Ubiquitin-proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation

Degradation of proteins mediated by ubiquitin-proteasome pathway (UPP) plays important roles in the regulation of eukaryotic cell cycle. In this study, the functional roles and regulatory mechanisms of UPP in mouse oocyte meiotic maturation, fertilization, and early embryonic cleavage were studied by drug-treatment, Western blot, antibody microinjection, and confocal microscopy. The meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated by two potent, reversible, and cell-permeable proteasome inhibitors, ALLN and MG-132. The metaphase I spindle assembly was prevented, and the distribution of ubiquitin, cyclin B1, and polo-like kinase 1 (Plk1) was also distorted. When UPP was inhibited, mitogen-activated protein kinase (MAPK)/p90rsk phosphorylation was not affected, but the cyclin B1 degradation that occurs during normal metaphase-anaphase transition was not observed. During oocyte activation, the emission of second polar body (PB2) and the pronuclear formation were inhibited by ALLN or MG-132. In oocytes microinjected with ubiquitin antibodies, PB2 emission and pronuclear formation were also inhibited after in vitro fertilization. The expression of cyclin B1 and the phosphorylation of MAPK/p90rsk could still be detected in ALLN or MG-132-treated oocytes even at 8 h after parthenogenetic activation or insemination, which may account for the inhibition of PB2 emission and pronuclear formation. We also for the first time investigated the subcellular localization of ubiquitin protein at different stages of oocyte and early embryo development. Ubiquitin protein was accumulated in the germinal vesicle (GV), the region between the separating homologous chromosomes, the midbody, the pronuclei, and the region between the separating sister chromatids. In conclusion, our results suggest that the UPP plays important roles in oocyte meiosis resumption, spindle assembly, polar body emission, and pronuclear formation, probably by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.     

Species-crossreactive scFv against the tumor stroma marker "fibroblast activation protein" selected by phage display from an immunized FAP-/- knock-out mouse.

BACKGROUND: Fibroblast activation protein (FAP) is a type II membrane protein expressed on tumor stroma fibroblasts in more than 90% of all carcinomas. FAP serves as a diagnostic marker and is potential therapeutic target for treatment of a wide variety of FAP+ carcinomas. Murine tumor stroma models and FAP-specific antibodies are required to investigate the functional role of FAP in tumor biology and its usefulness for drug targeting. We here describe the development of antibodies with crossreactivity for human (hFAP) and murine FAP (mFAP), which share 89% amino acid identity. MATERIAL AND METHODS: An FAP-/- mouse was sequentially immunized with recombinant murine and human FAP-CD8 fusion proteins. Immunoglobulin cDNA derived from hyperimmune spleen cells was used for the construction of a combinatorial single chain Fv (scFv) library. Phage display selection of FAP-specific scFv was performed on immobilized hFAP followed by selection on cells expressing murine FAP. RESULTS: High-affinity, species-crossreactive, FAP-specific scFv were isolated upon sequential phage display selection. A bivalent derivative (minibody M036) constructed thereof was applied for immunohistochemical analyses and allowed detection of FAP expression on stroma cells of different human carcinomas as well as on murine host stroma in a tumor xenograft model. CONCLUSIONS: MB M036, derived from phage display selected species crossreactive scFv, is suitable for tumor stroma targeting and will be a valuable tool in the analyses of the functional role of FAP in tumor biology as well as in the evaluation of the suitability of FAP for drug targeting.     

Eicosanoids in insect immunity: bacterial infection stimulates hemocytic phospholipase A2 activity in tobacco hornworms

Intracellular phospholipase A(2) (PLA(2)) is responsible for releasing arachidonic acid from cellular phospholipids, and is thought to be the first step in eicosanoid biosynthesis. Intracellular PLA(2)s have been characterized in fat body and hemocytes from tobacco hornworms, Manduca sexta. Here we show that bacterial challenge stimulated increased PLA(2) activity in isolated hemocyte preparations, relative to control hemocyte preparations that were challenged with water. The increased activity was detected as early as 15 s post-challenge and lasted for at least 1 h. The increased activity depended on a minimum bacterial challenge dose, and was inhibited in reactions conducted in the presence of oleyoxyethylphosphorylcholine, a site-specific PLA(2) inhibitor. In independent experiments with serum prepared from whole hemolymph, we found no PLA(2) activity was secreted into serum during the first 24 h following bacterial infection. We infer that a hemocytic intracellular PLA(2) activity is increased immediately an infection is detected. The significance of this enzyme lies in its role in launching the biosynthesis of eicosanoids, which mediate cellular immune reactions to bacterial infection.     

