RESUMO
BACKGROUND: Orchid seeds are reputed to be relatively short-lived, although comparative studies across a wide range of storage temperatures and moisture contents are few. OBJECTIVE: To explore how a wide range of temperature and moisture contents affects seed longevity in the orchid genus Cattleya. MATERIALS AND METHODS: Seeds of three Brazilian orchid species in the genus Cattleya (C. amethystoglossa, C. kautskyana and C. tigrina) were subjected to controlled deterioration (CD) and seeds at three moisture levels were stored for up to 270 days at temperatures as low as -196 degree C. Seed ageing curves were constructed and seed quality assessed as germination level and speed index in vitro, and by tetrazolium staining. Seed morphometry, lipid content and composition were also determined. RESULTS: Seeds were found to be lipid rich (54-70% DW) and short-lived under CD, with P50s varying less than two-fold (42 to 69 h) at 41 degree C. At cold (5 degree C) to ultracold (-196 degree C) temperatures longevity was greatest after pre-drying seeds to 15% RH, with germination varying by c. 6% after 270 days storage at these temperature limits. However, storage at -20 degree C resulted in a 5-46% reduction in germination, indicating a dry-seed sensitivity to conventional seed bank temperature. Ultra-dry (silica gel) and high humidity (82% RH) storage, in combination with various temperatures, compromised viability in one or more species. Overall, seed longevity in C. kautskyana > C. tigrina > C. amethystoglossa, which also reflected the pattern of embryo volume. The prospect that the generally shorter lifespans in orchid seeds may relate to stochastic processes the negative impact of which reach criticality sooner in smaller embryos requires further testing. CONCLUSION: Further evidence is provided that cryobiotechnology provides a biobanking option for orchid seeds with relatively short lifespans.
Assuntos
Longevidade , Orchidaceae , Bancos de Espécimes Biológicos , Criopreservação/veterinária , SementesRESUMO
Ulcerative colitis (UC) is a chronic inflammatory bowel disease featuring infiltration by plasma cells producing immunoglobulins. We have reported previously the specific and significant proliferation of immature plasma cells in the inflamed colonic and pouch mucosa of UC patients. The aim of this study was to characterize peripheral blood immature plasma cells and the migration mechanisms of such immature plasma cells to inflamed sites in UC. The characteristics of peripheral blood immature plasma cells and chemokine receptor expression were examined by flow cytometry. Expression of mucosal chemokine was quantified using real-time reverse transcription-polymerase chain reaction and immunohistochemistry. The number of peripheral blood immature plasma cells was significantly higher in patients with active UC and active Crohn's disease (CD) than in healthy controls. The proportion of immature plasma cells was correlated positively with clinical activities of UC and CD. Many peripheral blood immature plasma cells were positive for CXCR3, CXCR4, CCR9 and CCR10. Expression of CXCR3 and CXCR4 in UC patients was significantly higher than in controls. CXCL9, CXCL10 and CXCL11 mRNA levels in colonic mucosa of inflamed IBD were higher than in controls. Immunofluorescence study also showed abundant CXCR3-positive immature plasma cells in the inflamed colonic mucosa of UC. Increased numbers of immature plasma cells may migrate towards inflammatory sites of UC via the CXCR3 axis, and may participate in UC pathogenesis.
Assuntos
Movimento Celular , Colite Ulcerativa/imunologia , Plasmócitos/imunologia , Receptores CXCR3/imunologia , Receptores CXCR4/imunologia , Adulto , Antígenos CD19/análise , Antígenos CD19/imunologia , Quimiocinas/análise , Quimiocinas/imunologia , Colite Ulcerativa/patologia , Doença de Crohn/imunologia , Doença de Crohn/patologia , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Contagem de Linfócitos , Pessoa de Meia-Idade , Receptores CCR/análise , Receptores CCR/imunologia , Receptores CCR/metabolismo , Receptores CXCR3/análise , Receptores CXCR4/análiseRESUMO
Upon addition of NADP+, the rose bengal-sensitized photoinactivation of D-erythrulose reductase from beef liver is prevented to a remarkable extent. Adenosine 2',5'-diphosphate (2',5'-ADP) also has a protective effect, but to a lesser extent. On the other hand, 2'-AMP markedly enhances the photoinactivation. Other nucleotides which have no 2'-phosphoryl group, such as NAD+, 3'-AMP, 5'-AMP, ADP, and NMN, are ineffective. Further, only 2'-AMP derivatives (NADP+, 2',5'-ADP, and 2'-AMP) among these nucleotides were found to be potent competitive inhibitors of the enzyme with small Ki's (6--13 muM). Photooxidation of some methionine residues in the enzyme is prevented by the addition of NADP+ and accelerated in the presence of 2'-AMP. Photooxidation products(s) of 2'-AMP derivatives have no effect upon the enzymatic activity. Although NADP+ and 2'-AMP induce detectable conformational changes of the enzyme, the changes are not characteristic to the compounds. Based on these observations, we present a possible action mechanism of 2'-AMP derivatives on the photoinactivation of D-erythrulose reductase.
