Effects of Age on Inflammatory Profiles and Nutrition/Energy Metabolism in Domestic Cats.

BACKGROUND: Animals tend to increase in body weight and body condition score (BCS) with aging. Serum diagnostic markers related to energy metabolism may show changes even in healthy cats with aging. MATERIALS AND METHODS: Seventy domestic cats were recruited for this study. Based upon the modified AAFP-AAHA Feline Life Stage Guidelines, animals were divided into six groups: Junior (7 months-2 years), Prime (3 -6 years), Mature (7-10 years), Senior (11-14 years), Geriatric-obese (15 years ≤) and Geriatric-thin (15 years ≤). Their body condition scores (BCS) ranged from 3/9 to 9/9. Changes in metabolites, inflammatory markers, hormone concentrations and enzyme activities related to energy metabolism were investigated in serum of 70 domestic cats of various ages. RESULTS: Serum glucose (GLU) concentrations in the Mature, Senior, and Geriatric-obese groups were significantly higher than those in the Junior group. Serum amyloid A (SAA) concentrations in the Geriatric-thin group were significantly increased compared with the Junior group. SAA concentrations in the Geriatric-obese group tended to increase although there were no statistically significant differences. In the Mature, Senior, Geriatric-obese and Geriatric-thin groups, malate dehydrogenase/lactate dehydrogenase (M/L) ratio, an energy metabolic indicator, tended to decrease compared with the Junior group. In the Senior group, triglyceride (TG) concentrations were significantly increased compared with the Junior group. In the Geriatric-obese and Geriatric-thin groups, blood urea nitrogen (BUN) concentrations were significantly increased compared with the Junior group. In the Geriatric-obese group, albumin (ALB) concentrations were decreased compared with the Junior group. CONCLUSION: Aged domestic cats tend to increase in body weight and BCS. In addition, serum GLU, TG, SAA, and BUN concentrations increased and serum ALB concentrations and M/L ratio decreased. These diagnostic markers may be useful to detect small changes related to energy metabolism with aging that may cause obesity with light inflammation in healthy cats.

Propagermanium administration for patients with type 2 diabetes and nephropathy: A randomized pilot trial.

AIMS: We assessed the potential efficacy and safety of propagermanium (PG), an organic compound that inhibits the C-C chemokine receptor type 2, administration in patients with type 2 diabetes and nephropathy. Furthermore, we assessed the feasibility of future studies. MATERIALS AND METHODS: We recruited patients from nine medical institutions in Japan for this randomized, open-label, parallel two-arm pilot trial. Inclusion criteria were diagnosis of type 2 diabetes, age 30-75 years, dipstick proteinuria of ≥1+ or urinary albumin-to-creatinine ratio (UACR) of ≥30 mg/g and estimated glomerular filtration rate of ≥30 mL/min/1.73 m2. Patients were randomly assigned (1:2) using a minimization algorithm to either continuing usual care or concomitant administration of 30 mg PG per day for 12 months. The primary outcome was the change in UACR from baseline to 12 months. We also collected safety information for all patients who received at least one dose of PG. RESULTS: We enrolled 29 patients, 10 were assigned to continue usual care and 19 to receive PG. Changes in UACR by PG in addition to the usual care were 25.0% (95% CI -20.4%, 96.5%, P = .33). No severe adverse events or renal events were observed during the study. CONCLUSION: Although the treatment with PG was generally well tolerated, the dosage of 30 mg/d for 12 months did not reduce albuminuria when used in addition to usual care in patients with type 2 diabetes and nephropathy. Efficacy of PG should be verified in future definitive trials.

Cloning and heterologous expression of P450Lent4B11, a novel bacterial P450 gene, for hydroxylation of an antifungal agent sordaricin.

