Effect of long-term isolation of the rat on in vitro biosynthesis of gonadal steroids from dehydroepiandrosterone.

In vitro biosynthesis of gonadal steroids from dehydroepiandrosterone was studied in isolated and in socially reared male and female rats. Acetone-dried powder of gonadal tissue incubated with dehydroepiandrosterone-4-14C yielded androstenedione, androst-5-ene-3beta, 17beta-diol, 11beta-hydroxyandrostenedione and testosterone. In the male, conversion to androstenedione was significantly increased after isolation and conversion to androst-5-ene-3beta, 17beta-diol was significantly lowered. In the female, conversion to androstenedione and androstenediol was significantly lowered by isolation. Testosterone and 11beta-hydroxyandrostenedione were not affected by isolation. Gonadal tissue of isolated and of socially reared male and female rats metabolizes dehydroepiandrosterone in a different way. These findings support the view that the conditions of housing affect the production of sex steroids.     

SOX9 and SF1 are involved in cyclic AMP-mediated upregulation of anti-Mullerian gene expression in the testicular prepubertal Sertoli cell line SMAT1

In Sertoli cells, anti-Müllerian hormone (AMH) expression is upregulated by FSH via cyclic AMP (cAMP), although no classical cAMP response elements exist in the AMH promoter. The response to cAMP involves NF-κB and AP2; however, targeted mutagenesis of their binding sites in the AMH promoter do not completely abolish the response. In this work we assessed whether SOX9, SF1, GATA4, and AP1 might represent alternative pathways involved in cAMP-mediated AMH upregulation, using real-time RT-PCR (qPCR), targeted mutagenesis, luciferase assays, and immunocytochemistry in the Sertoli cell line SMAT1. We also explored the signaling cascades potentially involved. In qPCR experiments, Amh, Sox9, Sf1, and Gata4 mRNA levels increased after SMAT1 cells were incubated with cAMP. Blocking PKA abolished the effect of cAMP on Sox9, Sf1, and Gata4 expression, inhibiting PI3K/PKB impaired the effect on Sf1 and Gata4, and reducing MEK1/2 and p38 MAPK activities curtailed Gata4 increase. SOX9 and SF1 translocated to the nucleus after incubation with cAMP. Mutations of the SOX9 or SF1 sites, but not of GAT4 or AP1 sites, precluded the response of a 3,063-bp AMH promoter to cAMP. In conclusion, in the Sertoli cell line SMAT1 cAMP upregulates SOX9, SF1, and GATA4 expression and induces SOX9 and SF1 nuclear translocation mainly through PKA, although other kinases may also participate. SOX9 and SF1 binding to the AMH promoter is essential to increase the activity of the AMH promoter in response to cAMP.     

Bombesin receptor subtype-3 agonists stimulate the growth of lung cancer cells and increase EGF receptor tyrosine phosphorylation

The effects of bombesin receptor subtype-3 (BRS-3) agonists were investigated on lung cancer cells. The BRS-3 agonist (DTyr⁶, (Ala¹¹, Phe¹³, Nle¹⁴) bombesin^6-14 (BA1), but not gastrin releasing peptide (GRP) or neuromedin B (NMB) increased significantly the clonal growth of NCI-H1299 cells stably transfected with BRS-3 (NCI-H1299-BRS-3). Also, BA1 addition to NCI-H727 or NCI-H1299-BRS-3 cells caused Tyr¹⁰⁶⁸ phosphorylation of the epidermal growth factor receptor (EGFR). Similarly, (DTyr⁶, R-Apa¹¹, Phe¹³, Nle¹⁴) bombesin^6-14 (BA2) and (DTyr⁶, R-Apa¹¹, 4-Cl,Phe¹³, Nle¹⁴) bombesin^6-14 (BA3) but not gastrin releasing peptide (GRP) or neuromedin B (NMB) caused EGFR transactivation in NCI-H1299-BRS-3 cells. BA1-induced EGFR or ERK tyrosine phosphorylation was not inhibited by addition of BW2258U89 (BB₂R antagonist) or PD168368 (BB₁R antagonist) but was blocked by (DNal-Cys-Tyr-DTrp-Lys-Val-Cys-Nal)NH₂ (BRS-3 ant.). The BRS-3 ant. reduced clonal growth of NCI-H1299-BRS-3 cells. BA1, BA2, BA3 and BRS-3 ant. inhibit specific ¹²⁵I-BA1 binding to NCI-H1299-BRS-3 cells with an IC₅₀ values of 1.1, 21, 15 and 750 nM, respectively. The ability of BRS-3 to regulate EGFR transactivation in NCI-H1299-BRS-3 cells was reduced by AG1478 or gefitinib (EGFR tyrosine kinase inhibitors), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), N-acetylcysteine (anti-oxidant), Tiron (superoxide scavenger) and DPI (NADPH oxidase inhibitor). These results demonstrate that BRS-3 agonists may stimulate lung cancer growth as a result of EGFR transactivation and that the transactivation is regulated by BRS-3 in a Src-, reactive oxygen and matrix metalloprotease-dependent manner.     

