A Novel Peroxisome Proliferator-activated Receptor (PPAR)α Agonist and PPARγ Antagonist,Z-551, Ameliorates High-fat Diet-induced Obesity and Metabolic Disorders in Mice

A novel peroxisome proliferator-activated receptor (PPAR) modulator, Z-551, having both PPARα agonistic and PPARγ antagonistic activities, has been developed for the treatment of obesity and obesity-related metabolic disorders. We examined the effects of Z-551 on obesity and the metabolic disorders in wild-type mice on the high-fat diet (HFD). In mice on the HFD, Z-551 significantly suppressed body weight gain and ameliorated insulin resistance and abnormal glucose and lipid metabolisms. Z-551 inhibited visceral fat mass gain and adipocyte hypertrophy, and reduced molecules involved in fatty acid uptake and synthesis, macrophage infiltration, and inflammation in adipose tissue. Z-551 increased molecules involved in fatty acid combustion, while reduced molecules associated with gluconeogenesis in the liver. Furthermore, Z-551 significantly reduced fasting plasma levels of glucose, triglyceride, free fatty acid, insulin, and leptin. To elucidate the significance of the PPAR combination, we examined the effects of Z-551 in PPARα-deficient mice and those of a synthetic PPARγ antagonist in wild-type mice on the HFD. Both drugs showed similar, but weaker effects on body weight, insulin resistance and specific events provoked in adipose tissue compared with those of Z-551 as described above, except for lack of effects on fasting plasma triglyceride and free fatty acid levels. These findings suggest that Z-551 ameliorates HFD-induced obesity, insulin resistance, and impairment of glucose and lipid metabolisms by PPARα agonistic and PPARγ antagonistic activities, and therefore, might be clinically useful for preventing or treating obesity and obesity-related metabolic disorders such as insulin resistance, type 2 diabetes, and dyslipidemia.     

Risk Score to Predict 1-Year Mortality after Haemodialysis Initiation in Patients with Stage 5 Chronic Kidney Disease under Predialysis Nephrology Care

Background

Few risk scores are available for predicting mortality in chronic kidney disease (CKD) patients undergoing predialysis nephrology care. Here, we developed a risk score using predialysis nephrology practice data to predict 1-year mortality following the initiation of haemodialysis (HD) for CKD patients.

Methods

This was a multicenter cohort study involving CKD patients who started HD between April 2006 and March 2011 at 21 institutions with nephrology care services. Patients who had not received predialysis nephrology care at an estimated glomerular filtration rate (eGFR) of approximately 10 mL/min per 1.73 m² were excluded. Twenty-nine candidate predictors were selected, and the final model for 1-year mortality was developed via multivariate logistic regression and was internally validated by a bootstrapping technique.

Results

A total of 688 patients were enrolled, and 62 (9.0%) patients died within one year of HD initiation. The following variables were retained in the final model: eGFR, serum albumin, calcium, Charlson Comorbidity Index excluding diabetes and renal disease (modified CCI), performance status (PS), and usage of erythropoiesis-stimulating agent (ESA). Their β-coefficients were transformed into integer scores: three points were assigned to modified CCI≥3 and PS 3–4; two to calcium>8.5 mg/dL, modified CCI 1–2, and no use of ESA; and one to albumin<3.5 g/dL, eGFR>7 mL/min per 1.73 m², and PS 1–2. Predicted 1-year mortality risk was 2.5% (score 0–4), 5.5% (score 5–6), 15.2% (score 7–8), and 28.9% (score 9–12). The area under the receiver operating characteristic curve was 0.83 (95% confidence interval, 0.79–0.89).

Conclusions

We developed a simple 6-item risk score predicting 1-year mortality after the initiation of HD that might help nephrologists make a shared decision with patients and families regarding the initiation of HD.     

