Changes in various plasma lipid components, glucose, and insulin in Spermophilus lateralis during hibernation

This study evaluated fat mobilization as related to gluconeogenesis in two age groups of hibernating golden-mantled ground squirrels (Spermophilus lateralis). Our experimental group consisted of a total of 16 male and 15 female squirrels. Plasma samples were collected from selected animals being killed weekly from January to March, and the concentration of triglycerides, glycerol (GY), free fatty acids (FFA), total cholesterol, lipase activity, glucose, and insulin, were determined by biochemical assays and radioimmunoassay. Our results showed a mean FFA/GY ratio of five, which was higher than the predicted value of three, suggesting a significant depletion of GY and an enhanced rate of gluconeogenesis via GY to maintain glucose homeostasis in the hibernating animals. The factor of age did not significantly affect plasma lipid components. However, in the male group, plasma glucose levels were significantly higher for adults than for juveniles. Overall, females had significantly higher plasma glucose levels than males (150 ± 11 vs. 110 ± 8 mg%, P < 0.05). In the adult group, a gender influence was also seen on plasma insulin levels, with females' being higher than males' (66 ± 13 vs. 25 ± 3 μIU/ml, P < 0.01). We suggest that during hibernation, female squirrels may have a higher rate of lipolysis and gluconeogenesis along with a lower glucose utilization than their male counterparts. Additionally, adult females may exhibit a higher peripheral insulin resistance during hibernation than adult males, a possibility which merits further study.     

The ionic basis of electrical activity in embryonic cardiac muscle

The intracellular sodium concentration reported for young, embryonic chick hearts is extremely high and decreases progressively throughout the embryonic period, reaching a value of 43 mM immediately before hatching. This observation suggested that the ionic basis for excitation in embryonic chick heart may differ from that responsible for electrical activity of the adult organ. This hypothesis was tested by recording transmembrane resting and action potentials on hearts isolated from 6-day and 19-day chick embryos and varying the extracellular sodium and potassium concentrations. The results show that for both young and old embryonic cardiac cells the resting potential depends primarily on the extracellular potassium concentration and the amplitude and rate of rise of the action potential depend primarily on the extracellular sodium concentration.     

Selective single cell isolation for genomics using microraft arrays

Genomic methods are used increasingly to interrogate the individual cells that compose specific tissues. However, current methods for single cell isolation struggle to phenotypically differentiate specific cells in a heterogeneous population and rely primarily on the use of fluorescent markers. Many cellular phenotypes of interest are too complex to be measured by this approach, making it difficult to connect genotype and phenotype at the level of individual cells. Here we demonstrate that microraft arrays, which are arrays containing thousands of individual cell culture sites, can be used to select single cells based on a variety of phenotypes, such as cell surface markers, cell proliferation and drug response. We then show that a common genomic procedure, RNA-seq, can be readily adapted to the single cells isolated from these rafts. We show that data generated using microrafts and our modified RNA-seq protocol compared favorably with the Fluidigm C1. We then used microraft arrays to select pancreatic cancer cells that proliferate in spite of cytotoxic drug treatment. Our single cell RNA-seq data identified several expected and novel gene expression changes associated with early drug resistance.     

SH3‐like motif‐containing C‐terminal domain of staphylococcal teichoic acid transporter suggests possible function

The negatively charged bacterial polysaccharides—wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two‐component transporter, TagGH, comprising a transmembrane subunit TagG and an ATPase subunit TagH. We determined the crystal structure of the C‐terminal domain of TagH (TagH‐C) to investigate its function. The structure shows an N‐terminal SH3‐like subdomain wrapped by a C‐terminal subdomain with an anti‐parallel β‐sheet and an outer shell of α‐helices. A stretch of positively charged surface across the subdomain interface is flanked by two negatively charged regions, suggesting a potential binding site for negatively charged polymers, such as WTAs or acidic peptide chains. Proteins 2016; 84:1328–1332. © 2016 Wiley Periodicals, Inc.     

In Vivo Neurochemical Characterization of Developing Guinea Pigs and the Effect of Chronic Fetal Hypoxia

Neurochemical Research - The guinea pig is a frequently used animal model for human pregnancy complications, such as oxygen deprivation or hypoxia, which result in altered brain development. To...     

Isomerization of (3S)-2,3-dihydro-5-fluoro-L-tryptophan and of 5-fluoro-L-tryptophan catalyzed by tryptophan synthase: studies using fluorine-19 nuclear magnetic resonance and difference spectroscopy

We are exploring the active site and the mechanism of the pyridoxal phosphate dependent reactions of the bacterial tryptophan synthase alpha 2 beta 2 complex by use of substrate analogues and of reaction intermediate analogues. Fluorine-19 nuclear magnetic resonance studies and absorption spectroscopy are used to study the binding and reactions of the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)- and (3R)-2,3-dihydro-5-fluorotryptophan. Tryptophan synthase specifically and tightly binds the 3S diastereoisomer of both 2,3-dihydro-5-fluoro-D-tryptophan and 2,3-dihydro-5-fluoro-L-tryptophan, whereas it binds 5-fluoro-D-tryptophan more tightly than 5-fluoro-L-tryptophan. Unexpectedly, we find that the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)-2,3-dihydro-5-fluorotryptophan are slowly interconverted by isomerization reactions. Since these isomerization reactions are 10(3)-10(5) times slower than the beta-replacement and beta-elimination reactions catalyzed by tryptophan synthase, they have no biochemical significance in vivo. However, the occurrence of these slow reactions does throw some light on the nature of the active site of tryptophan synthase and its requirements for substrate binding. Our results raise the interesting question of whether tryptophan synthase itself serves a catalytic role in these slow reactions or whether the enzyme simply binds the substrate and pyridoxal phosphate stereospecifically and thus promotes the intrinsic catalytic activity of pyridoxal phosphate.