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1.
Several authors have demonstrated the involvement of tyrosine kinases during sperm capacitation and acrosome reaction. Shc proteins (p46Shc, p52Shc, and p66Shc) are cytoplasmic substrates of activated tyrosine kinases and are widely expressed in mammalian somatic tissues. Experiments were designed to demonstrate the presence of Shc in spermatozoa and to study its involvement in the signal transduction events leading to acrosome reaction. Anti-Shc antibodies strongly reacted with the acrosomal region of methanol-fixed human sperm. Only one Shc isoform (p52Shc) was detected on Western blot. To study the degree of phosphorylation of Shc during capacitation and acrosome reaction, sperm samples were divided into two groups: noncapacitated and capacitated/progesterone treated. Lysates from both groups were immunoprecipitated with anti-phosphotyrosine antibodies and the precipitated (ie, phosphorylated) proteins were tested with anti-Shc antibodies. The intensity of p52Shc was clearly increased in capacitated/progesterone-stimulated cells. Mol. Reprod. Dev. 50:113–120, 1998. © 1998 Wiley-Liss, Inc. 相似文献
2.
The umuDC locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli umuDC genes cloned on pBR322-derived plasmids wer e isolated by two methods. First, spontaneously-arising mutant umuDC plasmids that failed to confe cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant umuDC plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD+C+ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD+C+ plasmids. pBR322-derived umuD+C+ plasmids inhibited the induction of the SOS response of lexA+ strains as measured by expression of din::Mu dl(lac) Ap) fusionsbut most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA+ strainsto killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis. pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA+ strains. The effect of pLM109 on the UV sensitivity of lexA(Def)strains was similar to that of the parental umuD+C+ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125mutation was a G:C → A:T transition that changed codon 39 of umuC from GCC → GTC thus changing Ala39 to Val39. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed. 相似文献
3.
Subsite mapping is a crucial procedure in the characterization of α-amylases (EC 3.2.1.1), which are extensively used in starch-based industries and in diagnosis of pancreatic and salivary glands disorders. A computer-aided method has been developed for subsite mapping of α-amylases, which substitutes the difficult, expensive, and time-consuming experimental determination of action patterns to crystal structures based energy calculations. Interaction energies between enzymes and carbohydrate substrates were calculated after short energy minimization by a molecular mechanics program. A training set of wild type and mutant amylases with known experimental action patterns of 13 enzymes of wide range of origin was used to set up the procedure. Calculations for training set resulted in good correlation in case of subsite binding energies (r2 = 0.827–0.929) and bond cleavage frequencies (r2 = 0.727–0.835). A set of eight novel barley amylase 1 mutants was used to test our model. Subsite binding energies were predicted with r2 = 0.502 correlation coefficient, while bond cleavage frequency prediction resulted in r2 = 0.538. Our computer-aided procedure may supplement the experimental subsite mapping methods to predict and understand characteristic features of α-amylases. 相似文献
4.
Karnam R. Purushotham Tivadar Zelles Michael G. Humphreys-Beher 《Molecular and cellular biochemistry》1991,102(1):19-33
The involvement of protein phosphorylation in isoproterenol (ISO)-mediated proliferation in the rat parotid gland was investigated by labeling the cells with [32P] orthophosphate. An increased (4–6 fold) incorporation of the radiolabel was noted in the total parotid gland homogenates of ISO-treated animals when compared to controls. Plasma membrane, nuclear membrane and cytoplasm were isolated, the proteins separated by SDS/PAGE and the phosphoproteins detected by autoradiography. Two phosphoproteins with apparent Mr of 45 and 170 kDa were identified in the cytoplasm while the 170 kDa phosphoprotein also appeared as part of plasma membrane. Transfer of these proteins to nitrocellulose followed by Western blot detection with an antiphosphotyrosine monoclonal antibody showed reactivity with the 170 kDa region of the plasma membrane and cytoplasm. Separate in vitro studies involving incubations of rat parotid slices with 0.2 mM ISO and [3H] myo-inositol for 1 min induced inositol phosphate hydrolysis resulting in a significant increase in inositol-bis and -tris phosphate production. Inositol phosphate production can be blocked by pre-incubation with a mixed -adrenergic receptor antagonist but not with physiological concentrations of - or 1-specific adrenergic receptor antagonists, indicating the ISO effects are mediated through the 2-adrenergic receptors. The inclusion of calmodulin antagonists along with ISO prevented the expression of cell-surface galactosyltransferase and retarded gland hypertrophy and hyperplasia. These results suggest that ISO treatment leads to the phosphorylation of target proteins which may be involved in signal transduction pathways leading to cell proliferation.Abbreviations InsP1, InsP2, InSP3
inositol mono-, bis-, and tris-phosphates
- UDP
Uridine diphosphate
- PMSF
phenylmethylsulfonylfluoride
- SDS
sodium dodecyl sulfate
- TFP
Trifluoperazine
- P-tyr
phosphotyrosine
- Gal Tase
galactosyltransferase 相似文献
5.
