Resistance to thyroid hormone and down syndrome: coincidental association or genetic linkage?

We report the first case of RTH and DS. Although this congruence could be coincidental, we cannot exclude a possible linkage between both syndromes.

Glut2-dependent glucose-sensing controls thermoregulation by enhancing the leptin sensitivity of NPY and POMC neurons.

The physiological contribution of glucose in thermoregulation is not completely established nor whether this control may involve a regulation of the melanocortin pathway. Here, we assessed thermoregulation and leptin sensitivity of hypothalamic arcuate neurons in mice with inactivation of glucose transporter type 2 (Glut2)-dependent glucose sensing. Mice with inactivation of Glut2-dependent glucose sensors are cold intolerant and show increased susceptibility to food deprivation-induced torpor and abnormal hypothermic response to intracerebroventricular administration of 2-deoxy-d-glucose compared to control mice. This is associated with a defect in regulated expression of brown adipose tissue uncoupling protein I and iodothyronine deiodinase II and with a decreased leptin sensitivity of neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons, as observed during the unfed-to-refed transition or following i.p. leptin injection. Sites of central Glut-2 expression were identified by a genetic tagging approach and revealed that glucose-sensitive neurons were present in the lateral hypothalamus, the dorsal vagal complex, and the basal medulla but not in the arcuate nucleus. NPY and POMC neurons were, however, connected to nerve terminals from Glut2-expressing neurons. Thus, our data suggest that glucose controls thermoregulation and the leptin sensitivity of NPY and POMC neurons through activation of Glut2-dependent glucose-sensing neurons located outside of the arcuate nucleus.

Autoimmunity does not contribute to the highly prevalent glucose metabolism disturbances in a Japanese Brazilian population

The Japanese Brazilian population has one of the highest prevalences of diabetes worldwide. Despite being non-obese according to standard definitions, their body fat distribution is typically central. We investigated whether a subset of these subjects had autoantibodies that would suggest a slowly progressive form of type 1 diabetes. A total of 721 Japanese Brazilians (386 men) in the 30- to 60-year age group underwent clinical examination and laboratory procedures, including a 75-g oral glucose tolerance test and determinations of serum autoantibodies. Antibodies to glutamic acid decarboxylase (GADab) were determined by radioimmunoassay and to thyroglobulin (TGab) and thyroperoxidase (TPOab) by flow-cytometry assays. Mean body mass index was 25.2 ± 3.8 kg/m2, but waist circumference was elevated according to the Asian standards. Diabetes, impaired glucose tolerance, and impaired fasting glycemia were found in 31%, 22%, and 22%, respectively, and 53% of the subjects had metabolic syndrome. Glutamic acid decarboxylase (GADab) was positive in 4.72%, TGab in 9.6%, and TPOab in 10% of the whole sample. When participants were stratified according to the presence of thyroid antibodies, similar frequencies of GADab were found in positive and negative groups. The prevalence rates of glucose metabolism disturbances did not differ between GADab positive and negative groups. Our data did not support the view that autoimmune injury could contribute to the high prevalence of diabetes seen in Japanese Brazilians, and the presence of co-morbidities included in the spectrum of metabolic syndrome favors the classification as type 2 diabetes.

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10 mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10 mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing apoptosis. (C) 2010 Elsevier Ltd. All rights reserved.

Thyroid peroxidase activity is inhibited by amino acids

Normal in vitro thyroid peroxidase (TPO) iodide oxidation activity was completely inhibited by a hydrolyzed TPO preparation (0.15 mg/ml) or hydrolyzed bovine serum albumin (BSA, 0.2 mg/ml). A pancreatic hydrolysate of casein (trypticase peptone, 0.1 mg/ml) and some amino acids (cysteine, tryptophan and methionine, 50 µM each) also inhibited the TPO iodide oxidation reaction completely, whereas casamino acids (0.1 mg/ml), and tyrosine, phenylalanine and histidine (50 µM each) inhibited the TPO reaction by 54% or less. A pancreatic digest of gelatin (0.1 mg/ml) or any other amino acid (50 µM) tested did not significantly decrease TPO activity. The amino acids that impair iodide oxidation also inhibit the TPO albumin iodination activity. The inhibitory amino acids contain side chains with either sulfur atoms (cysteine and methionine) or aromatic rings (tyrosine, tryptophan, histidine and phenylalanine). Among the amino acids tested, only cysteine affected the TPO guaiacol oxidation reaction, producing a transient inhibition at 25 or 50 µM. The iodide oxidation inhibitory activity of cysteine, methionine and tryptophan was reversed by increasing iodide concentrations from 12 to 18 mM, while no such effect was observed when the cofactor (H2O2) concentration was increased. The inhibitory substances might interfere with the enzyme activity by competing with its normal substrates for their binding sites, binding to the free substrates or reducing their oxidized form.

