螯合细胞外钙离子对体外培养的大鼠卵巢颗粒细胞成熟的影响

在发现利用钙离子螯合剂EGTA螯合细胞外钙离子(Ca 2 )后,可以显著抑制促滤泡(激)素(FSH)刺激体外培养的颗粒细胞合成和分泌雌激素,并且该抑制作用呈剂量依赖性.假设该特异性反应是通过Ca2 影响细胞内腺苷酸环化酶(AC)发挥作用的,因为Ca 2 具有激活ACVIII的作用.通过RT-PCR和Northern印迹检测大鼠不同阶段卵巢组织中ACVIII的表达.结果表明,虽然Ca2 可以调控颗粒细胞类固醇激素的合成,但不同阶段的卵巢组织中均检测不到ACVIII的mRNA.实验间接提示了Ca2 促进颗粒细胞成熟的作用不是通过ACVIII发挥作用的,而可能是通过其他AC同工酶或其他Ca2 信号通路发挥作用.     

Post-translational modifications of adiponectin: mechanisms and functional implications

Adiponectin is an insulin-sensitizing adipokine with anti-diabetic, anti-atherogenic, anti-inflammatory and cardioprotective properties. This adipokine is secreted from adipocytes into the circulation as three oligomeric isoforms, including trimeric, hexameric and the HMW (high-molecular-mass) oligomeric complex consisting of at least 18 protomers. Each oligomeric isoform of adiponectin exerts distinct biological properties in its various target tissues. The HMW oligomer is the major active form mediating the insulin-sensitizing effects of adiponectin, whereas the central actions of this adipokine are attributed primarily to the hexameric and trimeric oligomers. In patients with Type 2 diabetes and coronary heart disease, circulating levels of HMW adiponectin are selectively decreased due to an impaired secretion of this oligomer from adipocytes. The biosynthesis of the adiponectin oligomers is a complex process involving extensive post-translational modifications. Hydroxylation and glycosylation of several conserved lysine residues in the collagenous domain of adiponectin are necessary for the intracellular assembly and stabilization of its high-order oligomeric structures. Secretion of the adiponectin oligomers is tightly controlled by a pair of molecular chaperones in the ER (endoplasmic reticulum), including ERp44 (ER protein of 44 kDa) and Ero1-Lalpha (ER oxidoreductase 1-Lalpha). ERp44 inhibits the secretion of adiponectin oligomers through a thiol-mediated retention. In contrast, Ero1-Lalpha releases HMW adiponectin trapped by ERp44. The PPARgamma (peroxisome-proliferator-activated receptor gamma) agonists thiazolidinediones selectively enhance the secretion of HMW adiponectin through up-regulation of Ero1-Lalpha. In the present review, we discuss the recent advances in our understanding of the structural and biological properties of the adiponectin oligomeric isoforms and highlight the role of post-translational modifications in regulating the biosynthesis of HMW adiponectin.     

Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitro

Members of the RNase III family are the primary cellular agents of dsRNA (double-stranded RNA) processing. Bacterial RNases III function as homodimers and contain two dsRBDs (dsRNA-binding domains) and two catalytic sites. The potential for functional cross-talk between the catalytic sites and the requirement for both dsRBDs for processing activity are not known. It is shown that an Escherichia coli RNase III heterodimer that contains a single functional wt (wild-type) catalytic site and an inactive catalytic site (RNase III[E117A/wt]) cleaves a substrate with a single scissile bond with a k(cat) value that is one-half that of wt RNase III, but exhibits an unaltered K(m). Moreover, RNase III[E117A/wt] cleavage of a substrate containing two scissile bonds generates singly cleaved intermediates that are only slowly cleaved at the remaining phosphodiester linkage, and in a manner that is sensitive to excess unlabelled substrate. These results demonstrate the equal probability, during a single binding event, of placement of a scissile bond in a functional or nonfunctional catalytic site of the heterodimer and reveal a requirement for substrate dissociation and rebinding for cleavage of both phosphodiester linkages by the mutant heterodimer. The rate of phosphodiester hydrolysis by RNase III[E117A/wt] has the same dependence on Mg(2+) ion concentration as that of the wt enzyme, and exhibits a Hill coefficient (h) of 2.0+/-0.1, indicating that the metal ion dependence essentially reflects a single catalytic site that employs a two-Mg(2+)-ion mechanism. Whereas an E. coli RNase III mutant that lacks both dsRBDs is inactive, a heterodimer that contains a single dsRBD exhibits significant catalytic activity. These findings support a reaction pathway involving the largely independent action of the dsRBDs and the catalytic sites in substrate recognition and cleavage respectively.     

