Clostridium sordellii phospholipase C: gene cloning and comparison of enzymatic and biological activities with those of Clostridium perfringens and Clostridium bifermentans phospholipase C

The gene encoding Clostridium sordellii phospholipase C (Csp) was cloned and expressed as a histidine-tagged (His-tag) protein, and the protein was purified to compare its enzymatic and biological activities with those of Clostridium perfringens phospholipase C (Cpa) and Clostridium bifermentans phospholipase C (Cbp). Csp was found to consist of 371 amino acid residues in the mature form and to be more homologous to Cbp than to Cpa. The egg yolk phospholipid hydrolysis activity of the His-tag Csp was about one-third of that of His-tag Cpa, but the hemolytic activity was less than 1% of that of His-tag Cpa. His-tag Csp was nontoxic to mice. Immunization of mice with His-tag Cbp or His-tag Csp did not provide effective protection against the lethal activity of His-tag Cpa. These results indicate that Csp possesses similar molecular properties to Cbp and suggest that comparative analysis of toxic and nontoxic clostridial phospholipases is helpful for characterization of the toxic properties of clostridial phospholipases.     

The effect of pitch in multislice spiral/helical CT

The purpose of this study is to understand the effect of pitch on raw data interpolation in multislice spiral/helical computed tomography (CT) and provide guidelines for scanner design and protocol optimization. Multislice spiral CT is mainly characterized by the three parameters: the number of detector arrays, the detector collimation, and the table increment per x-ray source rotation. The pitch in multislice spiral CT is defined as the ratio of the table increment over the detector collimation in this study. In parallel to the current framework for studying longitudinal image resolution, the central fan-beam rays of direct and opposite directions are considered, assuming a narrow cone-beam angle. Generally speaking, sampling in the Radon domain by the direct and opposite central rays is nonuniform along the longitudinal axis. Using a recently developed methodology for quantifying the sensitivity of signal reconstruction from nonuniformly sampled finite points, the effect of pitch on raw data interpolation is analyzed in multislice spiral CT. Unlike single-slice spiral CT, in which image quality decreases monotonically as the pitch increases, the sensitivity of raw data interpolation in multislice spiral CT increases in an alternating way as the pitch increases, suggesting that image quality does not decrease monotonically in this case. The most favorable pitch can be found from the sensitivity-pitch plot for any given set of multislice spiral CT parameters. An example for four-slice spiral CT is provided. The study on the effect of pitch using the sensitivity analysis approach reveals the fundamental characteristics of raw data interpolation in multislice spiral CT, and gives insights into interaction between pitch and image quality. These results may be valuable for design of multislice spiral CT scanners and imaging protocol optimization in clinical applications.     

Education modifies the effect of apolipoprotein epsilon 4 on cognitive decline

OBJECTIVES: To assess the influence of education on the association between apolipoprotein E and cognitive change. DESIGN: Prospective cohort. PARTICIPANTS: HMO-based sample of 2168 non-demented community-dwelling elderly followed over 6 years. MEASUREMENTS: Generalized estimating equations were used with the difference between baseline and follow-up cognitive abilities screening instrument (CASI) as the outcome variable. RESULTS: At follow-up, 6% of the sample had a decline of 1.5 S.D. or greater on the CASI. Compared to individuals without an APOE4 allele, individuals with a single APOE4 allele did not have greater CASI decline. By contrast, individuals with two APOE4 alleles experienced greater decline in cognitive performance and the magnitude of that decline decreased as years of educational attainment increased. These relationships held after adjusting for age, gender, ethnicity, depression, diabetes, and history of vascular disease. CONCLUSION: Lower education was associated with steep 4-year cognitive decline for APOE4 homozygotes but not for APOE4 heterozygotes. Potentially modifiable host factors such as education could influence the association of high-risk genotypes and cognitive decline.     

