Testosterone and the sex difference in blood pressure in mice

Blood pressure of adult male SWR/J mice is approximately 20 mm Hg higher than that of females. Castrating males at weaning eliminated this sex difference. Testosterone restored blood pressure of castrated mice to the level of the intact males within 10 days indicating a pressor effect of testosterone. Ovariectomy had no effect on blood pressure.     

Analytical techniques for cell fractions. XXIII. A stable thermal gradient device for heat denaturation studies on proteins

The ISO-DALT two-dimensional electrophoretic system (1,2), based on the method of O'Farrell (3), is capable of performing large numbers of analysis on complex mixtures of proteins. However, both separations employed are carried out under dissociating or denaturing conditions and no enzyme activities are readily observable in the analyzed proteins. In order to identify the spots corresponding to particular enzymes, it is therefore necessary to employ some nondestructive resolving technique first and as a second step to perform both enzyme and two-dimensional electrophoretic analyses on the fractions generated. By correlating enzyme activity with intensity of various spots on the two-dimensional gels throughout the series of initial fractions, identifications, can be made. This approach, unlike the more direct immunoprecipitation methods (4), requires the running of large numbers of enzyme analyses and two-dimensional gels and some convenient initial resolving procedure. Convenient and rapid techniques for the analyses (5,6) and gels (1,2) have been described previously in this series and elsewhere. This paper deals with the use of selective denaturation in a temperature gradient as an initial resolving procedure and describes a simple thermal gradient device for generating such a gradient.     

Measured change in protein solvation with substrate binding and turnover

Osmotic stress is used to measure solvation changes that accompany the conformational changes of an active enzyme. For hexokinase both the equilibrium dissociation constant and the kinetic Michaelis-Menten constant for glucose vary linearly, and to the same extent, with the activity of water in the protein medium, as adjusted with large molecular weight (> 2000) osmolytes. The variation over the whole osmotic pressure range studied indicates that glucose binding is accompanied by the release of at least 65 +/- 10 water molecules, and this is reversed on enzyme turnover. The results indicate that near the physiological range of pressures the number may be higher. Most of this water, which behaves like an inhibitor, likely comes from the cleft which is induced to close around the substrate. Such large dehydration/rehydration reactions during turnover imply a significant contribution of solvation to the energetics of the conformational changes. Osmotic stress is a method of general applicability to probe water's contribution to functioning molecules.     

EEG pharmacodynamics upon single administration of seduxen in healthy volunteers

We have examined the type I collagen protein, RNA, and cDNA of 2 children with moderately severe (type IV) osteogenesis imperfecta (OI). They have in common a non-lethal form of OI with ambulatory potential, overmodification of type I collagen protein, and a substitution of serine for glycine in the collagen chain produced by one alpha 1(I) allele. The first child (Marini et al.: J Biol Chem 264:11893-11900, 1989) is now 7 years old, with the height of a 3-year-old. Her course includes significant remodeling of lower long bones and 4 femur fractures. She walks independently. A mishmatch was detected in her alpha 1(I) mRNA using RNA/RNA hybrids; it was demonstrated to be due to a G-->A point mutation in one allele of alpha 1(I), resulting in the substitution of serine for glycine 832. The second child is now 6 1/2 years old, with the height of 1 1/2-year-old. Her history includes significant bowing of femurs and tibias, 6 femur fractures, S-curve scoliosis, compression of all lumbar vertebrae, and limited short-distance walking with braces. Her alpha 1(I) mRNA has also been studied by RNA hybrid analysis; there is a single G-->A change in one alpha 1(I) allele causing the substitution of serine for gly 352. Both children have moderately severe OI. However, the serine substitution at gly 352 is associated with a more severe phenotype then is the serine substitution at gly 832. Compared to substitutions described in other cases of OI, the serine 352 is located in the middle of a cluster of cysteine substitutions associated with non-lethal OI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chiroinositol deficiency and insulin resistance. I. Urinary excretion rate of chiroinositol is directly associated with insulin resistance in spontaneously diabetic rhesus monkeys

