A retrospective study of Babesia macropus associated with morbidity and mortality in eastern grey kangaroos (Macropus giganteus) and agile wallabies (Macropus agilis)

This is a retrospective study of 38 cases of infection by Babesia macropus, associated with a syndrome of anaemia and debility in hand-reared or free-ranging juvenile eastern grey kangaroos (Macropus giganteus) from coastal New South Wales and south-eastern Queensland between 1995 and 2013. Infection with B. macropus is recorded for the first time in agile wallabies (Macropus agilis) from far north Queensland. Animals in which B. macropus infection was considered to be the primary cause of morbidity had marked anaemia, lethargy and neurological signs, and often died. In these cases, parasitised erythrocytes were few or undetectable in peripheral blood samples but were sequestered in large numbers within small vessels of visceral organs, particularly in the kidney and brain, associated with distinctive clusters of extraerythrocytic organisms. Initial identification of this piroplasm in peripheral blood smears and in tissue impression smears and histological sections was confirmed using transmission electron microscopy and molecular analysis. Samples of kidney, brain or blood were tested using PCR and DNA sequencing of the 18S ribosomal RNA and heat shock protein 70 gene using primers specific for piroplasms. The piroplasm detected in these samples had 100 sequence identity in the 18S rRNA region with the recently described Babesia macropus in two eastern grey kangaroos from New South Wales and Queensland, and a high degree of similarity to an unnamed Babesia sp. recently detected in three woylies (Bettongia penicillata ogilbyi) in Western Australia.

GPRA and the asthma locus on chromosome 7p14-p15

The basis of this work was the identification of a genomic region on chromosome 7p14-p15 that strongly associated with asthma and high serum total immunoglobulin E in a Finnish founder population from Kainuu. Using a hierarchical genotyping approach the linkage region was narrowed down until an evolutionary collectively inherited 133-kb haplotype block was discovered. The results were confirmed in two independent data sets: Asthma families from Quebec and allergy families from North-Karelia. In all the three cohorts studied, single nucleotide polymorphisms tagging seven common gene variants (haplotypes) were identified. Over half of the asthma patients carried three evolutionary closely related susceptibility haplotypes as opposed to approximately one third of the healthy controls. The risk effects of the gene variants varied from 1.4 to 2.5. In the disease-associated region, there was one protein-coding gene named GPRA (G Protein-coupled Receptor for Asthma susceptibility also known as NPSR1) which displayed extensive alternative splicing. Only the two isoforms with distinct intracellular tail sequences, GPRA-A and -B, encoded a full-length G protein-coupled receptor with seven transmembrane regions. Using various techniques, we showed that GPRA is expressed in multiple mucosal surfaces including epithelial cells throughout the respiratory tract. GPRA-A has additional expression in respiratory smooth muscle cells. However, in bronchial biopsies with unknown haplotypes, GPRA-B was upregulated in airways of all patient samples in contrast to the lack of expression in controls. Further support for GPRA as a common mediator of inflammation was obtained from a mouse model of ovalbumin-induced inflammation, where metacholine-induced airway hyperresponsiveness correlated with elevated GPRA mRNA levels in the lung and increased GPRA immunostaining in pulmonary macrophages. A novel GPRA agonist, Neuropeptide S (NPS), stimulated phagocytosis of Esterichia coli bacteria in a mouse macrophage cell line indicating a role for GPRA in the removal of inhaled allergens. The suggested GPRA functions prompted us to study, whether GPRA haplotypes associate with respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in infants sharing clinical symptoms with asthma. According to the results, near-term RDS and asthma may also share the same susceptibility and protective GPRA haplotypes. As in asthma, GPRA-B isoform expression was induced in bronchial smooth muscle cells in RDS and BPD suggesting a role for GPRA in bronchial hyperresponsiveness. In conclusion, the results of the present study suggest that the dysregulation of the GPRA/NPS pathway may not only be limited to the individuals carrying the risk variants of the gene but is also involved in the regulation of immune functions of asthma.

