RESUMO
One of the promising approaches in the therapy of ulcerative colitis is administration of butyrate, an energy source for colonocytes, into the lumen of the colon. This study investigates the effect of butyrate producing bacterium Clostridium tyrobutyricum on dextran sodium sulphate (DSS)-induced colitis in mice. Immunocompetent BALB/c and immunodeficient severe combined immunodeficiency (SCID) mice reared in specific-pathogen-free (SPF) conditions were treated intrarectally with C. tyrobutyricum 1 week prior to the induction of DSS colitis and during oral DSS treatment. Administration of DSS without C. tyrobutyricum treatment led to an appearance of clinical symptoms - bleeding, rectal prolapses and colitis-induced increase in the antigen CD11b, a marker of infiltrating inflammatory cells in the lamina propria. The severity of colitis was similar in BALB/c and SCID mice as judged by the histological damage score and colon shortening after 7 days of DSS treatment. Both strains of mice also showed a similar reduction in tight junction (TJ) protein zonula occludens (ZO)-1 expression and of MUC-2 mucin depression. Highly elevated levels of cytokine tumour necrosis factor (TNF)-α in the colon of SCID mice and of interleukin (IL)-18 in BALB/c mice were observed. Intrarectal administration of C. tyrobutyricum prevented appearance of clinical symptoms of DSS-colitis, restored normal MUC-2 production, unaltered expression of TJ protein ZO-1 and decreased levels of TNF-α and IL-18 in the descending colon of SCID and BALB/c mice, respectively. Some of these features can be ascribed to the increased production of butyrate in the lumen of the colon and its role in protection of barrier functions and regulation of IL-18 expression.
Assuntos
Butiratos/metabolismo , Clostridium tyrobutyricum/fisiologia , Colite Ulcerativa/microbiologia , Interleucina-18/biossíntese , Probióticos/uso terapêutico , Fator de Necrose Tumoral alfa/biossíntese , Doença Aguda , Administração Retal , Animais , Translocação Bacteriana , Antígeno CD11b/biossíntese , Antígeno CD11b/genética , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/genética , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Sulfato de Dextrana/toxicidade , Ácidos Graxos/metabolismo , Imunocompetência , Interleucina-18/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Mucina-2/biossíntese , Mucina-2/genética , Mucinas/biossíntese , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Organismos Livres de Patógenos Específicos , Fator de Necrose Tumoral alfa/genética , Proteína da Zônula de Oclusão-1RESUMO
This study was aimed to evaluate the role of commensal Gram-negative bacterium Bacteroides ovatus in murine model of chronic intestinal inflammation. The attempt to induce chronic colitis was done in Bacteroides ovatus-monoassociated, germ-free and conventional mice either in immunocompetent (BALB/c) mice or in mice with severe combined immunodeficiency (SCID), using 2.5 % dextran-sodium sulfate (DSS) in drinking water (7 days DSS, 7 days water, 7 days DSS). Conventional mice developed chronic colitis. Some of germ-free BALB/c and the majority of germ-free SCID mice did not survive the long-term treatment with DSS due to massive bleeding into the intestinal lumen. However, monocolonization of germ-free mice of both strains with Bacteroides ovatus prior to long-term treatment with DSS protected mice from bleeding, development of intestinal inflammation and precocious death. We observed that though DSS-treated Bacteroides ovatus-colonized SCID mice showed minor morphological changes in colon tissue, jejunal brush-border enzyme activities such as gamma-glutamyltranspeptidase, lactase and alkaline phosphatase were significantly reduced in comparison with DSS-untreated Bacteroides ovatus-colonized mice. This modulation of the enterocyte gamma-glutamyltranspeptidase localized to the brush border membrane has been described for the first time. This enzyme is known to reflect an imbalance between pro-oxidant and anti-oxidant mechanisms, which could be involved in protective effects of colonization of germ-free mice with Bacteroides ovatus against DSS injury.
