Elastase-1 concentration was < 200 mu g/g in 23% of the patients. Chymotrypsin activity was < 6 U/g in 26% of the patients. In 66% of the patients elastase-1 concentration was > 200 mu g/g and chymotrypsin activity > 6U/g. One test was below threshold in 19%, both in 15%. In patients with Type 1 diabetes, the three groups defined by results of elastase-1 concentration and chymotrypsin activity differed with regard to duration of diabetes and prevalence of glutamic acid decarboxylase antibodies, but not BMI or HbA1c, or prevalence of retinopathy, neuropathy, nephropathy or vascular disease. In patients with Type Screening Library clinical trial 2 diabetes, the three

groups differed with regard to BMI, use of insulin and vascular disease, but not known duration. Conclusion Factors associated with pancreatic exocrine failure differ in patients with Type 1 diabetes compared with patients with type 2 diabetes. In patients with Type 2 diabetes, association of decreased pancreatic exocrine function with BMI and vascular disease suggests a role of pancreatic arteriopathy.”
“The objective of the current study was to investigate the regulation of VEGF signaling and tumor angiogenesis by gamma-secretase inhibitor DAPT in glioblastoma. Selleckchem YH25448 Effects of DAPT on VEGFR1, VEGFR2, endothelial cell proliferation and vessel function were evaluated

using mouse microvascular endothelial H5V cell line and U87MG xenograft mouse models. We found that DAPT efficiently inhibited Notch signaling, increased VEGFR2 expression,

but decreased VEGFR1 expression. DAPT treatment enhanced endothelial cell proliferation when used combined with VEGF, but exerted no effect if used alone. In U87MG xenograft mouse models, DAPT treatment increased tumor vessel density but compromised vessel JNJ-26481585 chemical structure function, as evidenced by poor perfusion and aggravated hypoxia. Therefore, DAPT treatment results in an uncoupling of tumor vessel density from vessel function and suppresses glioblastoma growth; disturbance of angiogenesis with DAPT presents a novel therapeutic approach for glioblastoma.”
“PURPOSE. To examine the relationship between the optic disc torsion and peripapillary retinal nerve fiber layer (RNFL) thickness through a comparison with the macular ganglion cell inner plexiform layer complex (GCIPL) thickness measured by Cirrus optical coherence tomography (OCT). METHODS. Ninety-four eyes of 94 subjects with optic disc torsion and 114 eyes of 114 subjects without optic disc torsion were enrolled prospectively. The participants underwent fundus photography and OCT imaging in peripapillary RNFL mode and macular GCIPL mode. The participants were divided into groups according to the presence or absence of optic disc torsion. The eyes with optic disc torsion were further divided into supranasal torsion and inferotemporal torsion groups according to the direction of optic disc torsion.

InvE is normally repressed at 30 degrees C because of decreased messenger RNA (mRNA) stability, but rodZ mutants markedly increase invE-mRNA stability. Importantly, the inhibition of InvE production by RodZ can be genetically separated LY2090314 cost from its role in cell-shape maintenance, indicating that these functions are distinguishable. Thus, we propose that RodZ is a new membrane-bound RNA-binding protein that provides a scaffold for post-transcriptional regulation.”
“Glycerophosphocholines (GPChos) are known to cause matrix ionization effects during the analysis of biological samples (i.e. plasma, urine, etc.) in LC-MS/MS.

In general, such matrix effect is directly related to an insufficient sample clean-up of the biofluids. In addition to GPCho; design of ionization source and/or LC also plays a very important role in matrix effects. In this research paper, different types of matrix effects, i.e. ion suppression or enhancement were observed in differently designed ion sources coupled with different LCs, from the same molecule,

acamprosate (ACM). under the same chromatographic conditions. ACM was analyzed in a negative polarity in electrospray ionization interface using Z-spray and orthogonal spray ion source design. The analyte showed almost complete ion suppression in Selleck IPI 145 the Z-spray ionization source coupled with UPLC/HPLC, whereas there was very little ion enhancement in the orthogonal spray ionization source coupled with HPLC. In both the cases different GPChos were responsible, as evident from the presence of m/z 815.4 in Z-spray ion source and m/z 759.0 in orthogonal spray ion source. Hence, this approach can be used to evaluate the matrix effects in plasma samples during development and validation of LC-MS/MS method of drugs and their metabolites in different biological matrixes. (C) 2012 Elsevier B.V. All rights reserved.”
“Physiological and pathological roles for small non-encoding

miRNAs (nnicroRNAs) ACY-738 research buy in the cardiovascular system have recently emerged and are now widely studied. The discovery of widespread functions of miRNAs has increased the complexity of gene-regulatory processes and networks in both the cardiovascular system and cardiovascular diseases. Indeed, it has recently been shown that miRNAs are implicated in the regulation of many of the steps leading to the development of cardiovascular disease. These findings represent novel aspects in miRNA biology and, therefore, our understanding of the role of these miRNAs during the pathogenesis of cardiovascular disease is critical for the development of novel therapies and diagnostic interventions. The present review will focus on understanding how miRNAs are involved in the onset and development of cardiovascular diseases.”
“Background: Weight loss may improve glucose control in persons with type 2 diabetes. The effects of fat quality, as opposed to quantity, on weight loss are not well understood.

\n\nObjective: The aim of this study was to describe the features and clinical course of APS1 and correlate them with AIRE and HLA class II genotypes in a large homogeneous cohort

of Sardinian patients followed for up to 25 yr.\n\nPatients: Twenty-two pediatric APS1 patients were studied prospectively.\n\nResults: This Sardinian series(female/male ratio, 1.44; median current age, 30.7yr; range, 1.8-46yr) showed early disease onset (age range, 0.3-10 yr; CT99021 manufacturer median, 3.5 yr) and severe phenotype (on average, seven mani-festations per patient). Besides the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism, and Addison’s disease, autoimmune hepatitis was a serious and surprisingly common/early/presenting feature (27%; two deaths), with a 5: 1 female bias (median age, 6 yr; range, 2.5-11 yr). By contrast, type 1 diabetes was rare (one patient), and hypothyroidism was not seen. Additional disease components(several

of them potentially life-threatening) appeared in adulthood. The major BTSA1 in vitro nonsense mutation, R139X, was found in 93% of the mutant AIRE alleles. High-titer interferon (IFN)-omega and IFN-alpha autoantibodies were detected in all patients tested, even preclinically at 4 months of age in one sibling. HLA alleles appear to influence the exact phenotype-the most interesting apparent association being between HLA-DRB1*0301-DQB1* 0201, liver-kidney microsome autoantibodies (anti-CYP1A2), and autoimmune hepatitis.\n\nConclusion: APS1 in Sardinia is characterized by severe phenotype, marked clinical heterogeneity, and relative genetic homogeneity. The single AIRE mutation, R139X, and the anti-IFN-omega and IFN-alpha autoantibodies are helpful for earlier diagnosis, especially when APS1 presents unusually. HLA genotypes can modify the phenotype. (J Clin Endocrinol Metab

97: 1114-1124, 2012)”
“This systematic narrative review of randomised controlled trials (RCTs) identifies and evaluates the efficacy of behaviour-change techniques explicitly aimed at walking in individuals with intermittent claudication. An electronic database search was conducted up SYN-117 to December 2012. RCTs were included comparing interventions incorporating behaviour-change techniques with usual care, walking advice or exercise therapy for increasing walking in people with intermittent claudication. Studies were evaluated using the Cochrane Collaboration risk of bias tool. The primary outcome variable was maximal walking ability at least 3 months after the start of an intervention. Secondary outcome variables included pain-free walking ability, self-report walking ability and daily walking activity. A total of 3,575 records were retrieved. Of these, six RCTs met the inclusion criteria. As a result of substantial heterogeneity between studies, no meta-analysis was conducted.

