The p.A2215D Thyroglobulin Gene Mutation Leads to Deficient Synthesis and Secretion of the Mutated Protein and Congenital Hypothyroidism with Wide Phenotype Variation

Context: Thyroglobulin (TG) is a large glycoprotein and functions as a matrix for thyroid hormone synthesis. TG gene mutations give rise to goitrous congenital hypothyroidism (CH) with considerable phenotype variation. Objectives: The aim of the study was to report the genetic screening of 15 patients with CH due to TG gene mutations and to perform functional analysis of the p. A2215D mutation. Design: Clinical evaluation and DNA sequencing of the TG gene were performed in all patients. TG expression was analyzed in the goitrous tissue of one patient. Human cells were transfected with expression vectors containing mutated and wild-type human TG cDNA. Results: All patients had an absent rise of serum TG after stimulation with recombinant human TSH. Sequence analysis revealed three previously described mutations (p. A2215D, p. R277X, and g. IVS30 + 1G > T), and two novel mutations (p. Q2142X and g. IVS46-1G > A). Two known (g. IVS30 + 1G/p. A2215D and p. A2215D/p. R277X) and one novel (p. R277X/g. IVS46-1G > A) compound heterozygous constellations were also identified. Functional analysis indicated deficiency in TG synthesis, reduction of TG secretion, and retention of the mutant TG within the cell, leading to an endoplasmic reticulum storage disease, whereas small amounts of mutant TG were still secreted within the cell system. Conclusion: All studied patients were either homozygous or heterozygous for TG gene mutations. Two novel mutations have been detected, and we show that TG mutation p. A2215D promotes the retention of TG within the endoplasmic reticulum and reduces TG synthesis and secretion, causing mild hypothyroidism. In the presence of sufficient iodine supply, some patients with TG mutations are able to compensate the impaired hormonogenesis and generate thyroid hormone. (J Clin Endocrinol Metab 94: 2938-2944, 2009)

SINGLE MEASUREMENTS OF C-REACTIVE PROTEIN AND DISEASE ACTIVITY SCORES ARE NOT PREDICTORS OF CAROTID ATHEROSCLEROSIS IN RHEUMATOID ARTHRITIS PATIENTS

Background: Although inflammation has a defined role in the pathogenesis of atherosclerosis, the link between rheumatoid arthritis (RA) parameters of disease activity and atherosclerotic findings are not defined. Objective: To investigate the association between subclinical carotid atherosclerosis and clinical/laboratorial parameters of RA systemic inflammatory activity. Methods: Seventy-one RA patients were consecutively selected and compared to 53 healthy controls. Smoking, diabetes and hypertension were excluded, as well as the use of statins or fibrates. B-mode carotid ultrasound was performed in all subjects. CRP, ESR and fibrinogen were determined in both groups. Clinical assessment of RA activity included DAS 28 and SDAI. Correlation between plaques and intima-media thickness (IMT) of common carotid arteries and inflammatory parameters was evaluated. Results: Carotid plaques were more prevalent in RA patients than in controls (14.1% vs. 1.9 %, p=0.02) and marginally increased IMT was observed (0.72 +/- 0.17 vs. 0.67 +/- 0.15mm, p=0.07). RA patients with plaques had older age (p=0.001) and increased IMT (p<0.001), but low SDAI (p=0.025) compared to those without plaques. RA patients with plaques had also longer disease duration, although this difference did not reach statistical significance (p=0.06). No significant correlations were found between IMT and ESR (p=0.80), CRP (p=0.75), fibrinogen (p=0.94), HAQ (p=0.89) and DAS 28 (p=0.13). Conclusions: Carotid atherosclerosis is more frequently detected in RA but its prevalence was not correlated with isolated inflammatory markers measurement or noncumulative activity scores. These findings reinforce the need to evaluate subclinical atherosclerosis in RA patients, and to find predictors of atherosclerotic lesions.

