Glycosidases that convert natural glycosides to bioactive compounds.
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Biotransformation↗
Biomedical subjects
C K Sung
Publications and source records attributed to C K Sung.
Missed injuries in abdominal trauma.
Injuries missed at initial diagnoses or operations have the potential to cause disastrous complications in abdominal trauma patients. The aim of this retrospective study is to assess the causes and the outcomes of missed abdominal injuries. Twelve patients (2%) with missed injuries were identified among 607 abdominal trauma patients operated on from 1985 to 1993. Ten patients were male and two were female. The modes of the trauma were ten blunt injuries (83%) and two stab penetrating injuries (17%). Five cases had delayed operations because of clinical errors in the initial diagnosis. Their causative factors were obscured trauma history (two cases), radiologic misinterpretation (two cases), no reliable radiologic finding (one case), and admission to inappropriate department (one case). Missed injured organs were spleen (two cases), liver (one case), diaphragm (one case), and rectum (one case). Median delayed time was 7 days (3 to 96 days). Another seven cases of injuries were missed at the time of initial operation because of incomplete exploration. Their causative factors were surgical inexperience (two cases), severe peritoneal adhesions (one case), neglected exploration of retroperitoneal hematoma (two cases), underestimated mesocolic vascular injury (one case), and early contraction of the perforating wound with intraoperative hypotension (one case). Missed injured organs were stomach (two cases), duodenum (one case), rectum (one case), pancreas (one case), urinary bladder (one case), and rectosigmoid mesocolon (one case). Median time interval between initial and second operation was 9 days (4 to 32 days). Two patients died of complications directly related to their missed injuries. Major complication and mortality rates of missed injuries were 83 and 17%, respectively. These were significantly higher compared with those (39 and 6.3%, respectively) of detected abdominal injuries. We conclude that missed abdominal injuries can cause high mortality and morbidity, and, therefore, a systematic approach, including careful history taking, complete diagnostic procedure, complete surgical explorations, and early reoperation are mandatory for patients with multiple trauma.
Abdominal Injuries↗
Insulin-like growth factor-1 stimulation of cells induces formation of complexes containing phosphatidylinositol-3-kinase, guanosine triphosphatase-activating protein (GAP), and p62 GAP-associated protein.
The insulin-like growth factor-1 (IGF-1) receptor is structurally related to the insulin receptor and shares common features in receptor signaling. These features include receptor autophosphorylation, phosphorylation of insulin receptor substrate-1, and activation of Ras and phosphatidylinositol-3-kinase (PI3K). Previously, we reported that after insulin treatment of rat HTC cells expressing human insulin receptors, a unique insulin receptor signaling complex was formed that contained the insulin receptor, the p85 subunit of PI3K, GTPase-activating protein (GAP), and p62 GAP-associated protein. In the present study, using wild type HTC cells, we investigated whether the activated IGF-1 receptor also forms a similar signaling complex. To study the proteins present in IGF-1 receptor signaling complexes, we used immunoprecipitation and Western blotting analysis with appropriate antibodies. In response to IGF-1, insulin receptor substrate-1 was tyrosine phosphorylated and formed a complex with the PI3K heterodimer that consists of a p85 regulatory subunit and a p110 catalytic subunit. In addition, a separate complex was formed, consisting of p85, p62 GAP-associated protein and GAP. The p62 in this complex was tyrosine phosphorylated. These studies suggest, therefore, that the IGF-1 receptor, like the insulin receptor, induces the formation of multiple signaling complexes that most likely mediate the proliferative effects of these receptors.
Animals↗
Role of p85 subunit of phosphatidylinositol-3-kinase as an adaptor molecule linking the insulin receptor to insulin receptor substrate 1.