Microbial transformation is known to be one of promising options to add functional groups such as a hydroxyl moiety to active base compounds to generate their derivatives. Sordaricin, a diterpene aglycone of the natural product sordarin, is an antifungal agent to selectively inhibit fungal protein synthesis by stabilizing the ribosome/EF-2 (elongation factor 2) complex. We screened actinomycetes to catalyze hydroxylation of sordaricin on the basis that the hydroxyl moiety would make it easier to generate derivatives of sordaricin. As a result of the screening, 6-hydroxylation of sordaricin was found to be catalyzed by Lentzea sp. 7887. We found that the cytochrome P450 inhibitor metyrapone inhibited this reaction, suggesting that a cytochrome P450 may be responsible for the biotransformation. As a next step, we cloned multiple cytochrome P450 genes, one of which were named P450Lent4B11, using degenerate PCR primers. The expressed cytochrome P450 derived from the P450Lent4B11 gene provided a different absorbance spectrum pattern from original one when it was incubated with sordaricin. Moreover, in cell-free conditions, the corresponding cytochrome P450 displayed the 6-hydroxylation activity toward sordaricin. Taken together, these results indicate that P450Lent4B11, derived from Lentzea sp. 7887, should be responsible for catalyzing 6-hydroxylation of sordaricin.

Effects of Supplementation with Anti-Inflammatory Compound Extracted from Herbs in Healthy and Obese Cats.

BACKGROUND: Obesity has become a serious public health problem all over the world, and prevalence of obesity has increased in cats. Obesity is characterized by continuous low-grade inflammation based on oxidative stress by excessively produced reactive oxygen species (ROS). Supplementation with anti-oxidant and anti-inflammatory compounds is very effective to relieve the obesity condition. A plant extract mixture containing Rhus verniciflua and some other herbs, Rv-PEM01-99, shows anti-oxidant and anti-inflammatory effects in animals. The aim of this study was to evaluate the effects of supplementation with Rv-PEM01-99 as an anti-inflammatory compound in healthy and obese cats. MATERIALS AND METHODS: Ten healthy mix breed cats and four obesity disease cats were used. The healthy cats were randomly divided into control and test groups. Anti-inflammatory compound, Rv-PEM01-99, in which quercetin derivative is the main component, was supplemented to the healthy test group and the obesity disease cats at the dose of 100-120 mg/kg/day (2.5-3.0 mg/kg/day as quercetin) for 4 weeks. Metabolites, hormones and enzymes were measured before and after the compound supplementation. RESULTS: The anti-inflammatory compound supplementation decreased serum amyloid A (SAA) concentrations as inflammatory markers in both healthy and obesity disease cats. In obesity disease cats, plasma total cholesterol concentrations and AST and ALT activities decreased significantly after the compound supplementation. CONCLUSION: Quercetin derivative seems to have strong anti-inflammatory activities. In the healthy cats, anti-inflammatory compound supplementation decreased plasma NEFA and SAA concentrations. In the obesity disease cats, the compound supplementation may have alleviated obesity disease by relieving inflammation and improvement of lipid metabolism in livers.

Effects of astaxanthin supplementation in healthy and obese dogs.

BACKGROUND: Since astaxanthin (ASX) has potent anti-oxidative effects with inhibitory action of lipid peroxidation and singlet oxygen quenching activity, it is widely used as a functional food for keeping good health in human. Obesity is a risk factor for various metabolic disorders. It is characterized by low-grade chronic inflammation based on oxidative stress by excessively produced ROS. From the point of preventive medicine, natural compounds have been proposed as potential therapeutic agents in the prevention of metabolic disorder in companion animals. The purpose of this study is to evaluate the effects of ASX supplementation in healthy and obese dogs. MATERIALS AND METHODS: Ten healthy beagle dogs and 5 clinically obese dogs were used in this study. The healthy beagle dogs were randomly divided into 2 groups as follows: control and test groups. The test group dogs received ASX supplementation mixed with the food for 6 weeks. Five clinically obese dogs received ASX supplementation for 8 weeks. Metabolites, hormones and enzymes were measured before and after ASX supplementation. RESULTS: In the healthy dog groups, after 6 weeks, plasma triglyceride (TG) and malondialdehyde concentrations and lactate dehydrogenase (LDH) values significantly decreased in the test group. There was no significant difference in the control group. In clinically obese dogs, plasma TG concentration decreased after 8 weeks of ASX supplementation. Plasma alanine aminotransferase and LDH values clearly decreased in all 5 dogs and 4 dogs out of 5 dogs, respectively. CONCLUSION: ASX supplementation (0.3 mg/kg body weight/day) for 6 weeks in healthy dogs and 8 weeks in obese dogs induced the elevation of antioxidant function and of liver function by ameliorating lipid metabolism.