Assuntos
Fígado/enzimologia , NADP/análogos & derivados , NADP/farmacologia , Rosa Bengala/farmacologia , Desidrogenase do Álcool de Açúcar/antagonistas & inibidores , Monofosfato de Adenosina , Animais , Bovinos , Dicroísmo Circular , Cinética , Luz , Oxirredução , Conformação Proteica , Relação Estrutura-Atividade , TetrosesRESUMO
D-Erythrulose reductase from chicken liver has been purified to homogeneity as judged by acrylamide gel electrophoresis and ultracentrifugation. The overall purification of the enzyme was 164-fold from a crude extract. The enzyme was crystallized from ammonium sulfate solution at pH 7.0 to give hexagonal plates. The molecular weight determined by sedimentation equilibrium analysis was 94,600 and that by SDS-polyacrylamide gel electrophoresis was 22,400, which suggests a tetrameric structure for the native enzyme. The enzyme was found to contain up to 3 molecules of NADP+ per enzyme; this high amount of NADP+ resulted in a higher absorption at 260 nm than at 280 nm. The extinction coefficient of the enzyme at 290 nm was found to be 4.0. The contents of various amino acids were very similar to those of the beef liver enzyme formerly crystallized in our laboratory. The isoelectric point of the enzyme determined by Ampholine isoelectric focusing was pH 6.43. The enzyme was shown to catalyze the reduction of D-erythrulose to D-threitol with the concomitant oxidation of NAD(P)H to NAD(P)+, and was highly specific to D-erythrulose with an apparent Km of 0.38 mM. NADH was less effective than NADPH and the Km's for NADH and NADPH were 67 micrometers and 7.9 micrometers, respectively. D-Threitol was slightly oxidized by the enzyme with either NADP+ or NAD+ as a cofactor at pH's 7.5 and 9.0.
Assuntos
Fígado/enzimologia , Desidrogenase do Álcool de Açúcar/metabolismo , Aminoácidos/análise , Animais , Galinhas , Cristalização , Cinética , Peso Molecular , Espectrofotometria Ultravioleta , Especificidade por Substrato , Desidrogenase do Álcool de Açúcar/isolamento & purificação , Tetroses/isolamento & purificação , Tetroses/metabolismoRESUMO
We have discovered new characteristics of D-erythrulose reductase, namely, that it can catalyze reduction of not only D-erythrulose but also such diketones as diacetyl. These substrates have a common structure with two neighboring carbonyls possibly in s-cis plane structure, showing that the enzyme may rigorously distinguish between substrates and other compounds. D-Erythrulose reductase was predominantly located in the kidney and the liver of the chicken. The obtained results suggest that D-erythrulose reductase plays an important role in metabolizing alpha-dicarbonyls in animal organs, because these diketones widely occur in natural foods.
Assuntos
Diacetil/metabolismo , Fígado/enzimologia , Desidrogenase do Álcool de Açúcar/metabolismo , Animais , Galinhas , Cromatografia Líquida , Masculino , Oxirredução , Especificidade por Substrato , Desidrogenase do Álcool de Açúcar/isolamento & purificaçãoRESUMO
An aldehyde reductase catalyzing the NADPH-dependent reduction of D-erythrose 4-phosphate to D-erythritol 4-phosphate was purified from beef liver. It was proved to be homogeneous by polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugation analysis. The enzyme was proved to be a monomeric enzyme and its molecular weight was about 40,000. The enzyme was able to reduce not only tetroses but also trioses, aromatic aldehydes, D-glucuronate and succinic semialdehyde. Apparent Km-values for aromatic aldehydes were lower than those for tetroses, trioses, D-glucuronate and succinic semi-aldehyde. Barbiturates and valproate were potent inhibitors of the enzyme and their apparent K1-values were in the range of 80-180 microM. Quercitrin was the most potent inhibitor and its K1-value was about 7 microM. From the viewpoint of substrate specificity and inhibitor sensitivity, it seems that the enzyme belongs to the high-Km type aldehyde reductases.
Assuntos
Oxirredutases do Álcool/metabolismo , Fígado/enzimologia , Fosfatos Açúcares/metabolismo , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/isolamento & purificação , Aminoácidos/análise , Animais , Bovinos , Cromatografia , Precipitação Fracionada , Ponto Isoelétrico , Peso Molecular , NADP/farmacologia , Especificidade por SubstratoRESUMO
Thioltransferase, an enzyme which catalyzes the thiol/disulfide exchange reaction in the presence of GSH, was purified to homogeneity on 15% SDS-PAGE from human (36,000-fold purification) and bovine (23,000-fold) erythrocyte hemolysates. These enzymes had similar properties in their monomeric structures (M(r) = 11,000) and broad specificities for substrates ranging from low-molecular disulfides (S-sulfocysteine, cystamine, and cystine) to protein disulfides (trypsin and insulin). They were highly sensitive to SH-reagents (monoiodoacetic acid and mercuric chloride), but were protected from inactivation by the presence of disulfides (GSSG, cystamine, and cystine). Phosphofructokinase and pyruvate kinase that had been inactivated by disulfides were reactivated effectively by the addition of thioltransferase with GSH. In addition, disulfides in membrane proteins of human erythrocytes that have been oxidatively damaged by diamide treatment were reduced to the SH-free form more effectively by incubation with thioltransferase.
