Fusion and Matrix Protein Gene Sequence Analysis of Paramyxoviruses of Type 1(PMV-1) Isolated from Pigeons in Slovenia

Paramyxoviruses of type 1 (PMV-l) isolated from pigeons were genetically analyzed. A part of the fusion and the matrix protein genes were amplified and sequenced, Typical amino acid sequences associated with virulence were determined at the fusion protein cleavage site in all PMV-1 isolates. All Slovene pigeon PMV-1 strains share high amino acid sequence similarity with other pigeon strains. In the phylogenetic tree, they are clustered together with pigeon PMV-1 isolates with moderate pathogenicity. Phylogenetic analysis obtained from the fusion and the matrix protein gene alignments showed the same branching order. Viruses circulating among pigeons were found to form quite unique lineage of virulent NDV strains.     

人乳腺癌MDR1基因稳定沉默细胞的筛选及其生物学特性

目的筛选MDR1沉默的人乳腺癌细胞并比较其生物学特性。方法采用BLOCK-iT Lentiviral RNAi Expres-sion System生产表达MDR1基因shRNA的慢病毒载体,转导敏感人乳腺癌细胞MCF-7和耐阿霉素人乳腺癌细胞MCF-7/ADM,杀稻瘟菌素(blasticidin)筛选获得MDR1稳定沉默细胞MCF-7/RNAi和MCF-7/ADM/RNAi。定量RT-PCR检测MDR1mRNA表达,流式细胞术检测P-GP蛋白表达和功能,MTT法检测药物敏感性。结果经10 mg/L blasticidin筛选12 d获得MCF-7/RNAi和MCF-7/ADM/RNAi细胞。MCF-7、MCF-7/RNAi、MCF-7/ADM和MCF-7/ADM/RNAi细胞的MDR1mRNA相对表达水平分别为1、0.13、17.14和2.01;P-GP表达分别为(1.5±0.3)%、(1.2±0.2)%、(89.4±3.6)%和(16.3±1.9)%;细胞内Rh123的潴留分别为(92.4±3.1)%、(90.6±4.0)%、(13.6±1.6)%、(72.4±2.8)%;IC50值分别为0.90、0.92、19.61和4.04 mg/L。结论应用慢病毒载体稳定沉默MDR1基因可有效逆转人乳腺癌细胞耐药性。     

Distribution of thietazole in organs and tissues of rats after single and repeated administration

We studied the kinetics of thietazole distribution in the liver, brain, kidneys, spleen, heart, skeletal muscles, lungs, adipose tissue, and testicles after single and repeated administration of this drug. Single and repeated administration of thietazole was followed by elimination of this drug from the blood into organs and tissues. After repeated administration, thietazole was selectively accumulated in the spleen. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 555–557, May, 2008     

Eleven single nucleotide polymorphisms and one triple nucleotide insertion of the human TGF-β III receptor gene

We found 11 single nucleotide polymorphisms and one triple nucleotide insertion in the cDNA of the human transforming growth factor β (TGF-β) III receptor gene (TGFBR3) located on 1p33–p32, encoding betaglycan, a component of the TGF-β receptor system. Inside the 5′ untranslated region (UTR), a G→A polymorphism was identified at position 311. In the open reading frame (ORF), a non-conservative T→C polymorphism was identified at position 392, and three conservative polymorphisms were found at positions 563 (G→A), 1548 (G→A), and 2370 (C→T). A triple nucleotide insertion (GCA) was identified at position 1419. Inside the 3′ UTR, six polymorphisms were identified: four G→A, at positions 2918, 3055, 3098, and 3355; one T→A, at position 3183; and one G→C, at position 3966. In addition to these changes, some divergences from the published sequence were observed in all 12 chromosomes tested. These included, in the ORF, an additional C after position 555, two additional G after position 563, and an additional T after position 1388. No T was found at position 1394. The alterations translate to a changed amino acid sequence. Inside the 3′ UTR, additional discrepancies were identified. The discovered changes and polymorphisms may be useful for further genetic studies of TGFBR3 receptor deficiencies. Received: December 22, 1999 / Accepted: February 25, 2000     