Production of recombinant bovine lactoferrin N-lobe in insect cells and its antimicrobial activity

Lactoferrin is a multifunctional, iron-binding glycoprotein found in physiological fluids of mammals. In the present study, a gene encoding the N-terminal half (N-lobe) of bovine lactoferrin was cloned and expressed in cultured insect cells using a baculovirus expression system. One mutation was found in the lactoferrin N-lobe gene, but it resulted in no amino acid substitution. The recombinant lactoferrin N-lobe was secreted into the culture medium and partially purified by means of an immobilized heparin column. The recombinant lactoferrin N-lobe secreted was not glycosylated, but it possessed antimicrobial activity toward Escherichia coli O111. The recombinant product synthesized and accumulated in the host cells exhibited greater electrophoretic mobility on SDS-PAGE than the secreted product and showed no potency to inhibit the growth of bacteria. It is thought that the product accumulated intracellularly lacks antimicrobial ability due to its degradation in the host cells or due to disruption of the active conformation.     

Metabolic regulation analysis of wild-type and arcA mutant Escherichia coli under nitrate conditions using different levels of omics data

It is of practical interest to investigate the effect of nitrates on bacterial metabolic regulation of both fermentation and energy generation, as compared to aerobic and anaerobic growth without nitrates. Although gene level regulation has previously been studied for nitrate assimilation, it is important to understand this metabolic regulation in terms of global regulators. In the present study, therefore, we measured gene expression using DNA microarrays, intracellular metabolite concentrations using CE-TOFMS, and metabolic fluxes using the (13)C-labeling technique for wild-type E. coli and the ΔarcA (a global regulatory gene for anoxic response control, ArcA) mutant to compare the metabolic state under nitrate conditions to that under aerobic and anaerobic conditions without nitrates in continuous culture conditions at a dilution rate of 0.2 h(-1). In wild-type, although the measured metabolite concentrations changed very little among the three culture conditions, the TCA cycle and the pentose phosphate pathway fluxes were significantly different under each condition. These results suggested that the ATP production rate was 29% higher under nitrate conditions than that under anaerobic conditions, whereas the ATP production rate was 10% lower than that under aerobic conditions. The flux changes in the TCA cycle were caused by changes in control at the gene expression level. In ΔarcA mutant, the TCA cycle flux was significantly increased (4.4 times higher than that of the wild type) under nitrate conditions. Similarly, the intracellular ATP/ADP ratio increased approximately two-fold compared to that of the wild-type strain.     

Processing pathway deduced from the structures of N-glycans in Carica papaya

A processing The processing pathway of N-glycans in Carica papaya was deduced from the structures of N-glycans. The N-glycans were liberated by hydrazinolysis followed by N-acetylation. Their reducing-end sugar residues were tagged with 2-aminopyridine and the pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Eleven PA-sugar chains were found, and their structures were analyzed by two-dimensional sugar mapping combined with partial acid hydrolysis and exoglycosidase digestion. The structures of the N-glycans were of the highmannose types with xylose and fucose; however, among them two new N-glycans, Manalpha1-6(Manalpha1-3)Manalpha1-6(Xylbeta1-2)+ ++Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-3)GlcNAc and Manalpha1-3Manalpha1-6(Xylbeta1-2)Manbeta1-4G lcNAcbeta1-4(Fucalpha1-3 )GlcNAc, were found. Judging from these structures together with Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) (Xylbeta1-2)Manbeta1- 4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc reported previously [Shimazaki, A., Makino, Y., Omichi, K., Odani, S., and Hase, S. (1999) J. Biochem. 125, 560- 565], a processing pathway for N-glycans in C. papaya is inferred in which the activity of Golgi alpha-mannosidase II is incomplete.     

