β-hydroxyacyl-coa-dehydrogenase (HADH) activity of unfrozen and frozen—thawed frog (Rana esculenta) legs

The β-hydroxyacyl-CoA-dehydrogenase (HADH) activity of unfrozen and thawed frog legs was investigated. The enzyme was extracted by either immersing frog legs in phosphate buffer 0.1 M, pH 6.0 at 25°C for 15 min or pressing them between trichinoscopy glasses. The enzyme activity was assayed using acetoacetyl-CoA as substrate and measured spectrophotometrically at 340 nm. It was possible by both extraction methods to distinguish between thawed and unfrozen samples although when the juice was obtained by pressing the HADH activity of the dilution was ～ 1.5 times higher than that obtained by immersion. The HADH activity was significantly higher (P≤0·001) in frozen-thawed frogs than in unfrozen legs because during freezing there is a release of HADH. No significative differences were found in the HADH activity in samples frozen in the temperature range -10 to -196°C. HADH activity was not affected by the storage time in crushed ice up to 6 days.     

Progesterone, but not 17beta-estradiol, up-regulates erythropoietin (EPO) production in human amniotic epithelial cells

Human amniotic epithelial (HAE) cells have great potential for successful use in cell therapy, since they do not cause acute rejection upon allotransplantation. However, to date, HAE cells have not well been studied. We previously reported that HAE cells produce erythropoietin (EPO), which is known to be a regulator of hematopoiesis, and that the induction mechanism of HAE cells is unknown, although EPO production from HAE cells is not increased by hypoxia which induces several cell types to produce EPO. In this study, we determined whether female sex hormones, including progesterone and 17beta-estradiol, affect the EPO production of HAE cells. Bioactive measurement of EPO activity in the culture supernatants of HAE-SV40 cells, which were immortalized by transfection with a simian virus 40 large T antigen, revealed that EPO bioactivity was significantly increased by treatment with progesterone, but not 17beta-estradiol. Treatment of HAE-SV40 cells with progesterone transiently increased the EPO mRNA level by fivefold, while there was no change in response to 17beta-estradiol. Furthermore, the progesterone receptor (PR)-B was detected in both HAE cells and HAE-SV40 cells by Western blotting. These results suggest that EPO synthesis in HAE-SV40 cells is stimulated by progesterone, but not by 17beta-estradiol, and thus it is highly likely that the EPO synthesis of HAE cells is also regulated by progesterone.     

The c-kit receptor protein in the testis of green frog Rana esculenta: seasonal changes in relationship to testosterone titres and spermatogonial proliferation

The green frog Rana esculenta is a seasonal breeder. The cyclic changes between almost arrested and highly activated spermatogenesis offer an ideal model to study basic mechanisms of spermatogenesis. In this study, we demonstrated, to our knowledge for the first time, c-kit receptor positive cells in the testis of this amphibian. The presence of c-kit receptor protein was confirmed by western blotting (Wb) analyses carried out in the testis during all the three main phases of the sexual cycle. The antibody recognized a band of about 150 kDa that was correlated with the positive staining in the germinal epithelium. The immunolabelling for c-kit receptor, evaluated by immunohistochemistry (IHC), was localized in I and II spermatogonia (SPG), in I and II spermatocytes, in both elongating spermatids and spermatozoa and in the Leydig cells. Furthermore, c-kit expression showed a seasonal pattern connected with both testicular and plasma profiles of testosterone during the reproductive cycle. The highest expression of c-kit receptor occurred during the reproductive period, when the testis exhibited the maximum concentration of testosterone. In this period, the mitotic activity of germ cell, assessed by both Wb and IHC analyses for proliferating cell nuclear antigen (PCNA), was intensive. Indeed, during the post-reproductive period, testosterone titres were the lowest and the expression of both PCNA and c-kit receptor protein in the testis, although present, is minor when compared with the reproductive phase. This evidence suggests that cell division can continue sufficiently to accumulate SPG for the next spring, when new germinal cells undergo multiplication. Finally, during the pre-reproductive period, testosterone levels begin to increase and mitotic activity of germinal epithelium is comparably enhanced. These events seem to precede the period of maximum stimulated spermatogonial proliferation, i.e. the reproductive period. These results suggest that the c-kit receptor may play a role in germ cell proliferation and provide a basis for future detailed investigation of regulatory factors of the proliferation of SPG.     

