AlphaLISA for detection of staphylococcal enterotoxin B free from interference by protein A.

Staphylococcal enterotoxin B (SEB) is an important enterotoxin which is a major reason for food poisoning and a potential biologic agent. Hence, rapid and accurate detection is very important. An amplified luminescent proximity homogeneous assay (AlphaLISA) for SEB detection was established here. Its performance was evaluated on mock specimen and culture supernatant. Its free from the effect of protein A was compared with ELISA. Results showed that the linear range for SEB detection was 25 pg/mL to 25 ng/mL. The detection limitation is 25 pg/mL in buffer and 50 pg/mL in specimen respectively. There was no cross-reaction with other classical SEs or botulinum toxin. AlphaLISA was also tolerant to the matrix of sample and showed good repeatability. The inter-assay coefficient of variation (CV) and intra-assay CV were<10% for buffer and mock specimens, respectively. Besides AlphaLISA detection was free of the interference of protein A (which is the obstacle for immune-based detection of SEs in Staphylococcus aureus culture supernatants): this feature is very important for food-poisoning confirmation caused by SEB contamination. These data suggest that the AlphaLISA established here is well suited for SEB detection in food samples and S. aureus culture supernatants.

Development of a TaqMan Array card to target 21 purulent meningitis-related pathogens.

BACKGROUND: Purulent meningitis (PM) is a serious life-threatening infection of the central nervous system (CNS) by bacteria or fungi and associated with high mortality and high incidence of CNS sequelae in children. However, the conventional cerebrospinal fluid (CSF) culture method is time-consuming and has a low sensitivity. METHODS: Our study developed a real-time PCR-based purulent meningitis-TaqMan array card (PM-TAC) that targeted 21 PM-related pathogens and could produce results within 3 h. Primers and probes were adapted from published sources possibly. The performance of them were evaluated and optimized and then they were spotted on TAC. RESULTS: The PM-TAC showed a sensitivity and specificity of 95 and 96%, respectively. For all of the 21 targeted pathogens, the PM-TAC assay had a LOD ranging from 5 copies/reaction to 100 copies/reaction, an intra-assay variation of 0.07-4.45%, and an inter-assay variation of 0.11-6.81%. Of the 15 CSF samples collected from patients with PM after empiric antibiotic therapies, the positive rate was 53.3% (8/15) for our PM-TAC assay but was only 13.3% (2/15) for the CSF culture method. Of the 17 CSF samples showing negative CSF culture, the PM-TAC assay identified a case of Neisseria meningitidis infection. Furthermore, all of the 10 CSF samples from patients without CNS infection showed negative for the PM-TAC assay. CONCLUSIONS: Our PM-TAC assay also demonstrated that the pathogen loads in the CSF samples correlated with the severity of PM. Thus, the PM-TAC may be helpful to improve the prognosis of PM and clinical outcomes from antibiotic therapies.

TAp73 promotes cell survival upon genotoxic stress by inhibiting p53 activity.

p53 plays a key role in regulating DNA damage response by suppressing cell cycle progression or inducing apoptosis depending on extent of DNA damage. However, it is not clear why mild genotoxic stress favors growth arrest, whereas excessive lesions signal cells to die. Here we showed that TAp73, a p53 homologue thought to have a similar function as p53, restrains the transcriptional activity of p53 and prevents excessive activation of its downstream targets upon low levels of DNA damage, which results in cell cycle arrest. Extensive DNA damage triggers TAp73 depletion through ubiquitin/proteasome-mediated degradation of E2F1, leading to enhanced transcriptional activation by p53 and subsequent induction of apoptosis. These findings provide novel insights into the regulation of p53 function and suggest that TAp73 keeps p53 activity in check in regulating cell fate decisions upon genotoxic stress.

Regulation of early signaling and gene expression in the alpha-particle and bystander response of IMR-90 human fibroblasts.

BACKGROUND: The existence of a radiation bystander effect, in which non-irradiated cells respond to signals from irradiated cells, is well established. To understand early signaling and gene regulation in bystander cells, we used a bio-informatics approach, measuring global gene expression at 30 minutes and signaling pathways between 30 minutes and 4 hours after exposure to alpha-particles in IMR-90 fibroblasts. METHODS: We used whole human genome microarrays and real time quantitative PCR to measure and validate gene expression. Microarray analysis was done using BRB-Array Tools; pathway and ontology analyses were done using Ingenuity Pathway Analysis and PANTHER, respectively. We studied signaling in irradiated and bystander cells using immunoblotting and semi-quantitative image analysis. RESULTS: Gene ontology suggested signal transduction and transcriptional regulation responding 30 minutes after treatment affected cell structure, motility and adhesion, and interleukin synthesis. We measured time-dependent expression of genes controlled by the NF-kappaB pathway; matrix metalloproteinases 1 and 3; chemokine ligands 2, 3 and 5 and interleukins 1beta, 6 and 33. There was an increased response of this set of genes 30 minutes after treatment and another wave of induction at 4 hours. We investigated AKT-GSK3beta signaling and found both AKT and GSK3beta are hyper-phosphorylated 30 minutes after irradiation and this effect is maintained through 4 hours. In bystander cells, a similar response was seen with a delay of 30 minutes. We proposed a network model where the observed decrease in phosphorylation of beta-catenin protein after GSK3beta dependent inactivation can trigger target gene expression at later times after radiation exposure CONCLUSIONS: These results are the first to show that the radiation induced bystander signal induces a widespread gene expression response at 30 minutes after treatment and these changes are accompanied by modification of signaling proteins in the PI3K-AKT-GSK3beta pathway.

Sp1 and p73 activate PUMA following serum starvation.

p53-upregulated modulator of apoptosis (PUMA) plays an essential role in p53-dependent apoptosis following DNA damage. PUMA also mediates apoptosis independent of p53. In this study, we investigated the role and mechanism of PUMA induction in response to serum starvation in p53-deficient cancer cells. Following serum starvation, the binding of Sp1 to the PUMA promoter significantly increased, whereas inhibition of Sp1 completely abrogated PUMA induction. p73 was found to be upregulated by serum starvation and mediate PUMA induction through the p53-binding sites in the PUMA promoter. Sp1 and p73beta appeared to cooperatively activate PUMA transcription, which is inhibited by the phosphoinsitide 3-kinase (PI3K)-protein kinase B (AKT) pathway. Furthermore, knockdown of PUMA suppressed serum starvation-induced apoptosis in leukemia cells. Our results suggest that transcription factors Sp1 and p73 mediate p53-independent induction of PUMA following serum starvation to trigger apoptosis in human cancer cells.

BH3 mimetics to improve cancer therapy; mechanisms and examples.

Tumor cell survival is highly dependent on the expression of certain pro-survival Bcl-2 family proteins. An attractive therapeutic approach is to inhibit these proteins using agents that mimic the Bcl-2 homology 3 (BH3) domains of the proapoptotic Bcl-2 family members, which neutralize these proteins by binding to their surface hydrophobic grooves. A number of BH3 mimetic peptides and small molecules have been described, a few of which have advanced into clinical trials. Recent studies have highlighted ABT-737, a bona fide BH3 mimetic and potent inhibitor of antiapoptotic Bcl-2 family members, as a promising anticancer agent. This review summarizes recent advances in understanding the mechanisms of action of BH3 domains and several classes of BH3 mimetics, as well as the prospects of using these agents to improve cancer therapy.

PUMA Dissociates Bax and Bcl-X(L) to induce apoptosis in colon cancer cells.

PUMA is a BH3-only Bcl-2 family protein that plays an essential role in DNA damage-induced apoptosis. PUMA interacts with anti-apoptotic Bcl-2 and Bcl-X(L) and is dependent on Bax to induce apoptosis. In this study, we investigated how the interactions of PUMA with the antiapoptotic proteins coordinate with Bax to initiate apoptosis in HCT116 colon cancer cells. We found that Bcl-X(L) was most effective among several antiapoptotic proteins in suppressing PUMA-induced apoptosis and PUMA-dependent apoptosis induced by the DNA-damaging agent adriamycin. Mutant Bcl-X(L) that cannot interact with Bax was unable to protect cells from PUMA-mediated apoptosis. Knockdown of Bcl-X(L) by RNA interference significantly enhanced PUMA-mediated apoptosis in HCT116 cells but not in PUMA-knockout cells. Furthermore, Bax was found to be dissociated preferentially from Bcl-X(L) in HCT116 cells but not in the PUMA-knockout cells, in response to PUMA induction and adriamycin treatment. PUMA inhibited the association of Bax and Bcl-X(L) in vitro by directly binding to Bcl-X(L) through its BH3 domain. Finally, we found that wild-type Bax, but not mutant Bax deficient in either multimerization or mitochondrial localization, was able to restore PUMA-induced apoptosis in the BAX-knockout cells. Together, these results indicate that PUMA initiates apoptosis in part by dissociating Bax and Bcl-X(L), thereby promoting Bax multimerization and mitochondrial translocation.

Tight association of hepatocellular carcinoma with HBV infection in North China.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second most common cancer in China. Hepatitis B and C viruses (HBV and HCV) and aflatoxins are known risk factors for HCC, but the etiological status of these factors in HCC development is not clear. This study was undertaken to define the absolute importance of HBV in hepatocarcinogenesis of North China. METHODS: A consecutive series of 119 patients with pathologically proven HCC were collected from North China during January 1998 to December 2000 by the Cancer Hospital of the Chinese Academy of Medical Sciences, Beijing. Serum HBsAg, anti-HBc and anti-HCV were negative HBV sero-markers. The HBV X gene was analyzed for its expression by PCR, DNA sequencing, and immunohistochemistry. RESULTS: In the 119 HCC patients, 82.4% (98/119) were HBsAg seropositive. When a comprehensive set of HBV markers were detected, the HBV infection rate in these HCC patients was 99.2% (118/119). Of the patients, 11.8%(14/119) were found to be anti-HCV positive. But all the anti-HCV positive HCC patients were co-infected with HBV. CONCLUSIONS: HBV infection is virtually ubiquitous in HCC patients in North China. The tight association of HBV with HCC strongly suggests the dominant role of HBV infection in causing hepatocellular carcinoma. About 11.8% of HCC patients being HCV-related are co-infected with HBV.

Dominant role of hepatitis B virus and cofactor role of aflatoxin in hepatocarcinogenesis in Qidong, China.

We assessed the separate and combined effects of hepatitis B virus (HBV), hepatitis C virus (HCV), and aflatoxin in causing hepatocellular carcinoma (HCC) in Qidong, China. A consecutive series of 181 pathologic-diagnosed HCC cases were studied for hepatitis B surface antigen (HBsAg), anti-HBc, HBV X gene sequence, anti-HCV, the 249ser-p53 mutation, and chronic hepatitis pathology. Each of the 181 incident HCC cases had markers for HBV infection and hepatitis pathology; only 6 of 119 cases were coinfected with HCV. The 249ser-p53 mutation was found in 54% (97/181) of HCC cases and in all 7 cases with tissue for analysis from the hepatitis cohort but in none of 42 matched cases from Beijing. The estimated cumulative dose of aflatoxin B1 in these 7 cases ranged from 0.13 to 0.49 mg/kg. Follow-up data through 13.25 years on a cohort of 145 men with chronic HBV hepatitis showed that the relative risk from aflatoxin exposure was 3.5 (1.5-8.1). A similar relative risk was found using 249ser-p53 mutation as a marker for aflatoxin exposure. In conclusion, HBV hepatitis is ubiquitous in Qidong HCC cases, whereas HCV contributes little to its risk. The 249ser-p53 mutation appears to result from coexposure to aflatoxin and HBV infection. Even modest levels of aflatoxin exposure tripled the risk of HCC in HBV-infected men.

Prevention and control of hepatitis B in China.

About 170 million Chinese are infected chronically with HBV and 10% suffer from chronic hepatitis. Around half a million Chinese die from hepatitis B caused hepatocellular carcinoma and endstage cirrhosis each year. From 1983 to the present, a controlled clinical trial involving 80,000 children on a universal hepatitis B vaccination programme to prevent chronic hepatitis, hepatocellular carcinoma, and endstage cirrhosis was implemented in Qidong, China. A pilot study demonstrated that the HBsAg rate reached the adult level before the fifth year of age, and neonatal vaccination with either plasma-derived or recombinant hepatitis B vaccines provided a similar 75% protective efficacy against HBV infection. The high rate of follow-up and blood tests coverage of the cohorts provided data to show 75% protection at the tenth to eleventh years of age against serum HBsAg and also against prolonged hepatic dysfunction. The strategy of controlling hepatitis B nationwide was based on the universal immunisation of newborns, beginning in cities and then the rural areas. The large-scale vaccine source was provided by domestic plants through technology transfer, first providing plasma-derived vaccine replaced completely by recombinant DNA vaccine in 1997. An official survey in 1999 using a cluster sampling of 25,878 children from 31 provinces reported an average coverage rate of three dose of hepatitis B vaccination of 70.7%, being higher in urban areas. The Ministry of Public Health of China has planned to integrate hepatitis B vaccination into the nationwide EPI program with Government-provided vaccines starting January 1, 2002.