Assuntos
Bacteroides/crescimento & desenvolvimento , Colite/prevenção & controle , Colo/microbiologia , Fosfatase Alcalina/metabolismo , Animais , Doença Crônica , Colite/induzido quimicamente , Colite/enzimologia , Colite/microbiologia , Colite/patologia , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Mucosa Intestinal/enzimologia , Jejuno/enzimologia , Lactase/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Microvilosidades/enzimologia , Índice de Gravidade de Doença , Fatores de Tempo , gama-Glutamiltransferase/metabolismoRESUMO
The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Mucosa Intestinal/citologia , Lacticaseibacillus casei , Lacticaseibacillus rhamnosus , Probióticos/farmacologia , Animais , Caspase 1/biossíntese , Citocinas/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-18/biossíntese , Mucosa Intestinal/efeitos dos fármacos , Proteínas dos Microfilamentos/biossíntese , RNA Mensageiro/biossíntese , Ratos , Frações Subcelulares/metabolismo , Receptores Toll-Like/biossíntese , Receptores Toll-Like/efeitos dos fármacos , beta Catenina/biossínteseRESUMO
The alpha 2,6-sialyltransferase (alpha 2,6-ST) acting on N-acetyllactosamine is the major sialyltransferase of suckling rat jejunum. Jejunal explants of 7-day-old rats maintained in serum-free or serum-containing organ culture secreted alpha 2,6-ST into the cultivation medium. Dexamethasone (80 nM) stimulates primarily the secreted pool of alpha 2,6-ST. Fetal calf serum promotes the stimulatory effect of dexamethasone also on the bound form of alpha 2,6-ST.
Assuntos
Dexametasona/farmacologia , Jejuno/enzimologia , Sialiltransferases/análise , Animais , Animais Recém-Nascidos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Ratos , Sialiltransferases/metabolismo , Tripsina , Regulação para CimaRESUMO
Sialyltransferase activity was assayed in rat intestinal cells isolated as fractions reflecting the villus-crypt axis of differentiation. In 13-day-old rats both endo- and exogenous sialyltransferase activity reached their maximum in undifferentiated crypt cells and their peaks overlapped. In contrast, sialyltransferase of the adult intestine was 4-fold lower than that of sucklings in the crypts, with slight tendency to be transferred to the villus cells. Hydrocortisone applied to 10-day-old rats caused three days later a precocious drop of sialyltransferase activity in the crypt cells. Unlike in vivo, glucocorticoid responsiveness was accompanied by increased sialyltransferase activity in fetal small intestine cultivated for 17 days.
Assuntos
Feto/enzimologia , Hidrocortisona/farmacologia , Intestino Delgado/crescimento & desenvolvimento , Sialiltransferases/metabolismo , Animais , Diferenciação Celular , Dexametasona/farmacologia , Intestino Delgado/embriologia , Intestino Delgado/enzimologia , Jejuno/embriologia , Jejuno/enzimologia , Microvilosidades/enzimologia , Ácido N-Acetilneuramínico , Técnicas de Cultura de Órgãos , Ratos , Ácidos Siálicos/metabolismo , Sialiltransferases/antagonistas & inibidores , Distribuição Tecidual , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
Hydrocortisone acetate or hemisuccinate (75 mg/kg body mass) applied to rats i.m. and/or s.c. on the 9th and 10th postnatal days causes a precocious decrease of sialic acid content of the small intestinal brush-border membrane. On the 15th postnatal day the bound sialic acid of the whole membrane fraction drops to almost half of the values of control animals and to one third of the control values for the papain-solubilized membrane proteins. The hydrocortisone effect is manifested on isoelectric focusing zymograms by a faster increase of pI of the solubilized brush-border enzymes on the 12th and 15th postnatal days.
Assuntos
Grupos de População Animal/crescimento & desenvolvimento , Animais Lactentes/crescimento & desenvolvimento , Glucosidases/análise , Hidrocortisona/farmacologia , Jejuno/ultraestrutura , Ácidos Siálicos/análise , alfa-Glucosidases/análise , Animais , Hidrocortisona/análogos & derivados , Ponto Isoelétrico , Proteínas de Membrana/análise , Microvilosidades/enzimologia , Ácido N-Acetilneuramínico , Neuraminidase/metabolismo , RatosRESUMO
Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are associated with regulation of various gastrointestinal functions. In order to better understand their role in developing small intestine EGF, TGF-alpha and EGF-R steady-state mRNA levels and transcript stability were determined. Reverse transcription (RT) competitive-polymerase chain reaction (PCR) revealed that intestinal TGF-alpha mRNA levels were 10-fold higher in comparison with EGF mRNA. The primary intestinal culture technique was used to evaluate mRNA stability. The stability of TGF-alpha mRNA was remarkably lower than the stability of EGF mRNA. High levels of TGF-alpha mRNA accompanied by high degradation rate of this mRNA suggested a rapid turnover of intestinal TGF-alpha mRNA.
