Directed evolution of formate dehydrogenase from Candida boidinii for improved stability during entrapment in polyacrylamide

In two cycles of an error-prone PCR process, variants of formate dehydrogenase from Candida boidinii were created which revealed an up to 4.4-fold (440%) higher residual activity after entrapment in polyacrylamide gels than the wild-type enzyme. These were identified in an assay using single precursor molecules of polyacrylamide instead of the complete gel for selection. The stabilization resulted from an exchange of distinct lysine, glutamic acid, and cysteine residues remote from the active site, which did not affect the kinetics of the catalyzed reaction. Thermal stability increased at the exchange of lysine and glutamic acid, but decreased due the exchange of cysteine. Overall, the variants reveal very suitable properties for application in a technical synthetic process, enabling use of entrapment in polyacrylamide as an economic and versatile immobilization method.     

On the origin of the Synodontis catfish species flock from Lake Tanganyika

Species flocks within Great Lakes provide unique insights into the factors affecting diversification. Lake Tanganyika (LT) is of particular interest because it contains many endemic groups for which general factors affecting diversification can be discerned. Here, we present the first phylogenetic study of the LT Synodontis (Siluriformes, Mochokidae) species flock using mtDNA sequence data. Our data reveal some previously unrecognized species diversity and indicate that the LT species flock is not monophyletic, and that two closely related clades of endemics may have independently colonized LT. Other comparable small species flocks are characterized by a single colonization event. Molecular date estimates of the timing of the initial within-lake diversification of the LT endemics, based on a fossil calibration, are comparable to those reported for other groups, suggesting that extrinsic factors maybe important common causes of clade diversification. The basal divergence in the sampled Synodontis reveals an East-West African faunal split seen in many terrestrial, but few aquatic groups, the timing of which coincides with East African rifting events.     

Non-SELEX: selection of aptamers without intermediate amplification of candidate oligonucleotides

Aptamers are typically selected from libraries of random DNA (or RNA) sequences through systematic evolution of ligands by exponential enrichment (SELEX), which involves several rounds of alternating steps of partitioning of candidate oligonucleotides and their PCR amplification. Here we describe a protocol for non-SELEX selection of aptamers--a process that involves repetitive steps of partitioning with no amplification between them. Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), which is a highly efficient affinity method, is used for partitioning. NECEEM also facilitates monitoring of bulk affinity of enriched libraries at every step of partitioning and screening of individual clones for their affinity to the target. NECEEM allows all clones to be screened prior to sequencing, so that only clones with suitable binding parameters are sequenced. The entire protocol can be completed in 1 wk, whereas conventional SELEX protocols take several weeks even in a specialized industrial facility.     

Eating in the Absence of Hunger: A Genetic Marker for Childhood Obesity in Prepubertal Boys?

Objective: Eating in the absence of hunger (EAH) may be a behavioral trait through which obesity‐promoting genes promote positive energy balance. The primary aim of this study was to compare children born at high vs. low risk for obesity with respect to EAH at 5 years of age. Research Methods and Procedures: This was an observational investigation of families enrolled in the University of Pennsylvania and The Children's Hospital of Philadelphia's Infant Growth Study. Five‐year‐old children born at high (N = 28) or low (N = 25) risk for obesity on the basis of maternal prepregnancy body weight were evaluated at a hospital‐based laboratory. Children consumed 11 snack foods ad libitum after consuming an ad libitum dinner and reporting fullness. Parents reported on snack foods at home and their own eating styles. Nutritive sucking at 3 months of age was evaluated by computerized apparatus. Results: EAH in high‐risk boys (mean ± standard error = 326 ± 66 kJ] was more than twice that of low‐risk boys (mean ± standard error = 151 ± 39 kJ), p = 0.03. Among girls, there was a trend for EAH to be associated with increased parental limitations on daughter snack food consumption at home (p = 0.06). EAH was unrelated to 3‐month sucking behavior. Discussion: Genes that promote childhood obesity may partially exert their influence through EAH, an effect that was limited to boys born at risk for obesity. The unique influences of genes and home environment on this trait should be disaggregated in subsequent studies.     