Metabolism of triglyceride-rich lipoproteins and lipid transfer to high-density lipoprotein in young obese and normal-weight patients with polycystic ovary syndrome

Objective: To clarify whether the metabolism of triglyceride-rich lipoproteins and lipid transfer to high-density lipoprotein (HDL) are altered in patients with polycystic ovary syndrome (PCOS). Design: Case control study. Setting: Endocrinology clinics. Patient(s): Eight normal-weight (NW) and 15 obese (013) patients with PCOS were compared with 10 NW and 10 Ob women without PCOS paired for age and body mass index. Intervention(s): Determination of triglyceride-rich lipoprotein metabolism and lipid transfer to HDL. Main Outcome Measure(s): Participants were injected triglyceride-rich emulsions labeled with (14)C-cholesteryl esters and (3)H-triglycerides and the fractional clearance rate (FCR, in min(-1)) of labels was determined. Lipid transfer from artificial nanoemulsions to HDL was performed by incubating radioactively labeled lipid nanoemulsions with plasma during 1 hour, followed by radioactive counting of HDL-containing supernatant after chemical precipitation. Result(s): Lipolysis estimated by triglyceride FCR was equal in PCOS groups (NW = 0.043 +/- 0.032, Ob = 0.033 +/- 0.009) and respective controls (NW = 0.039 +/- 0.015, Ob = 0.044 +/- 0.019). However, the remnant removal as estimated by cholesteryl ester FCR was reduced in both PCOS groups (NW = 0.005 +/- 0.006, Ob = 0.005 +/- 0.005) compared with controls (NW = 0.016 +/- 0.006, Ob = 0.011 +/- 0.072). Lipid transfer rates were not different among groups, but triglyceride transfer rates were positively correlated with homeostasis model assessment estimate of insulin resistance in PCOS. Conclusion(s): PCOS patients showed decreased removal of atherogenic remnants even when fasting glucose was <100 mg/dL. This reinforces the usefulness of the measures taken to prevent cardiovascular events in PCOS patients. (Fertil Steril (R) 2010;93:1948-56. (C)2010 by American Society for Reproductive Medicine.)

Body Mass Index Increase, Serum Leptin, Adiponectin, Neuropeptide Y and Lipid Levels during Treatment with Olanzapine and Haloperidol

Introduction: Body mass index (BMI) increase is an undesired effect associated with antipsychotics, and crucial for patients` global health and treatment compliance. We aimed to investigate the relation between BMI during olanzapine or halopericlol treatments and leptin, neuropeptide Y (NPY), adiponectin and lipid serum levels. Methods: In this 9-month, randomized and naturalist study, 34 male patients, 18 on olanzapine and 16 on haloperidol group were enrolled, all were under monotherapy. Patient outcome was evaluated with positive and negative syndrome scale (PANSS) at every 3-month period. In each visit, BMI, leptin, NPY, lipid, olanzapine or haloperidol levels were also monitored. Results and Discussion: Leptin levels positively correlated with BMI in olanzapine (r = 0.64, p < 0.001) and haloperidol (r = 0.73, p < 0.001) groups; only in olanzapine patients, the former also correlated with PANSS score (r = 0.54, p < 0.05). NPY levels negatively correlated with olanzapine levels (r = -0.65, p < 0.01). Adiponectin levels had not significantly varied. Conclusion: Antipsychotics probably interfere on leptin and NPY signalling ways and disturb these hormones in eating behaviour control.

A subunit vaccine based on biodegradable microspheres carrying rHsp65 protein and KLK protects BALB/c mice against tuberculosis infection

Of the hundreds of new tuberculosis ( TB) vaccine candidates some have therapeutic value in addition to their prophylactic properties. This is the case for the DNA vaccine encoding heat-shock protein 65 (DNAhsp65) from Mycobacterium leprae. However, there are concerns about the use of DNA vaccines in certain populations such as newborns and pregnant women. Thus, the optimization of vaccination strategies that circumvent this limitation is a priority. This study evaluated the efficacy of a single dose subunit vaccine based on recombinant Hsp65 protein against infection with M. tuberculosis H37Rv. The Hsp65 protein in this study was either associated or not with immunostimulants, and was encapsulated in biodegradable PLGA microspheres. Our results demonstrate that the protein was entrapped in microspheres of adequate diameter to be engulfed by phagocytes. Mice vaccinated with a single dose of Hsp65-microspheres or Hsp65 + CpG-microspheres developed both humoral and cellular-specific immune responses. However, they did not protect mice against challenge with M. tuberculosis. By contrast, Hsp65+KLK-microspheres induced specific immune responses that reduced bacilli loads and minimized lung parenchyma damage. These data suggest that a subunit vaccine based on recombinant protein Hsp65 is feasible.

Chronic Acetonemia Alters Liver Oxidative Balance and Lipid Content in Rats. A Model of Nash?

