Rosiglitazone ameliorates palmitic acid-induced cytotoxicity in TM4 Sertoli cells.

The Sertoli cell is the only somatic cell within the seminiferous tubules, and is vital for testis development and spermatogenesis. Rosiglitazone (RSG) is a member of the thiazolidinedione family and is a peroxisome proliferator-activated receptor-γ (PPARγ) agonist. It has been reported that RSG protects various types of cells from fatty acid-induced damage. However, whether RSG serves a protective role in Sertoli cells against palmitic acid (PA)-induced toxicity remains to be elucidated. Therefore, the aim of the present study was to investigate the effect of RSG on PA-induced cytotoxicity in Sertoli cells. MTT assay and Oil Red O staining revealed that RSG ameliorated the PA-induced decrease in TM4 cell viability, which was accompanied by an alleviation of PA-induced lipid accumulation in cells. In primary mouse Sertoli cells, RSG also showed similar protective effects against PA-induced lipotoxicity. Knockdown of PPARγ verified that RSG exerted its protective role in TM4 cells through a PPARγ-dependent pathway. To evaluate the mechanism underlying the protective role of RSG on PA-induced lipotoxicity, the present study analyzed the effects of RSG on PA uptake, and the expression of genes associated with both fatty acid oxidation and triglyceride synthesis. The results demonstrated that although RSG did not affect the endocytosis of PA, it significantly elevated the expression of carnitine palmitoyltransferase (CPT)-1A, a key enzyme involved in fatty acid oxidation, which indicated that the protective effect of RSG may have an important role in fatty acid oxidation. On the other hand, the expression of CPT1B was not affected by RSG. Moreover, the expression levels of diacylglycerol O-acyltransferase (DGAT)-1 and DGAT2, both of which encode enzymes catalyzing the synthesis of triglycerides, were not suppressed by RSG. The results indicated that RSG reduced PA-induced lipid accumulation by promoting fatty acid oxidation mediated by CPT1A. The effect of RSG in protecting cells from lipotoxicity was also found to be specific to Sertoli cells and hepatocytes, and not to other cell types that do not store excess lipid in large quantities, such as human umbilical vein endothelial cells. These findings provide insights into the cytoprotective effects of RSG on Sertoli cells and suggest that PPARγ activation may be a useful therapeutic method for the treatment of Sertoli cell dysfunction caused by dyslipidemia.

Nrf2 inhibition affects cell cycle progression during early mouse embryo development.

Brusatol, a quassinoid isolated from the fruit of Bruceajavanica, has recently been shown to inhibit nuclear factor erythroid 2-related factor 2 (Nrf2) via Keap1-dependent ubiquitination and proteasomal degradation or protein synthesis. Nrf2 is a transcription factor that regulates the cellular defense response. Most studies have focused on the effects of Nrf2 in tumor development. Here, the critical roles of Nrf2 in mouse early embryonic development were investigated. We found that brusatol treatment at the zygotic stage prevented the early embryo development. Most embryos stayed at the two-cell stage after 5 days of culture (P < 0.05). This effect was associated with the cell cycle arrest, as the mRNA level of CDK1 and cyclin B decreased at the two-cell stage after brusatol treatment. The embryo development potency was partially rescued by the injection of Nrf2 CRISPR activation plasmid. Thus, brusatol inhibited early embryo development by affecting Nrf2-related cell cycle transition from G2 to M phase that is dependent on cyclin B-CDK1 complex.

Clarithromycin Synergistically Enhances Thalidomide Cytotoxicity in Myeloma Cells.

Clarithromycin (CAM) is a macrolide antibiotic that is widely used in the treatment of respiratory tract infections, sexually transmitted diseases and infections caused by the Helicobacter pylori and Mycobacterium avium complex. Recent studies showed that CAM was highly effective against multiple myeloma (MM) when used in combination with immunomodulatory drugs and dexamethasone. However, the related mechanism is still unknown. As 3 immunomodulatory agents are all effective in the respective regimen, we postulated that CAM might enhance the effect of immunomodulatory drugs. We evaluated the interaction effects of CAM and thalidomide on myeloma cells. Taking into consideration that thalidomide did not affect the proliferation of myeloma cells in vitro, we cocultured myeloma cells with peripheral blood monocytes and evaluated the effects of CAM and thalidomide on the cocultured cell model. Data showed that thalidomide and CAM synergistically inhibited the proliferation of the cells. On this same model, we also found that thalidomide and CAM synergistically decreased the secretion of tumor necrosis factor-α and interleukin-6. This might be caused by the effect of the 2 drugs on inhibiting the activation of ERK1/2 and AKT. These data suggest that the efficacy of CAM against MM was partly due to its synergistic action with the immunomodulatory agents.

