Estudo da sinalização da insulina nos tecidos muscular e adiposo de ratos submetidos à desnutrição materna no início da lactação

Vários estudos sugerem que a desnutrição materna no período pós-natal poderia causar alterações na homeostase glicêmica da prole na vida adulta. Neste trabalho objetivamos investigar a interferência da programação metabólica induzida pela desnutrição protéica materna durante o início da lactação sobre a homeostase glicêmica e a sinalização da insulina nos tecidos muscular e adiposo. Animais desnutridos (D-dieta da mãe contendo 0% de proteína nos primeiros 10 dias de lactação) ou controle (C-dieta da mãe contendo 22% de proteína) foram estudados do nascimento até a vida adulta. Em resumo, observamos uma diminuição na insulina plasmática acompanhada de normoglicemia nos animais adultos desnutridos. A ativação do receptor de insulina (IR), após a estimulação com o hormônio apresentou-se diminuída durante o período de restrição protéica em músculo isolado destes animais experimentais. Durante o período da lactação, observamos uma diminuição na captação de glicose, na fosforilação do substrato para o receptor de insulina (IRS 1) e na translocação do GLUT 4 no tecido muscular. Na idade adulta, entretanto, houve aumento significativo na captação de glicose e translocação do GLUT 4 no músculo, associado com o aumento na expressão da PI3 quinase associada ao IRS 1. No tecido adiposo de ratos desnutridos adultos observamos menor fosforilação em tirosina tanto do IR quanto do IRS 1, que foi compensada pela maior ativação do IRS 2 e da PI3 quinase. Os níveis basais de pAkt e de GLUT 4 na membrana estavam aumentados, culminando em um aumento na captação de glicose. Observamos também uma redistribuição do citoesqueleto de actina e maior resistência aos efeitos da Ltrunculina B nos adipócitos dos ratos desnutridos. Em conclusão, este estudo demonstrou que a desnutrição materna no início da lactação é capaz de causar alterações na prole na vida adulta, o que parece estar relacionado com a expressão e ativação de proteínas chave na cascata da sinalização da insulina nos tecidos periféricos, importantes na regulação do metabolismo da glicose.

Preservation of Otolithus argenteus at low temperature

The studies provided data on the spoilage pattern of Otolithus argenteus during low temperature preservation. Changes in the total volatile bases, hypoxanthine, tyrosine, salt soluble nitrogen, non-protein nitrogen, pH, peroxide value, free fatty acids and thiobarbituric acid number along with organoleptic score have been reported. Organoleptically, fish stored at +20 degree C remained in acceptable condition upto 12 days while for those stored at 0 degree C in ice upto 19 days. Of the various indices tested Hypoxanthine, salt soluble nitrogen and total volatile bases nitrogen, in the order of merit can be used as freshness tests for refrigerated fish.

Effect of aureomycin on the behaviour of certain free amino acids in oil sardine (Sardinella longiceps) held in ice storage

The course of development of a few free amino acids under the influence of aureomycin in oil sardine (Sardinella lingiceps) held in ice storage was investigated. The levels of leucines and valine regularly increased in the control and aureomycin treated fush throughout the storage period. Alanines and threonine showed similar trend in both control and fish treated with 20ppm aureomycin. These amino acids however showed a gradual fall in fish treated at 5 ppm level. The changes in tyrosine+tryptophane were found to be irregular. Most of the amino acids studied indicated a remarkable change in trend by about the 16th day of ice storage in the case of fish treated with 50ppm aureimycin.

