Protein kinases: Signaling molecules controlling ovarian functions

The present focus survey represents a review of current knowledge concerning involvement of protein kinases in control of basic ovarian functions in mammals. Ovarian cells produce a number of protein kinases, whose expression depends on type of cells, their state and action of hormones and other protein kinases. A number of protein kinases are involved in control of ovarian cell proliferation, apoptosis, oocyte maturation, hormone release, reception and response to hormones, as well as in mediating action of hormones on these ovarian functions. Protein kinases and their regulators could be used for characterization, prediction and control of ovarian folliculogenesis and atresia, corpus luteum functions, oocyte maturation, fertility, release of hormones, response of ovarian structures to hormonal regulators, as well as for treatment of some reproductive disorders.     

银杏叶提取物对过氧化氢刺激下的血管内皮细胞的影响

目的:探讨银杏叶提取物对血管内皮细胞的保护作用及可能的保护机制。方法:进行血管内皮细胞培养。用过氧化氢(H2O2)处理血管内皮细胞建立细胞凋亡模型。将细胞分为四组:空白对照组、H2O2处理组、单独银杏叶提取物处理组、银杏叶提取物预处理组(提前2h给药后H2O2处理)。进行MTT检测细胞的相对活力、RT-PCR检测目的基因CHOP的表达、Western Blot分析目的蛋白CHOP的表达等。结果:与对照处理组比较,H2O2处理组细胞凋亡率、CHOPmRNA相对表达及CHOP蛋白表达量明显升高。与H2O2处理组比较,银杏叶提取物预处理组细胞凋亡指数、CHOPmRNA相对表达及CHOP蛋白表达量明显降低(P0.05)。结论:作为一种清除自由基、抗氧化、抗衰老药物,银杏叶提取物可能通过调节CHOP蛋白选择性地抑制过度的内质网应激来保护血管内皮细胞。     

Protective effects of Ginkgo biloba extract (EGb761) and its constituents quercetin and ginkgolide B against β-amyloid peptide-induced toxicity in SH-SY5Y cells

Ginkgo biloba extract EGb761 has been shown to protect against β-amyloid peptide (Aβ)-induced neurotoxicity but the specific mechanisms remain unclear. In the present study, effects of EGb761 and two of its constituents, quercetin and ginkgolide B, on the cytotoxic action of Aβ (1-42) were tested with human neuroblastoma SH-SY5Y cells. We found that EGb761 was able to block Aβ (1-42)-induced cell apoptosis, reactive oxygen species (ROS) accumulation, mitochondrial dysfunction and activation of c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt signaling pathways. Both quercetin and ginkgolide B may be involved in the inhibitory effects of EGb761 on JNK, ERK1/2 and Akt signaling pathways. Ginkgolide B also helped to improve mitochondrial functions but quercetin failed to show this effect. Additional experiments suggest that, protective effects of EGb761 against Aβ toxicity may be associated with its antioxidant and platelet activating factor (PAF) antagonist activities. Quercetin but not ginkgolide B is one of the constituents responsible for the antioxidant action of EGb761. Both quercetin and ginkgolide B may be involved in the PAF antagonist activity of EGb761. Overall, actions of individual EGb761 components provide further insights into direct mechanisms underlying the neuroprotective effects of EGb761.     

Protein kinases and ovarian functions

The present focus survey represents a short review of current knowledge concerning involvement of protein kinases in control of basic ovarian functions. Ovarian cells produce a number of protein kinases, whose expression depends on type of cells, their state and action of hormones and other protein kinases. A number of protein kinases are involved in control of ovarian cell proliferation, apoptosis, oocyte maturation, hormone release, reception and response to hormones, as well as in mediating action of hormones on these ovarian functions. Complexity of interrelationships between different protein kinase‐dependent signaling pathways occurs. Protein kinases and their regulators could be used for characterization, prediction and control of ovarian folliculogenesis and atresia, Corpus luteum functions, oocyte maturation, fertility, release of hormones, response of ovarian structures to hormonal regulators, as well as for treatment of some reproductive disorders. The present data demonstrate importance of protein kinases in control of basic ovarian function and potential usage of protein kinases for characterization, prediction and control of these functions. J. Cell. Physiol. 226: 37–45, 2010. © 2010 Wiley‐Liss, Inc.     

