Functional studies of yeast actin mutants corresponding to human cardiomyopathy mutations

The molecular mechanisms by which different mutations in actin lead to distinct cardiomyopathies are unknown. Here, actin mutants corresponding to α-cardiac actin mutations causing hypertrophic cardiomyopathy [(HCM) P164A and A331P] and dilated cardiomyopathy [(DCM) R312H and E361G] were expressed in yeast and purified for in vitro functional studies. While P164A appeared unaltered compared to wild-type (WT) actin, A331P function was impaired. A331P showed reduced stability in circular dichroism melting experiments; its monomer unfolding transition was 10°C lower compared to WT actin. Additionally, in vitro filament formation was hampered, and yeast cell cultures were temperature sensitive, implying perturbations in actin–actin interactions. Filament instability of the A331P mutant actin could lead to actomyosin dysfunction observed in HCM. Yeast strains harboring the R312H mutation did not grow well in culture, suggesting that cell viability is compromised. The E361G substitution is located at an α-actinin binding region where the actin filament is anchored. The mutant actin, though unaltered in the in vitro motility and standard actomyosin functions, had a threefold reduction in α-actinin binding. This could result in impairment of force-transduction in muscle fibers, and a DCM phenotype. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of quantitative PCR and branched-chain DNA assay for detection of hepatitis B virus DNA in sera from hepatocellular carcinoma and liver transplant patients

This study evaluated the applicability of quantitative PCR (Q-PCR) and branched-chain DNA assays for detection of hepatitis B virus (HBV) DNA in sera. For 42 samples, the detection rates were 81 and 41%, respectively, with a correlation coefficient of 0.633. The Q-PCR is useful for early monitoring of HBV load in high-risk patients.     

Recurrent and novel mutations of GCDH gene in Chinese glutaric acidemia type I families

Glutaric acidemia type I is caused by mutations of the glutaryl-CoA dehydrogenase (GCDH) gene resulting in loss of GCDH enzyme activity. Patients present with progressive dystonia and lesions in basal ganglia. Dietary treatment, when instituted from the early neonatal period, markedly reduces dystonia and morbidity. Early diagnosis and prenatal diagnosis will be facilitated by knowledge of locally prevalent GCDH mutations. Several common GCDH mutations have been found in different ethnic groups. GCDH mutations were studied in 5 Chinese glutaric acidemia type I families. We detected two novel recurrent mutations (A219T and IVS10-2A>C) which were found in two unrelated families. An asymptomatic carrier of IVS10-2A>C was also found on screening of 120 individuals. Other mutations were identified, including two other novel (R386G & IVS3+1G>A) and two known mutations (G178R & R355H). Fibroblasts from patients carrying the novel mutations were confirmed to be deficient for GCDH activity. This is the first report of GCDH mutations describing recurrent mutations in Chinese patients. The carrier rate of IVS10-2A>C may be particularly high in Chinese.     

Detection and deletion of motion artifacts in electrogastrogram using feature analysis and neural networks

Electrogastrogram is a surface measurement of gastric myoelectrical activity, and electrogastrography has been an attractive method for physiological and pathophysiological studies of the stomach due to its nonivasive nature. Motion artifacts, however, ruin the electrogastrogram (EGG), and make the analysis very difficult and sometimes even impossible. They must be eliminated from EGG signals before analysis. Up to now, this can only be done by visual inspection, which is not only time-consuming but also subjective. In this study, a method using feature analysis and neural networks has been developed to realize automatic detection and elimination of the motion artifacts in EGG recordings by computer. Experiments were conducted to investigate the characteristics of different motion artifacts. Useful features were extracted, and different combinations of the features used as the input of the neural network were compared to obtain the optimal performance for the detection of motion artifacts using the artificial neural network.     

Distribution of type I, II, III and V in the pepsin solubilized collagens in bovine menisci

The inner one-third (IM) of both lateral and medial menisci resembles hyaline cartilage, both in gross appearance and histological examination, while the outer two-thirds (OM) is fibrocartilaginous in appearance. Collagen was extracted with pepsin, purified with anion and cation exchange column chromatographies and examined by differential salt precipitation, cyanogen bromide-peptide analysis and SDS gel electrophoresis. IM constitutes approximately 10% of the wet weight of whole meniscus, is made up of 70% collagen of which 34% is pepsin soluble. IM is composed of 60% type II and 40% type I collagen. OM is made up of 80% collagen of which 17% is pepsin soluble. The predominant collagen of OM is type I with a trace amount of types III and V (less than 1%).     

Pharmacokinetic analysis of pegylated megakaryocyte growth and development factor in humans

Phase I studies with pegylated megakaryocyte growth and development factor (PEG-rHuMGDF), a c-Mpl ligand that stimulates megakaryopoiesis, have demonstrated that PEG-rHuMGDF is biologically active alone and causes a dose-related enhancement of platelet recovery when administered after chemotherapy. Here we report the dose-ranging pharmacokinetics of PEG-rHuMGDF. Pre-injection blood samples were drawn daily for pharmacokinetic studies on 43 patients. An ELISA, established using PEG-rHuMGDF as the standard, was able to quantitate Mpl ligand at concentrations > 0.02 ng/mL. Over the dose range 0.03 to 5.0 microg/kg/day, subcutaneous administration produced linear increases in steady-state serum levels. Maximum levels of PEG-rHuMGDF attained after 5.0 microg/kg/day were 5.88 to 10.9 ng/mL. After discontinuation of PEG-rHuMGDF, concentrations of Mpl ligand returned to baseline within 5 days. The pharmacokinetics were best described by a one-compartment model with first-order absorption, an absorption delay, and non linear clearance over the first 48 hours. The mean terminal half-life was 33.3 + 16.7 hours, and the average apparent at steady state was 27.7 + 14.0 mL/h/kg; both were independent of administered dose. The apparent clearance of PEG-rHuMGDF was not predicted by platelet count. Administration of chemotherapy and Filgrastim did not alter the pharmacokinetics of PEG-rHuMGDF.     

Development and validation of a CGH microarray for clinical cytogenetic diagnosis.

PURPOSE: We developed a microarray for clinical diagnosis of chromosomal disorders using large insert genomic DNA clones as targets for comparative genomic hybridization (CGH). METHODS: The array contains 362 FISH-verified clones that span genomic regions implicated in over 40 known human genomic disorders and representative subtelomeric clones for each of the 41 clinically relevant human chromosome telomeres. Three or four clones from almost all deletion or duplication genomic regions and three or more clones for each subtelomeric region were included. We tested chromosome microarray analysis (CMA) in a masked fashion by examining genomic DNA from 25 patients who were previously ascertained in a genetic clinic and studied by conventional cytogenetics. A novel software package implemented in the R statistical programming language was developed for normalization, visualization, and inference. RESULTS: The CMA results were entirely consistent with previous cytogenetic and FISH findings. For clone by clone analysis, the sensitivity was estimated to be 96.7% and the specificity was 99.1%. Major advantages of this selected human genome array include the following: interrogation of clinically relevant genomic regions, the ability to test for a wide range of duplication and deletion syndromes in a single analysis, the ability to detect duplications that would likely be undetected by metaphase FISH, and ease of confirmation of suspected genomic changes by conventional FISH testing currently available in the cytogenetics laboratory. CONCLUSION: The array is an attractive alternative to telomere FISH and locus-specific FISH, but it does not include uniform coverage across the arms of each chromosome and is not intended to substitute for a standard karyotype. Limitations of CMA include the inability to detect both balanced chromosome changes and low levels of mosaicism.