After insulin stimulation of cells, signaling complexes are formed, containing the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), and phosphatidylinositol-3-kinase. To study the nature of these complexes, we employed purified IR, recombinant IRS-1, antibodies to IR and IRS-1, and fusion proteins containing the two SH2 domains of p85. In intact cells, insulin increased tyrosine phosphorylation of both the IR and IRS-1. Both of these proteins were immunoprecipitated with antibodies to p85. Also, fusion proteins containing the two SH2 domains of p85 directly precipitated both the IR and IRS-1. Next, these signaling complexes were reconstituted in vitro with purified IR, recombinant IRS-1, and the two SH2 domains of p85. In the presence of both SH2 domains of p85, the IR associated with IRS-1. Other data, both in intact cells and in vitro, demonstrated that N- and C-terminal SH2 domains of p85 had preferential binding affinities for the IR and IRS-1, respectively. Studies with an IR mutant truncated in the C terminus indicated that the C-terminal phosphotyrosines of the IR play a major role in interacting with the SH2 domains of p85. In conclusion, both in vivo and in vitro data support a role for p85 in directly linking the IR to IRS-1 via its SH2 domains. The formation of these complexes, therefore, may provide a mechanism for the translocation to the plasma membrane of phosphatidylinositol-3-kinase and other molecules that are involved in IR signaling.
Animals↗
Molecular cloning of cDNA encoding human lanosterol synthase.
A cDNA encoding human lanosterol synthase, the enzyme responsible for the backbone formation step in sterol biosynthesis, was cloned by extensive application of PCRs. Five degenerate oligonucleotide primers (139S, 440S, 528A, 575A and 712A) corresponding to the homologous amino acid sequences among the known 2,3-oxidosqualene cyclase(OSC) were designed. PCR with one pair(440S and 528A) of five primers yielded a 285-bp fragment. PCRs with the primers based on the obtained fragment and the degenerate primers (139S and 712A) gave longer fragments. Finally, full nucleotide sequence of cDNA was obtained by a "rapid amplification of cDNA ends" (RACE) method.
Amino Acid Sequence↗
Role of phosphatidylinositol-3-kinase in insulin receptor signaling: studies with inhibitor, LY294002.
In order to study the role of phosphatidylinositol-3-kinase (PI3K) in insulin action, we employed a specific inhibitor of PI3K, LY294002, and measured five biological functions of insulin in mouse 3T3 fibroblasts overexpressing human insulin receptors. LY294002 had no effect on tyrosine phosphorylation of both the insulin receptor beta-subunit and insulin receptor substrate 1 (IRS-1) and did not influence the association of the p85 subunit of PI3K with IRS-1. However, LY294002 partially inhibited insulin stimulated glucose uptake, amino acid uptake and protein synthesis, while it completely inhibited insulin stimulation of DNA synthesis and p70 S6 kinase activation. These data suggest that: 1) PI3K plays a crucial role in various functions of insulin; and 2) there exist multiple signaling pathways (both PI3K dependent and PI3K independent) for the insulin receptor.
3T3 Cells↗
Role of p85 subunit of phosphatidylinositol-3-kinase as an adaptor molecule linking the insulin receptor, p62, and GTPase-activating protein.
After insulin stimulation of rat HTC hepatoma cells overexpressing normal human insulin receptors (IR), an antiserum to the p85 subunit of phosphatidylinositol-3-kinase (PIK) (alpha-p85) immunoprecipitated three major tyrosine-phosphorylated proteins: IR, insulin receptor substrate-1 (IRS-1), and a new 62-kDa protein (p62). Studies with antibodies to GTPase activating protein (alpha-GAP) and p62 GAP-associated protein suggested that p62 was the same as (or closely related to) p62 GAP-associated protein. In order to understand how p62 interacts with p85, we employed: 1) antibodies to the p110 subunit of PIK (alpha-p110); and 2) antiserum to IRS-1. To determine which subunit of PIK (p110 or p85) p62 associates with, we first immunoprecipitated insulin-treated cell lysates with alpha-p110 and subsequently immunoprecipitated with alpha-p85 followed by Western blotting analysis with anti-phosphotyrosine antibody (alpha-PY). In response to insulin, most of the tyrosine-phosphorylated p62 was complexed to p85 alone rather than with the PIK heterodimer. Moreover, p62 was absent in alpha-IRS-1 immunoprecipitates. These data suggest that: 1) p62 GAP-associated protein is tyrosine phosphorylated after insulin stimulation of cells; 2) p62 and IRS-1 form separate complexes with p85; 3) p62-GAP complex may be linked to p85 that is not bound to p110; 4) p85 may serve as an adaptor molecule in insulin receptor signaling, interacting with and regulating other intracellular proteins via SH2 domains.