Inhibition of the basal and oestradiol-stimulated mitotic activity of primary spermatogonia by melatonin in the testis of the frog,Rana esculenta,in vivo and in vitro

Melatonin has a direct inhibitory effect on the basal and oestradiol-stimulated mitotic activity of primary spermatogonia in the testis of the frog, Rana esculenta. In this study oestradiol was used to induce spermatogonial proliferation to verify the anti-proliferative effect of melatonin. The colchicine metaphase arrest technique was used. The results obtained from in vivo experiments confirm that oestradiol increases the mitotic index of primary spermatogonia and, for the first time, indicate that melatonin has an inhibitory role on the proliferation of primary spermatogonia in the frog testis. Similar results were obtained from testes of melatonin-injected frogs that were exposed to oestradiol in vitro; in fact spermatogonia were unresponsive to hormonal stimulation. In addition, in short-term cultured testes, melatonin (at physiological concentration) interferes with the effects of oestradiol on spermatogonial proliferation, supporting the hypothesis that melatonin exerts the inhibitory effect directly via its local action on the frog gonads. Morphological observation after in vivo or in vitro melatonin treatments indicates that Leydig cells display degenerative features, whereas in adjacent germinal tubules, Sertoli cells show heterochromatic nuclei. These results indicate that melatonin may act on Leydig cells and confirm that there is a paracrine interaction between interstitial and germinal compartments. The results of the present study indicate, for the first time, that melatonin may be directly involved in the inhibitory control of spermatogonial proliferation in the testis of the frog, R. esculenta.     

Sexual differentiation in sensitivity to male body odor(1)

We have confirmed that more female subjects than male subjects evaluate male body odor as significantly unpleasant. Through an investigation on sexual differentiation in sensitivity to male body odor, we concluded that one of the volatile steroids, androstenone, had two effects on female olfactory sense. First, female subjects perceived androstenone itself to be more unpleasant than male subjects. Second, for only female subjects, androstenone, at a concentration of one-tenth of detection threshold, enhanced the intensity and unpleasantness of body-odor constituents such as short-chain fatty acids.     

Characterization of gametogenetin 1 (GGN1) and its potential role in male fertility through the interaction with the ion channel regulator, cysteine-rich secretory protein 2 (CRISP2) in the sperm tail

Cysteine-rich secretory protein 2 (CRISP2) is a testis-enriched protein localized to the sperm acrosome and tail. CRISP2 has been proposed to play a critical role in spermatogenesis and male fertility, although the precise function(s) of CRISP2 remains to be determined. Recent data have shown that the CRISP domain of the mouse CRISP2 has the ability to regulate Ca(2+) flow through ryanodine receptors (RyR) and to bind to MAP kinase kinase kinase 11 (MAP3K11). To further define the biochemical pathways within which CRISP2 is involved, we screened an adult mouse testis cDNA library using a yeast two-hybrid assay to identify CRISP2 interacting partners. One of the most frequently identified CRISP2-binding proteins was gametogenetin 1 (GGN1). Interactions occur between the ion channel regulatory region within the CRISP2 CRISP domain and the carboxyl-most 158 amino acids of GGN1. CRISP2 does not bind to the GGN2 or GGN3 isoforms. Furthermore, we showed that Ggn1 is a testis-enriched mRNA and the protein first appeared in late pachytene spermatocytes and was up-regulated in round spermatids before being incorporated into the principal piece of the sperm tail where it co-localized with CRISP2. These data along with data on RyR and MAP3K11 binding define the CRISP2 CRISP domain as a protein interaction motif and suggest a role for the GGN1-CRISP2 complex in sperm tail development and/or motility.     