The testis-specific apoptosis related gene TTL.6 underwent adaptive evolution in the lineage leading to humans

The TTL.6 gene is a member of the tubulin-tyrosine ligase (TTL) family involved in apoptosis and preferentially expressed in the testis. We sequenced the coding region and part of the introns of TTL.6 in world wide human populations and five representativ

Stejnulxin, a novel snake C-type lectin-like protein from Trimeresurus stejnegeri venom is a potent platelet agonist acting specifically via GPVl

Stejnulxin, a novel snake C-type lectin-like protein with potent platelet activating activity, was purified and characterized from Trimeresurus stejnegeri venom. Under non-reducing conditions, it migrated on a SDS-polyacrylamide gel with an apparent molecular mass of 120 kDa. On reduction, it separated into three polypeptide subunits with apparent molecular masses of 16 kDa (alpha), 20 kDa (beta(1)) and 22 kDa (beta(2)), respectively. The complete amino acid sequences of its subunits were deduced from cloned cDNAs. The N-terminal sequencing and cDNA cloning indicated that beta(1) and beta(2) subunits of stejnulxin have identical amino acid sequences and each contains two N-glycosylation sites. Accordingly, the molecular mass difference between 1 and 2 is caused by glycosylation heterogenity. The subunit amino acid sequences of stejnulxin are similar to those of convulxin, with sequence identities of 52.6% and 66.4% for the U. and beta, respectively. Stejnulxin induced human platelet aggregation in a dose-dependent manner. Antibodies against UNA inhibited the aggregation response to stejnulxin, indicating that activation of alpha(IIb)beta(3) and binding of fibrinogen are involved in stejnulxin-induced platelet aggregation. Antibodies against GPIbalpha or alpha(2)beta(1) as well as echicetin or rhodocetin had no significant effect on stejnulxin-induced platelet aggregation. However, platelet activation induced by stejnulxin was blocked by anti-GPVI antibodies. In addition, stejnulxin induced a tyrosine phosphorylation profile in platelets that resembled that produced by convulxin. Biotinylated stejnulxin bound specifically to platelet membrane GPVI.

GPIb is involved in platelet aggregation induced by mucetin, a snake C-type lectin protein from Chinese habu (Trimeresurus mucrosquamatus) venom

Mucetin (Trimeresurus mucrosquamatus venom activator, TMVA) is a potent platelet activator purified from Chinese habu (Trimeresurus mucrosquamatus) venom. It belongs to the snake venom heterodimeric C-type lectin family and exists in several multimeric forms. We now show that binding to platelet glycoprotein (GP) lb is involved in mucetin-induced platelet aggregation. Antibodies against GPIb as well as the GPIb-blocking C-type lectin echicetin inhibited mucetin-induced platelet aggregation. Binding of GPIb was confirmed by affinity chromatography and Western blotting. Antibodies against GPVI inhibited convulxin- but not mucetin-induced aggregation. Signalling by mucetin involved rapid tyrosine phosphorylation of a number of proteins including Syk, Src, LAT and PLCgamma2. Mucetininduced phosphorylation of the Fcgamma chain of platelet was greatly promoted by inhibition of alpha(llb)beta(3) by the peptidomimetic EMD 132338, suggesting that phosphatases downstream Of alpha(llb)beta(3) activation are involved in dephosphorylation of Fcgamma. Unlike other multimeric snake C-type lectins that act via GPIb and only agglutinate platelets, mucetin activates alpha(llb)beta(3). Inhibition Of alpha(llb)beta(3) strongly reduced the aggregation response to mucetin, indicating that activation Of alpha(llb)beta(3) and binding of fibrinogen are involved in mucetin-induced platelet aggregation. Apyrase and aspirin also inhibit platelet aggregation induced by mucetin, suggesting that ADP and thromboxaneA(2) are involved in autocrine feedback. Sequence and structural comparison with closely related members of this protein family point to features that may be responsible for the functional differences.

Studies on phenolase enzyme in prawns. II. The inhibitory action on the enzyme by certain chemical agents

The effect of certain chemical agents on dopa oxidation by phenolases has been examined. Sulphur containing amino carboxylic acids are inhibitory agents for dopa oxidation. Tyrosine, a substrate for the enzyme also acts as an inhibitor for dopa oxidation by the enzyme. The possible mode of action has been discussed. The function of diethyl dithiocarbamate in suppressing the display of enzyme activity has been detailed and its behaviour has been compared to the other chemical agents studied.