Kisspeptin as autocrine/paracrine regulator of human ovarian cell functions: Possible interrelationships with FSH and its receptor

The present study aims to examine the role of kisspeptin (KP), FSH, and its receptor (FSHR), and their interrelationships in the control of basic human ovarian granulosa cells functions. We investigated: (1) the ability of granulosa cells to produce KP and FSHR, (2) the role of KP in the control of ovarian functions, and (3) the ability of KP to affect FSHR and to modify the FSH action on ovarian functions. The effects of KP alone (0, 10 and 100 ng/mL); or of KP (10 and 100 ng/mL) in combination with FSH (10 ng/mL) on cultured human granulosa cells were assessed. Viability, markers of proliferation (PCNA and cyclin B1) and apoptosis (bax and caspase 3), as well as accumulation of KP, FSHR, and steroid hormones, IGF-I, oxytocin (OT), and prostaglandin E2 (PGE2) release were analyzed by the Trypan blue exclusion test, quantitative immunocytochemistry, and ELISA. KP given at a low dose (10 ng/mL) stimulated viability, proliferation, inhibited apoptosis, promoted the release of progesterone (P4), estradiol (E2), IGF-I, OT, and PGE2, the accumulation of FSHR, but not testosterone (T) release. KP given at a high dose (100 ng/mL) had the opposite, inhibitory effect. FSH stimulated cell viability, proliferation and inhibited apoptosis, promoted P4, T, E2, IGF-I, and OT, but not PGE2 release. Furthermore, KP at a low dose promoted the stimulatory effect of FSH on viability, proliferation, P4, E2, and OT release, promoted its inhibitory action on apoptosis, but did not modify its action on T, IGF-I, and PGE2 output. KP at a high dose prevented and inverted FSH action. These results suggest an intra-ovarian production and a functional interrelationship between KP and FSH/FSHR in direct regulation of basic ovarian cell functions (viability, proliferation, apoptosis, and hormones release). The capability of KP to stimulate FSHR, the ability of FSH to promote ovarian functions, as well as the similarity of KP (10 ng/mL) and FSH action on granulosa cells’ viability, proliferation, apoptosis, steroid hormones, IGF-I, OT, and PGE2 release, suggest that FSH influence these cells could be mediated by KP. Moreover, the capability of KP (100 ng/mL) to decrease FSHR accumulation, basal and FSH-induced ovarian parameters, suggest that KP can suppress some ovarian granulosa cell functions via down-regulation of FSHR. These observations propose the existence of the FSH-KP axis up-regulating human ovarian cell functions.     

Tetrahymena cells and the quantitative two-phase assay: visual assessment of chemokinesis and gravikinesis

Ginkgo biloba extract (GBE), which is one of the most sold herbal extracts in the world, is considered as a multifunctional material, which can promote radical scavenging activity and improve brain functioning. Although much research has carried out on its mechanisms against diseases, there is a need for further investigation for understanding molecular mechanisms, potential health benefits, and possible health risks. Recently, we have been developing an alternative screening method for studying phytochemical materials bioactivities with Tetrahymena thermophila as experimental organism. In this paper, the effects of GBE and its constituents were systematically investigated on chemoattraction and cGMP-dependent protein kinase (PKG) in T. thermophila . GBE and its constituents exerted significant inhibitions of chemoattraction and PKG. The minimal concentrations to completely inhibit chemotaxis of T. thermophila were 2 mg/ml, 50, 25, 12, 100, 100, 400, 500, 300 and 400 μM for GBE, quercetin, kaempferol, isorhamnetin, myricetin, genistein, rutin, quercitrin, isoquercitrin and quercetin-3- d -galactoside, respectively. The IC₅₀ values for PKG were 0.15 mg/ml, 26, 22, 19, 160, 132, 81 and 186 μM for GBE, myricetin, kaempferol, quercetin, isorhamnetin, genistein, rutin and isoquercitrin, respectively. The results indicate that the inhibitions of GBE and its constituents of chemotaxis of T. thermophila and their effects on PKG are rather similar. This suggests that the ciliate of T. thermophila may be a potential experimental organism in screening such bioactive phytochemicals.     