Kaempferol induces apoptosis in ovarian cancer cells through activating p53 in the intrinsic pathway

Ovarian cancer is a significant malignancy for women in the western world, and its death rate has remained unchanged over the past 50 years, leaving room for proper chemoprevention. Kaempferol is a natural flavonoid widely distributed in fruits and vegetables, and epidemiological studies have found a negative correlation between kaempferol consumption and ovarian cancer risk. To understand the mechanism behind this negative correlation, we investigated kaempferol's ability to induce apoptosis in A2780/CP70, A2780/wt, and OVCAR-3 ovarian cancer cell lines. Kaempferol inhibited cell proliferation but did not cause necrosis in all 3 cell lines. For the apoptosis, caspase 3/7 levels were induced in a concentration-dependent manner by kaempferol treatment, with A2780/wt cells being the most responsive. This induction can be diminished by pre-treatment with a caspase-9 inhibitor, indicating an intrinsic apoptosis pathway. Western blot analysis revealed that protein levels of Bcl-x(L) were decreased in ovarian cancer cells, while p53, Bad, and Bax proteins were up-regulated by kaempferol treatment. Our data indicate that kaempferol induces apoptosis in ovarian cancer cells through regulating pro-apoptotic and anti-apoptotic protein expressions in the intrinsic apoptosis pathways, and is a good candidate for the chemoprevention of ovarian cancers in humans. Further studies in animal models and clinical trials are therefore warranted.     

Assessment of the risk of solar ultraviolet radiation to amphibians. I. Dose-dependent induction of hindlimb malformations in the northern leopard frog (Rana pipiens)

A number of environmental stressors have been hypothesized as responsible for recent increases in limb malformations in several species of North American amphibians. The purpose of this study was to generate dose-response data suitable for assessing the potential role of solar ultraviolet (UV) radiation in causing limb malformations in a species in which this phenomenon seemingly is particularly prevalent, the northern leopard frog (Rana pipiens). Frogs were exposed from early embryonic stages through complete metamorphosis to varying natural sunlight regimes, including unaltered (100%) sunlight, sunlight subjected to neutral density filtration to achieve relative intensities of 85%, 75%, 65%, 50%, and 25% of unaltered sunlight, and sunlight filtered with glass or acrylamide to attenuate, respectively, the UVB (290-320 nm) and UVB plus UVA (290-380 nm) portions of the spectrum. The experiments were conducted in a controlled setting, with continual monitoring of UVB, UVA, and visible light to support a robust exposure assessment. Full sunlight caused approximately 50% mortality of the frogs during early larval development; no significant treatment-related mortality occurred under any of the other exposure regimes, including 100% sunlight with glass or acrylamide filtration. There was a dose-dependent (p < 0.0001) induction of hindlimb malformations in the frogs, with the percentage of affected animals ranging from about 97% under unaltered sunlight to 0% in the 25% neutral density treatment. Malformations were comprised mostly of missing or truncated digits, and generally were bilateral as well as symmetrical. Filtration of sunlight with either glass or acrylamide both significantly reduced the incidence of malformed limbs. The estimated sunlight dose resulting in a 50% limb malformation rate (ED50) was 63.5%. The limb ED50 values based on measured sunlight intensities corresponded to average daily doses of 4.5 and 100 Wh x m(-2) for UVB and UVA, respectively. Exposure to sunlight also resulted in increased eye malformations in R. pipiens, however, the dose-response relationship for this endpoint was not monotonic. The results of this study, in conjunction with measured or predicted exposure data from natural settings, provide a basis for quantitative prediction of the risk of solar UV radiation to amphibians.     

Stage and season effects on cell cycle and apoptotic activities of germ cells and Sertoli cells during spermatogenesis in the spiny dogfish (Squalus acanthias)