<Emphasis Type="Italic">Narcissus tazetta</Emphasis> lectin shows strong inhibitory effects against respiratory syncytial virus,influenza A (H1N1, H3N2, H5N1) and B viruses

A mannose-binding lectin (Narcissus tazetta lectin [NTL]) with potent antiviral activity was isolated and purified from the bulbs of the Chinese daffodil Narcissus tazetta var. chinensis, using ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, affinity chromatography on mannose-agarose and fast protein liquid chromatography (FPLC)-gel filtration on Superose 12. The purified lectin was shown to have an apparent molecular mass of 26 kDa by gel filtration and 13 kDa by SDS-PAGE, indicating that it is probably a dimer with two identical subunits. The cDNA-derived amino acid sequence of NTL as determined by molecular cloning also reveals that NTL protein contains a mature polypeptide consisting of 105 amino acids and a C-terminal peptide extension. Three-dimensional modelling study demonstrated that the NTL primary polypeptide contains three subdomains, each with a conserved mannose-binding site. It shows a high homology of about 60%–80% similarity with the existing monocot mannose-binding lectins. NTL could significantly inhibit plaque formation by the human respiratory syncytial virus (RSV) with an IC₅₀ of 2.30 μg/ml and exhibit strong antiviral properties against influenza A (H1N1, H3N2, H5N1) and influenza B viruses with IC₅₀ values ranging from 0.20 μg/ml to 1.33 μg/ml in a dose-dependent manner. It is worth noting that the modes of antiviral action of NTL against RSV and influenza A virus are significantly different. NTL is effective in the inhibition of RSV during the whole viral infection cycle, but the antiviral activity of NTL is mainly expressed at the early stage of the viral cycle of influenza A (H1N1) virus. NTL with a high selective index (SI=CC₅₀/IC₅₀≥141) resulting from its potent antiviral activity and low cytotoxicity demonstrates a potential for biotechnological development as an antiviral agent.     

Fate of intraluminal glutamine in the perfused renal tubule

To determine the fate of intraluminal glutamine and specifically the role of brush border gamma glutamyltransferase in its hydrolysis and reabsorption, proximal convoluted tubules of rabbits were isolated and perfused with an artificial ultrafiltrate containing 1 mM 14C-glutamine and 3H-PEG as a volume absorption marker. The tubules, average length 0.80 +/- 0.09 mm, were bathed in perfusate containing albumin, 6.5 percent but no glutamine. Aliquots of collectate and bathing media were monitored for total 14C counts while the distribution of radioactive 14C between glutamine and glutamate in the collectate was determined by separation on a Dowex X8 formate form ion-exchange column. After 3 ten minute control periods the perfusate was switched to one containing 1 mM AT-125 in addition to glutamine and after equilibration an additional 3 collections were obtained. Control period glutamine load averaged 16.1 +/- 2.4 pmole/min of which 35 percent was absorbed and 38 and 27 percent excreted as glutamine and glutamate respectively; of the absorbed glutamine 25 percent was metabolized. During AT-125 administration, glutamine delivery averaged 15.0 +/- 2.1 pmole/min of which 57 percent was absorbed; increased absorption occurred at the expence of intraluminal glutamate formation which fell to less than 10 percent. Thus luminal transport and gamma glutamyltransferase mediated hydrolysis appear to compete for available glutamine. Significantly, reducing intraluminal glutamine hydrolysis doubles the cellular metabolism of absorbed glutamine suggesting that extracellular conversion of glutamine to glutamate alters the metabolic fate of filtered glutamine.     

Epitope-specific regulation in Ir gene systems. I. Conditions for the induction of epitope-specific suppressors to poly(Glu60Ala30Tyr10)

Injection of responder mice with poly(Glu60Ala30Tyr10) (GAT) followed by immunization with GAT-methylated bovine serum albumin (GATMBSA) selectively suppresses anti-MBSA plaque-forming cell (PFC) and delayed hypersensitivity (DTH) reactions. Conversely, MBSA injection followed by GATMBSA immunization suppresses anti-GAT PFC and DTH, while anti-MBSA responses remain intact. Suppression occurs for doses of antigen which are optimally immunogenic. The suppression is specific and does not act in a bystander fashion. These results demonstrate that epitope-specific regulation is reciprocal, is not limited to humoral responses, and is not limited to molecules of low molecular weight.     

盐胁迫下大豆根组织定量PCR分析中内参基因的选择

实时荧光定量PCR已广泛用于基因表达的分析, 适当的内参基因选择是获得准确分析结果的关键。在大豆(Glycine max)分子生物学研究中, 逆境响应基因和microRNA (miRNA)表达的内参辅助检测基因均有哪些目前尚不清楚。该研究选用不同盐梯度和时间点组合处理的大豆根组织为材料, 对已报道的其它条件下表达相对稳定的内参基因(ACT、ACT2/7、CYP2、ELF1A、ELF1B、F-Box、TUA和UBC2)以及miRNA内参基因(U6、miR1515a、miR1520c、miR1520d、miR171a和miR171b)的表达情况进行了全面检测; 并采用Δ-Ct、Bestkeeper、NormFinder和Genorm四种方法对检测结果进行了综合分析, 发现ELF1B和CYP2适合作为大豆根系盐胁迫响应基因研究的内参基因, miR1515a和U6适合作为盐胁迫下大豆根组织miRNA研究的内参。上述研究结果为大豆盐胁迫响应基因和miRNA表达及其进一步的功能研究奠定了基础。