Comparison of Visceral Fat Accumulation and Metabolome Markers among Cats of Varying BCS and Novel Classification of Feline Obesity and Metabolic Syndrome.

As in humans, obesity and its associated diseases represent the most significant threat to the health of veterinary populations. Previous human studies have provided insights into the risk factors of obesity, complex pathogenesis of obesity-associated diseases, and their life-threatening consequences. In humans, the "metabolic syndrome" represents a cluster of metabolic risk factors associated with the development of cardiovascular disease. Risk factors for metabolic syndrome, such as diabetes, obesity, high blood pressure, and its complications increase health-care utilization and medical expenses. Early diagnosis and intervention through preemptive approach is in need, and the new International Diabetes Federation definition of MS serves as the universally accepted diagnostic tool that is accessible in clinical settings. In veterinary populations, especially in cats, similar pathophysiological path and disease progression to the development of MS, such as adipokine dysregulations, chronic inflammation, lipotoxicity, are expected. The aim of this manuscript is twofold. First of all, it presents our preliminary feline obesity study that serves as the basis for discussion of obesity and its metabolic impact on feline population. In this study, we observed the effects of weight gain on energy metabolism using metabolome markers, such as adiponectin (ADN) and proinflammatory cytokines, in correlation with other common biochemical parameters in 14 clinically healthy cats of varying weight status. Further, we evaluated the visceral fat accumulation in three subjects of varying Body Condition Scores via computed tomography imaging and laparoscopic examination, and assessed the adipocyte type and size histologically. Mutually antagonizing changes in ADN and visceral adipose tissue (VAT) reflected the pathophysiological derangements leading to MS earlier than the common biochemical predictors such as glucose, liver values, and lipid markers. Second, it proposes the novel diagnostic and classification method of feline obesity and MS, based on the established diagnostic criteria of human MS and the presented study results. The results supported our novel "classification of feline obesity" and "Feline MS diagnostic criteria," suggesting the need to complement ADN measurement with VAT volume to better understand the pathogenesis of metabolic disturbances in the feline population.

Bioconversion of FR901459, a novel derivative of cyclosporin A, by Lentzea sp. 7887.

FR901459, a product of the fungus Stachybotrys chartarum No. 19392, is a derivative of cyclosporin A (CsA) and a powerful immunosuppressant that binds cyclophilin. Recently, it was reported that CsA was effective against hepatitis C virus (HCV). However, FR901459 lacks active moieties, which are essential for synthesizing more potent and safer derivatives of this anti-HCV agent. Here we identified an actinomycete strain (designated 7887) that was capable of efficient bioconversion of FR901459. Structural elucidation of the isolated bioconversion products (1-7) revealed that compounds 1-4 were mono-hydroxylated at the position of 1-MeBmt or 9-MeLeu, whereas compounds 5-7 were bis-hydroxylated at both positions. The results of morphological and chemical characterization, as well as phylogenetic analysis of 16S ribosomal DNA (rDNA), suggested that strain 7887 belonged to the genus Lentzea. Comparison of the FR901459 conversion activity of strain 7887 with several other Lentzea strains revealed that although all examined strains metabolized FR901459, strain 7887 had a characteristic profile with respect to bioconversion products. Taken together, these findings suggest that strain 7887 can be used to derivative FR901459 to produce a chemical template for further chemical modifications that may provide more effective and safer anti-HCV drugs.

Fibrocytes are involved in the pathogenesis of human chronic kidney disease.

