Effect of glucocorticoids pretreatment on steroidogenic capacity of adrenocortical cells isolated from Meishan piglets

The present in vitro experiment was designed to test whether 48 h of pretreatment with glucocorticoids, cortisol, or dexamethasone (DEX), would affect basal and corticotrophin (ACTH) stimulated (24 h) cortisol secretion from primary cultures of pig adrenocortical cells. Cells were divided into six groups: control pretreatment with or without ACTH challenge, cortisol pretreatment with or without ACTH challenge, and DEX pretreatment with or without ACTH. The culture medium and cells were collected at the end of treatment. Cortisol concentration in medium was measured by radioimmunoassay, and protein content of glucocorticoid receptor (GR) and key regulatory factors for steroidogenesis, including melanocortin type 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochrome P450 (P450scc), were detected by Western blot analysis. The results showed that glucocorticoid pretreatment did not affect cortisol secretion under basal condition without ACTH challenge, but significantly enhanced ACTH-stimulated cortisol secretion. Furthermore, the protein content of GR, MC2R, StAR, and P450scc was all increased in groups pretreated with glucocorticoids. These results indicate that adrenocortical cells pretreated with glucocorticoids display higher steroidogenic capacity under ACTH challenge, through the upregulation of GR and other steroidogenic regulatory factors.     

Intracellular serine protease inhibitor SERPINB4 inhibits granzyme M-induced cell death

Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1' triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×10(4) M(-1) s(-1). SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death.     

Apelin-13 Inhibits Apoptosis of Cortical Neurons Following Brain Ischemic Reperfusion Injury in a Transient Model of Focal Cerebral Ischemia

Stroke is the third leading cause of death world-wide, affecting 15 million people annually. Diminished blood supply to the brain cells is the main cause of damage following stroke. When focal ischemia occurs, the core of brain tissue influenced by reduced blood supply undergoes necrotic cell death. The adipocytokine Apelin is a peptide that was isolated from a bovine stomach for the first time. This peptide and its receptor are abundantly expressed in the nervous and cardiovascular systems. According to previous studies, Apelin-13 protects cardiomyocytes from ischemic injury and apoptosis. In addition, this peptide has neuroprotective effect on hippocampal and cultured mouse cortical neurons against NMDA receptor-mediated excitotoxicity as well as cortical neurons from ischemic injury. The present study was conducted to determine whether Apelin-13 inhibits apoptosis in the ischemic penumbra in transient focal cerebral ischemia. Focal cerebral ischemia was induced in male Wistar rats by 60 min middle cerebral artery occlusion (MCAO) using a filament method, followed by 23-h reperfusion. Saline as a vehicle and Apelin-13 at doses of 50 and 100 μg were injected intracerebro-ventriculary (ICV) at the beginning of ischemia. Apoptosis and neurological dysfunction were assessed 24-h after MCAO. Our results indicated that administration of Apelin-13 at doses of 50 and 100 μg ICV markedly reduced apoptosis by decreasing positive TUNEL cells (P < 0.001). In addition, Apelin-13 at doses of 100 μg significantly change neurological dysfunction (P < 0.05). Our findings demonstrate that treatment by Apelin-13 exerts its protective effects in ischemic models via blocking programmed cell-death. We suggest that Apelin-13 might be a promising therapeutic target for stroke, although more researches are necessary to take into account the potential therapeutic effects of Apelin-13 in stroke patients.     