Short-term enzyme replacement in the murine model of Sanfilippo syndrome type B

The Sanfilippo syndrome type B (MPS III B) is an autosomal recessive disease caused by deficiency of alpha-N-acetylglucosaminidase (EC 3. 2.1.50), one of the lysosomal enzymes required for the degradation of heparan sulfate. The disease is characterized by profound neurodegeneration but relatively mild somatic manifestations, and is usually fatal in the second decade. A mouse model had been generated by disruption of the Naglu gene in order to facilitate the study of pathogenesis and the development of therapy for this currently untreatable disease. Recombinant human alpha-N-acetylglucosaminidase (rhNAGLU) was prepared from secretions of Lec1 mutant Chinese hamster ovary cells. The enzyme, which has only unphosphorylated high-mannose carbohydrate chains, was endocytosed by mouse peritoneal macrophages via mannose receptors, with half-maximal uptake at ca. 10(-7) M. When administered intravenously to 3 month-old mice, rhNAGLU was taken up avidly by liver and spleen but marginally if at all by thymus, lung, kidney, heart, and brain (in order of diminishing uptake). The half-life of the enzyme was 2.5 days in liver and spleen. Immunohistochemistry and electron microscopy showed that only macrophages were involved in enzyme uptake and correction in these two organs, yet the storage of glycosaminoglycan was reduced to almost normal levels. The results show that the macrophage-targeted rhNAGLU can substantially reduce the body burden of glycosaminoglycan storage in the mouse model of Sanfilippo syndrome III B.     

Clinical utility of an enhanced human immunodeficiency virus type 1 p24 antigen capture assay

The presence of p24 core antigen in the serum of individuals with human acquired immunodeficiency syndrome has been used as one of the important prognostic markers of HIV-1 infection and also as an end point in evaluating antiviral drugs and vaccines. Unfortunately the majority of p24 antigen present in serum exists as an antigenantibody complex and is not detected with the commercial kits currently available to measure p24 antigen. In this study, we report a simple procedure utilizing treatment of serum samples with glycine buffer (pH 1.85) to dissociate antigen-antibody complexes prior to assaying for p24 antigen. A 300% increase in the number of p24-reactive samples and a 3- to 12-fold increase in the quantity of antigen detected were observed when samples were pretreated with 1.5M glycine buffer (pH 1.85) for 1 hr. Glycine treatment of samples did not result in nonspecific positive tests and samples previously shown to be reactive remained positive. In reconstruction experiments the release of antigen was found to be inversely proportional to the amount of p24 antibody present in the serum. The percentage of HIV-1-infected patients positive for p24 antigen was clearly a function of CD4 count. Forty-nine percent of patients with more than 500 CD4 cells and 100% of patients with less than 200 CD4 were p24 positive. The improved sensitivity for detection of p24 provided by this procedure enhances our understanding of the pathogenesis of AIDS by showing that the majority of patients with HIV-1 infection is p24 positive and facilitates the analysis of data obtained in clinical trials involving anti-HIV compounds.     

Solid phase C1q microassay for the rapid detection of circulating immune complexes

A C1q solid phase microassay was designed for the rapid detection of circulating immune complexes. Its level of sensitivity is comparable to that of the Raji cell and greater than the C1q binding assay; furthermore, it is faster and low in cost. These conditions make it more practical and applicable in the clinical setting.     

