A novel ELISA for measuring CD36 protein in human adipose tissue

CD36 is a transmembrane protein present in many tissues that is believed to facilitate inward fatty acid transport. Western blotting is the most widely used method to measure tissue CD36 protein content, but it is time consuming, technically demanding, and semiquantitative. To more precisely measure adipose tissue CD36 content we developed an enzyme linked immunosorbent assay (ELISA) after establishing that: 1) the anti-CD36 antibodies gave a single distinct band on traditional Western blots, and 2) the vast majority of adipocyte CD36 resides in the plasma membrane. By using serial dilutions of each sample and including a calibrator sample and quality control sample on each plate, we could achieve inter- and intra-assay variability of ～ 10%. We found that CD36 content in omental and abdominal subcutaneous adipose tissue varied over a 2-5-fold range depending upon the means of data expression (per units of tissue protein, weight, or lipid). Omental CD36 content in women decreased markedly (P = 0.01) as a function of fat cell size. For the most part, tissue CD36 content was not correlated with CD36 mRNA. This ELISA method for tissue CD36 content should enhance research into the role of this protein on tissue fatty acid uptake.     

Lycopene is superior to moringa in improving fertility markers in diet-induced obesity male rats

Obesity is a condition of chronic tissue inflammation and oxidative stress that poses as a risk factor for male infertility. Moringa oleifera oil extract is known to have cholesterol-lowering properties and a potential to treat obesity, while lycopene is a potent antioxidant. We hypothesize that Moringa or lycopene may improve male fertility markers in an animal model of diet-induced obesity. Male Albino rats (n = 60) were randomized to receive regular chow (RC) or high-fat diet (HFD) for 12 weeks (n = 30 each). Animals in each arm were further randomized to receive gavage treatment with corn oil (vehicle), lycopene (10 mg/kg), or Moringa (400 mg/kg) for four weeks starting on week 9 (n = 10 each). Animals were sacrificed at 12 weeks, and blood was collected to assess lipid profile, serum testosterone, and gonadotropin levels. The testes and epididymides were removed for sperm analysis, oxidative stress and inflammatory markers, and histopathological assessment. In comparison to their RC littermates, animals on HFD showed an increase in body weights, serum lipids, testosterone and gonadotrophin levels, testicular oxidative stress and inflammatory markers, as well as sperm abnormalities and disrupted testicular histology. Moringa or lycopene reduced body weight, improved oxidative stress, and male fertility markers in HFD-fed animals with lycopene exhibiting better anti-antioxidant and anti-lipidemic effects. Lycopene is superior to Moringa in improving male fertility parameters, possibly by attenuating oxidative stress.     

The proto-Nucleic Acid Builder: a software tool for constructing nucleic acid analogs

The helical structures of DNA and RNA were originally revealed by experimental data. Likewise, the development of programs for modeling these natural polymers was guided by known structures. These nucleic acid polymers represent only two members of a potentially vast class of polymers with similar structural features, but that differ from DNA and RNA in the backbone or nucleobases. Xeno nucleic acids (XNAs) incorporate alternative backbones that affect the conformational, chemical, and thermodynamic properties of XNAs. Given the vast chemical space of possible XNAs, computational modeling of alternative nucleic acids can accelerate the search for plausible nucleic acid analogs and guide their rational design. Additionally, a tool for the modeling of nucleic acids could help reveal what nucleic acid polymers may have existed before RNA in the early evolution of life. To aid the development of novel XNA polymers and the search for possible pre-RNA candidates, this article presents the proto-Nucleic Acid Builder (https://github.com/GT-NucleicAcids/pnab), an open-source program for modeling nucleic acid analogs with alternative backbones and nucleobases. The torsion-driven conformation search procedure implemented here predicts structures with good accuracy compared to experimental structures, and correctly demonstrates the correlation between the helical structure and the backbone conformation in DNA and RNA.     

Steroidogenesis in granulosa cells during follicular maturation: evidence for desensitization-resensitization during the ovulation cycle

Output of progesterone, the end product of the steroidogenic pathway, was studied in isolated chicken granulosa cells in relation to follicular maturation and during the ovulation cycle with particular reference to the period between the LH peak and ovulation. The evidence gathered from a series of experiments conducted during the past 2-3 years in the authors' laboratory indicate that the steroidogenic capacity of granulosa cells during follicular maturation is regulated not so much by receptor-coupled adenylate cyclase activity as has been proposed by other investigators, but by the activity of key steroidogenic enzymes, particularly the cholesterol 20,22 desmolase. Furthermore, granulosa cells undergo cyclic sensitization following the endogenous LH surge reaching maximal responsiveness about 1 hr before oviposition. This is followed by a rapid desensitization shortly before ovulation. This desensitization extends to the second and subsequent developing follicles probably in proportion to the evolving LH receptors. It is suggested that granulosa cells remain in such a desensitized state for several hours postovulation, during which time progesterone responses to LH are attenuated and consequently ovulation does not occur prematurely. It is proposed that this intraovarian mechanism is an important component of the physiological events that regulate the ovulation cycle in the domestic fowl.     