Abiotic Formation of Valine Peptides Under Conditions of High Temperature and High Pressure

We investigated the oligomerization of solid valine and the stabilities of valine and valine peptides under conditions of high temperature (150–200 °C) and high pressure (50–150 MPa). Experiments were performed under non-aqueous condition in order to promote dehydration reaction. After prolonged exposure of monomeric valine to elevated temperatures and pressures, the products were analyzed by liquid chromatography mass spectrometry comparing their retention times and masses. We identified linear peptides that ranged in size from dimer to hexamer, as well as a cyclic dimer. Previous studies that attempted abiotic oligomerization of valine in the absence of a catalyst have never reported valine peptides larger than a dimer. Increased reaction temperature increased the dissociative decomposition of valine and valine peptides to products such as glycine, β-alanine, ammonia, and amines by processes such as deamination, decarboxylation, and cracking. The amount of residual valine and peptide yields was greater at higher pressures at a given temperature, pressure, and reaction time. This suggests that dissociative decomposition of valine and valine peptides is reduced by pressure. Our findings are relevant to the investigation of diagenetic processes in prebiotic marine sediments where similar pressures occur under water-poor conditions. These findings also suggest that amino acids, such as valine, could have been polymerized to peptides in deep prebiotic marine sediments within a few hundred million years.     

Collagen adhesin–nanoparticle interaction impairs adhesin's ligand binding mechanism

Background

Pathogenic bacteria specifically recognize extracellular matrix (ECM) molecules of the host (e.g. collagen, fibrinogen and fibronectin) through their surface proteins known as MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) and initiate colonization. On implantation, biomaterials easily get coated with these ECM molecules and the MSCRAMMs mediate bacterial adherence to biomaterials. With the rapid rise in antibiotic resistance, designing alternative strategies to reduce/eliminate bacterial colonization is absolutely essential.

Methods

The Rhusiopathiae surface protein B (RspB) is a collagen‐binding MSCRAMM of Erysipelothrix rhusiopathiae. It also binds to abiotic surfaces. The crystal structure of the collagen‐binding region of RspB (rRspB_31–348) reported here revealed that RspB also binds collagen by a unique ligand binding mechanism called “Collagen Hug” which is a common theme for collagen‐binding MSCRAMMs of many Gram-positive bacteria. Here, we report the interaction studies between rRspB_31–348 and silver nanoparticles using methods like gel shift assay, gel permeation chromatography and circular dichroism spectroscopy.

Results

The “Collagen Hug” mechanism was inhibited in the presence of silver nanoparticles as rRspB_31–348 was unable to bind to collagen. The total loss of binding was likely because of rRspB_31–348 and silver nanoparticle protein corona formation and not due to the loss of the structural integrity of rRspB_31–348 on binding with nanoparticles as observed from circular dichroism experiments.

General significance

Interaction of rRspB_31–348 with silver nanoparticle impaired its ligand binding mechanism. Details of this inhibition mechanism may be useful for the development of antimicrobial materials and antiadhesion drugs.     

Serum stability of selected decapeptide agonists of KISS1R using pseudopeptides

Metastin/kisspeptin, a 54-amino acid peptide, is the ligand of the G-protein-coupled receptor KISS1R which plays a key role in pathways that regulate reproduction and cell migration in many endocrine and gonadal tissues. The N-terminally truncated decapeptide, metastin(45–54), has 3–10 times higher receptor affinity and intracellular calcium ion-mobilizing activity but is rapidly inactivated in serum. In this study we designed and synthesized stable KISS1R agonistic decapeptide analogs with selected substitutions at positions 47, 50, and 51. Replacement of glycine with azaglycine (azaGly) in which the α-carbon is replaced with a nitrogen atom at position 51 improved the stability of amide bonds between Phe⁵⁰-Gly⁵¹ and Gly⁵¹-Leu⁵² as determined by in vitro mouse serum stability studies. Substitution for tryptophan at position 47 with other amino acids such as serine, threonine, β-(3-pyridyl)alanine, and d-tryptophan (d-Trp), produced analogs that were highly stable in mouse serum. d-Trp⁴⁷ analog 13 showed not only high metabolic stability but also excellent KISS1R agonistic activity. Other labile peptides may have increased serum stability using amino acid substitution.     

A biochemically defined system for mammalian nonhomologous DNA end joining

Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ.