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1.
Transplantation ; 72(11): 1730-6, 2001 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-11740381

RESUMO

BACKGROUND: Difficulties with recovering and preserving pancreatic islets have hampered progress in islet transplantation. In previous in vitro studies, our laboratory successfully demonstrated that using serum-free medium for prolonged pancreatic islet culture allows postculture recovery ratios greater than those obtained with standard media with sustained in vitro islet function. The goal of this study was to determine whether culturing of islets in a modified serum-free medium (M-SFM) would sustain function in vivo. METHODS: Islets were isolated from pancreata procured from 12 cadaveric organ donors and cultured in the M-SFM for up to 2 months, cryopreserved at -70 degrees C within 1-3 days of isolation for 2 months, or placed in short-term culture (3-5 days) before their transplantation under the kidney capsule of nonobese diabetic-severe combined immunodeficient mice (n=4-7 per group/time point). In vivo islet function was assessed by measuring the production of human insulin and C-peptide over a period of 3-15 months. RESULTS: After extended culture of islets in M-SFM for 1 or 2 months, transplanted islets maintained their viability, and in some instances in vivo function improved when compared with short-term cultured islets transplanted from the same preparation (P<0.01). Improvement was particularly evident for islets cultured for 1 month. Furthermore, when compared with cryopreserved preparations, early function (postoperative day 7) of islets from 1-month culture preparations was statistically better (P<0.05). Prolonged culture in M-SFM had no significant impact on long-term function, inasmuch as cultured islets functioned for more than 120 days. CONCLUSION: These data demonstrate that prolonged islet culture in M-SFM sustained viability and function, and in some instances had a positive effect on in vivo islet function, particularly in the 1-month cultures. No negative effect on long-term in vivo function was demonstrated in this study. Confirmation in clinical models utilizing extended (1-2 months) islet culture in M-SFM could significantly enhance islet transplantation by allowing the identification of best-matched recipients, pretransplantation recipient conditioning, and possible pretransplantation islet modifications to promote engraftment and prolonged graft function.


Assuntos
Técnicas de Cultura , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/fisiologia , Transplante Heterólogo , Animais , Criopreservação , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fatores de Tempo
2.
Scand J Infect Dis ; 33(3): 227-9, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11303816

RESUMO

Infections with Group C Streptococci can lead to severe disease, particularly in individuals with underlying illnesses such as cardiovascular disease, malignancy or immunosuppression. We report the first case of rhabdomyolysis and disseminated intravascular coagulation secondary to Group C Streptococcus in a previous healthy male. A toxic shock-like syndrome associated with Group C and Group G Streptococci has been reported. However, unlike with Group A Streptococci, production of endotoxins by these organisms is less well defined. We tested the patient's isolate for its ability to produce superantigenic toxins and to induce a mitogenic response. Although it is not known whether Group C Streptococci require special growth conditions for the production of superantigens, we could not demonstrate either the production of exotoxins or the induction of a mitogenic response.


Assuntos
Coagulação Intravascular Disseminada/complicações , Rabdomiólise/complicações , Infecções Estreptocócicas/complicações , Streptococcus/isolamento & purificação , Adulto , Bacteriemia/complicações , Bacteriemia/microbiologia , Exotoxinas/biossíntese , Humanos , Masculino , Choque Séptico/complicações , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/microbiologia , Streptococcus/metabolismo
3.
Arch Biochem Biophys ; 345(1): 97-102, 1997 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-9281316

RESUMO

The effect of the polyamines, putrescine, spermine, and spermidine, on the activity of extrahepatic methionine adenosyltransferase (MAT II) was studied. The polyamines inhibited MAT II activity at concentrations equal to or greater than 5 mm. Combinations of polyamines were more effective than individual polyamines in inhibiting MAT activity; maximum inhibition approached 80% with combinations of all three polyamines. S-Adenosylmethionine (AdoMet), Pi, and PPi, the products of the MAT reaction, are known to be synergistic inhibitors of the nonhepatic form of the enzyme. Combinations of polyamines plus Pi and/or PPi induced an additive inhibition of the enzyme. AdoMet plus polyamines also resulted in significant inhibition, but inhibition plateaued at about 80%, indicating the presence of a protective mechanism to maintain AdoMet synthesis. Extrahepatic MAT from human and rat tissues was inhibited by the polyamines, indicating that this phenomenon is not species specific. In addition, we examined the effect of polyamines on MAT activity in resting and activated human lymphocytes that were shown to differ in the relative expression of MAT II subunits. Although MAT from mitogen (phytohemagglutinin, PHA)- and superantigen (Staphylococcal enterotoxin B, SEB)-stimulated lymphocytes were similarly inhibited by 10 mM polyamines, at lower concentrations of polyamines (1-5 mM), MAT from SEB-stimulated cells appeared to be more susceptible to inhibition by the polyamines. Inasmuch as SEB is a more physiological stimulator of T cells than PHA, the data suggest a possible role of polyamines in regulating MAT activity.


