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Sorbitol dehydrogenase inhibitors have been found to prevent, or alleviate, various secondary complications of diabetes mellitus. In the present study, the effects of nucleosides and nucleotides on the rate of sorbitol oxidation catalyzed by the sheep liver enzyme were studied by steady-state kinetics at pH 7.4. Various such compounds, including ATP and the 2'-deoxy-analogues of ATP, ADP and AMP, reversibly inhibit enzyme activity by formation of enzyme-coenzyme-inhibitor ternary complexes. In each case, no deviations from linearity were seen in the double-reciprocal plots using sorbitol or NAD(+) as the varied substrate and there was a linear relationship between inhibitor concentration and the observed inhibitory effects. Sorbitol was docked into a model of the sheep SDH-NAD(+) complex based upon the structure of the human SDH-NAD(+) holoenzyme. The resulting structure of the ternary complex of sheep SDH, NAD(+) and sorbitol (PMDB ID code PM 0078068) shows that the reactive C-2 hydroxyl group of sorbitol is oriented toward the 4'-position of the nicotinamide moiety of the coenzyme, and that the adjacent primary hydroxyl group of sorbitol interacts with the catalytic zinc. The results indicate that the ribose moiety of the inhibitor structures is an important determinant for the observed effects. Specifically, the 2'-position of the ribose ring exerts an effect with respect to inhibitor potency.
Assuntos
L-Iditol 2-Desidrogenase/antagonistas & inibidores , Fígado/enzimologia , Nucleosídeos/química , Nucleotídeos/química , Ovinos/metabolismo , Animais , Ativação Enzimática , Estabilidade EnzimáticaRESUMO
AIM: The aim of this study was to obtain a classification of peristomal bulging based on findings at ultrasonography in patients with a sigmoid colostomy. METHODS: The patient material comprised 30 men and 33 women. The median age was 69 years (28-90) and the median time between stoma creation and investigation was 68 months (3-426). Any bulging was measured, and the abdominal opening for the stoma bowel was evaluated at the clinical examination. At the ultrasonographic investigation, the patients were first investigated in the supine position to measure the transverse and vertical diameter of the abdominal opening and the thickness of the abdominal muscles. RESULTS: Three types of ultrasonographic findings were identified. In ultra-I, the stoma bowel was completely fixed or showed telescoping-like movement through the abdominal opening. In ultra-II, fatty tissue was prolapsed together with the stoma bowel forming a bend in the subcutaneous tissue. In ultra-III, another bowel segment or fatty tissue passed beside the stoma bowel through the abdominal opening into the abdominal wall. A normal finding without any bulging at the clinical examination was associated with a smaller area and a smaller diameter of the abdominal opening than the area and diameter in patients with a visible peristomal bulging. There was no difference in the thickness of the muscle layer of the abdominal wall between patients with and without bulging. CONCLUSIONS: Ultrasonography can make a dynamic diagnosis of parastomal hernia. In patients with visible peristomal bulging, the area of the abdominal opening is increased but there is no decrease in the thickness of the muscles of the abdominal wall.
Assuntos
Parede Abdominal/diagnóstico por imagem , Colostomia/efeitos adversos , Hérnia Abdominal/diagnóstico por imagem , Estomas Cirúrgicos , Músculos Abdominais/diagnóstico por imagem , Músculos Abdominais/patologia , Parede Abdominal/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Hérnia Abdominal/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Prolapso de Órgão Pélvico/diagnóstico por imagem , Prolapso de Órgão Pélvico/patologia , UltrassonografiaRESUMO
OBJECTIVE: Peristomal bulging caused by hernia or prolapse is common in patients with a sigmoidostomy. It is not known whether and to what extent peristomal bulging influences various daily activities. The purpose of this study was to evaluate the effects of bulging by using a general and disease-specific health scale (Short Health Scale, SHS) and a stoma-specific quality of life (Stoma-QoL) questionnaire in patients with and without peristomal bulging. MATERIAL AND METHODS: Seventy patients with sigmoidostomies were examined to identify peristomal bulging. The mean (SD) age was 71.7 (13.7) years and the patients had had their sigmoidostomies for a mean of 8.1 (7.9) years. Bulging was noticed in 46 patients (66%) while 24 had no bulging. RESULTS: It was found that patients with bulging were at a disadvantage. In the SHS, patients with bulging reported significantly impaired QoL in 3 out of 4 scales regarding symptom load, worry and general sense of well-being. Also, in the Stoma-QoL questionnaire there was a significant difference between patients with and those without bulging. CONCLUSIONS: QoL evaluated with a general and disease-specific instrument (SHS) was significantly impaired in patients with bulging around a sigmoidostomy. The Stoma-QoL questionnaire showed a small but statistically significant difference between patients with and those without bulging but the clinical significance is uncertain. Further studies are required to evaluate the role of some of the individual items in the Stoma-QoL questionnaire.
