L-Arginine improves multiple physiological parameters in mice exposed to diet-induced metabolic disturbances

L-Arginine (L-Arg) is a conditionally essential amino acid and a natural constituent of dietary proteins. Studies in obese rats and type 2 diabetic humans have indicated that dietary supplementation with L-Arg can diminish gain in white adipose tissue (WAT) and improve insulin sensitivity. However, the effects of L-Arg on glucose homeostasis, body composition and energy metabolism remain unclear. In addition, no studies have, to our knowledge, examined whether L-Arg has beneficial effects as a dietary supplement in the mouse model. In the present study, we investigated the effects of L-Arg supplementation to male C57BL/6 mice on an array of physiological parameters. L-Arg supplemented mice were maintained on a low-protein diet and body composition, appetite regulation, glucose tolerance, insulin sensitivity and energy expenditure were evaluated. A significant reduction in epididymal WAT was observed in L-Arg supplemented mice compared with mice fed an isocaloric control diet. Surprisingly, the L-Arg supplemented animals were hyperphagic corresponding to a highly significant decrease in feed efficiency, as body weight developed in a similar pattern in both experimental groups. Glucose homeostasis experiments revealed a major effect of L-Arg supplementation on glucose tolerance and insulin sensitivity, interestingly, independent of a parallel regulation in whole-body adiposity. Increased L-Arg ingestion also raised energy expenditure; however, no concurrent effect on locomotor activity, substrate metabolism or expression of uncoupling proteins (UCP1 and UCP2) in adipose tissues was displayed. In conclusion, dietary L-Arg supplementation substantially affects an array of metabolic-associated parameters including a reduction in WAT, hyperphagia, improved insulin sensitivity and increased energy expenditure in mice fed a low-protein diet.     

Long-term effect of apatite on ectomycorrhizal growth and community structure

Ectomycorrhizal (ECM) fungi are efficient at taking up phosphorus (P) from mineral sources, such as apatite, which are not easily available to the host trees. Since ECM fungal species differ in P uptake rates, it can be expected that the composition of the ECM fungal community will change upon exposure to apatite, provided that the P transfer is rewarded by more carbon being transferred to the fungal symbiont. Control and apatite-amended mesh bags were buried in pairs in the humus layer of a P-poor Norway spruce forest. The ECM fungal community that colonized these bags was analyzed by DNA extraction, PCR amplification of the internal transcribed spacer (ITS) region, cloning, and random sequencing. Fungal biomass was estimated by ergosterol analysis. No change in the ECM fungal community structure was seen after 5?years of apatite exposure, although the fungal biomass increased threefold upon apatite amendment. Our results indicate that host trees enhance carbon allocation to ECM fungi colonizing P sources in P-poor forests but the lack of change in the composition of the ECM fungal community suggests that P transfer rates were similar among the species. Alternatively, higher P transfer among certain species was not rewarded with higher carbon transfer from the host.     

A polarized-light spectroscopy study of interactions of a hairpin polyamide with DNA

We here study the interactions of a polyamide with large DNA, and compare to those of minor groove binder distamycin (DST), including high ligand/DNA binding ratios. Specific as well as nonspecific binding is probed using polarized-light spectroscopy combined with singular value decomposition analysis. Circular and linear dichroism data confirm binding geometries consistent with minor groove binding for both of the ligands. Interestingly, at high and intermediate ligand/DNA ratios the polyamide exhibits no significant sequence discrimination between mixed-sequence (calf thymus) and AT DNA as compared to DST. Each ligand is concluded to exhibit two different binding modes depending upon ligand/DNA ratio and nucleo-base sequence. At high binding ratios, distinct differences between the ligands are observed: circular dichroism spectra exciton effects provide evidence of bimolecular interactions of the polyamide when bound to AT-DNA, whereas no effects are seen with DST or mixed-sequence DNA. Also linear dichroism indicates that a change in binding geometry occurs at high polyamide/AT ratios, and that the effect occurs only with polyamide in contrast to DST. Since the effect is insignificant with DST, or with calf thymus DNA, it is concluded that it relates to the sizes of the ligands and the minor grooves, becoming critical in the limit of crowding.     

Conserved conformation of RecA protein after executing the DNA strand-exchange reaction. A site-specific linear dichroism structure study

RecA protein and its eukaryotic homologue Rad51 protein catalyzes the DNA strand exchange, which is a key reaction of homologous recombination. At the initial step of the reaction, RecA proteins form a helical filament on a single-stranded DNA (ssDNA). Binding of double-stranded DNA (dsDNA) to the filament triggers the homology search; as homology is found, the exchange of strands occurs, and the displaced DNA is released. These are the principal steps of genetic recombination; however, despite many years of extensive study of RecA activities, the details of the mechanism are still obscure. A high-resolution structure of the active nucleoprotein filament could provide information to help understand this process. Using a linear dichroism polarized-light spectroscopy technique, in combination with protein engineering (the site-specific linear dichroism method), we have previously studied the arrangement of RecA in complex with ssDNA. In the present study, we have used this approach to search for structural variations of RecA at the atomic level as the DNA in the complex is changed from ssDNA to dsDNA. The structural data of the RecA-dsDNA filament are found to be very similar to the data previously obtained for the RecA-ssDNA complex, indicating that the overall orientation and also the internal structure of RecA in the active filament are not markedly altered when the bound DNA changes from single- to double-stranded. The implications of the structural similarities as well as the significance of some conformational variations observed for a few amino acid residues that may be involved in interactions with DNA are discussed.     