The presence of chronic kidney disease in humans is associated with a risk of kidney function loss as well as the development of cardiovascular disease. Fibrocytes have been shown to contribute to organ fibrosis. In this study, the presence of fibrocytes was investigated immunohistochemically in kidney biopsy specimens from 100 patients with chronic kidney disease. In addition, 6 patients with thin basement membrane disease were studied as a disease control. In patients with chronic kidney disease, the infiltration of fibrocytes was observed mainly in the interstitium. The number of interstitial fibrocytes in patients with chronic kidney disease was higher than that in patients with thin basement membrane disease. The number of infiltrated fibrocytes in the interstitium correlated well with the severity of tubulointerstitial lesions, such as interstitial fibrosis, in patients with chronic kidney disease. In addition, there were significant correlations between the number of interstitial fibrocytes and the number of CD68-positive macrophages in the interstitium as well as urinary monocyte chemoattractant protein-1/CCL2 levels. In particular, there was an inverse correlation between the number of interstitial fibrocytes and kidney function at the time of biopsy. Finally, the numbers of interstitial fibrocytes and macrophages as well as urinary CCL2 levels were significantly decreased during convalescence induced by glucocorticoid therapy. These results suggest that fibrocytes may be involved in the pathogenesis of chronic kidney disease through the interaction with macrophages as well as CCL2.

Effect of sevelamer hydrochloride on markers of bone turnover in Japanese dialysis patients with low biointact PTH levels.

BACKGROUND: In hemodialysis patients, adynamic bone disease has been reported to be closely associated with low levels of parathyroid hormone (PTH) due to exposure to high levels of serum calcium following the administration of calcium carbonate (CaCO3) or vitamin D agents. This study was conducted to clarify the therapeutic effect of a non-calcemic phosphate binder, sevelamer hydrochloride (sevelamer), for hypoparathyroidism in hemodialysis patients with or without diabetes mellitus. METHODS: Based on entry criteria, 40 Japanese chronic hemodialysis patients (22 males and 18 females with a mean age of 60.6, 14 diabetic patients and 26 non-diabetic patients) were switched from CaCO3 to sevelamer for 48 weeks. Serum calcium, phosphate, intact (i) PTH and PTH-(1-84) were analyzed. Bone remodeling activity was evaluated by determining intact osteocalcine (iOC), bone-specific alkaline phosphatase (BAP). RESULTS: The switch from CaCO3 to sevelamer significantly decreased the serum levels of calcium, resulting in the elevation of iPTH levels from 31+/-18 pg/mL to 95+/-96 pg/mL by 48 weeks. In contrast, serum phosphate levels remained similar to those in patients with CaCO3 treatment. Concomitantly, the levels of BAP and iOC were elevated. Further, these beneficial effects on bone turnover were observed in both diabetic and non-diabetic patients. CONCLUSION: Sevelamer reduced the calcium concentration and thereby increased PTH levels, resulting in the improvement of markers of bone turnover. The administration of sevelamer is of therapeutic benefit for the improvement of bone remodeling activity even in hemodialysis patients with diabetes.

FR227673 and FR190293, novel antifungal lipopeptides from Chalara sp. No. 22210 and tolypocladium parasiticum No. 16616.

Novel antifungal lipopeptides, FR227673 and FR190293, were isolated from the fermentation broths of fungal strains Chalara sp. No. 22210 and Tolypocladium parasiticum No. 16616, respectively. These compounds have the same cyclic peptide nuclear structure as FR901379, with different side chains, and showed antifungal activity against Aspergillus fumigatus and Candida albicans attributed to inhibition of 1,3-beta-glucan synthesis.

FR220897 and FR220899, novel antifungal lipopeptides from Coleophoma empetri no. 14573.

Novel antifungal lipopeptides, FR220897 and FR220899, were isolated from the fermentation broth of a fungal strain No. 14573. This strain was identified as Coleophoma empetri No. 14573 from morphological and physiological characteristics. FR220897 and FR220899 showed antifungal activities against Aspergillus fumigatus and Candida albicans attributed to inhibition of 1,3-beta-glucan synthesis. Furthermore, FR220897 was effective in a murine model of systemic candidiasis.

FR207944, an antifungal antibiotic from Chaetomium sp. No. 217 II. Isolation and structure elucidation.

We discovered FR207944 produced by Chaetomium sp. No. 217 in the course of screening for antifungal antibiotics from natural products. FR207944 is identical with fuscoatroside, described in the preceding paper as an anti-Aspergillus flavus agent. Determination of the relative stereochemistry of fuscoatroside was made formally by comparison with WF11605 (16-Oxo-FR207944). We confirmed the stereochemistry on the basis of single crystal X-ray analysis.