Assuntos
Animais Lactentes/genética , Fator de Crescimento Epidérmico/genética , Jejuno/metabolismo , RNA Mensageiro/biossíntese , Fator de Crescimento Transformador alfa/genética , Animais , Animais Lactentes/crescimento & desenvolvimento , Fator de Crescimento Epidérmico/biossíntese , Feminino , Jejuno/crescimento & desenvolvimento , Masculino , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transcrição Gênica , Fator de Crescimento Transformador alfa/biossínteseRESUMO
The progressive loss of sialic acids of the brush-border membrane glycoproteins is one of the major biochemical changes which occur in the rat small intestine during the transition from suckling to weaning, and this process is speeded up by an injection of glucocorticoids to the suckling animals. We used the rat liver beta-galactoside alpha2,6-sialyltransferase (ST6(N), EC 2.4.99.1) cDNA as a probe to examine the mRNA level of this enzyme in the small intestine of both suckling (13-day-old) and weaned (25-day-old) rats. In the ileum of suckling rats, the ST6(N) mRNA level was about four times higher than in the jejunum, whereas the membrane-bound enzyme activity was less than two times higher. In comparison with the controls, hydrocortisone treatment significantly decreased the level of this transcript and of the corresponding enzyme activity in both segments of the small intestine of suckling rats. Additionally, the antiglucocorticoid mifepristone (RU-38.486) suppressed the effect of hydrocortisone. The expression of ST6(N) mRNA in the small intestine of weaned (25-day-old) rats was several times lower than that in suckling (13-day-old) rats, and was unresponsive to hydrocortisone as well as to mifepristone. These results indicate that the glucocorticoid-induced transcriptional down-regulation of ST6(N) expression in the small intestine of suckling rats is mediated via the glucocorticoid receptor pathway, and support the notion that alterations in sialylation of brush-border membrane glycoconjugates occurring upon weaning are the result of a lower expression of ST6(N).
Assuntos
Hidrocortisona/farmacologia , Intestino Delgado/enzimologia , Mifepristona/farmacologia , Sialiltransferases/efeitos dos fármacos , Comportamento de Sucção/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Regulação para Baixo , Feminino , Antagonistas de Hormônios/farmacologia , Íleo/efeitos dos fármacos , Íleo/enzimologia , Mucosa Intestinal/enzimologia , Intestino Delgado/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Jejuno/enzimologia , Microvilosidades/efeitos dos fármacos , Microvilosidades/enzimologia , Gravidez , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Glucocorticoides/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismo , Solubilidade , Transcrição Gênica/efeitos dos fármacos , Desmame , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
In attempting to elucidate the molecular basis of the expression of alpha 2,6-sialyltransferase (alpha 2,6-ST) in jejunal explants of 7-day-old rats during cultivation, the total jejunal RNA was analysed by hybridization using a cDNA clone encoding rat liver alpha 2,6-ST. Under cultivation in both serum-free and serum-containing media jejunal alpha 2,6-ST mRNA closely paralleled the bound (100,000 g pellet) as well as the soluble (100,000 g supernatant) alpha 2,6-ST activity, the correlation coefficients being 0.976 and 0.816, respectively. Dexamethasone (Dx) treatment enhanced alpha 2,6-ST mRNA and membrane-bound alpha 2,6-ST activity in close correlation. Jejunal alpha 2,6-ST mRNA is sensitive to actinomycin D and is lost with apparently identical kinetics in Dx-stimulated and control explants, suggesting that regulation by Dx may be exerted by altering the rate of mRNA synthesis. Dx-dependent activation resulted in elevation of the 4.3-Kb mRNA and can be inhibited by the antiglucocorticoid onapristone, demonstrating the participation of the glucocorticoid receptor pathway.