Numerical alterations of ageing B lymphocyte subsets

The global population is ageing. Elderly people suffer from more severe infections than younger persons. The major reason for the increased susceptibility to infections in the elderly is the deregulated functions of the immune system. Immunosenescence affects both innate and adaptive immune reactions. Among these, quantitative alterations of B lymphocyte subsets determine outcome of infections and vaccination. The overall number of B cells seems to be stable or the decrease is moderate. Reduced input of naive B lymphocytes is compensated by anergic, exhausted memory cells. Concerning B lymphocyte subsets, experimental data obtained in the mouse model and in vivo studies conducted in old-age humans are frequently controversial. Further analysis of human B lymphocyte subpopulations is required that could be regarded as an important biomarker of human life span.     

Adsorption of Adenine, Cytosine, Thymine, and Uracil on Sulfide-Modified Montmorillonite: FT-IR, M?ssbauer and EPR Spectroscopy and X-Ray Diffractometry Studies

In the present work the interactions of nucleic acid bases with and adsorption on clays were studied at two pHs (2.00, 7.00) using different techniques. As shown by Mössbauer and EPR spectroscopies and X-ray diffractometry, the most important finding of this work is that nucleic acid bases penetrate into the interlayer of the clays and oxidize Fe²⁺ to Fe³⁺, thus, this interaction cannot be regarded as a simple physical adsorption. For the two pHs the order of the adsorption of nucleic acid bases on the clays was: adenine????cytosine?>?thymine?>?uracil. The adsorption of adenine and cytosine on clays increased with decreasing of the pH. For unaltered montmorillonite this result could be explained by electrostatic forces between adenine/cytosine positively charged and clay negatively charged. However for montmorillonite modified with Na₂S, probably van der Waals forces also play an important role since both adenine/cytosine and clay were positively charged. FT-IR spectra showed that the interaction between nucleic acid bases and clays was through NH⁺ or NH ₂ ⁺ groups. X-ray diffractograms showed that nucleic acid bases adsorbed on clays were distributed into the interlayer surface, edge sites and external surface functional groups (aluminol, silanol) EPR spectra showed that the intensity of the line g????2 increased probably because the oxidation of Fe²⁺ to Fe³⁺ by nucleic acid bases and intensity of the line g?=?4.1 increased due to the interaction of Fe³⁺ with nucleic acid bases. Mössbauer spectra showed a large decreased on the Fe²⁺ doublet area of the clays due to the reaction of nucleic acid bases with Fe²⁺.     

How will fish that evolved at constant sub‐zero temperatures cope with global warming? Notothenioids as a case study

Current climate change has raised concerns over the fate of the stenothermal Antarctic marine fauna (animals that evolved to live in narrow ranges of cold temperatures). The present paper focuses on Notothenioidei, a taxonomic group that dominates Antarctic fish. Notothenioids evolved in the Southern Ocean over the last 20 million years, providing an example of a marine species flock with unique adaptations to the cold at morphological, physiological and biochemical levels. Their phenotypic modifications are often accompanied by ‘irreversible’ genomic losses or gene amplifications. On a micro‐evolutionary scale, relatively ‘shallow’ genetic variation is observed, on account of past fluctuations in population size, and a significant genetic structure is evident, suggesting low population connectivity. These features suggest that Antarctic fish might have relatively little potential to adapt to global warming, at least at a genetic level. The extent of their phenotypic plasticity, which is evident to some degree, awaits further research.