreases in maximum diastolic potential, overshoot, rising velocity of phase 0, and duration of the action potential. The slope of depolarization during phase 4 increased markedly. Arrhythmias characterized by escape rhythms or single and multiple bursts of premature excitations occurred in greater than 90% of the experiments. None of these changes was noted when identical levels of hypoxia were applied for a similar period to normal cells. Blockade of the beta-adrenergic receptors with propranolol, 0.3 mg/L, did not alter the response of ouabain-pretreated cells to hypoxia in any manner, ruling out release of endogenous catecholamines as essential for the observed effects. These results suggest that ouabain and hypoxia have a synergistic effect directly on the cells and produce the observed changes in membrane potential. The ventricular arrhythmias observed in digitalized humans or animals that become hypoxemic may result either from the induction of oscillatory after potentials in Purkinje cells causing triggered spontaneous excitations or from reentry of excitation.

Effects of flecainide on the electrophysiologic properties of isolated canine and rabbit myocardial fibers.

The electrophysiologic properties of flecainide, a new potent antiarrhythmic drug, are poorly defined. In this study, they were investigated by standard microelectrode technique in isolated cardiac muscle from rabbit and dog hearts. The concentrations of flecainide used were between 0.1 and 10.0 micrograms/ml. Flecainide produced a concentration-dependent decrease in maximal rate of rise of phase 0 of the action potential (Vmax), action potential amplitude and overshoot potential with an increase in the effective refractory period in ventricular muscle. Vmax was reduced by 52.5% after 1 microgram/ml of flecainide (p less than 0.001) and by 79.8% after 10.0 micrograms/ml (p less than 0.001). The corresponding values for Purkinje fibers were 18.6% (p less than 0.01) and 70.8% (p less than 0.001), respectively, but in these fibers the effective refractory period was shortened at the lower concentration and restored to control value at the higher concentration. The depression of Vmax by flecainide was frequency-dependent. The action potential duration was lengthened by flecainide in ventricular muscle and shortened in Purkinje fibers. At high concentrations (10 micrograms/ml), flecainide depressed slow channel-dependent fibers. Purkinje fiber automaticity induced by isoproterenol was slowed by flecainide. The data indicate that the overall electrophysiologic effects of flecainide in isolated cardiac muscle are complex with a major depressant action on Vmax that may account for its dominant antiarrhythmic effects. It is also possible that the differential effects of the compound on the action potential duration and refractoriness in ventricular muscle and Purkinje fibers contribute to the known arrhythmogenic potential of the drug.

H+-induced membrane depolarization in canine cardiac Purkinje fibers.

The H+-induced membrane depolarization in canine cardiac Purkinje cells in false tendons was studied. In some electrically paced Purkinje cells ("sensitive" cells), exposure to a pH 6.0 superfusate produced a large membrane depolarization [44.5 +/- 6.7 (SD) mV], whereas other Purkinje cells ("resistant" cells) developed only a small depolarization (9.8 +/- 5.6 mV) even after 60 min of exposure to the low-pH superfusate. Cs+, Ba2+, tetraethylammonium, 9-aminoacridine, verapamil, or exposure to Ca2+- or K+-deficient or hypertonic solutions were capable of converting resistant cells to sensitive cells. Increasing extracellular K+ concentration [( K+]) or rapid electrical pacing failed to convert resistant cells to sensitive cells. Membrane depolarizations of approximately equal magnitude produced in Purkinje cells by either increasing [K+] to 18.2 mM, decreasing [K+] to 0.5 mM, reducing extracellular pH to 4.1, or ouabain administration were associated with membrane resistances of approximately 45, 377, 386, or 45%, respectively, of the membrane resistances in the control solution. The results suggest that the H+-induced membrane depolarization in sensitive Purkinje cells is caused by a mechanism similar to that responsible for a low [K+]-induced depolarization.

Demonstration of beta-adrenergic receptors in rat bronchiolar epithelial cells employing 9-amino-acridyl propranolol fluorescent microscopy.

Prior work has provided ultrastructural evidence that beta-adrenergic agonists stimulate secretion by nonciliated bronchiolar epithelial (Clara) cells of the rat (J Clin Invest 67:345, 1981). However, since the lung is a multicellular organ it is not clear if the beta-agonists act directly on the Clara cell. The absence in Clara cells of beta-adrenergic receptors would indicate an indirect action of the beta-adrenergic agonists. In the present study, we used 9-amino-acridyl propranolol in an attempt to determine if beta-adrenergic receptors are present in rat bronchiolar Clara cells. Discrete, intense yellow fluorescent dots were identified microscopically in ciliated and in Clara cells of the rat. This anatomical localization of beta-adrenergic receptors supports the notion that beta-adrenergic agonists stimulate secretion by acting directly on Clara cells.

Synthesis, resolution, absolute stereochemistry, and enantioselectivity of 3',4'-dihydroxynomifensine.

