RESUMO
A minority of collodion babies, called 'self-healing collodion babies', heal spontaneously. We describe a novel clinical phenotype of acral self-healing collodion baby caused by a new TGM1 mutation. The proband, born to healthy parents, presented at birth as a collodion baby strictly localized to the extremities. The skin condition returned to normal at the age of 3 weeks. The older sister was born as a generalized collodion baby; the condition then developed into lamellar ichthyosis. Molecular analysis of TGM1 revealed three novel mutations in the family. The proband was compound heterozygous for the p.Val359Met and p.Arg396His mutations, whereas the older sister was compound heterozygous for p.Arg396His and a deletion mutation c.1922_1926+2delGGCCTGT. Structural modelling of the p.Val359Met mutation suggested a minor disruption of the protein structure, whereas a modification of protein-protein interaction was predicted for p.Arg396His. These predictions corroborated the analysis of recombinant transglutaminase (TGase)-1 proteins carrying the p.Val359Met and p.Arg396His mutations. Both showed decreased levels of protein expression: p.Val359Met displayed residual activity (12.8%), while p.Arg396His caused a dramatic loss of activity (3.3%). These observations demonstrate for the first time that TGM1 mutations can be associated with acral self-healing collodion baby, and expand the clinical spectrum of TGase-1 deficiency.
Assuntos
Ictiose/genética , Deleção de Sequência/genética , Transglutaminases/genética , Feminino , Dedos , Predisposição Genética para Doença , Heterozigoto , Humanos , Recém-Nascido , Fenótipo , Remissão Espontânea , Dedos do PéRESUMO
The NOD mouse is a model of human IDDM, which is characterized by a cell-mediated autoimmune process resulting in spontaneous diabetes. Alpha-interferon (IFN-alpha) is thought to play a pathogenic role in this autoimmune process. We report that recombinant alpha-interferon (rIFN-alpha) administration decreases the development of spontaneous diabetes and the passive transfer of diabetes in NOD mice. Spontaneous diabetes was inhibited by IFN-alpha in a dose-dependent fashion. A dose of as little as 20 x 10(3) U inhibited diabetes development, while a dose of 100 x 10(3) U potently prevented diabetes (14% incidence vs. 70% incidence in control mice). Even at the termination of the experiment, nondiabetic mice administered rIFN-alpha maintained normal glucose tolerance. Islet inflammation was 65% lower in the pancreases of rIFN-alpha mice. rIFN-alpha administration decreased anti-islet effector cell bioactivity of spleen cells without inducing generalized immunosuppression. Passive transfer experiments demonstrated that the decreased anti-islet effector cell activity was not a direct action of rIFN-alpha on these cells. In conclusion, rIFN-alpha potently and paradoxically prevents diabetes by indirectly decreasing anti-islet effector cell activity and in turn the development of insulitis without inducing generalized immunosuppression. This work, which goes against our current understanding of the role of rIFN-alpha in autoimmunity, may have significant implications to further our understanding of the pathogenesis of IDDM and to further the development of novel modes to prevent the disease.
Assuntos
Antineoplásicos/uso terapêutico , Diabetes Mellitus Tipo 1/prevenção & controle , Interferon Tipo I/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Divisão Celular/efeitos dos fármacos , Concanavalina A/farmacologia , Feminino , Interferon Tipo I/administração & dosagem , Ilhotas Pancreáticas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Proteínas Recombinantes , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologiaRESUMO
The 9-cis retinoic acid receptor, RXR, binds DNA effectively as a homodimer or as a heterodimer with other nuclear receptors. The DNA-binding sites for these RXR complexes are direct repeats of a consensus sequence separated by one to five base-pairs of intervening space. Here, we report the 2.1 A crystal structure of the RXR-DNA-binding domain as a homodimer in complex with its idealized direct repeat DNA target. The structure shows how a gene-regulatory site can induce conformational changes in a transcription factor that promote homo-cooperative assembly. Specifically, an alpha-helix in the T-box is disrupted to allow efficient DNA-binding and subunit dimerization. RXR displays a relaxed mode of sequence recognition, interacting with only three base-pairs in each hexameric half-site. The structure illustrates how site selection is achieved in this large eukaryotic transcription factor family through discrete protein-protein interactions and the use of tandem DNA binding sites with characteristic spacings.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Pareamento de Bases/genética , Sequência de Bases , Sítios de Ligação , Sequência Consenso/genética , Cristalização , Cristalografia por Raios X , DNA/genética , Dimerização , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Elementos de Resposta/genética , Receptores X de Retinoides , Especificidade por SubstratoRESUMO