Previously, we demonstrated that nondiabetic insulin-resistant monkeys had reduced covalent insulin activation of muscle glycogen synthase (GS) compared to normal monkeys and that covalent insulin activation of adipose tissue GS was absent in these monkeys. Covalent insulin activation of muscle and adipose tissue GS in monkeys with impaired glucose tolerance and noninsulin-dependent diabetes (NIDDM) was also absent. As in humans, monkeys with NIDDM have a lower urinary excretion rate of chiroinositol (CI), a component of a putative mediator of insulin action, compared to normal monkeys. To determine whether the urinary excretion rate of CI was related to insulin resistance, which develops naturally in many obese rhesus monkeys, we examined the relationships between 24-h urinary CI excretion rate and 1) whole body insulin-mediated glucose disposal rates (M) and insulin-mediated changes in 2) the skeletal muscle GS activity ratio (sm delta GSAR), 3) the skeletal muscle glycogen phosphorylase activity ratio, and 4) the adipose tissue GS activity ratio (at delta GSAR) in 27 monkeys ranging from normal (n = 12) to insulin resistant (n = 8) to overtly diabetic (n = 7). The urinary CI excretion rate was significantly correlated with M (r = 0.47; P < 0.02), sm delta GSAR (r = 0.38; P < 0.05), skeletal muscle glycogen phosphorylase activity ratio (r = -0.49; P < 0.01), and at delta GSAR (r = 0.46; P < 0.02). The urinary CI excretion rate was also correlated with glucose tolerance (r = 0.39; P < 0.05). There was a wide range of urinary CI excretion rates (0.42-5.17 mumol/day) in monkeys with normal fasting plasma glucose concentrations. However, of the 7 diabetic monkeys, 6 had a urinary CI excretion rate below 2.0 mumol/day, and in the subgroup of 16 monkeys with a urinary CI excretion rate less than 2.0 mumol/day, the associations of urinary CI with M rate (r = 0.65; P < 0.005), glucose tolerance (r = 0.63; P < 0.01), and sm delta GSAR (r = 0.73; P < 0.001) increased in strength and significance. We propose that the urinary CI excretion rate may be 1) a biochemical indicator of both in vivo and in vitro insulin resistance and 2) a noninvasive diagnostic tool with potential for the identification of those individuals at risk for NIDDM and other related diseases with insulin resistance.     

Temperature effect on the action of corticotropin-releasing hormone (CRH) on secretion of immunoreactive beta-endorphin (IR beta EP) in TM3 and AtT-20 cell lines

Testicular function is sensitive to chemical and thermal stresses. To investigate the effects of small temperature changes on CRH-stimulated beta EP release, we employed TM3 cells, a mouse prepubertal Leydig cell line that secretes ir beta EP. To monitor beta EP secretion from these cells we used the reverse hemolytic plaque assay. After 3.5 hr incubation of cells with hormone, the EC50 of the CRH dose-response curve at 34 degrees C and 37 degrees C were 0.1 nM and 1 nM, respectively. For comparison, we also investigated the effect of temperature on CRH-stimulated beta EP release from a non-testicular cell line, AtT-20, a mouse anterior pituitary cell line. Using radioimmunoassay to measure ir beta EP levels in the media of AtT-20 cells, the EC50s for the CRH dose-response curve at 34 degrees C and 37 degrees C were 0.2 nM and 2 nM, respectively, at 1 h. After 3.5 h this temperature dependent difference in EC50 was still observed. These results suggest that CRH receptors or post-receptor actions in Leydig cells and anterior pituitary corticotropes are sensitive to small temperature changes.     