Comparative and functional genome analysis of fungi for development of the protein production host Trichoderma reesei

Filamentous fungi of the subphylum Pezizomycotina are well known as protein and secondary metabolite producers. Various industries take advantage of these capabilities. However, the molecular biology of yeasts, i.e. Saccharomycotina and especially that of Saccharomyces cerevisiae, the baker's yeast, is much better known. In an effort to explain fungal phenotypes through their genotypes we have compared protein coding gene contents of Pezizomycotina and Saccharomycotina. Only biomass degradation and secondary metabolism related protein families seem to have expanded recently in Pezizomycotina. Of the protein families clearly diverged between Pezizomycotina and Saccharomycotina, those related to mitochondrial functions emerge as the most prominent. However, the primary metabolism as described in S. cerevisiae is largely conserved in all fungi. Apart from the known secondary metabolism, Pezizomycotina have pathways that could link secondary metabolism to primary metabolism and a wealth of undescribed enzymes. Previous studies of individual Pezizomycotina genomes have shown that regardless of the difference in production efficiency and diversity of secreted proteins, the content of the known secretion machinery genes in Pezizomycotina and Saccharomycotina appears very similar. Genome wide analysis of gene products is therefore needed to better understand the efficient secretion of Pezizomycotina. We have developed methods applicable to transcriptome analysis of non-sequenced organisms. TRAC (Transcriptional profiling with the aid of affinity capture) has been previously developed at VTT for fast, focused transcription analysis. We introduce a version of TRAC that allows more powerful signal amplification and multiplexing. We also present computational optimisations of transcriptome analysis of non-sequenced organism and TRAC analysis in general. Trichoderma reesei is one of the most commonly used Pezizomycotina in the protein production industry. In order to understand its secretion system better and find clues for improvement of its industrial performance, we have analysed its transcriptomic response to protein secretion stress conditions. In comparison to S. cerevisiae, the response of T. reesei appears different, but still impacts on the same cellular functions. We also discovered in T. reesei interesting similarities to mammalian protein secretion stress response. Together these findings highlight targets for more detailed studies.

Pathophysiology of Congenital Nephrotic Syndrome of the Finnish type

Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disease which is highly enriched in the Finnish population. It is caused by mutations in the NPHS1 gene encoding for nephrin, which is a major component of the glomerular filtration barrier in the kidney. Patients with NPHS1 have heavy proteinuria and nephrotic syndrome (NS) from birth and develop renal fibrosis in early childhood. Renal transplantation (TX) is the only curative treatment for NPHS1. These patients form the largest group of pediatric kidney transplant children in our country. The NPHS1 kidneys are removed in infancy and they serve as an excellent human material for studies of the pathophysiology of proteinuric kidney diseases. Sustained proteinuria is a major factor leading to end-stage renal failure and understanding this process is crucial for nephrology. In this study we investigated the glomerular and tubulointerstitial changes that occur in the NPHS1 kidneys during infancy as well as the expression of nephrin in non-renal tissues. We also studied the pathology and management of recurrent proteinuria in kidney grafts transplanted to NPHS1 children. Severe renal lesions evolved in patients with NPHS1 during the first months of life. Glomerular sclerosis developed through progressive mesangial sclerosis, and capillary obliteration was an early consequence of this process. Shrinkage of the glomerular tuft was common, whereas occlusion of tubular opening or protrusion of the glomerular tuft into subepithelial space or through the Bowman's capsule were not detected. Few inflammatory cells were detected in the mesangial area. The glomerular epithelial cells (podocytes) showed severe ultrastructural changes and hypertrophy. Podocyte proliferation and apoptosis were rare, but moderate amounts of podocytes were detached and ended up in the urine. The results showed that endocapillary lesions not extracapillary lesions, as generally believed were important for the sclerotic process in the NPHS1 glomeruli. In the tubulointerstitium, severe lesions developed in NPHS1 kidneys during infancy. Despite heavy proteinuria, tubular epithelial cells (TECs) did not show transition into myofibroblasts. The most abundant chemokines in NPHS1 tissue were neutrophil activating protein-2 (NAP-2), macrophage inhibiting factor (MIF), and monocyte chemoattractant protein-1 (MCP-1). Interstitial inflammation and fibrosis were first detected in the paraglomerular areas and the most abundant inflammatory cells were monocytes/macrophages. Arteries and arterioles showed intimal hypertrophy, but the pericapillary microvasculature remained quite normal. However, excessive oxidative stress was evident in NPHS1 kidneys. The results indicated that TECs were relatively resistant to the heavy tubular protein load. Nephrin was at first thought to be podocyte specific, but some studies especially in experimental animals have suggested that nephrin might also be expressed in non-renal tissues such as pancreas and central nervous system. The knowledge of nephrin biology is important for the evaluation of nephrin related diseases. In our study, no significant amounts of nephrin protein or mRNA were detected in non-renal tissues of man and pig as studied by immunohistochemistry and in situ hybridization. The phenotype analysis of NPHS1 children, who totally lack nephrin, revealed no marked impairment in the neurological, testicular, or pancreatic function speaking against the idea that nephrin would play an important functional role outside the kidney. The NPHS1 kidneys do not express nephrin and antibodies against this major glomerular filter protein have been observed in NPHS1 children after renal TX most likely as an immune reaction against a novel antigen. These antibodies have been associated with the development of recurrent NS in the kidney graft of NPHS1 patients. In our study, a third of the NPHS1 patients homozygous for Fin-Major mutation developed recurrent NS in the transplanted graft. Re-transplantations were performed to patients who lost their graft due to recurrent NS and heavy proteinuria immediately developed in all cases. While 73% of the patients had detectable serum anti-nephrin antibodies, the kidney biopsy findings were minimal. Introduction of plasma exchange (PE) to the treatment of recurrent nephroses increased the remission rate from 54% to 89%. If remission was achieved, recurrent NS did not significantly deteriorate the long term graft function. In conclusion, the results show that the lack of nephrin in podocyte slit diaphragm in NPHS1 kidneys induces progressive mesangial expansion and glomerular capillary obliteration and inflicts interstitial fibrosis, inflammation, and oxidative stress with surprisingly little involvement of the TECs in this process. Nephrin appears to have no clinical significance outside the kidney. Development of antibodies against nephrin seems to be a major cause of recurrent NS in kidney grafts of NPHS1 patients and combined use of PE and cyclophosphamide markedly improved remission rates.