Assuntos
Dexametasona/farmacologia , Regulação Enzimológica da Expressão Gênica , Glucocorticoides/farmacologia , Jejuno/enzimologia , Sialiltransferases/biossíntese , Ativação Transcricional/efeitos dos fármacos , Animais , Células Cultivadas , Meios de Cultura , Indução Enzimática/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Sialiltransferases/genética , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
The antigestagen-antiglucocorticoid onapristone (ZK 98.299) was tested on three glucocorticoid-sensitive systems after hydrocortisone (HC) administration to suckling male rats, by determining onapristone (ZK)-induced inhibition of HC-provoked (1) increase of activities of intestinal brush-border enzymes, (2) desialylation of brush-border components and (3) thymolysis. HC acetate (75 mg/kg body weight (b.w.)) was injected s.c. on postnatal days 9 and 10, and ZK (150 mg/kg b.w.) on days 9, 10 and 11. The animals were killed on day 12 for assessing the early effect, or on days 15-17 for determining the delayed effect of HC and ZK. In all three systems the glucocorticoid effects were antagonized by ZK. The most sensitive to HC were systems 1 and 3, which exhibited both the early and the delayed effects. The most sensitive to the counteraction of ZK against administered HC was system 1, where HC was antagonized in both its early and delayed effects, whereas only delayed antagonistic action against administered HC was found in system 2. ZK alone had an early inhibitory effect on the activities of several brush-border enzymes and produced an early increase in thymus weight, accompanied by an increased DNA-protein ratio. No delayed effects of ZK alone on the three systems were observed.
Assuntos
Gonanos/farmacologia , Hidrocortisona/farmacologia , Jejuno/enzimologia , Progesterona/antagonistas & inibidores , Timo/efeitos dos fármacos , Animais , Animais Lactentes , DNA/biossíntese , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Masculino , Microvilosidades/efeitos dos fármacos , Microvilosidades/enzimologia , Microvilosidades/metabolismo , Ácido N-Acetilneuramínico , Tamanho do Órgão , Biossíntese de Proteínas , Ratos , Ratos Wistar , Ácidos Siálicos/metabolismoRESUMO
From a database of 133 patients (98 children and 35 adults) who underwent multiple blood sampling for 99Tcm-mercaptoacetyltriglycine (MAG3) clearance, we determined simplified algorithms allowing the estimation of clearance. A one-compartment model with two blood samples was applied. The best choices for the adult population were the 12 and 90 min blood samples, giving a standard error of the estimate (S.E.E.) of less than 10 ml min-1 1.73 m-2; for the children, the 10 and 80 min blood samples gave a S.E.E. of 20 ml min-1 1.73 m-2; for both the adults and the children, the 10 and 70 min blood samples gave the best results with, however, a S.E.E. of 19 ml min-1 1.73 m-2. The use of such a combined algorithm will therefore result in a degradation of the results in adults, suggesting that a separate algorithm for each group is preferable. We compared the accuracy of the two blood sample method to the one blood sample method based on previously published algorithms for children and adults, respectively. The S.E.E. was significantly lower, in adults as well as in children, using the empirical two blood sample method. This two blood sample method seems potentially useful for routine practice in adult patients. The advantages of using such a method in children is balanced by the practical problems inherent in the need to take a second blood sample during the first 10 min, at a time when the plasma activity is rapidly decreasing.
Assuntos
Coleta de Amostras Sanguíneas , Tecnécio Tc 99m Mertiatida/farmacocinética , Adulto , Criança , Bases de Dados Factuais , Humanos , Taxa de Depuração Metabólica , Análise de Regressão , Tecnécio Tc 99m Mertiatida/sangue , Fatores de TempoRESUMO
The aim of this study was to evaluate the levels of disaccharidase and dipeptidyl peptidase i.v. activities in rat jejunal enterocytes under the influence of long-term germ-free conditions. We found that the brush-border lactase and dipeptidyl peptidase i.v. activities were two to three times higher in 2-month-old germ-free rats in comparison with their conventional counterparts. The highest effect of germ-free condition was observed on lactase activity in 6-month-old and dipeptidyl peptidase i.v. in 2-month-old rats. No difference between germ-free and conventional rats in sucrase and glucoamylase activities was found in 2-month-old rats. The difference develops with increasing age, sucrase activity becoming significantly higher in 6- and 12-month-old rats and glucoamylase in 12-month-old germ-free rats.