Selective ligand purification using high-performance affinity beads.

Since the development of affinity chromatography, affinity purification technology has been applied to many aspects of biological research, becoming an indispensable tool. Efficient strategies for the identification of biologically active compounds based on biochemical specificity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that specifically interact with a target biomolecule using reverse affinity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was efficiently purified in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse affinity beads facilitated identification of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were purified from a crude cell extract, respectively. These data indicate that reverse affinity beads are powerful tools for identification of both specific ligands and proteins that interact with receptor/ligand complexes.

FR207944, an antifungal antibiotic from Chaetomium sp. no. 217 I. Taxonomy, fermentation, and biological properties.

An antifungal antibiotic, FR207944, was isolated from the culture broth of a fungal strain Chaetomium sp. no. 217. FR207944 is a triterpene glucoside with antifungal activity against Aspergillus fumigatus and Candida albicans. Specifically, FR207944 exhibits in vitro and in vivo antifungal activity against A. fumigatus. The effects of FR207944 on the morphology of A. fumigatus were shown to be similar to those of FR901379, a known 1,3-beta-glucan synthase inhibitor. The MECs of FR207944 against A. fumigatus FP1305 and C. albicans FP633 in micro-broth dilution test were 0.039 and 1.6 mug/ml respectively. FR207944 showed good potency by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED(50)s of 5.7 and 17 mg/kg respectively.

Involvement of extracellular signal-regulated kinase and p38 in human diabetic nephropathy.

BACKGROUND: The involvement of mitogen-activated protein kinase (MAPK) in human diabetic nephropathy has not been fully investigated. METHODS: The presence of cells positive for the phosphorylated MAPK family (phosphorylated extracellular signal-regulated kinase [p-ERK], phosphorylated p38MAPK [p-p38MAPK]) was investigated immunohistochemically in kidneys of 30 patients with diabetic nephropathy. In addition, 10 patients with minimal change nephrotic syndrome, 10 patients with thin basement membrane disease, and 5 patients with benign nephrosclerosis were studied as disease controls. The presence of activated nuclear factor-kappaB (p65)-positive cells also was evaluated in kidney specimens. RESULTS: In patients with diabetic nephropathy, p-ERK, p-p38MAPK, and p65 were observed in mesangial cells, endothelial cells, podocytes, tubular epithelial cells, and mononuclear infiltrates in interstitium. Numbers of p-ERK-, p-p38MAPK-, and p65-positive cells in both glomeruli and interstitium in patients with diabetic nephropathy were higher than those in controls. In particular, the number of glomerular p-ERK-positive cells in patients with diabetic nephropathy increased in accordance with the progression of glomerular lesions and correlated well with the number of glomerular p65-positive cells (r = 0.654; P < 0.01; n = 30). Conversely, the number of p-p38MAPK-positive cells in glomeruli did not correlate with glomerular lesions. However, the number of tubulointerstitial p-p38MAPK-positive cells in patients with diabetic nephropathy reflected the severity of tubulointerstitial lesions, and numbers of those in the interstitium increased with good correlation to numbers of tubulointerstitial p65-positive cells (r = 0.757; P < 0.01; n = 30) and interstitial CD68-positive macrophages (r = 0.647; P < 0.05; n = 30) and urinary monocyte chemoattractant protein-1 levels (r = 0.605; P < 0.05; n = 30). CONCLUSION: These results suggest that MAPK phosphorylation contributes to human diabetic nephropathy. In particular, ERK and p38MAPK may be distinctly involved in glomerular and tubulointerstitial lesions in human diabetic nephropathy.

FR177391, a new anti-hyperlipidemic agent from Serratia. IV. Target identification and validation by chemical genetic approaches.