Optical amplification of ligand-receptor binding using liquid crystals

Liquid crystals (LCs) were used to amplify and transduce receptor-mediated binding of proteins at surfaces into optical outputs. Spontaneously organized surfaces were designed so that protein molecules, upon binding to ligands hosted on these surfaces, triggered changes in the orientations of 1- to 20-micrometer-thick films of supported LCs, thus corresponding to a reorientation of approximately 10(5) to 10(6) mesogens per protein. Binding-induced changes in the intensity of light transmitted through the LC were easily seen with the naked eye and could be further amplified by using surfaces designed so that protein-ligand recognition causes twisted nematic LCs to untwist. This approach to the detection of ligand-receptor binding does not require labeling of the analyte, does not require the use of electroanalytical apparatus, provides a spatial resolution of micrometers, and is sufficiently simple that it may find use in biochemical assays and imaging of spatially resolved chemical libraries.     

Recombinant adeno-associated virus vector: use for transgene expression and anterograde tract tracing in the CNS

Several signal transduction pathways have been implicated in the mechanism of protection induced by ischemic preconditioning (PC). For example, stimulation of a variety of G-protein coupled receptors results in stimulation of protein kinase C (PKC) which has been suggested to act as common denominator in eliciting protection. PC also significantly attenuated cAMP accumulation during sustained ischemia, suggesting involvement of an anti-adrenergic mechanism. The aim of this study was to evaluate the beta-adrenergic signal transduction pathway (as evidenced by changes in tissue cAMP and cAMP- and cGMP-phosphodiesterase) during the PC protocol as well as during sustained ischemia. Isolated perfused rat hearts were preconditioned by 3 x 5 min global ischemia (PC1,2,3) interspersed by 5 min reperfusion, followed by 25 min global ischemia. Tissue cAMP- and cGMP-PDE activity as well as cAMP and cGMP levels were determined at different time intervals during the PC protocol and sustained ischemia. Tissue cAMP increased with each PC ischemic event and normalized upon reperfusion, while PDE activity showed the opposite, viz a reduction during ischemia and an increase during reperfusion. Except for PC1, tissue cGMP showed similar fluctuations. Throughout 25 min sustained ischemia, cAMP- and cGMP-PDE activities were higher in PC than in nonpreconditioned hearts, associated with a significantly lesser accumulation in cAMP and higher cGMP levels in the former. Fluctuations in cyclic nucleotides during preconditioning were associated with concomitant changes in PDE activity, while the attenuated beta-adrenergic response of preconditioned hearts during sustained ischemia may partially be due to increased PDE activity.     

Dimerization of recombinant horseradish peroxidase in a reversed micellar system

Recombinant horseradish peroxidase reactivated from E. coli inclusion bodies was studied in a reversed micellar system of AOT in octane. The ability of the recombinant enzyme, in contrast to native horseradish peroxidase, to form a dimeric structure was found. The existence of the dimer was proved by results of sedimentation analysis. Dimer/monomer ratio in the enzyme-containing micelles and dimer catalytic activity were found to depend on the substrate used (pyrogallol, guaiacol, o-dianisidine, o-phenylenediamine). Computer modelling was used to describe possible structures of the dimeric recombinant horseradish peroxidase.     

Antibiotic phenol nor-triterpenes from Maytenus canariensis

OBJECTIVE: To calculate and compare the costs of the treatment of varicocele by antegrade scrotal sclerotherapy with other modalities. PATIENTS AND METHODS: A total of 2305 operations using antegrade scrotal sclerotherapy to treat varicocele in childhood and adolescence were analysed for cost factors and compared with different surgical treatment methods for varicocele. RESULTS: Calculation of the pre-, intra- and post-operative costs showed that antegrade scrotal sclerotherapy was the most economically effective of all forms of surgical management for varicocele. CONCLUSIONS: Because antegrade scrotal sclerotherapy is a cost-effective treatment for varicocele, the indications for treatment may be widened to include more men with potential infertility, and thus avoid the need for expensive methods of artificial fertilization.