Increases in renal ε-(γ-glutamyl)-lysine crosslinks result from compartment-specific changes in tissue transglutaminase in early experimental diabetic nephropathy:Pathologic implications

Diabetic nephropathy (DN) is characterized by an early, progressive expansion and sclerosis of the glomerular mesangium leading to glomerulosclerosis. This is associated with parallel fibrosis of the renal interstitium. In experimental renal scarring, the protein cross-linking enzyme, tissue transglutaminase (tTg), is up-regulated and externalized causing an increase in its crosslink product, e-(γ-glutamyl)-lysine, in the extracellular space. This potentially contributes to the extracellular matrix (ECM) accumulation central to tissue fibrosis by increasing deposition and inhibiting breakdown. We investigated if a similar mechanism may contribute to the ECM expansion characteristic of DN using the rat streptozotocin model over 120 days. Whole kidney e-(γ-glutamyl)-lysine (HPLC analysis) was significantly increased from Day 90 (+337%) and peaked at Day 120 (+650%) (p <0.05). Immunofluorescence showed this increase to be predominantly extracellular in the peritubular interstitial space, but also in individual glomeruli. Total kidney transglutaminase (Tg) was not elevated. However, using a Tg in situ activity assay, increased Tg was detected in both the extracellular interstitial space and glomeruli by Day 60, with a maximal 53% increase at Day 120 (p <0.05). Using a specific anti-tTg antibody, immunohistochemistry showed a similar increase in extracellular enzyme in the interstitium and glomeruli. To biochemically characterize glomerular changes, glomeruli were isolated by selective sieving. In line with whole kidney measurement, there was an increase in glomerular e-(γ-glutamyl) lysine (+ 361%); however, in the glomeruli this was associated with increases in Tg activity (+228%) and tTg antigen by Western blotting (+215%). Importantly, the ratio of glomerular e-(γ-glutamyl) lysine to hydroxyproline increased by 2.2-fold. In DN, changes in the kidney result in increased translocation of tTg to the extracellular environment where high Ca2+ and low GTP levels allow its activation. In the tubulointerstitium this is independent of increased tTg production, but dependent in the glomerulus. This leads to excessive ECM cross-linking, contributing to the renal fibrosis characteristic of progressive DN.

Fructose Alters Adiponectin, Haptoglobin and Angiotensinogen Gene Expression in 3T3-L1 Adipocytes

Fructose- or sucrose-rich diets can cause insulin resistance and increase the risk of cardiovascular disease. Adipokines are correlated with the development of these diseases in obesity. We hypothesize that fructose and sucrose induce insulin resistance via effects on adipokine gene expression in adipocytes. This study analyzed the effect of fructose or glucose on adiponectin, haptoglobin, and angiotensinogen gene expression in 3T3-L1 adipocytes. Ten days after differentiation, the cells were pretreated with serum- and glucose-free medium. Twenty-four hours later, fructose or glucose (0, 5, 10, or 20 mmol) was added into the medium, and the cells were collected after a further 24 hours. Adiponectin, haptoglobin, and angiotensinogen gene expression were determined. Adiponectin gene expression increased when 10 or 20 mmol glucose was added compared with that observed for the non–hexose-treated cells. A similar effect occurred when 5 mmol fructose was added. Glucose (10 mmol) and fructose (20 mmol) stimulated haptoglobin gene expression in 3T3-L1 adipocytes compared with 0 mmol, with glucose producing a more pronounced effect. Although 20 mmol fructose caused an increase in angiotensinogen gene expression, glucose did not. In conclusion, in this study of 2 hexoses revealed an increase in adiponectin gene expression, suggesting that the effect of a glucose-rich diet on the development of insulin resistance is not related to the effect of these hexoses on adipocyte adiponectin gene expression. However, insulin resistance and cardiovascular disease promoted by fructose-rich diets could be partially related to the effect of fructose on adiponectin and angiotensinogen gene expression.

Quantitative Proteomic Analysis Reveals Metabolic Alterations, Calcium Dysregulation, And Increased Expression Of Extracellular Matrix Proteins In Laminin α2 Chain-deficient Muscle.

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain-deficient dy(3K)/dy(3K) mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).

Augmented β-cell Function And Mass In Glucocorticoid-treated Rodents Are Associated With Increased Islet Ir-β /akt/mtor And Decreased Ampk/acc And As160 Signaling.