The frequency of occurrence of autoantibodies against beta1-adrenoceptors and its clinical relevance in patients with hepatitis virus myocarditis

The aim of this study was to examine the frequency of occurrence of autoantibodies against beta1-adrenoceptors in patients with hepatitis virus myocarditis (HVM) and its possible correlation with clinical characteristics. A total of 103 patients with viral myocarditis were divided into a positive group (HVM group, n=29) and a negative group (Non-HVM group, n=74) according to the laboratory findings regarding their type of hepatitis virus. The study parameters included UCG, ECG, biochemical findings and screening of autoantibodies against beta1-adrenoceptor. It was shown that the positive rate of the hepatitis virus was 28.16% (29/103) in patients with viral myocarditis. The severity of myocardial or liver injuries and the frequency of occurrence of autoantibodies against beta1-adrenoceptors in patients with viral myocarditis were more pronounced, before treatment, in the HVM group than in the Non-HVM group. The positive rates of the antibodies against the hepatitis virus and the autoantibodies against beta1-adrenoceptors were highly consistent in patients with HVM (p<0.05). In conclusion, the frequency of occurrence of the autoantibodies against beta1-adrenoceptors may be one important marker of HVM and, thus, possibly involved in the pathogenesis of the HVM.     

Comparison of in vitro and in vivo alpha/beta ratios for prostate cancer

Parallel in vitro and in vivo studies provide insight into the relationship between clinical response and intrinsic cellular radiosensitivity and may aid in the development of predictive assays. Compilations of radiosensitivity parameters from in vitro experiments can also be used to examine the potential effectiveness of alternative or new treatment plan designs until enough clinical data become available to directly estimate the requisite radiosensitivity parameters. In this work, survival data for six prostate cancer cell lines (ten datasets total) have been extracted from the literature and re-analysed using the linear-quadratic (LQ) survival model. The paired bootstrap technique for regression is used to compute 95% confidence intervals for the estimated radiosensitivity parameters. LQ radiosensitivity parameters derived from the in vitro data are then compared to radiosensitivity parameters derived from clinical data for prostate cancer. Estimates of alpha range from 0.09 to 0.35 Gy(-1) (all cell lines), and the alpha/beta ratio ranges from 1.09 to 6.29 Gy (all cell lines). Point estimates of the repair half-time (PPC-1, TSU-Pr1, PC-3 and DU-145 cell lines) range from 5.7 to 8.9 h (95% confidence interval from 0.26 h to 10.7 h). Differences in the radiosensitivity parameters determined from the data reported by different laboratories are as large as or larger than the differences in radiosensitivity parameters observed among the various prostate cell lines. The reported studies demonstrate that even seemingly small corrections for dose rate effects, such as those expected in high dose rate (HDR) experiments, can sometimes have a significant impact on estimates of alpha and alpha/beta. By neglecting dose rate effects in the analysis of HDR experiments, estimates of the alpha/beta, ratio may be too high by factors as large as 1.3 to 6.2. The half-time for repair derived from the in vitro experiments appears significantly larger (slower repair rate) than estimates derived from the clinical data. However, the prostate radiosensitivity parameters alpha and alpha/beta may be approximately the same in vitro and in vivo. Most of the in vitro data are consistent with an alpha/beta ratio for prostate cancer less than 3 or 4 Gy.     

Urachal malignant fibrous histiocytoma: a case report and review of the literature

Pathologic processes involving the urachus are usually related to inflammatory or sinofistular conditions. Neoplasms rarely arise within this structure, and when they do occur, they are typically epithelial, with mucinous adenocarcinoma being the most common. Mesenchymal lesions, both benign and malignant, have rarely been described in this location. We report the case of a 66-year-old white man who presented with a primary urachal malignant fibrous histiocytoma and died of metastatic disease 20 months after the initial diagnosis. This is an unusual case of malignant fibrous histiocytoma arising in a urachal remnant.     

Optimization of plasmid maintenance in the attenuated live vector vaccine strain Salmonella typhi CVD 908-htrA

The broad objective of the research presented here is to develop a noncatalytic plasmid maintenance system for the stabilization of multicopy expression plasmids encoding foreign antigens in a Salmonella typhi live-vector vaccine strain such as CVD 908-htrA. We have enhanced the maintenance of expression plasmids at two independent levels. First, we removed dependence upon balanced-lethal maintenance systems that involve catalytic enzymes expressed from multicopy plasmids; we accomplished this through incorporation into expression plasmids of a postsegregational killing system based on the noncatalytic hok-sok plasmid addiction system from the antibiotic resistance factor pR1. We also included at least one naturally occurring plasmid partition function in our expression plasmids, which eliminates random segregation of these plasmids, thereby enhancing their inheritance and stability; to accomplish this, we incorporated either the par locus from pSC101, the parA locus from pR1, or both. We monitored the stability of optimized expression plasmids within CVD 908-htrA by quantitating expression of a variant of green fluorescent protein (GFPuv) by using flow cytometry. In this report, we demonstrate the utility of this novel plasmid maintenance system in enhancing the stability of our expression plasmids and go on to show that as the copy number of stabilized plasmids increases, the toxicity of GFPuv synthesis also increases. The implications of these observations for the rational design of immunogenic and protective bacterial live vector vaccines are discussed.     

