pcDNA3.1/hIL-18真核表达载体的构建及表达

目的 :构建重组真核表达质粒pcDNA3.1/IL 18,并在哺乳动物细胞COS 7和Rlc310中进行瞬时和稳定性表达。方法 :从含hIL 18基因的中介载体 pGEM TEasy( pGEM T/hIL 18)中 ,以限制性内切酶酶切方法获得目的片段 ,克隆入真核表达质粒 pcDNA3.1( )中。以脂质体法转染COS 7和Rlc310细胞 ,用RT PCR检测IL 18mRNA的水平 ,免疫组化染色法检测蛋白表达。结果 :构建了hIL 18基因的重组真核表达质粒pcDNA3.1/IL 18,并可在哺乳动物细胞中瞬时、稳定表达 ,获得了可稳定表达hIL 18基因的Rlc310细胞株。结论 :pcDNA3.1/IL 18的构建及表达 ,为IL 18抗肿瘤作用的研究奠定了基础     

Expression of Mcl-1 in mantle cell lymphoma is associated with high-grade morphology,a high proliferative state,and p53 overexpression

Mantle cell lymphoma (MCL) is a distinct type of B-cell non-Hodgkin's lymphoma characterized by the t(11;14)(q13;q32) and cyclin D1 overexpression. Defects in apoptosis may contribute to pathogenesis. This study evaluated the expression of the anti-apoptotic protein Mcl-1 in two MCL cell lines and five frozen MCL tumours (four small-cell, one blastoid/large-cell) using western blot analysis. Mcl-1 expression was also assessed in 36 formalin-fixed, paraffin wax-embedded MCL tumours (24 small-cell, 12 blastoid/large-cell) by immunohistochemistry. Western blot analysis revealed the expected 37 kD protein product in both MCL cell lines and in five frozen tumours, with the blastoid case having the highest expression level. Using a cut-off of >10% immunolabelled cells for Mcl-1, it was found that 12 of 36 MCL tumours were positive. Mcl-1-positive tumours had a higher frequency of blastoid/large-cell morphology (8/12 versus 4/24, p = 0.009), p53 overexpression (3/10 versus 1/23, p = 0.04), and higher Ki67 immuno-labelling (p = 0.002). It is concluded that expression of Mcl-1 in MCL is heterogeneous. A relatively high level of Mcl-1 expression correlates with high-grade morphology, a high proliferative state, and p53 overexpression.     

A PKC epsilon-ENH-channel complex specifically modulates N-type Ca2+ channels

Multiple protein kinase C (PKC) isozymes are present in neurons, where they regulate a variety of cellular functions. Due to the lack of specific PKC isozyme inhibitors, it remains unknown how PKC acts on its selective target(s) and achieves its specific actions. Here we show that a PKC binding protein, enigma homolog (ENH), interacts specifically with both PKCepsilon and N-type Ca2+ channels, forming a PKCepsilon-ENH-Ca2+ channel macromolecular complex. Coexpression of ENH facilitated modulation of N-type Ca2+ channel activity by PKC. Disruption of the complex reduced the potentiation of the channel activity by PKC in neurons. Thus, ENH, by interacting specifically with both PKCepsilon and the N-type Ca2+ channel, targets a specific PKC to its substrate to form a functional signaling complex, which is the molecular mechanism for the specificity and efficiency of PKC signaling.     

Occult hepatitis B virus infection and clinical outcomes of patients with chronic hepatitis C

Although occult hepatitis B virus (HBV) infections in individuals without detectable hepatitis B surface antigen (HBsAg) may occur and have been reported to be common in patients with chronic hepatitis C, the clinical relevance remains controversial. We searched for serum HBV DNA in 210 HBsAg-negative patients with hepatitis C virus (HCV)-related liver disease (110 patients with chronic hepatitis, 50 patients with cirrhosis, and 50 patients with hepatocellular carcinoma) by PCR. Most of the patients had detectable antibodies to HBsAg or HBV core antigen. All of the 110 chronic hepatitis C patients were treated with a combination therapy consisting of interferon plus ribavirin. In addition, 100 HBsAg-negative healthy adults served as controls. Thirty-one of the 210 patients (14.8%) had HBV DNA in their sera, as did 15 of the 100 healthy controls (15%). HBV DNA was not detected in the sera of those negative for serological markers of HBV infection. In patients with chronic HCV infection, the prevalence of occult HBV infection did not parallel the severity of liver disease (14.5% in patients with chronic hepatitis, 8% in patients with liver cirrhosis, and 22% in patients with hepatocellular carcinoma). In addition, the sustained response to combination therapy against hepatitis C was comparable between patients with and without occult HBV infection (38 versus 39%). In conclusion, these data suggest that occult HBV infection does not have clinical significance in chronic hepatitis C patients residing in areas where HBV infection is endemic.     