Examination of the proliferative responses in vitro to mitogens (concanavalin A, phytohemagglutinin, lipopolysaccharide) of spleen cells recovered from C57BL/6 mice during blood-stage Plasmodium chabaudi AS infection revealed that the most severe suppression occurred during the first 14 days post infection, that is, during the acute phase of infection. Coincidently, inducible nitric oxide synthase gene expression was found to be up-regulated in the spleens of infected mice, and both splenic and peritoneal macrophages produced high levels of NO in vitro in response to stimulation with lipopolysaccharide (LPS). The roles of NO, a molecule recently found to mediate immunosuppression during parasitic infections, and of the well-recognized immunosuppressive molecule prostaglandin were, therefore, investigated in the suppression of proliferation to mitogens and specific antigen of spleen cells from 7- and 14-day P. chabaudi AS-infected mice. Addition of either 0.5 mM NG-monomethyl-L-arginine (L-NMMA) or 0.5 mM aminoguanidine (AG), inhibitors of NO synthase, or 10 micrograms/ml indomethacin (INDO), a prostaglandin inhibitor, partially but significantly abrogated the suppression in response to concanavalin A (Con A) and phytohemagglutinin (PHA). Only the addition of INDO significantly increased the responses to LPS. Addition of L-NMMA or AG in combination with INDO partially but significantly abrogated the suppression in response to Con A and completely abrogated the suppression in response to PHA. The addition of L-NMMA or AG also significantly increased proliferation in response to parasite antigen. The contribution of NO to suppression of lymphoproliferation was confirmed by adding 3-morpholino-sydnonimine-hydrochloride (SIN-1), a chemical generator of NO, to mitogen-stimulated splenocyte cultures prepared from normal mice. The mechanism of NO-mediated suppression was investigated in coculture experiments using spleen cells from normal mice and peritoneal macrophages from either normal or day 7 infected mice. The addition of 5-10 x 10(4) peritoneal macrophages from infected mice significantly and consistently suppressed Con A- or PHA-stimulated proliferation of normal splenocytes. Moreover, suppression correlated with production of NO and could be reversed by the addition of L-NMMA or AG. These results suggest that, in addition to prostaglandin, increased NO production by macrophages within the first 2 weeks after infection with P. chabaudi AS contributes to immunosuppression associated with blood-stage malaria.
Assuntos
Ativação Linfocitária , Macrófagos Peritoneais/imunologia , Malária/imunologia , Óxido Nítrico/fisiologia , Aminoácido Oxirredutases/biossíntese , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Sequência de Bases , Primers do DNA/química , Feminino , Guanidinas/farmacologia , Tolerância Imunológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Óxido Nítrico Sintase , Plasmodium chabaudi , Prostaglandinas/fisiologia , Baço/citologia , ômega-N-MetilargininaRESUMO
Lamellar ichthyosis, one form of congenital autosomal recessive ichthyosis, is caused by mutations in the gene (TGM1) encoding the transglutaminase 1 enzyme. Mutations, deletions, or insertion of TGM1 have been reported so far. Here we report that three novel mutations of TGM1, D101V, N288T, and R306W, cause lamellar ichthyosis in two different families. The patient in family LI-KD has N288T and R306W mutations, and the patient in family LI-LK has D101V and R306W mutations. The activity of the transglutaminase 1 enzyme of the patient in family LI-LK was only about 15% of normal. Also, three-dimensional structural prediction analyses revealed that the N288T and R306W mutations, and possibly the D101V mutation, cause misfolding in the central catalytic core domain of the transglutaminase 1 enzyme that would probably result in reduced enzyme activity. Our data suggest that the greatly reduced transglutaminase 1 activities are due to disruptions of the native folding of transglutaminase 1, and that these mutations may play a critical role in the pathology of lamellar ichthyosis.
