Flightless anchors IQGAP1 and R-ras to mediate cell extension formation and matrix remodeling.

Tractional remodeling of collagen fibrils by fibroblasts requires long cell extensions that mediate fibril alignment. The formation of these cell extensions involves flightless I (FliI), an actin-binding protein that contains a leucine-rich-repeat (LRR), which binds R-ras and may regulate cdc42. We considered that FliI interacts with small GTPases and their regulators to mediate assembly of cell extensions. Mass spectrometry analyses of FliI immunoprecipitates showed abundant Ras GTPase-activating-like protein (IQGAP1), which in immunostained samples colocalized with FliI at cell adhesions. Knockdown of IQGAP1 reduced the numbers of cell extensions and the alignment of collagen fibrils. In experiments using dominant negative mutants, cdc42 activity was required for the formation of short extensions while R-ras was required for the formation of long extensions. Immunoprecipitation of wild-type and mutant constructs showed that IQGAP1 associated with cdc42 and R-ras; this association required the GAP-related domain (1004-1237 aa) of IQGAP1. In cells transfected with FliI mutants, the LRR of FliI, but not its gelsolin-like domains, mediated association with cdc42, R-ras, and IQGAP1. We conclude that FliI interacts with IQGAP1 and co-ordinates with cdc42 and R-ras to control the formation of cell extensions that enable collagen tractional remodeling.

Compartmentalised MAPK pathways.

The mitogen-activated protein kinase (MAPK) pathway provides cells with the means to interpret external signal cues or conditions, and respond accordingly. This cascade regulates many cell functions such as differentiation, proliferation and migration. Through modulation of both the amplitude and duration of MAPK signalling, cells can control their responses to the multiple activators of the pathway. In addition, recent work has highlighted the importance of the cellular compartment from which the signalling occurs. Cells have developed intricate systems that enable them to localise MAPK components to specific subcellular domains in response to a particular stimulus. Consequently, different factors can activate the same kinase in separate locations. Crucial to this ability are molecular scaffolds, which act as signalling modules for MAPKs, confining them to the desired compartment. The participation of the MAPK network in fundamental physiological processes, such as cell proliferation and inflammation, and the derangement of the homeostasis that occurs in disease processes, renders MAPK a highly desirable target for therapeutic intervention. As we enhance our comprehension of scaffolds and other regulatory molecules, novel targets for drug design may be discovered that will afford selective and specific MAPK modulation.

Alteration of calmodulin-protein interactions by a monoclonal antibody to calmodulin.

The effects of specific anti-calmodulin monoclonal antibodies on the conformation and interaction of calmodulin with two enzymes, the insulin receptor tyrosine kinase and casein kinase II, are examined. Addition of the anti-calmodulin antibody 2D1 in vitro augments phosphorylation of calmodulin by rat hepatocyte insulin receptors 4.9 +/- 0.5-fold (n = 7). Nonimmune immunoglobulin has no effect. Maximal phosphorylation is observed at a molar ratio of calmodulin:antibody of approx. 2:1, with higher concentrations of antibody producing lesser enhancement. Increasing Ca2+ concentrations in the physiological range progressively inhibit phosphorylation both in the absence and presence of antibody 2D1. Phosphate is incorporated predominantly on Tyr-99, which is distant from the antibody binding site. Enhancement of casein kinase II-catalyzed calmodulin phosphorylation is also produced by the antibody 2D1, implying that antibody binding induces a change in calmodulin conformation. In contrast, two other anti-calmodulin monoclonal antibodies, 4F4 and 4G2, decrease phosphorylation of calmodulin by both the insulin receptor kinase and casein kinase II. These data indicate that secondary and tertiary structures are important in enzyme-substrate interactions and suggest that the antibodies may be useful in investigating the mechanism of calmodulin function.

Calmodulin as substrate for insulin-receptor kinase. Phosphorylation by receptors from rat skeletal muscle.