Molecular analyses of the rice glutamate dehydrogenase gene family and their response to nitrogen and phosphorous deprivation.

Glutamate dehydrogenases (GDH, EC 1.4.1.2 approximately 4) are ubiquitous enzymes encoded by GDH genes. So far, at least two GDH members have been characterized in plants, but most members of this family in rice remains to be characterized. Here, we show that four putative GDH genes (OsGDH1-4) are present in the rice genome. The GDH sequences from rice and other species can be classified into two types (I and II). OsGDH1-3 belonged to type II genes, whereas OsGDH4 belonged to type I like gene. Our data implied that the expansion rate of type I genes was much slower than that of type II genes and species-specific expansion contributed to the evolution of type II genes in plants. The expression levels of the different members of GDH family in rice were evaluated using quantitative real-time PCR and microarray analysis. Gene expression patterns revealed that OsGDH1, OsGDH2, and OsGDH4 are expressed ubiquitously in various tissues, whereas OsGDH3 expression is glumes and stamens specific. The expression of the OsGDH family members responded differentially to nitrogen and phosphorus-deprivation, indicating their roles under such stress conditions. Implications of the expression patterns with respect to the functions of these genes were discussed.

The effect of vitamin D on sperm motility and the underlying mechanism.

Vitamin D deficiency is a common health issue around the world. We therefore evaluated the associations of semen quality with both serum and seminal plasma vitamin D levels and studied the mechanisms underlying these by incubating spermatozoa with 1,25(OH)2D In vitro. Two hundred and twenty-two men were included in our study. Vitamin D was detected using an electrochemiluminescence method. Spermatozoa used for In vitro experiments were isolated by density gradient centrifugation. Positive relationships of serum 25(OH)D with semen volume and seminal plasma fructose were identified. Seminal plasma 25(OH)D level showed no relationship with serum 25(OH)D level, while it was inversely associated with sperm concentration and positively correlated with semen volume and sperm kinetic values. In vitro, sperm kinetic parameters increased after incubation with 1,25(OH)2D, especially upon incubation for 30 min with it at a concentration of 0.1 nmol l-1. Under these incubation conditions, the upward migration of spermatozoa increased remarkably with increasing adenosine triphosphate (ATP) concentration. The concentration of cyclic adenosine monophosphate (cAMP) and the activity of protein kinase A (PKA) were both elevated, and the PKA inhibitor, N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89) reversed the increase of ATP production. The concentrations of cytoplasmic calcium ions and nicotinamide adenine dinucleotide (NADH) were both enhanced, while mitochondrial calcium uniporter (MCU) inhibitor, Ruthenium 360 (Ru360) did not reverse the increase of ATP production. Therefore, seminal plasma vitamin D may be involved in regulating sperm motility, and 1,25(OH)2D may enhance sperm motility by promoting the synthesis of ATP both through the cAMP/PKA pathway and the increase in intracellular calcium ions.

Sirt1/Nrf2 pathway is involved in oocyte aging by regulating Cyclin B1.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is capable of inducing a variety of biological effects, and the regulation of the Nrf2 signaling pathway is closely related to longevity. To find out whether the nuclear factor erythroid 2-related factor 2 (Nrf2) is involved in oocyte aging or not which may cause reduced female fertility, a series of biological methods was applied, including oocyte collection and culture, micro injection, RNA interference, western blotting, immunofluorescence and confocal microscopy, and quantitative real-time PCR.Our data demonstrated that Nrf2 depletion disrupted oocyte maturation and spindle/chromosome organization by suppressing Cyclin B1 expression. Sirtuin 1 (Sirt1) depletion reduced Nrf2 expression, which indicated the existence of the Sirt1-Nrf2-Cyclin B1 signaling pathway in mouse oocytes. Additionally, immunoblotting results reflected a lower Nrf2 protein level in oocytes from aged mice, and maternal age-associated meiotic defects can be ameliorated through overexpression of Nrf2, which supported the hypothesis that decreased Nrf2 is an important factor contributing toward oocyte age-dependent deficits. Furthermore, we show that the expression of Nrf2 is related to female age in ovarian granular cells, suggesting that the decreased expression of Nrf2 may be related to the decline in the reproductive capacity of older women.

