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There is evidence that the total cellular content of placental cord blood (PCB) grafts is related to the speed of engraftment,though the total nucleated cell (TNC) dose is not a precise predictor of the time of neutrophil or platelet engraftment. It is important to understand the reasons for the quantitative association and to improve the criteria for selecting PCB grafts by using indices more precisely predictive of engraftment. The posttransplant course of 204 patients who received grafts evaluated for hematopoietic colony-forming cell (CFC) content among 562 patients reported previously were analyzed using univariate and multivariate life-table techniques to determine whether CFC doses predicted hematopoietic engraftment speed and risk for transplant-related events more accurately than the TNC dose. Actuarial times to neutrophil and platelet engraftment were shown to correlate with the cell dose,whether estimated as TNC or CFC per kilogram of recipient's weight. CFC association with the day of recovery of 500 neutrophils/microL,measured as the coefficient of correlation,was stronger than that of the TNC (R = -0.46 and -0.413,respectively). In multivariate tests of speed of platelet and neutrophil engraftment and of probability of posttransplantation events,the inclusion of CFC in the model displaced the significance of the high relative risks associated with TNC. The CFC content of PCB units is associated more rigorously with the major covariates of posttransplantation survival than is the TNC and is,therefore,a better index of the hematopoietic content of PCB grafts. (Blood. 2000;96:2717-2722) View Publication

Incubation of skeletal muscle with 5-aminoimidazole-4carboxamide ribonucleoside (AICAR),a compound that activates 5'-AMP-activated protein kinase (AMPK),has been demonstrated to stimulate glucose transport and GLUT4 translocation to the plasma membrane. In this study,we characterized the AMPK cascade in 3T3-L1 adipocytes and the response of glucose transport to incubation with AICAR. Both isoforms of the catalytic alpha-subunit of AMPK are expressed in 3T3-L1 adipocytes,in which AICAR stimulated AMPK activity in a time- and dose-dependent fashion. AICAR stimulated 2-deoxy-D-glucose transport twofold and reduced insulin-stimulated uptake to 62% of the control transport rate dose-dependently,closely correlating with the activation of AMPK. AICAR also inhibited insulin-stimulated GLUT4 translocation,assessed using the plasma membrane lawn assay. The effects of AICAR on insulin-stimulated glucose transport are not mediated by either adenosine receptors or nitric oxide synthase and are mediated downstream of phosphatidylinositol 3'-kinase stimulation. We propose that in contrast to skeletal muscle,in which AMPK stimulation promotes glucose transport to provide ATP as a fuel,AMPK stimulation inhibits insulin-stimulated glucose transport in adipocytes,inhibiting triacylglycerol synthesis,to conserve ATP under conditions of cellular stress. Investigation of the mode of action of AICAR and AMPK may,therefore,give insight into the mechanism of insulin action. View Publication

The bis-indole indirubin is an active ingredient of Danggui Longhui Wan,a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC(50): 50-100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC(50): 5-50 nm) of an evolutionarily related kinase,glycogen synthase kinase-3beta (GSK-3 beta). Testing of a series of indoles and bis-indoles against GSK-3 beta,CDK1/cyclin B,and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3 beta's ATP binding pocket in a way similar to their binding to CDKs,the details of which were recently revealed by crystallographic analysis. GSK-3 beta,along with CDK5,is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3'-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3'-monoxime also inhibits the in vivo phosphorylation of DARPP-32 by CDK5 on Thr-75,thereby mimicking one of the effects of dopamine in the striatum. Finally,we show that many,but not all,reported CDK inhibitors are powerful inhibitors of GSK-3 beta. To which extent these GSK-3 beta effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3 beta to be described. View Publication

Cyclic AMP (cAMP) and cyclic GMP (cGMP) are key second messengers involved in a multitude of cellular events. From the wealth of synthetic analogs of cAMP and cGMP,only a few have been explored with regard to their therapeutic potential. Some of the first-generation cyclic nucleotide analogs were promising enough to be tested as drugs,for instance N(6),O(2)'-dibutyryl-cAMP and 8-chloro-cAMP (currently in clinical Phase II trials as an anticancer agent). Moreover,8-bromo and dibutyryl analogs of cAMP and cGMP have become standard tools for investigations of biochemical and physiological signal transduction pathways. The discovery of the Rp-diastereomers of adenosine 3',5'-cyclic monophosphorothioate and guanosine 3',5'-cyclic monophosphorothioate as competitive inhibitors of cAMP- and cGMP-dependent protein kinases,as well as subsequent development of related analogs,has proven very useful for studying the molecular basis of signal transduction. These analogs exhibit a higher membrane permeability,increased resistance against degradation,and improved target specificity. Furthermore,better understanding of signaling pathways and ligand/protein interactions has led to new therapeutic strategies. For instance,Rp-8-bromo-adenosine 3',5'-cyclic monophosphorothioate is employed against diseases of the immune system. This review will focus mainly on recent developments in cyclic nucleotide-related biochemical and pharmacological research,but also highlights some historical findings in the field. View Publication

