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31.
Swf1p, a member of the DHHC-CRD family of palmitoyltransferases, regulates the actin cytoskeleton and polarized secretion independently of its DHHC motif 下载免费PDF全文
Rho and Rab family GTPases play a key role in cytoskeletal organization and vesicular trafficking, but the exact mechanisms by which these GTPases regulate polarized cell growth are incompletely understood. A previous screen for genes that interact with CDC42, which encodes a Rho GTPase, found SWF1/PSL10. Here, we show Swf1p, a member of the DHHC-CRD family of palmitoyltransferases, localizes to actin cables and cortical actin patches in Saccharomyces cerevisiae. Deletion of SWF1 results in misorganization of the actin cytoskeleton and decreased stability of actin filaments in vivo. Cdc42p localization depends upon Swf1p primarily after bud emergence. Importantly, we revealed that the actin regulating activity of Swf1p is independent of its DHHC motif. A swf1 mutant, in which alanine substituted for the cysteine required for the palmitoylation activity of DHHC-CRD proteins, displayed wild-type actin organization and Cdc42p localization. Bgl2p-marked exocytosis was found wild type in this mutant, although invertase secretion was impaired. These data indicate Swf1p has at least two distinct functions, one of which regulates actin organization and Bgl2p-marked secretion. This report is the first to link the function of a DHHC-CRD protein to Cdc42p and the regulation of the actin cytoskeleton. 相似文献
32.
Blanchard V Gadkari RA George AV Roy S Gerwig GJ Leeflang BR Dighe RR Boelens R Kamerling JP 《Glycoconjugate journal》2008,25(3):245-257
The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating
hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using
NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for
an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the
site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed
in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type
N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor
assays, and in vitro bioassays.
Véronique Blanchard and Rupali A. Gadkari contributed equally. 相似文献
33.
Mourya DT Rohankhedkar MS Yadav P Dighe V Deobagkar DN 《Indian journal of experimental biology》2003,41(1):91-93
Mosquitoes were infected by intrathoracic inoculation. About 95% head squashes were positive for dengue virus antigen on the 15th post infection day (PID). Esterase activity was determined in the homogenates prepared from the salivary glands and midguts on different PIDs of dengue virus inoculated and control mosquitoes showed that it was consistently higher in the virus-infected batches. 相似文献
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Jagatpala Shetty Gopal K Marathe Suresh Ramaswamy Rajan R Dighe 《Journal of biosciences》1996,21(1):81-92
The relative regulatory roles of the pituitary gonadotropins, luteinizing hormone and follicle stimulating hormone in the
spermatogonial proliferation has been studied using specific antibodies against these hormones in the immature rats. Immunoneutralization
of lu teinizing hormone for 7 days resulted in significant reduction in tetraploid cells and total absence of haploid cells,while there was a relative increase in the diploid population. This was also accomopanied by a decrease in spermatogonial
proliferation as indicated by a decrease in [3H] thymidine incorpor-ation into DNA by purified spermatogonia. Administration of follicle stimulating hormone a/s for 7 days
also caused a significant decrease in the rate of spermatogonial proliferation. Withdrawal of follicle stimulating hormone
led to a significant reduction in tetraploid and haploid cells. However interestingly,it failed to totally abolish the appearance of these cells. Administration of testosterone (3 mg/day/rat) for 2 days along
with the gonadotropin a/s could partially reverse the effect on spermatogonial proliferation. It is concluded that (i) both luteinizing hormone and follicle stimulating hormone are involved in spermatogonial proliferation, (ii) lack of
testosterone consequent of the neutralization of luteinizing hormone prevented the entry of spermatogonial cells into meiosis,
(iii) testosterone may be involved in spermatogonia] proliferation providing a mitotic signal and (v) both follicle stimulating
hormone and testosterone act synergistically and lack of any one of the hormones results in impairment of spermatogonial proliferation.
A part of the data was presented at the 16th International Congress of Biochemistry and Molecular Biology, New Delhi, September
1994. 相似文献
36.