Glucocorticoid (GC) therapies may adversely cause insulin resistance (IR) that lead to a compensatory hyperinsulinemia due to insulin hypersecretion. The increased β-cell function is associated with increased insulin signaling that has the protein kinase B (AKT) substrate with 160 kDa (AS160) as an important downstream AKT effector. In muscle, both insulin and AMP-activated protein kinase (AMPK) signaling phosphorylate and inactivate AS160, which favors the glucose transporter (GLUT)-4 translocation to plasma membrane. Whether AS160 phosphorylation is modulated in islets from GC-treated subjects is unknown. For this, two animal models, Swiss mice and Wistar rats, were treated with dexamethasone (DEX) (1 mg/kg body weight) for 5 consecutive days. DEX treatment induced IR, hyperinsulinemia, and dyslipidemia in both species, but glucose intolerance and hyperglycemia only in rats. DEX treatment caused increased insulin secretion in response to glucose and augmented β-cell mass in both species that were associated with increased islet content and increased phosphorylation of the AS160 protein. Protein AKT phosphorylation, but not AMPK phosphorylation, was found significantly enhanced in islets from DEX-treated animals. We conclude that the augmented β-cell function developed in response to the GC-induced IR involves inhibition of the islet AS160 protein activity.

Frequency Of The Allelic Variant C.1150t > C In Exon 10 Of The Fibroblast Growth Factor Receptor 3 (fgfr3) Gene Is Not Increased In Patients With Pathogenic Mutations And Related Chondrodysplasia Phenotypes.

Mutations in the FGFR3 gene cause the phenotypic spectrum of FGFR3 chondrodysplasias ranging from lethal forms to the milder phenotype seen in hypochondroplasia (Hch). The p.N540K mutation in the FGFR3 gene occurs in ∼70% of individuals with Hch, and nearly 30% of individuals with the Hch phenotype have no mutations in the FGFR3, which suggests genetic heterogeneity. The identification of a severe case of Hch associated with the typical mutation c.1620C > A and the occurrence of a c.1150T > C change that resulted in a p.F384L in exon 10, together with the suspicion that this second change could be a modulator of the phenotype, prompted us to investigate this hypothesis in a cohort of patients. An analysis of 48 patients with FGFR3 chondrodysplasia phenotypes and 330 healthy (control) individuals revealed no significant difference in the frequency of the C allele at the c.1150 position (p = 0.34). One patient carrying the combination `pathogenic mutation plus the allelic variant c.1150T > C' had a typical achondroplasia (Ach) phenotype. In addition, three other patients with atypical phenotypes showed no association with the allelic variant. Together, these results do not support the hypothesis of a modulatory role for the c.1150T > C change in the FGFR3 gene.

Increased Arterial Stiffness In Resistant Hypertension Is Associated With Inflammatory Biomarkers.

Increased levels of inflammatory biomarkers such as interleukin-6 (IL-6), 10 (IL-10), 1β (IL-1β), tumor necrosis factor-α (TNF-α) high-sensitivity C-reactive protein (hs-CRP) are associated with arterial stiffness in hypertension. Indeed, resistant hypertension (RHTN) leads to unfavorable prognosis attributed to poor blood pressure (BP) control and target organ damage. This study evaluated the potential impact of inflammatory biomarkers on arterial stiffness in RHTN. In this cross-sectional study, 32 RHTN, 20 mild hypertensive (HTN) and 20 normotensive (NT) patients were subjected to office BP and arterial stiffness measurements assessed by pulse wave velocity (PWV). Inflammatory biomarkers were measured in plasma samples. PWV was increased in RHTN compared with HTN and NT (p < 0.05). TNF-α levels were significantly higher in RHTN and HTN than NT patients. No differences in IL-6 levels were observed. RHTN patients had a higher frequency of subjects with increased levels of IL-10 and IL-1β compared with HTN and NT patients. Finally, IL-1β was independently associated with PWV (p < 0.001; R(2) = 0.5; β = 0.077). RHTN subjects have higher levels of inflammatory cytokines (TNF-α, IL-1β and IL-10) as well as increased arterial stiffness, and detectable IL-1β levels are associated arterial stiffness. These findings suggest that inflammation plays a possible role in the pathophysiology of RHTN.

