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1.
Scand J Immunol ; 69(2): 110-8, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19170963

RESUMO

Artocarpus tonkinenesis (Moraceae) has been used in Vietnamese traditional medicine for the treatment of backache and joint diseases since many 100 years. We have previously shown that a crude extract of A. tonkinensis elicited anti-inflammatory effects in rat collagen-induced arthritis (CIA), with significant improvement of disease symptoms. However, the pharmacological basis of the bioactivity of A. tonkinensis extract is not known. In the present study, we have isolated four individual active components from A. tonkinensis extract by reverse phase high-pressure liquid chromatography. The structures of the compounds were determined by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry and their biological effects investigated. A novel biologically active flavonoid glucoside (5-hydroxy-8-hydroxymethyl-8-methyl-2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-phenyl]-8H-pyrano[3,2-g]chromen-4-one) with an average molecular mass of 514.49 Da was isolated.We have named the compound artonkin-4'-O-glucoside. The name 'artonkin' for the novel flavonoid part of the compound was coined from the Latin name of its source Artocarpus tonkinensis. The three other active flavonoid glucosides isolated and characterized were alphitonin-4-O-beta-D-glucoside, maesopsin-4-O-beta-D-glucoside and kaempherol-3-O-beta-D-glucoside. All four compounds were found to cause anti-inflammatory effect with different potencies. The anti-inflammatory effects demonstrated in the rat model of arthritis correlate well with the inhibition of mitogen-induced T-cell proliferation. Furthermore, the compounds inhibit production of cytokines, such as tumour necrosis factor-a and interferon-c, in mitogen-stimulated T cells in a concentration-dependent manner. We postulate that the isolated flavonoids suppress T-cell proliferation as well as cytokine expression and thereby contribute to an amelioration of arthritis severity in CIA.


Assuntos
Anti-Inflamatórios/isolamento & purificação , Artrite Experimental/tratamento farmacológico , Artocarpus/química , Flavonoides/isolamento & purificação , Glucosídeos/isolamento & purificação , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Flavonoides/química , Flavonoides/uso terapêutico , Glucosídeos/química , Glucosídeos/uso terapêutico , Interferon gama/biossíntese , Interleucina-6/biossíntese , Ativação Linfocitária/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Folhas de Planta/química , Ratos , Fator de Necrose Tumoral alfa/biossíntese
2.
Cell Mol Biol (Noisy-le-grand) ; 49(5): 763-8, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-14528913

RESUMO

Metallothionein-3 (MT-3) is a brain-specific isoform of metallothioneins, which is down-regulated in Alzheimer's disease (AD), inhibits the growth of neurons in vitro, and differs from common MTs also in gene regulation. To elucidate the differences in structure and function between MT-3 and common MTs, Zn2+ and Cd2+ binding to MT-3 and MT-1 were studied using electrospray ionization time of flight mass spectrometry (ESI TOF MS) at pH values between 7.5 and 2.7. The metal binding properties of MT-3 differ considerably from those of MT-1. After reconstitution with a metal excess, metallated MT-3 exists as a mixture of Zn7MT-3 (or Cd7MT-3, respectively) and several metalloforms with stoichiometries below and above seven. In contrast, MT-1 exists as a single Zn7MT-1 (or Cd7MT-1). Lowering of pH leads to a stepwise release of metals from metallated MT-3, first from extra sites, then from the 3-metal cluster and finally from the 4-metal cluster. At acidic pH values the 4-metal cluster of MT-3 is slightly more stable than that of MT-1. The results demonstrate higher structural plasticity, dynamics and metal binding capacity of MT-3 than of MT-1, which makes MT-3 suitable as a zinc buffer-transfer molecule in zinc-enriched neurons functioning at conditions of fluctuating zinc concentrations.


Assuntos
Cádmio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Zinco/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Cádmio/química , Humanos , Concentração de Íons de Hidrogênio , Metalotioneína/química , Metalotioneína/isolamento & purificação , Metalotioneína/metabolismo , Metalotioneína 3 , Proteínas do Tecido Nervoso/química , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Zinco/química
3.
Arch Biochem Biophys ; 385(2): 276-82, 2001 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-11368008