《Critical reviews in biochemistry and molecular biology》2013,48(5-6):339-359
AbstractVanadium and its compounds exhibit a wide variety of insulin-like effects. In this review, these effects are discussed with respect to the treatment of type I and type II diabetes in animal models, in vitro actions, antineoplastic role, treatment of IDDM and NIDDM patients, toxicity, and the possible mechanism(s) involved. Newly established CytPTK plays a major role in the bioresponses of vanadium. It has a molecular weight of approximately 53 kDa and is active in the presence of Co2+ rather than Mn2+. Among the protein-tyrosine kinase blockers, staurosporine is found to be a potent inhibitor of Cyt PTK but a poor inhibitor of InsRTK. Vanadium inhibits PTPase activity, and this in turn enhances the activity of protein tyrosine kinases. Our data show that inhibition of PTPase and protein tyrosine kinase activation has a major role in the therapeutic efficacy of vanadium in treating diabetes mellitus. 相似文献
6.
E. D. Badaeva A. V. Amosova O. V. Muravenko T. E. Samatadze N. N. Chikida A. V. Zelenin B. Friebe B. S. Gill 《Plant Systematics and Evolution》2002,231(1-4):163-190
Six polyploid Aegilops species containing the D genome were studied by C-banding and fluorescence in situ hybridization (FISH) using clones pTa71
(18S-5.8S-26S rDNA), pTa794 (5S rDNA), and pAs1 (non-coding repetitive DNA sequence) as probes. The C-banding and pAs1-FISH
patterns of Ae. cylindrica chromosomes were identical to those of the parental species. However, inactivation of the NOR on chromosome 5D with a simultaneous
decrease in the size of the pTa71-FISH site was observed. The Nv and Dv genomes of Ae. ventricosa were somewhat modified as compared with the N genome of Ae. uniaristata and the D genome of Ae. tauschii. Modifications included minor changes in the C-banding and pAs1-FISH patterns, complete deletion of the NOR on chromosome
5Dv, and the loss of several minor 18S-5.8S-26S rDNA loci on Nv genome chromosomes. According to C-banding and FISH analyses, the Dcr1 genome of Ae. crassa is more similar to the Dv genome of Ae. ventricosa than to the D genome of Ae. tauschii. Mapping of the 18S-5.8S-26S rDNA and 5S rDNA loci by multicolor FISH suggests that the second (Xcr) genome of tetraploid Ae. crassa is a derivative of the S genome (section Emarginata of the Sitopsis group). Both genomes of Ae. crassa were significantly modified as the result of chromosomal rearrangements and redistribution of highly repetitive DNA sequences.
Hexaploid Ae. crassa and Ae. vavilovii arose from the hybridization of chromosomal type N of tetraploid Ae. crassa with Ae. tauschii and Ae. searsii, respectively. Chromosomal type T1 of tetraploid Ae. crassa and Ae. umbellulata were the ancestral forms of Ae. juvenalis. The high level of genome modification in Ae. juvenalis indicates that it is the oldest hexaploid species in this group. The occurrence of hexaploid Ae. crassa was accompanied by a species-specific translocation between chromosomes 4Dcr1 and 7Xcr. No chromosome changes relative to the parental species were detected in Ae. vavilovii, however, its intraspecific diversity was accompanied by a translocation between chromosomes 3Xcr and 3Dcr1.
Received July 24, 2001 Accepted October 1, 2001 相似文献
7.