Calmodulin is a substrate for insulin-receptor kinase obtained from rat adipocytes and hepatocytes and human placenta. In this study, we demonstrate that insulin stimulates the phosphorylation of calmodulin via insulin receptors partially purified from rat skeletal muscle. Phosphorylation of calmodulin was maximal in the presence of Mg2+ and insulin and the absence of Ca2+. Free-Ca2+ concentrations greater than 0.1 microM progressively inhibited phosphorylation with almost total inhibition at 200 microM Ca2+. Insulin-stimulated phosphorylation of calmodulin was dose dependent and saturable with half-maximal effect obtained at approximately 5 x 10(-10) M insulin. There was an absolute requirement for certain basic proteins, e.g., polylysine or protamine sulfate, to obtain phosphate incorporation into calmodulin. Polylysine stimulated the phosphorylation of calmodulin independently of insulin, but this was increased up to sixfold by the addition of insulin. Phosphate incorporation into calmodulin increased with increasing concentration of the substrate up to a saturating concentration of 2.4 microM. The Km for calmodulin was approximately 0.2 microM. Up to 0.15 mol of phosphate was incorporated per mole of calmodulin with tyrosine the predominant amino acid phosphorylated. The observations that calmodulin is phosphorylated by insulin-receptor kinase from all three classic target organs for insulin confirm that calmodulin is a general substrate for this kinase and suggest that Ca2+ and calmodulin may be components of the insulin-signaling mechanism.

Time to positivity of a rapid bedside assay for cardiac-specific troponin T predicts prognosis in acute coronary syndromes: a Thrombolysis in Myocardial Infarction (TIMI) 11A substudy.

OBJECTIVES: We sought to determine whether the rapid bedside assay for troponin T identified patients at risk for a more complicated hospital stay and a higher rate of adverse clinical events. BACKGROUND: In patients with an acute coronary syndrome, the amount of cardiac-specific troponin T released bears a stoichiometric relation to the extent of myocardial damage. METHODS: In 597 patients with unstable angina or non-Q wave myocardial infarction participating in the Thrombolysis in Myocardial Infarction (TIMI) 11A substudy, a rapid bedside assay and simultaneous quantitative serum measurement for troponin T were obtained at enrollment. RESULTS: The composite end point of the sum of death, nonfatal myocardial infarction or recurrent ischemia through day 14 occurred in 33.6% of patients with a positive assay compared with only 22.5% of patients with a negative assay (p = 0.01). Those patients in whom the rapid assay became positive in < or = 10 min had the highest mortality rate of 4.2% through day 14 compared with 1.1% in those patients who had either a late-appearing positive assay (> 10 min) or a negative assay. The duration of hospital stay in the 116 patients (19%) with a positive rapid assay at enrollment was a median of 5 days compared with only 3 days in the 481 patients (81%) with a negative rapid assay at enrollment (p = 0.002). CONCLUSIONS: A positive rapid assay for troponin T at presentation identifies those patients at risk for higher rates of adverse clinical events and longer, more complicated hospital stays. Stratification of patients by time to development of a positive rapid assay identifies those patients at highest mortality risk.

Prognostic value of cardiac troponin T after noncardiac surgery: 6-month follow-up data.

OBJECTIVES: We sought to evaluate the prognostic significance of cardiac troponin T (TnT) serum levels after noncardiac surgery. BACKGROUND: Cardiac TnT has been found to be marker for myocardial injury, but elevations of TnT are common in patients undergoing noncardiac surgery without clinical evidence of severe ischemia. METHODS: We studied 772 patients who underwent major noncardiac procedures and did not have major cardiovascular complications during their inpatient course. Total serum creatine kinase (CK) and cardiac TnT were measured according to a protocol that included sampling in the recovery room and during the next 2 days. A 6-month follow-up interview was performed for 722 (94%) of the patients. RESULTS: Elevated cardiac TnT and CK-MB results were detected for 92 (12%) and 211 (27%) patients, respectively. During the follow-up period, there were 19 (2.5%) major cardiac complications, including 14 cardiac deaths, 3 nonfatal myocardial infarctions and 2 admissions for unstable angina. Compared with patients with cardiac TnT values < 0.1 ng/ml, patients with elevated TnT had a relative risk for cardiac events of 5.4 (95% confidence interval: 2.2 to 13, p = 0.001), whereas CK-MB was not correlated with postdischarge cardiac events. In multivariate logistic regression analysis adjusting for preoperative clinical and CK-MB data, a cardiac TnT value > 0.1 ng/ml was in independent correlate of cardiac events (adjusted odds ratio 4.6, p < 0.05). This correlation was a function of the relation of elevated TnT levels with postoperative in-hospital congestive heart failure and new sustained arrhythmias, suggesting that elevated postoperative TnT levels detected myocardial ischemia during these clinical events. CONCLUSIONS: We conclude that an abnormal TnT level in patients undergoing noncardiac surgery may be a useful marker of ischemic disease and a predictor of 6-month prognosis.

Thrombin-induced phosphorylation of MARCKS does not alter its interactions with calmodulin or actin.