Animals↗
Deletion of residues 485-599 from the human insulin receptor abolishes antireceptor antibody binding and influences tyrosine kinase activation.
We have studied insulin and antireceptor antibody binding to mutated human insulin receptors deleted of residues 485-599 in the alpha-subunit by site-directed mutagenesis. Both normal and mutated receptors were expressed in rat HTC hepatoma cells. Cells expressing either the normal receptor or the mutated receptor retained the ability to bind insulin. In contrast to the normal receptor, however, the mutated receptor failed to interact with antireceptor alpha-subunit antibodies. The inability of the mutated receptor to interact with various antireceptor antibodies was further documented by photoaffinity labeling studies. In intact HTC cells expressing mutated receptors, basal insulin receptor tyrosine autophosphorylation was 2-fold elevated when compared to cells expressing normal receptors. In these cells, however, the response of this function to insulin was blunted. When receptors were isolated from these cells and assayed for both autophosphorylation and phosphotransferase activities toward the synthetic substrate poly(Glu, Tyr), the response to insulin was also blunted. To study the ability of the mutated receptor to transmembrane signal, insulin stimulation of S6 kinase activity was measured. In cells with mutated receptors, in concert with the insulin receptor kinase data, basal S6 kinase activity was elevated, and the response to insulin was blunted. The data suggest, therefore, that residues 485-599 in the alpha-subunit of the insulin receptor are critical for antireceptor antibody binding, but not for insulin binding. Moreover, these data suggest that residues 485-599 contain a regulatory domain for insulin regulation of receptor beta-subunit functions.
Animals↗
Phosphatidylinositol-3-kinase is a non-tyrosine phosphorylated member of the insulin receptor signalling complex.
In rat HTC cells expressing a large number of human insulin receptors, insulin stimulated phosphatidylinositol-3-kinase (PI-3-kinase) activity. This activity was more effectively immunoprecipitated with anti-phosphotyrosine antibody (alpha-PY) than with anti-insulin receptor antibody (alpha-IR), suggesting that PI-3-kinase was not directly associated with the insulin receptor. alpha-PY immunoprecipitable PI-3 kinase activity, which was regulated by insulin, corresponded to a small pool of the total cellular PI-3-kinase activity. PI-3-kinase was not directly tyrosine phosphorylated by insulin treatment. A comparison of both catalytic activity and content of PI-3-kinase in alpha-PY immunoprecipitates indicated that after insulin treatment PI-3-kinase activity was enhanced by its association with tyrosine phosphorylated proteins. These studies suggest therefore that PI-3-kinase is a non-tyrosine phosphorylated member of the insulin receptor signalling complex.
Animals↗
Regulation of biological functions by an insulin receptor monoclonal antibody in insulin receptor beta-subunit mutants.