Improvement Of The Physical Performance Is Associated With Activation Of No/pgc-1α/mttfa Signaling Pathway And Increased Protein Expressions Of Electron Transport Chain In Gastrocnemius Muscle From Rats Supplemented With L-arginine.

To examine the influence of l-arginine supplementation in combination with physical training on mitochondrial biomarkers from gastrocnemius muscle and its relationship with physical performance. Male Wistar rats were divided into four groups: control sedentary (SD), sedentary supplemented with l-arginine (SDLA), trained (TR) and trained supplemented with l-arginine (TRLA). Supplementation of l-arginine was administered by gavage (62.5mg/ml/day/rat). Physical training consisted of 60min/day, 5days/week, 0% grade, speed of 1.2km/h. The study lasted 8weeks. Skeletal muscle mitochondrial enriched fraction as well as cytoplasmic fractions were obtained for Western blotting and biochemical analyses. Protein expressions of transcriptor coactivator (PGC-1α), transcriptor factors (mtTFA), ATP synthase subunit c, cytochrome oxidase (COXIV), constitutive nitric oxide synthases (eNOS and nNOS), Cu/Zn-superoxide dismutase (SOD) and manganese-SOD (Mn-SOD) were evaluated. We also assessed in plasma: lipid profile, glycemia and malondialdehyde (MDA) levels. The nitrite/nitrate (NOx(-)) levels were measured in both plasma and cytosol fraction of the gastrocnemius muscle. 8-week l-arginine supplementation associated with physical training was effective in promoting greater tolerance to exercise that was accompanied by up-regulation of the protein expressions of mtTFA, PGC-1α, ATP synthase subunit c, COXIV, Cu/Zn-SOD and Mn-SOD. The upstream pathway was associated with improvement of NO bioavailability, but not in NO production since no changes in nNOS or eNOS protein expressions were observed. This combination would be an alternative approach for preventing cardiometabolic diseases given that in overt diseases a profound impairment in the physical performance of the patients is observed.

Increased Circulating Pedf And Low Sicam-1 Are Associated With Sickle Cell Retinopathy.

Sickle cell retinopathy (SCR) develops in up to 30% of sickle cell disease patients (SCD) during the second decade of life. Treatment for this affection remains palliative, so studies on its pathophysiology may contribute to the future development of novel therapies. SCR is more frequently observed in hemoglobin SC disease and derives from vaso-occlusion in the microvasculature of the retina leading to neovascularization and, eventually, to blindness. Circulating inflammatory cytokines, angiogenic factors, and their interaction may contribute to the pathophysiology of this complication. Angiopoietin (Ang)-1, Ang-2, soluble vascular cell adhesion molecule-1, intercellular adhesion molecule (ICAM)-1, E-selectin, P-selectin, IL1-β, TNF-α, pigment epithelium derived factor (PEDF) and vascular endothelial growth factor plasmatic levels were determined in 37 SCD patients with retinopathy, 34 without retinopathy, and healthy controls. We observed that sICAM-1 is significantly decreased, whereas PEDF is elevated in HbSC patients with retinopathy (P=0.012 and P=0.031, respectively). Ang-1, Ang-2 and IL1-β levels were elevated in SCD patients (P=0.001, P<0.001 and P=0.001, respectively), compared to controls, and HbSS patients presented higher levels of Ang-2 compared to HbSC (P<0.001). Our study supports the possible influence of sICAM-1 and PEDF on the pathophysiology of retinal neovascularization in SCD patients.

Increased Expression Of Hes5 Protein In Notch Signaling Pathway In The Hippocampus Of Mice Offspring Of Dams Fed A High-fat Diet During Pregnancy And Suckling.