Posttranscriptional regulation of sodium-iodide symporter mRNA expression in the rat thyroid gland by acute iodide administration

Serrano-Nascimento C, Calil-Silveira J, Nunes MT. Posttranscriptional regulation of sodium-iodide symporter mRNA expression in the rat thyroid gland by acute iodide administration. Am J Physiol Cell Physiol 298: C893-C899, 2010. First published January 27, 2010; doi:10.1152/ajpcell.00224.2009.-Iodide is an important regulator of thyroid activity. Its excess elicits the Wolff-Chaikoff effect, characterized by an acute suppression of thyroid hormone synthesis, which has been ascribed to serum TSH reduction or TGF-beta increase and production of iodolipids in the thyroid. These alterations take hours/days to occur, contrasting with the promptness of Wolff-Chaikoff effect. We investigated whether acute iodide administration could trigger events that precede those changes, such as reduction of sodium-iodide symporter (NIS) mRNA abundance and adenylation, and if perchlorate treatment could counteract them. Rats subjected or not to methylmercaptoimidazole treatment (0.03%) received NaI (2,000 mu g/0.5 ml saline) or saline intraperitoneally and were killed 30 min up to 24 h later. Another set of animals was treated with iodide and perchlorate, in equimolar doses. NIS mRNA content was evaluated by Northern blotting and real-time PCR, and NIS mRNA poly(A) tail length by rapid amplification of cDNA ends-poly(A) test (RACE-PAT). We observed that NIS mRNA abundance and poly(A) tail length were significantly reduced in all periods of iodide treatment. Perchlorate reversed these effects, indicating that iodide was the agent that triggered the modifications observed. Since the poly(A) tail length of mRNAs is directly associated with their stability and translation efficiency, we can assume that the rapid decay of NIS mRNA abundance observed was due to a reduction of its stability, a condition in which its translation could be impaired. Our data show for the first time that iodide regulates NIS mRNA expression at posttranscriptional level, providing a new mechanism by which iodide exerts its autoregulatory effect on thyroid.

Differential gene expression analysis of iodide-treated rat thyroid follicular cell line PCCl3

The inhibitory effect of supraphysiological iodide concentrations on thyroid hormone synthesis (Wolff - Chaikoff effect) and on thyrocyte proliferation is largely known as iodine autoregulation. However, the molecular mechanisms by which iodide modulates thyroid function remain unclear. In this paper, we analyze the transcriptome profile of the rat follicular cell lineage PCCl3 under untreated and treated conditions with 10 (- 3) M sodium iodide (NaI). Serial analysis of gene expression (SAGE) revealed 84 transcripts differentially expressed in response to iodide (p <= 0.001). We also showed that iodide excess inhibits the expression of essential genes for thyroid differentiation: Tshr, Nis, Tg, and Tpo. Relative expression of 14 of 20 transcripts selected by SAGE was confirmed by real-time PCR. Considering the key role of iodide organification in thyroid physiology, we also observed that both the oxidized form of iodide and iodide per se are responsible for gene expression modulation in response to iodide excess. (c) 2008 Elsevier Inc. All rights reserved.