Assuntos
Envelhecimento , Vida Livre de Germes , Mucosa Intestinal/enzimologia , Jejuno/enzimologia , Microvilosidades/enzimologia , Animais , Dipeptidil Peptidase 4/metabolismo , Dissacaridases/metabolismo , Feminino , Glucana 1,4-alfa-Glucosidase/metabolismo , Mucosa Intestinal/ultraestrutura , Jejuno/ultraestrutura , Lactase , Masculino , Ratos , Ratos Wistar , Sacarase/metabolismo , beta-Galactosidase/metabolismoRESUMO
Data are summarized about digestion and absorption of carbohydrates, lipids and proteins during mammalian perinatal development including human fetuses. Corresponding with the high fat intake in suckling rats, absorption of triglycerides was found to be approximately 2-3 times higher in suckling than in adult rats. Carnitine contents of the small intestinal mucosa of rats decrease postnatally, reaching adult levels at the time of weaning. Other studies suggested that gluconeogenesis may occur in the small intestine in the neonatal period. The intestinal mucosa of infant rats produces ketones; it was suggested that ketone production is to a large extent due to a breakdown of long-chain fatty acids. Studies dealing with the development of colonic sodium transport in rats are described. Other studies on the developing colon showed that the proximal colon resembles ileum during the early postnatal period. Developmental changes of the "specialization" of intestinal segments are reviewed. In all studies attention is given to the maturative effects of hormones of the adrenal cortex and thyroid gland (88 references).
Assuntos
Sistema Digestório/crescimento & desenvolvimento , Adulto , Animais , Sistema Digestório/embriologia , Fenômenos Fisiológicos do Sistema Digestório , Humanos , RatosRESUMO
1. The action in Onapristone infant male rats displays short-term and delayed effects. 2. Suppression of intestinal brush-border enzymes and increase of thymus mass were observed only immediately after 3-day treatment with Onapristone. After an additional 3 days its effect disappeared. There was no immediate or delayed effect of Onapristone on the desialylation of brush-border enzymes. 3. In the short-term and delayed effects, Onapristone suppressed the HC-provoked induction of several intestinal brush-border enzymes, especially alpha-glycosidases. In the delayed effect the drug also suppressed thymolysis induced by the exogeneously given glucocorticoid, and suppressed the HC-induced desialylation of a brush-border enzyme DP IV, which serves as a marker of the desialylation process. 4. These experiments seem to support a conclusion that the postnatal development of intestinal brush-border enzymes and the development of thymus in infant rats are controlled by endogeneously secreted glucocorticoids. 5. The control of sialylation of intestinal brush-border proteins by endogeneously secreted glucocorticoids during the postnatal development of the rat remains debatable.
Assuntos
Gonanos/farmacologia , Antagonistas de Hormônios/farmacologia , Hidrocortisona/antagonistas & inibidores , Intestino Delgado/efeitos dos fármacos , Ácidos Siálicos/metabolismo , Timo/efeitos dos fármacos , Amilases/metabolismo , Animais , Animais Lactentes , Dipeptidil Peptidase 4/metabolismo , Indução Enzimática/efeitos dos fármacos , Glucana 1,4-alfa-Glucosidase/metabolismo , Glicosilação/efeitos dos fármacos , Hidrocortisona/farmacologia , Intestino Delgado/enzimologia , Intestino Delgado/crescimento & desenvolvimento , Intestino Delgado/ultraestrutura , Lactase , Masculino , Microvilosidades/efeitos dos fármacos , Microvilosidades/enzimologia , Ácido N-Acetilneuramínico , Tamanho do Órgão/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Sacarase/metabolismo , Timo/crescimento & desenvolvimento , beta-Galactosidase/metabolismoRESUMO
The aerobic uptake and metabolism of D-fructose in the rings of the small intestine of conventional and germ-free piglets during development was studied. No accumulation of fructose in the intracellular water against its concentration gradient was found. In both groups of animals the intracellular concentration of fructose decreases with increasing age, being highest in the newborn intestine and the sharptest decrease being observed on the first two days of post-natal life. Lactic acid was found to be the main metabolic product of fructose; a considerable amount of it was formed also from endogenous sources of the small intestine. In the newborn piglet fructose does not affect the formation of lactic acid, on the first two days after birth this effect of fructose is already apparent and more pronounced in germ-free piglets than in conventional ones. Conventional and germ-free animals differ markedly in endogenous lactic acid production. While in the former with increasing age endogenous lactate formation decreases, in the latter it rises after a sharp drop on the first post-natal day.