Myristoylated alanine-rich C kinase substrate (MARCKS) is a calmodulin (CaM)- and actin-binding protein and prominent protein kinase C (PKC) substrate. In vitro phosphorylation of MARCKS by PKC has been shown to induce the release of both CaM and actin, leading to the suggestion that MARCKS may regulate CaM availability during agonist-induced signalling. In support of this hypothesis we previously demonstrated that thrombin-induced MARCKS phosphorylation in endothelial cells (EC) parallels activation of myosin light chain kinase, a CaM-dependent enzyme. To test this theory further, we transfected CHO cells, which normally do not express significant levels of MARCKS, with a MARCKS cDNA. The thrombin-stimulated phosphorylation of myosin light chains and the sensitivity to CaM antagonists in the MARCKS overexpressing cells was the same as that in control CHO cells. MARCKS associated with the actin cytoskeleton in EC was markedly increased upon treatment with the PKC activator, PMA, but only modestly enhanced by thrombin treatment. Similarly, colocalisation of MARCKS with actin was enhanced when the EC were challenged with PMA but not thrombin. These data may be partially explained by PKC-independent phosphorylation of MARCKS in response to thrombin stimulation.

Calmodulin antagonists inhibit insulin-stimulated GLUT4 (glucose transporter 4) translocation by preventing the formation of phosphatidylinositol 3,4,5-trisphosphate in 3T3L1 adipocytes.

It has been previously reported that calmodulin plays a regulatory role in the insulin stimulation of glucose transport. To examine the basis for this observation, we examined the effect of a panel of calmodulin antagonists that demonstrated a specific inhibition of insulin-stimulated glucose transporter 4 (GLUT4) but not insulin- or platelet-derived growth factor (PDGF)-stimulated GLUT1 translocation in 3T3L1 adipocytes. These treatments had no effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate 1 (IRS1). Furthermore, IRS1 or phosphotyrosine antibody immunoprecipitation of phosphatidylinositol (PI) 3-kinase activity was not affected. Despite the marked insulin and PDGF stimulation of PI 3-kinase activity, there was a near complete inhibition of protein kinase B activation. Using a fusion protein of the Grp1 pleckstrin homology (PH) domain with the enhanced green fluorescent protein, we found that the calmodulin antagonists prevented the insulin stimulation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] formation in vivo. Similarly, although PDGF stimulation increased PI 3-kinase activity in in vitro immunoprecipitation assays, there was also no significant formation of PI(3,4,5)P3 in vivo. These data demonstrate that calmodulin antagonists prevent insulin-stimulated GLUT4 translocation by inhibiting the in vivo production of PI(3,4,5)P3 without directly affecting IRS1- or phosphotyrosine-associated PI 3-kinase activity. This phenomenon is similar to that observed for the PDGF stimulation of 3T3L1 adipocytes.

Characteristics of calmodulin phosphorylation by the insulin receptor kinase.

Calmodulin is a substrate for the insulin receptor kinase. The time sequence of events resulting in insulin-stimulated phosphorylation of calmodulin was analyzed at a number of different insulin concentrations using partially purified solubilized insulin receptor preparations from rat adipocytes. The respective insulin concentrations needed to reach half-maximal binding, phosphorylation of the beta-subunit of the insulin receptor, and phosphorylation of calmodulin were 4.5 X 10(-10), 4.3 X 10(-10), and 3.9 X 10(-10) M, respectively. At all insulin concentrations, the time to reach 50% of the maximum (defined as the value obtained at 60 min) occurred in the sequence: insulin binding less than beta-subunit phosphorylation less than calmodulin phosphorylation. Insulin binding and beta-subunit phosphorylation occurred almost immediately, whereas there was a lag phase preceding calmodulin phosphorylation. Although stoichiometry was generally low under routine assay conditions (0.01-0.10 mol phosphate/mol calmodulin), it could be increased 4.3 +/- 0.5-fold (n = 5) by pretreating the calmodulin with 0.1 N NaOH. Insulin-stimulated phosphorylation of calmodulin was exclusively on tyrosine residues. The calmodulin molecule in animals contains only two tyrosine residues, located at positions 99 and 138. The amount of phosphate incorporation into a semisynthetic calmodulin (VU1) which contains only one of these tyrosine residues (tyrosine-138) was half that obtained with porcine or chicken calmodulin. Therefore, insulin, via its receptor kinase, stimulates the phosphorylation of calmodulin; calmodulin can be phosphorylated on both tyrosine residues 99 and 138.

Troponin T as a marker for myocardial ischemia in patients undergoing major noncardiac surgery.

To assess the diagnostic performance of cardiac troponin T as a marker for myocardial injury in patients undergoing major noncardiac surgery, we prospectively collected preoperative and postoperative clinical data, including measurements for creatine kinase (CK), CK-MB, and troponin T for 1,175 patients undergoing major noncardiac surgery. Acute myocardial infarction was diagnosed in 17 patients (1.4%) by a reviewer who was blinded to troponin T data and who used CK-MB and electrocardiographic criteria to define acute myocardial infarction. Other predischarge major cardiac complications were detected for another 17 patients. Troponin T elevations (>0.1 ng/ml) occurred in 87% of patients with and in 16% of patients without myocardial infarction. Among patients without myocardial infarction, troponin T was elevated in 62% of patients with and in 15% of patients without major cardiac complications. Receiver-operating characteristic analysis indicated that troponin T had a performance for the diagnosis of acute myocardial infarction similar to CK-MB, and a significantly better correlation with other major cardiac complications in patients without definitive infarction. Future research should seek to determine the significance of troponin T elevations in patients without complications.

The pathogenesis of type II diabetes mellitus. A polygenic disease.

Diabetes mellitus is the most common endocrine disease, consuming almost 15% of annual health care expenditures in the United States. The focus of this review is on type II (non-insulin-dependent) diabetes mellitus, which accounts for approximately 90% of diabetic patients. The current understanding of the pathogenesis of type II diabetes is multifaceted. Complex defects in both insulin action and insulin secretion produce the metabolic derangements responsible for the disease. The pathophysiology is the result of a combination of polygene defects and environmental factors. The search for the responsible gene or genes is described and several candidate genes are discussed. Diabetogenes are addressed in the context of type II diabetes representing a prototype for the identification of the genetic basis of common diseases.

Radial partition fluorescent immunoassay of thyrotropin. Analytic evaluation and clinical correlation.

The authors evaluated the analytic and clinical performance of a sensitive radial partition fluorescent enzyme immunoassay for thyrotropin (TSH) performed on Stratus and compared it with a nonsensitive radioimmunoassay (RIA) method. Sensitivity of 0.15 mIU/L was obtained, and precision, specificity, and linearity were acceptable. A good correlation was observed between the two assays in samples from 311 hospitalized patients (r = 0.976). Stratus TSH results were outside the reference range for 20% of clinically euthyroid patients (n = 126), and 2.4% had undetectable levels. The clinically hyperthyroid group (n = 11) with the exception of one patient had TSH values below 0.2 mIU/L. Only 39% of hypothyroid patients on thyroid hormone replacement (n = 74) had TSH values in the reference range, with 38% and 23% exhibiting low and high values, respectively. All untreated primary hypothyroid patients (n = 8) had elevated TSH concentrations. The authors conclude that this sensitive TSH assay is useful for diagnosing hyperthyroidism when there is a clinical suspicion but cannot be recommended for thyroid screening in hospitalized patients.

The utility of patient age in evaluating prostate cancer.

The utility of age in examining patients for prostate cancer was assessed. Of the 462 patients in the study, 138 had prostate cancer. The age distribution of the patients with cancer was similar to that found in patients with prostate cancer in the US population, and a correlation between age and the serum prostate-specific antigen (PSA) value was noted (r = .4, P < .002). Selection of reference intervals had a significant effect on test performance. Using an interval of 0 to 4.0 ng/mL, sensitivity of the PSA assay was 90% overall and varied from 78% (patients aged 50-59 years) to 94% (patients aged 70-79 years). In contrast, age-adjusted reference ranges yielded corresponding sensitivities of 84%, 78%, and 88%. With a single, fixed reference range, specificity decreased with advancing patient age (P < .001). This trend was eliminated by adjusting the cutoff in different age groups. In addition, age-adjusted reference ranges improved specificity by 10%, and by using the results of examination of a biopsy specimen as the "gold standard," the total number of patients classified correctly by PSA increased from 226 to 250 (49%-54%). For staging before treatment, patient age, clinical stage, and Gleason score were combined to yield a single probability estimate for organ-confined disease (P < .001). The use of age-adjusted reference ranges is supported by this study, which demonstrates that assay efficiency and specificity improve and sensitivity, although decreased overall, becomes more uniform across age groups. In this patient population, age was useful in determining the probability of organ-confined prostate cancer. Use of this model in clinical decision making should await evaluation in a prospective trial.

The mechanism of insulin action.

Insulin was isolated over 70 years ago, but the intracellular transduction of the insulin signal has not been elucidated. Significant progress has been made, particularly in the last 10 years, with the characterization of the insulin receptor and its intrinsic tyrosine kinase. However, no mechanism has been proposed that accounts for all the actions of insulin. Furthermore, all the mechanisms discussed in this brief overview contain major inadequacies. Despite these gaps in our knowledge, substantial evidence indicates that receptor tyrosine kinase activity is essential for insulin action and multiple pathways almost certainly participate. Hopefully, continued dissection of the signalling pathways will soon yield the mechanism of insulin action.

Predictive value of creatine kinase (CK)-MB for diagnosis of acute myocardial infarction after major noncardiac surgery.

BACKGROUND: The aim of this study was to provide insight into the interpretation of CK-MB data after major noncardiac surgery. METHODS: Some 3,321 patients who underwent major non-emergent noncardiac procedures (orthopedic 31%, intrathoracic 12%, vascular 22%, other 35%) were studied. All patients had at least two CK samples measured postoperatively. RESULTS: Acute myocardial infarction was diagnosed in 43 (1.3%) patients using study criteria including CK-MB and electrocardiographic data. All of the various threshold values of peak CK-MB values and peak CK-MB as a percentage of total CK had poor positive predictive values because of high false positive rates and the low rate of acute myocardial infarction. CONCLUSION: These data demonstrate the need for markers of myocardial injury with greater cardiac specificity after noncardiac surgery.

Haemoglobin A1c analysis in the management of patients with diabetes: from chaos to harmony.

Effective management of patients with diabetes mellitus requires accurate assessments of blood glucose control. The best characterised marker of long term glycaemic control is whole blood haemoglobin A(1c) (HbA(1c)). Published clinical trials have identified quantitative and direct relationships between the HbA(1c) concentration and risks of diabetic microvascular complications. However, in order to practice evidence-based medicine, assays used to measure patient samples should ideally produce values comparable to the assays used in these trials. Numerous assays using chromatographic and immunological detection methods are used around the world. This paper briefly reviews the scientific evolution of HbA(1c) and its analysis, discusses the reasons why HbA(1c) assay standardisation is a challenge, describes the approaches that have been adopted to harmonise HbA(1c) assays, and addresses the current initiatives to standardise HbA(1c) globally. These efforts have established HbA(1c) as an essential component in the management of patients with diabetes mellitus and are likely to lead to the use of HbA(1c) in the screening/diagnosis of diabetes.

The role of scaffold proteins in MEK/ERK signalling.

Signal transduction networks allow cells to recognize and respond to changes in the extracellular environment. All eukaryotic cells have MAPK (mitogen-activated protein kinase) pathways that participate in diverse cellular functions, including differentiation, survival, transformation and movement. Five distinct groups of MAPKs have been characterized in mammals, the most extensively studied of which is the Ras/Raf/MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]/ERK cascade. Numerous stimuli, including growth factors and phorbol esters, activate MEK/ERK signalling. How disparate extracellular signals are translated by MEK/ERK into different cellular functions remains obscure. Originally identified in yeast, scaffold proteins are now recognized to contribute to the specificity of MEK/ERK pathways in mammalian cells. These scaffolds include KSR (kinase suppressor of Ras), beta-arrestin, MEK partner-1, Sef and IQGAP1. Scaffolds organize multiprotein signalling complexes. This targets MEK/ERK to specific substrates and facilitates communication with other pathways, thereby mediating diverse functions. The adaptor proteins regulate the kinetics, amplitude and localization of MEK/ERK signalling, providing an efficient mechanism that enables an individual extracellular stimulus to promote a specific biological response.

Acute coronary ischemia: troponin I and T.

Despite advances in diagnosis and management, ischemic heart disease remains the leading cause of death in the USA. Serum cardiac enzymes, one of the three fundamental criteria for establishing the diagnosis of myocardial infarction, are not specific for cardiac muscle and have a narrow time-window. The recent development of monoclonal antibodies to cardiac troponin I and troponin T has resulted in cardiac-specific assays. Several published studies have documented the utility of troponin proteins in the evaluation of myocardial necrosis. A brief overview of the characteristics and clinical utility of troponin T and I is presented here.

Assessment of the interaction between calmodulin and casein kinase II.

Calmodulin is an in vitro substrate for casein kinase II and is also reported to inhibit casein kinase II activity in rat liver nuclear extracts. Here we demonstrate that in the presence or absence of Ca2+, calmodulin did not significantly alter either in vitro casein kinase II activity or autophosphorylation of its beta-subunit. In contrast, Ca2+ inhibited in a dose-dependent manner both casein kinase II activity and casein kinase II-catalysed calmodulin phosphorylation. These data indicate that calmodulin does not directly inhibit casein kinase II activity, but casein kinase II is sensitive to physiologic Ca2+ concentrations.