70 resultados para Substrate boundaries


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The Nuclear factor (NF)-kappaB signalling pathway plays a critical role in the regulation and coordination of a wide range of cellular events such as cell growth, apoptosis and cell differentiation. Activation of the IKK (inhibitor of NF-kappaB kinase) complex is a crucial step and a point of convergence of all known NF-kappaB signalling pathways. To analyse bovine IKKalpha (IKK1), IKKbeta (IKK2) and IKKgamma (or NF-kappaB Essential MOdulator, NEMO) and their substrate IkappaBalpha (Inhibitor of NF-kappaB), the corresponding cDNAs of these molecules were isolated, sequenced and characterized. A comparison of the amino acid sequences with those of their orthologues in other species showed a very high degree of identity, suggesting that the IKK complex and its substrate IkappaBalpha are evolutionarily highly conserved components of the NF-kappaB pathway. Bovine IKKalpha and IKKbeta are related protein kinases showing 50% identity which is especially prominent in the kinase and leucine zipper domains. Co-immunoprecipitation assays and GST-pull-down experiments were carried out to determine the composition of bovine IKK complexes compared to that in human Jurkat T cells. Using these approaches, the presence of bovine IKK complexes harbouring IKKalpha, IKKbeta, NEMO and the interaction of IKK with its substrate IkappaBalpha could be demonstrated. Parallel experiments using human Jurkat T cells confirmed the high degree of conservation also at the level of protein-protein interactions. Finally, a yeast two-hybrid analysis showed that bovine NEMO molecules, in addition to the binding to IKKalpha and IKKbeta, also strongly interact with each other.

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BACKGROUND: Starches are the major source of dietary glucose in weaned children and adults. However, small intestine alpha-glucogenesis by starch digestion is poorly understood due to substrate structural and chemical complexity, as well as the multiplicity of participating enzymes. Our objective was dissection of luminal and mucosal alpha-glucosidase activities participating in digestion of the soluble starch product maltodextrin (MDx). PATIENTS AND METHODS: Immunoprecipitated assays were performed on biopsy specimens and isolated enterocytes with MDx substrate. RESULTS: Mucosal sucrase-isomaltase (SI) and maltase-glucoamylase (MGAM) contributed 85% of total in vitro alpha-glucogenesis. Recombinant human pancreatic alpha-amylase alone contributed <15% of in vitro alpha-glucogenesis; however, alpha-amylase strongly amplified the mucosal alpha-glucogenic activities by preprocessing of starch to short glucose oligomer substrates. At low glucose oligomer concentrations, MGAM was 10 times more active than SI, but at higher concentrations it experienced substrate inhibition whereas SI was not affected. The in vitro results indicated that MGAM activity is inhibited by alpha-amylase digested starch product "brake" and contributes only 20% of mucosal alpha-glucogenic activity. SI contributes most of the alpha-glucogenic activity at higher oligomer substrate concentrations. CONCLUSIONS: MGAM primes and SI activity sustains and constrains prandial alpha-glucogenesis from starch oligomers at approximately 5% of the uninhibited rate. This coupled mucosal mechanism may contribute to highly efficient glucogenesis from low-starch diets and play a role in meeting the high requirement for glucose during children's brain maturation. The brake could play a constraining role on rates of glucose production from higher-starch diets consumed by an older population at risk for degenerative metabolic disorders.

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The most important early pathomechanism in traumatic brain injury (TBI) is alteration of the resting membrane potential. This may be mediated via voltage, or agonist-dependent ion channels (e.g. glutamate-dependent channels). This may result in a consequent increase in metabolism with increased oxygen consumption, in order to try to restore ionic balance via the ATP-dependent pumps. We hypothesize that glutamate is an important agonist in this process and may induce an increase in lactate, potassium and brain tissue CO2, and hence a decrease in brain pH. Further we propose that an increase in lactate is thus not an indicator of anaerobic metabolic conditions as has been thought for many years. We therefore analyzed a total of 85 patients with TBI, Glasgow Coma Scale (GCS) < 8 using microdialysis, brain tissue oxygen, CO2 and pH monitoring. Cerebral blood flow studies (CBF) were performed to test the relationship between regional cerebral blood flow (rCBF) and the metabolic determinants. Glutamate was significantly correlated with lactate (p < 0.0001), potassium (p < 0.0001), brain tissue pH (p = 0.0005), and brain tissue CO2 (p = 0.006). rCBF was inversely correlated with glutamate, lactate and potassium. 44% of high lactate values were observed in brain with tissue oxygen values, above the threshold level for cell damage. These results support the hypothesis of a glutamate driven increase in metabolism, with secondary traumatic depolarization and possibly hyperglycolysis. Further, we demonstrate evidence for lactate production in aerobic conditions in humans after TBI. Finally, when reduced regional cerebral blood flow (rCBF) is observed, high dialysate glutamate, lactate and potassium values are usually seen, suggesting ischemia worsens these TBI-induced changes.

