Molecular typing of echovirus serotype 4 isolates

We report on clinical samples Stuttgart/97, Berlin/99 and Jasi/99 associated with aseptic meningitis. All three samples contained echovirus 4 (E4) but Stuttgart/97 was simultaneous infected with echovirus 30 (E30). The genetic relationship of the E4 strains was assessed using RT-PCR and direct sequencing of amplicons derived from the genomic region encoding the capsid protein VP1. The sequences have been compared with each other and with sequences of further E4 strains obtained from GenBank. The analysis confirms that sequences of recent isolates have drifted away from elderly strains over a longer period of time. Several amino acid changes in assumed antigenic sites of the VP1 gene may be sufficient to cause changes in antigenic specificity and therefore they may be a reason for failure of serological typing of some new antigenic E4 variants.     

冈比亚按蚊嗅觉结合蛋白基因agCP1588的克隆鉴定及其组织特异性表达谱分析

昆虫主要依靠嗅觉发现寄主 ,嗅觉在按蚊的寄主搜索行为中起关键作用。本文基于冈比亚按蚊的全基因组序列 ,设计特异引物 ,采用RT -PCR克隆了该按蚊嗅觉结合蛋白候选基因agCP15 88。测序分析结果表明该基因具有嗅觉蛋白的标志性结构域 ,通过半定量RT PCR技术研究了该基因的组织特异性表达谱 ,发现该基因只在雌蚊触角中表达。这一发现为进一步研究该嗅觉蛋白基因的功能奠定了基础     

Expression of mucin genes in the human testis and its relationship to spermatogenesis

In this study we investigate the expression pattern of mucin genes in the human testis and evaluate the relationship between the expression of mucin genes and impaired spermatogenesis in the human testis. Thirty human testis tissues were collected from patients undergoing diagnostic testicular biopsy to investigate the cause of infertility. One part of the tissue underwent histological observation, and the other part of the tissue was subjected to semiquantitative RT-PCR of mucin genes, that is, mucin1, 2, 3, 4, and 9. The relative amount of mucin mRNAs was calculated by densitometry using glyceraldehydes-3-phosphate dehydrogenase (GAPDH) as an internal control. The samples were histologically diagnosed as either obstructive azoospermia with normal spermatogenesis (n = 13) or non-obstructive azoospermia with impaired spermatogenesis (n = 17). In the human testis with normal spermatogenesis, mRNA expression of mucin1, 9, 13 and GAPDH were found, but RT-PCR products of mucin 2, 3 and 4 were not detected. In the testis with impaired spermatogenesis, however, RT-PCR product of mucin1 was not found. There was no difference in the other mucin mRNA expression patterns between the testis with either normal or impaired spermatogenesis. To our knowledge, this study is the first that has detected the mRNA of mucin9 and 13 in human testis. This study also shows that mucin1 expression might be closely related to spermatogenesis. Our findings should be substantiated by more direct evidence, such as mucin protein expression and localization.     

5′-Cytosine DNA-methyltransferase mRNA levels in hereditary colon carcinoma

 DNA methylation plays an important part in the regulation of gene expression. Alterations in DNA methylation in tumours have been reported and have been used to generate hypotheses about mutagenesis and silencing of tumour suppressor genes. However, the underlying mechanism is still poorly understood, and conflicting data on the levels of overexpression of 5′-cytosine DNA methyltransferase in sporadic colon carcinoma have been published. We used a competitive RT-PCR assay for quantification of mRNA of 5′-cytosine DNA methyltransferase in colon biopsies obtained from patients with hereditary colon carcinoma syndromes and compared the results with those obtained in a control group. No significant difference was found between the flat mucosa of FAP patients and the mucosa of the control group. In FAP and HNPCC patients, the 5′-cytosine DNA methyltransferase mRNA levels of adenomas were significantly higher (P<0.05) than of flat mucosa in the same group, but both showed great variability from patient to patient. Our findings suggest that the mRNA levels of methyltransferase cannot be used as predictive marker for screening in families affected by hereditary colon carcinoma. Received: 20 July 1998 / Accepted: 21 September 1998     

Large Envelope Glycoprotein and Nucleocapsid Protein of Equine Arteritis Virus (EAV) Induce an Immune Response in Balb/c Mice by DNA Vaccination; Strategy for Developing a DNA-Vaccine Against EAV-Infection

