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.     

Synthesis of a Nano-Silver Metal Ink for Use in Thick Conductive Film Fabrication Applied on a Semiconductor Package

The success of printing technology in the electronics industry primarily depends on the availability of metal printing ink. Various types of commercially available metal ink are widely used in different industries such as the solar cell, radio frequency identification (RFID) and light emitting diode (LED) industries, with limited usage in semiconductor packaging. The use of printed ink in semiconductor IC packaging is limited by several factors such as poor electrical performance and mechanical strength. Poor adhesion of the printed metal track to the epoxy molding compound is another critical factor that has caused a decline in interest in the application of printing technology to the semiconductor industry. In this study, two different groups of adhesion promoters, based on metal and polymer groups, were used to promote adhesion between the printed ink and the epoxy molding substrate. The experimental data show that silver ink with a metal oxide adhesion promoter adheres better than silver ink with a polymer adhesion promoter. This result can be explained by the hydroxyl bonding between the metal oxide promoter and the silane grouping agent on the epoxy substrate, which contributes a greater adhesion strength compared to the polymer adhesion promoter. Hypotheses of the physical and chemical functions of both adhesion promoters are described in detail.     

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.     

Topological studies on the enzymes catalyzing the biosynthesis of Glc-P- dolichol and the triglucosyl cap of Glc3Man9GlcNAc2-P-P-dolichol in microsomal vesicles from pig brain: use of the processing glucosidases I/II as latency markers

In the current model for Glc3Man9GlcNAc2-P-P-Dol assembly, Man5GlcNAc2- P-P-Dol, Man-P-Dol, and Glc-P-Dol are synthesized on the cytoplasmic face of the ER and diffuse transversely to the lumenal leaflet where the synthesis of the lipid-bound precursor oligosaccharide is completed. To establish the topological sites of Glc-P-Dol synthesis and the lipid-mediated glucosyltransfer reactions involved in Glc3Man9GlcNAc2-P-P-Dol synthesis in ER vesicles from pig brain, the trypsin-sensitivity of Glc-P-Dol synthase activity and the Glc-P- Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) was examined in sealed microsomal vesicles. Since ER vesicles from brain do not contain glucose 6-phosphate (Glc 6-P) phosphatase activity, the latency of the lumenally oriented, processing glucosidase I/II activities was used to assess the intactness of the vesicle preparations. Comparative enzymatic studies with sealed ER vesicles from brain and kidney, a tissue that contains Glc 6-P phosphatase, demonstrate the reliability of using the processing glucosidase activities as latency markers for topological studies with microsomal vesicles from non-gluconeogenic tissues lacking Glc 6-P phosphatase. The results obtained from the trypsin-sensitivity assays with sealed microsomal vesicles from brain are consistent with a topological model in which Glc-P-Dol is synthesized on the cytoplasmic face of the ER, and subsequently utilized by the three Glc-P-Dol-mediated GlcTases after "flip-flopping" to the lumenal monolayer.      

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.