3',4'-Dihydroxynomifensine, 8-amino-1,2,3,4-tetrahydro-4-(3,4-dihydroxyphenyl)-2-methylisoquinoli ne (1a), is an agonist of dopamine receptors in central and peripheral systems. Since this dopamine receptor agonist bears an asymmetric center at position 4, its synthesis and resolution were undertaken as part of a study directed toward determining the mode of interaction of these agents with the receptor(s). The enantiomers of 3',4'-dihydroxynomifensine are of particular interest, as they provide additional probes of present conceptual models of the dopamine receptor(s). Initial attempts to prepare 1a were inefficient or unsuccessful; instead, an isomeric compound, 1,2,4,5-tetra-hydro-2-(3,4-dihydroxyphenyl)-4- methyl-3H-1,4-benzodiazepine (9), was obtained. For this reason, a new route to 3',4'-dihydroxynomifensine was employed. The racemic dimethoxy intermediate 1d, thus obtained, was resolved. Methoxyl cleavage of the isomers of 1d afforded the enantiomers of 1a. Enantiomeric excess of these antipodes or appropriate derivatives was examined by NMR, CD, and HPLC methods. CD analysis suggests an enantiomeric excess greater than 99%. Determination of the absolute configuration of the enantiomers of 1a was determined by single-crystal X-ray diffractometric analysis. Examination of the isomers in several pharmacological test systems revealed a high degree of enantioselectivity. D-1 dopaminergic activity resides almost exclusively in the S enantiomer. The findings of the study have been employed to suggest an accessory binding site on the dopamine receptor(s) that differs from that advanced earlier. This accessory binding site may be specific for the D-1 subpopulation of dopamine receptors.

Effects of glucose on the transmembrane potential of hypoxic canine ventricular muscle fibers.

Since the introduction of glucose-insulin-potassium therapy for acute myocardial infarction 20 years ago, there have been many attempts to determine the efficacy of this treatment. Some trials have shown that this therapy is antiarrhythmic and others have found no benefit. In this study we demonstrate that concentrations of glucose found to be antiarrhythmic in recent clinical trials exert favorable effects on resting membrane potential, overshoot, and rising velocity in hypoxic canine heart cells. The refractory period, shortened by hypoxia, is also significantly lengthened by extra glucose. All of these single-cell effects are potentially antiarrhythmic and may explain the effectiveness of this therapy.

The subcellular localization of the beta-galactoside-binding protein of rat lung.

The subcellular localization of the beta-galactoside-binding protein, or lectin, from rat lung was investigated by the specific binding of anti-lectin immunoglobulin G to subcellular fractions. We used both adult and immature (12-day-old) rats; the immature rat lungs have an 8-10-fold greater concentration than adult rat lungs [Powell & Whitney (1980) Biochem. J. 188, 1-8]. In both groups of animals we observed greater specific binding of anti-lectin immunoglobulin G to intracellular membrane (mitochondrial and microsomal fractions) than to plasma membranes. Pre-incubation of membrane fractions with lactose resulted in a marked diminution of anti-lectin immunoglobulin G binding. In the adult rat lung most (approx. 80%) of the lectin activity was membrane-associated. In the immature rat lung only approx. 30% of the lectin activity was membrane associated and most of the beta-galactoside-binding protein appeared to be a soluble cytoplasmic component. The rat lung beta-galactoside-binding protein appeared to have a broad but predominantly intracellular location, being associated with membranes through one of its galactoside-binding sites.

Characterization of unusual intermediate density lipoproteins.

We report on the physicochemical properties of unusual lipoproteins isolated from both lymph and blood of ruminating cattle. The densities of most of these particles fall within the range between 1.006 and 1.020 g/ml, although densities of 0.97-0.99 g/ml are calculated from chemical composition, assuming a liquid core. The triglycerides of these particles have a high content of saturated fatty acids. The major apoprotein has a mobility on polyacrylamide-SDS gels consistent with a molecular weight of 40,000. The negatively-stained particles appear flattened and asymmetric in electron micrographs. The particles are very large, with molecular weights in the 20 to 250 million dalton range, and they scatter light strongly. The hydrodynamic frictional ratio is about 1.4, consistent with oblate ellipsoids with axial ratios of about 8 to 1. The flat appearance, asymmetric shape, and anomalous densities of the particles would be explained if these lipoproteins consisted of a core of crystallized triglycerides encapsulated within a phospholipid monolayer. Crystallization of the saturated triglycerides could occur during routine lipoprotein isolation, in which temperatures much lower than the melting points of their core lipids are employed. when protocols are done entirely at 37 degrees C, the unusual structures are not observed in the intermediate density class. Although the saturated fats in these bovine lipoproteins are derived from ruminal fermentation, we feel that any triglyceride-rich lipoprotein highly enriched in saturated fats will behave similarly if isolation temperatures are well below the melting points of the core lipids.

Orally active 7-phenylglycyl cephalosporins. Structure-activity studies related to cefatrizine (SK&F 60771).

The synthesis of a series of related broad-spectrum 7-phenylglycyl cephalosporins with 3-heterocyclicthiomethyl substituents is described. The effects of benzene-ring hydroxylation and 3-substituent variation on the in vitro antibacterial activity, height and duration of mouse serum levels, and effectiveness in protecting against bacterial infection in the mouse are examined. Included for comparison are cephalexin, cephaloglycin and their ortho-, meta- and para-hydroxy derivatives. The biological properties examined were influenced by the position of the hydroxyl group and by the nature of the 3-substituent. The 7-(p-hydroxyphenylglycyl)-3-heterocyclicthiomethyl analogs were found to produce significantly higher serum levels on oral administration to mice than their unhydroxylated counterparts. This effect was not observed with the 7-(m-hydroxyphenylglycyl)-3-heterocyclicthiomethyl cephalosporins, nor with the p-hydroxyphenylglycyl analog of cephalexin. While m- and p-hydroxylation had little effect on in vitro activity and o-hydroxyphenylglycyl cephalosporins tested had very low antibacterial activities and were not examined further. One derivative, 7-[R-2-amino-2-(4-hydroxyphenyl)acetamido]-3-(1H-1, 2, 3-triazole-4(5)-ylthiomethyl)-3-cephem-4-carboxylic acid (SK&F 60771) was found to have outstanding in vitro and in vivo activities along with oral and subcutaneous serum levels in the mouse that were significantly higher than those obtained with cephalexin. This derivative which has been given the generic name cefatrizine was selected for extensive additional biological evaluation.