Molecular and developmental studies of a sperm acrosome antigen recognized by HS-63 monoclonal antibody.

A conserved mouse sperm antigen (MSA-63) recognized by a monoclonal antibody (HS-63) was isolated from mouse testes by single-step immunoaffinity chromatography. Isolated MSA-63 preparation was shown to be a group of proteins ranging from 24-84 kDa and with isoelectric points (pIs) ranging from 4.0-6.0 when analyzed by two-dimensional (2-D) gel electrophoresis. Microsequencing techniques were employed to determine the relationships of various protein spots on 2-D gels. Partial amino acid sequences of some protein spots in isolated MSA-63 preparation were shown to be homologous to mouse actins, while others revealed homology only to the SP-10 protein. Rabbit antisera raised against isolated MSA-63 antigen preparation were used to immunoscreen a mouse testis cDNA library. Isolated cDNA clones carrying a 1.2-kb insert were used to obtain nucleotide sequences containing open-reading frames and to deduce the corresponding amino acid sequence of MSA-63. A high degree of homology was observed between MSA-63 and a known human sperm antigen, SP-10, at DNA/protein levels. Amino acid sequences of tryptic peptides derived from protein spots of 24-47 kDa and pIs of 4.2-4.4 were found to be identical to those deduced from isolated cDNA clones. The gene expression of MSA-63 during spermatogenesis in mice was studied using a specific cDNA probe as well as HS-63. It was observed that MSA-63 was not expressed until the postmeiotic stages of spermatogenesis.     

The association of leptin and C-reactive protein with the cardiovascular risk factors and metabolic syndrome score in Taiwanese adults

ABSTRACT: BACKGROUND: Serum C-reactive protein (CRP) and leptin levels have been independently associated with the cardiovascular risk factors. The aim of the present study was to determine if their serum levels were associated with cardiovascular risk factors or metabolic syndrome as well as their correlation in the Taiwanese population. METHODS: This retrospective study included 999 subjects (> 18 y), who underwent a physical examination in Chang-Gung Memorial Hospital-Linkou and Chiayi in Taiwan. The associations between CRP and/or leptin levels and cardiovascular risk factors and metabolic syndrome were determined using independent two sample t-tests to detect gender differences and chi-square tests to evaluate differences in frequencies. To compare the means of the variables measured among the four groups (high and low leptin and high and low CRP), analysis of variance (ANOVA) was used. RESULTS: Both CRP and leptin levels were independently associated with several cardiovascular risk factors, including diabetes, hypercholesterolemia and metabolic syndrome in both men and women (P < 0.05). In addition, a positive correlation between leptin and CRP levels was observed in both genders. Both high-CRP and high-leptin were associated with high blood glucose, waist circumference and serum triglyceride. Whereas increased metabolic syndrome incidence was observed in males with elevated leptin regardless of CRP levels, females with elevated CRP or leptin had increased incidence of metabolic syndrome. CONCLUSION: Both leptin and CRP levels were associated with cardiovascular risk factors as well as metabolic syndrome score in both men and women although gender-specific differences were observed. Thus, CRP and leptin may represent useful biomarkers for predicting the onset of cardiovascular disease or metabolic syndrome in Taiwanese adults. Trial registration IRB/CGMH 100-3514B.     

Molecular and structural evolution of Citrus satellite DNA

Highly repeated satellite DNA (stDNA) of citric plants was characterized by cloning and sequencing 10–14 repeats of each plant (Citrus limon, C. sinensis, C. ichangensis, Poncirus trifoliata). The monomers are mostly 181 bp in length with a GC-content between 60% and 68% (significantly higher than the average GC-content of the citrus group genomes). Similarity among the repeats indicates that they belong to a satellite family that underwent species-specific modifications, which are reflected in the phylogenetic relationships. Curvature provoked by dA-stretches of the repeats analyzed by gel shifts revealed structural conservation, even though the nucleotide sequences vary among species, thereby probably supporting the heterochromatic structure of stDNA. We show that the species-specific modification of the satellite consensus involves changes in the position and number of dA tracts. The molecule shapes of satellite oligomeres predicted by computer modelling indicate a superhelical structure of the tandem repeats which is in a good agreement with the satellite sequence dendrogram. The contribution of DNA bending elements to the evolution of plant satellite repeats is discussed. Received: 27 November 2000 / Accepted: 12 January 2001     

Phenoxyl free radical formation during the oxidation of the fluorescent dye 2',7'-dichlorofluorescein by horseradish peroxidase. Possible consequences for oxidative stress measurements.