Novel flutamide regulated genes in the rat ventral prostate: differential modulation of their expression by castration and flutamide treatments

Aim: To identify flutamide regulated genes in the rat ventral prostate. Methods: Total RNA from ventral prostates control and flutamide treated rats were isolated. Differentially expressed transcripts were identified using display reverse transcriptase polymerase chain reaction. The effect of castration on the expression of regulated transcripts was studied. Results: We have identified beta 2-microglobulin, cytoplasmic FMR1 protein 2 and pumilio 1 as flutamide induced and spermine binding protein and ribophorin II as flutamide targets in the rat ventral prostate. Although flutamide treatment caused an induction of pumilio I mRNA, had no effect. Conclusion: Castration and flutamide treatments exert differential effects on gene expression. might also have direct AR independent effects, which might have implications in the emergence of androgen dent prostate cancer and the failure of flutamide therapy.

ErbB-2 expression and its association with other biological parameters of breast cancer among Indian women

Overexpression of the epidermal growth factor receptor family genes, which include ErbB-1, 2, 3 and 4, has been implicated in a number of cancers. We have studied the extent of ErbB-2 overexpression among Indian women with sporadic breast cancer. Methods: Immmunohistochemistry and genomic polymerase chain reaction (PCR) were used to study the ErbB2 overexpression. ErbB2 status was correlated with other clinico-pathological parameters, including patient survival. Results: ErbB-2 overexpression was detected in 43.2% (159/368) of the cases by immunohistochemistry. For a sub-set of patients (n = 55) for whom total DNA was available, ErbB-2 gene amplification was detected in 25.5% (14/55) of the cases by genomic PCR. While the ErbB2 overexpression was significantly higher in patients with lymphnode (χ2 = 12.06, P≤ 0.001), larger tumor size (χ2 = 8.22, P = 0.042) and ductal carcinoma (χ2 = 15.42, P ≤ 0.001), it was lower in patients with disease-free survival (χ2 = 22.13, P ≤ 0.001). Survival analysis on a sub-set of patients for whom survival data were available (n = 179) revealed that ErbB-2 status (χ2 =25.94, P ≤ 0.001), lymphnode status (χ2 = 12.68, P ≤ 0.001), distant metastasis (χ2 = 19.49, P ≤ 0.001) and stage of the disease (χ2 = 28.04, P ≤0.001) were markers of poor prognosis. Conclusions: ErbB-2 overexpression was significantly greater compared with the Western literature, but comparable to other Indian studies. Significant correlation was found between ErbB-2 status and lymphnode status, tumor size and ductal carcinoma. ErbB-2 status, lymph node status, distant metastasis and stage of the disease were found to be prognostic indicators.