The present study analyzed the distribution pattern of the Ae. speltoides–derived repetitive clone pGc1R-1 in the Triticum/Aegilops complex. Fluorescence in situ hybridization analysis showed that clone pGc1R-1 is a S-genome-specific repetitive sequence
that hybridized to the S-genome of three species in the section Sitopsis, Aegilops speltoides (S), Ae. longissima (Sl), and Ae. sharonensis (Ssh), but not to Ae. bicornis (Sb) and Ae. searsii (Ss), nor to any other diploid Aegilops species. This clone also hybridized to the very closely related G-genome of T. timopheevii subsp. armeniacum and T. timopheevii ssp. timopheevii, but not to the B-genome of T. turgidum and T. aestivum. Hybridization also was observed in the polyploid Aegilops species, Ae. kotschyi (UkSk), Ae. peregrina (UpSp), and Ae. vavilovii (XvaDvaSva). Large inter- and intraspecific variations were observed. Our results confirm that the S genome is related more to the Sl and Ssh genomes than to the Sb and Ss genomes; there is a greater affinity between the G and S genomes than between the B and S genomes. Mechanisms to account
for the variation in the FISH pattern with different genomes include sequence amplification and deletion. Variation in the
distribution of this genome-specific DNA sequence, pGc1R-1, on chromosomes can be used to reveal evolutionary relationships
in the Triticum and Aegilops complex.
Received April 10, 2002; accepted July 12, 2002 Published online: November 28, 2002
Address of the authors: Peng Zhang, Bernd Friebe (e-mail: friebe@ksu.edu), Bikram S. Gill, Wheat Genetics Resource Center,
Department of Plant Pathology, 4024 Throckmorton, Plant Sciences Center, Kansas State University, Manhattan, KS 66506-5502,
USA. 相似文献
8.
Waraich RS Zaidi N Moeschel K Beck A Weigert C Voelter W Kalbacher H Lehmann R 《Biochemical and biophysical research communications》2008,376(1):26-31
Antibodies that recognize specifically phosphorylated sites on proteins are widely utilized for studying the regulation and biological function of phosphoproteins. The proposed strategy is a powerful, analytical tool allowing the generation of phospho-site specific antibodies albeit adjacent phosphorylation sites are present. Here, we demonstrate the assessment and elimination of cross reactivity of phospho-site-specific-Ser357 IRS-1 antibody. While determining the specificity of p-Ser357 antiserum we came across the cross reactivity of the antiserum with adjacent Ser358 which was successfully abolished by an improved immuno-purification method. The specificity of the purified antiserum was then verified by indirect ELISA, results of ELISA were also mirrored in the experiments carried out in BHK-IR cells using different mutants of IRS-1 carrying mutations at either Ser357/Ser358/Ser357/358. Immuno-purified-p-Ser357 did not react with IRS-1 Ala357 and IRS-1 Ala357/358. In conclusion, the present study describes generation and characterization of p-Ser357 IRS-1 antibody, which reacts with IRS-1 in site specific and phosphorylation state-dependent manner without showing cross reactivity to adjacent Ser358. This antibody can be effectively used to further clarify the inhibitory role of Ser357 in insulin signal transduction. 相似文献
9.
An in vitro procedure for large scale multiplication of Sterculia urens Roxb. (Gum Kadaya Tree) has been developed using cotyledonary node segments. An average of 4.0 shoots per node were obtained on Murashige and Skoog's (MS) medium containing 2.0 mgl–1 6-benzyl amino-purine (BAP) within 21 days of initial culture. Upon subsequent subculture 16 shoots/node could be harvested every three weeks and upto three times. Sixty per cent of the shoots were successfully rooted. Rooted plantlets were transferred to plastic pots containing soil under mist house conditions before they were finally exposed to an external environment. Fifty seven per cent of the plantlets survived in nursery sheds. 相似文献
10.
Nanthawan Mekha Masako Takashima Jotika Boon‐long Otomi Cho Takashi Sugita 《Microbiology and immunology》2014,58(1):9-14
11.