Amifostine has antiangiogenic properties in vitro by changing the redox status of human endothelial cells

Amifostine is a broad-spectrum cytoprotective agent, selective for normal tissues. It is a pro-drug metabolised to the free thiol WR-1065 that may act as a scavenger of free radicals, generated in tissues exposed to chemotherapeutic agents or radiation. WR-1065 can be further oxidized to its symmetric disulfide WR-33278 or degraded to hydrogen peroxide (H₂O₂). Both WR-1065 and WR-33278 resemble endogenous polyamines. Although amifostine is used in some cases in the clinic, there are only few studies concerning its actions at the cellular level. We have previously shown that amifostine inhibits angiogenesis in vivo, affecting the expression of several angiogenic genes. In the present work, we studied the effect of amifostine on human umbilical vein endothelial cell (HUVEC) functions in vitro, in order to further clarify its mechanism(s) of action. Amifostine increased HUVEC proliferation, an effect that was reversed by the intracellular H₂O₂ scavenger sodium pyruvate, agents that increase intracellular cAMP levels and L-valine. On the other hand, amifostine decreased HUVEC migration, an effect that was reversed by L-valine or L-arginine but not sodium pyrouvate. The decrease in migration was in line with decreased tube formation on matrigel and decreased amounts of metalloproteinase-2 released into the culture medium of HUVEC. Finally, amifostine reduced tyrosine nitration of the cytoskeletal proteins actin and α-tubulin in a time dependent manner. This last action could be due to the reduced production of nitric oxide (NO) or to other not yet identified mechanisms. Collectively, our results suggest that amifostine acts on endothelial cells through pathways that affect the redox status of the cells, either by producing H₂O₂ or by modulating NO production.     

中药银杏制剂的心血管药理效应:机制与展望

银杏是传统的活血化瘀中药,银杏制剂是现代科技开发的植物药中最为成功的案例之一.目前研究较多的银杏制剂是银杏酮酯,其主要活性成分为银杏黄酮、银杏内酯等,具有较为广泛的心血管药理效应,对多种心血管疾病具有较好的预防和治疗效果.本文对中药银杏制剂的主要活性成分、心血管药理效应及其机制、研究展望做一述评.     

TEL/JAK2 tyrosine kinase inhibits DNA repair in the presence of amifostine

The TEL/JAK2 chromosomal translocation (t(9;12)(p24;p13)) is associated with T cell childhood acute lymphoblastic leukemia. The TEL/JAK2 fusion protein contains the JAK2 catalytic domain and the TEL-specific oligomerization domain. TEL-mediated oligomerization of the TEL/JAK2 proteins results in the constitutive activation of the tyrosine kinase activity. Leukemia cells expressing TEL/JAK2 tyrosine kinase become resistant to anti-neoplastic drugs. Amifostine is a pro-drug which can selectively protect normal tissues against the toxicity of anticancer drugs and radiation. We investigated the effects of amifostine on idarubicin-induced DNA damage and repair in murine pro-B lymphoid BaF3 cells and BaF3-TEL/JAK2-transformed cells using alkaline single cell gel electrophoresis (comet assay). Idarubicin induced DNA damage in both cell types but amifostine reduced its extent in control non-transformed BaF3 cells and enhanced it in TEL/JAK2-transformed cells. The transformed cells did not show measurable DNA repair after exposure to amifostine and idarubicin, but cells treated only with idarubicin were able to recover within a 60-min incubation. Because TEL/JAK2-transformed cells can be considered as model cells for certain human leukemias and lymphomas we anticipate an enhancement of idarubicin cytotoxicity by amifostine in these diseases. Moreover, TEL/JAK2 tyrosine kinase might be involved in cellular response to DNA damage. Amifostine could promote apoptosis or lower the threshold for apoptosis induction dependent on TEL/JAK2 activation.     