To understand the processes involved in the spatial and temporal maturation of testicular cells in Squalus acanthias, we used standard morphometry, proliferating-cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) immunohistochemistry. Except for immature spermatocysts (germinal zone, GZ; early-stage pre-meiotic, E-PrM), the number of cysts in all subsequent stages and the total number of cysts in the spermatogenic progression varied seasonally. The spermatogenic cycle spans about 2 years and is interrupted by germcell clone deletion via apoptosis at the mitosis-meiosis transition in April/May, manifesting as a zone of degeneration (ZD). Rate of displacement of the ZD across the testis diameter indicates that late-stage premeiotic (L-PrM) generations 12-13 require 9-10 months to reach the mature-spermatid stage. Also, the number of cysts completing spermatogenesis is approximately 4-5-fold less than the number that entered spermatogenesis proper 2 years earlier. Pronounced gonocytogenesis in the germinal ridge was coincident with ZD formation in April/May, but it was absent in the fall when mature spermatogonial and meiotic activities had resumed. Whereas strong Sertoli cell PCNA immunoreactivity dominated the GZ cyst cell-cycle activities throughout the year, except during the spring/summer months, the spermatogonial- and Sertoli-cell PCNA indices in E-PrM cysts were inversely related. PCNA immunoreactivity in spermatocytes was seasonal and dependent on the stage of meiosis. TUNEL labelling was limited to spermatogonia and increased stage-dependently in the PrM region (L-PrM = mid-stage PrM >E-PrM >GZ), correlating with ZD formation, in a season-dependent manner. Results imply that effects of normal regulatory factors in Squalus are stage- and process-specific.     

Arabinogalactan-type polysaccharides (APS) from Cordyceps militaris grown on germinated soybeans (GSC) induces innate immune activity of THP-1 monocytes through promoting their macrophage differentiation and macrophage activity

In this study, the immunemodulatory activities of arabinogalactans-type polysaccharide (APS) from Cordyceps militaris grown on germinated soybeans was evaluated in THP-1 human monocytes. The cellular size and the number of intracellular organelles of THP-1 monocytes were increased by APS. Also, APS-treated THP-1 cells significantly developed cellular adherence and macrophagic differentiation to the culture plate surface. APS noticeably enhanced phagocytic activity of THP-1 cells against IgG-FITC-latex beads. APS induced the level of TNF-α, IL-12 p40, and IL-8 as well as TLR2 and TLR4 mRNAs. APS can be developed as a promising immunmodulating agent with macrophage-activating properties.     

Chrysophanol induces necrosis through the production of ROS and alteration of ATP levels in J5 human liver cancer cells

Anthraquinone compounds have been shown to induce apoptosis in different cancer cell types. Effects of chrysophanol, an anthraquinone compound, on cancer cell death have not been well studied. The goal of this study was to examine if chrysophanol had cytotoxic effects and if such effects involved apoptosis or necrosis in J5 human liver cancer cells. Chrysophanol induced necrosis in J5 cells in a dose‐ and time‐dependent manner. Non‐apoptotic cell death was induced by chrysophanol in J5 cells and was characterized by caspase independence, delayed externalization of phosphatidylserine and plasma membrane disruption. Blockage of apoptotic induction by a general caspase inhibitor (z‐VAD‐fmk) failed to protect cells against chrysophanol‐induced cell death. The levels of reactive oxygen species production and loss of mitochondrial membrane potential (ΔΨ_m) were also determined to assess the effects of chrysophanol. However, reductions in adenosine triphosphate levels and increases in lactate dehydrogenase activity indicated that chrysophanol stimulated necrotic cell death. In summary, human liver cancer cells treated with chrysophanol exhibited a cellular pattern associated with necrosis and not apoptosis.     

Effect of processing on the retention of carotenoids in yellow‐fleshed cassava (Manihot esculenta Crantz) roots

The retention of carotenoids was studied in roots from yellow‐fleshed, high carotene cassava clones in four different processing methods. The results indicated that the extent of retention varied with the method of processing. The highest retention was observed in oven drying (total carotenoids 54.70–84.01% and β‐carotene 63.90–94.53%) followed by boiling (total carotenoids 47.87–83.79% and β‐carotene 51.31–81.04%) and frying (total carotenoids 48.76–79.77% and β‐carotene 44.11–83.87%). The lowest retention of total carotenoids (32.86–56.40%) and β‐carotene (21.47–56.68%) was recorded in the sun drying method. The variation in the total carotenoids and β‐carotene content depends on variety, processing method and initial carotene content of the fresh root.     