Acetone is considered to be a substance that can disturb cellular oxidative status, being also associated with the production of glucose during its metabolization. The objective of the present study was to determine the effects of chronic treatment with acetone in oxidative stress and metabolic parameters in rats. Twenty male Wistar rats were divided into two groups: control (CG) and chronic acetone group (CAG). After 28 days of acetone ingestion in a 5% aqueous solution (CAG) or water (CG) the animals were euthanized and urine, plasma and liver were collected for the determination of acetone, glucose, lipemia, hepatic fat, malondialdehyde (MDA), reduced glutathione (GSH), and vitamin E. As expected, urinary and plasma acetone levels were higher in CAG. There was no difference in hepatic MDA values between groups, whereas hepatic GSH was lower in CAG than in CG and hepatic vitamin E was higher in CAG than in CG. There was also an increase in glycemia, cholesterolemia and hepatic fat in CAG compared to CG. Chronic treatment with a 5% acetone solution produced an increase in acetonemia that was able to promote changes in hepatic oxidative metabolism and in lipid content in rats similar to those observed in nonalcoholic steatohepatitis.

Effects of creatine supplementation on homocysteine levels and lipid peroxidation in rats

Hyperhomocysteinaemia is an independent risk factor for CVD. Recent data show a relationship between homocysteine (Hcy) and free radical formation. Since creatine synthesis is responsible for most of the methyl group transfers that result in Hcy formation, creatine supplementation might inhibit Hcy production and reduce free radical formation. The present study investigated the effects of creatine supplementation on Hcy levels and lipid peroxidation biomarkers. Thirty rats were divided into three groups: control group; diet with creatine group (DCr; 2% creatine in the diet for 28 d); creatine overload plus diet with creatine group (CrO + D; 5 g creatine/kg by oral administration for 5 d + 2 % in the diet for 23 d). Plasma Hcy was significantly lower (P<0.05) in DCr (7.5 (SD 1.2) mu mol/l) and CrO + D (7.2 (SD 1.7) mu mol/l) groups compared with the control group (12.4 (SD 2.2) mu mol/l). Both plasma thiobarbituric acid-reactive species (TBARS) (control, 10 (SD 3.4); DCr, 4.9 (So 0.7); CrO + D, 2.4 (SD 1) mu mol/l) and plasma total glutathione (control, 4.3 (SD 1.9); DCr, 2.5 (SD 0.8); CrO + D, 1.8 (SD 0.5) mu mol/l) were lower in the groups that received creatine (P<0.05). In addition, Hcy showed significant negative correlation (P<0.05) with plasma creatine (r - 0.61) and positive correlation with plasma TBARS (r 0.74). Plasma creatine was negatively correlated with plasma TBARS (r - 0.75) and total peroxide (r - 0.40). We conclude that creatine supplementation reduces plasma Hcy levels and lipid peroxidation biomarkers, suggesting a protective role against oxidative damage. Modulating Hcy fort-nation may, however, influence glutathione synthesis and thereby affect the redox state of the cells.

Proteomic analysis of low- to high-grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non-neoplastic brain tissue as control using 2-DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up-regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p < 0.05), whereas four proteins were down-regulated, among them raf kinase inhibitor protein (RKIP) (p < 0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real-time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.

Determination of P-glycoprotein, MDR-related protein 1, breast cancer resistance protein, and lung-resistance protein expression in leukemic stem cells of acute myeloid leukemia

Background: The most primitive leukemic precursor in acute myeloid leukemia (AML) is thought to be the leukemic stem cell (LSC), which retains the properties of self-renewal and high proliferative capacity and quiescence of the hematopoietic stem cell. LSC seems to be immunophenotypically distinct and more resistant to chemotherapy than the more committed blasts. Considering that the multidrug resistance (MDR) constitutive expression may be a barrier to therapy in AML, we have investigated whether various MDR transporters were differentially expressed at the protein level by different leukemic subsets. Methods: The relative expression of the drug-efflux pumps P-gp, MRP, LRP, and BCRP was evaluated by mean fluorescence index (MFI) and the Kolmogorov-Smirnov analysis (D values) in five leukemic subpopulations: CD34(+)CD38(-)CD123(+) (LSCs), CD34(+)CD38(+)CD123(-), CD34(+)CD38(+)CD123(+), CD34(+)CD38(+)CD123(-), and CD34(-) mature cells in 26 bone marrow samples of CD34(+) AML cases. Results: The comparison between the two more immature subsets (LSC versus CD34(+)CD38(-)CD123(-) cells) revealed a higher P-gp, MRP, and LRP expression in LSCs. The comparative analysis between LSCs and subsets of intermediate maturation (CD34(+)CD38(+)) demonstrated the higher BCRP expression in the LSCs. In addition, P-gp expression was also significantly higher in the LSC compared to CD34(+)CD38(+)CD123(-) subpopulation. Finally, the comparative analysis between LSC and the most mature subset (CD34(-)) revealed higher MRP and LRP and lower P-gp expression in the LSCs. Conclusions: Considering the cellular heterogeneity of AML, the higher MDR transporters expression at the most immature, self-renewable, and quiescent LSC population reinforces that MDR is one of the mechanisms responsible for treatment failure. (C) 2008 Clinical Cytometry Society.

Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface-exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

P-T-Fluid evolution and graphite deposition during retrograde metamorphism in Ribeira Fold Belt, SE Brazil: Oxygen fugacity, fluid inclusions and C-O-H isotopic evidence

Combined fluid inclusion (FI) microthermometry, Raman spectroscopy, X-ray diffraction, C-O-H isotopes and oxygen fugacities of granulites from central Ribeira Fold Belt, SE Brazil, provided the following results: i) Magnetite-Hematite fO(2) estimates range from 10(-11.5) bar (QFM + 1) to 10(-18.3) bar (QFM - 1) for the temperature range of 896 degrees C-656 degrees C, implying fO(2) decrease from metamorphic peak temperatures to retrograde conditions; ii) 5 main types of fluid inclusions were observed: a) CO(2) and CO(2)-N(2) (0-11 mol%) high to medium density (1.01-0.59 g/cm(3)) FI; b) CO(2) and CO(2)-N(2) (0-36 mol%) low density (0.19-0.29 g/cm(3)) FI; c) CO(2) (94-95 mol%)-N(2) (3 mol%)-CH(4) (2-3 mol%)-H(2)O (water phi(v) (25 degrees C) = 0.1) FI; d) low-salinity H(2)O-CO(2) FI; and e) late low-salinity H(2)O FI; iii) Raman analyses evidence two graphite types in khondalites: an early highly ordered graphite (T similar to 450 degrees C) overgrown by a disordered kind (T similar to 330 degrees C); iv) delta(18)O quartz results of 10.3-10.7 parts per thousand, imply high-temperature CO(2) delta(18)O values of 14.4-14.8 parts per thousand, suggesting the involvement of a metamorphic fluid, whereas lower temperature biotite delta(18)O and delta D results of 7.5-8.5 parts per thousand and -54 to -67 parts per thousand respectively imply H(2)O delta(18)O values of 10-11 parts per thousand and delta D(H2O) of -23 to -36 parts per thousand suggesting delta(18)O depletion and increasing fluid/rock ratio from metamorphic peak to retrograde conditions. Isotopic results are compatible with low-temperature H(2)O influx and fO(2) decrease that promoted graphite deposition in retrograde granulites, simultaneous with low density CO(2), CO(2)-N(2) and CO(2)-N(2)-CH(4)-H(2)O fluid inclusions at T = 450-330 degrees C. Graphite delta(13)C results of -10.9 to -11.4 parts per thousand imply CO(2) delta(13)C values of -0.8 to -1.3 parts per thousand suggesting decarbonation of Cambrian marine carbonates with small admixture of lighter biogenic or mantle derived fluids. Based on these results, it is suggested that metamorphic fluids from the central segment of Ribeira Fold Belt evolved to CO(2)-N(2) fluids during granulitic metamorphism at high fO(2), followed by rapid pressure drop at T similar to 400-450 degrees C during late exhumation that caused fO(2) reduction induced by temperature decrease and water influx, turning carbonic fluids into CO(2)-H(2)O (depleting biotite delta(18)O and delta D values), and progressively into H(2)O. When fO(2) decreased substantially by mixture of carbonic and aqueous fluids, graphite deposited forming khondalites. (C) 2010 Elsevier Ltd. All rights reserved.

Salinity influences glutathione S-transferase activity and lipid peroxidation responses in the Crassostrea gigas oyster exposed to diesel oil

Biochemical responses in bivalve mollusks are commonly employed in environmental studies as biomarkers of aquatic contamination. The present study evaluated the possible influence of salinity (35, 25,15 and 9 ppt) in the biomarker responses of Crassostrea gigas oysters exposed to diesel at different nominal concentrations (0.01, 0.1 and 1 mLL(-1)) using a semi-static exposure system. Salinity alone did not resulted in major changes in the gill`s catalase activity (CAT), glutathione S-transferase activity (GST) and lipid peroxidation levels (measured as malondialdehyde. MDA), but influenced diesel related responses. At 25 ppt salinity, but not at the other salinity levels, oysters exposed to diesel showed a strikingly positive concentration-dependent GST response. At 25 ppt and 1 mLL(-1) diesel, the GST activity in the gills remained elevated, even after one week of depuration in clean water. The increased MDA levels in the oysters exposed to diesel comparing to control groups at 9, 15 and 35 ppt salinities suggest the occurrence of lipid peroxidation in those salinities, but not at 25 ppt salinity. The MDA quickly returned to basal levels after 24 h of depuration. CAT activity was unaltered by the treatments employed. High toxicity for 1 mLL(-1) diesel was observed only at 35 ppt salinity, but not in the other salinities. Results from this study strongly suggest that salinity influences the diesel related biomarker responses and toxicity in C. gigas, and that some of those responses remain altered even after depuration. (C) 2011 Elsevier B.V. All rights reserved.