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720,Rottlerin and quercetin were found to inhibit many protein kinases,sometimes much more potently than their presumed targets,and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides,as well as H89,HA1077 and Y 27632,were more selective inhibitors,but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62,PD 98059,U0126,PD 184352,rapamycin,wortmannin,SB 203580 and SB 202190. U0126 and PD 184352,like PD 98059,were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1),and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352,even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays. View Publication

There is no known treatment for fatty liver,a ubiquitous cause of chronic liver disease. However,because it is associated with hyperinsulinemia and insulin-resistance,insulin-sensitizing agents might be beneficial. To evaluate this possibility,insulin-resistant ob/ob mice with fatty livers were treated with metformin,an agent that improves hepatic insulin-resistance. Metformin improved fatty liver disease,reversing hepatomegaly,steatosis and aminotransferase abnormalities. The therapeutic mechanism likely involves inhibited hepatic expression of tumor necrosis factor (TNF) alpha and TNF-inducible factors that promote hepatic lipid accumulation and ATP depletion. These findings suggest a mechanism of action for metformin and identify novel therapeutic targets in insulin-resistant states. View Publication

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The synthetic retinoid 6-[3-(adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437),which can bind to and activate the nuclear retinoic acid receptors beta and gamma (RARbeta/gamma),is a potent inducer of apoptosis in various cancer cell lines. However,this effect was reported to be independent of RARs. In this study,we compared and contrasted the potencies and mechanisms of action of CD437 and several other receptor-selective retinoids in induction of apoptosis and modulation of squamous differentiation in UMSCC22B human head and neck squamous cell carcinoma cell line. CD437 and the structurally related retinoid CD2325 exhibited almost equal potency in inducing apoptosis,whereas several other retinoids failed to induce apoptosis. The RAR-specific pan antagonist AGN193109 failed to suppress CD437-induced apoptosis,indicating that the induction of apoptosis by CD437 was RAR-independent. c-Fos expression was induced by CD437 and CD2325 that induced apoptosis in the cell line but not by other retinoids that failed to induce apoptosis,suggesting a role for c-Fos in CD437-induced apoptosis. At low concentration (0.01 microM),CD437 shared with several other receptor-selective retinoids the ability to suppress the mRNA levels of the squamous differentiation markers Spr1,involucrin,and cytokeratin 1. This effect of CD437 could be blocked by AGN193109. We conclude that CD437 can exert its effects in UMSCC22B human human head and neck squamous cell carcinoma cells by at least two mechanisms: RAR-mediated suppression of squamous differentiation and RAR-independent induction of apoptosis. View Publication

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* The high rate of proliferation required of the bone marrow renders it highly susceptible to the influence of external factors. * Anaemia is the most common haematological abnormality seen in systemic disorders. * In the anaemia of chronic disease,erythropoietin production is reduced and proliferation of erythroid progenitor cells is also impaired; this anaemia can generally be alleviated by correction of the underlying disease process. * The status of the endocrine system must always be considered in evaluation of a normocytic,normochromic anaemia. * Anaemia in infection can be due to host or parasite factors or to the treatment administered. * Anaemia due to malignant disease responds to erythropoietin therapy in many cases; failure to respond is a poor prognostic sign. View Publication

OBJECT In Japan fasudil hydrochloride (HA1077),a protein kinase inhibitor,is widely administered to prevent vasospasm in patients after subarachnoid hemorrhage. The effects of fasudil on experimental spinal cord injury (SCI) were investigated and compared with those obtained using methylprednisolone. METHODS Spinal cord contusion was induced in rats by applying an aneurysm clip extradurally to the spinal cord at T-3 for 1 minute. After injury three groups of rats were treated with intravenously administered saline (control),intraperitoneally administered fasudil (10 mg/kg),or intravenously administered methylprednisolone (four 30 mg/kg injections). Neurological recovery was evaluated periodically over 1 month by using a modified combined behavioral scale and histopathological examination. Leukocyte infiltration near the injury site was evaluated by measuring myeloperoxidase (MPO) activity at 24 hours. Spinal cord blood flow was measured at intervals up to 3 hours after injury by using laser Doppler flowmetry. In rats in the fasudil-treated group significant improvement in modified combined behavioral score was demonstrated at each time point,whereas in the methylprednisolone-treated rats no beneficial effects were shown. In the fasudil-treated group,reduction of traumatic spinal cord damage was evident histologically in the caudal portion of the injured areas,and tissue MPO activity in tissue samples was reduced. Spinal cord blood flow was not significantly different between fasudil-treated and control group rats. CONCLUSIONS Fasudil hydrochloride showed promise of effectiveness in promoting neurological recovery after traumatic SCI. Possible mechanisms of this effect include protein kinase inhibition and decreased infiltration by neutrophils. View Publication