Carbohydrate- and lipid-enriched meals acutely disrupt glycemic homeostasis by inducing transient insulin resistance in rats

Chronic intake of high-carbohydrate or high-lipid diets is a well-known insulin resistance inducer. This study investigates the immediate effect (1-6 h) of a carbohydrate- or lipid-enriched meal on insulin sensitivity. Fasted rats were refed with standard, carbohydrate-enriched (C), or lipid-enriched (L) meal. Plasma insulin, glucose, and non-esterified fatty acids (NEFA) were measured at 1, 2, 4, and 6 h of refeeding. The glucose-insulin index showed that either carbohydrates or lipids decreased insulin sensitivity at 2 h of refeeding. At this time point, insulin tolerance tests (ITTs) and glucose tolerance tests (GTTs) detected insulin resistance in C rats, while GTT confirmed it in L rats. Reduced glycogen and phosphorylated AKT and GSK3 content revealed hepatic insulin resistance in C rats. Reduced glucose uptake in skeletal muscle subjected to the fatty acid concentration that mimics the high NEFA level of L rats suggests insulin resistance in these animals is mainly in muscle. In conclusion, carbohydrate- or lipid-enriched meals acutely disrupt glycemic homeostasis, inducing a transient insulin resistance, which seems to involve liver and skeletal muscle, respectively. Thus, the insulin resistance observed when those types of diets are chronically consumed may be an evolution of repeated episodes of this transient insulin resistance.     

Purification and characterization of aprotinin from porcine lungs

Aprotinin, the most studied serine proteinase inhibitor, was isolated from porcine lung for the first time. The purified porcine aprotinin had an Mr value of ∼7 kDa. It cross-reacted with polyclonal serum anti-commercial aprotinin. About 1 μg porcine aprotinin inhibited 6 μg trypsin whereas 1 μg commercial soybean inhibitor inhibited only 1 μg trypsin. The aprotinin gene was also isolated from porcine lung: the deduced amino acid sequence showed 74% identity to bovine aprotinin.     

Effects of long-term fixation on histological quality of undecalcified murine bones embedded in methylmethacrylate

While long-term fixation and storage of specimens is common and useful for many research projects, it is particularly important for space flight investigations where samples may not be returned to Earth for several months (International Space Station) or years (manned mission to Mars). We examined two critical challenges of space flight experimentation: the effect of long-term fixation on the quality of mouse bone preservation and the preservation of antigens and enzymes for both histochemical and immunohistochemical analyses, and how the animal/sample processing affects the preservation. We show that long-term fixation minimally affects standard histological staining, but that enzyme histochemistry and immunolabeling are greatly compromised. Further, we demonstrate that whole animal preservation is not as suitable as whole leg or stripped leg preservation for long-term fixation and all histological analyses. Overall, we recommend whole leg processing for long-term storage of bone specimens in fixative prior to embedding in plastic for histological examination.     

Crystal structure of Escherichia coli ketopantoate reductase at 1.7 A resolution and insight into the enzyme mechanism.

Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate on the pantothenate (vitamin B(5)) biosynthetic pathway. The Escherichia coli panE gene encoding KPR was cloned and expressed at high levels as the native and selenomethionine-substituted (SeMet) proteins. Both native and SeMet recombinant proteins were purified by three chromatographic steps, to yield pure proteins. The wild-type enzyme was found to have a K(M)(NADPH) of 20 microM, a K(M)(ketopantoate) of 60 microM, and a k(cat) of 40 s(-1). Regular prismatic KPR crystals were prepared using the hanging drop technique. They belonged to the tetragonal space group P4(2)2(1)2, with cell parameters: a = b = 103.7 A and c = 55.7 A, accommodating one enzyme molecule per asymmetric unit. The structure of KPR was determined by the multiwavelength anomalous dispersion method using the SeMet protein, for which data were collected to 2.3 A resolution. The native data were collected to 1.7 A resolution and used to refine the final structure. The secondary structure comprises 12 alpha-helices, three 3(10)-helices, and 11 beta-strands. The enzyme is monomeric and has two domains separated by a cleft. The N-terminal domain has an alphabeta-fold of the Rossmann type. The C-terminal domain (residues 170-291) is composed of eight alpha-helices. KPR is shown to be a member of the 6-phosphogluconate dehydrogenase C-terminal domain-like superfamily. A model for the ternary enzyme-NADPH-ketopantoate ternary complex provides a rationale for kinetic data reported for specific site-directed mutants.     