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The detailed mechanistic aspects for the final starch digestion process leading to effective alpha-glucogenesis by the 2 mucosal alpha-glucosidases, human sucrase-isomaltase complex (SI) and human maltase-glucoamylase (MGAM), are poorly understood. This is due to the structural complexity and vast variety of starches and their intermediate digestion products, the poorly understood enzyme-substrate interactions occurring during the digestive process, and the limited knowledge of the structure-function properties of SI and MGAM. Here we analyzed the basic catalytic properties of the N-terminal subunit of MGAM (ntMGAM) on the hydrolysis of glucan substrates and compared it with those of human native MGAM isolated by immunochemical methods. In relation to native MGAM, ntMGAM displayed slower activity against maltose to maltopentose (G5) series glucose oligomers, as well as maltodextrins and alpha-limit dextrins, and failed to show the strong substrate inhibitory "brake" effect caused by maltotriose, maltotetrose, and G5 on the native enzyme. In addition, the inhibitory constant for acarbose was 2 orders of magnitude higher for ntMGAM than for native MGAM, suggesting lower affinity and/or fewer binding configurations of the active site in the recombinant enzyme. The results strongly suggested that the C-terminal subunit of MGAM has a greater catalytic efficiency due to a higher affinity for glucan substrates and larger number of binding configurations to its active site. Our results show for the first time, to our knowledge, that the C-terminal subunit of MGAM is responsible for the MGAM peptide's "glucoamylase" activity and is the location of the substrate inhibitory brake. In contrast, the membrane-bound ntMGAM subunit contains the poorly inhibitable "maltase" activity of the internally duplicated enzyme.

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Human maltase-glucoamylase (MGAM) is one of the two enzymes responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM is anchored to the small-intestinal brush-border epithelial cells and contains two homologous glycosyl hydrolase family 31 catalytic subunits: an N-terminal subunit (NtMGAM) found near the membrane-bound end and a C-terminal luminal subunit (CtMGAM). In this study, we report the crystal structure of the human NtMGAM subunit in its apo form (to 2.0 A) and in complex with acarbose (to 1.9 A). Structural analysis of the NtMGAM-acarbose complex reveals that acarbose is bound to the NtMGAM active site primarily through side-chain interactions with its acarvosine unit, and almost no interactions are made with its glycone rings. These observations, along with results from kinetic studies, suggest that the NtMGAM active site contains two primary sugar subsites and that NtMGAM and CtMGAM differ in their substrate specificities despite their structural relationship. Additional sequence analysis of the CtMGAM subunit suggests several features that could explain the higher affinity of the CtMGAM subunit for longer maltose oligosaccharides. The results provide a structural basis for the complementary roles of these glycosyl hydrolase family 31 subunits in the bioprocessing of complex starch structures into glucose.

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Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIR delta JM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIR delta JM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation.

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OBJECTIVE: Transgression of boundaries in the relationship between physician and patient is commonly studied with patient as victim and physician as transgressor. A recent survey in the U.S. reported that almost 90% of physicians face transgression by patients over one year. Incidents happened mainly through verbal abuse, disregarding privacy, and overly affectionate behavior. Since this incidence seems to be alarmingly high, we were interested to analyze how often general practitioners in Switzerland experience transgression by patients. METHODS: 24% of the members of the Swiss Society of Internal Medicine (SGIM) and of the Swiss Society of General Medicine (SGAM) (n=675/2781) responded to an internet-based survey which asked for experiences of transgression by patients and for physicians' responses to transgression in the last 12 months. RESULTS: 81% of responding physicians experienced transgression over the period of one year. Analyzing the frequency of incidents per physician per year, the most common forms of transgression were 'use of physician's first name' (7.7/y), 'asking personal questions' (1.8/y), 'being verbally abusive' (1.5/y), and 'being overly affectionate' (1.4/y). Calculated incidence of transgression was 3 per 1000 patient contacts. 39% of physicians decided to ignore the incident, 37% discussed the event openly. Transgression led to dismissal of patients in 13% of events. CONCLUSION: Transgression even in mild and modest form is a rare phenomenon in Swiss practices. PRACTICE IMPLICATION: The Swiss data do not suggest that there is a specific risk for Swiss practitioners to be exposed to major transgression for which they should specifically be prepared for example in communication skills trainings.