Tyrosine kinase-independent inhibition by genistein on spermatogenic T-type calcium channels attenuates mouse sperm motility and acrosome reaction

Although the protein tyrosine kinase (PTK) inhibitor, genistein, has been widely used to investigate the possible involvement of PTK during reproductive functions, it is unknown whether it modulates sperm calcium channel activity. In the present study, we recorded T-type calcium currents (I(Ca,T)) in mouse spermatogenic cells using whole-cell patch clamp and found that extracellular application of genistein reversibly decreased I(Ca,T) in a concentration-dependent manner (IC(50) approximately 22.7 microM). To determine whether TK activity is required for I(Ca,T) inhibition, we found that peroxovanadate, a tyrosine phosphatase inhibitor, was ineffective in preventing the inhibitory effect of genistein. Furthermore, intracellular perfusion of the cells with ATP-gamma-S also did not alter the inhibitory effect of genistein. To further reveal the direct inhibitory mechanism of genistein on I(Ca,T), we applied into the bath lavendustin A, a PTK inhibitor structurally unrelated to genistein, and found that the current amplitude remained unchanged. Moreover, daidzein, an inactive structural analog of genistein, robustly inhibited the currents. The inhibitory effect of genistein on T-type calcium channels was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Genistein was observed to decrease sperm motility and to significantly inhibit sperm acrosome reaction (AR) evoked by zona pellucida. Using transfected HEK293 cells system, only Cav3.1 and Cav3.2, instead of Cav3.3, channels were inhibited by genistein. Since T-type calcium channels are the key components in the male reproduction, such as in AR and sperm motility, our data suggest that this PTK-independent inhibition of genistein on I(Ca,T) might be involved in its anti-reproductive effects.     

Karyotypic, palynological, and RAPD study on 12 taxa from two subsections of section Idaeobatus in Rubus L. and taxonomic treatment of R. ellipticus, R. pinfaensis, and R. ellipticus var. obcordatus

Twelve taxa belonging to two subsections of section Idaeobatus in Rubus L. from southwestern China were characterized by karyotypic, palynological, and random amplified polymorphic DNA (RAPD) data as follows: (1) The 12 taxa were all diploid species (2n = 2x = 14), among which the chromosome counts for R. mesogaeus var. oxycomus, R. subtibetanus, R. ellipticus var. obcordatus, R. inopertus var. echinocalyx, and R. stans were reported for the first time; (2) All taxa except for R. ellipticus and R. pinfaensis could be distinguished from each other by karyotype, pollen morphology, and RAPD markers. Karyotypes were mainly characterized by the difference in numbers and positions of submetacentric chromosomes and chromosomes with satellited pair, the index of the karyotypic asymmetry, and the ratio of the longest to the shortest chromosome. Pollen morphology were mainly characterized by the discrepancy in specific pollen size, P/E ratio, colpi width, distance between the apices of two ectocolpi, and exine ornamentation characters. The cluster results based on RAPD markers were consistent with morphology classification except for R. pinfaensis; (3) Based on the general data of karyotypic, palynological, and RAPD, R. ellipticus var. obcordatus should be treated as a species R. obcordatus, R. ellipticus and R. pinfaensis should be combined as R. ellipticus, and it was more reasonable to place the combinants and R. obcordatus into subsection Stimulantes rather than into subsection Pungentes.     

Cloning and characterization of the ddsA gene encoding decaprenyl diphosphate synthase from Rhodobacter capsulatus B10

A decaprenyl diphosphate synthase gene (ddsA, GenBank accession No. DQ191802) was cloned from Rhodobacter capsulatus B10 by constructing and screening the genome library. An open reading frame of 1002 bp was revealed from sequence analysis. The deduced polypeptide consisted of 333 amino acids residues with an molecular mass of about 37 kDa. The DdsA protein contained the conserved amino acid sequence (DDXXD) of E-type polyprenyl diphosphate synthase and showed high similarity to others. In contrast, DdsA showed only 39% identity to a solanesyl diphosphate synthase cloned from R. capsulatus SB1003. DdsA was expressed successfully in Escherichia coli. Assaying the enzyme in vivo found it made E.coli synthesize UQ-10 in addition to the endogenous production UQ-8.     

基于系统安全性的死锁避免算法

死锁是多用户操作系统的一个重要事件,本文着重介绍了系统的安全状态,并对操作系统的避免死锁算法——Dijkstra银行家算法进行了研究分析。     

Understanding the “SMART” features of hematopoietic stem cells and beyond

Science China Life Sciences - Since the huge success of bone marrow transplantation technology in clinical practice, hematopoietic stem cells (HSCs) have become the gold standard for defining the...     