DNA Condensation by the Rat Spermatidal Protein TP2 Shows GC-Rich Sequence Preference and Is Zinc Dependent

Transition protein-2 (TP2), isolated from rat testes, was recently shown to be a zinc metalloprotein. We have now carried out a detailed analysis of the DNA condensing properties of TP2 with various polynucleotides using circular dichroism spectroscopy. The condensation of the alternating copolymers by TP2 (incubated with 10 mu M ZnSO4), namely, poly(dG-dC). poly(dG-dC) and poly(dA-dT). poly(dA-dT), was severalfold higher than condensation of either of the homoduplexes poly(dG). poly-(dC) and poly(dA). poly(dT) or rat oligonucleosomal DNA. Between the two alternating copolymers, poly(dG-dC). poly(dG-dC) was condensed 3.2-fold more effectively than poly(dA-dT). poly(dA-dT). Preincubation of TP2 with 5 mM EDTA significantly reduced its DNA-condensing property. Interestingly, condensation of the alternating copolymer poly(dI-dC). poly(dI-dC) by TP2 was much less as compared to that of poly(dG-dC). poly(dG-dC). The V8 protease-derived N-terminal fragment (88 aa) condensed poly(dA-dT). poly(dA-dT) to a very small extent but did not have any effect on poly(dG-dC). poly-(dG-dC). The C-terminal fragment (28 aa) was able to condense poly(dA-dT) . poly(dA-dT) more effectively than poly(dG-dC). poly(dG-dC). These results suggest that TP2 in its zinc-coordinated form condenses GC-rich polynucleotides much more effectively than other types of polynucleotides. Neither the N-terminal two-thirds of TP2 which is the zinc-binding domain nor the C-terminal basic domain are as effective as intact TP2 in bringing about condensation of DNA.

Development of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of sugarcane streak mosaic virus isolates from Southern India

Sugarcane streak mosaic virus (SCSMV), causes mosaic disease of sugarcane and is thought to belong to a new undescribed genus in the family Potyviridae. The coat protein (CP) gene from the Andhra Pradesh (AP) isolate of SCSMV (SCSMV AP) was cloned and expressed in Escherichia coli. The recombinant coat protein was used to raise high quality antiserum. The CP antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) based assay for the detection and discrimination of SCSMV isolates in South India. The sequence of the cloned PCR products encoding 3'untranslated region (UTR) and CP regions of the virus isolates from three different locations in South India viz. Tanuku (Coastal Andhra Pradesh), Coimbatore (Tamil Nadu) and Hospet (Karnataka) was compared with that of SCSMV AP The analysis showed that they share 89.4, 89.5 and 90% identity respectively at the nucleotide level. This suggests that the isolates causing mosaic disease of sugarcane in South India are indeed strains of SCSMV In addition, the sensitivity of the IC-RT-PCR was compared with direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA) and dot-blot immunobinding assays and was found to be more sensitive and hence could be used to detect the presence of virus in sugarcane breeding, germplasm centres and in quarantine programs.

Unique Utilization of a Phosphoprotein Phosphatase Fold by a Mammalian Phosphodiesterase Associated with WAGR Syndrome

Metallophosphoesterase-domain-containing protein 2 (MPPED2) is a highly evolutionarily conserved protein with orthologs found from worms to humans. The human MPPED2 gene is found in a region of chromosome 11 that is deleted in patients with WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome, and MPPED2 may function as a tumor suppressor. However, the precise cellular roles of MPPED2 are unknown, and its low phosphodiesterase activity suggests that substrate hydrolysis may not be its prime function. We present here the structures of MPPED2 and two mutants, which show that the poor activity of MPPED2 is not only a consequence of the substitution of an active-site histidine residue by glycine but also due to binding of AMP or GMP to the active site. This feature, enhanced by structural elements of the protein, allows MPPED2 to utilize the conserved phosphoprotein-phosphatase-like fold in a unique manner, ensuring that its enzymatic activity can be combined with a possible role as a scaffolding or adaptor protein. (C) 2011 Elsevier Ltd. All rights reserved.

Sesbania Mosaic Virus (SeMV) Infectious Clone: Possible Mechanism of 3 ` and 5 ` End Repair and Role of Polyprotein Processing in Viral Replication

Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3' and 5' terminal deletion mutants, we propose a possible mechanism for 3' and 5' end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.