The toxic effects and possible mechanisms of Brusatol on mouse oocytes.

Brusatol is a natural quassinoid that shows a potential therapeutic use in cancer models by the inhibition of Nuclear factor erythroid 2-related factor 2 (Nrf2) and is capable of inducing a variety of biological effects. The effects of Brusatol on oocyte meiosis has not been addressed. In this study, we investigated the impact of Brusatol treatment on mouse oocyte maturation and its possible mechanism. Our data demonstrated that Brusatol treatment disrupted oocyte maturation and spindle/chromosome organization by modulating Nrf2-Cyclin B1 pathway, as the influence of Brusatol was compensated by the addition of Nrf2 activation plasmid, and the mRNA and protein levels of Cyclin B1 were severely reduced in oocytes following Nrf2 decline. In summary, our data support a model that Brusatol, through the inhibition of Nrf2, modulate Cyclin B1 levels, consequently disturbing proper spindle assembly and chromosome condensation in meiotic oocytes.

Activity of fibroblast growth factor receptor inhibitors TKI258, ponatinib and AZD4547 against TPR­FGFR1 fusion.

8p11 myeloproliferative syndrome (EMS) is a rare disease characterized by the constitutive activation of fibroblast growth factor receptor 1 (FGFR1). To date, four cases of EMS with the chromosomal translocation, t(1;8)(q25;p11.2), have been reported. In the present study, TPR­FGFR1­expressing Baf3 cells were established and confirmed by polymerase chain reaction. To identify the most promising drug for EMS, the activities and associated mechanism of three tyrosine kinase inhibitors (TKIs), TKI258, ponatinib and AZD4547, against TPR­FGFR1 were tested by MTT assay, flow cytometry and western blot. The data demonstrated that TPR­FGFR1 was localized in the cytoplasm, and was able to transform interleukin-3-dependent hematopoietic Baf3 cells into growth factor­independent cells. All of the three TKIs markedly inhibited the proliferation of TPR­FGFR1­expressing Baf3 cells, and the activation of FGFR1 and the downstream signaling molecules, extracellular signal­regulated kinase 1/2, phospholipiase Cγ and signal transducer and activator of transcription 5. AZD4547 was the most efficient drug, and TKI258 was the least. By contrast, no significant difference was found among the three drugs on their effect on cell apoptosis. Taken together, the data obtained in the present study suggested that AZD4547 had increased potency, compared with TKI258 and ponatinib, for the treatment of EMS.

Maternal diabetes impairs the initiation of meiosis in murine female germ cells.

Gestational diabetes mellitus (GDM) is characterized by an initial diagnosis of glucose intolerance during pregnancy. There is increasing evidence supporting the association between GDM and the inhibited development of several organs in offspring. In the present study, a murine GDM model was established in mice by intraperitoneal injection of streptozotocin to evaluate the effect of maternal diabetes on the initiation of meiosis in female germ cells of offspring. The effect of GDM on the initiation of meiosis in the offspring was evaluated by reverse transcription-quantitative polymerase chain reaction, flow cytometry and hematoxylin and eosin staining. The results showed that, compared with the control group, fetal ovary growth was inhibited, the expression levels of meiosis­specific genes, stimulated by retinoic acid gene 8, synaptonemal complex protein, and DNA meiotic recombinase were inhibited, and the number of primordial/primary follicles was reduced in the GDM group. These may have been induced by an increase of apoptosis and inhibition of growth, as the mRNA levels of p21, a vital G1 cell cycle inhibitor, and apoptotic genes were upregulated, whereas the expression levels of genes important in folliculogenesis were decreased in the GDM group. In conclusion, the data obtained in the present study suggested that maternal diabetes may impair the initiation of meiosis and ovarian growth via growth inhibition, cell cycle arrest and the induction of apoptosis.

[Progress of research on clarithromycin for treatment of multiple myeloma].

Clarithromycin is a 14-membered ring macrolide antibiotics that is widely used in the treatment of infectious disease. Several clinical investigations showed that clarithromycin was highly efficient for multiple myeloma in improving response rate and survival when used in combination with the conventional chemotherapy since 1997. This finding highlights the importance of clarithromycin on the treatment of multiple myeloma. It offers a new regimen for the relapsed/refractory multiple myeloma patients, and provids a new thought for the treatment of multiple myeloma. However, its related mechanism is still unclear, and more investigations are needed. This review summerizes the recent research progress of clarithromycin for treatment of multiple myeloma and its potential mechanisms.