Prion Protein and Its Ligand Stress Inducible Protein 1 Regulate Astrocyte Development

Prion protein (PrP(C)) interaction with stress inducible protein 1 (STI1) mediates neuronal survival and differentiation. However, the function of PrP(C) in astrocytes has not been approached. In this study, we show that STI1 prevents cell death in wild-type astrocytes in a protein kinase A-dependent manner, whereas PrP(C)-null astrocytes were not affected by STI1 treatment. At embryonic day 17, cultured astrocytes and brain extracts derived from PrP(C)-null mice showed a reduced expression of glial fibrillary acidic protein (GFAP) and increased vimentin and nestin expression when compared with wild-type, suggesting a slower rate of astrocyte maturation in PrP(C)-null animals. Furthermore, PrP(C)-null astrocytes treated with STI1 did not differentiate from a flat to a process-bearing morphology, as did wild-type astrocytes. Remarkably, STI1 inhibited proliferation of both wild-type and PrP(C)-null astrocytes in a protein kinase C-dependent manner. Taken together, our data show that PrP(C) and STI1 are essential to astrocyte development and act through distinct signaling pathways.(C) 2009 Wiley-Liss, Inc.

Adding value to the meat of spent laying hens manufacturing sausages with a healthy appeal

The aim of this study was to evaluate the viability of the use of spent laying hens' meat in the manufacturing of mortadella-type sausages with healthy appeal by using vegetable oil instead of animal fat. 120 Hy-line® layer hens were distributed in a completely randomized design into two treatments of six replicates with ten birds each. The treatments were birds from light Hy-line® W36 and semi-heavy Hy-line® Brown lines. Cold carcass, wing, breast and leg fillets yields were determined. Dry matter, protein, and lipid contents were determined in breast and leg fillets. The breast and legg fillets of three replicates per treatment were used to manufacture mortadella. After processing, sausages were evaluated for proximal composition, objective color, microbiological parameters, fatty acid profile and sensory acceptance. The meat of light and semi-heavy spent hens presented good yield and composition, allowing it to be used as raw material for the manufacture of processed products. Mortadellas were safe from microbiological point of view, and those made with semi-heavy hens fillets were redder and better accepted by consumers. Values for all sensory attributes were evaluated over score 5 (neither liked nor disliked). Both products presented high polyunsaturated fatty acid contents and good polyunsaturated to saturated fatty acid ratio. The excellent potential for the use of meat from spent layer hens of both varieties in the manufacturing of healthier mortadella-type sausage was demonstrated.

A New Approach to Ammonium Sulphate Feeding for Fed-Batch Arthrospira (Spirulina) platensis Cultivation in Tubular Photobioreactor

Arthrospira platensis was cultivated in tubular photobioreactor using different photosynthetic photon flux densities (PPFD) and protocols of (NH(4))(2)SO(4) fed-hatch supply. Results were evaluated by variance analysis selecting maximum cell concentration (X(m)), cell productivity (P(x)), nitrogen-to-cell conversion factor (Y(X/N)) and biomass, protein and lipid contents as responses. At PPFD of 120 and 240 mu mol-photons/m(2) s, a parabolic profile of (NH(4))(2)SO(4) addition aiming at producing biomass with 7% nitrogen content ensured X(m) values (14.1 and 12.2 g/L, respectively) comparable to those obtained with NaNO(3). At PPFD of 240 mu mol-photons/m(2) s, P(x) (1.69 g/Ld) was 36% higher, although the photosynthetic efficiency (3.0%) was less than one-half that at PPFD of 120 mu mol-photons/m(2) s. Biomass was shown to be constituted by about 35% proteins and 10% lipids, without any dependence on PPFD or kind of nitrogen source. These results highlight the possible use of (NH(4))(2)SO(4) as alternative, cheap nitrogen source for A. platensis cultivation in tubular photobioreactors. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1271-1277, 2010