Osteopontin Deletion Prevents the Development of Obesity and Hepatic Steatosis via Impaired Adipose Tissue Matrix Remodeling and Reduced Inflammation and Fibrosis in Adipose Tissue and Liver in Mice

Osteopontin (OPN) is a multifunctional extracellular matrix (ECM) protein involved in multiple physiological processes. OPN expression is dramatically increased in visceral adipose tissue in obesity and the lack of OPN protects against the development of insulin resistance and inflammation in mice. We sought to unravel the potential mechanisms involved in the beneficial effects of the absence of OPN. We analyzed the effect of the lack of OPN in the development of obesity and hepatic steatosis induced by a high-fat diet (HFD) using OPN-KO mice. OPN expression was upregulated in epididymal white adipose tissue (EWAT) and liver in wild type (WT) mice with HFD. OPN-KO mice had higher insulin sensitivity, lower body weight and fat mass with reduced adipose tissue ECM remodeling and reduced adipocyte size than WT mice under a HFD. Reduced MMP2 and MMP9 activity was involved in the decreased ECM remodeling. Crown-like structure number in EWAT as well as F4/80-positive cells and Emr1 expression in EWAT and liver increased with HFD, while OPN-deficiency blunted the increase. Moreover, our data show for the first time that OPN-KO under a HFD mice display reduced fibrosis in adipose tissue and liver, as well as reduced oxidative stress in adipose tissue. Gene expression of collagens Col1a1, Col6a1 and Col6a3 in EWAT and liver, as well as the profibrotic cytokine Tgfb1 in EWAT were increased with HFD, while OPN-deficiency prevented this increase. OPN deficiency prevented hepatic steatosis via reduction in the expression of molecules involved in the onset of fat accumulation such as Pparg, Srebf1, Fasn, Mogat1, Dgat2 and Cidec. Furthermore, OPN-KO mice exhibited higher body temperature and improved BAT function. The present data reveal novel mechanisms of OPN in the development of obesity, pointing out the inhibition of OPN as a promising target for the treatment of obesity and fatty liver.     

A highly sensitive DNA bead-based suspension array for the detection and species identification of bovine piroplasms

Piroplasms are among the most harmful tick-borne pathogens for livestock and sensitive and specific diagnostic methods for rapid detection and identification of the different species are needed for effective control. Reverse Line Blot has been the molecular technique of choice but it is laborious, time-consuming and highly susceptible to subjective variation in the interpretation of the hybridisation signal. Here, an oligonucleotide multiplex suspension microarray (Luminex® microsphere system) was developed for bovine piroplasms. Probes previously used in Reverse Line Blot for Babesia divergens, Babesia bovis, Babesia occultans, Babesia bigemina and Theileria buffeli, and a catch-all Theileria and Babesia control probe, were included in the Luminex assay together with newly designed probes for Theileria annulata and Babesia major. An internal amplification control that was detected with a Luminex probe was included to monitor for inhibition. Serially diluted linearised recombinant plasmids of the different species were used to assess the analytical sensitivity and specificity, and the detection limit of the Luminex assay was determined using serial dilutions of infected blood from an animal with a known level of T. annulata parasitaemia. The assay was then validated on 214 bovine blood samples analysed in parallel by Reverse Line Blot and Luminex. The Luminex assay proved to be highly specific and more sensitive than Reverse Line Blot, detecting 0.05 parasites/μl of blood. Technically, the Luminex procedure was rapid, provided high throughput screening, transformed the subjective interpretation of Reverse Line Blot results into numerical objective values, and allowed more flexibility in array preparation than Reverse Line Blot. The method described herein can substantially improve the detection of piroplasm carriers and thus better protect livestock trade and facilitate preventive control programs.     

myo-Inositol uptake is essential for bulk inositol phospholipid but not glycosylphosphatidylinositol synthesis in Trypanosoma brucei

myo-Inositol is an essential precursor for the production of inositol phosphates and inositol phospholipids in all eukaryotes. Intracellular myo-inositol is generated by de novo synthesis from glucose 6-phosphate or is provided from the environment via myo-inositol symporters. We show that in Trypanosoma brucei, the causative pathogen of human African sleeping sickness and nagana in domestic animals, myo-inositol is taken up via a specific proton-coupled electrogenic symport and that this transport is essential for parasite survival in culture. Down-regulation of the myo-inositol transporter using RNA interference inhibited uptake of myo-inositol and blocked the synthesis of the myo-inositol-containing phospholipids, phosphatidylinositol and inositol phosphorylceramide; in contrast, it had no effect on glycosylphosphatidylinositol production. This together with the unexpected localization of the myo-inositol transporter in both the plasma membrane and the Golgi demonstrate that metabolism of endogenous and exogenous myo-inositol in T. brucei is strictly segregated.     