A touch of history and a peep into the future of the lipid-quinone known as coenzyme Q and ubiquinone

Coenzyme Q (ubiquinone), a fully substituted benzoquinone with polyprenyl side chain, participates in many cellular redox activities. Paradoxically it was discovered only in 1957, albeit being ubiquitous. It required a person, F. L. Crane, a place, Enzyme Institute, Madison, USA, and a time when D. E. Green was directing vigorous research on mitochondria. Located at the transition of 2-electron flavoproteins and 1-electron cytochrome carriers, it facilitates electron transfer through the elegant Q-cycle in mitochondria to reduce O-2 to H2O, and to H2O2, now a significant signal-transducing agent, as a minor activity in shunt pathway (animals) and alternative oxidase (plants). The ability to form Q-radical by losing an electron and a proton was ingeniously used by Mitchell to explain the formation of the proton gradient, considered the core of energy transduction, and also in acidification in vacuoles. Known to be a mobile membrane constituent (microsomes, plasma membrane and Golgi apparatus), allowing it to reach multiple sites, coenzyme Q is expected to have other activities. Coenzyme Q protects circulating lipoproteins being a better lipid antioxidant than even vitamin E. Binding to proteins such as QPS, QPN, QPC and uncoupling protein in mitochondria, QA and QB in the reaction centre in R. sphaeroides, and disulfide bond-forming protein in E. coli (possibly also in Golgi), coenzyme Q acquires selective functions. A characteristic of orally dosed coenzyme Q is its exclusive absorption into the liver, but not the other tissues. This enrichment of Q is accompanied by significant decrease of blood pressure and of serum cholesterol. Inhibition of formation of mevalonate, the common precursor in the branched isoprene pathway, by the minor product, coenzyme Q, decreases the major product, cholesterol. Relaxation of contracted arterial smooth muscle by a side-chain truncated product of coenzyme Q explains its effect of decreasing blood pressure. Extensive clinical studies carried out on oral supplements of coenzyine Q, initially by K. Folkers and Y. Yamamura and followed many others, revealed a large number of beneficial effects, significantly in cardiovascular diseases. Such a variety of effects by this lipid quinone cannot depend on redox activity alone. The fat-soluble vitamins (A, D, E and K) that bear structural relationship with coenzyme Q are known to be active in their polar forms. A vignette of modified forms of coenzyme Q taking active role in its multiple effects is emerging.

Crystal Structure of a Monomeric Thiolase-Like Protein Type 1 (TLP1) from Mycobacterium smegmatis

An analysis of the Mycobacterium smegmatis genome suggests that it codes for several thiolases and thiolase-like proteins. Thiolases are an important family of enzymes that are involved in fatty acid metabolism. They occur as either dimers or tetramers. Thiolases catalyze the Claisen condensation of two acetyl-Coenzyme A molecules in the synthetic direction and the thiolytic cleavage of 3-ketoacyl-Coenzyme A molecules in the degradative direction. Some of the M. smegmatis genes have been annotated as thiolases of the poorly characterized SCP2-thiolase subfamily. The mammalian SCP2-thiolase consists of an N-terminal thiolase domain followed by an additional C-terminal domain called sterol carrier protein-2 or SCP2. The M. smegmatis protein selected in the present study, referred to here as the thiolase-like protein type 1 (MsTLP1), has been biochemically and structurally characterized. Unlike classical thiolases, MsTLP1 is a monomer in solution. Its structure has been determined at 2.7 angstrom resolution by the single wavelength anomalous dispersion method. The structure of the protomer confirms that the N-terminal domain has the thiolase fold. An extra C-terminal domain is indeed observed. Interestingly, it consists of six beta-strands forming an anti-parallel beta-barrel which is completely different from the expected SCP2-fold. Detailed sequence and structural comparisons with thiolases show that the residues known to be essential for catalysis are not conserved in MsTLP1. Consistent with this observation, activity measurements show that MsTLP1 does not catalyze the thiolase reaction. This is the first structural report of a monomeric thiolase-like protein from any organism. These studies show that MsTLP1 belongs to a new group of thiolase related proteins of unknown function.