Assuntos
Colostomia/efeitos adversos , Qualidade de Vida , Estomas Cirúrgicos/patologia , Idoso , Feminino , Humanos , Masculino , Inquéritos e QuestionáriosRESUMO
Using the nonaccelerated murine nephrotoxic nephritis (NTN) as a model of chronic kidney disease (CKD) could provide an easily inducible model that enables a rapid test of treatments. Originally, the NTN model was developed as an acute model of glomerulonephritis, but in this study we evaluate the model as a CKD model and compare CD1 and C57BL/6 female and male mice. CD1 mice have previously showed an increased susceptibility to CKD in other CKD models. NTN was induced by injecting nephrotoxic serum (NTS) and evaluated by CKD parameters including albuminuria, glomerular filtration rate (GFR), mesangial expansion, and renal fibrosis. Both strains showed significant albuminuria on days 2-3 which remained significant until the last time point on days 36-37 supporting dysfunctional filtration also observed by a significantly declined GFR on days 5-6, 15-17, and 34-37. Both strains showed early progressive mesangial expansion and significant renal fibrosis within three weeks suggesting CKD development. CD1 and C57BL/6 females showed a similar disease progression, but female mice seemed more susceptible to NTS compared to male mice. The presence of albuminuria, GFR decline, mesangial expansion, and fibrosis showed that the NTN model is a relevant CKD model both in C57BL/6 and in CD1 mice.
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AIMS: Lamin A/C mutations are generally believed to be associated with a severe prognosis. The aim of this study was to investigate disease expression in three affected families carrying different LMNA missense mutations. Furthermore, the potential molecular disease mechanisms of the mutations were investigated in fibroblasts obtained from mutation carriers. METHODS AND RESULTS: A LMNA-p.Arg216Cys missense mutation was identified in a large family with 36 mutation carriers. Disease expression was unusual with a late onset and a favourable prognosis. Two smaller families with severe disease expression were shown to carry a LMNA-p.Arg471Cys and LMNA-p.Arg471His mutation, respectively. LMNA gene and protein expression was investigated in eight different mutation carriers by quantitative reverse transcriptase polymerase chain reaction, Western blotting, immunohistochemistry, and protein mass spectrometry. The results showed that all mutation carriers incorporated mutated lamin protein into the nuclear envelope. Interestingly, the ratio of mutated to wild-type protein was only 30:70 in LMNA-p.Arg216Cys carriers with a favourable prognosis while LMNA-p.Arg471Cys and LMNA-p.Arg471His carriers with a more severe outcome expressed significantly more of the mutated protein by a ratio of 50:50. CONCLUSION: The clinical findings indicated that some LMNA mutations may be associated with a favourable prognosis and a low risk of sudden death. Protein expression studies suggested that a severe outcome was associated with the expression of high amounts of mutated protein. These findings may prove to be helpful in counselling and risk assessment of LMNA families.
Assuntos
DNA/genética , Predisposição Genética para Doença , Insuficiência Cardíaca/genética , Lamina Tipo A/genética , Mutação de Sentido Incorreto , Miocárdio/patologia , Adolescente , Adulto , Idoso , Western Blotting , Células Cultivadas , Análise Mutacional de DNA , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Genótipo , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/metabolismo , Humanos , Imuno-Histoquímica , Lamina Tipo A/metabolismo , Masculino , Pessoa de Meia-Idade , Miocárdio/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
In contrast to the prevalent view in the literature hitherto, the present study shows that pancreatic trypsin can activate human promatrix metalloproteinase-2 (proMMP-2). It is shown that trypsin's ability to activate proMMP-2 is dependent on various environmental factors such as the level of exogenously added Ca(2+) and Brij-35, temperature, as well as trypsin concentration. The activation occurred as a sequential processing of the proenzyme, initially generating an active 62kDa species. This was followed by successive truncation of the C-terminal domain, giving rise to active species of 56kDa, 52kDa and 50kDa. Tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) prevented the trypsin-mediated C-terminal truncation, without affecting the generation of the 62kDa species, while the presence of EDTA increased the rate of the trypsin-mediated activation of proMMP-2. MALDI-TOF MS analysis of the 50kDa form indicated that trypsin generated active forms with either Lys87 or Trp90 as the N-terminal residue and Arg538 as a C-terminal residue. The trypsin-activated MMP-2 was active in solution against both synthetic and physiologic substrates, and the steady-state kinetic coefficients k(cat), K(m) and k(cat)/K(m) were determined for the enzyme activated either by APMA, membrane-type 1 matrix metalloproteinase (MT1-MMP) or trypsin. The trypsin-activated MMP-2 exhibited slightly lower k(cat) and k(cat)/K(m) values as well as a slightly higher K(i) value against TIMP-1 compared to the enzyme activated by APMA or MT1-MMP. Docking studies of TIMP-1 revealed that the slightly weaker binding of the inhibitor to the trypsin-activated MMP-2 could be attributed to its shorter N terminus (Lys87/Trp90 versus Tyr81), as Phe83 and Arg86 interacted directly with the inhibitor. Our results suggest that the trypsin-activated MMP-2 possesses the catalytic and regulatory potential to be of significance in vivo.