Equine arteritis virus (EAV) is a member of the Arteriviridae family, that includes lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian haemorrhagic fever virus (SHFV). Equine arteritis is a contagious disease of horses and is spread via respiratory or reproductive tract. The objective of the present study is to evaluate the possibility for developing a model system for prevention horses against an EAV infection by DNA vaccination. A cDNA bank from the RNA of EAV was established. This gene library contains the translation unit of the EAV open reading frames (ORF) 1 to 7. The identity of the cDNA was confirmed by nucleotide sequence analysis. Using this defined EAV cDNA gene library the cDNA sequence of the viral ORFs were molecularly cloned into the corresponding sites of well characterized and powerful expression vectors (pCR3.1, pDisplay, and/or pcDNA3.1/HisC).The capability of these recombinant plasmids expressing the gene products of the individual viral ORFs 3 to 5, and 7 in induction of an immune response in mouse system was investigated. The Balb/c mice (ten mice per assay) were inoculated with the DNA of the constructed expression vectors harboring and expressing the EAV cDNA of the viral ORFs. The Balb/c mice were injected with about 100 g DNA diluted in 100 l PBS. The DNA was injected subcutaneously and into the tibialis cranialis muscle (Musculus gastrocnemius). The mice were boosted 3 to 5 times with the same quantities of DNA and under the same conditions at about two week intervals. Control mice received the same amount of parental expression vectors via an identical route and frequency.The pre- and post-vaccinated sera of the individual animals were screened by neutralization tests (NT). Neutralizing antibodies against EAV were detected when the animals were inoculated with the DNA of the expression vectors harboring cDNA of the EAV ORFs 5 and 7. Highest NT-titers were observed when the animals were administered with the cDNA of ORF 5 and/or with the cDNA of the neutralization determinants of EAV that is located on the N-terminal ectodomain of the gene product of ORF 5 between the amino acid positions 1–121. These results obtained from these studies justified proofing the capability of the EAV cDNA sequences of the viral genes including ORFs 5 and 7 in the autologous animal system horse.     

SHP-1和JAK1基因在初治急性白血病患者中的表达

目的：探讨SHP-1和JAK1基因在初治急性白血病（AL）细胞的转录表达及其与初治AL患者化疗效果的关系。方法：采用半定量逆转录-聚合酶链反应（RT-PCR）方法检测60例初治AL患者骨髓单个核细胞及20例健康人外周血单个核细胞中SHP-1和JAK1 mRNA的表达。结果：60例初治AL患者SHP-1和JAK1的mRNA表达阳性率分别为30%，100%；初治AL患者SHP-1 mRNA表达水平明显低于正常对照组（P<0.001），JAK1 mRNA表达水平较正常对照组略增高，但差异无统计学意义（P=0.180）,初治AL患者SHP-1 mRNA阳性组的诱导化疗完全缓解（CR）率为88.9%，阴性患者组CR率为54.8%，差异有统计学意义（P=0.005）。SHP-1与JAK1 mRNA表达呈负相关（P=0.046）。结论：SHP-1可能是白血病潜在的抑制基因，可望作为判断初治急性白血病患者疗效和预后的指标。     

Bronchiolar expression of aquaporin-3 (AQP3) in rat lung and its dynamics in pulmonary oedema

Aquaporins (AQPs) are water channel proteins that permit osmotically driven water movement. To determine their dynamics in pulmonary oedema, we examined the expression of mRNA and protein for AQP1, AQP3, AQP4, and AQP5 in the lungs of normal and thiourea-treated rats. In the thiourea group, lung water content increased significantly (vs. controls) with the peak at around 4 h. Semi-quantitative RT-PCR showed that AQP3 mRNA in the thiourea group rose significantly, peaking at around 4–8 h. The expression of AQP1, AQP4, AQP5, ENaC and CFTR mRNA each decreased significantly some time after the peak in lung water content. Immunoblot analysis showed that glycosylated AQP3 protein was increased 4–10 h after treatment. Expression of the other AQP proteins was not significantly altered, except for that of AQP4. Immunohistochemical examination revealed that AQP1 was expressed in endothelia, AQP3 in the basal cells of the large airways and in cuboidal cells in the bronchioles, AQP4 in the basolateral membrane of airway cells and AQP5 in type-I pneumocytes. Our results suggest that AQP3 is expressed not only in large airways, but also in bronchioles, and is related to water movement in pulmonary oedema.     

Macrophage mannose receptor in chronic sinus disease

BACKGROUND: The role of infectious agents in the onset and maintenance of chronic sinus disease is still not fully understood. Macrophage mannose receptor (MMR), an innate pattern recognizing receptor, capable of phagocytosis of invaders and signal transduction for proinflammatory mechanisms, might be of importance in immune interactions in chronic sinus disease. OBJECTIVE: We examined the MMR in sinonasal airway mucosa to evaluate its possible role in chronic rhinosinusitis (CS) and nasal polyposis (NPs). METHODS: Surgical samples from patients with sinonasal disease were investigated with real-time RT-PCR for quantification of MMR mRNA expression, and the presence and location of MMR-positive cells was analysed by immunohistochemistry. RESULTS: Quantification of MMR mRNA showed a statistically significant higher expression in NPs compared to CS without NP and controls. Immunohistochemistry revealed expression of MMR in all tissue samples; however, in NP we found an enhanced positive cellular staining including cell aggregates. CONCLUSIONS: We could demonstrate for the first time that the expression of MMR is significantly upregulated in NP compared to patients with CS without NP or turbinate tissue of controls. Macrophages expressing MMR, accumulated in cell aggregates in NPs, play a possible key role in pathogen-macrophage interaction in NP disease.