ZmPUMP encodes a fully functional monocot plant uncoupling mitochondrial protein whose affinity to fatty acid is increased with the introduction of a His pair at the second matrix loop

Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins that uncouple respiration from ATP synthesis. In this study, cDNA encoding maize uncoupling protein (ZmPUMP) was expressed in Escherichia coli and recombinant ZmPUMP reconstituted in liposomes. ZmPUMP activity was associated with a linoleic acid (LA)-mediated H+ efflux with Km of 56.36 ± 0.27 μM and Vmax of 66.9 μmol H+ min-1 (mg prot)-1. LA-mediated H+ fluxes were sensitive to ATP inhibition with Ki of 2.61 ± 0.36 mM (at pH 7.2), a value similar to those for dicot UCPs. ZmPUMP was also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in plant UCPs. ZmPUMP with introduced His pair (Lys155His and Ala157His) displayed a 1.55-fold increase in LA-affinity while its activity remained unchanged. Our data indicate conserved properties of plant UCPs and suggest an enhancing but not essential role of the histidine pair in proton transport mechanism. © 2006 Elsevier Inc. All rights reserved.

Exercise training improves relaxation response and SOD-1 expression in aortic and mesenteric rings from high caloric diet-fed rats

Background. Obesity has been associated with a variety of disease such as type II diabetes mellitus, arterial hypertension and atherosclerosis. Evidences have shown that exercise training promotes beneficial effects on these disorders, but the underlying mechanisms are not fully understood. The aim of this study was to investigate whether physical preconditioning prevents the deleterious effect of high caloric diet in vascular reactivity of rat aortic and mesenteric rings. Methods. Male Wistar rats were divided into sedentary (SD); trained (TR); sedentary diet (SDD) and trained diet (TRD) groups. Run training (RT) was performed in sessions of 60 min, 5 days/week for 12 weeks (70-80% VO2max). Triglycerides, glucose, insulin and nitrite/nitrate concentrations (NOx -) were measured. Concentration- response curves to acetylcholine (ACh) and sodium nitroprusside (SNP) were obtained. Expression of Cu/Zn superoxide dismutase (SOD-1) was assessed by Western blotting. Results. High caloric diet increased triglycerides concentration (SDD: 216 ± 25 mg/dl) and exercise training restored to the baseline value (TRD: 89 ± 9 mg/dl). Physical preconditioning significantly reduced insulin levels in both groups (TR: 0.54 ± 0.1 and TRD: 1.24 ± 0.3 ng/ml) as compared to sedentary animals (SD: 0.87 ± 0.1 and SDD: 2.57 ± 0.3 ng/ml). On the other hand, glucose concentration was slightly increased by high caloric diet, and RT did not modify this parameter (SD: 126 ± 6; TR: 140 ± 8; SDD: 156 ± 8 and TRD 153 ± 9 mg/dl). Neither high caloric diet nor RT modified NO x - levels (SD: 27 ± 4; TR: 28 ± 6; SDD: 27 ± 3 and TRD: 30 ± 2 μM). Functional assays showed that high caloric diet impaired the relaxing response to ACh in mesenteric (about 13%), but not in aortic rings. RT improved the relaxing responses to ACh either in aortic (28%, for TR and 16%, to TRD groups) or mesenteric rings (10%, for TR and 17%, to TRD groups) that was accompanied by up-regulation of SOD-1 expression and reduction in triglycerides levels. Conclusion. The improvement in endothelial function by physical preconditioning in mesenteric and aortic arteries from high caloric fed-rats was directly related to an increase in NO bioavailability to the smooth muscle mostly due to SOD-1 up regulation. © 2008 de Moraes et al; licensee BioMed Central Ltd.

The Rv1712 locus from Mycobacterium tuberculosis H37Rv codes for a functional CMP kinase that preferentially Phosphorylates dCMP

The Mycobacterium tuberculosis cmk gene, predicted to encode a CMP kinase (CMK), was cloned and expressed, and its product was purified to homogeneity. Steady-state kinetics confirmed that M. tuberculosis CMK is a monomer that preferentially phosphorylates CMP and dCMP by a sequential mechanism. A plausible role for CMK is discussed. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Functional activity of neutrophils and systemic inflammatory response of Down's syndrome patients with periodontal disease

