Steroidogenesis in isolated adrenocortical cells. Correlation with receptor-bound adenosine 3′:5′-cyclic monophosphate

Because several groups have recently questioned a mediating role for cyclic AMP in adrenocortical steroidogenesis, we analysed the problem in more detail by measuring three different cyclic AMP pools in cells isolated from decapsulated rat adrenals. Extra-cellular, total intracellular and bound intracellular cyclic AMP were determined by radioimmunoassay in comparison with corticosterone production induced by low corticotropin concentrations. The increase in extracellular and total intracellular cyclic AMP with low corticotropin concentrations was dependent on the presence of a phosphodiesterase inhibitor and short incubation times. Bound intracellular cyclic AMP was less dependent on these two parameters. In unstimulated cells cyclic AMP bound to its receptor represents only a small fraction of the total intracellular cyclic AMP. After stimulation by a concentration of corticotropin around the threshold for corticosterone production, an increase in bound cyclic AMP was observed which correlated very well with steroidogenesis both temporally and with respect to corticotropin concentration. This finding was complemented by measuring a concomitant decrease in free receptor sites. Full occupancy of the receptors was not necessary for maximal steroidogenesis. Binding kinetics of cyclic [(3)H]AMP in concentrations equivalent to the intracellular cyclic AMP concentration suggest the presence of at least three different intracellular cyclic AMP pools. These observations are in agreement with a possible role for cyclic AMP as a mediator of acute steroidogenesis induced by low corticotropin concentrations.     

A DNA Vaccine against Yellow Fever Virus: Development and Evaluation

Attenuated yellow fever (YF) virus 17D/17DD vaccines are the only available protection from YF infection, which remains a significant source of morbidity and mortality in the tropical areas of the world. The attenuated YF virus vaccine, which is used worldwide, generates both long-lasting neutralizing antibodies and strong T-cell responses. However, on rare occasions, this vaccine has toxic side effects that can be fatal. This study presents the design of two non-viral DNA-based antigen formulations and the characterization of their expression and immunological properties. The two antigen formulations consist of DNA encoding the full-length envelope protein (p/YFE) or the full-length envelope protein fused to the lysosomal-associated membrane protein signal, LAMP-1 (pL/YFE), aimed at diverting antigen processing/presentation through the major histocompatibility complex II precursor compartments. The immune responses triggered by these formulations were evaluated in H2b and H2d backgrounds, corresponding to the C57Bl/6 and BALB/c mice strains, respectively. Both DNA constructs were able to induce very strong T-cell responses of similar magnitude against almost all epitopes that are also generated by the YF 17DD vaccine. The pL/YFE formulation performed best overall. In addition to the T-cell response, it was also able to stimulate high titers of anti-YF neutralizing antibodies comparable to the levels elicited by the 17DD vaccine. More importantly, the pL/YFE vaccine conferred 100% protection against the YF virus in intracerebrally challenged mice. These results indicate that pL/YFE DNA is an excellent vaccine candidate and should be considered for further developmental studies.     

The flavodoxin from Helicobacter pylori: structural determinants of thermostability and FMN cofactor binding

Flavodoxin has been recently recognized as an essential protein for a number of pathogenic bacteria including Helicobacter pylori, where it has been proposed to constitute a target for antibacterial drug development. One way we are exploring to screen for novel inhibitory compounds is to perform thermal upshift assays, for which a detailed knowledge of protein thermostability and cofactor binding properties is of great help. However, very little is known on the stability and ligand binding properties of H. pylori flavodoxin, and its peculiar FMN binding site together with the variety of behaviors observed within the flavodoxin family preclude extrapolations. We have thus performed a detailed experimental and computational analysis of the thermostability and cofactor binding energetics of H. pylori flavodoxin, and we have found that the thermal unfolding equilibrium is more complex that any other previously described for flavodoxins as it involves the accumulation of two distinct equilibrium intermediates. Fortunately the entire stability and binding data can be satisfactorily fitted to a model, summarized in a simple phase diagram, where the cofactor only binds to the native state. On the other hand, we show how variability of thermal unfolding behavior within the flavodoxin family can be predicted using structure-energetics relationships implemented in the COREX algorithm. The different distribution and ranges of local stabilities of the Anabaena and H. pylori apoflavodoxins explain the essential experimental differences observed: much lower Tm1, greater resistance to global unfolding, and more pronounced cold denaturation in H. pylori. Finally, a new strategy is proposed to identify using COREX structural characteristics of equilibrium intermediate states populated during protein unfolding.     

