Identification of a C-terminal poly(A)-binding protein (PABP)-PABP interaction domain: role in cooperative binding to poly (A) and efficient cap distal translational repression

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.     

Interaction of secretory immunoglobulin A antibodies with Naegleria fowleri trophozoites and collagen type I

In this work, we analyzed the in vitro interaction of human secretory immunoglobulin A (sIgA) antibodies with Naegleria fowleri trophozoites and the capacity of these antibodies to inhibit amoeba adherence to collagen type I. We also studied N. fowleri antigens that are recognized by sIgA, using immunoblot assays. Immunocytochemical analysis of the interaction showed a redistribution of antigens on the surface of trophozoites by sIgA antibodies. Ultrastructural analysis of antibody-amoeba interaction showed that besides the patching and cap formation, parasites were capable of eliminating the antigen-antibody complex produced on the surface. sIgA antibodies were capable of inhibiting the in vitro adhesion of trophozoites to collagen type I. We suggest that nonsymptomatic infections by N. fowleri may stimulate a local specific immunity that prevents trophozoite adhesion and invasion of nasal mucosa.     

Dynamics of genetic variation of Sartov cultivars of common wheat Triticum aestivum L. (from the gliadin-coding locus) after a an 80-year period of scientific selection

Based on analysis of gliadin patterns in common wheat cultivars developed at the Research Institute of Agriculture of the Southeast, profile dynamics in gliadin loci has been surveyed for the period of over eight decades. It was shown that long-term breeding of the wheat cultivars involved gradual replacement of alleles characteristic of ancient cultivars for those widely spread in the world, which are probably linked with alleles that currently confer advantage to their carriers. The process of reduction of inter-population genetic diversity in wheat (with special reference to the allele frequency dynamics at gliadin loci) is discussed. This process is responsible for genetic erosion of the species.     

Prognostic significance of nuclear morphometry in superficial bladder cancer

OBJECTIVE: To evaluate the significance of nuclear morphometry in predicting the clinical course in superficial (pTa and pT1) bladder cancer. STUDY DESIGN: The study included 73 patients with superficial transitional cell carcinoma of the bladder who were followed for a median of 21 months (range, 1-90). Nuclear morphometry was performed by a computer-assisted image analyzer system on hematoxylineosin-stained histologic sections and characterized by five nuclear variables: area, perimeter, major and minor diameter, and form factor. Patient charts and microscopic slides were reviewed to record tumor stage, grade and size. Tumor proliferative activity was assessed by immunohistochemical staining with Ki-67 antibody. RESULTS: None of the morphometric variables showed a significant relation to tumor progression and recurrence. Higher values of mean nuclear area, perimeter, and major and minor diameter were significantly related to higher grade and proliferative activity. Mean nuclear area and minor diameter were associated with advanced stage. Of established prognostic factors, only histologic grade was significant in predicting progression. CONCLUSION: The results suggest that nuclear morphometry may be valuable in determining proliferative activity and may be well correlated with histologic grade in superficial bladder cancer. However, like many other potential prognostic factors, it seems to be unreliable in predicting clinical behavior.     

大鼠卵巢不同功能阶段时c-fos表达的动态变化

目的和方法：本文用免疫组织化学方法检测了成年大鼠卵巢于不同的功能阶段和未成年大鼠外源性激素诱导的卵巢于不同的功能阶段c-fos表达的变化以及与血清E2和P含量的相关关系。结果：在成年大鼠动情前期、动情期卵巢的间质腺和基质中及动情间期和妊娠期卵巢的黄体细胞和基质中有c-fos表达,c-fos蛋白阳性信号的表达范围和表达强度在动情前期卵巢较大,动情间期和动情期卵巢较低,妊娠期卵巢最大,这种变化与血清E2和P含量的变化呈明显的正相关关系。未成年大鼠,用DES使卵泡发育至窦前期时,在卵巢中未检测到有c-fos蛋白的表达,用PMSG使贸泡发育至排卵前期时在卵巢的间质腺和基质中有c-fos表达,用PMSG和hCG后4天形成的早期黄体化卵巢c-fos在黄体细胞和基质中的表达明显升高,而于用hCG后9天形成的晚期黄体化卵巢c-fos表达明显下降,这种变化也与血清E2和P含量的变化呈明显的正相关关系。结论：c-fos与卵泡发育、排卵、黄体形成和退化等功能密切相关。     