Cytocidal and apoptotic effects of the ClyA protein from Escherichia coli on primary and cultured monocytes and macrophages

Cytolysin A (ClyA) is a newly discovered cytolytic protein of Escherichia coli K-12 that mediates a hemolytic phenotype. We show here that highly purified ClyA and ClyA-expressing E. coli were cytotoxic and apoptogenic to fresh as well as cultured human and murine monocytes/macrophages.     

Wheezing in Chinese schoolchildren: disease severity distribution and management practices, a community-based study in Hong Kong and Guangzhou

BACKGROUND: Asthma is a common chronic disease and information on its management practices at the community level is helpful in identifying problems and improving asthma care. OBJECTIVE: To assess the severity status and management of the asthma symptom of wheeze of children at the community level in Hong Kong (HK) and Guangzhou (GZ). METHODS: Cross-sectional study of children aged 10 years using the International Study of Asthma and Allergic disease in Childhood (ISAAC Phase II protocol). Asthma management and lung function were assessed in 178 (98 from HK and 80 from GZ) randomly selected children with wheeze over the past 12 months. RESULTS: Eighty-three percent, 11%, 6% and 0% of children suffered from intermittent, mild persistent, moderate persistent and severe persistent asthma, respectively, according to the frequency of their symptoms. Addition of spirometric parameter only changed the asthma severity classification in 2.8% of children. Medications were used by 30.6% and 71.3% of children for wheeze in HK and GZ, respectively. In HK, inhaled beta(2)-agonist (73.3% among the drug users) was the commonest medication used followed by inhaled corticosteroid (ICS) (23.3%). In GZ, inhaled beta(2)-agonist was used by 75.4% of children, but use of ICS (26.3%), oral beta(2)-agonist (26.3%), oral theophylline (45.6%), oral ketotifen (36.8%) and oral steroid (35.1%) were also common. ICS was only used by 11.4% of children with persistent asthma. Ten percent and 18.7% of children in HK and GZ, respectively, had emergency department visits, while 16.3% and 11.6% of children in HK and GZ, respectively, had missed school secondary to asthma over the past 1 year. CONCLUSIONS: Most children in the community had intermittent asthma and addition of lung function to symptoms did not significantly affect classification of asthma severity. Significant morbidity was seen even in this group of children with mostly intermittent and mild persistent asthma.     

Early phylogenetic estimate of the effective reproduction number of SARS-CoV-2

To reconstruct the evolutionary dynamics of the 2019 novel-coronavirus recently causing an outbreak in Wuhan, China, 52 SARS-CoV-2 genomes available on 4 February 2020 at Global Initiative on Sharing All Influenza Data were analyzed. The two models used to estimate the reproduction number (coalescent-based exponential growth and a birth-death skyline method) indicated an estimated mean evolutionary rate of 7.8 × 10⁻⁴ subs/site/year (range, 1.1 × 10⁻⁴-15 × 10⁻⁴) and a mean tMRCA of the tree root of 73 days. The estimated R value was 2.6 (range, 2.1-5.1), and increased from 0.8 to 2.4 in December 2019. The estimated mean doubling time of the epidemic was between 3.6 and 4.1 days. This study proves the usefulness of phylogeny in supporting the surveillance of emerging new infections even as the epidemic is growing.     