Discovery of novel aminothiadiazole amides as selective EP3 receptor antagonists

This Letter discloses a series of 2-aminothiadiazole amides as selective EP₃ receptor antagonists. SAR optimization resulted in compounds with excellent functional activity in vitro. In addition, efforts to optimize DMPK properties in the rat are discussed. These efforts have resulted in the identification of potent, selective EP₃ receptor antagonists with excellent DMPK properties suitable for in vivo studies.     

Antiglycation Effects of Carnosine and Other Compounds on the Long-Term Survival of Escherichia coli

Glycation, or nonenzymatic glycosylation, is a chemical reaction between reactive carbonyl-containing compounds and biomolecules containing free amino groups. Carbonyl-containing compounds include reducing sugars such as glucose or fructose, carbohydrate-derived compounds such as methylglyoxal and glyoxal, and nonsugars such as polyunsaturated fatty acids. The latter group includes molecules such as proteins, DNA, and amino lipids. Glycation-induced damage to these biomolecules has been shown to be a contributing factor in human disorders such as Alzheimer''s disease, atherosclerosis, and cataracts and in diabetic complications. Glycation also affects Escherichia coli under standard laboratory conditions, leading to a decline in bacterial population density and long-term survival. Here we have shown that as E. coli aged in batch culture, the amount of carboxymethyl lysine, an advanced glycation end product, accumulated over time and that this accumulation was affected by the addition of glucose to the culture medium. The addition of excess glucose or methylglyoxal to the culture medium resulted in a dose-dependent loss of cell viability. We have also demonstrated that glyoxylase enzyme GloA plays a role in cell survival during glycation stress. In addition, we have provided evidence that carnosine, folic acid, and aminoguanidine inhibit glycation in prokaryotes. These agents may also prove to be beneficial to eukaryotes since the chemical processes of glycation are similar in these two domains of life.One factor that may affect the long-term survival of bacterial cells in a population is the level of damage incurred by macromolecules via the nonenzymatic process of glycation, first described by Louis-Camille Maillard (16). The Maillard reaction is responsible for the formation of several compounds identified as advanced glycation end products (AGEs) (9). In vivo this reaction appears to play a role in the aging process, as it leads to slow degradation of molecules. The principal mechanisms of glycation-related damage involve cross-links between proteins and/or DNA, modifying or destroying their functional properties (2, 8, 38). Most studies of glycation have been performed with eukaryotes because of its relationship to aging and disorders such as Alzheimer''s disease and diabetes (6, 21, 30, 42). However, several studies (32, 33) have shown that glycation also takes place in Escherichia coli, affecting protein and DNA of this prokaryote.Many biochemical pathways produce reactive dicarbonyl intermediates, such as glyoxal and methylglyoxal (MG), which can further react with DNA, proteins, or other biomolecules to form AGEs (8, 36). Reaction of glucose with amino groups of proteins and subsequent formation of reactive dicarbonyls via a series of reactions involving Schiff base and Amadori product intermediates have been well documented (40). Methylglyoxal can be formed by spontaneous decomposition of glycolytic triose phosphates such as dihydroxyacetone phosphate (DHAP) (1) or can be produced enzymatically from DHAP by the E. coli enzyme methylglyoxal synthase (MgsA) (12). MG synthesis usually requires an environment low in phosphate and high in DHAP, a situation that occurs most frequently under high-glucose conditions (25, 26). If MG is not degraded, MG accumulation will lead to cell death (12). E. coli maintains pathways for the detoxification of methylglyoxal, including glyoxalase enzymes I and II (encoded by gloA and gloB, respectively), which convert MG to S-lactoyl glutathione and then to d-lactate (12). This system has been proposed to be the predominant MG detoxification system in E. coli (12, 29).Glyoxal is also a toxic dicarbonyl compound capable of damaging cells via AGE formation. One of the AGEs formed in the presence of glyoxal is carboxymethyl lysine (CML), which has been used extensively as a biomarker for aging (11, 20, 31, 39). CML can be formed by different pathways: glucose can be oxidized to glyoxal, which can react with protein to form CML (1, 17); glucose can also react with protein to form fructoselysine (an Amadori product), which can undergo oxidative cleavage to form CML (1). In this study, we investigated CML formation in E. coli growing under standard and glycation-prone laboratory conditions. Since AGE formation may negatively affect cell survival and reproduction during long-term batch culture (35), we hypothesized that CML would accumulate in these cultures as cells progress through stationary phase.One product that may interfere with AGE formation is carnosine (β-alanyl-l-histidine), a naturally occurring dipeptide in many organisms. Although its mechanism of action has not been fully determined, there is evidence that both the free amino group derived from the β-alanine and the imidazole ring of histidine compete with amino groups of proteins in the presence of reactive dicarbonyl compounds (7, 24). In this study we designed assays to determine the effect of carnosine (and other compounds) on survival of cultures of E. coli under a variety of experimental conditions. Additionally, since strains lacking glyoxalase enzymes I and II have a reduced ability to detoxify methylglyoxal, we hypothesized that gloA and/or gloB mutants would require larger amounts of carnosine than would wild-type strains to survive in the presence of this toxic electrophile.