Menopausal estrogen deprivation activates steroid sensitive stem cells (3SC) and local estrogen biosynthesis: A model for breast cancer development

We propose a hypothesis for breast cancer (BC) development and its implications for BC prevention. We describe a model in which some breast cells function as both stem cells and steroid sensors (steroid sensitive stem cells). Estrogen receptors on those cells could be upregulated in women who had increased cumulative exposure to estrogen, leading to their progressive sensitization. At menopause, such women experience considerable decline of estrogen concentration in their blood. Consequently, the sensitized stem cells activate mechanisms of local estrogen synthesis including the activation of aromatase. The intracrine build-up of estrogen and its metabolites induces proliferation and genetic dysfunction. Eventually, a normal stem cell transforms into an estrogen-sensitive cancer stem cell that is capable of tumor initiation and delineation into other phenotypes of cancer cells. This hypothesis is supported by significant in-vitro and clinical research evidence. According to this model, we suggest that estrogen therapy could be protective against BC. Alternatively, aromatase inhibitors are expected to be effective in BC prevention. A combination of AIs and estrogen might augment the preventative merits of both drugs and maintain a good tolerability profile for long-term prevention protocols.     

Hormone stimulated steroid biosynthesis in granulosa cells studied with a fluorogenic probe for cytochrome P-450SCC.

The regulation of steroidogenesis by luteinizing hormone (LH) was studied in granulosa cells during follicular development using a fluorescent reporter assay based on the metabolism of a fluorescent probe specific for cytochrome P-450SCC (cholesterol side-chain cleavage enzyme). Intact granulosa cells or mitochondria were obtained from the first (F1) second (F2) and third (F3) largest preovulatory follicles of the hen ovary and incubated with the fluorogenic substrate. Metabolism of this substrate by cytochrome P-450SCC generates the highly fluorescent resorufin anion (the fluorescent reporter). In both mitochondria and intact granulosa cells, incubated with the fluorescent substrate, an increase in resorufin fluorescence was observed and the increase was greater in samples derived from F1 than in samples from F2 or F3. In cells, LH added simultaneously with the P-450SCC substrate significantly increased resorufin fluorescence above control values in a time- and dose-dependent manner up to 2-3 h after the incubation was initiated. Forskolin and 8-bromo-cAMP also stimulated metabolism of the P-450SCC substrate significantly by 15 min. When granulosa cells were preincubated with LH before exposure to the P-450SCC substrate resorufin fluorescence was significantly attenuated compared to controls (not exposed to LH in the preincubation period). The decrease in resorufin fluorescence observed when cells were pretreated with LH, may be due to the release of cholesterol from endogenous pools and its competition with the exogenous fluorogenic for the substrate P-450SCC enzyme. In granulosa cells that were preloaded with the P-450SCC substrate, the stimulatory effect of LH treatment remained constant from 30 min to 2 h after hormone addition. The results show that this fluorescent probe can be used in a rapid assay for the continuous measurement of the acute effects of hormone agonists on cholesterol conversion to pregnenolone in steroidogenic cells.     

Sodium-dependent regulation of steroidogenesis in rat granulosa cells: possible involvement of a Na+/H+ antiport

The possible role of Na+/H+ antiport in the gonadotropic regulation of steroidogenesis was examined in rat granulosa cells incubated for up to 6 h in a chemically defined medium in the absence or presence of Na+ (128 mM), gonadotropin (FSH or LH; 0-500 ng/ml), dibutyryl cyclic AMP [Bu)2cAMP; 2 mM) and amiloride (0-1 mM). Replacement of Na+ (Na+0) in the incubation medium with choline chloride resulted in a marked decrease in basal and LH-, FSH- and (Bu)2cAMP-stimulated progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) synthesis in vitro. The Na+/H+ exchange inhibitor, amiloride significantly suppressed basal and hormone-stimulated progestin production dose-dependently in the presence of Na+0. However, it was without effect in Na+-deficient medium. The effect of the inhibitor on progestin production appeared to be directed at specific step(s) involved in the synthesis of pregnenolone, as concentrations of amiloride which inhibited progesterone production failed to influence the metabolism of exogenous pregnenolone to progestins. Cell viability and the incorporation of [3H]leucine into acid-precipitable material were not affected by amiloride. Our findings support the contention that extracellular sodium is important for steroidogenesis in rat granulosa cells. The inhibition by amilordie indicates an involvement of the Na+/H+ exchange in the regulation of this granulosa cell function.