Expression of enzymatically active enkephalinase (neutral endopeptidase) in mammalian cells

A cDNA encoding the rat enkephalinase protein (neutral endopeptidase; EC 3.4.24.11) has been constructed from overlapping lambda gt10 cDNA clones. This cDNA was inserted into an expression plasmid containing the cytomegalovirus enhancer and promoter. When transfected with this plasmid, Cos 7 cells transiently expressed the enkephalinase protein in a membrane-bound state. Recombinant enkephalinase recovered in solubilized extracts from transfected Cos 7 cells was enzymatically active and displayed properties similar to those of the native enzyme with respect to sensitivity to classical enkephalinase inhibitors.     

Antiproliferative Action of Benzodiazepines in Cultured Brain Cells Is Not Mediated Through the Peripheral-Type Benzodiazepine Acceptor

The benzodiazepines, Ro 5-4864, diazepam, clonazepam, and also PK-11195, inhibited, at micromolar concentrations, the proliferation of rat C6 glioma and mouse neuro-2A neuroblastoma cells in culture. The cells possessed high levels of "peripheral-type" high-affinity benzodiazepine binding sites as judged by binding assays and displacement potencies. However, the different potencies and specificities of compounds for the antiproliferative actions and binding affinities for the binding site suggest that the antiproliferative actions were not mediated through the peripheral-type binding site. In support of this, these compounds have also been shown to inhibit proliferation of some nonneuronal cultured cell lines, e.g., mouse SP2/O-Ag 14 hybridoma and rat NCTC epithelial cells, which have no detectable high-affinity peripheral-type benzodiazepine binding sites.     

The radial forearm skin graft-fascial flap

The radial forearm flap has become a versatile flap for upper extremity reconstruction. The use of the forearm flap for hand reconstruction in the patient with previously burned forearms has not been widely appreciated. In those patients whose forearms have been previously split-thickness skin-grafted on fascia, we have employed the reverse radial forearm flap as a skin graft-fascial flap for hand reconstruction and have obtained excellent functional results. Three patients at various intervals postburn are presented to demonstrate use of this flap for wrist contracture release, coverage of arthroplasties, first web space contracture release, and acute salvage of phalanges and tendons. Assessment of the hand's vascular anatomy and careful treatment of the donor area have contributed to no added morbidity and an excellent aesthetic result at the donor site.     

Polymorphisms and linkage disequilibrium in the COL6A1 and COL6A2 gene cluster: novel DNA polymorphisms in the region of a candidate gene for congenital heart defects in Down's syndrome

The COL6A1 and COL6A2 (collagen VI) gene cluster on chromosome 21 is a candidate region for defects leading to congenital heart anomalies in Down's syndrome. We report a variable number of tandem repeats (VNTR) and a restriction fragment length polymorphism (RFLP) in this gene region, detected using a COL6A1 cDNA probe. Linkage disequilibrium relationships were studied among the RFLPs of this gene cluster. The RFLP reported here shows no significant linkage disequilibrium with any others in the region. It has a polymorphism information content value of 0.27, raising the informativity of the locus.     

Experimental evidence of size-selective harvest and environmental stochasticity effects on population demography,fluctuations and non-linearity

Theory and analyses of fisheries data sets indicate that harvesting can alter population structure and destabilise non-linear processes, which increases population fluctuations. We conducted a factorial experiment on the population dynamics of Daphnia magna in relation to size-selective harvesting and stochasticity of food supply. Harvesting and stochasticity treatments both increased population fluctuations. Timeseries analysis indicated that fluctuations in control populations were non-linear, and non-linearity increased substantially in response to harvesting. Both harvesting and stochasticity induced population juvenescence, but harvesting did so via the depletion of adults, whereas stochasticity increased the abundance of juveniles. A fitted fisheries model indicated that harvesting shifted populations towards higher reproductive rates and larger-magnitude damped oscillations that amplify demographic noise. These findings provide experimental evidence that harvesting increases the non-linearity of population fluctuations and that both harvesting and stochasticity increase population variability and juvenescence.     

Faster induction of photosynthesis increases biomass and grain yield in glasshouse-grown transgenic Sorghum bicolor overexpressing Rieske FeS

Sorghum is one of the most important crops providing food and feed in many of the world's harsher environments. Sorghum utilizes the C₄ pathway of photosynthesis in which a biochemical carbon-concentrating mechanism results in high CO₂ assimilation rates. Overexpressing the Rieske FeS subunit of the Cytochrome b₆f complex was previously shown to increase the rate of photosynthetic electron transport and stimulate CO₂ assimilation in the model C₄ plant Setaria viridis. To test whether productivity of C₄ crops could be improved by Rieske overexpression, we created transgenic Sorghum bicolor Tx430 plants with increased Rieske content. The transgenic plants showed no marked changes in abundances of other photosynthetic proteins or chlorophyll content. The steady-state rates of electron transport and CO₂ assimilation did not differ between the plants with increased Rieske abundance and control plants, suggesting that Cytochrome b₆f is not the only factor limiting electron transport in sorghum at high light and high CO₂. However, faster responses of non-photochemical quenching as well as an elevated quantum yield of Photosystem II and an increased CO₂ assimilation rate were observed from the plants overexpressing Rieske during the photosynthetic induction, a process of activation of photosynthesis upon the dark–light transition. As a consequence, sorghum with increased Rieske content produced more biomass and grain when grown in glasshouse conditions. Our results indicate that increasing Rieske content has potential to boost productivity of sorghum and other C₄ crops by improving the efficiency of light utilization and conversion to biomass through the faster induction of photosynthesis.     

Evidence of Commingling in Human Eating Behavior

This investigation tested whether distributions of certain aspects of eating behavior were consistent with the notion of a “mixture model;” that is, two or more distinct Commingled component distributions, consistent with the possibility of major gene action. Undergraduates (n=901) completed self-report trait measures of hunger, disinhibition, and dietary restraint. Variables were residualized for gender and age and transformed to remove skewness. Residualized transformed distributions were tested for departure from unimodality with Hartigan's (14) dip statistic. The distributions of all three aspects of eating behavior were significantly non-unimodal. Next, component multivariate normal distributions were estimated via maximum likelihood. Likelihood ratio tests were employed to compare nested models. A mixture of four distributions with unequal variance-covariance matrices tit significantly better than any more parsimonious model. In sum, these data strongly suggest that the distributions of several measures of eating behavior are composed of four component distributions. This finding is consistent with the possibility of major gene effects for eating behavior.     

Simultaneous determination of azathioprine and 6-mercaptopurine by high-performance liquid chromatography

A specific, sensitive, single-step solid-phase extraction and reversed-phase high-performance liquid chromatographic method for the simultaneous determination of plasma 6-mercaptopurine and azathioprine concentrations is reported. Following solid-phase extraction, analytes are separated on a C₁₈ column with mobile phase consisting of 0.8% acetonitrile in 1 mM triethylamine, pH 3.2, run on a gradient system. Quantitation limits were 5 ng/ml and 2 ng/ml for azathioprine and 6-mercaptopurine, respectively. Peak heights correlated linearly to known extracted standards for 6-mercaptopurine and azathioprine (r = 0.999) over a range of 2–200 ng/ml. No chromatographic interferences were detected.