On the Forkker-Planck equation in the stochastic theory of mortality: I

This paper, consisting of two parts, gives all the mathematical details that were omitted in a previous work by G. A. Sacher and E. Trucco (“The Stochastic Theory of Mortality.”Ann. N. Y. Acad. Sci.,96, 985–1007, cited here as ST). We assume that the reader is familiar with ST, where the stochastic theory of mortality, originally proposed by Sacher, is discussed at length. We recall that the basic model presented there refers to an ensemble of particles performing Brownian motion in one dimension, with the added constraint of two absorbing barriers. These two points, collectively, are designated as the “lethal bound.” Part I (section 1 to 4) deals with the special case in which the two absorbing barriers are symmetrically located at a finite distance from the origin. The solution of the Fokker-Planck equation is obtained from the theory of eigenvalue problems. Quite generally, the eigenfunctions functions belong to the family of Kummer's confluent hypergeometric functions, but the symmetry condition imposed here results in considerable simplification and makes it possible to estimate the first few eigenvalues by a graphical procedure. In section 3 we show how perturbation theory can be applied in the limiting case of “weak homeostasis,” and section 4 deals with the opposite extreme of “strong homeostasis.” A rigorous proof is given for the result corresponding to equation (28) of ST (asymptotic or quasi-static approximation for the “force of mortality”). This work was performed under the auspices of the U.S. Atomic Energy Commission.     

Altered infant feeding patterns in boys with acquired nonsyndromic cryptorchidism

BACKGROUND : Genetic and environmental factors likely influence susceptibility to nonsyndromic cryptorchidism, a common disease presenting at birth or in later childhood. We compared cases and controls to define differential risk factors for congenital versus acquired cryptorchidism. METHODS : We compared questionnaire and clinical data from cases of congenital cryptorchidism (n = 230), acquired cryptorchidism (n = 182) and hernia/hydrocele (n = 104) with a group of healthy male controls (n = 358). Potential predictor variables (p < 0.2 in univariable analysis) were included in stepwise multivariable logistic regression models. RESULTS : Temporary (odds ratio [OR], 0.5; 95% confidence interval [CI], 0.4–0.8) or exclusive (OR, 0.6; 95% CI, 0.4–0.9) breastfeeding was reduced and soy formula feeding increased (OR, 1.8; 95% CI, 1.2–2.9) in acquired but not congenital or hernia/hydrocele groups. The highest risk estimates were observed for primary soy formula feeding with limited or no breastfeeding (OR 2.5; 95% CI, 1.4–4.3; adjusted OR, 2.7; 95% CI, 1.4–5.4) in the acquired group. Primary feeding risk estimates were equivalent or strengthened when multivariable models were limited to age greater than 2 years, full‐term or not small for gestational age, or Caucasian subjects. Pregnancy complications and increased maternal exposure to cosmetic or household chemicals were not consistently associated with either form of cryptorchidism in these models. CONCLUSIONS : Our data support reduced breastfeeding and soy formula feeding as potential risk factors for acquired cryptorchidism. Although additional studies are needed, hormonally active components of breast milk and soy formula could influence the establishment of normal testis position in the first months of life, leading to apparent ascent of testes in childhood. Birth Defects Research (Part A), 2012. © 2012 Wiley Periodicals, Inc.     

Structural and Functional Alterations of Subcutaneous Small Resistance Arteries in Severe Human Obesity

Obese persons are at increased cardiovascular risk and exhibit increased arterial stiffness and impaired endothelial function of large‐ and medium‐size arteries. We hypothesized that normotensive subjects suffering from severe obesity would also present remodeling and endothelial dysfunction of small resistance arteries. A total of 16 lean (age: 49.6 ± 2.9 years, BMI: 22.9 ± 0.3 kg/m², mean ± s.e.m.) and 17 age‐matched severely obese (BMI: 41.1 ± 2.3 kg/m²) normotensive subjects were investigated. None had glucose or lipid metabolic abnormalities except for insulin resistance. Resistance arteries, dissected from abdominal subcutaneous tissue, were assessed on a pressurized myograph. For superimposable blood pressure, the media thickness, media cross‐sectional area (CSA), and media‐to‐lumen ratio values of resistance arteries were markedly and significantly greater in obese compared to lean subjects (media thickness 26.3 ± 0.6 vs. 16.2 ± 0.6 µm, CSA 22,272 ± 1,339 vs. 15,183 ± 1,186 µm², and media‐to‐lumen ratio 0.113 ± 0.006 vs. 0.059 ± 0.001, respectively, P < 0.01). Acetylcholine‐induced relaxation was impaired in vessels from obese subjects compared to the lean individuals (?40.4 ± 1.3%, P < 0.01), whereas endothelium‐independent vasorelaxation was similar in all groups. Stiffness of small arteries as assessed by the stress/strain relationship was similar in lean and severely obese subjects. We conclude that severe human obesity is associated with profound alterations in structural and functional characteristics of small arteries, which may be responsible for the presence of elevated cardiovascular risk and increased incidence of coronary, cerebrovascular and renal events reported in obesity.     

Effect of goniothalamin on the development of Ehrlich solid tumor in mice

In this work the antiproliferative activity of goniothalamin (1), both in racemic and in its enantiomeric pure forms, in a solid tumor experimental model using laboratory animals is described. The antiedematogenic activity displayed by racemic 1 in the carrageenan edema model in mice together with the reduction of Ehrlich solid tumor model suggest a relationship between anticancer and antiinflammatory activities with the antiinflammatory activity favoring the antiproliferative activity itself.