Airway responses to antigen challenge in allergic rhinitis and allergic asthma

The density dependence of the maximum expiratory flow-volume curve, functional residual capacity (FRC), and specific airway conductance (SGaw) were determined before and during bronchial provocation with ragweed extract in 27 subjects with ragweed hypersensitivity and a history of either bronchial asthma (16 subjects) or allergic rhinitis (11 subjects). Mean baseline SGaw was significantly lower while mean volume of isoflow (Visov) and FrC were significantly higher in subjects with bronchial asthma. During antigen challenge, 10 of 16 subjects with bronchial asthma (63%) and five of 11 subjects with allergic rhinitis (45%) showed a greater than 35% decrease in SGaw ("reactors"): mean relative decreases in SGaw from baseline were 46% and 53%, respectively. The remaining subjects showed a less than 35% decrease in SGaw ("nonreactors") with mean relative decreases of 9% (allergic asthma) and 6% (allergic rhinitis). Mean Visov increased in all subjects with bronchial asthma and in eight of 11 subjects with allergic rhinitis. A significant increase in FRC (6%) was seen only in the "reactors" with bronchial asthma. Following antigen challenge, the beta adrenergic agonist, isoetharine, increased SGaw and decreased Visov. We conclude that in asymptomatic subjects with ragweed hypersensitivity, (1) central and peripheral airway function is more abnormal in subjects with bronchial asthma than in subjects with allergic rhinitis, (2) subjects of both groups show quantitatively and qualitatively comparable airway responses during antigen challenge with a decrease in SGaw or an increase in Visov, possibly representing increase in central and/or peripheral airflow resistance, respectively, (3) Visov may be a more sensitive indicator of airway response to antigen challenge than SGaw, and (4) the bronchodilator effects of a beta adrenergic agonist on antigen-induced bronchospasm are similar in both groups.     

Are transforming properties of the bovine papillomavirus E5 protein shared by E5 from high-risk human papillomavirus type 16?

The E5 proteins of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 16 (HPV-16) are small (44-83 amino acids), hydrophobic polypeptides that localize to membranes of the Golgi apparatus and endoplasmic reticulum, respectively. While the oncogenic properties of BPV-1 E5 have been characterized in detail, less is known about HPV-16 E5 due to its low expression in mammalian cells. Using codon-optimized HPV-16 E5 DNA, we have generated stable fibroblast cell lines that express equivalent levels of epitope-tagged BPV-1 and HPV-16 E5 proteins. In contrast to BPV-1 E5, HPV-16 E5 does not activate growth factor receptors, phosphoinositide 3-kinase or c-Src, and fails to induce focus formation, although it does promote anchorage-independent growth in soft agar. These variant activities are apparently unrelated to differences in intracellular localization of the E5 proteins since retargeting HPV-16 E5 to the Golgi apparatus does not induce focus formation.     

Overexpression of Ets-1 proto-oncogene in latent and clinical prostatic carcinomas

AIMS: The high incidence of clinically diagnosed prostatic cancer is exceeded by the frequency of tumours detected at autopsy. The Ets-1 proto-oncogene is expressed by a variety of malignant and normal tissues. Therefore, in this study, expression of Ets-1 protein was investigated in 'latent' prostatic cancer detected at autopsy, compared with benign prostatic hyperplasia, normal prostatic tissues and clinical prostatic cancer. METHODS AND RESULTS: Using immunohistochemistry, we analysed Ets-1 expression in 95 prostatic specimens including 19 cases of latent prostatic carcinoma (LPC) and 55 cases of clinical prostatic carcinoma (CPC), 11 cases of benign prostatic hyperplasia (BPH) and 10 cases of normal prostate (NP). Differences in the incidence of LPC and CPC suggest different courses for the biological progression of prostatic cancer. There was a significant difference in the degree of Ets-1 expression in CPC and LPC (P < 0.05). Ets-1 was not expressed in BPH and NP, but in malignant cases (57 of 74; 77.0%) commonly demonstrated immunoreactivity in the tumour cells. In our study the expression of Ets-1 between benign and malignant, and well, moderately and poorly differentiated adenocarcinomas of prostatic cancer showed significant differences. The presence of Ets-1 mRNA was confirmed by in-situ hybridization in human prostatic tissues. CONCLUSION: Our results suggest that Ets-1 might play an important role in carcinogenesis and/or the progression of human prostatic carcinomas.