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

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

New insights about the posttranscriptional mechanisms triggered by iodide excess on sodium/iodide symporter (NIS) expression in PCCl3 cells

Iodide excess acutely downregulates NIS mRNA expression, as already demonstrated. PCCl3 cells treated or not with Nal, Nal + NaClO4 or Nal + Methimazole, for 30 min to 24 h, were used to further explore how iodide reduces NIS gene expression. NIS mRNA expression was evaluated by Real-Time PCR; its poly(A) tail length, by RACE-PAT; its translation rate, by polysome profile; total NIS content, by Western blotting. NIS mRNA decay rate was evaluated in actinomycin-D-treated cells, incubated with or without Nal for 0-6 h. Iodide treatment caused a reduction in NIS mRNA expression, half-life, poly(A) tail length, recruitment to ribosomes, as well as NIS protein expression. Perchlorate, but not methimazole, prevented these effects. Therefore, reduced poly(A) tail length of NIS mRNA seems to be related to its decreased half-life, in addition to its translation impairment. These data provide new insights about the molecular mechanisms involved in the rapid and posttranscriptional inhibitory effect of iodide on NIS expression. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Extraction, purification and biochemical characterization of a peroxidase from Copaifera langsdorffii leaves

The aim of this work is to obtain, purify and characterize biochemically a peroxidase from Copaifera langsdorffii leaves (COP). COP was obtained by acetone precipitation followed by ion-exchange chromatography. Purification yielded 3.5% of peroxidase with the purification factor of 46.86. The COP optimum pH is 6.0 and the temperature is 35 ºC. COP was stable in the pH range of 4.5 to 9.3 and at temperatures below 50.0 ºC. The apparent Michaelis-Menten constants (Km) for guaiacol and H2O2 were 0.04 mM and 0.39 mM respectively. Enzyme turnover was 0.075 s-1 for guaiacol and 0.28 s-1 for hydrogen peroxide. Copaifera langsdorffii leaves showed to be a rich source of active peroxidase (COP) during the whole year. COP could replace HRP, the most used peroxidase, in analytical determinations and treatment of industrial effluents at low cost.

Ecdysteroid-Dependent Expression of the Tweedle and Peroxidase Genes during Adult Cuticle Formation in the Honey Bee, Apis mellifera

Cuticle renewal is a complex biological process that depends on the cross talk between hormone levels and gene expression. This study characterized the expression of two genes encoding cuticle proteins sharing the four conserved amino acid blocks of the Tweedle family, AmelTwdl1 and AmelTwdl2, and a gene encoding a cuticle peroxidase containing the Animal haem peroxidase domain, Ampxd, in the honey bee. Gene sequencing and annotation validated the formerly predicted tweedle genes, and revealed a novel gene, Ampxd, in the honey bee genome. Expression of these genes was studied in the context of the ecdysteroid-coordinated pupal-to-adult molt, and in different tissues. Higher transcript levels were detected in the integument after the ecdysteroid peak that induces apolysis, coinciding with the synthesis and deposition of the adult exoskeleton and its early differentiation. The effect of this hormone was confirmed in vivo by tying a ligature between the thorax and abdomen of early pupae to prevent the abdominal integument from coming in contact with ecdysteroids released from the prothoracic gland. This procedure impaired the natural increase in transcript levels in the abdominal integument. Both tweedle genes were expressed at higher levels in the empty gut than in the thoracic integument and trachea of pharate adults. In contrast, Ampxd transcripts were found in higher levels in the thoracic integument and trachea than in the gut. Together, the data strongly suggest that these three genes play roles in ecdysteroid-dependent exoskeleton construction and differentiation and also point to a possible role for the two tweedle genes in the formation of the cuticle (peritrophic membrane) that internally lines the gut.

Extraction of manganese peroxidase produced by Lentinula edodes

Lentinula edodes, commonly called shiitake, is considered a choice edible mushroom with exotic taste and medicinal quality. L. edodes grows very well and produces a range of enzymes when cultivated on eucalyptus residues. Development of appropriate experimental procedures for recovery and determination of enzymes became a widely important cash crop. In this work, enzymes produced by L. edodes were extracted using different pH buffer and determined regarding peroxidases and proteases. Lignin peroxidase (LiP) was not detected in the extracts based on veratryl alcohol or azure B oxidation. Proteases were very low while Mn-peroxidases (MnP) predominated. The optimal pH for MnP recovery was 5.0, under agitation at 25 degrees C. The oxidation of phenol red decreased after dark-colored small compounds or ions were eliminated by dialysis. The extract of L. edodes contained components of high molecular weight, such as proteases or high polyphenol, that could be involved in the LiP inactivation. L. edodes sample previously submitted to dialysis was also joined to UP of Phanerochaete chrysosporium and a total inhibition of UP was observed. (C) 2007 Elsevier Ltd. All rights reserved.