We investigated the effects of MA-5, a human-specific monoclonal antibody to the insulin receptor alpha-subunit, on transmembrane signaling in cell lines transfected with and expressing both normal human insulin receptors and receptors mutated in their beta-subunit tyrosine kinase domains. In cell lines expressing normal human insulin receptors, MA-5 stimulated three biological functions: aminoisobutyric acid (AIB) uptake, thymidine incorporation, and S6 kinase activation. Under conditions where these biological functions were stimulated, there was no detectable stimulation of receptor tyrosine kinase. We then combined the use of this monoclonal antibody with cells expressing insulin receptors with mutations in the beta-subunit tyrosine kinase domain; two of ATP binding site mutants V1008 (Gly----Val) and M1030 (Lys----Met) and one triple-tyrosine autophosphorylation site mutant F3 (Tyr----Phe at 1158, 1162, and 1163). In cells expressing V1008 receptors, none of the three biological functions of insulin was stimulated. In cells expressing M1030 receptors, AIB uptake was stimulated to a small, but significant, extent whereas the other two functions were not. In cells expressing F3 receptors, AIB uptake and S6 kinase activation, but not thymidine incorporation, were fully stimulated. The data suggest, therefore, that (1) activation of insulin receptor tyrosine kinase may not be a prerequisite for signaling of all the actions of insulin and (2) there may be multiple signal transduction pathways to account for the biological actions of insulin.
Aminoisobutyric Acids↗
Insulin receptor signaling through non-tyrosine kinase pathways: evidence from anti-receptor antibodies and insulin receptor mutants.
Although there is general agreement that insulin receptor tyrosine kinase activity mediates many of the actions of insulin, two types of studies suggest that non-tyrosine kinase dependent pathways may also exist. First, both monoclonal and polyclonal antibodies to the receptor have been shown to mediate many of insulin's actions with little or no stimulation of receptor kinase. Second, insulin receptor mutants, with reduced or no tyrosine kinase activity, have been shown to mediate several actions of insulin. Non-tyrosine kinase pathways that could signal insulin effects through the insulin receptor include non-covalent activation of G proteins, phospholipase Cs, or docking proteins such as IRS-1. Further studies on the chemical structures of phospholipids and their hydrolysis products involved in insulin action will be required to sort out the underlying mechanisms of insulin action via non-tyrosine kinase dependent pathways.
Animals↗
Monoclonal antibody to the human insulin receptor, but not insulin, stimulates S6 kinase via human insulin receptors mutated at three major tyrosine autophosphorylation sites.
Studies were carried out to examine the role of the major insulin receptor tyrosine autophosphorylation sites in stimulation of S6 kinase activity. For these studies, we employed HTC rat hepatoma cells transfected with and expressing human insulin receptors. In cells transfected with and expressing a large number of normal human insulin receptors (HTC-IR cells), the sensitivity of cells to insulin to stimulate S6 kinase was increased tenfold when compared to untransfected wild type HTC cells (HTC-WT cells). However, in cells transfected with and expressing a large number of mutated human insulin receptors where the tyrosines at three major autophosphorylation sites (1158, 1162, and 1163) were mutated to phenylalanines (HTC-F3 cells), there was no change in insulin sensitivity when compared to HTC-WT cells. We next studied the effect of a human-specific monoclonal antibody to the human insulin receptor, MA-5, on S6 kinase activation. In HTC-WT cells, MA-5 did not interact with endogenous rat insulin receptors and thus did not stimulate S6 kinase. In HTC-IR cells expressing normal human insulin receptors, MA-5 stimulated S6 kinase. Interestingly, MA-5, unlike insulin, was also able to stimulate S6 kinase in HTC-F3 cells expressing mutated receptors. In order to further understand the signaling mechanisms by MA-5 and insulin, two potential intermediate protein kinases were investigated. Neither insulin nor MA-5 appears to activate either microtubule-associated protein 2 (MAP-2) kinase or protein kinase C in these cells. These studies suggest therefore that: 1) insulin and MA-5 may signal S6 kinase activation by independent mechanisms that do not employ either MAP-2 kinase or protein kinase C; and 2) under certain circumstances, S6 kinase appears to be activated by mechanisms that are independent of insulin receptor tyrosine autophosphorylation.
Animals↗
Energy restriction and testosterone implants alter ornithine decarboxylase gene expression in kidney of female rats.
