The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.     

人晶状体上皮细胞蛋白质组的双向电泳和质谱鉴定

目的： 建立人晶状体上皮细胞蛋白质组研究体系，探讨双向电泳和质谱鉴定技术在人晶状体上皮细胞蛋白质组研究中的作用。 方法: 体外培养人晶状体上皮细胞株，用两种不同方法提取总蛋白，进行固相pH梯度(IPG)等电聚焦双向凝胶电泳，凝胶通过GS-800扫描仪（Bio-Rad）获取图像并使用PDQuest专业图像分析软件分析。在此基础之上，胰酶消化蛋白质斑点并进行质谱分析。 结果: 获得了重复性较好的人晶状体上皮细胞蛋白质组电泳图谱。晶状体蛋白质斑点在等电点pH值为4-7、相对分子质量为17-72 kD之间均有分布。其中高丰度蛋白点主要分布于分子量19-50 kD、PI 5-7范围内。2个蛋白点通过质谱分析和数据库的检索得到了初步的鉴定。 结论: 建立起了一个稳定的分析人晶状体上皮细胞蛋白质组学实验体系；为进一步研究人类晶状体在生理状态及白内障等病理条件下的改变提供了蛋白质组学的研究方法和途径。     

17βE2对切除卵巢兔动脉粥样硬化与血液流变学的作用

目的: 探讨17βE₂对卵巢切除(OVX)兔的AS斑块、血脂代谢及血液流变学的影响。方法: 34只成熟未孕雌兔分为4组:A为NC;B为Sham+CHO;C为OVX+CHO;D为OVX+CHO+17βE₂。喂养12周,喂养前与12周后测定血脂TC、TG、HDL-C、LDL-C、ApoA1、ApoB;测定全血粘度、血浆粘度、AIRC及纤维蛋白原。喂养12周后处死,测定AS斑块面积与主动脉总面积百分比。结果: ①AS斑块面积与主动脉总面积之比,A、B、C、D组分别为0.02±0.00、0.42±0.15、0.67±0.23、0.12±0.11,B组小于C组(P＜0.05)、D组明显小于C组(P＜0.01)。②血脂TC、TG、LDL-C、ApoB,B、D组明显小于C组(P＜0.01)。③血液流变学:全血粘度、血浆粘度AIRC与纤维蛋白原D组明显低于C组(P＜0.01)。结论: 17βE₂能抑制脂质斑块沉积,改善血脂代谢,减轻高脂血症,改善血液流变学。这可能是雌激素抑制AS形成的部份机理。     

Changes in the withdrawal bleeding pattern and endometrial histology during 17β-estradiol —dydrogesterone therapy in postmenopausal women: a 2 year prospective study

Objective: To describe changes in the withdrawal bleeding pattern and endometrial histology during a sequential 17β-estradiol —dydrogesterone regimen in postmenopausal women. Design: Open-label, non-comparative, prospective study. Setting: Gynecological outpatient department of a university hospital. Patients: Twenty-seven healthy nonhysterectomized postmenopausal women. Interventions: Continuous micronized 17β-estradiol supplementation, 2 mg daily, and cyclic administration of dydrogesterone, 10 mg daily for the first half of each 28 day treatment cycle. Main Outcome Measures: Changes in the characteristics of the withdrawal bleeding pattern and the endometrial biopsy histology during 2 years of treatment. Results: The initial withdrawal bleeding was comparable to normal menstruation with respect to amount and duration. During the 2 years of treatment the bleeding showed a significant tendency to become shorter with less blood loss. This was mainly the result of the decrease (P < 0.001) in the number of days per cycle with bleeding grade II (normal menstruation). None of the women developed endometrial hyperplasia, and in almost all women the given hormone replacement therapy regimen induced secretory or atrophic changes of the endometrium. Conclusions: This sequential 17β-estradiol —dydrogesterone regimen can be regarded as safe with respect to the prevention of endometrial disease and appeared to foster patient compliance.     

Alert surveillance of intensive care unit-acquired Acinetobacter infections in a Sicilian hospital

The epidemiological impact of Acinetobacter baumannii nosocomial infections in a Sicilian intensive care unit (ICU) was investigated to determine the Acinetobacter-specific infection rates, to estimate the preventable proportion of Acinetobacter infections, i.e., those resulting from cross-transmission, and to investigate the molecular epidemiology of antimicrobial resistance in Acinetobacter. The impact of Acinetobacter nosocomial infection in the ICU was determined to be 3.0 new cases per 100 admissions. Site-specific rates confirmed that ICU-acquired pneumonia was the most important infection type. The incidence rate, adjusted by the number of patient-days, was 3.3 infections/1000 patient-days. The estimated preventable proportion of A. baumannii nosocomial infections in the ICU was 66.7%. A class 1 integron, characterised by its gene cassette content, was present in all A. baumannii isolates of four different pulsed-field gel electrophoresis types, and was associated significantly with clones implicated in cross-transmission episodes. Furthermore, the same integron was detected in two genetically distinct isolates responsible for recurrent infection in the same patient, suggesting the occurrence of horizontal gene transfer in vivo. Even in an endemic setting with low infection rates, spread of A. baumannii was caused mainly by infection control shortcomings that require appropriate surveillance and control policies.     

