Novel chemical class of pUL97 protein kinase-specific inhibitors with strong anticytomegaloviral activity

Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC(50)s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC(50)s of these three quinazoline compounds (2.4 +/- 0.4, 3.4 +/- 0.6, and 3.9 +/- 1.1 microM, respectively) were in the range of the IC(50) of ganciclovir (1.2 +/- 0.2 microM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.     

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。     

Age‐related macular degeneration and functional promoter and coding variants of the apolipoprotein E gene

Age‐related macular degeneration (AMD) is a frequent, multifactorial disease of the central retina and a major cause of irreversible vision loss in industrialized countries. Apolipoprotein E (APOE) has been consistently associated with AMD, particularly its two functional isoforms E2 (predisposing) and E4 (protective). The biological correlate of this association, however, is still unclear. In this study, we have defined an extended haplotype block encompassing the entire APOE gene locus, including known coding as well as cis‐regulatory promoter variants. Of the five extended APOE haplotypes common in the general population, two were found to be significantly associated with AMD, namely G‐G‐G‐G‐ε2 (odds ratio [OR], 1.59; 95% confidence interval [CI], 1.19–2.12) and T‐G‐A‐G‐ε4 (OR, 0.76; 95% CI, 0.58–0.99). When analyzing common extended haplotype combinations, T‐C‐G‐G‐ε3/T‐G‐A‐G‐ε4 exhibited the most prominent effect (OR, 0.32; 95% CI, 0.20–0.51). Intriguingly, we also found one extended ε3‐haplotype, G‐G‐G‐A‐ε3, to be protective in the homozygous state (OR, 0.65; 95% CI, 0.49–0.87). Since single nucleotide polymorphism (SNP) rs405509:G>T is a constituent of the extended ε‐haplotype block and is known to significantly influence APOE promoter activity, we hypothesize that both the relative rate of APOE isoform expression in conjunction with established functional differences of the respective isoforms may be crucial in mediating AMD pathology. This would also imply that genotyping of the core ε‐haplotypes alone is not sufficient to estimate AMD risk, but that determination of extended haplotype combinations, including the functional promoter SNP rs405509, is required instead. Hum Mutat 0:1–6, 2009. © 2009 Wiley‐Liss, Inc.     

Die sekundäre Gelenksteife als Hauptkomplikation nach Monteggia-Verletzungen

Introduction

Monteggia fractures represent a rare and often underestimated injury (2–5% of proximal forearm fractures). Even though there is good understanding of the biomechanics of the fracture and the advantage of the operative treatment, the rates of complications, revisions, and disappointing functional outcome results are high. The purpose of the study was to evaluate the complications after operative treatment of Monteggia injuries due to the fracture type, additional injuries, and initial operative treatment.

Patients and methods

During the period from January 2003 to December 2007, we treated 21 patients with a Monteggia fracture at our institution. There were 12 females and 9 males. The mean age was 51 years (range 23–77 years). The patients were followed up clinically and radiologically for a mean of 24 months (range 3–40 months).Clinical assessment was performed using the Morrey and the Leipziger Elbow Scores. Postoperative complications were evaluated and the patients were reevaluated clinically and radiologically after operative treatment of the complications.

Results

Complications were found in 6 of 21 patients. Four patients developed secondary stiffness (19%) and 2 patients developed a non-union of the proximal ulna (10%). The overall Morrey Score was 96.6 points in 17 patients (excluding the 4 patients with secondary stiffness) and the overall Leipziger Elbow Score was 85.2 points. After 6 months, the 4 patients after revision surgery due to secondary stiffness achieved slightly inferior results according to the Morrey and Leipziger Elbow Scores (Morrey Score: 89 points, Leipziger Elbow Score: 71 points).

Conclusion

Patients with secondary stiffness due to operative treatment of a Monteggia fracture benefit from open arthrolysis, despite the Morrey and Leipziger Elbow Scores showing slightly inferior results. Secondary stiffness is often associated with additional fractures of the radial head, intraarticular fractures, and soft tissue injuries. The patient must be informed about the severity of this type of fracture and its complications, including possible reoperation.     