The oxidation of the fluorescent dye 2',7'-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF(.)). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF(.) oxidizes GSH, generating the glutathione thiyl radical (GS(.)), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS(.) radical. Similarly, DCF(.) oxidized NADH, generating the NAD(.) radical that reduced oxygen to superoxide (O-(2)), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H(2)O(2), which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H(2)O(2) production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.     

Mutations in a BTB-Kelch Protein, KLHL7, Cause Autosomal-Dominant Retinitis Pigmentosa

Retinitis pigmentosa (RP) refers to a genetically heterogeneous group of progressive neurodegenerative diseases that result in dysfunction and/or death of rod and cone photoreceptors in the retina. So far, 18 genes have been identified for autosomal-dominant (ad) RP. Here, we describe an adRP locus (RP42) at chromosome 7p15 through linkage analysis in a six-generation Scandinavian family and identify a disease-causing mutation, c.449G→A (p.S150N), in exon 6 of the KLHL7 gene. Mutation screening of KLHL7 in 502 retinopathy probands has revealed three different missense mutations in six independent families. KLHL7 is widely expressed, including expression in rod photoreceptors, and encodes a 75 kDa protein of the BTB-Kelch subfamily within the BTB superfamily. BTB-Kelch proteins have been implicated in ubiquitination through Cullin E3 ligases. Notably, all three putative disease-causing KLHL7 mutations are within a conserved BACK domain; homology modeling suggests that mutant amino acid side chains can potentially fill the cleft between two helices, thereby affecting the ubiquitination complexes. Mutations in an identical region of another BTB-Kelch protein, gigaxonin, have previously been associated with giant axonal neuropathy. Our studies suggest an additional role of the ubiquitin-proteasome protein-degradation pathway in maintaining neuronal health and in disease.     

A comparison of major histocompatibility complex SNPs in Han Chinese residing in Taiwan and Caucasians

Summary Genetic dissection of complex diseases is both important and challenging. The human major histocompatibility complex is involved in many human diseases and genetic mechanisms. This highly polymorphic chromosome region has been extensively studied in Caucasians but not as well in Asians. Thus, we compared genotypic distributions, linkage disequilibria and haplotype blocks between Caucasian and Taiwan’s Han Chinese populations. Moreover, we investigated the population admixture and phylogenetic system in Han Chinese residing in Taiwan. The results show that Taiwan’s Han Chinese differ drastically in genotypic information compared with Caucasians but are relatively homogeneous among the three major ethnic subgroups, Minnan, Hakka and Mainlanders. Differences in allele frequency (AF) between Taiwanese and Caucasians in some disease-associated loci may reveal clues to differences in disease prevalence. The results of ethnic heterogeneity imply that public databases should be used with caution in cases where the study population(s) differs from the population characterized in the database. The high homogeneity we observed among the Taiwanese subpopulations mitigates the possibility of spurious association caused by ignoring population stratification in Taiwanese disease gene association studies. These results are useful for understanding our genetic background and designing future disease gene mapping studies.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

A genome-wide study of preferential amplification/hybridization in microarray-based pooled DNA experiments

Microarray-based pooled DNA methods overcome the cost bottleneck of simultaneously genotyping more than 100000 markers for numerous study individuals. The success of such methods relies on the proper adjustment of preferential amplification/hybridization to ensure accurate and reliable allele frequency estimation. We performed a hybridization-based genome-wide single nucleotide polymorphisms (SNPs) genotyping analysis to dissect preferential amplification/hybridization. The majority of SNPs had less than 2-fold signal amplification or suppression, and the lognormal distributions adequately modeled preferential amplification/hybridization across the human genome. Comparative analyses suggested that the distributions of preferential amplification/hybridization differed among genotypes and the GC content. Patterns among different ethnic populations were similar; nevertheless, there were striking differences for a small proportion of SNPs, and a slight ethnic heterogeneity was observed. To fulfill appropriate and gratuitous adjustments, databases of preferential amplification/hybridization for African Americans, Caucasians and Asians were constructed based on the Affymetrix GeneChip Human Mapping 100 K Set. The robustness of allele frequency estimation using this database was validated by a pooled DNA experiment. This study provides a genome-wide investigation of preferential amplification/hybridization and suggests guidance for the reliable use of the database. Our results constitute an objective foundation for theoretical development of preferential amplification/hybridization and provide important information for future pooled DNA analyses.