Opportunities of marker‐assisted selection for rice fragrance through marker–trait association analysis of microsatellites and gene‐based markers

Developing fragrant rice through marker‐assisted/aided selection (MAS) is an economical and profitable approach worldwide for the enrichment of an elite genetic background with a pleasant aroma. The PCR‐based DNA markers that distinguish the alleles of major fragrance genes in rice have been synthesised to develop rice scent biofortification through MAS. Thus, the present study examined the aroma biofortification potential of these co‐dominant markers in a germplasm panel of 189 F₂ progeny developed from crosses between a non‐aromatic variety (MR84) and a highly aromatic but low‐yielding variety (MRQ74) to determine the most influential diagnostic markers for fragrance biofortification. The SSRs and functional DNA markers RM5633 (on chromosome 4), RM515, RM223, L06, NKSbad2, FMbadh2‐E7, BADEX7‐5, Aro7 and SCU015RM (on chromosome 8) were highly associated with the 2AP (2‐acetyl‐1‐pyrroline) content across the population. The alleles traced via these markers were also in high linkage disequilibrium (R² > 0.70) and explained approximately 12.1, 27.05, 27.05, 27.05, 25.42, 25.42, 20.53, 20.43 and 20.18% of the total phenotypic variation observed for these biomarkers, respectively. F₂ plants harbouring the favourable alleles of these effective markers produced higher levels of fragrance. Hence, these rice plants can be used as donor parents to increase the development of fragrance‐biofortified tropical rice varieties adapted to growing conditions and consumer preferences, thus contributing to the global rice market.     

Hydrophilic carotenoids: surface properties and aggregation behavior of a highly unsaturated carotenoid lysophospholipid

The water dispersibility of a hydrophobic carotenoid has been greatly enhanced by using it as the acyl part in the synthesis of a highly unsaturated lysophospholipid. Dynamic light scattering has revealed the formation of stable aggregates with an average hydrodynamic radius of a few nanometers, and absorption spectra show that the aggregates can withstand the addition of ethanol or acetonitrile until the volume fraction of water falls below 70 and 62%, respectively. The properties of the carotenoid phospholipids have been characterized by determining surface tension, critical micelle concentration, surface concentration, molecular area, free energy of adsorption and micellation, adsorption-micellar energy relationship, and equilibrium constants.     

Discovery of human inversion polymorphisms by comparative analysis of human and chimpanzee DNA sequence assemblies

With a draft genome-sequence assembly for the chimpanzee available, it is now possible to perform genome-wide analyses to identify, at a submicroscopic level, structural rearrangements that have occurred between chimpanzees and humans. The goal of this study was to investigate chromosomal regions that are inverted between the chimpanzee and human genomes. Using the net alignments for the builds of the human and chimpanzee genome assemblies, we identified a total of 1,576 putative regions of inverted orientation, covering more than 154 mega-bases of DNA. The DNA segments are distributed throughout the genome and range from 23 base pairs to 62 mega-bases in length. For the 66 inversions more than 25 kilobases (kb) in length, 75% were flanked on one or both sides by (often unrelated) segmental duplications. Using PCR and fluorescence in situ hybridization we experimentally validated 23 of 27 (85%) semi-randomly chosen regions; the largest novel inversion confirmed was 4.3 mega-bases at human Chromosome 7p14. Gorilla was used as an out-group to assign ancestral status to the variants. All experimentally validated inversion regions were then assayed against a panel of human samples and three of the 23 (13%) regions were found to be polymorphic in the human genome. These polymorphic inversions include 730 kb (at 7p22), 13 kb (at 7q11), and 1 kb (at 16q24) fragments with a 5%, 30%, and 48% minor allele frequency, respectively. Our results suggest that inversions are an important source of variation in primate genome evolution. The finding of at least three novel inversion polymorphisms in humans indicates this type of structural variation may be a more common feature of our genome than previously realized.     