Effects of testosterone and energy restriction (30%) on enzyme activity and mRNA level of ornithine decarboxylase (ODC) were studied in 19-wk-old female Sprague-Dawley rats. Testosterone implantation for 1 wk elicited a sixfold increase in ODC activity and a fourfold increase in ODC mRNA transcripts in the kidney. Energy restriction also increased renal ODC activity. Further, the 56% increase in ODC enzyme activity observed in the testosterone-implanted, energy-restricted group was greater than the 24% increase in the placebo-implanted, energy-restricted group. No changes in renal ODC mRNA levels were observed in the energy-restricted groups. These observations suggest that translational or post-translational mechanism(s) are involved in the greater renal ODC activity in energy-restricted rats.
Animals↗
Transmembrane signalling by insulin via an insulin receptor mutated at tyrosines 1158, 1162, and 1163.
In order to study the role of tyrosine autophosphorylation in insulin receptor signalling, we investigated a mutant human insulin receptor whereby the three major tyrosine autophosphorylation sites at positions 1158, 1162, and 1163 in the receptor beta-subunit were mutated to phenylalanines. When these mutant receptors were expressed in HTC rat hepatoma cells, there was no enhanced beta-subunit autophosphorylation and tyrosine kinase activity. In these cells there was enhanced insulin stimulation of [3H]AIB uptake and [3H]thymidine incorporation when compared to wild type HTC cells. The present study suggests therefore that the presence of the major insulin autophosphorylation sites is not a requirement for insulin stimulation of amino acid transport and mitogenesis.
Aminoisobutyric Acids↗
Cholecystokinin stimulates a specific ribosomal S6 kinase in rat pancreatic acini.
Stimulation of intact rat pancreatic acini with cholecystokinin (CCK) enhances the phosphorylation of the ribosomal protein S6 in a dose-dependent manner with half maximal stimulation at 40 pM and maximal stimulation at 1 nM CCK octapeptide. Soluble cellular extracts contained S6 kinase activity assayed using purified rat pancreatic ribosomes as substrate. Stimulation by CCK of S6 kinase was concentration dependent, being half maximal at 50 pM and maximal at 1 nM CCK. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), an activator of protein kinase C, also increased both S6 phosphorylation in intact acini and soluble S6 kinase activity. In order to determine whether S6 kinase mediated S6 phosphorylation following CCK treatment of acini, two-dimensional phosphopeptide analysis was performed for S6 proteins phosphorylated under various conditions. These data suggest that a specific soluble S6 kinase, the activation of which appears to be directly or indirectly mediated by protein kinase C, is the functional enzyme in intact acini that mediates the action of CCK to increase S6 phosphorylation and may be involved in increased protein synthesis in pancreatic acini treated with CCK.
Animals↗
Monoclonal antibodies mimic insulin activation of ribosomal protein S6 kinase without activation of insulin receptor tyrosine kinase. Studies in cells transfected with normal and mutant human insulin receptors.
The effects of species-specific monoclonal antibodies to the human insulin receptor on ribosomal protein S6 phosphorylation were studied in rodent cell lines transfected with human insulin receptors. First, Swiss mouse 3T3 fibroblasts expressing normal human insulin receptors (3T3/HIR cells) were studied. Three monoclonal antibodies, MA-5, MA-20, and MA-51, activated S6 kinase in these cells but had no effects in untransfected 3T3 cells. Both insulin and MA-5, the most potent antibody, activated S6 kinase in a similar time- and dose-dependent manner. To measure S6 phosphorylation in vivo, 3T3/HIR cells were preincubated with [32P]Pi and treated with insulin and MA-5. Both agents increased S6 phosphorylation, and their tryptic phosphopeptide maps were similar. MA-5 and the other monoclonal antibodies, unlike insulin, failed to stimulate insulin receptor tyrosine kinase activity either in vitro or in vivo. Moreover, unlike insulin, they failed to increase the tyrosine phosphorylation of the endogenous cytoplasmic protein, pp 185. Next, HTC rat hepatoma cells, expressing a human insulin receptor mutant that had three key tyrosine autophosphorylation sites in the beta-subunit changed to phenylalanines (HTC-IR-F3 cells), were studied. In this cell line but not in untransfected HTC cells, monoclonal antibodies activated S6 kinase without stimulating either insulin receptor autophosphorylation or the tyrosine phosphorylation of pp 185. These data indicate, therefore, that monoclonal antibodies can activate S6 kinase and then increase S6 phosphorylation. Moreover, they suggest that activation of receptor tyrosine kinase and subsequent tyrosine phosphorylation of cellular proteins may not be crucial for activation of S6 kinase by the insulin receptor.
