Phenotypic differences in the GnRH neuronal system of deer mice Peromyscus maniculatus under a natural short photoperiod

The neural mechanisms by which short photoperiod induces gonadal regression among seasonally breeding mammals are not well understood. One hypothesis suggests that the proximate cause of seasonal gonadal regression is a photoperiod-induced modification in GnRH secretion. This hypothesis is indirectly supported by our recent findings using immunocytochemistry which identified specific photoperiod-induced adjustments in the number and morphology of GnRH containing neurones between reproductively competent and reproductively regressed laboratory housed male deer mice. Herein, we report that the GnRH neuronal system is similarly affected in reproductively responsive and nonresponsive wild male deer mice Peromyscus maniculatus exposed to a natural short photoperiod. The distribution of immunoreactive (IR)-GnRH neurones was nearly identical in field caught animals and those housed under artificial photoperiod in the laboratory. Compared with reproductively nonresponsive males, reproductively responsive mice from the field population possessed a greater total number of IR-GnRH neurones, a greater number of IR-GnRH neurones within the lateral hypothalamus, and a greater proportion of bipolar IR-GnRH neurones. Each of these distributional and morphological characters was consistent with our findings in laboratory housed male deer mice exposed to an artificial short photoperiod. Taken together, these data underscore the validity of using an artificial photoperiod to evaluate seasonal adjustments in reproductive function in the laboratory.     

Measuring the impact of menopausal symptoms on quality of life.

OBJECTIVE--To examine the impact of menopausal symptoms on the overall quality of life of women. DESIGN--Data collection with a questionnaire administered by an interviewer, incorporating two different quality of life measurement techniques (time trade off and rating scale). SETTING--Specialist menopause clinic and two general practices in Oxford. SUBJECTS--63 women aged 45-60 years recruited opportunistically during a clinic or appointment with a general practitioner; no exclusion criteria. RESULTS--Subjects gave very low quality of life ratings for health states with menopausal symptoms. The time trade off method of measuring preferences for these health states (on a scale from 0 to 1, where preference for full health is given as 1) yielded utility values of 0.64 for severe menopausal symptoms and 0.85 for mild symptoms. The rating scale measurement technique yielded even lower values: utilities of 0.30 and 0.65 were obtained for severe and mild symptoms respectively. Kappa scores indicated that the two methods produced results that were poorly related but not contradictory. Comparison of quality of life ratings before and after treatment with hormone replacement therapy showed significant improvements: with the rating scale measurement technique mean increases in utility values after the relief of severe and mild menopausal symptoms were 0.56 and 0.18 respectively. CONCLUSIONS--Quality of life may be severely compromised in women with menopausal symptoms, and perceived improvements in quality of life in users of hormone replacement therapy seem to be substantial. This emphasises the need to include quality of life measurements when assessing outcomes of hormone replacement therapy. Several limitations may exist with widely applied measurement techniques, calling for the development of appropriate and well validated instruments for measuring quality of life associated with reduced health states.     

Purification and characterization of cholyl-CoA: taurine N-acetyltransferase from the liver of domestic fowl (Gallus gallus).

The enzymological basis for the ability of mammalian liver to conjugate bile acids with both glycine and taurine, and for non-mammalian liver to make only taurine conjugates, was investigated. The taurine-conjugating enzyme has been purified 1200-fold from the liver of domestic fowl and its properties compared with those of the glycine/taurine-conjugating enzyme from bovine liver [Czuba & Vessey (1980) J. Biol. Chem. 255, 5296-5299]. The enzyme from both species followed a Ping Pong mechanism. The enzymes were also similar with respect to their affinity for taurine, although the enzyme from domestic fowl would not bind glycine. The affinity of both for cholyl-CoA was quite similar, too, and both enzymes were inhibited reversibly by p-mercuribenzoate. The enzymes, however, were quite different in size. The enzyme from domestic fowl had a mol.wt. of 63000-65000 by both gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This is approx. 15 000 mol.wt. units larger than the enzyme from bovine liver, and suggests a loss of genome over the course of evolution as the basis for the altered specificity at the amino-acid binding site.     

Inhibition of glutathione S-transferase by bile acids.

The effects of bile acids on the detoxification of compounds by glutathione conjugation have been investigated. Bile acids were found to inhibit the total soluble-fraction glutathione S-transferase activity from rat liver, as assayed with four different acceptor substrates. Dihydroxy bile acids were more inhibitory than trihydroxy bile acids, and conjugated bile acids were generally less inhibitory than the parent bile acid. At physiological concentrations of bile acid, the glutathione S-transferase activity in the soluble fraction was inhibited by nearly 50%. This indicates that the size of the hepatic pool of bile acids can influence the ability of the liver to detoxify electrophilic compounds. The A, B and C isoenzymes of glutathione S-transferase were isolated separately. Each was found to be inhibited by bile acids. Kinetic analysis of the inhibition revealed that the bile acids were not competitive inhibitors of either glutathione or acceptor substrate binding. The microsomal glutathione S-transferase from guinea-pig liver was also shown to be inhibited by bile acids. This inhibition, however, showed characteristics of a non-specific detergent-type inhibition.     