Assuntos
Ictiose Lamelar/genética , Mutação de Sentido Incorreto , Transglutaminases/química , Transglutaminases/genética , Substituição de Aminoácidos , Células Cultivadas , Éxons , Feminino , Humanos , Ligação de Hidrogênio , Recém-Nascido , Queratinócitos/citologia , Queratinócitos/enzimologia , Estrutura Terciária de Proteína , RNA Mensageiro/análise , Pele/citologia , Relação Estrutura-Atividade , Transglutaminases/metabolismoRESUMO
The fatty aldehyde dehydrogenase (Vh-ALDH) isolated from the luminescent bacterium, Vibrio harveyi, differs from other aldehyde dehydrogenases in its high affinity for NADP(+). The binding of NADP(+) appears to arise from the interaction of the 2'-phosphate of the adenosine moiety of NADP(+) with a threonine (T175) in the nucleotide recognition site just after the beta(B) strand as well as with an arginine (R210) that pi stacks over the adenosine moiety. The active site of Vh-ALDH contains the usual suspects of a cysteine (C289), two glutamates (E253 and E377) and an asparagine (N147) involved in the aldehyde dehydrogenase mechanism. However, Vh-ALDH has one polar residue in the active site that distinguishes it from other ALDHs; a histidine (H450) is in close contact with the cysteine nucleophile. As a glutamate has been implicated in promoting the nucleophilicity of the active site cysteine residue in ALDHs, the close contact of a histidine with the cysteine nucleophile in Vh-ALDH raises the possibility of alternate routes to increase the reactivity of the cysteine nucleophile. The effects of mutation of these residues on the different functions catalyzed by Vh-ALDH including acylation, (thio)esterase, reductase and dehydrogenase activities should help define the specific roles of the residues in the active site of ALDHs.
Assuntos
Aldeído Desidrogenase/química , Aldeído Desidrogenase/metabolismo , Vibrio/enzimologia , Aldeído Desidrogenase/genética , Domínio Catalítico , Ligação de Hidrogênio , Cinética , Modelos Moleculares , NADP/metabolismo , Mutação Puntual , Conformação Proteica , Especificidade por Substrato , Vibrio/genéticaRESUMO
A novel method that permits the quantitative detection and classification of various carbonyl groups in lignins has been developed. The proposed method was optimized with the quantitative trifluoromethylation of a series of carbonyl-containing lignin-like model compounds. This effort was followed by (19)F NMR spectral analyses of the resulting fluorine derivatives allowing for a thorough understanding of their structure/(19)F chemical shift relationships. The various carbonyl groups present in lignins were also investigated by trifluoromethylating them in the presence of catalytic amounts of tetramethylammonium fluoride (TMAF), followed by hydrolysis with TMAF in tetrahydrofuran. By using a variety of selective reactions, it became possible to assign a number of prominent (19)F NMR signals to a variety of carbonyl groups present in lignins. These studies demonstrated that the proposed method can be applied to the quantitative determination of carbonyl groups that are present in soluble native and technical lignins.
Assuntos
Lignina/química , Flúor , Cromatografia Gasosa-Espectrometria de Massas , Espectroscopia de Ressonância Magnética , Compostos de Amônio Quaternário/químicaRESUMO
The proton spin-lattice relaxation times (T1H) for isolated cuoxam lignin and fully bleached cellulose were measured as a function of pH in the solid-state. These experiments provided the opportunity to examine for possible macromolecular connectivities that may be present between lignin and carbohydrates within softwood. These studies have shown that the molecular mobilities of the isolated polymeric constituents of wood are affected by the ionization of their functional groups at different pHs. The shapes of the plots of T1H as a function of pH for the two isolated polymers were dramatically different, while those for softwood were similar. This fact provides evidence supporting the notion that in wood, lignin and carbohydrates are intimately associated with each other.
Assuntos
Madeira , Celulose/química , Celulose/isolamento & purificação , Concentração de Íons de Hidrogênio , Lignina/química , Lignina/isolamento & purificação , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética/métodos , PrótonsRESUMO
Myristoyl-ACP thioesterase (LuxD) from Vibrio harveyi causes the slow release of fatty acids for reduction into the aldehyde substrate required for the bacterial bioluminescence reaction. The active site Ser nucleophile (S(114)) of the LuxD thioesterase is in a gamma-turn with a sequence (AXS(114)XS) quite different from the standard motif of GXSXG found in almost all (thio) esterases and lipases. The presence of an Arg residue (R(118)) in the first turn of the helix after the gamma-turn also distinguishes LuxD from other enzymes. Mutation of R(118) to Leu inactivated the enzyme and prevented acylation of the Ser(114) nucleophile, while even a conservative replacement with Lys resulted in over 75% loss of the same functions, suggesting that R(118) helps maintain the configuration of the active site. In contrast, replacement of S(116) with Gly but not Ala stimulated the esterase and deacylation rates by over threefold. Purification of the S116G mutant to homogeneity and analyses of its intrinsic fluorescence on acylation with myristoyl-CoA clearly demonstrated that this mutant was much more active than wild-type LuxD. The presence of S(116) rather than the expected Gly residue in the gamma-turn containing the Ser nucleophile may function so that release of fatty acids from LuxD is restricted allowing a more efficient delivery of fatty acids to the luminescent system.