RESUMO

Chinese hamster ovary (CHO) cells are widely used as hosts for receptor expression and pharmacological studies. However, several endogenous receptor populations are present on these cells. Intestinal tissue extracts were found to induce strong extracellular acidification responses (ECAR) in CHO cells, yet several pure hormonal peptides, such as VIP, secretin, CCK, GIP, and galanin were ineffective. It is not known, which are the active compounds in the extracts that can stimulate the extracellular acidification in CHO cells. These active substances may be ligands for yet unknown receptors that are present natively in this cell type. We therefore decided to identify the active compound(s) by isolation from intestinal extract and structural characterization. Using chromatographic separations in combination with microphysiometry we have purified and characterized one such bioactive ligand. Structural analysis indicated that the isolated peptide was identical to insulin-like growth factor I (IGF-I). In the intestine, IGF-I is present in low amounts and has previously been detected only with radioimmunoassays. The results indicate that CHO cells express functional receptors for IGF-I. Among the peptides extracted from the intestine IGF-I is probably the strongest stimulator of ECAR in CHO cells. Moreover, IGF-I acts synergistically with other factors present in the crude tissue extract. Additionally, a fragment of calponin H1 (residues 1-43), previously not described at the protein level, was identified in the IGF-I containing fractions. The fragment was characterized by mass spectrometry and found to be N-terminally modified by acetylation suggesting that the whole protein bears the same posttranslational modification.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Fator de Crescimento Insulin-Like I/isolamento & purificação , Fator de Crescimento Insulin-Like I/farmacologia , Intestinos/química , Acetilação , Animais , Células CHO , Proteínas de Ligação ao Cálcio/isolamento & purificação , Extratos Celulares/química , Extratos Celulares/farmacologia , Cricetinae , Sinergismo Farmacológico , Concentração de Íons de Hidrogênio , Fator de Crescimento Insulin-Like I/química , Proteínas dos Microfilamentos , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Peptídeos/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos , Calponinas
4.
FEBS Lett ; 492(1-2): 119-22, 2001 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-11248248

RESUMO

We have isolated a posttranslationally modified form of peptide YY (PYY) from porcine intestine and shown by MALDI-TOF and electrospray tandem mass spectrometry that it is phosphorylated at Ser(13). Phospho-PYY exhibits high affinity for binding to neuropeptide Y (NPY) receptors Y1, Y2 and Y5. The IC(50) values with the Y1, Y2, and Y5 receptor subtypes were for NPY 2.4, 3.1, and 3.3 nM, for PYY 2.3, 0.94, and 3.2 nM, and for phospho-PYY 4.6, 2.2, and 5.5 nM, respectively. Phospho-PYY potently inhibits forskolin-stimulated cAMP accumulation in SK-N-MC cells with an IC(50) value of 0.5 nM compared to 0.15 nM for non-phosphorylated PYY. The finding of phosphorylation of PYY is unusual among hormonal peptides, and emphasizes the importance of direct protein analysis of gene products.


Assuntos
Mucosa Intestinal/metabolismo , Peptídeo YY/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Animais , AMP Cíclico/metabolismo , Humanos , Peptídeo YY/química , Fosforilação , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Suínos , Células Tumorais Cultivadas
6.
Cell Mol Life Sci ; 56(7-8): 709-13, 1999 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-11212317

RESUMO

A C-terminally elongated form of peptide histidine isoleucine amide (PHI) was isolated from porcine intestine based on its effect on cAMP production in IMR-32 cells. The structure was determined by amino acid sequence analysis of tryptic fragments and by mass spectrometry. The peptide has 42 amino acid residues like those described from human, rat and mouse, but the amino acid sequence of the C-terminal extension of pig PHI is unique. Unlike the other peptides, it has a C-terminal Ala and it differs at five positions from the human form and at six positions from the rat form, while the human and the rat forms differ by only two substitutions. To avoid confusion arising from different C-terminal residues, a unifying nomenclature is proposed: PHI-27 for the hormone and PHI-42 for the elongated product.


Assuntos
Intestinos/química , Peptídeo PHI/química , Peptídeo PHI/isolamento & purificação , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Peptídeo PHI/metabolismo , Peptídeo PHI/farmacologia , Alinhamento de Sequência , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos , Tripsina/metabolismo
8.
Neurosci Lett ; 240(1): 21-4, 1998 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-9488165

RESUMO

Recent experiments have shown that an ischemic insult can induce both necrosis and apoptosis. A series of experiments were designed to examine the potential induction of apoptosis by oxygen-glucose deprivation (OGD) in cerebellar granule cell culture. A medium OGD (90 min) induced apoptosis in cell culture, with maximal effect 12 h after exposure, as indicated by following morphological (TdT-mediated dUTP-biotin nick end-labeling) and biochemical markers (DNA oligonucleosomal fragmentation). Mitochondrial injury (MTT assay) was among the early effects we detected during and after OGD and it was correlated with the dynamics of TUNEL positive cells. The amount of LDH release from damaged cells, associated with necrosis was increased significantly 12 h after exposure. These results indicate that medium OGD induced a rapid (<12 h) mixture of apoptosis and necrosis, followed by mainly secondary necrosis.


Assuntos
Apoptose , Cerebelo/citologia , Cerebelo/patologia , Glucose/deficiência , Neurônios/citologia , Neurônios/patologia , Animais , Animais Recém-Nascidos , Hipóxia Celular , Células Cultivadas , Cerebelo/metabolismo , Fragmentação do DNA , Necrose , Neurônios/metabolismo , Ratos , Fatores de Tempo
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