Vaccination of full-length HPV16 E6 or E7 protein inhibits the growth of HPV16 associated tumors.

Cervical cancer is the second most common cancer in women worldwide. Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Two HPV16 proteins, E6 and E7, are consistently expressed in tumor cells. Most therapeutic vaccines target one or both of these proteins. Taking the advantages of safety and no human leukocyte antigen restriction, protein vaccine has become the most popular form of HPV therapeutic vaccines. Here we demonstrate that immunization with full-length HPV16 E6 or E7 protein elicited specific immunological effect and inhibition of TC-1 cell growth using TC-1 mouse model. HPV16 E6 and E7 genes were cloned into pET-28a(+) and introduced into E. coli Rosetta. Expression of the genes was induced by IPTG. Proteins were purified by Ni-NTA agarose and they were detected by SDS-PAGE and Western blotting. C57BL/6 mice were vaccinated with 1.5 nmol HPV16 E6 or E7 protein. Then they were implanted with 1x10(5) TC-1 cells. No tumor was detected in any mouse vaccinated with E7 protein. Forty days later, the tumor-free mice and control mice were challenged with 2x10(5) TC-1 cells. All control mice developed tumors 6 days later, but E7 immunized mice were tumor free until 90 days. Tumor growth was slow in the E6 immunized mice, but 83% of the mice developed tumors and the survival percentage was not significantly different from the control. An adoptive immune model was used to demonstrate the therapeutic effect. Results showed that the development of TC-1 cells was obviously reduced by transfusion of T-cells but not serum from mice immunized with E7 protein. T-cells from E7 immunized mice also induced the lysis of TC-1 cells in the cytotoxic T lymphocyte assay. These findings show that immunization with HPV16 E6 or E7 protein was able to elicit specific protective immunity against TC-1 tumor growth.

The expression profile of genes in rice roots under low phosphorus stress.

Phosphorus (P) is one of the most essential macronutrients required for plant growth. Although it is abundant in soil, P is often the limiting nutrient for crop yield potential because of the low concentration of soluble P that plants can absorb directly. The gene expression profile was investigated in rice roots at 6, 24 and 72 h under low P stress and compared with a control (normal P) profile, using a DNA chip of 60000 oligos (70 mer) that represented all putative genes of the rice genome. A total of 795 differentially expressed genes were identified in response to phosphate (Pi) starvation in at least one of the treatments. Based on the analysis, we found that: (i) The genes coding for the Pi transporter, acid phosphatase and RNase were up-regulated in rice roots; (ii) the genes involved in glycolysis were first up-regulated and then down-regulated; (iii) several genes involved in N metabolism and lipid metabolism changed their expression patterns; (iv) some genes involved in cell senescence and DNA or protein degradation were up-regulated; and (v) some transmembrane transporter genes were up-regulated. The results may provide useful information in the molecular process associated with Pi deficiency and thus facilitate research in improving Pi utilization in crop species.

KT/HAK/KUP potassium transporters gene family and their whole-life cycle expression profile in rice (Oryza sativa).

KT/HAK/KUP potassium transporter protein-encoding genes constitute a large family in the plant kingdom. The KT/HAK/KUP family is important for various physiological processes of plant life. In this study, we identified 27 potential KT/HAK/KUP family genes in rice (Oryza sativa) by database searching. Analysis of these KT/HAK/KUP family members identified three conserved motifs with unknown functions, and 11-15 trans-membrane segments, most of which are conserved. A total of 144 putative cis-elements were found in the 2 kb upstream region of these genes, of which a Ca2+-responsive cis-element, two light-responsive cis-elements, and a circadian-regulated cis-element were identified in the majority of the members, suggesting regulation of these genes by these signals. A comprehensive expression analysis of these genes was performed using data from microarrays hybridized with RNA samples of 27 tissues covering the entire life cycle from three rice genotypes, Minghui 63, Zhenshan 97, and Shanyou 63. We identified preferential expression of two OsHAK genes in stamen at 1 day before flowering compared with all the other tissues. OsHAK genes were also found to be differentially upregulated or downregulated in rice seedlings subjected to treatments with three hormones. These results would be very useful for elucidating the roles of these genes in growth, development, and stress response of the rice plant.