From genes to proteins: High‐throughput expression and purification of the human proteome

The development of high‐throughput methods for gene discovery has paved the way for the design of new strategies for genome‐scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high‐throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high‐throughput expression system for protein production. A prototype was designed to demonstrate the feasibility of our approach. Using a PCR‐based strategy, 72 unique IMAGE cDNA clones have been used to create an array of recombinant baculoviruses in a 96‐well microtiter plate format. Forty‐two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were performed in 96‐well microtiter plates, and are thus amenable to automation. Each recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expressed from this system are currently being analyzed for functional and biochemical properties. J. Cell. Biochem. 80:187–191, 2000. © 2000 Wiley‐Liss, Inc.     

Exercise training in heart failure

Chronic heart failure (CHF) is a common condition with a poor prognosis. It is associated with poor exercise tolerance and debilitating symptoms. These symptoms appear to be associated with pathophysiological changes that occur systemically in the patient with CHF. Exercise training in carefully selected patients has been shown to be safe and to improve exercise capacity. Many of the pathophysiological abnormalities of CHF are improved by training. Some studies have suggested a possible improvement in morbidity and mortality with training. This review analyzes the controlled clinical trials of exercise training in CHF published to date.     

Obesity and eating behaviour in a three-generation chilean family with carriers of the Thr150Ile mutation in the melanocortin-4 receptor gene

It has been proposed that functional mutations in the melanocortin 4 receptor (MC4R) gene have an important impact in body mass index, being considered as a major susceptibility gene for obesity. A number of mutations have been reported in the MC4R gene in subjects from different countries and ethnic groups. However, no reports of MC4R mutations are have been published for South American populations. In this study, DNA samples of thirty-two unrelated obese women of Chilean origin were examined to search for genetic variants in the single exon of the gene through the use of single strand conformational polymorphism techniques and direct sequencing, leading to the identification of a Thr150Ile mutation in heterozygous status. The evaluation of family relatives of the index case for this mutation using PCR-RFLP analysis, identified two additional carriers in a three-generation family. Obesity, eating behavior and body composition phenotypes in this family revealed a possible relation of this variant with obesity in the presence of reduced penetrance. According to our knowledge, this is the first report of MC4R mutations in South American populations.     

Spatial and temporal expression of histone demethylase,Kdm2a,during murine molar development

The histone demethylase, lysine (K)-specific demethylase 2A (Kdm2a), is highly conserved and expressed ubiquitously. Kdm2a can regulate cell proliferation and osteo/dentinogenic, adipogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs) derived from dental tissue. We used quantitative real-time RT-PCR analysis and immunohistochemistry to detect Kdm2a expression during development of the murine molar at embryonic days E12, E14, E16 and E17 and postnatal days P3 and P14. Immunohistochemistry results showed no positive staining of Kdm2a at E12. At E14, Kdm2a was expressed weakly in the inner enamel epithelium, stellate reticulum cells and dental sac. At E16, Kdm2a was expressed mainly in the inner and outer enamel epithelium, stratum intermedium and dental sac, but weaker staining was found in cervical loop and dental papilla cells adjacent to the basement membrane. At E17, the strongest Kdm2a staining was detected in the ameloblasts and stronger Kdm2a staining also was detected in the stratum intermedium, outer enamel epithelium and dental papilla cells compared to the expression at E16. Postnatally, we found that Kdm2a was localized in secretory and mature ameloblasts and odontoblasts, and dentin was unstained. Real-time RT-PCR showed that Kdm2a mRNA levels in murine germ cells increased from E12 to E14 and from E14 to E16; no significant change occurred at E16, E17 or P3, then the levels decreased at P14 compared to P3. Kdm2a expression may be closely related to cell proliferation, to ameloblast and odontoblast differentiation and to the secretion of extracellular enamel and dentin during murine tooth development.     

Trends in Height and BMI of 6‐Year‐Old Children during the Nutrition Transition in Chile

Objective: We analyzed trends in height and BMI and their interaction in 6‐year‐old Chilean children over the last 15 years. Research Methods and Procedures: We calculated height for age z‐score (HAZ), BMI z‐score, prevalence of obesity, underweight, and stunting from cross‐sectional national school‐based annual population surveys in 1987, 1990, 1993, 1996, 2000, and 2002. Using mixed model analysis, we determined the risk of obesity according to height over time as odds ratios (ORs) and 95% confidence interval and the potential influence of height and year of study on BMI z‐score. Results: Over the study period, height increased by 2.8 cm in boys and 2.6 cm in girls, whereas stunting declined from 5% to 2% in both. Tallness increased by ～2%, BMI z‐score increased from +0.3 to +0.65 in boys and to +0.62 in girls, and HAZ increased from ?0.47 in boys and ?0.45 in girls to 0 in 2002. Underweight declined from 4% to 3%, whereas obesity rose from 5% to ～14%. The probability of obesity among tall children was significantly greater than that for normal height children (OR, 2.3 to 3.5). The lowest obesity risk was observed between ?2 and ?1 HAZ. The OR for obesity in the stunted relative to normal height children was variable, ranging from 1.23 to 0.65, whereas it was significant and consistently positive (1.1 to 1.7) for boys and girls when it was compared with the lowest obesity risk according to height. Discussion: Tallness is significantly associated with increased obesity risk in children, while stunting is also associated, but to a lesser degree.