Characterization of growth hormone binding sites in granulosa and theca layers at different stages of follicular maturation and ovulatory cycle in the domestic hen

The currently available evidence points to a possible influence of growth hormone (GH) on avian folliculogenesis, which can be mediated by both hepatic- and ovarian-derived IGF-I. Therefore, the purpose of the present study was to reveal GH-binding sites in granulosa and theca layers of preovulatory follicles and to determine the binding characteristics depending on the degree of follicular maturation and the stage of the ovulatory cycle in the hen. Hens were killed 2 h (stage I), 9 h (stage II), 16 h (stage III), and 23 h (stage IV) after oviposition, and the five largest yellow follicles (from F1 to F5) were removed. GH-binding sites in granulosa and theca layers from F1 to F5 follicles were characterized using a radioreceptor assay. Equilibrium dissociation constants (K(d)) and binding capacities (B(max)) were determined by Scatchard analysis of saturation curves, which revealed a single class of high-affinity GH-binding sites in both theca tissue and granulosa cells. In F1, F2, and F5 follicles, B(max) and K(d) for GH-binding sites in the granulosa layer changed during the ovulatory cycle, decreasing between stages I and III, to increase again at stage IV, with alterations in K(d) being less profound. No significant differences in binding capacities and affinities of GH-binding sites in the theca layer were found between various stages of the cycle. Furthermore, the concentration of GH-binding sites in the granulosa layer rose, whereas that in the theca layer fell with follicular enlargement. These findings indicate the presence of high-affinity GH-binding sites in both granulosa and theca layers of hen preovulatory follicles. Data also demonstrate that GH-binding sites in these tissues are regulated in a tissue-specific manner. Furthermore, the regulation of binding capacity of GH binding in granulosa cells by hormonal factors associated with ovulatory cycle is apparently not dependent on the state of follicular maturation.     

Comparative study of integrating cavity absorption meters

Integrating spheres (IS) are widely used for recording spectra of scattering samples by placing the specimen inside or outside the sphere. An unusual application of integrating spheres has been also demonstrated earlier where the liquid sample completely filled the spherical cavity; such a device is often called an integrating cavity absorption meter (ICAM). In the present work, integrating cavities with different coatings are compared. The spheres were made of glass, covered by metal or white paint, and their surfaces were prepared for diffuse or specular reflectance. The spheres were evaluated by recording kinetic traces following a short light pulse with the aid of time-correlated single photon counting (TCSPC), and by recording steady-state spectra through single-photon counting (SPC) detection. The relative efficiencies of the spheres were determined by comparing the steady-state spectra. Possible reasons for differences in the performance are discussed.     

Heteroaromatic-aminomethyl quinolones: potent and selective iNOS inhibitors

The overproduction of nitric oxide during the biological response to inflammation by the nitric oxide synthase (NOS) enzymes have been implicated in the pathology of many diseases. By removal of the amide core from uHTS-derived quinolone 4, a new series highly potent heteroaromatic-aminomethyl quinolone iNOS inhibitors 8 were identified. SAR studies led to identification of piperazine 22 and pyrimidine 32, both of which reduced plasma nitrates following oral dosing in a mouse lipopolysaccharide challenge assay.     

DNA interaction studies of a copper (II) complex containing an antiviral drug, valacyclovir: the effect of metal center on the mode of binding

The water-soluble complex, [Cu(Val)(2)(NO(3))(2)]; in which Val = valacyclovir, an antiviral drug, has been synthesized and characterized by elemental analysis, furier transfer-infrared, hydrogen nuclear magnetic resonance (H NMR), and UV-Vis techniques. The binding of this Cu (II) complex to calf thymus DNA was investigated using fluorimetry, spectrophotometry, circular dichroism, and viscosimetry. In fluorimetric studies, the enthalpy and entropy of the reaction between the complex and calf-thymus DNA (CT-DNA) showed that the reaction is endothermic (ΔH = 208.22 kJ mol(-1); ΔS = 851.35 J mol(-1)K(-1)). The complex showed the absorption hyperchromism in its ultra violet-visible (UV-Vis) spectrum with DNA. The calculated binding constant, K(b), obtained from UV-Vis absorption studies was 2 × 10(5) M(-1). Moreover, the complex induced detectable changes in the circular dichroism spectrum of CT-DNA, as well as changes in its viscosity. The results suggest that this copper (II) complex interacts with CT-DNA via a groove-binding mode.