Plasticity of symbiont acquisition in new recruits of the massive coral Platygyra daedalea under ocean warming and acidification

Coral Reefs - Symbiosis establishment is a milestone in the life cycles of most broadcast-spawning corals; however, it remains largely unknown how initial symbiont infection is affected by ocean...     

Distinct roles of CysLT1 and CysLT2 receptors in oxygen glucose deprivation-induced PC12 cell death

Cysteinyl leukotrienes are involved in ischemic brain injury, and their receptors (CysLT(1) and CysLT(2)) have been cloned. To clarify which subtype mediates the ischemic neuronal injury, we performed permanent transfection to increase CysLT(1) and CysLT(2) receptor expressions in PC12 cells. Oxygen glucose deprivation (OGD)-induced cell death was detected by Hoechst 33258 and propidium iodide fluorescent staining as well as by flow cytometry. OGD induced late phase apoptosis mainly and necrosis minimally. Over-expression of CysLT(1) receptor decreased and over-expression of CysLT(2) receptor increased OGD-induced cell death. An agonist LTD(4) (10(-7)M) also induced apoptosis, especially in CysLT(2) receptor over-expressing cells. A selective CysLT(1) receptor antagonist montelukast did not affect OGD-induced apoptosis; while non-selective CysLT receptor antagonist Bay u9773 inhibited OGD-induced apoptosis, especially in CysLT(2) receptor over-expressing cells. Thus, CysLT(1) and CysLT(2) receptors play distinct roles in OGD-induced PC12 cell death; CysLT(1) attenuates while CysLT(2) facilitates the cell death.     

MicroRNA-99a-3p/GRP94 axis affects metastatic progression of human papillary thyroid carcinoma by regulating ITGA2 expression and localization

Papillary thyroid cancer (PTC) usually has favorable prognosis;however,distant metastasis is a leading cause of death associated with PTC.MicroRNA-99a-3p (miR-9...     

Characterization of streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots.

The actinomycete Streptomyces lydicus WYEC108 showed strong in vitro antagonism against various fungal plant pathogens in plate assays by producing extracellular antifungal metabolites. When Pythium ultimum or Rhizoctonia solani was grown in liquid medium with S. lydicus WYEC108, inhibition of growth of the fungi was observed. When WYEC108 spores or mycelia were used to coat pea seeds, the seeds were protected from invasion by P. ultimum in an oospore-enriched soil. While 100% of uncoated control seeds were infected by P. ultimum within 48 h after planting, less than 40% of coated seeds were infected. When the coated seeds were planted in soil 24 h prior to introduction of the pathogen, 96 h later, less than 30% of the germinating seeds were infected. Plant growth chamber studies were also carried out to test for plant growth effects and for suppression by S. lydicus WYEC108 of Pythium seed rot and root rot. When WYEC108 was applied as a spore-peat moss-sand formulation (10(8) CFU/g) to P. ultimum-infested sterile or nonsterile soil planted with pea and cotton seeds, significant increases in average plant stand, plant length, and plant weight were observed in both cases compared with untreated control plants grown in similar soils. WYEC108 hyphae colonized and were able to migrate downward with the root as it elongated. Over a period of 30 days, the population of WYEC108 colonized emerging roots of germinating seeds and remained stable (10(5) CFU/g) in the rhizosphere, whereas the nonrhizosphere population of WYEC108 declined at least 100-fold (from 10(5) to 10(3) or fewer CFU/g). The stability of the WYEC108 population incubated at 25 degrees C in the formulation, in sterile soil, and in nonsterile soil was also evaluated. In all three environments, the population of WYEC108 maintained its size for 90 days or more. When pea, cotton, and sweet corn seeds were placed into sterile and nonsterile soils containing 10(6) or more CFU of WYEC108 per g, it colonized the emerging roots. After a 1-week growing period, WYEC108 populations of 10(5) CFU/g (wet weight) of root were found on pea roots in the amended sterile soil environment versus 10(4) CFU/g in amended nonsterile soil. To further study the in vitro interaction between the streptomycete and P. ultimum, mycelia of WYEC108 were mixed with oospores of P. ultimum in agar, which was then used as a film to coat slide coverslips.(ABSTRACT TRUNCATED AT 400 WORDS)