Kinetic analysis of wild-type and YMDD mutant hepatitis B virus polymerases and effects of deoxyribonucleotide concentrations on polymerase activity

Mutations in the YMDD motif of the hepatitis B virus (HBV) DNA polymerase result in reduced susceptibility of HBV to inhibition by lamivudine, at a cost in replication fitness. The mechanisms underlying the effects of YMDD mutations on replication fitness were investigated using both a cell-based viral replication system and an in vitro enzyme assay to examine wild-type (wt) and YMDD-mutant polymerases. We calculated the affinities of wt and YMDD-mutant polymerases for each natural deoxyribonucleoside triphosphate (dNTP) and determined the intracellular concentrations of each dNTP in HepG2 cells under conditions that support HBV replication. In addition, inhibition constants for lamivudine triphosphate were determined for wt and YMDD-mutant polymerases. Relative to wt HBV polymerase, each of the YMDD-mutant polymerases showed increased apparent K(m) values for the natural dNTP substrates, indicating decreased affinities for these substrates, as well as increased K(i) values for lamivudine triphosphate, indicating decreased affinity for the drug. The effect of the differences in apparent K(m) values between YMDD-mutant polymerase and wt HBV polymerase could be masked by high levels of dNTP substrates (>20 microM). However, assays using dNTP concentrations equivalent to those measured in HepG2 cells under physiological conditions showed decreased enzymatic activity of YMDD-mutant polymerases relative to wt polymerase. Therefore, the decrease in replication fitness of YMDD-mutant HBV strains results from the lower affinities (increased K(m) values) of the YMDD-mutant polymerases for the natural dNTP substrates and physiological intracellular concentrations of dNTPs that are limiting for the replication of YMDD-mutant HBV strains.     

两种荧光显微成像系统亚细胞定位的对比

目的:应用高分辨率荧光显微成像系统采集细胞器探针图像,并与激光共聚焦显微成像系统进行对比。方法:实验于2003-05/2004-01在解放军总医院完成。①实验材料:鼠肺毛细血管内皮细胞株(1H11)由上海复旦张江生物公司提供;荧光探针Rhodamine-123,Lucifer Yellow,DiOC6[3],BODIPY(美国Sigma公司)。②细胞培养及荧光探针染色:细胞培养采用含体积分数为0.2胎牛血清的低糖DMEM培养基,密度5×107L-1。选择Rhodamine-123作为细胞线粒体特异性荧光探针,选择DiOC6[3]作为细胞内质网特异性荧光探针,选择BODIPY作为细胞高尔基体特异性荧光探针,选择Lucifer Yellow作为细胞溶酶体探针。前3个探针在完全避光条件下与培养的细胞共同孵育0.5h,后者则共同孵育15h。③高分辨率荧光成像系统的图像采集:线粒体荧光图像采集,选取经Rhodamine-123共孵育完成的细胞,选择激发滤色镜为BP460-490,吸收滤色镜为BA515,分光镜为DM500,另加一绿通道液晶滤光片,激发出Rhodamine-123的荧光。电荷耦合器件采集图像并送入计算机。重复上述步骤,采用DiOC6[3]标记内质网,BODIPY标记高尔基体,Lucifer Yellow标记细胞溶酶体,激发条件同Rhodamine-123。分别采集同一视野靶细胞DiOC6[3]、BODIPY或Lucifer Yellow的荧光图像,完成全部图像采集并储存在计算机中。④激光共聚焦显微成像系统的图像采集:选择经4种探针染色的靶细胞,使用氩离子激光器在488nm激发Rhodamine-123,Rhodamine-123荧光通过配置有530/60-G发射滤光片的通道1探测。重复上述步骤,在488nm激发DiOC6[3]和BODIPY,在457nm激发Lucifer yellow,3种荧光均由通道1探测,后2个探针的发射滤光片的配置为515/30-G,DiOC6[3]选择530/60-G。由光电倍增管接收信号并传输入计算机成像。结果:①高分辨率荧光成像系统所采集图像,靶细胞中由荧光探针Rhodamine-123染色的线粒体呈多个典型的小棒状或卵圆状,聚集在核周;Lucifer yellow染色的溶酶体呈多个非对称球型,在胞浆内随机分布,颗粒尺寸通常大于线粒体;荧光探针DiOC6[3]着色的内质网占据胞浆的很大空间,以囊状聚集为特征;BODIPY特异性地结合在高尔基体上,荧光图像显示围绕在细胞核周围呈条索状。②与高分辨率荧光成像系统比较,激光共聚焦显微成像系统所采集的图像其荧光强度基本相同,但分辨率低、细节显示模糊、胞浆中细胞器的准确分布信息和形态特征显示效果欠佳。结论:两种荧光显微成像系统均可采集到细胞器探针的荧光图像。但高分辨率荧光成像系统采集的荧光图像具有细节清晰、分辨度高、准确显示胞浆中细胞器的分布信息和形态特征等优点。     