Assessment of telomere length and factors that contribute to its stability.

Short strands of tandem hexameric repeats known as telomeres cap the ends of linear chromosomes. These repeats protect chromosomes from degradation and prevent chromosomal end-joining, a phenomenon that could occur due to the end-replication problem. Telomeres are maintained by the activity of the enzyme telomerase. The total number of telomeric repeats at the terminal end of a chromosome determines the telomere length, which in addition to its importance in chromosomal stabilization is a useful indicator of telomerase activity in normal and malignant tissues. Telomere length stability is one of the important factors that contribute to the proliferative capacity of many cancer cell types; therefore, the detection and estimation of telomere length is extremely important. Until relatively recently, telomere lengths were analyzed primarily using the standard Southern blot technique. However, the complexities of this technique have led to the search for more simple and rapid detection methods. Improvements such as the use of fluorescent probes and the ability to sort cells have greatly enhanced the ease and sensitivity of telomere length measurements. Recent advances, and the limitations of these techniques are evaluated. Drugs that assist in telomere shortening may contribute to tumor regression. Therefore, factors that contribute to telomere stability may influence the efficiency of the drugs that have potential in cancer therapy. These factors in relation to telomere length are also examined in this analysis.     

Recombination by resolvase is inhibited by lac repressor simultaneously binding operators between res sites

The Tn3 resolvase requires that the two recombination (res) sites be aligned as direct repeats on the same molecule for efficient recombination to occur. To test whether resolvase must contact the DNA between res sites as predicted by tracking models, we have determined the sensitivity of recombination to protein diffusion blockades. Recombination between two res sites is unaffected either by lac repressor or bacteriophage T7 RNA polymerase being bound between them. Yet recombination is inhibited by lac repressor if the res site is bounded by a lac operator on both sides. We demonstrate that lac repressor will bind to more than one DNA site under the conditions used to assay recombination. This result suggests that lac repressor can inhibit resolvase by forming a DNA loop that isolates a res site topologically. These results do not support a tracking model for resolvase but suggest that the structure and topology of the DNA substrate is important in the formation of a synapse between res sites.     

Acute and chronic effects of an aromatase inhibitor on territorial aggression in breeding and nonbreeding male song sparrows

Many studies have demonstrated that male aggression is regulated by testosterone. The conversion of testosterone to estradiol by brain aromatase is also known to regulate male aggression in the breeding season. Male song sparrows (Melospiza melodia morphna) are territorial not only in the breeding season, but also in the nonbreeding season, when plasma testosterone and estradiol levels are basal. Castration has no effect on nonbreeding aggression. In contrast, chronic (10 day) aromatase inhibitor (fadrozole) treatment decreases nonbreeding aggression, indicating a role for estrogens. Here, we show that acute (1 day) fadrozole treatment decreases nonbreeding territoriality, suggesting relatively rapid estrogen effects. In spring, fadrozole decreases brain aromatase activity, but acute and chronic fadrozole treatments do not significantly decrease aggression, although trends for some behaviors approach significance. In gonadally intact birds, fadrozole may be less effective at reducing aggression in the spring. This might occur because fadrozole causes a large increase in plasma testosterone in intact breeding males. Alternatively, estradiol may be more important for territoriality in winter than spring. We hypothesize that sex steroids regulate male aggression in spring and winter, but the endocrine mechanisms vary seasonally.