Molecular evolution of the keratin associated protein gene family in mammals, role in the evolution of mammalian hair

Background: Hair is unique to mammals. Keratin associated proteins (KRTAPs), which contain two major groups: high/ultrahigh cysteine and high glycine-tyrosine, are one of the major components of hair and play essential roles in the formation of rigid and

Identification of protein superfamily from structure-based sequence motif

The structure-based sequence motif of the distant proteins in evolution, protein tyrosine phosphatases (PTP) I and II superfamilies, as an example, has been defined by the structural comparison, structure-based sequence alignment and analyses on substitut

Tests of an adaptive QM/MM calculation on free energy profiles of chemical reactions in solution.

We present reaction free energy calculations using the adaptive buffered force mixing quantum mechanics/molecular mechanics (bf-QM/MM) method. The bf-QM/MM method combines nonadaptive electrostatic embedding QM/MM calculations with extended and reduced QM regions to calculate accurate forces on all atoms, which can be used in free energy calculation methods that require only the forces and not the energy. We calculate the free energy profiles of two reactions in aqueous solution: the nucleophilic substitution reaction of methyl chloride with a chloride anion and the deprotonation reaction of the tyrosine side chain. We validate the bf-QM/MM method against a full QM simulation, and show that it correctly reproduces both geometrical properties and free energy profiles of the QM model, while the electrostatic embedding QM/MM method using a static QM region comprising only the solute is unable to do so. The bf-QM/MM method is not explicitly dependent on the details of the QM and MM methods, so long as it is possible to compute QM forces in a small region and MM forces in the rest of the system, as in a conventional QM/MM calculation. It is simple, with only a few parameters needed to control the QM calculation sizes, and allows (but does not require) a varying and adapting QM region which is necessary for simulating solutions.

Waterborne exposure to fluorotelomer alcohol 6:2 FTOH alters plasma sex hormone and gene transcription in the hypothalamic-pituitary-gonadal (HPG) axis of zebrafish

Fluorotelomer alcohols (FTOHs) have shown estrogenic activity in vitro and in vivo, but the mechanism of this activity is not known. In this study, 18-week-old zebrafish (Danio rerio) were exposed to 0, 0.03, 0.3 and 3.0 mg/l 1H, 1H, 2H, 2H-perfluorooctan-1-ol (6:2 ETCH) for 7 days, and the effects on plasma sex hormone levels were measured followed by use of real-time PCR to examine selected gene expression in hypothalamic-pituitary-gonadal (HPG) axis and liver. Exposure to 6:2 FTOH significantly increased plasma estradiol (E2) and testosterone (T) levels in both males and females. Furthermore, the ratio of T/E2 was reduced in females while increased in males. In females, the increase of E2 was accompanied by up-regulated hepatic estrogenic receptor alpha (ER alpha) and vitellogenin (VTG1 and VTG3) expression. In males, the elevation of the T level is consistent with the up-regulation of cytochrome P450 c17 alpha-hydroxylase, 17, 20-lase (CYP17) and the down-regulation of cytochrome P450 aromatase A (CYP19A). The present study demonstrated that waterborne exposure to 6:2 FTOH alter plasma sex hormone levels and the ratio of T/E2, as well as the transcriptional profiles of some genes in the HPG axis and liver. The results suggested that FTOHs may disturb fish reproduction through endocrine disrupted activity. (C) 2009 Elsevier B.V. All rights reserved.

Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA Library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full Length cDNA of carp SLP-76 was 2007 bp, consisting of a T-terminal untranslated region (UTR) of 285 bp, a T-terminal. UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homotogues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2 k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts. (c) 2007 Published by Elsevier Ltd.