Inhibition of platelet activating factor (PAF)-induced aggregation of human thrombocytes by ginkgolides: considerations on possible bleeding complications after oral intake of Ginkgo biloba extracts

The special Ginkgo biloba leaf extract EGb 761 is marketed for more then two decades. During this time its therapeutic efficacy and favorable safety profile have been proven in numerous clinical trails as well as by postmarketing surveillance in accordance with German drug regulations. During recent years, however, several cases of hemorrhage have been reported to occur in coincidence with the use of Ginkgo products. Although a clear causality between Ginkgo intake and bleeding could not be established, these observations have generally been explained by the platelet-activating factor (PAF)-antagonistic action of ginkgolides, which represent characteristic constituents of Ginkgo extracts. PAF was originally characterized by inducing aggregation and secretion of serotonin and histamine from rabbit platelets. We now confirmed that induction of aggregation of human platelets by PAF requires at least 200 times higher concentration when compared to rabbit cells. Under the chosen experimental conditions, PAF-mediated aggregation of human platelets was half-maximally inhibited by ginkgolide B, A, C and J at concentrations of 2.5, 15.8, 29.8 and 43.5 microg/ml, respectively. These concentrations are generally more than 100 times higher as the peak plasma values measured after oral intake of EGb 761 at recommended doses between 120 and 240 mg. As PAF is a 'weak' platelet activator, which does not appear to be of importance for primary hemostasis, our results rise serious doubts that the PAF antagonistic effect of ginkgolides could be responsible for hemorrhage in patients taking EGb 761.     

银杏与玉米花粉肌动蛋白含量的比较研究

通过免疫印迹鉴定,证明银杏(Ginkgo biloba L.)花粉中存在肌动蛋白。同时对银杏和玉米(Zeamays L.)花粉肌动蛋白含量进行了比较。结果表明,银杏花粉肌动蛋白含量明显少于玉米花粉肌动蛋白含量。SDS-PAGE扫描图谱显示,银杏花粉肌动蛋白含量只有玉米花粉的1/6。两种花粉DNaseⅠ活性抑制结果表明,银杏花粉肌动蛋白含量约为玉米花粉的1/7。     

Primary cell cultures from sea urchin ovaries: a new experimental tool

In the present work, primary cell cultures from ovaries of the edible sea urchin Paracentrotus lividus were developed in order to provide a simple and versatile experimental tool for researches in echinoderm reproductive biology. Ovary cell phenotypes were identified and characterized by different microscopic techniques. Although cell cultures could be produced from ovaries at all stages of maturation, the cells appeared healthier and viable, displaying a higher survival rate, when ovaries at early stages of gametogenesis were used. In terms of culture medium, ovarian cells were successfully cultured in modified Leibovitz-15 medium, whereas poor results were obtained in minimum essential medium Eagle and medium 199. Different substrates were tested, but ovarian cells completely adhered only on poly-L-lysine. To improve in vitro conditions and stimulate cell proliferation, different serum-supplements were tested. Fetal calf serum and an originally developed pluteus extract were detrimental to cell survival, apparently accelerating processes of cell death. In contrast, cells cultured with sea urchin egg extract appeared larger and healthier, displaying an increased longevity that allowed maintaining them for up to 1 month. Overall, our study provides new experimental bases and procedures for producing successfully long-term primary cell cultures from sea urchin ovaries offering a good potential to study echinoid oogenesis in a controlled system and to investigate different aspects of echinoderm endocrinology and reproductive biology.     