Evaluation of ketone-oxime method for developing therapeutic on-demand cleavable immunoconjugates

The use of antibody molecules in immunoassay, molecular targeting, or detection techniques encompasses a broad variety of applications affecting nearly every field of medical science. In cancer therapy, monoclonal antibodies (mAb) have been used as vehicles to deliver radionuclides, toxins, or drugs to the target cancer cells. New conjugation methods are most needed to conjugate a wide variety of targeting small molecules and peptidomimatic compounds. Here, we exploited a keto-oxime method for conjugation of protease susceptible linkers to an antibody. This modified method involves two steps: (i) introduction of methyl ketone linkers (referred to as linker moiety) to the primary amines present in the antibody and (ii) conjugation of ketone linkers to aminoxy functional group present in the conjugated moiety (referred to as functional moiety). We have optimized this conjugation method and shown that approximately 10 functional moieties can be conjugated to antibody. Conjugation was verified by MALDI-TOF MS and Western blot analysis. The acidic pH conditions used in this method did not change the immune reactivity of the Ab. In addition, in vitro protease susceptibility assay was performed to validate this method for prodrug release assay as well as to remove excess radioimmune conjugates from circulation. This orthogonal method is compatible with peptides containing a thiol, amino, or carboxyl groups in the conjugation moiety.     

Effect of a traditional Chinese medicine preparation Xindi soft capsule on rat model of acute blood stasis: a urinary metabonomics study based on liquid chromatography-mass spectrometry

Xindi soft capsule is a traditional Chinese medicine preparation which consists of sea buckthorn flavonoids and sea buckthorn berry oil. In this study, a urinary metabonomics method based on the ultra-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS) was used to evaluate the efficacy and study the mechanism of traditional Chinese medicine preparation to blood stasis. With pattern recognition analysis (principal component analysis and partial least squares-discriminate analysis) of urinary metabolites, a clear separation of acute blood stasis model group and healthy control group was achieved, the dose groups were located between acute blood stasis model group and healthy control group showing a tendency of recovering to healthy control group, high dose and middle dose were more effective than low dose. Some significantly changed metabolites like cholic acid, phenylalanine and kynurenic acid have been found and identified and used to explain the mechanism. The work shows that the metabonomics method is a valuable tool in the research mechanism of traditional Chinese medicine.     

Isolation and characterization of microsatellite markers in black muntjac (Muntiacus crinifrons)

The black muntjac (Muntiacus crinifrons) is a vulnerable species found in the mountains of eastern China, about which little is known. Here we develop 11 polymorphic microsatellite loci from a (CA)(n) -enrichment library for the animal. In the analyses of 25 individuals sampled, unbiased expected heterozygosity levels varied from 0.686 to 0.876 and the number of alleles per locus varied from five to nine. Results that 11 microsatellite loci were highly polymorphic indicated that these markers are sufficiently powerful to address such questions as parentage, mating system and population genetic structure of M. crinifrons.     

Genomic instability is associated with natural life span variation in Saccharomyces cerevisiae

Increasing genomic instability is associated with aging in eukaryotes, but the connection between genomic instability and natural variation in life span is unknown. We have quantified chronological life span and loss-of-heterozygosity (LOH) in 11 natural isolates of Saccharomyces cerevisiae. We show that genomic instability increases and mitotic asymmetry breaks down during chronological aging. The age-dependent increase of genomic instability generally lags behind the drop of viability and this delay accounts for approximately 50% of the observed natural variation of replicative life span in these yeast isolates. We conclude that the abilities of yeast strains to tolerate genomic instability co-vary with their replicative life spans. To the best of our knowledge, this is the first quantitative evidence that demonstrates a link between genomic instability and natural variation in life span.