Assuntos
Precursores Enzimáticos/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Pâncreas/enzimologia , Tripsina/metabolismo , Alquilantes/farmacologia , Cálcio/metabolismo , Detergentes/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Ponto Isoelétrico , Isoenzimas , Cinética , Pâncreas/metabolismo , Polidocanol , Polietilenoglicóis/farmacologia , Estrutura Terciária de Proteína , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Titulometria , Tosilfenilalanil Clorometil Cetona/farmacologiaRESUMO
We here report molecular investigations of a missense mutation in the HSPE1 gene encoding the HSP10 subunit of the HSP60/ HSP10 chaperonin complex that assists protein folding in the mitochondrial matrix. The mutation was identified in an infant who came to clinical attention due to infantile spasms at 3 months of age. Clinical exome sequencing revealed heterozygosity for a HSPE1 NM_002157.2:c.217C>T de novo mutation causing replacement of leucine with phenylalanine at position 73 of the HSP10 protein. This variation has never been observed in public exome sequencing databases or the literature. To evaluate whether the mutation may be disease-associated we investigated its effects by in vitro and ex vivo studies. Our in vitro studies indicated that the purified mutant protein was functional, yet its thermal stability, spontaneous refolding propensity, and resistance to proteolytic treatment were profoundly impaired. Mass spectrometric analysis of patient fibroblasts revealed barely detectable levels of HSP10-p.Leu73Phe protein resulting in an almost 2-fold decrease of the ratio of HSP10 to HSP60 subunits. Amounts of the mitochondrial superoxide dismutase SOD2, a protein whose folding is known to strongly depend on the HSP60/HSP10 complex, were decreased to approximately 20% in patient fibroblasts in spite of unchanged SOD2 transcript levels. As a likely consequence, mitochondrial superoxide levels were increased about 2-fold. Although, we cannot exclude other causative or contributing factors, our experimental data support the notion that the HSP10-p.Leu73Phe mutation could be the cause or a strong contributing factor for the disorder in the described patient.
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To study the role of the metastasis associated protein S100A4, an osteosarcoma cell line (OHS) with a high level of this protein was transfected with a vector containing a ribozyme that degrades S100A4 mRNA and, as controls, OHS cells were transfected with the vector alone. We have followed up our previous investigation (Bjørnland et al. 1999) by a detailed investigation of these cell lines' synthesis of MMP and TIMP proteins at different cell densities. It is shown that the cell lines with a low S100A4 level produced a reduced amount of immunoreactive MMP-2 at cellular subconfluence, while at confluence there was no difference compared to the control cells. The cell lines with a reduced S100A4 level produced less of the activated form of MMP-2 (62-kDa) and less TIMP-1 than the corresponding control cells, independent of cell density. Isolated cell membranes from cell lines with a reduced S100A4 level contained less MT1-MMP, MMP-2 and TIMP-2 compared to the control cells. Activation of exogenously added proMMP-2 was less effective with the former membrane preparations. It appeared that the mechanism behind the S100A4 dependent activation of proMMP-2 varied with cell density, as SN50, a peptide inhibitor of NF-kappaB nuclear translocation reduced the activation of MMP-2 at low cell density, but had no effect at high cell density. Thus, one of the mechanisms by which S100A4 may exert its effect on metastasis of some tumors is by regulating the MMP-2 activity.