Biotechnological approaches to enhance the biosynthesis of ginkgolides and bilobalide in Ginkgo biloba

Ginkgo biloba is one of the oldest living tree species and its extracts or powdered leaves are one of the best selling herbal preparations. The main bioactive constituents are flavonoids and the terpene trilactones, ginkgolides and bilobalide, which are responsible for their pharmacological activity. However, there are many difficulties for ginkgo leaves supply and the chemical synthesis is far from of being applicable for commercial-scale production. G. biloba cell cultures have arisen as a useful alternative source of pharmacologically active terpene trilactones. This review sheds light on the chemistry and biosynthesis of terpene trilactones with the aim of increasing the production of these high value compounds by biotechnological approaches. Different biotechnological strategies to improve ginkgolides and bilobalide production will be discussed, including screening and selection of in vitro ginkgo cultures, cell differentiation levels of these cultures, optimization of culture conditions, feeding and elicitation strategies. Special attention will be paid in developing new methodologies to enhance ginkgo cell biomass and provide high amounts of these bioactive terpene trilactones using large-scale cell cultures.     

Nanos interacts with cup in the female germline of Drosophila

Nanos (Nos) is a translational regulator that governs abdominal segmentation of the Drosophila embryo in collaboration with Pumilio (Pum). In the embryo, the mode of Nos and Pum action is clear: they form a ternary complex with critical sequences in the 3'UTR of hunchback mRNA to regulate its translation. Nos also regulates germ cell development and survival in the ovary. While this aspect of its biological activity appears to be evolutionarily conserved, the mode of Nos action in this process is not yet well understood. In this report, we show that Nos interacts with Cup, which is required for normal development of the ovarian germline cells. nos and cup also interact genetically--reducing the level of cup activity specifically suppresses the oogenesis defects associated with the nos(RC) allele. This allele encodes a very low level of mRNA and protein that, evidently, is just below the threshold for normal ovarian Nos function. Taken together, these findings are consistent with the idea that Nos and Cup interact to promote normal development of the ovarian germline. They further suggest that Nos and Pum are likely to collaborate during oogenesis, as they do during embryogenesis.     

Cytokines: signalling molecules controlling ovarian functions

The present short review demonstrates an important role of different cytokines (colony stimulating factors, tumor necrosis factors, interleukins, anti-Mullerian hormone, inhibin, activin, follistatin, bone morphogenetic proteins, growth and differentiation factors) in the control of different ovarian functions - ovarian cell proliferation, apoptosis, folliculogenesis, luteogenesis, oogenesis, release of hormones, response to upstream hormonal regulators, fertility and, in some cases, in development of ovarian disorders. The possibility of practical application of these molecules for characterization, prediction and regulation of the ovarian state including treatment of ovarian disorders is demonstrated.     

Different effects of the constituents of EGb761 on apoptosis in rat cerebellar granule cells induced by hydroxyl radicals

The present study was conducted to evaluate the different effects of the constituents of EGb761 (Ginkgo biloba Extract) on apoptosis in cerebellar granule cells induced by hydroxyl radicals. The total flavonoid component of EGb761, two pure EGb761 components (rutin and quercetin), and a mixture of flavonoids and terpenes protected cerebellar granule cells from oxidative damage and apoptosis induced by hydroxyl radicals. ESR(electron spin resonance) results showed that the IC50 of the flavonoids for scavenging hydroxyl radicals was almost the same as that of EGb761, even though flavonoids make up only 24% of EGb761, implying that other constituents of EGb761 besides flavonoids can scavenge hydroxyl radicals. Total terpenes of EGb761 did not protect against apoptosis. Flavonoids and terpenes did not show a synergistic effect in this regard. Terpenes did not scavenge hydroxyl radicals directly, which might be related to their "cage-like" structures.