Deficient major histocompatibility complex-linked innate murine cytomegalovirus immunity in MA/My.L-H2b mice and viral downregulation of H-2k class I proteins 下载免费PDF全文
NK cells are key effectors of innate immunity and host survival during cytomegalovirus (CMV) infection. Innate murine CMV (MCMV) resistance in MA/My mice requires Ly49H/m157-independent H-2k-linked NK cell control. Here we show that replacement of MA/My H-2k with C57L H-2b susceptibility genes led to a remarkable loss of innate virus immunity, though NK gamma interferon was induced in H-2b and H-2k strains shortly after infection. Thus, H-2b genes expressed in C57L or MA/My.L-H2b are sufficient in alerting NK cells to intrusion but fail to support NK restraint of viral infection. In addition, novel H-2 recombinant strains were produced and utilized in a further refinement of a critical genetic interval controlling innate H-2k-linked MCMV resistance. Importantly, this analysis excluded the gene interval from Kk class I through class II. The responsible gene(s) therefore resides in an interval spanning Dk class Ia and more-distal major histocompatibility complex (MHC) nonclassical class Ib genes. Recently, the NK activation receptor Ly49P and MHC class I Dk proteins were genetically implicated in MCMV resistance, in part because Ly49P-expressing reporter T cells could specifically bind Dk molecules on MCMV-infected mouse embryonic fibroblasts (MEFs). However, as we found that H-2k innate resistance differs in the C57L or MA/My backgrounds and because MCMV very efficiently downregulates H-2k class I proteins in L929 cells and primary MEFs shortly after infection, a Ly49P/Dk model should not fully explain H-2k-linked MCMV resistance. 相似文献
37.
Jieun Jeong Kun-Hee Kim Dong-Young Kim Gopalakrishnan Chandrasekaran Minhee Kim Suvarna H. Pagire Mahesh Dighe Eun Young Choi Su-Min Bak Eun-Young Kim Myung-Geun Shin Seok-Yong Choi Jin Hee Ahn 《Bioorganic & medicinal chemistry》2019,27(19):115014
A new series of 1,3-diketone, heterocyclic and α,β-unsaturated derivatives were synthesized and evaluated for their AhR antagonist activity using zebrafish and mammalian cells. Compounds 1b, 2c, 3b and 5b showed significant AhR antagonist activity in a transgenic zebrafish model. Among them, compound 3b, and 5b were found to have excellent AhR antagonist activity with IC50 of 3.36 nM and 8.3 nM in a luciferase reporter gene assay. In stem cell proliferation assay, compound 5b elicited marked HSC expansion. 相似文献
38.
The strategy of translationally fusing the subunits of heterodimeric proteins into single chain molecules is often used to
overcome the mutagenesis-induced defects in subunit interactions. The approach of fusing the α and β subunits of human Chorionic
Gonadotropin (hCG) to produce a single chain hormone (phCGαβ) was used to investigate roles of critical residues of the α
subunit in hormone receptor interaction and biological activity. The α subunit was mutated using PCR-based site-directed mutagenesis,
fused to the wild type β subunit and the fusion protein was expressed using Pichia pastoris expression system. Following partial purification, the mutant proteins were extensively characterized using immunological
probes, receptor assays, and in vitro bioassays. The mutation hCGα P38A, which disrupts subunit interaction in the heterodimeric molecule, produced a fusion molecule
exhibiting altered subunit interactions as judged by the immunological criteria, but could bind to the receptor with lower
affinity and elicit biological response. Mutation of hCGα T54A disrupting the glycosylation at Asparagine 52, believed to
be important for bioactivity, also yielded a biologically active molecule suggesting that the glycosylation at this site is
not as critical for bioactivity as it is in the case of the heterodimer. The fusion protein approach was also used to generate
a superagonist of hormone action. Introduction of four lysine residues in the Loop 1 of the α subunit led to the generation
of a mutant having higher affinity for the receptor and enhanced bioactivity. Immunological characterization of single chain
molecules revealed that the interactions between the subunits were not identical to those seen in the heterodimeric hormone,
and the subunits appeared to retain their isolated conformations, and also retained the ability to bind to the receptors and
elicit response. These data suggest the plasticity of the hormone-receptor interactions. 相似文献
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Wilfredo F. Garcia-Beltran Evan C. Lam Michael G. Astudillo Diane Yang Tyler E. Miller Jared Feldman Blake M. Hauser Timothy M. Caradonna Kiera L. Clayton Adam D. Nitido Mandakolathur R. Murali Galit Alter Richelle C. Charles Anand Dighe John A. Branda Jochen K. Lennerz Daniel Lingwood Aaron G. Schmidt Alejandro B. Balazs 《Cell》2021,184(2):476-488.e11