Periodontal disease (PD) is characterized as an inflammatory process that compromises the support and protection of the periodontium. Patients with Down's syndrome (DS) are prone to develop PD. Neutrophils (NE) are the first line of defense against infection and their absence sets the stage for disease. Aim: To compare the activity and function of NE in the peripheral blood from DS patients with and without PD, assisted at the Center for Dental Assistance to Patients with Special Needs affiliated with the School of Dentistry of Araçatuba, Brazil. Methods: Purified NE were collected from peripheral blood of 22 DS patients. NE were used to detect the 5-lypoxigenase (5-LO) expression by RT-PCR. Plasma from peripheral blood was collected to measure tumor necrosis factor-a (TNF-α) and interleukin-8 (IL-8) by ELISA and nitrite (NO 3) using a Griess assay. Results: Data analysis demonstrated that DS patients with PD present high levels of TNF-a and IL-8 when compared with DS patients without PD. However, there was no statistically significant difference in the levels of NO 3 production between the groups. The levels of the inflammatory mediator 5-LO expression increased in DS patients with PD. Conclusions: According with these results, it was concluded that TNF-α and IL-8 are produced by DS patients with PD. Furthermore, DS patients with PD presented high levels of 5-LO expression, suggesting the presence of leukotriene B 4 (LTB 4) in PD, thus demonstrating that the changes in NE function due to the elevation of inflammatory mediators contribute to PD.

Involvement of the insular cortex in the consolidation and expression of contextual fear conditioning

The insular cortex (IC) has been reported to be involved in the modulation of memory and autonomic and defensive responses. However, there is conflicting evidence about the role of the IC in fear conditioning. To explore the IC involvement in both behavioral and autonomic responses induced by contextual fear conditioning, we evaluated the effects of the reversible inhibition of the IC neurotransmission through bilateral microinjections of the non-selective synapse blocker CoCl2 (1 mm) 10 min before or immediately after the conditioning session or 10 min before re-exposure to the aversive context. In the conditioning session, rats were exposed to a footshock chamber (context) and footshocks were used as the unconditioned stimulus. Forty-eight hours later, the animals were re-exposed to the aversive context for 10 min, but no shock was given. Behavioral (freezing) as well as cardiovascular (arterial pressure and heart rate increases) responses induced by re-exposure to the aversive context were analysed. It was observed that the local IC neurotransmission inhibition attenuated freezing and the mean arterial pressure and heart rate increase of the groups that received the CoCl2 either immediately after conditioning or 10 min before re-exposure to the aversive context, but not when the CoCl2 was injected before the conditioning session. These findings suggest the involvement of the IC in the consolidation and expression of contextual aversive memory. However, the IC does not seem to be essential for the acquisition of memory associated with aversive context. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Effects of BST and high energy diet on gene expression in mammary parenchyma of dairy heifers

The objective of this study was to determine the effects of dietary energy and recombinant bovine somatotropin (bST) injection to identify genes that might control mammogenesis. Total RNA was extracted from the parenchymal tissue of 32 heifers randomly assigned to one of four treatments: two diets (a standard diet and a high energy, high protein diet), each with or without bST. To perform microarray experiments, RNA samples were pooled (2 animals/pool) before reverse transcription and labeling with Cy3 or Cy5. A 4-node loop design was used to examine the differential gene expression among treatments using a bovine-specific cDNA micro array (National Bovine Functional Genomics Consortium Library, NBFGC) containing 18,263 unique expressed sequence tags (EST). Significance levels of differential gene expression among treatments were assessed using a mixed model approach. Injection of bST altered the expression of 12 % of the genes on NBFGC slide related to tissue development, whereas 6% were altered by diet. Administration of bST increases the expression of genes positively related to cell proliferation and mammary parenchyma to a greater extent than a high energy diet. © 2013 Sociedade Brasileira de Zootecnia.

Expression, purification and molecular analysis of the human ZNF706 protein

Background: The ZNF706 gene encodes a protein that belongs to the zinc finger family of proteins and was found to be highly expressed in laryngeal cancer, making the structure and function of ZNF706 worthy of investigation. In this study, we expressed and purified recombinant human ZNF706 that was suitable for structural analysis in Escherichia coli BL21(DH3). Findings. ZNF706 mRNA was extracted from a larynx tissue sample, and cDNA was ligated into a cloning vector using the TOPO method. ZNF706 protein was expressed according to the E. coli expression system procedures and was purified using a nickel-affinity column. The structural qualities of recombinant ZNF706 and quantification alpha, beta sheet, and other structures were obtained by spectroscopy of circular dichroism. ZNF706's structural modeling showed that it is composed of α-helices (28.3%), β-strands (19.4%), and turns (20.9%), in agreement with the spectral data from the dichroism analysis. Conclusions: We used circular dichroism and molecular modeling to examine the structure of ZNF706. The results suggest that human recombinant ZNF706 keeps its secondary structures and is appropriate for functional and structural studies. The method of expressing ZNF706 protein used in this study can be used to direct various functional and structural studies that will contribute to the understanding of its function as well as its relationship with other biological molecules and its putative role in carcinogenesis. © 2013 Colombo et al.; licensee BioMed Central Ltd.