Selective effects of PPARγ agonists and antagonists on human pre‐adipocyte differentiation

Aim: The insulin sensitizer rosiglitazone (RTZ) acts by activating peroxisome proliferator and activated receptor γ (PPAR γ), an effect accompanied in vivo in humans by an increase in fat storage. We hypothesized that this effect concerns PPARγ₁ and PPARγ₂ differently and is dependant on the origin of the adipose cells (subcutaneous or visceral). To this aim, the effect of RTZ, the PPARγ antagonist GW9662 and lentiviral vectors expressing interfering RNA were evaluated on human pre‐adipocyte models. Methods: Two models were investigated: the human pre‐adipose cell line Chub‐S7 and primary pre‐adipocytes derived from subcutaneous and visceral biopsies of adipose tissue (AT) obtained from obese patients. Cells were used to perform oil‐red O staining, gene expression measurements and lentiviral infections. Results: In both models, RTZ was found to stimulate the differentiation of pre‐adipocytes into mature cells. This was accompanied by significant increases in both the PPARγ₁ and PPARγ₂ gene expression, with a relatively stronger stimulation of PPARγ₂. In contrast, RTZ failed to stimulate differentiation processes when cells were incubated in the presence of GW9662. This effect was similar to the effect observed using interfering RNA against PPARγ₂. It was accompanied by an abrogation of the RTZ‐induced PPARγ₂ gene expression, whereas the level of PPARγ₁ was not affected. Conclusions: Both the GW9662 treatment and interfering RNA against PPARγ₂ are able to abrogate RTZ‐induced differentiation without a significant change of PPARγ₁ gene expression. These results are consistent with previous results obtained in animal models and suggest that in humans PPARγ₂ may also be the key isoform involved in fat storage.     

Evaluation of the SD BIOLINE TB Ag MPT64 Rapid® for the diagnosis of tuberculosis

The purpose of this study was to evaluate the SD Bioline Ag MPT64 Rapid^® for identification of the Mycobacterium tuberculosis complex. The method uses an immunochromatographic assay and needs 100 μl of sample taken from liquid culture or colonies suspended. The sensitivity was determined using 99 strains of M. tuberculosis complex and the specificity using 10 nontuberculous mycobacteria and 85 strains other than mycobacteria genus. The test showed excellent sensitivity (99%) and specificity (100%). This technique displays several advantages and is destined to spread in all laboratories and particularly in endemic areas.     

The fMRI success rate of children and adolescents: Typical development,epilepsy, attention deficit/hyperactivity disorder,and autism spectrum disorders

Functional magnetic resonance imaging (fMRI) in children is increasingly used in clinical application and in developmental research; however, little is known how pediatric patient and typically developing populations successfully complete studies. We examined pediatric success rates with epilepsy, attention deficit/hyperactivity disorder (ADHD), autism spectrum disorders (ASD), and typically developing children (TYP). We also examined the affect of age, and, for ADHD populations, medication status on success rates. We defined a successful fMRI individual run when the data were interpretable and included in group statistics. For unsuccessful runs, datasets with excessive motion or floor task performance were categorized when possible. All clinical groups scanned less successfully than controls; medication status did not affect ADHD success (epilepsy, 80%; ADHD (off methylphenidate), 77%; ADHD (on methylphenidate), 81%; ASD, 70%; TYP, 87%). Ten to 18‐year‐old had a significantly greater scan success rate than 4‐ to 6‐year‐old; adolescents (13‐ to 18‐year‐old) demonstrated greater scan success rates than 7‐ to 9‐year‐old. Success rate for completing an entire battery of experimental runs (n = 2–6), varied between 50–59% for patient populations and 69% for TYP (79% when excluding 4‐ to 6‐year‐old). Success rate for completing one run from a battery was greater than 90% for all groups, except for ASD (81%). These data suggest 20–30% more children should be recruited in these patient groups, but only 10–20% for TYP for research studies. Studies with 4‐ to 6‐year‐olds may require 20–40% additional participants; studies with 10‐ to 18‐year‐olds may require 10–15% additional participants. Hum Brain Mapp, 2009. © 2009 Wiley‐Liss, Inc.