Assuntos
Frutose/metabolismo , Vida Livre de Germes , Intestino Delgado/metabolismo , Suínos/metabolismo , Animais , Animais Recém-Nascidos , Biotransformação , Técnicas In Vitro , Lactatos/metabolismoRESUMO
The effect of intestinal colonization with Bifidobacterium bifidum (Gram-positive anaerobic bacterium colonizing the intestine of healthy new-born mammals, exhibiting a probiotic effect, protecting the intestinal mucosa against colonization by pathogenic microflora) on enterocyte brush-border enzymes was examined in weaned 23-d- and in 2-month-old gnotobiotic inbred mice and compared with that in corresponding germ-free (GF) and conventional (CV) controls. The two groups of GF mice were associated with human B. bifidum 11 d before the end of the experiment. Specific activity of enterocyte brush-border enzymes--lactase, alkaline phosphatase and gamma-glutamyltranspeptidase was significantly higher in both age groups of GF mice in comparison with CV ones; on the other hand, sucrase and glucoamylase activities were higher in CV mice. Monoassociation with B. bifidum accelerates biochemical maturation of enterocytes resulting in a shift of specific activities of brush-border enzymes between the values found for GF and CV mice. This effect of B. bifidum supplementation was less pronounced for alkaline phosphatase, sucrase, glucoamylase and dipeptidyl peptidase i.v. in immature gut of weaned mice than of 2-month-old ones.
Assuntos
Bifidobacterium , Vida Livre de Germes/fisiologia , Intestinos/microbiologia , Fosfatase Alcalina/análise , Animais , Feminino , Glucana 1,4-alfa-Glucosidase/análise , Intestinos/enzimologia , Lactase , Masculino , Sacarase/análise , beta-Galactosidase/análise , gama-Glutamiltransferase/análiseRESUMO
A nonpathogenic bacterium of external environment possessing remarkable immunomodulatory activity, Bacillus firmus (BF) inactivated with formaldehyde, was given intragastrically to two genetically different mouse strains BALB/c (H-2d) and B10.BR/SnPh (B10.BR, H-2k) reared in conventional (CV) and B10.BR strain also in germ-free (GF) conditions. Repeated intragastric administration of BF (500 micrograms every other day over two weeks, starting at the age of 3 months) significantly enhanced intestinal IgA levels in CV BALB/c mice but did not affect intestinal IgA in CV B10.BR mice. In GF B10.BR mice, IgG levels in sera and intestinal washings increased after BF administration compared to CV B10.BR mice. In CV BALB/c mice, specific activity of enterocyte brush-border enzymes (lactase, gamma-glutamyltransferase, alkaline phosphatase) decreased after BF treatment; sucrase (sucrose alpha-glucosidase) activity was not affected. On the other hand, in B10.BR mice, specific activity of gamma-glutamyltransferase and dipeptidyl peptidase IV were higher after administration of BF in both CV and GF groups relative to untreated controls. The activities of lactase and glucoamylase (glucan 1,4-alpha-glucosidase) were significantly stimulated only in the group of GF B10.BR mice treated with formolized BF. The stimulation of immunoglobulin production after BF treatment was accompanied by changes in the levels of enterocyte brush-border enzymes; this responsiveness to BF treatment was genetically regulated.