Association and linkage of allelic variants of the dopamine transporter gene in ADHD

Previously, we had reported a genome-wide scan for attention-deficit/hyperactivity disorder (ADHD) in 102 families with affected sibs of German ancestry; the highest multipoint LOD score of 4.75 was obtained on chromosome 5p13 (parametric HLOD analysis under a dominant model) near the dopamine transporter gene (DAT1). We genotyped 30 single nucleotide polymorphisms (SNPs) in this candidate gene and its 5' region in 329 families (including the 102 initial families) with 523 affected offspring. We found that (1) SNP rs463379 was significantly associated with ADHD upon correction for multiple testing (P=0.0046); (2) the global P-value for association of haplotypes was significant for block two upon correction for all (n=3) tested blocks (P=0.0048); (3) within block two we detected a nominal P=0.000034 for one specific marker combination. This CGC haplotype showed relative risks of 1.95 and 2.43 for heterozygous and homozygous carriers, respectively; and (4) finally, our linkage data and the genotype-IBD sharing test (GIST) suggest that genetic variation at the DAT1 locus explains our linkage peak and that rs463379 (P<0.05) is the only SNP of the above haplotype that contributed to the linkage signal. In sum, we have accumulated evidence that genetic variation at the DAT1 locus underlies our ADHD linkage peak on chromosome 5; additionally solid association for a single SNP and a haplotype were shown. Future studies are required to assess if variation at this locus also explains other positive linkage results obtained for chromosome 5p.     

Pre-incisional epidural ropivacaine, sufentanil, clonidine, and (S)+-ketamine does not provide pre-emptive analgesia in patients undergoing major pancreatic surgery

BACKGROUND: The concept of pre-emptive analgesia remains controversial. This prospective, randomized, and double-blind study compared epidural administration of ropivacaine 2 mg ml(-1), sufentanil 0.5 microg ml(-1), clonidine 3 microg ml(-1), and S(+)-ketamine 0.25 mg ml(-1) (study solution) given before incision with the same combination started at the end of the operation. METHODS: After testing the stability of the solution using high performance liquid chromatography (HPLC) and examining 12 patients for possible side-effects in comparison with the epidural infusion of ropivacaine 2 mg ml(-1) and sufentanil 0.5 microg ml(-1), 30 patients undergoing major pancreatic surgery were recruited into the study. Before induction of anaesthesia, an epidural catheter was inserted (TH6-8). Patients in Group 1 received a bolus of 8 ml followed by a continuous infusion (8 ml h(-1)) of the study solution before induction of anaesthesia. In Group 2, patients received the same volume of saline before operation, the study solution was started at the end of surgery. After operation, the infusion was maintained for at least 96 h using a patient-controlled epidural analgesia (PCEA) pump in both groups. Patients were evaluated up to the seventh postoperative day for pain and side-effects. RESULTS: Visual analogue scale (VAS) values at rest were as follows: G1 vs G2: 24 h, 19 (sd 23) vs 6 (13); 48 h, 4 (10) vs 11 (21); and 72 h, 12 (22) vs 13 (21). VAS values during coughing and mobilization were also comparable. Total volume of epidural infusion was 904 (114) ml in G1 vs 892 (154) ml in G2. The incidence of side-effects (nausea, vomiting, and motor block) was low and not different between the groups. CONCLUSIONS: Pre-incisional epidural analgesic infusion did not provide pre-emptive analgesia compared with administration started at the end of surgery, but both groups had low pain scores.     

Analysis of pooled data from two pivotal controlled trials on the efficacy of topiramate in the prevention of migraine

CONTEXT: A substantial proportion of the patient population with migraine headache should be considered for preventive treatment based on the frequency and disability associated with this disorder. Use of the anticonvulsant topiramate was previously examined in two large, double-blind, randomized, placebo-controlled clinical trials of a subset of patients who have 3 to 12 migraine episodes per month. OBJECTIVE: To better characterize the efficacy of topiramate for prevention of migraine, with or without aura, by pooling and analyzing data from the two large clinical trials. METHODS: The pooled intent-to-treat population included 937 patients receiving topiramate at one of three dosages (50 mg/d, 100 mg/d, 200 mg/d) or placebo. Outcome measures included change in mean monthly migraine frequency and categorical responder rate throughout the 26-week doubleblind phase. Results: At daily doses of 100 and 200 mg, topiramate was associated with significant reductions in mean monthly migraine frequency throughout the double-blind phase compared with placebo (P<.001). Significantly more patients treated with these topiramate doses exhibited high-percentage reductions in monthly migraine frequency (>/=50% [P<.001], >/=75% [P<.001], 100% [P=.049]) versus placebo. The most common adverse events included anorexia, cognitive deficits, diarrhea, fatigue, nausea, and paresthesia. Topiramate (100 mg/d, 200 mg/d) was associated with significant and sustained reductions in mean monthly migraine frequency beginning as early as 1 week into therapy. CONCLUSION: Pooled efficacy data from two large, similarly designed, placebo-controlled migraine-prevention trials demonstrated that a statistically significant proportion of patients using topiramate met or exceeded two main outcome guidelines recommended by the International Headache Society (>/=50% and >/=75% reduction in frequency of monthly attacks). Based on efficacy and tolerability, topiramate at a dosage of 100 mg per day (50 mg twice daily) should be the target dosage for most patients with migraine.