Assuntos
Aciltransferases/genética , Proteínas de Bactérias/genética , Vibrio/enzimologia , Aciltransferases/química , Proteínas de Bactérias/química , Mutagênese Sítio-Dirigida , Conformação ProteicaRESUMO
Aldehyde dehydrogenase from the bioluminescent bacterium, Vibrio harveyi, catalyses the oxidation of long-chain aliphatic aldehydes to acids. The enzyme is unique compared with other forms of aldehyde dehydrogenase in that it exhibits a very high specificity and affinity for the cofactor NADP(+). Structural studies of this enzyme and comparisons with other forms of aldehyde dehydrogenase provide the basis for understanding the molecular features that dictate these unique properties and will enhance our understanding of the mechanism of catalysis for this class of enzyme. The X-ray structure of aldehyde dehydrogenase from V. harveyi has been solved to 2.5-A resolution as a partial complex with the cofactor NADP(+) and to 2. 1-A resolution as a fully bound 'holo' complex. The cofactor preference exhibited by different forms of the enzyme is predominantly determined by the electrostatic environment surrounding the 2'-hydroxy or the 2'-phosphate groups of the adenosine ribose moiety of NAD(+) or NADP(+), respectively. In the NADP(+)-dependent structures the presence of a threonine and a lysine contribute to the cofactor specificity. In the V. harveyi enzyme an arginine residue (Arg-210) contributes to the high cofactor affinity through a pi stacking interaction with the adenine ring system of the cofactor. Further differences between the V. harveyi enzyme and other aldehyde dehydrogenases are seen in the active site, in particular a histidine residue which is structurally conserved with phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This may suggest an alternative mechanism for activation of the reactive cysteine residue for nucleophilic attack.
Assuntos
Aldeído Desidrogenase/química , NADP/metabolismo , Vibrio/enzimologia , Aldeído Desidrogenase/metabolismo , Cristalografia por Raios X , Conformação Proteica , Especificidade por SubstratoRESUMO
AIMS/HYPOTHESIS: Cyclophosphamide has been shown to augment the diabetic process in NOD mouse and BB rat models of Type I (insulin-dependent) diabetes mellitus. Because cyclophosphamide has, however, been shown to increase immunoregulatory cell activity, we examined if cyclophosphamide treatment increases immunoregulatory cell activity and inhibits the diabetic process in BB rats. METHODS: The development of insulitis and diabetes was explored in BB rats treated with saline and cyclophosphamide (60 to 175 mg/kg body weight). Subsets of spleen cells were assessed by flow cytometry and cytokine gene expression by RT-PCR. To determine if cyclophosphamide induces immunoregulatory cell activity, the development of diabetes was assessed in BB rats injected with spleen cells from rats treated with saline and cyclophosphamide. RESULTS: All dosages of cyclophosphamide decreased the development of diabetes. The degree of insulitis was lower in pancreata from 55-day-old rats treated with cyclophosphamide than those from controls. Cyclophosphamide caused no alterations in the numbers of NK cells, T-cell subsets, or RT6.1+ T cells. The adoptive transfer of spleen cells from cyclophosphamide-treated rats to BB rats inhibited the development of diabetes. Cyclophosphamide treatment decreased IL-12, IL-1beta, IL-2, IFN-gamma and TNF-alpha gene expressions in mononuclear spleen cells but IL-4 gene expression increased. CONCLUSION/INTERPRETATION: These findings show that cyclophosphamide treatment decreases the development of diabetes by inhibiting the development of insulitis. This inhibitory action of cyclophosphamide on the diabetic process seems to be mediated by the induction of immunoregulatory cell activity. The suppression of cytokines that promote Thl cell differentiation by cyclophosphamide treatment could also play a part in the diabetes sparing effect of cyclophosphamide.