Maternal high-fat diet (HFD) impairs hippocampal development of offspring promoting decreased proliferation of neural progenitors, in neuronal differentiation, in dendritic spine density and synaptic plasticity reducing neurogenic capacity. Notch signaling pathway participates in molecular mechanisms of the neurogenesis. The activation of Notch signaling leads to the upregulation of Hes5, which inhibits the proliferation and differentiation of neural progenitors. This study aimed to investigate the Notch/Hes pathway activation in the hippocampus of the offspring of dams fed an HFD. Female Swiss mice were fed a control diet (CD) and an HFD from pre-mating until suckling. The bodyweight and mass of adipose tissue in the mothers and pups were also measured. The mRNA and protein expression of Notch1, Hes5, Mash1, and Delta1 in the hippocampus was assessed by RT-PCR and western blotting, respectively. Dams fed the HFD and their pups had an increased bodyweight and amount of adipose tissue. Furthermore, the offspring of mothers fed the HFD exhibited an increased Hes5 expression in the hippocampus compared with CD offspring. In addition, HFD offspring also expressed increased amounts of Notch1 and Hes5 mRNA, whereas Mash1 expression was decreased. However, the expression of Delta1 did not change significantly. We propose that the overexpression of Hes5, a Notch effector, downregulates the expression of the proneural gene Mash1 in the offspring of obese mothers, delaying cellular differentiation. These results provide further evidence that an offspring's hippocampus is molecularly susceptible to maternal HFD and suggest that Notch1 signaling in this brain region is important for neuronal differentiation.

EFEITO DA ASSOCIAÇÃO DE PECTINASE, INVERTASE E GLICOSE ISOMERASE NA QUALIDADE DO SUCO DE BANANA

The association of 0,03 % v/w pectinase (Clarex), 0,6 % v/w invertase (Invertase-S) and 0,5 % w/w glucose isomerase (Taka-sweet) in industrialized banana (Musa cavendishii) pulp, under conditions of hydrolysis 40oC, 15 minutes, was observed and compared to other three enzymatic treatements: 0,03 % v/w pectinase (Clarex); 0,03 % v/w pectinase (Clarex) associated to 0,6 % v/w invertase (Invertase-S); and 0,03 % v/w pectinase (Sigma) associated to 0,03 % cellulase (Sigma) to determine the quality using a group of physical, physico-chemical, chemical, microbiological and sensory properties of the banana juices obtained. These properties had not differ significantly in function of pectinases and celulase employed. The addition of invertase had increased sweetness and decreased viscosity in juice. On the other side, the addition of glucose isomerase in inverted juice was not able in increasing significantly fructose content.

Abnormally increased iron concentration in basal ganglia in Shy-Drager syndrome MR imaging and autonomic study: RM imagem e estudo autonômico

Report of an early case of Shy-Drager syndrome in a 67 year-old woman patient. Autonomic failure was diagnosed by functional evaluation as well as laboratory tests. MR imaging disclosed a prominent putamina hypodensity in T2-weighted images at high field strength due to iron increased depositing in this basal ganglia. MR imaging evidences confirm Shy-Drager syndrome diagnosis, and contributes for differential diagnosis of idiopathic hypotension (pure autonomic failure) in special in SDS early cases.

Bottle feeding, increased overjet and Class 2 primary canine relationship: is there any association?

The aim of this study was to investigate the association between bottle feeding and prevalence rates of increased overjet and Class 2 primary canine relationship. The sample consisted of 911 children (461 boys, 450 girls) aged 3 (13.9%), 4 (40.8%), 5 (34%) and 6 (11.3%) years, with complete primary dentition. Information about nutritive and nonnutritive (pacifier and/or digit) sucking habits was collected through questionnaires. Three calibrated dentists (κ: 0.9-1.0 and Rs > 0.90) performed the clinical assessments. The children were divided into four groups: G1 - not bottle-fed; G2 - exclusively bottle-fed; G3 - breast- and bottle-fed, bottle feeding ceased before 3 years of age; and G4 - breast- and bottle-fed, bottle feeding ceased between 3 and 4 years of age. Associations between nutritive and nonnutritive sucking behaviors and the malocclusions studied were analyzed by multiple binary logistic regression (α= 0.05). The frequencies of increased overjet were: 25.3% (G1), 38.8% (G2), 39.2% (G3) and 47.8% (G4). The percentages of Class 2 canine relationship were: 27.9% (G1), 48.8% (G2), 43.4% (G3) and 43% (G4). No significant effect of bottle feeding was found. The chances of diagnosing increased overjet (O.R. = 4.42, p < 0.001) and Class 2 canine relationship (O.R. = 4.02, p < 0.001) were greater for children with pacifier and/or digit-sucking habits, compared to those without a history of nonnutritive sucking behavior. It may be suggested that bottle feeding alone is not directly associated with higher prevalence rates of increased overjet and Class 2 canine relationship in the primary dentition.