Broadbridge Robert J. Sharma Ram P. 《International journal of peptide research and therapeutics》1999,6(5-6):335-341
Summary Phosphorylation reactions are key meditors in a variety of biochemical signal processes. Research into the selective inhibition
of protein tyrosine kinases to generate anticancer agents has madeO-phosphotyrosyl analogues important pharmacological tools. The simple procedures reported here involving the formation of
interative peptide libraries together with the development of a selective and sensitive bead-binding assay have made it possible
to rapidly screen peptides incorporatingO-phosphotyrosyl surrogates (includingO-phospho-2,3,5,6-tetrafluorotyrosine, 4-(phosphono)hydroxymethyl-phenylalanine and 4-(phosphono)fluoromethyl-phenylalanine)
for their potential to inhibit the protein tyrosine kinase p56lck. These procedures can be easily adapted to combinatorical peptide libraries. 相似文献
12.
13.
Sabine Elowe 《Cell cycle (Georgetown, Tex.)》2017,16(8):746-748
Tyrosine phosphorylation is rare, representing only about 0.5% of phosphorylations in the cell under basal conditions. While mitogenic tyrosine kinase signaling has been extensively explored, the role of phosphotyrosine signaling across the cell cycle and in particular during mitosis is poorly understood.
Two recent, independent studies tackled this question from different angles to reveal exciting new insights into the role of this modification during cell division. Caron et al.1 exploited mitotic phosphoproteomics data sets to determine the extent of mitotic tyrosine phosphorylation, and St-Denis et al.2 identified protein tyrosine phosphatases from all subfamilies as regulators of mitotic progression or spindle formation. These studied collectively revealed that tyrosine phosphorylation may play a more prominent and active role in mitotic progression than previously appreciated. 相似文献
14.
用差异显示反转录PCR银染技术研究植物基因表达的差异 总被引:5,自引:0,他引:5
通过调整差异显示反转录PCR(DDRT-PCR)中总RNA、锚定引物、随机引物、cDNA和dNTP等关键试剂的用量,优化了适用于银染检测的DDRT-PCR方法.PCR扩增产物经6%变性聚丙烯酰胺凝胶垂直电泳分离后,银染能检测到多而清晰的条带.泳道中的条带数最少为40个,最多达80个,平均为60个,条带大小分布在100~900 bp范围,灵敏度为5 pg/mm2 .此方法操作简便快速,灵敏度高,重复性好.采用这个改良的方法,分析了拟南芥野生型和ast突变型基因表达的差异.从16 000个cDNA扩增产物条带中筛选出28个差异条带.二次PCR扩增后,进一步筛选出13个差异条带,其中7个是野生型特异表达的,6个是突变型特异表达的,为进一步认识ast突变表型的产生机制奠定了基础. 相似文献
15.
芦竹(Arundo donax)对多种重金属都有较好的耐受性,是植物修复技术较理想的选择,而关于芦竹对Cd和Tl胁迫生理反应的相关研究却较少,为了有效治理Cd和Tl的污染,本研究以芦竹为材料,通过添加不同浓度重金属Tl(4,10和20 mg·kg~(-1))、Cd(50,100和200 mg·kg~(-1))进行芦竹盆栽试验,测定芦竹的株高、分蘖数、叶绿素含量、光合生理指标以及Tl和Cd在芦竹中的累积量,探讨芦竹对Tl和Cd胁迫的响应机制。结果表明:Tl(4~20 mg·kg~(-1))和Cd(50~200 mg·kg~(-1))对芦竹株高、分蘖数以及叶绿素含量均无显著影响(P0.05);芦竹体内Tl和Cd含量随着Tl和Cd浓度的升高呈上升趋势,芦竹体内Tl含量的分布规律为根茎叶,Cd含量的分布规律:Cd浓度50 mg·kg~(-1)时为茎叶根,Cd浓度100和200 mg·kg~(-1)时为根茎叶,表明Tl和Cd主要分布在根部,芦竹对Tl、Cd有一定的富集能力。Cd和Tl处理均显著降低芦竹叶片的胞间CO2浓度,在Tl浓度为10 mg·kg~(-1)时,净光合速率、气孔导度和蒸腾速率得到显著提高,当Cd浓度为50 mg·kg~(-1)时,净光合速率、气孔导度和蒸腾速率得到显著提高。这表明芦竹对重金属Cd和Tl有较强的耐受性,可为Cd和Tl污染土壤的治理和修复提供参考。 相似文献
16.