Assuntos
Ciclofosfamida/uso terapêutico , Diabetes Mellitus Tipo 1/prevenção & controle , Envelhecimento , Animais , Citocinas/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Imunossupressores/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Ratos , Ratos Endogâmicos BB , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/efeitos dos fármacos , Baço/imunologiaRESUMO
Aldehyde dehydrogenases (ALDHs) catalyze the transfer to NAD(P) of a hydride ion from a thiohemiacetal derivative of the aldehyde coupled with a cysteine residue in the active site. In Vibrio harveyi aldehyde dehydrogenase (Vh-ALDH), a histidine residue (H450) is in proximity (3.8 A) to the cysteine nucleophile (C289) and is thus capable of increasing its reactivity in sharp contrast to other ALDHs in which more distantly located glutamic acid residues are proposed to act as the general base. Mutation of H450 in Vh-ALDH to Gln and Asn resulted in loss of dehydrogenase, (thio)esterase, and acyl-CoA reductase activities; the residual activity of H450Q was higher than that of the H450N mutant in agreement with the capability of Gln but not Asn to partially replace the epsilon-imino group of H450. Coupled with a change in the rate-limiting step, these results indicate that H450 increases the reactivity of C289. Moreover, for the first time, the acylated enzyme intermediate could be directly monitored after reaction with [(3)H]tetradecanoyl-CoA showing that the H450Q mutant was acylated more rapidly than the H450N mutant. Inactivation of the wild-type enzyme with N-ethylmaleimide was much more rapid than the H450Q mutant which in turn was faster than the H450N mutant, demonstrating directly that the nucleophilicity of C289 was affected by H450. As the glutamic acid residue implicated as the general base in promoting cysteine nucleophilicity in other ALDHs is conserved in Vh-ALDH, elucidation of why a histidine residue has evolved to assist in this function in Vh-ALDH will be important to understand the mechanism of ALDHs in general, as well as help delineate the specific roles of the active site glutamic acid residues.
Assuntos
Aldeído Desidrogenase/metabolismo , Domínio Catalítico , Histidina/metabolismo , Vibrio/enzimologia , Acil Coenzima A/metabolismo , Acilação , Aciltransferases/metabolismo , Aldeído Desidrogenase/genética , Animais , Sítios de Ligação/genética , Domínio Catalítico/genética , Cisteína/metabolismo , Ativação Enzimática/genética , Esterases/metabolismo , Etilmaleimida/farmacologia , Histidina/genética , Cinética , Família Multigênica , Mutagênese Sítio-Dirigida , NADP/metabolismo , Niacinamida/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Tioléster Hidrolases/metabolismo , Vibrio/genéticaRESUMO
The fatty aldehyde dehydrogenase from the luminescent bacterium, Vibrio harveyi (Vh-ALDH), is unique with respect to its high specificity for NADP(+) over NAD(+). By mutation of a single threonine residue (Thr175) immediately downstream of the beta(B) strand in the Rossmann fold, the nucleotide specificity of Vh-ALDH has been changed from NADP(+) to NAD(+). Replacement of Thr175 by a negatively charged residue (Asp or Glu) resulted in an increase in k(cat)/K(m) for NAD(+) relative to that for NADP(+) of up to 5000-fold due to a decrease for NAD(+) and an increase for NADP(+) in their respective Michaelis constants (K(a)). Differential protection by NAD(+) and NADP(+) against thermal inactivation and comparison of the dissociation constants of NMN, 2'-AMP, 2'5'-ADP, and 5'-AMP for these mutants and the wild-type enzyme clearly support the change in nucleotide specificity. Moreover, replacement of Thr175 with polar residues (N, S, or Q) demonstrated that a more efficient NAD(+)-dependent enzyme T175Q could be created without loss of NADP(+)-dependent activity. Analysis of the three-dimensional structure of Vh-ALDH with bound NADP(+) showed that the hydroxyl group of Thr175 forms a hydrogen bond to the 2'-phosphate of NADP(+). Replacement with glutamic acid or glutamine strengthened interactions with NAD(+) and indicated why threonine would be the preferred polar residue at the nucleotide recognition site in NADP(+)-specific aldehyde dehydrogenases. These results have shown that the size and the structure of the residue at the nucleotide recognition site play the key roles in differentiating between NAD(+) and NADP(+) interactions while the presence of a negative charge is responsible for the decrease in interactions with NADP(+) in Vh-ALDH.