Apparent digestibility of diets with different concentrations of lysine and energy in piglets with different body weights and post-weaning age

The effects of body weight or age and dietary digestible lysine and metabolizable energy on apparent digestibility of energy and dry matter were evaluated in piglets after weaning. The animals were weaned at 21 days of age and distributed in two groups: 8.68 ± 0.76 kg at 28 days of age (weaned 7 days earlier); and 12.73 ± 0.99 kg at 35 days of age (weaned 14 days earlier). The pigs were allotted in digestibility cages in a completely randomized block design with the following factorial arrangements: 2 × 4 composed of two weight categories and four levels of digestible lysine (1.222; 1.305; 1.390 and 1.497%); and 2 × 3 composed of two weight categories and three levels of metabolizable energy (3,510; 3,700 and 3,830 kcal/kg rations). Digestible lysine was evaluated in six replications and metabolizable energy in eight replications and each animal constituted an experimental unit. Piglets with higher body weight and age were more efficient in nitrogen retention and energetic balance, compared to lighter and younger piglets, particularly those given lower concentration of lysine in the diet. The energy increase favored nitrogen retention by the heavier and older piglets. However, coefficients of dry matter and energy apparent digestibility did not differ among weight categories. Older and heavier piglets were more efficient in nitrogen retention, although this efficacy depended on concentration of the energy in the diet. This better use of protein and energy suggest differences on nutritional requirements.

Lisina digestível e zinco quelado para frangos de corte machos: desempenho e retenção de nitrogênio na fase pré-inicial

Em dois ensaios experimentais, avaliaram-se níveis de lisina digestível (0,90; 1,00; 1,10; 1,20 e 1,40%) combinados a zinco quelado (43 e 253 ppm) para frangos de corte machos. Os períodos considerados foram: 1 a 11 dias de idade (desempenho) e 1 a 7 dias de idade (balanço de nitrogênio). O delineamento experimental utilizado foi de blocos ao acaso, em esquema fatorial 5 × 2. No ensaio de desempenho, utilizaram-se 7 repetições e 15 aves por unidade experimental e, no ensaio de digestibilidade, 6 repetições e 6 aves por unidade experimental. As dietas continham 2.960 de EM/kg e 21% de proteína bruta. Na fase de 1 a 11 dias de idade, não houve efeito de interação lisina digestível × zinco quelado no desempenho, mas verificou-se efeito quadrático dos níveis de lisina digestível sobre o peso final, o ganho de peso e o ganho de peso relativo e efeito linear crescente no consumo de ração. A conversão alimentar não diferiu com as variações dos níveis de lisina digestível e zinco na dieta. Nas condições de avaliação do desempenho, o nível ótimo de lisina digestível para frangos de corte machos mantidos em piso de concreto é de 1,19%. Na fase de 1 a 7 dias de idade, não houve influência da relação entre níveis de lisina digestível e zinco quelado no balanço de nitrogênio. A retenção de nitrogênio aumentou de forma linear crescente com o aumento da concentração de lisina digestível na dieta, o que está de acordo com o aumento linear do ganho de peso e da eficiência alimentar. A exigência de lisina digestível para frangos de corte machos de 1 a 7 dias de idade é igual ou superior a 1,40%.

Dietary levels of lysine for male broilers from 23 to 36 days of age: performance and body composition

One thousand and fifty (1,050) Ross male broilers from 23 to 36 days of age were used to evaluate the dietary effects of levels of lysine. It was used a random block design with 5 levels of digestible lysine (0.95; 1.00; 1.05; 1.10 and 1.15%). Each level was applied to seven replicates and the experimental unit was composed of 30 birds. Diets were offered in seven replicates and the experimental unit was represented by 30 birds. Weight gain, feed intake, feed conversion, carcass traits, cut yields, composition and deposition of body nutrients were evaluated. The concentration of 1.09% of digestible lysine optimized performance without significant effects on feed intake. Carcass traits and cut yields had no significant differences among levels of lysine. On the other hand, there was a quadratic effect on carcass weight, which was the best at the level of 1.07% of digestible lysine. On crude protein content of the empty body, the best response was observed at the level of 1.10% of dietary lysine. This same level corresponded to a quadratic response in the deposition of water from carcass and empty body. The level of digestible lysine for better performance, chemical composition and body nutrient deposition in broilers from 23 to 36 days of age is between 1.07 and 1.10%.