为探明华重楼离体胚培养及植株再生的基本体系,该文以华重楼离体幼胚为试验材料,以MS培养基为基本培养基,研究不同光照、不同浓度梯度组合的植物生长调节剂对华重楼离体幼胚萌发、成苗的影响。结果表明:培养到60 d时,暗培养条件下华重楼离体幼胚的生长率和萌发率分别比光培养条件下高45.25%、19.17%,故暗培养比光培养更有利于华重楼离体幼胚生长发育。当GA3浓度相同时,离体幼胚萌发所需时间随IAA浓度增加而延长。不同浓度的GA3都可促进离体幼胚萌发,促进作用由强到弱依次为5 mg·L-1GA3>1 mg·L-1GA3> 10 mg·L-1GA3。在黑暗条件下,优选得到最适华重楼离体幼胚生长发育配方为1/2 MS+30g·L-1蔗糖+7 g·L-1琼脂+0.5 g·L-1活性炭+5 mg·L-1GA3 相似文献
17.
A large-scale transformation procedure handling an adequate number of stable transformants with highly efficient positive-negative selection is a necessary prerequisite to successful gene targeting by homologous recombination, as the integration of a transgene by somatic homologous recombination in higher plants has been reported to be 10-3 to 10-5 compared with random integration by non-homologous end joining. We established an efficient and large-scale Agrobacterium-mediated rice transformation protocol that generated around 103 stable transformants routinely from 150 seeds and a strong positive-negative selection procedure that resulted in survivors at 10-2 using the gene for diphtheria toxin A fragment as a negative marker. The established transformation procedure provides a basis for efficient gene targeting in rice.Abbreviations
AS:
Acetosyringone
-
5-FU:
5-Fluorouracil
-
FW:
Fresh weight
-
GT:
Gene targeting
-
HR:
Homologous recombination
-
NHEJ:
Non-homologous end joining
Communicated by H. Ebinuma 相似文献
18.
With the help of a ribonucleoprotein it is possible to precipitate collagen in a layer of fibers with a 700 Å period. As collagen is a constituent of many membrane systems in the body, it seemed interesting to investigate the permeability of ions and water through a native collagen membrane.The experiments were carried out with the help of an acryl glass apparatus, where an osmotic pressure, a hydrostatic pressure difference or both can be maintained between the two bulk phases separated by the membrane. The diffusion coefficients for NaCl and KCl were found to be comparable with those in other biological membranes (Ds = 9 · 10−7cm2 · s−1) whereas there is difference of more than three orders of magnitude in the hydraulic permeability (Lp = 6 cm4 · J−1 · s−1).Volume flow measurements caused by an osmotic gradient indicated that the reflection coefficient for NaCl and KCl is very small. In hydrostatic pressure experiments, the membrane shows a preferred direction for volume flows which seems to have something to do with the mode of preparation of the membrane. 相似文献
19.
20.
Mesenchymal stem cells (MSCs) are regarded as a promising source of cell-based therapy for heart injury. In fact, less than 30% of MSCs contribute to cardiomyocytes differentiation, and the isolation procedure and biological characteristics of this population of cells remain unknown. Here we isolate and investigate the biological characteristics of this subpopulation of MSCs. Twenty four MSC clones were randomly selected using single-cell monoclonal technology. After induced with 5-azacytidine, eight clones displayed cardiomyocyte-like morphologies, and highly (over 90%) expressed cardiac-specific markers cTnT and α-actin, and displayed transient outward K+ current (Ito), inwardly rectifying K+ current (IK1) and delayed rectifier K+ current (IKDR), which were typical of cardiomocytes. Other clones merely showed Ito current, and the current densities were different from those of cardiomyocytes. In contrast to the other clones, before induced with 5-azacytidine, the eight clones expressed early cardiac markers GATA4 and NKX2.5, but not cTnT, α-actin, CD44 and CD90, and had no potentials for adiopogenesis, osteogenesis or chondrogenesis after induction. Our data suggest that the subgroup of MSCs that contributes to cardiomyocytes differentiation is cardiac progenitor cells. Moreover, we show the preliminary purification of this population of cells with a high potential for cardiomyocytes differentiation using single-cell monoclonal technology. 相似文献