Methionine and valine activate the mammalian target of rapamycin complex 1 pathway through heterodimeric amino acid taste receptor (TAS1R1/TAS1R3) and intracellular Ca2+ in bovine mammary epithelial cells

Amino acids play a key role in regulating milk protein synthesis partly through activation of the mammalian target of rapamycin (mTOR) signaling pathway. However, the involvement of extracellular AA sensing receptors in this process is not well understood. In nonruminants, it is well established that the AA taste 1 receptor member 1/3 (TAS1R1/TAS1R3) heterodimer contributes to the sensing of most l-AA. Whether this receptor is functional in bovine mammary cells is unknown. The objective of this study was to determine essential AA signaling through TAS1R1/TAS1R3 and their roles in regulating mTOR signaling pathway and casein mRNA abundance in primary bovine mammary epithelial cells and the Mac-T cell line. The bovine mammary epithelial cells were stimulated with complete Dulbecco's modified Eagle's medium (+EAA), medium without EAA (?EAA), or medium supplemented with only 1 of the 10 essential AA, respectively. The nonessential AA levels were the same across all treatments. Small interference RNA targeting TAS1R1 were designed and transfected into bovine primary mammary epithelial cells (bPMEC). Supplementation of a complete mixture of essential AA or Arg, Val, Leu, His, Phe, Met, and Ile individually led to greater mTOR phosphorylation. Phosphorylation of ribosomal protein S6 kinase β-1 was greater in the presence of Val, Leu, Trp, Met, and Ile. Valine, Leu, Met, and Ile led to greater eIF4E-binding protein 1 phosphorylation. Although +EAA and a few individual AA tested induced increases in intracellular calcium, Met and Val were the most potent. Knockdown of TAS1R1 decreased intracellular calcium in bPMEC cultured with both Val and Met. Phosphorylation of mTOR, ribosomal protein S6 kinase β-1, and eIF4E-binding protein 1 was lower when TAS1R1 was knocked-down in bPMEC supplemented with Val and Met. In addition, small interference RNA silencing of TAS1R1 resulted in lower β-casein (CSN2) abundance. The TAS1R1/TAS1R3 receptor may sense extracellular AA and activate mTOR signaling in bovine mammary cells, likely by elevating intracellular calcium concentration. This mechanism appears to have a role in Met- and Val-induced changes in CSN2 mRNA abundance. Further in vivo studies will have to be performed to assess the relevance of this mechanism in the mammary gland.     

磷脂酶A1(Lecitase Ultra)水解磷脂酰胆碱的动力学和热力学研究

本论文研究了磷脂酶A1(Lecitase Ultra)在水相体系中水解磷脂酰胆碱(PC)的动力学和热力学性质。依据Arrhenius经验方程式,计算出了催化水解反应的活化能为5.96 kJ/mol;并进行了Lecitase Ultra水解PC的Briggs-Haldane稳态法酶催化模型动力学模拟,利用底物抑制原理进行了模型修正;采用Lineweaver-Burk作图法,计算出反应的动力学参数Km,Vmax和kI分别为4.02 × 10-2 mol/L、10.05 mol/(L.min)和1.33 × 102 mol/L。研究结果为进一步探讨Lecitase Ultra催化机理及其变性机理提供了基础数据和理论支撑。     

Rosmarinic acid attenuated SIN-1-induced cytotoxicity in HepG2 cells through the HO-1 induction and radical scavenging activity

Rosmarinic acid (RA), a food-derived polyphenolic compound, has been reported to possess anti-oxidant, antiallergic, and anti-inflammatory activities. In this study, we investigated whether RA has cytoprotective activity in SIN-1 induced HepG2 cells and how to achieve it. The results show that RA attenuates SIN-1 induced cytotoxicity in HepG2 cells. RA significantly reduces the intracellular reactive oxygen species (ROS) generated from SIN-1 and induced heme oxygenase-1 (HO-1) expression. RA-induced HO-1 expression was attenuated by a pretreatment with specific inhibitors for JNK, ERK, and phosphoinositide 3-kinase (PI3K). The addition of N-acetyl-cysteine (NAC) also reduced HO-1 induction in RA-induced cells. Furthermore, RA induced the translocation of NF-E2-related factor 2 (Nrf2). Taken together, we conclude that RA attenuates cytotoxicity in SIN-1-induced HepG2 cells by direct radical scavenging activity and HO-1 induction through a ROS/PI3K/MAPKs/Nrf2 pathway.