Molecular cloning and characterization of common carp (Cyprinus carpio L.) TCR gamma and CD3 gamma/delta chains

Partial cDNA sequences of TCR gamma and CD3 gamma/delta were isolated from the thymus of common carp (Cyprinus carpio L.) by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp TCR gamma and CD3 gamma/delta were obtained by means of 3' RACE and 5' RACE, respectively. The full length of carp TCR gamma chain is 1368 bp and encodes 326 amino acids including a signal peptide region of 19 amino acids and a transmembrane region of 23 amino acids at the C-terminal region from aa 291 to 313. The V region of carp TCR gamma contains 109 amino acids, the core motif FGXG in J segment was also found in carp TCR gamma. The C region of carp TCR gamma contains the characteristic CX6PX6WX45C motif. The CP region of carp TCR C gamma contains 37 amino acids. The full length of carp CD3 gamma/delta is 790 bp and encodes 175 amino acids including a signal peptide region of 17 amino acids and a transmembrane region of 23 amino acids from aa 93 to 115. Similar to other known CD3 gamma/delta s, four cysteine residues in the extracellular domain and an immunoreceptor tyrosine-based activation motif ITAM (YxxL/Ix6-8YxxL/I) in the intracellular domain are also included in carp CD3 gamma/delta. Differing from other known CD3 gamma/delta s, carp CD3 gamma/delta tacks the CXXCXE motif in the extracellular domain. RTPCR analysis demonstrated that the expression of TCR gamma gene was mainly in the thymus and gill of 6-month carp, but in 18-month carp, TCR gamma gene was detected in all the examined tissues. The expression of CD3 gamma/delta gene was detected in all examined tissues of 6 and 18-month carp; among them, the highest expression level was in the thymus of 6-month carp. In situ hybridization showed that CD3 gamma/delta-expressing cells were widely distributed in the head kidney, spleen and kidney of carp, whereas in the thymus, they were densely distributed in the lymphoid outer zone and scattered in the epithelioid inner zone. (c) 2007 Published by Etsevier Ltd.

Molecular cloning and characterization of carp (Cyprinus carpio L.) CD8 beta and CD4-like genes

Partial cDNA sequences of both CD8 beta and CD4-like (CD4L) genes of common carp (Cyprinus carpio L.) were isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp CD8 and CD4L were obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp CD8 is 1164 bp and encodes 207 amino acids including a signal peptide region of 24 amino acids, a transmembrane region of 23 amino acids from aa 167 to aa189 and an immunoglobulin V-set from aa 19 to aa 141. Similar to other species CD8 beta s,carp CD8 beta also lacks p56(lck) domain in the cytoplasmic region. The full length cDNA of carp CD4L is 2001 bp and encodes 458 amino acids including four immunoglobulin (Ig)-like domains in the extracellular region, a transmembrane region of 23 amino acids at the C-terminal region from aa 402 to aa 424 and a cytoplasmic tail. Similar to mammalian, avian CD4s and fugu CD4L, carp CD4L also has the conserved p56(lck) tyrosine kinase motif (C-X-C) in the cytoplasmic region. RT-PCR analysis demonstrated that carp CD8 beta and CD4L genes were both expressed predominantly in thymus. The results from this study can be used to understand the evolution of both the CD8 beta and CD4 molecules which can be used as markers for cytotoxic and helper T cells in carp. (c) 2007 Published by Elsevier Ltd.

Transgenes in F-4 pMThGH-transgenic common carp (Cyprinus carpio L.) are highly polymorphic

To gain information on the integration pattern of pMThGH-transgene, 50 transgenes were recovered from F-4 generation of pMThGH transgenic common carp (Cyprinus carpio L,) and 33 recovered genes were analyzed. The restriction maps of these recovered genes were constructed by digestion with five kinds of enzymes. These transgenes can be classified into 4 types according to their restriction maps. Only one type of transgenes maintains its original molecular form, whereas the other three types are very different from the original one and vary each other on both molecular weight and restriction maps. This implies that the sequences of most transgenes have been deleted and/or rearranged during integration and inheritance. The results of PCR amplification and Southern blot hybridization indicate that MThGH in Type I transgene keeps intact but most of its sequence has been deleted in other three types. All these results suggest that transgenes in F-4 generation of transgenic carp are highly polymorphic. Two DNA fragments concerning integration site of transgenes were cloned from recovered transgenes, and found to be homologous to the 5'UTR of beta -actin gene of common carp and mouse mRNA for receptor tyrosine kinase (RTK), respectively.