Activities of antioxidant and redox enzymes in human normal hepatic and hepatoma cell lines

The cellular defense system (including glutathione, glutathione-related enzymes, antioxidant and redox enzymes) plays a crucial role in cell survival and growth in aerobic organisms. To understand its physiological role in tumor cells, the glutathione content and related enzyme activities in the human normal hepatic cell line, Chang and human hepatoma cell line, HepG2, were systematically measured and compared. Superoxide dismutase, catalase, and glutathione peroxidase activities are 2.8-, 4.3-, and 2.9-fold higher in HepG2 cells than in Chang cells. Total glutathione content is also about 1.4-fold higher in HepG2, which is supported by significant increases in gamma-glutamylcysteine synthetase and glutathione synthetase activities. Two other glutathione-related enzymes, glutathione reductase and gamma-glutamyltranspeptidase, are upregulated in HepG2 cells. However, thioredoxin reductase and glutathione S-transferase activities are significantly lower in HepG2 cells. These results propose that defense-related enzymes are largely modulated in tumor cells, which might be linked to their growth and maintenance.     

Pancreatic enzyme activity in early phases of acute experimental pancreatitis in rats

Experiments were performed on laboratory rats with acute pancreatitis caused by local freezing the pancreas with chlorethyl. An active action of enzymes alpha-amilase, lipase, phospholipase A2, was revealed. During the first hours, an increase in action of all three enzymes, particularly that of phospholipase A2, was found. It was established that the lipid spectrum of pancreas had changed. It shows that cell membranes were destroyed. Experiments revealed an activating role of Ca2+ ions for all the enzymes and a correcting action of chlorpromasine.     

Further characterization of a CD99-related 21-kDa transmembrane protein (VAP21) expressed in Syrian hamster cells and its possible involvement in vesicular stomatitis virus production

The VAP21, a CD99-related 21-kDa transmembrane protein, was first detected in the enveloped virions that were grown in a Syrian hamster-derived cell line, BHK-21 (Sagara et al., 1997; Yamamoto et al., 1999). We further tried to elucidate the nature and properties of VAP21. The VAP21 was detected in various organs of the Syrian hamster as well as in the Syrian hamster-derived cell lines (BHK-21 and HmLu-1). We could not detect the VAP21 antigen in other cell lines derived from other animal species we examined, including a Chinese hamster (CHO-K1), mouse (neuroblastoma C1300, clone NA), dog (MDCK), monkey (COS-7), and human (HeLa, HepG2). We tried to introduce the VAP21 gene into VAP21-negative cell lines using a tetracycline-regulated gene expression system. All of our trials, however, resulted in failure to establish stably positive inducible cell lines. To the contrary, we could easily establish the VAP21-overexpressing cell lines from the Syrian hamster cell lines, which were successfully grown and maintained without any loss of VAP21 expression even under the induced culture conditions. In such VAP21-overexpressing cells, production of the vesicular stomatitis virus (VSV) was increased several-fold, while suppression of the VAP21 expression resulted in reducing the VSV yields. From these results, we conclude that the VAP21 is a physiologically active cell membrane component of some animal species including the Syrian hamster, and might positively be involved in the VSV replication.     

The site-directed mutagenesis of gastrodia anti-fungal protein mannose-binding sites and its expression in Escherichia coli

Gastrodia anti-fungal protein (GAFP) displays strong inhibitory activity against certain fungal pathogens. Five GAFP analogues with different mutations at mannose-binding sites and the wild-type one were expressed and purified in Escherichia coli. The inhibitory analysis of the purified various GAFPs against the growth of Trichoderma viride indicates that single amino acid mutated-type GAFPs have inhibitory activity, but its activity is much less than the wild-type one. The double and triplicate amino acids mutated GAFPs have very low inhibitory activity. For the first time it was proved that GAFP mannose-binding sites play key role in anti-fungi process.