Interaction of salicylate and ibuprofen with the carboxylic acid: CoA ligases from bovine liver mitochondria

Neither salicylate nor ibuprofen was a substrate or inhibitor of the long-chain fatty acid: CoA ligase. In contrast, all three xenobiotic-metabolizing medium-chain fatty acid:CoA ligases (XL-I, XL-II, and XL-III) had activity toward salicylate. The K_m value for salicylate was similar for all three forms (2 to 3 μM), but XL-II and XL-III had higher activity at V_max. For ibuprofen, only XL-III catalyzed its activation, and it had a K_m for ibuprofen of 36 μM. Studies of salicylate inhibition of XL-I, XL-II, and XL-III revealed that it inhibited the benzoate activity of all three forms with K₁ values of ca. 2 μM, which is in agreement with the K_m values obtained with salicylate as substrate. Kinetic analysis revealed that salicylate conjugation by all three forms is characterized by substrate inhibition when salicylate exceeds ca. 20 μM. Substrate inhibition was more extensive with XL-I and XL-III. Previous work on the ligases employed assay concentrations of salicylate in the range of 0.1 to 1.0 mM, which are clearly inhibitory, particularly toward XL-I and XL-III. Thus, activity was not properly measured in previous studies, which accounts for the fact that salicylate conjugation was only found with one form, which is most likely XL-II since it has the highest V_max activity and shows the least amount of substrate inhibition. Studies with ibuprofen indicated that it inhibited XL-I, XL-II, and XL-III, with K₁ values being in the range of 75–125 μM. The short-chain ligase was inhibited by both salicylate and ibuprofen with K₁ values of 93 and 84 μM, respectively. It was concluded that pharmacological doses of salicylate, but not ibuprofen, will affect the metabolism of medium-chain fatty acids and carboxylic acid xenobiotics and that the previously described mitochondrial ibuprofen:CoA ligase activity is attributable to XL-III. © 1996 John Wiley & Sons, Inc.     

Characterization of the CoA ligases of human liver mitochondria catalyzing the activation of short- and medium-chain fatty acids and xenobiotic carboxylic acids.

Two distinct forms of xenobiotic/medium-chain fatty acid:CoA ligase (XM-ligase) were isolated from human liver mitochondria. They were referred to as HXM-A and HXM-B based on their order of elution from a DEAE-cellulose column. Activity of the two ligases was determined toward 15 different carboxylic acids. HXM-A represented 60-80% of the benzoate activity in the lysate, and kinetic analysis revealed that benzoate was the best substrate (highest V(max)/K(m)). The enzyme also had medium-chain fatty acid:CoA ligase activity. HXM-B had the majority of the hexanoate activity and hexanoate was its best substrate. It was, however, also active toward many xenobiotic carboxylic acids. Comparison of these two human XM-ligases with the previously characterized bovine XM-ligases indicated that they were kinetically distinct. When assayed with benzoic acid as substrate, both HXM-A and HXM-B had an absolute dependence on either Mg(2+) or Mn(2+) for activity. Further, addition of monovalent cation (K(+), Rb(+), or NH(4)(+)) stimulated HXM-A activity by >30-fold and HXM-B activity by 4-fold. For both forms, activity toward straight-chain fatty acids was stimulated less by K(+) than was activity toward benzoate or phenylacetate. A 60 kDa short-chain fatty acid:CoA ligase was also isolated. It had activity toward propionate and butyrate, but not acetate, hexanoate or benzoate. The K(m)(app) values were high but similar for propionate and butyrate (285 microM and 250 microM, respectively) but the V(max)(app) was nearly 6-fold greater with propionate as substrate. While the K(m) values are somewhat high, the enzyme is still more efficient with these substrates than either of the XM-ligases.     

Characterization of triacsin C inhibition of short-, medium-, and long-chain fatty acid: CoA ligases of human liver

Short-, medium-, and long-chain fatty acid:CoA ligases from human liver were tested for their sensitivity to inhibition by triacsin C. The short-chain fatty acid:CoA ligase was inhibited less than 10% by concentrations of triacsin C as high as 80 microM. The two mitochondrial xenobiotic/medium-chain fatty acid:CoA ligases (XM-ligases), HXM-A and HXM-B, were partially inhibited by triacsin C, and the inhibitions were characterized by low affinity for triacsin C (K(I) values > 100 microM). These inhibitions were found to be the result of triacsin C competing with medium-chain fatty acid for binding at the active site. The microsomal and mitochondrial forms of long-chain fatty acid:CoA ligase (also termed long-chain fatty acyl-CoA synthetase, or long-chain acyl-CoA synthetase LACS) were potently inhibited by triacsin C, and the inhibition had identical characteristics for both LACS forms. Dixon plots of this inhibition were biphasic. There is a high-affinity site with a K(I) of 0.1 microM that accounts for a maximum of 70% of the inhibition. There is also a low affinity site with a K(I) of 6 microM that accounts for a maximum of 30% inhibition. Kinetic analysis revealed that the high-affinity inhibition of the mitochondrial and microsomal LACS forms is the result of triacsin C binding at the palmitate substrate site.The high-affinity triacsin C inhibition of both the mitochondrial and microsomal LACS forms was found to require a high concentration of free Mg(2+), with the EC(50) for inhibition being 3 mM free Mg(2+). The low affinity triacsin C inhibition was also enhanced by Mg(2+). The data suggests that Mg(2+) promotes triacsin C inhibition of LACS by enhancing binding at the palmitate binding site. In contrast, the partial inhibition of the XM-ligases by triacsin C, which showed only a low-affinity component, did not require Mg(2+).