Assuntos
Inibidores Enzimáticos/farmacologia , Metionina Adenosiltransferase/antagonistas & inibidores , Poliaminas/farmacologia , Animais , Humanos , Isoenzimas/antagonistas & inibidores , Cristalino/enzimologia , Leucemia/enzimologia , Ativação Linfocitária , Mitógenos/farmacologia , Fosfatos/farmacologia , Putrescina/farmacologia , Ratos , Ratos Sprague-Dawley , Retina/enzimologia , S-Adenosilmetionina/farmacologia , Espermidina/farmacologia , Espermina/farmacologia , Linfócitos T/enzimologia
4.
Anal Biochem ; 180(1): 120-5, 1989 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2510543

RESUMO

A sensitive assay for measuring serine hydroxymethyltransferase activity has been developed, based on the binding of N5,N10-[14C]methylene tetrahydrofolate (THF) to DEAE-cellulose paper. The complete assay requires THF, pyridoxal 5'-phosphate, [14C]serine, and enzyme. The reaction is stopped by streaking an aliquot of the reaction mixture onto a square of DEAE-cellulose paper, washing the paper with water to remove unreacted serine, drying the paper, and counting the bound N5,N10-[14C]methylene-THF. To determine that the labeled product was N5,N10-methylene-THF, unlabeled formaldehyde, which exchanges with the labeled methylene carbon, was added after the product had accumulated; 2 min after the addition of formaldehyde the amount of labeled product was reduced by 50%, and by 85% after 10 min. In addition, glycine, which reverses the reaction, and hydroxylamine, which reacts with the methylene carbon, reduced the number of counts bound to the paper. Binding of product to the filter is proportional to both enzyme concentration and assay time. No counts were retained on phosphocellulose filters. This assay represents a new and simple method for measuring serine hydroxymethyltransferase activity, which can be used to measure enzyme activity in tissue homogenates and for screening large numbers of samples.


Assuntos
Glicina Hidroximetiltransferase/metabolismo , Cristalino/enzimologia , Tetra-Hidrofolatos , Transferases/metabolismo , Animais , DEAE-Celulose , Ácido Edético/farmacologia , Glicina/farmacologia , Concentração de Íons de Hidrogênio , Hidroxilamina , Hidroxilaminas/farmacologia , Troca Iônica , Cinética , Fígado/enzimologia , Ratos , Ratos Endogâmicos
5.
Exp Eye Res ; 47(2): 197-204, 1988 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-3409991

RESUMO

Methionine adenosyltransferase (MAT) activity, and the concentration of its reaction product, S-adenosylmethionine (AdoMet), were measured in the lenses of rats of different ages; ranging from 1 day of age to over 1 yr. The highest specific activity of MAT was found in the lenses of the one day old rats (sp. ac. 0.327 units/mg-1 protein). After 1 week the specific activity had dropped to 0.067, and by 6 weeks had declined to adult levels (0.02 units/mg-1 protein). AdoMet concentrations were measured by HPLC in perchloric acid extracts. The highest concentration of AdoMet was found in the lenses of day-old rats (48.2 microM), and gradually declined with increasing age, reaching 5.5 microM in the oldest rats. In addition, the specific activity of MAT was found to be higher in the lens epithelium than in the cortex plus nucleus. The specific activity of MAT is almost an order of magnitude higher in the lens epithelial fraction (0.099 units mg-1 protein) than in the combined cortex plus nucleus fraction (0.011 units mg-1 protein).


Assuntos
Cristalino/enzimologia , Metionina Adenosiltransferase/metabolismo , S-Adenosilmetionina/metabolismo , Transferases/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Cristalino/crescimento & desenvolvimento , Ornitina Descarboxilase/metabolismo , Ratos , Ratos Endogâmicos
6.
Exp Eye Res ; 43(6): 997-1008, 1986 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-3817036

RESUMO

Methionine adenosyltransferase (MAT) has been partially purified from rat lenses using a combination of ammonium sulfate fractionation and hydrophobic chromatography on phenyl Sepharose columns. The partially purified enzyme resembles purified Type II MAT from non-hepatic tissues. The Km for methionine is 3.0 microM, and the Km for ATP is 80 microM. The enzyme is activated by potassium ions (25-50 mM), and inhibited by higher concentrations of potassium. A divalent cation (magnesium or manganese) is essential for activity. The Vmax with magnesium is about five times higher than with manganese, but the optimal manganese concentration is around 2.0 mM, compared with 10-20 mM for magnesium. The enzyme is active over a broad pH range, with optimal activity between pH 7.0 and 8.0. The enzyme is inhibited by all three of its products, phosphate, pyrophosphate, and S-adenosylmethionine. Individually phosphate and pyrophosphate are weak inhibitors, but in combination they show a marked synergistic inhibitory effect. Tripolyphosphate is also an effective inhibitor. The inhibition of the enzyme by the cataractogenic agent, dimethylsulfoxide, further confirmed the similarity to Type II MAT.


Assuntos
Cristalino/enzimologia , Metionina Adenosiltransferase/isolamento & purificação , Transferases/isolamento & purificação , Animais , Anticorpos/imunologia , Ativação Enzimática , Concentração de Íons de Hidrogênio , Magnésio/farmacologia , Manganês/farmacologia , Metionina/metabolismo , Metionina Adenosiltransferase/imunologia , Metionina Adenosiltransferase/metabolismo , Fosfatos/metabolismo , Potássio/farmacologia , Ratos , Ratos Endogâmicos , S-Adenosilmetionina/metabolismo
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