Animals↗
Insulin and ribosomal protein S6 kinase in rat pancreatic acini.
Treatment of pancreatic acini from diabetic rats with insulin resulted in a dose-dependent increase in the phosphorylation of ribosomal protein S6 when analyzed by two-dimensional gel electrophoresis. To study the presence of the protein kinase mediating this phosphorylation, soluble extracts of intact acini that had been previously treated with insulin were prepared and assayed for protein kinase activity with rat pancreatic ribosomes as a substrate. Activation of S6 kinase activity, observed in a time-dependent manner, was maximal after 20-30 min and, in a dose-dependent manner, was half-maximal at 1 nM and maximal at 10 nM insulin concentration. Based on cofactor requirements, substrate specificity, and a slow activation of the enzyme, the S6 kinase was distinct from cAMP-dependent, Ca2+-calmodulin-dependent, and Ca2+-phospholipid-dependent protein kinases and protease-activated kinase II. The S6 kinase activated by insulin was highly specific for the ribosomal protein S6 when compared with various substrates, including casein, glycogen synthase, phosphorylase b, phosvitin, histone HIII-S, and histone HVIII-S. Protein S6 phosphorylation in intact acini and activation of the S6 kinase by insulin showed similar dose-response curves, consistent with the S6 kinase being responsible for the protein S6 phosphorylation in intact acini. The comparison of the dose-response curves for S6 phosphorylation and protein synthesis in acini suggests that there is a close correlation between these two insulin actions.
Animals↗
Regulation of protein phosphorylation in pancreatic acini by cyclic AMP-mediated secretagogues: interaction with carbamylcholine.
The effects on protein phosphorylation in mouse pancreatic acini of cyclic AMP-mediated secretagogues and the Ca2+-mediated agonist carbamylcholine were compared. Under the conditions adopted for the study of protein phosphorylation, carbamylcholine (3 microM) stimulated amylase release from pancreatic acini 6-fold, whereas vasoactive intestinal polypeptide (VIP) (100 nM) and the cyclic AMP analogue 8-bromo-cyclic AMP (1 mM) caused little or no increase in secretion. However, VIP and 8-bromo-cyclic AMP, when added in combination with carbamylcholine, potentiated the stimulation of amylase release to 170-180% of that caused by carbamylcholine alone. As assessed by two-dimensional gel electrophoresis, VIP reproduced four of the ten changes in protein phosphorylation elicited by carbamylcholine, these changes being the increased phosphorylation of one soluble protein and the decreased phosphorylation of three soluble proteins. VIP enhanced the carbamylcholine-induced changes in phosphorylation for three proteins. In addition, VIP increased the phosphorylation of a unique protein of Mr 52,000 and pI 5.66 which was not affected by carbamylcholine. All of the effects on protein phosphorylation exerted by VIP in the presence or absence of carbamylcholine were mimicked by 8-bromo-cyclic AMP. Secretin also reproduced most of the changes in protein phosphorylation caused by VIP, although concentrations of secretin of at least 100-fold higher were required to elicit a maximal response. It is concluded that cyclic AMP-mediated secretagogues alter the phosphorylation of a unique protein as well as of several pancreatic proteins affected by carbamylcholine. Moreover, these effects appear to be mediated primarily by VIP-preferring receptors and may be involved in the synergistic action of VIP to promote carbamylcholine-induced amylase release.
8-Bromo Cyclic Adenosine Monophosphate↗