Assuntos
Bacillus/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Fosfatase Alcalina/metabolismo , Animais , Dipeptidil Peptidase 4/metabolismo , Enterócitos/enzimologia , Enterócitos/microbiologia , Feminino , Predisposição Genética para Doença , Vida Livre de Germes , Glucana 1,4-alfa-Glucosidase/metabolismo , Imunoglobulina A/biossíntese , Imunoglobulina A/sangue , Imuno-Histoquímica , Mucosa Intestinal/enzimologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Intestinos/enzimologia , Lactase , Camundongos , Camundongos Endogâmicos BALB C , Microvilosidades/enzimologia , Sacarase/metabolismo , beta-Galactosidase/metabolismoRESUMO
Coeliac disease is a human, genetically linked, disorder which develops in gluten-sensitive persons. The aim of this study was to investigate the effect of prolonged feeding of gliadin, a major fraction of gluten, on enzyme activities of enterocyte brush border membrane enzymes in rats, mice and pigs. Brush-border membranes were isolated from mucosal scrapings of the small intestine of 21-d-old rat pups hand-fed with formula milk diet, two-month-old nu/nu and +/+ BALB/c mice and two-month-old piglets fed three times a week starting at birth with high doses of gliadin. Activities of lactase, sucrase and dipeptidyl peptidase IV (DPP IV) were determined. Individual animal models differed in their response to gliadin feeding. In comparison with albumin fed controls the activities of DPP IV and lactase were decreased in rat pups, nu/nu BALB/c mice and piglets. DPP IV activity was mostly affected in the ileum of rats and piglets fed with gliadin starting at birth. On the other hand, lactase and sucrase activities of nu/nu BALB/c mice and piglets decreased to the largest extent in jejunum.
Assuntos
Doença Celíaca/enzimologia , Modelos Animais de Doenças , Gliadina/administração & dosagem , Mucosa Intestinal/enzimologia , Intestino Delgado/enzimologia , Animais , Dipeptidil Peptidase 4/metabolismo , Humanos , Lactase , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microvilosidades/enzimologia , Ratos , Ratos Wistar , Sacarase/metabolismo , Suínos , Fatores de Tempo , beta-Galactosidase/metabolismoRESUMO
The authors studied the separation of glucoamylase and beta-galactosidases from the interstinal mucosa of young rats by affinity chromatography. They tested the following chromatographic materials: p-aminophenyl-beta-D-thioglucoside bound to Sepharose 4B via hexamethylenediamine, gluconate and galactonate bound in different ways to Sepharose 4B and phlorizin bound by an azo-coupling reaction to a spacer attached to Sepharose 4B. The conditions of the adsorption of glycosidases to these materials and their subsequent elution were studied. Using chromatography on Sepharose 4B with a beta-thioglucoside affinant, we succeeded in purifying lactase preparation so that, in electrophoresis on polyacrylamide gel, it formed a single zone identical with 1-naphthyl-beta-glucosidase activity.
Assuntos
Glicosídeo Hidrolases/isolamento & purificação , Cromatografia de Afinidade/métodos , GlucosídeosRESUMO
The presence of antigenic determinants of the following enzymes was detected in enterocytes by the indirect immunofluorescence method: 1. lactase in human biopsy material, 2. sucrase-isomaltase during ontogenesis in the rat. 1. Lactase: The antigenic relationship between rat and human lactase, demonstrated with the isolated enzymes, was utilized for the histochemical localization of human lactase. The indirect immunofluorescence method, using guinea pig antiserum to rat lactase, demonstrated the presence of human lactase in the enterocyte brush border. The usefulness of this method for clinical practice resides in the possibility of detecting enzymatically inactive protein immunologically related to lactase in cases of lactase deficiency, thereby facilitating more detailed classification of these diseases. 2. Sucrase-isomaltase: Guinea pig antiserum to rat sucrase-isomaltase (SI) was prepared. It was used to demonstrate antigenic determinants of the enzyme in the enterocyte brush border of the rat during ontogenesis. Structural SI protein is already present in 3-day-old rats, whereas enzyme activity can first be demonstrated histochemically from the 11th day of life and biochemically, in vitro, not until about the 18th day. We consider that this technique can be used for studying the biogenesis of membrane-bound enzymes.