Effects of pollution on human growth and development: an introduction

Pollution is a worldwide problem and its potential to influence the physiology of human populations is great. Studies of human growth and development in relation to pollution have increased in number and quality since the mid-twentieth century. Many studies have found that some pollutants have detrimental effects on human growth, particularly prenatal growth. The heavy metal, lead, is commonly found in human populations and is related to smaller size at birth and studies have reported decrements that range up to about 200 grams. Noise stress from transportation sources also is related to reduced prenatal growth with somewhat smaller decrements reported. Studies of humans exposed to polychlorinated biphenyls, one of the persistent organic pollutants, have reduced size at birth, advanced sexual maturation and altered hormone levels related to thyroid regulation. Thus different pollutants exert effects through different physiological pathways. However, some studies have not observed these effects, which indicates that the situation is complex and requires further study with better study designs. Determining the effects of pollutants on human physiology and growth is difficult as it requires fairly large numbers of subjects who are not purposely exposed but for whom exposure can be measured. These effects of pollutants and the mechanisms of effect require further study to understand and, it is hoped, to blunt or block any detrimental effects on human health and well-being.     

Faster nerve regeneration after sciatic nerve injury in mice over‐expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF‐2) is expressed in the peripheral nervous system and is up‐regulated after nerve lesion. It has been demonstrated that administration of FGF‐2 protects neurons from injury‐induced cell death and promotes axonal regrowth. Using transgenic mice over‐expressing FGF‐2 (TgFGF‐2), we addressed the importance of endogenously generated FGF‐2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild‐type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF‐2. Morphometric evaluation of intact nerves from TgFGF‐2 mice revealed no difference in number and size of myelinated fibers compared to wild‐type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF‐2 over‐expression on Schwann cell proliferation during the early regeneration process, we used BrdU‐labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild‐types. We propose that endogenously synthesized FGF‐2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Spatiotemporal expression of connexin 39 and -43 during myoblast differentiation in cultured cells and in the mouse embryo

Connexin39 (Cx39) and connexin43 (Cx43) are known to be expressed during development of skeletal muscles. Here we have compared the expression pattern of both connexins during differentiation of established C(2)C(12) mouse myoblasts and in the mouse embryo. Cx43 is highly abundant in undifferentiated myoblasts, but no Cx39 protein was detected in these cells. Upon differentiation into myotubes, Cx39 expression increased. The consecutive expression of these connexins was also observed in the mouse embryo. Cx39 and Cx43 were found in different plaques in accordance with the notion that Cx43 is exclusively expressed in myoblasts and Cx39 in myotubes. Thus, differentiating C(2)C(12) cells in culture can serve to study the involvement of gap junctions in myogenesis, since expression of corresponding Cx39 and Cx43 proteins appears to be very similar as in the mouse embryo.     

Spatiotemporal Expression of Connexin 39 and -43 During Myoblast Differentiation in Cultured Cells and in the Mouse Embryo

Connexin39 (Cx39) and connexin43 (Cx43) are known to be expressed during development of skeletal muscles. Here we have compared the expression pattern of both connexins during differentiation of established C₂C₁₂ mouse myoblasts and in the mouse embryo. Cx43 is highly abundant in undifferentiated myoblasts, but no Cx39 protein was detected in these cells. Upon differentiation into myotubes, Cx39 expression increased. The consecutive expression of these connexins was also observed in the mouse embryo. Cx39 and Cx43 were found in different plaques in accordance with the notion that Cx43 is exclusively expressed in myoblasts and Cx39 in myotubes. Thus, differentiating C₂C₁₂ cells in culture can serve to study the involvement of gap junctions in myogenesis, since expression of corresponding Cx39 and Cx43 proteins appears to be very similar as in the mouse embryo.