Optimal recovery process conditions for manganese-peroxidase obtained by solid-state fermentation of eucalyptus residue using Lentinula edodes

Enzyme production is a growing field in biotechnology and increasing attention has been devoted to the solid-state fermentation (SSF) of lignocellulosic biomass for production of industrially relevant lignocellulose deconstruction enzymes, especially manganese-peroxidase (MnP), which plays a crucial role in lignin degradation. However, there is a scarcity of studies regarding extraction of the secreted metabolities that are commonly bound to the fermented solids, preventing their accurate detection and limiting recovery efficiency. In the present work, we assessed the effectiveness of extraction process variables (pH, stirring rate, temperature, and extraction time) on recovery efficiency of manganese-peroxidase (MnP) obtained by SSF of eucalyptus residues using Lentinula edodes using statistical design of experiments. The results from this study indicated that of the variables studied, pH was the most significant (p < 0.05%) parameter affecting MnP recovery yield, while temperature, extraction time, and stirring rate presented no statistically significant effects in the studied range. The optimum pH for extraction of MnP was at 4.0-5.0, which yielded 1500-1700 IU kg (1) of enzyme activity at extraction time 4-5 h, under static condition at room temperature. (C) 2011 Elsevier Ltd. All rights reserved.

Thermal inactivation of polyphenoloxidase and peroxidase in green coconut (Cocos nucifera) water

P>Coconut water is an isotonic beverage naturally obtained from the green coconut. After extracted and exposed to air, it is rapidly degraded by enzymes peroxidase (POD) and polyphenoloxidase (PPO). To study the effect of thermal processing on coconut water enzymatic activity, batch process was conducted at three different temperatures, and at eight holding times. The residual activity values suggest the presence of two isoenzymes with different thermal resistances, at least, and a two-component first-order model was considered to model the enzymatic inactivation parameters. The decimal reduction time at 86.9 degrees C (D(86.9 degrees C)) determined were 6.0 s and 11.3 min for PPO heat labile and heat resistant fractions, respectively, with average z-value = 5.6 degrees C (temperature difference required for tenfold change in D). For POD, D(86.9 degrees C) = 8.6 s (z = 3.4 degrees C) for the heat labile fraction was obtained and D(86.9 degrees C) = 26.3 min (z = 6.7 degrees C) for the heat resistant one.

Photochemical damage and comparative performance of superoxide dismutase and ascorbate peroxidase in sugarcane leaves exposed to paraquat-induced oxidative stress

The physiological responses of sugarcane (Succharion officinarum L.) to oxidative stress induced by methyl viologen (paraquat) were examined with respect to photochemical activity, chlorophyll content, lipid peroxidation and superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities. Thirty-day-old sugarcane plants were sprayed with 0, 2, 4, 6 and 8 mM methyl viologen (MV). Chlorophyll fluorescence was measured after 18 It and biochemical analyses were performed after 24 and 48 h. Concentrations of MV above 2 mM caused significant damage to photosystem II (PSII) activity. Potential and effective quantum efficiency of PSII and apparent electron transport rate were greatly reduced or practically abolished. Both chlorophyll and soluble protein contents steadily decreased with MV concentrations above 2 mM after 24 It of exposure, which became more pronounced after 48 It, achieving a 3-fold decrease. Insoluble protein contents were little affected by MV. Oxidative stress induced by MV was evidenced by increases in lipid peroxidation. Specific activity of SOD increased, even after 48 h of exposure to the highest concentrations of MV, but total activity on a fresh weight basis did not change significantly. Nondenaturing YAGE assayed with H2O2 and KCN showed that treatment with MV did not change Cu/Zn-SOD and MnSOD isoform activities. In contrast, APX specific activity increased at 2 mM MV but then dropped at higher doses. Oxidative damage induced by MV was inversely related to APX activity. It is suggested that the major MV-induced oxidative damages in sugarcane leaves were related to excess H2O2, probably in chloroplasts, caused by an imbalance between SOD and APX activities, in which APX was a limiting step. Reduced photochemical activity allowed the early detection of the ensuing oxidative stress. (c) 2007 Elsevier Inc. All rights reserved.