Diverged evolution of recent equine-2 influenza (H3N8) viruses in the Western Hemisphere

Summary.  We reported previously that equine-2 influenza A virus (H3N8) had evolved into two genetically and antigenically distinct “Eurasian” and “American” lineages. Phylogenetic analysis, using the HA1 gene of more recent American isolates, indicated a further divergence of these viruses into three evolution lineages: A South American lineage, a Kentucky lineage, and a Florida lineage. These multiple evolution pathways were not due to geographic barriers, as viruses from different lineages co-circulated. For the Kentucky lineage, the evolution rate was estimated to be 0.89 amino acid substitutions per year, which agreed with the previously estimated rate of 0.8. For the South American lineage, the evolution rate was estimated to be only 0.27 amino acid substitutions per year. This low evolution rate was probably due to a unique alternating Ser138 to Ala138 substitutions at antigenic site A. For the Kentucky lineage, there was a preference for sequential nonsynonymous substitutions at antigenic site B, which was also a “hot spot” for amino acid substitutions. Convalescent sera had minimal cross-reactivity to viruses of different lineages, indicating antigenic distinctions among these viruses. In contrast to human H3N2 viruses, our results suggested that the evolution of equine-2 influenza virus resembled the multiple evolution pathways of influenza B virus. Accepted December 14, 2000 Received September 19, 2000     

Cyclin D1 expression in dysplastic nevi: an immunohistochemical study

OBJECTIVE: We previously surveyed cyclin D1 expression in common acquired nevi, Spitz nevi, and malignant melanomas and reported that benign nevi maintain a zonal pattern of cyclin D1 expression, in contrast with malignant melanomas. Our aim was to extend those observations by examining cyclin D1 expression in dysplastic nevi. METHODS: Cyclin D1 overexpression in 23 dysplastic nevi was detected by an immunohistochemical technique. The extent of atypia of the nevi was graded as mild, moderate, or severe, using previously established criteria. RESULTS: Cyclin D1 overexpression in dysplastic nevi maintained a zonal pattern, similar to Spitz nevi. Cyclin D1 overexpression was greatest in the region of the epidermal-dermal junction and was significantly less prominent in the papillary and reticular dermis, suggesting that cyclin D1 expression is under cell control and correlates with maturation of nevus cells. Cyclin D1 overexpression also correlated with cytologic atypia, as dysplastic nevi with moderate or severe cytologic atypia contained a greater percentage of cyclin D1-positive cells than did nevi with mild atypia. Six dysplastic nevi with many cyclin D1--positive cells were assessed by fluorescence in situ hybridization studies using cyclin D1--specific and chromosome 11 centromeric probes. In all cases, there was no evidence of 11q13 translocation, amplification, or trisomy of chromosome 11. CONCLUSIONS: Cyclin D1 may be involved in the pathogenesis of dysplastic nevi. Cyclin D1 overexpression does not appear to be explained by cyclin D1 locus amplification or translocation in most cases, and it may be a result of other cell abnormalities that up-regulate the protein level of cyclin D1.     

抗血小板糖蛋白Ⅱb/Ⅲa Fab抗体的原核表达及其生物活性

目的：研制抗血小板膜糖蛋白Ⅱb／Ⅲa Fab抗体。方法：通过问接免疫荧光试验和血小板聚集抑制试验，选取鼠源抗血小板糖蛋白Ⅱb／Ⅲa单克隆抗体(mAb P140)。从分泌该mAb的杂交瘤细胞株(P140)中，克隆到抗体轻链基因和重链Fd段基因，构建原核表达重组质粒p3MH／P140x-Fd，并在XLI-Blu菌株中进行表达。采用钴亲和层析法对P140 Fab抗体进行纯化，用SDS-PAGE、ELISA和Western blot等方法，对P140 Fab抗体进行检测，并通过血小板聚集抑制试验，观察P140 Fab抗体的抗栓活性。结果：SDS-PAGE和Western blot表明，纯化的P140Fab抗体的相对分子质量(Mr)约为47000。ELISA的结果显示，P140 Fab抗体可与人血小板特异性结合。在体外ADP诱导的血小板聚集试验中，P140 Fab抗体对血小板聚集的抑制作用成剂量依赖性，IC50的平均值为16．85mg/L。结论：成功地研制出具有抗栓活性的抗血小板膜糖蛋白Ⅱb／Ⅲa的Fab抗体。