Assuntos
Nucleotídeos de Adenina/farmacologia , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Mutação Puntual , Vibrio/enzimologia , Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/farmacologia , Aldeído Desidrogenase/antagonistas & inibidores , Aldeído Desidrogenase/isolamento & purificação , Sequência de Aminoácidos , Catálise , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NAD/metabolismo , NADP/metabolismo , Mononucleotídeo de Nicotinamida/farmacologia , Especificidade por Substrato , Vibrio/genéticaRESUMO
The application of tunable diode lasers for in situ diagnostics in laminar hydrocarbon diffusion flames is demonstrated. By the use of both direct-absorption and wavelength-modulation (second-derivative) techniques, carbon monoxide concentrations and the local flame temperature are determined for a laminar methane-air diffusion flame supported on a Wolfhard-Parker slot burner. In both cases the results are found to be in excellent agreement with prior measurements of these quantities using bothrobe and optical techniques.
RESUMO
Poly I:C, an inducer of IFN-alpha and other cytokines, has been used to study the development of diabetes in both the BioBreeding (BB) diabetes prone rat and non-obese diabetic (NOD) mouse animal models of insulin-dependent diabetes mellitus (IDDM). Surprisingly, poly I:C accelerates the disease in the BB rat while inhibiting it in the NOD mouse. Since cytokines can have dose related opposing effects on immune responses, we hypothesized that the paradoxical effect of polyinosinic polycytidylic acid (poly I:C) on diabetes in the two animal models is dose related. Accordingly, we compared the incidence of diabetes and degree of insulitis in diabetes prone BB rats administered saline and poly I:C at doses (0.05 microg/g body weight and 0.1 microg/g body weight) up to 100-fold lower than doses (poly-5 microg/g) previously found to accelerate diabetes. In addition, the non-specific suppressor activity of mononuclear splenocytes from BB rats administered low dose (poly-0.05 microg/g body weight), high dose (poly-5 microg/g body weight), and saline were compared. The development of diabetes was inhibited in rats treated with each dose of poly I:C. The degree of insulitis in poly-I:C treated animals was also less severe. The total white blood cell count and proportion of RT6+ T-cells and each T-cell subset were unaltered by poly I:C. When compared to splenocytes of control animals, splenocytes from poly I:C (0.05 microg/g body weight) treated rats suppressed responder cell proliferation to concanavalin A and alloantigen. However, spleen cells from high dose poly-I:C did not suppress responder cell proliferation to alloantigen. In adoptive transfer studies, the administration of spleen cells from poly-0.05 treated rats decreased the development of diabetes in recipient BB rats. In vitro studies also demonstrated that poly-I:C inhibits the proliferative response of BB rat spleen cells to concanavalin A. The administration of poly-0.05, but not poly-5.0, decreased TNF-alpha mRNA and IL-10 mRNA content in spleen cells. We conclude that poly I:C, at a dose 100 times lower than that required to accelerate diabetes prevents the development of diabetes in BB rates by interfering with the development of insulitis. The induction of suppressor cell activity induced by low dose poly-I:C in vivo and the inhibition of T-cell responses by poly-I:C in vitro suggests that the diabetes sparing activity of poly I:C is mediated by augmented immunoregulatory cell activity. Further studies with poly I:C may be important in increasing our understanding of the pathogenesis of IDDM and provide a means to prevent it.
Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Indutores de Interferon/uso terapêutico , Poli I-C/uso terapêutico , Animais , Peso Corporal/efeitos dos fármacos , Concanavalina A/farmacologia , Citocinas/biossíntese , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Relação Dose-Resposta a Droga , Citometria de Fluxo , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/biossíntese , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/patologia , Contagem de Leucócitos/efeitos dos fármacos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Masculino , Fenótipo , Ratos , Ratos Endogâmicos BB , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologiaRESUMO
Transglutaminases(TGases; protein-glutamine-glutamyl-transferases) are a large family of calcium-dependent acyl-transfer enzymes that catalyze the formation of covalent cross links in proteins. Of these, the "epidermal" or "hair follicle" TGase 3 isoform is critically involved in barrier formation in epithelia. It is a zymogen, requiring proteolytic activation to achieve maximal specific activity. In order to understand its structure and function, we have devised methods for the rapid large-scale expression of the TGase 3 zymogen in the baculovirus system, and here we describe the purification of the zymogen and activated forms. We describe methods for the formation of high-quality, well-diffracting crystals within 3-5 days, using both dioxane and beta-octylglucoside to overcome severe twinning problems. The crystal of the zymogen belongs to the triclinic space group P1 and diffracts to 2.2-A resolution, and the crystal of the active form belongs to the P2(1) space group at 2.7-A resolution.