Assuntos
Neoplasias Ósseas/enzimologia , Proteínas de Ligação ao Cálcio/farmacologia , Metaloproteinase 2 da Matriz/biossíntese , Osteossarcoma/enzimologia , Proteínas S100/farmacologia , Membrana Celular/metabolismo , Meios de Cultivo Condicionados , Ativação Enzimática , Humanos , Proteína A4 de Ligação a Cálcio da Família S100 , Inibidores Teciduais de Metaloproteinases/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Like the metastasis-associated protein S100A4, matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in physiological and pathological conditions. Previously, we showed that S100A4 is involved in the regulation of MMPs and TIMPs, and in the present work we have investigated whether the anti-inflammatory and microtubule-disrupting drug colchicine has an effect on the expression of these proteins in osteosarcoma cell lines (OHS) with high and low levels of S100A4. Colchicine treatment of the various OHS cells resulted in an increased expression of MT1-MMP and TIMP-2 mRNA, and a corresponding increase of these two proteins in isolated cell membranes. Colchicine-treated cells produced more of the activated form of MMP-2 than control cells. However, the drug did not affect the amount of MMP-2 and TIMP-1 mRNA or protein, and it reduced the S100A4 mRNA expression. Isolated cell membranes from the colchicine-treated cells were more effective in activating exogenous proMMP-2 than membranes from control cells, and inhibitory studies indicated that it was the colchicine-induced increase in MT1-MMP that caused the increased activation of endogenous MMP-2. A peptide inhibitor of nuclear factor kappaB nuclear translocation, SN50, blocked the colchicine-induced activation of proMMP-2 and reduced the synthesis of MMP-2 in colchicine-treated cells, but not in control cells. It can be concluded that colchicine modulates the expression of MT1-MMP and TIMP-2 and hence the activation of proMMP-2 independently of the S100A4 level in osteosarcoma cells.
Assuntos
Colchicina/farmacologia , Metaloproteinase 2 da Matriz/biossíntese , Osteossarcoma/enzimologia , Proteínas S100/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Indução Enzimática/efeitos dos fármacos , Supressores da Gota/farmacologia , Humanos , NF-kappa B/antagonistas & inibidores , Osteossarcoma/patologia , Peptídeos/farmacologia , Proteína A4 de Ligação a Cálcio da Família S100 , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Células Tumorais CultivadasRESUMO
Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1ß (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1ß proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1ß monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes.
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Ampicillin has been shown to improve glucose tolerance in mice. We hypothesized that this effect is present only if treatment is initiated prior to weaning and that it disappears when treatment is terminated. High-fat fed C57BL/6NTac mice were divided into groups that received Ampicillin at different ages or not at all. We found that both diet and Ampicillin significantly changed the gut microbiota composition in the animals. Furthermore, there was a significant improvement in glucose tolerance in Ampicillin-treated, five-week-old mice compared to nontreated mice in the control group. At study termination, expressions of mRNA coding for tumor necrosis factor, serum amyloid A, and lactase were upregulated, while the expression of tumor necrosis factor (ligand) superfamily member 15 was downregulated in the ileum of Ampicillin-treated mice. Higher dendritic cell percentages were found systemically in high-fat diet mice, and a lower tolerogenic dendritic cell percentage was found both in relation to high-fat diet and late Ampicillin treatment. The results support our hypothesis that a "window" exists early in life in which an alteration of the gut microbiota affects glucose tolerance as well as development of gut immunity and that this window may disappear after weaning.
Assuntos
Ampicilina/uso terapêutico , Glicemia/efeitos dos fármacos , Intolerância à Glucose/prevenção & controle , Obesidade/tratamento farmacológico , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Dieta Hiperlipídica , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/microbiologia , Intolerância à Glucose/sangue , Intolerância à Glucose/imunologia , Teste de Tolerância a Glucose , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Microbiota/efeitos dos fármacos , Obesidade/sangue , Obesidade/etiologia , Obesidade/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/patologiaRESUMO
The citric acid cycle (CAC) metabolite fumarate has been proposed to be cardioprotective; however, its mechanisms of action remain to be determined. To augment cardiac fumarate levels and to assess fumarate's cardioprotective properties, we generated fumarate hydratase (Fh1) cardiac knockout (KO) mice. These fumarate-replete hearts were robustly protected from ischemia-reperfusion injury (I/R). To compensate for the loss of Fh1 activity, KO hearts maintain ATP levels in part by channeling amino acids into the CAC. In addition, by stabilizing the transcriptional regulator Nrf2, Fh1 KO hearts upregulate protective antioxidant response element genes. Supporting the importance of the latter mechanism, clinically relevant doses of dimethylfumarate upregulated Nrf2 and its target genes, hence protecting control hearts, but failed to similarly protect Nrf2-KO hearts in an in vivo model of myocardial infarction. We propose that clinically established fumarate derivatives activate the Nrf2 pathway and are readily testable cytoprotective agents.