Natural products with distinct biological activities are very promising molecular probes to dissect the novel pathways of biology. FR177391, a product of bacteria, was obtained as a natural compound possessing anti-hyperlipidemic effects. FR177391 enhances differentiation of mouse 3T3-L1 fibroblasts to adipocytes and reduces the circulating levels of triglyceride in C57BL/KsJ-db/db mice, a obese non-insulin-dependent diabetes mellitus animal model, although its mechanism of actions remained to be unknown. We report here that the target protein for FR177391 was identified to be protein phosphatase 2A (PP2A) by employing the method of affinity chromatography. FR177391 potently inhibited PP2A activity at nano molar concentration, and shared its binding pocket with a phosphatase inhibitor, okadaic acid. In addition to the phenotypic alterations, the enhancement for phosphorylation of extracellular signal-regulated kinase (ERK) protein was observed in the FR177391-treated 3T3-L1 cells. These results suggest that prolonged activation of ERK protein due to inhibition of its dephosphorylation by PP2A plays an important role in adipocyte maturation and regulation of the blood revels of lipids.

FR177391, a new anti-hyperlipidemic agent from Serratia. III. Microbial conversion of FR177391 and synthesis of FR177391 derivatives for its target protein screening by chemical genetic approaches.

FR177391 produced by Serratia liquefaciens No. 1821 enhances differentiation of mouse 3T3-L1 fibroblasts to adipocytes and reduces the circulating levels of triglyceride in C57BL/KsJ-db/bd mice, an obese non-insulin-dependent diabetes mellitus animal model, although its mechanism of actions remained to be unknown. Its active derivative, 20-hydroxy FR177391, and its inactive derivative, 3-hydroxy FR177391 were produced by microbial conversion of FR177391, and biotin-labeled FR177391 was synthesized from 20-hydroxy FR177391 as an active affinity ligand to identify target molecules of FR177391 by chemical genetic approaches.

The outcome and a new ISN/RPS 2003 classification of lupus nephritis in Japanese.

BACKGROUND: A considerable diversity in prognosis is seen with lupus glomerulonephritis (LGN). Hence, the clinical usefulness of a recent International Society of Nephrology/Renal Pathology Society (ISN/RPS) 2003 classification to judge the long-term outcome of human LGN has been investigated. METHODS: We studied retrospectively 60 subjects with LGN (7 males, 53 females, mean age of 33 years old) who underwent renal biopsies and were followed from 1 to 366 months, with a mean of 187 months. We diagnosed renal pathology as classes, active and sclerosing lesions, according to the new and WHO1995 classification of LGN, and analyzed the clinicopathologic factors affecting to the prognosis of LGN. RESULTS: New classification got much higher consensus in the judgment of classes (98% vs. 83%, P = 0.0084). The group of Class IV-S (N = 6) or IV-G (N = 17) at initial biopsies showed higher rate of end-stage renal failure (ESRF) compared with that of Class I, II, III or V (40.9% vs. 2.6%, P < 0.001). The mean 50% renal survival time of Class IV was 189 +/- 29 months, and patients with Class IV-S tended to have a poorer prognosis (95 +/- 22 months for IV-S vs. 214 +/- 35 months for IV-G, P = 0.1495). Class IV was also selected as the most significant risk factor for ESRF by stepwise model (P = 0.002). In subanalysis for ESRF in Class IV (-S or -G), treatment including methylprednisolone pulse therapy was only selected as a significant improving factor for primary outcome (P = 0.034). In addition, activity index was the significant risk factor of death and/or ESRF after initial renal biopsies (P = 0.043). As for actuarial patient death during all follow-up periods, complications with anti-phospholipid syndrome or nephrotic syndrome were significant risk factors (P = 0.013, P = 0.041, respectively). CONCLUSION: New ISN/RPS 2003 classification provided beneficial pathologic information relevant to the long-term renal outcome and the optimal therapy preventing ESRF and/or death in patients with LGN.

FR225659-binding proteins: identification as serine/threonine protein phosphatase PP1 and PP2A using high-performance affinity beads.

FR225659 was originally isolated as a novel gluconeogenesis inhibitor produced by fungal strain Helicomyces sp. No. 19353. To identify the target protein of FR225659, we synthesized high-performance affinity latex beads that immobilized FR225659 derivative FR253761 or FR259383. Using these beads, we identified FR225659 binding proteins as serine/threonine protein phosphatase type1 (PP1) and type2A (PP2A) from rat hepatocyte crude extract. FR225659 and its synthetic derivatives were strongly inhibited the enzyme activities of purified catalytic subunits of PP1 and PP2A in vitro.