Neuroprotective effects of IGF-I against TNFalpha-induced neuronal damage in HIV-associated dementia

Human immunodeficiency virus type 1 (HIV-1) infection often results in disorders of the central nervous system, including HIV-associated dementia (HAD). It is suspected that tumor necrosis factor-alpha (TNFalpha) released by activated and/or infected macrophages/microglia plays a role in the process of neuronal damage seen in AIDS patients. In light of earlier studies showing that the activation of the insulin-like growth factor I receptor (IGF-IR) exerts a strong neuroprotective effect, we investigated the ability of IGF-I to protect neuronal cells from HIV-infected macrophages. Our results demonstrate that the conditioned medium from HIV-1-infected macrophages, HIV/CM, causes loss of neuronal processes in differentiated PC12 and P19 neurons and that these neurodegenerative effects are associated with the presence of TNFalpha. Furthermore, we demonstrate that IGF-I rescues differentiated neurons from both HIV/CM and TNFalpha-induced damage and that IGF-I-mediated neuroprotection is strongly enhanced by overexpression of the wt IGF-IR cDNA and attenuated by the antisense IGF-IR cDNA. Finally, IGF-I-mediated antiapoptotic pathways are continuously functional in differentiated neurons exposed to HIV/CM and are likely supported by TNFalpha-mediated phosphorylation of I(kappa)B. All together these results suggest that the balance between TNFalpha and IGF-IR signaling pathways may control the extent of neuronal injury in this HIV-related experimental setting.     

Leiomyosarcoma of the breast: a pathologic and comparative genomic hybridization study of two cases

Leiomyosarcoma is an extremely rare form of primary breast sarcoma. We present the pathologic and genetic findings of two cases of leiomyosarcoma of the breast. The patients were 44 and 52 years of age and they presented with circumscribed masses of 3.0 and 4.5 cm, (greatest dimension) respectively. Microscopically, the two tumors showed diffuse proliferation of spindle cells with oval and blunt-ended nuclei arranged in short fascicles or bundles. There was moderate cytologic atypia in both cases, and 6 and 12 mitotic figures per 10 high power fields, respectively. No epithelial component was identified. The tumor cells were strongly immunoreactive for markers of smooth-muscle differentiation, including desmin, muscle-specific actin, and smooth-muscle actin. Comparative genomic hybridization analysis showed losses of 10q (two of two cases), 13q (two of two cases), 17p (one of two cases), and gains of 1q (one of two cases) and 17p (one of two cases). The patterns of chromosomal imbalances identified in leiomyosarcoma of the breast are similar to those reported in leiomyosarcoma of soft tissue and uterus and are different from those reported for leiomyoma, indicating that these alterations may be important for development of malignant smooth-muscle tumors regardless of site or organ of origin.     

Haplotype analysis in multiple crosses to identify a QTL gene

Identifying quantitative trait locus (QTL) genes is a challenging task. Herein, we report using a two-step process to identify Apoa2 as the gene underlying Hdlq5, a QTL for plasma high-density lipoprotein cholesterol (HDL) levels on mouse chromosome 1. First, we performed a sequence analysis of the Apoa2 coding region in 46 genetically diverse mouse strains and found five different APOA2 protein variants, which we named APOA2a to APOA2e. Second, we conducted a haplotype analysis of the strains in 21 crosses that have so far detected HDL QTLs; we found that Hdlq5 was detected only in the nine crosses where one parent had the APOA2b protein variant characterized by an Ala61-to-Val61 substitution. We then found that strains with the APOA2b variant had significantly higher (P < or = 0.002) plasma HDL levels than those with either the APOA2a or the APOA2c variant. These findings support Apoa2 as the underlying Hdlq5 gene and suggest the Apoa2 polymorphisms responsible for the Hdlq5 phenotype. Therefore, haplotype analysis in multiple crosses can be used to support a candidate QTL gene.