Differential proteomic and genomic profiling of mouse striatal cell model of Huntington's disease and control; probable implications to the disease biology

Huntington's disease (HD) is an autosomal dominant disorder of central nervous system caused by expansion of CAG repeats in exon1 of the huntingtin gene (Htt). Among various dysfunctions originated from the mutation in Htt gene, transcriptional deregulation has been considered to be one of the most important abnormalities. Large numbers of investigations identified altered expressions of genes in brains of HD patients and many models of HD. In this study we employed 2D SDS-PAGE/MALDI-MS coupled with 2D-DIGE and real-time PCR experiments of an array of genes focused to HD pathway to determine altered protein and gene expressions in STHdh(Q111)/Hdh(Q111) cells, a cell model of HD and compared with STHdh(Q7)/Hdh(Q7) cells, its wild type counterpart. We annotated 76 proteins from these cells and observed differential expressions of 31 proteins (by 2D-DIGE) involved in processes like unfolded protein binding, negative regulation of neuron apoptosis, response to superoxides etc. Our PCR array experiments identified altered expressions of 47 genes. Altogether significant alteration of 77 genes/proteins could be identified in this HD cell line with potential relevance to HD biology. Biological significance: In this study we intended to find out differential proteomic and genomic profiles in HD condition. We used the STHdh cells, a cellular model for HD and control. These are mouse striatal neuronal cell lines harboring 7 and 111 knock -in CAG repeats in their two alleles. The 111Q containing cell line (STHdh(Q111)/Hdh(Q111)) mimics diseased condition, whereas the 7Q containing ones (STHdh(Q7)/Hdh(Q7)), serves as the proper control cell line. Proteomic experiments were performed earlier to obtain differential expressions of proteins in R6/2 mice models, Hdh(Q) knock -in mice and in plasma and CSF from HD patients. However, no earlier report on proteomic alterations in these two HD cell lines and control was available in literature. It was, therefore, an important objective to find out differential expressions of proteins in these two cell lines. In this study, we annotated 76 proteins from STHdh(Q7)/Hdh(Q7) and STHdh(Q111)/Hdh(Q111) cells using 2D-gel/mass spectrometry. Next, by performing 2D-DIGE, we observed differential expressions of 31 proteins (16 upregulated and 15 downregulated) between these two cell lines. We also performed customized qRT-PCR array focused to HD pathway and found differential expressions of 47 genes (8 gene exptessions increased and 39 genes were decreased significantly). A total of 77 genes/proteins (Htt downregulated in both the studies) were found to be significantly altered from both the experimental paradigms. We validated the differential expressions of Vim, Hypk, Ran, Dstn, Hspa5 and Sod2 either by qRT-PCR or Western blot analysis or both. Out of these 77, similar trends in alteration of 19 out of 31 and 38 out of 47 proteins/genes were reported in earlier studies. Thus our study confirmed earlier observations on differential gene/protein expressions in HD and are really useful. Additionally, we observed differential expression of some novel genes/proteins. One of this was Hypk, a Htt-interacting chaperone protein with the ability to solubilize mHtt aggregated structures in cell lines. We propose that downregulation of Hypk in STHdh-Qm (Q111)/Hdh(Q111) has a causal effect towards HD pathogenesis. Thus the novel findings from our study need further research and might be helpful to understand the molecular mechanism behind HD pathogenesis. (C) 2015 Elsevier B.V. All rights reserved.

Efeitos pleiotrópicos da telmisartana nos tecidos adiposos branco e marrom: aumento da expressão gênica e proteica pan-PPAR em camundongos obesos