Assuntos
Eritrócitos/enzimologia , Oxirredutases/sangue , Proteína Dissulfeto Redutase (Glutationa) , Aminoácidos/análise , Animais , Bovinos , Dissulfetos/sangue , Membrana Eritrocítica/metabolismo , Glutarredoxinas , Humanos , Proteínas de Membrana/sangue , Peso Molecular , Oxirredução , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Especificidade da Espécie , Especificidade por Substrato , Compostos de Sulfidrila/sangueAssuntos
Fígado/enzimologia , Desidrogenase do Álcool de Açúcar/isolamento & purificação , Animais , Bovinos , Fenômenos Químicos , Físico-Química , Cromatografia de Afinidade/métodos , Cromatografia DEAE-Celulose , Cristalização , Durapatita , Hidroxiapatitas , Técnicas In Vitro , Cinética , Desnaturação ProteicaAssuntos
Adenina , DNA Bacteriano/efeitos da radiação , Fotoquímica , Riboflavina , Bacillus subtilis , Corantes , Mononucleotídeo de Flavina , Flavina-Adenina Dinucleotídeo , Flavinas , Fluoresceínas , Azul de Metileno , Purinas , Pirimidinas , Compostos de Amônio Quaternário , Efeitos da Radiação , Temperatura , XantenosAssuntos
Oxirredutases do Álcool/metabolismo , Dissulfetos/farmacologia , Fígado/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases , Reagentes de Sulfidrila/farmacologia , Oxirredutases do Álcool/antagonistas & inibidores , Animais , Soluções Tampão , Citosol/enzimologia , Dissulfetos/metabolismo , Cinética , Masculino , Ratos , Ratos Endogâmicos , Especificidade por Substrato , Sulfetos/metabolismoRESUMO
The specific determinations of D-erythrulose by enzymatic assay or colorimetric method, which permit the quantitative determination of between 20 and 400 nmol of the sugar, are described. Enzymatic determination of D-erythrulose made use of the D-erythrulose reductase purified from beef or chicken liver, which catalyzes specifically the reduction of D-erythrulose with concomitant conversion of NADH to NAD+. The colorimetric microdetermination of erythrulose could be carried out by utilizing the phenol-sulfuric acid reaction under low temperature. These methods are simple, rapid, and sensitive, and give reproducible results.
Assuntos
Colorimetria/métodos , Desidrogenase do Álcool de Açúcar , Tetroses/análise , Animais , Carboidratos/análise , Bovinos , Galinhas , Fígado/enzimologia , Fenol , Fenóis , Ácidos SulfúricosRESUMO
The rate of the conversion of ribose 5-phosphate to hexose 6-phosphates by reaction of the non-oxidative pentose phosphate pathway was measured in the presence of various biological materials. Of 22 compounds tested, PRPP and ADP markedly inhibited the formation of hexose 6-phosphates from ribose 5-phosphate. The transketolase activity in beef liver enzyme preparation was extremely inhibited by PRPP and ADP, but the transaldolase activity was not inhibited. The mode of inhibition of transketolase by PRPP and ADP was a competitive one. The Ki value for PRPP was 0.14 mM and that for ADP 0.54 mM with respect to transketolase. We discuss the possible regulatory roles of ADP and PRPP on pentose phosphate metabolism in the pentose phosphate pathway.
Assuntos
Difosfato de Adenosina/farmacologia , Fígado/enzimologia , Via de Pentose Fosfato/efeitos dos fármacos , Pentosefosfatos/farmacologia , Fosforribosil Pirofosfato/farmacologia , Animais , Bovinos , Cromatografia Gasosa , Cinética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Transcetolase/metabolismoRESUMO
The enzyme which catalyzes the conversion of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate has been purified to homogeneity from a crude extract of beef liver. Analysis of the purified enzyme by Sephadex G-100 gel filtration and sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed it to be a dimer of relative molecular mass 43 000. From the gas chromatography/mas spectrometry analyses of the enzymatic reaction products, it appeared that about 90% of the total amount of tetrose 4-phosphate was present as D-erythrulose 4-phosphate after equilibration. The purified enzyme, which is tentatively called 'erythrose-4-phosphate isomerase' had no significant isomerase activities on D-glyceraldehyde 3-phosphate, D-ribose 5-phosphate, D-glucose 6-phosphate and D-fructose 6-phosphate, but a strong D-ribulose-5-phosphate 3-epimerase activity was co-purified with the erythrose-4-phosphate isomerase activity through every step in the isolation. Both the erythrose-4-phosphate isomerase and D-ribulose-5-phosphate 3-epimerase activities were inactivated at the same rate at the elevated temperature, and also inhibited to the same extent by various inhibitors. It is likely, that both activities are catalyzed by the single enzyme protein.