Characterization of a monoclonal anti-Bw4 antibody (Tü109): Evidence for similar epitopes on the Bw4 and Bw6 antigens

The production and serologic, as well as immunochemical properties of a cytotoxic murine IgG monoclonal antibody (Tü109) that precipitates HLA-class I molecules, are described. In the microcytotoxicity assay Tü109 supernatant was demonstrated on a panel of 424 HLA-ABC, -DR, -DQ, -MT typed normal Caucasian blood donors to define an epitope on HLA-B locus molecules in great association with the supertypic specificity Bw4. Reactivity of supernatant showed MHC linked inheritance of the Tü109 determinant and discriminated the HLA-Bw4/Bw6 associated HLA-B locus split antigens. Weak or lack of binding on lymphocytes from some HLA-Bw4 heterozygous individuals, particularly typing for HLA-Bw44, appeared to be due to qualitative and/or quantitative variations of HLA-B locus molecules on the cell surface. With Tü109 ascites fluid, however, extra-reactivity on all HLA-Bw6⁺ cells was demonstrated. Preferential binding of supernatant to HLA-Bw4, but reactivity of ascites fluid with HLA-Bw6⁺ molecules in addition, was furthermore confirmed by IEF analysis of antigens immunoprecipitated with Tü109 from cell lysates. Thus the antibody may help to analyze the evolutionary relationship of the diallelic specificities Bw4 and Bw6.     

巨噬细胞源性神经营养因子的初步分离

本文制备了巨噬细胞条件培养基（ＭφＣＭ），并应用快速自动比色微量分析法检测ＭφＣＭ对体外培养的生后７ｄＳＤ大鼠小脑皮质神经元的作用。结果表明，分子量大于１０ｋＤ的ＭφＣＭ对神经元（细胞密度１×１０６／ｍｌ）的作用，与对照组比较有明显的神经营养活性（Ｆ＜０．０５）；用盖玻片培养法表明，该组份有促进神经元突起生长的作用。ＭφＣＭ经ＳｅｐｈａｃｒｙｌＳ－１００－ＨＲ凝胶层析和生物活性鉴定，获得了具有神经营养活性的第二峰洗脱液。此洗脱液经ＳＤＳ－聚丙烯酰胺凝胶电泳分析，证明ＭφＣＭ中神经营养活性成份的蛋白质其分子量为３１ｋＤ－６８ｋＤ之间。     

Isolation and immunochemical characterization of the major allergen of birch pollen (Betula verrucosa)

The classification of some of the extractable birch pollen antigens as allergens was established by crossed radioimmunoelectrophoresis (CRIE). In CRIE the major allergen (antigen 23) exhibited the strongest “radiostaining” and only a few other components of birch pollen extract were visibly radiostained. The major allergen and a preparation containing mainly the minor allergens, antigens 25 and 19, were isolated from a crude aqueous birch pollen extract by a combination of anion-exchange, size-exclusion, and chelate chromatography. Antigen 23 was purified to near homogeneity. The molecular weights and the pIs of antigens 23, 25, and 19 were determined to be 17,000 daltons, pI 5.25 (5.5, 5.0); 25,000 daltons, pI 5.0 (4.9, 5.4); and 29,000 daltons, pI 6.2 (5.4), respectively. The classification of antigen 23 as the major allergen in birch pollen was supported by results of RAST inhibition experiments, RAST screening, and skin prick testing.     

Effects of aging on luteinizing hormone release in different physiological states of the female golden hamster

Effects of aging on estrous cycles and LH release in response to luteinizing hormone releasing hormone (LHRH), castration, and estradiol benzoate were studied in the female golden hamster (Mesocricetus auratus). About 80% to 90% of female golden hamsters still cycled regularly when reaching 19–22 months of age. However, some animals showed age-induced irregularity of the estrous cycle which included an interruption of complete absence of estrous vaginal discharge. Young female hamsters (3–5 months) had significantly (p<0.01) higher basal LH concentration than old animals (19–22 months) in the morning of each stage of estrous cycle. LHRH elicited about 20–30 fold increase in serum LH concentrations in both young and old hamsters. No significant difference in LH release was observed between young and old hamsters in response to LHRH. In acyclic hamsters, the peak of LH release in response to LHRH was delayed. LHRH-induced LH release was greater in the morning of proestrus than during diestrus in both young and old hamsters. LH increase was significantly greater in the young than in old hamsters on the 13th and 15th day after castration. However, positive feedback stimulation of LH release by estradiol benzoate was the same in both young and old hamsters. These results indicate that in the female hamster, LH response to acute stimuli such as LHRH and estrogens is the same in the young as in the old animal and that circulating basal LH concentration may decrease or its degradation or clearance may increase during the aging process in female golden hamsters. Irregularity of estrous cycles in aging hamsters may be related to delayed responsiveness of pituitary LH to LHRH stimulation.