Receptores ativadores de proliferação perixossomal(PPARs) são fatores de transcrição envolvidos com a oxidação dos ácidos graxos e proliferação celular, mediando diversas vias, o que representa uma estratégia promissora para enfrentar as características da síndrome metabólica. Existem três isoformas de PPARs(PPARalfa, beta/delta e gama), que são diferencialmente expressos em diferentes tecidos.No presente estudo, objetivou-se avaliar os efeitos pleiotrópicos da telmisartana, um anti-hipertensivo, bloqueador do receptor AT1 da angiotensina e agonista parcial PPAR gama, no tecido adiposo branco (TAB) e marrom (TAM) em camundongos obesos induzido por dieta.Camundongos machos, da linhagem C57BL/6 foram alimentados com uma dieta padrão (standard-chow, 10% da energia proveniente de lipídios) ou com uma dieta com alto teor lipídico (high fat, 49% de energia proveniente de lipídios) durante 10 semanas. Em seguida, os animais foram distribuídos aleatoriamente em quatro grupos: SC, SC-T, HF e HF-T (n=10). O fármaco foi administrado (10mg/kg de dieta) durante 4 semanas para os grupos SC-T e HF-T.O grupo HF apresentou sobrepeso, hipertensão arterial sistêmica, perfil de adipocinas pró-inflamatórias, resistência insulínica, diminuição do gasto energético, comprometimento do metabolismo da glicose e distribuição anormal da massa adiposa. Além disso, a obesidade ocasionou diminuição da expressão de PPARalfa, beta/delta e gama noTAB e TAM, resultando na inadequação da captação de glicose e termogênese insuficiente. Por outro lado,a ativação das três isoformas de PPARs, a melhora do perfil inflamatório das adipocinas, o aumento da sensibilidade à insulina e a melhora da captação de glicose, foi vistaapós o tratamento com telmisartana. A ativação dos PPARs no TAB trouxe muitos benefícios. No TAM, resultados surpreendentes foram que a telmisartana provocou o aumento da expressão do recepetor adrenérgico beta 3 (RAβ3), induzido pela ativação de PPARbeta/delta e maior termogênese comaumento da expressão da proteína desacopladora1 (UCP1). Em conclusão, nossos resultados mostram que telmisartanaaumenta a expressão gênica e proteica PAN-PPAR no TAB e TAM em camundongos obesos induzidos por dieta. Nossas observações mostram que, apesar do grupo HF-T ter reduzido a ingestão energética, os efeitossão explicados pela ativação PAN-PPAR da telmisartana, causando a ativação da termogênese e resultando num balanço energético negativo.

Dietas ricas em gordura, frutose e sacarose: alterações no fígado

Hábitos inadequados no estilo de vida, pelo consumo exacerbado de dietas ricas em gorduras e açúcares (frutose e sacarose), correlacionam-se positivamente com o desenvolvimento da obesidade, da resistência à insulina (RI) e da esteatose hepática não alcoólica (NAFLD). O estudo teve como objetivo avaliar a magnitude dos efeitos da administração crônica de dietas ricas em gordura e/ou frutose, e ainda, comparar os efeitos dos açúcares isoladamente (frutose e sacarose) sob as alterações bioquímicas, o perfil inflamatório, as respostas morfofuncionais e as expressões proteicas e gênicas de fatores de transcrição envolvidos na lipogênese, na beta-oxidação, na gliconeogênese e no estresse oxidativo no fígado. Camundongos machos C57BL/6 foram divididos em dois experimentos: 1) Dieta controle/standard chow (SC), dieta high fat (HF 42%), dieta high frutose (HFr 34%) e dieta high fat + high frutose (HFHFr - 42% fat + 34% frutose) por 16 semanas; 2) Dieta controle/standard chow (SC), dieta high frutose (HFru 50%) e dieta high sacarose (HSu 50%) por 15 semanas. Ao final dos experimentos foram observados: 1) Não houve diferença na massa corporal entre os animais HFr e SC, só foi observado ganho de peso nos grupos HF e HFHFr. Houve ainda aumento do colesterol total, dos triglicerídeos plasmáticos e hepáticos e RI nos grupos HF, HFr e HFHFr. No fígado, foi observado NAFLD com aumento na expressão de SREBP-1c e PPAR-γ, e redução de PPAR-α. A gliconeogênese mediada pelo GLUT-2 e PEPCK também foi aumentada nos grupos HF, HFr e HFHFr em relação ao grupo SC. Áreas de necroinflamação também foram observadas nos animais HFr e HFHFr; 2) Não houve diferença na massa corporal entre os grupos SC, HFru e HSu. Porém, houve aumento do colesterol total, dos triglicerídeos plasmáticos e hepáticos, da RI, das adipocinas (IL-6, resistina, MCP-1 e leptina), e redução da adiponectina. No fígado, abundante NAFLD com predominância da expressão proteica e gênica de SREBP-1c, PPAR-γ e redução de PPAR-α; e desequilíbrio antioxidante com redução da SOD, da Catalase e da GRx nos grupos HFru e HSu quando comparados ao SC. Não houve diferença na GPx entre os três grupos. Ainda foi observado aumento na expressão proteica de G6Pase, PEPCK e GLUT-2, envolvidos na gliconeogênese hepática nos grupos HFru e HSu. Áreas de necroinflamação, característico da transição NAFLD-NASH, também foram observados. Os resultados permitem concluir que, independente do aumento da massa corporal, a administração crônica de dietas ricas em frutose e sacarose tem efeitos similares aos observados com o consumo de dieta hiperlipídica. Parece que a RI e a NAFLD sejam os precursores destas alterações.