Time-course morphological and functional disorders of the kidney induced by long-term high-fat diet intake in female rats

BackgroundEvidence is emerging that highlights the far-reaching consequences of a high-fat diet (HFD) on kidney morphology and function disorders.MethodsThe present study was performed on 3-, 5-, 7- and 9-week-old HFD female rats compared with the appropriate gender and age-matched animals. We evaluated the kidney expression of angiotensin type II receptor and fibrotic and epithelial-to-mesenchymal transition (EMT) markers, by immunoblotting and immunohistochemical and histological techniques, in parallel with kidney function.ResultsIn the current study, the time-course HFD-treated group showed, by immunoblotting and immunohistochemical analysis, an early time-course increase in the expression of transforming growth factor β-1 (TGFβ-1) in the entire kidney of HFD-treated rats, compared with that observed in the control group. Simultaneously, the study shows a transient increase in the expression of ZEB2 in the HFD whole kidney accompanied by a fall in the E-cadherin expression and increased collagen and fibronectin deposition. A pronounced decrease in fractional urinary sodium excretion was also demonstrated in the long-term HFD-treated rats. The decreased FENa + was accompanied by a fall in FEPNa + and FEPP Na +, which occurred in association with significantly decreased CCr and, certainly on the sodium-filtered load. The reduction in the glomerular filtration rate (GFR) occurred in parallel to proteinuria and glomerular desmin overexpression.ConclusionsThe results of the current study suggest that podocyte injury in parallel with observed proteinuria and evidence of EMT transformation are associated with long-term loss of kidney function and renal sodium and water retention. © 2013 The Author. All rights reserved.

Influence of experimental canine ehrlichiosis on the E-ADA activity and purine levels in serum and possible functional correlations with pathogenesis

The aim of this study was to evaluate adenosine deaminase activity and purines levels in serum of dogs experimentally infected by Ehrlichia canis. Banked serum samples of dogs divided into two groups with five animals each: healthy animals and animals infected by E. canis. The concentration of purines (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine, inosine, hypoxanthine, xanthine and uric acid), and adenosine deaminase (E-ADA) activity in sera were evaluated. Samples were collected on days 12 and 30 post-infection (PI). The E-ADA activity showed a significant reduction on day 12 PI, and increased on day 30 PI in dogs infected with E. canis. On day 12, an increase in seric concentration of ATP, ADP and adenosine was verified, and different levels of hypoxanthine, xanthine and uric acid had a drastic reduction in infected compared healthy dogs (P< 0.05). However, on day 30 PI, the levels of seric ADP and AMP decreased, unlike the concentration of xanthine and uric acid that increased significantly in infected dogs (P< 0.05). Therefore, the activity of E-ADA and purine levels are altered in experimental canine ehrlichiosis, probably with the purpose of modulating the pathogenesis of the disease related to immune response, oxidative stress and coagulation disorders in acute phase. © 2013 Elsevier B.V.

Structural and functional characterization of the chaperone Hsp70 from sugarcane. Insights into conformational changes during cycling from cross-linking/mass spectrometry assays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Post-partum anoestrus in tropical beef cattle: A systems approach combining gene expression and genome-wide association results

The length of the post-partum anoestrous interval affects reproductive efficiency in many tropical beef cattle herds. In this study, results from genome-wide association studies (Experiment 1: GWAS) and gene expression (Experiment 2: microarray) were combined in a systems approach to reveal genetic markers, genes and pathways underlying the physiology of post-partum anoestrus in tropically adapted cattle. The microarray study measured the expression of 13,964 genes in the hypothalamus of Brahman cows. A total of 366 genes were differentially expressed (DE) in the post-partum period, when acyclic cows were compared to cows that had resumed ovarian cycles. Associated markers (P < 0.05) from a high density GWAS pointed to 2829 genes that were associated with post-partum anoestrous interval (PPAI) in two populations of beef cattle: Brahman and Tropical composite. Together the experiments provided evidence for 63 genes that are likely to influence the resumption of ovulation post-partum in tropically adapted beef cattle. Functional annotation analysis revealed that some of the 63 genes have known roles in hormonal activity, energy balance and neuronal synapse plasticity. Polymorphisms within candidate genes identified by this systems approach could have biological significance in post-partum anoestrus and help select Zebu (Bos indicus) influenced cattle with genetic potential for shorter post-partum anoestrus. Crown Copyright (C) 2014 Published by Elsevier B.V. All rights reserved.

Cryotolerance and global gene-expression patterns of Bos taurus indicus and Bos taurus taurus in vitro- and in vivo-produced blastocysts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phenotypic and functional evaluations of peripheral blood monocytes from chronic-form paracoccidioidomycosis patients before and after treatment

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gene expression profiling in leiomyosarcomas and undifferentiated pleomorphic sarcomas: SRC as a new diagnostic marker

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression Pattern and Promoter Analysis of a Eucalyptus grandis Germin-like Gene

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)