Discrimination of Aspergillus niger and other Aspergillus species belonging to section Nigri by PCR assays

Aspergillus species included in section Nigri are common in plant products and processed food, such as grapes, cereals, coffee and derivatives, particularly in warm and tropical climates. Two of these species, A. carbonarius and A. niger, are known to produce ochratoxin A (OTA), a potent nephrotoxin and carcinogenic to human (group 2B). Recognition of the several species of this section is difficult and requires considerable expertise using conventional methods based on morphological features. In this work we describe rapid, sensitive and robust assays based on the PCR technique to discriminate the main species included in section Nigri: A. japonicus, A. heteromorphus, A. ellipticus and the two morphologically indistinguishable species of the A. niger aggregate: A. niger and A. tubingensis. The species-specific primers have been designed on the basis of ITS (internal transcribed spacers of rDNA units) sequence comparisons obtained from several Aspergillus strains and have been tested in a number of strains from different origins and hosts. These PCR assays, based on multi-copy sequences, are highly sensitive and specific and represent a good tool for an early detection of OTA-producing Aspergillus species in order to prevent OTA from entering the food chain.     

Molecular cloning of an IL-8-like CXC chemokine and tissue factor in rainbow trout (Oncorhynchus mykiss) by use of suppression subtractive hybridization

Suppression subtractive hybridization (SSH) was performed to construct a Rainbow trout cDNA library enriched in sequences up-regulated in head kidney leukocytes after lipopolysaccharide (LPS) and tumour necrosis factor alpha (TNFalpha) stimulation. Random sequencing of fifty clones allowed the identification of a Rainbow trout interleukin 8 (IL-8)-related CXC chemokine, as well as the Rainbow trout tissue factor (TF) precursor. Expression of both the IL-8-like chemokine and TF is induced after LPS and TNFalpha stimulation, indicating that they are associated with inflammatory responses in fish, as has been suggested in mammals. These results confirm the potential of SSH to identify cytokines and immuno-regulatory genes in fish.     

The role of carotenoids in preventing oxidative damage in the pigmented yeast, Rhodotorula mucilaginosa

Rhodotorula mucilaginosa is an obligate aerobic yeast which contains a high concentration of carotenoid pigment. To test whether carotenoids are able to protect R. mucilaginosa against oxidative injury, yeast cells in liquid culture were incubated with duroquinone (DQ) (100 microM), a redox-cycling quinone known to generate intracellular O2-. or were grown in a hyperoxic atmosphere (80% O2) under conditions where carotenoid concentrations were altered either intracellularly or extracellularly. Neither of these oxidative challenges affected cell growth unless carotenogenesis was blocked by the addition of diphenylamine (50 microM). In the diphenylamine-treated nonpigmented cells, growth was completely inhibited by DQ and by hyperoxia. In normoxia, however, diphenylamine alone reduced growth by only 30%. The growth inhibition observed in diphenylamine-treated cells exposed to hyperoxia was primarily mycocidal rather than mycostatic since plating of these cells onto solid media revealed that only 25% of the cells were viable after 50 h of incubation when compared to plated control cells. Addition of 10 microM beta-carotene to diphenylamine-treated cells completely prevented the growth inhibition caused by either hyperoxia or DQ. Carotenoids, therefore, are able to prevent oxidant-induced cytotoxicity in R. mucilaginosa. Analysis of the absorption spectra of chloroform extracts of beta-carotene-supplemented cells showed that beta-carotene, not the endogenous carotenoid, torularhodin, was the major carotenoid present in these cells. Superoxide dismutase (SOD) activity in R. mucilaginosa was compared with that of another yeast, Saccharomyces cerevisiae by two methods: (i) activity staining of proteins separated by gel electrophoresis and (ii) measurement of inhibition of ferricytochrome c reduction. By these techniques, the R. mucilaginosa SOD activity had the characteristics of Mn-SOD. No Cu/ZnSOD activity was detected. Thus, the apparent absence of Cu/ZnSOD may make the antioxidant capability of endogenous carotenoids even more critical in preventing oxidative damage in R. mucilaginosa.     

Synthetic chalcones, flavanones, and flavones as antitumoral agents: biological evaluation and structure-activity relationships

A series of synthetic chalcones, flavanones, and flavones has been synthesized and evaluated for antitumor activity against the human kidney carcinoma cells TK-10, human mammary adenocarcinoma cells MCF-7 (estrogen receptor-positive), and human colon adenocarcinoma cells HT-29. The most active series is the chalcone ones with the best results against TK-10 and HT-29 cells. Fourteen out of 53 analyzed compounds resulted very active against at least two of the studied tumoral cells. Alkaline single cell gel electrophoresis, comet assay, was performed as a study of the chromosomal aberrations promoted by the compounds on normal cells. Four active and two inactive chalcones were studied in the comet assay against normal human kidney cells (HK-2). A structure-activity relationship analysis of these compounds was performed and for 4- and 3,4-disubstituted derivatives a quantitative correlation was obtained in the case of anti-HT-29 activity.