Importantly, immune reconstitution treatment with IVIg partially restored the CD4 T cell and mDC compartments. Treatment furthermore reduced the levels of CD8 Nutlin-3 purchase T cell activation and mDC activation, whereas levels of Treg cells and iNKT cells remained low. Thus, primary deficiency in humoral immunity with impaired control of microbial infections is associated with significant pathological changes in cell-mediated immunity. Furthermore, therapeutic enhancement of humoral immunity with IVIg infusions alleviates several of these defects, indicating a relationship between poor antibody-mediated immune control

of infections and the occurrence of abnormalities in the T cell and mDC compartments. These findings help our understanding of the immunopathogenesis of primary immunodeficiency, as well as acquired immunodeficiency caused by HIV-1 infection.”
“Background: Depression and anxiety are the most common mood symptoms and psychological consequences of stroke. This study aimed to examine the influence

of acute depression and anxiety symptoms on functional recovery and health-related quality of life (HRQoL) one year after stroke.\n\nMethods: At one month and one year after stroke, the prevalence and severity of depression and anxiety symptoms were this website examined in consecutively admitted patients, using the Hospital Anxiety and Depression Scale (HADS). Functional recovery was assessed using the Nottingham Extended Activities of Daily Living (NEADL) and HRQoL using the Stroke-Specific Quality of Life scale (SSQOL).\n\nResults: In 107 patients, the prevalence of depression and anxiety

symptoms was 35% at one month and 36% and 34%, respectively, at one year. Depression symptoms were significantly associated with functional ability (r = 0.19, p < 0.05) and HRQoL (r = -0.41, p < 0.001) at one year. Anxiety symptoms were significantly associated with HRQoL (r = -0.33, p < 0.001) only. Multivariate analyses indicated that both depression (beta = -0.33, p < 0.001) and anxiety (beta = -0.26, p < 0.01) symptoms explained some KU-55933 cell line variance in HRQoL at one month and did not predict functional recovery or HRQoL at one year, after controlling for other independent variables such as stroke severity and pre-morbid conditions.\n\nDiscussion: Mood symptoms following acute stroke were associated with a poorer HRQoL one year later but only depression symptoms influenced functional recovery. Other clinical factors such as pre-morbid conditions may need to be taken into consideration when determining the effect of mood symptoms on stroke recovery. Copyright (C) 2010 John Wiley & Sons, Ltd.”
“Background and Objective Goal was to evaluate the potential of in vivo optical coherence tomography (OCT) imaging to determine the response of patients with xerostomia to a dry mouth toothpaste versus fluoride toothpaste placebo.

(c) 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.”
“Connexin26 (Cx26) mutation is the most common cause for non-syndromic hereditary deafness. Different congenital Cx26 null mouse models revealed

a profound hearing loss pattern and developmental defect in the cochlea. Our study aimed at establishing a Cx26 knocking down mouse model at different postnatal time points and to investigate the time course and pattern of the hearing loss and cell degeneration in these models. Morphologic changes were observed for 5 months to detect long-term diversities among these models. Depending on the time point when Cx26 expression was reduced, mild to profound hearing loss patterns were found in different groups. Malformed organ of Corti with distinct RG7321 cell loss in middle turn was observed only in early Cx26 reduction group while mice in late Cx26 reduction group developed normal organ of Corti and only suffered a few hair loss in the basal turn. These results indicated that Cx26 may play essential roles in the postnatal maturation of the cochlea, and its role in normal hearing at more mature stage may be replaceable. (C) 2014 Elsevier Inc. All rights reserved.”
“Very late antigen-4 (VLA-4), a member of integrin superfamily, interacts

with its major counter ligand vascular cell adhesion molecule-1 (VCAM-1) and ARN-509 plays an important role in leukocyte adhesion to vascular endothelium and immunological synapse formation. However, irregular

expressions of these proteins may also lead to several autoimmune diseases and metastasis cancer. Thus, quantifying the interaction affinity of the VCAM-1/VLA-4 interaction is of fundamental importance in further understanding the nature of this interaction and drug discovery. In this study, we report an ‘in solution’ steady state organic fluorophore based quantitative fluorescence resonance energy transfer (FRET) assay to quantify this interaction in terms of the dissociation constant (K-d). We have used, in our FRET assay, the Alexa Fluor 488-VLA-4 conjugate as the donor, and Alexa Fluor 546-VCAM-1 as the acceptor. From the FRET signal analysis, K-d of this interaction was determined to be 41.82 +/- 2.36 nM. To further confirm our estimation, we have PXD101 cost employed surface plasmon resonance (SPR) technique to obtain K-d = 39.60 +/- 1.78 nM, which is in good agreement with the result obtained by FRET. This is the first reported work which applies organic fluorophore based ‘in solution’ simple quantitative FRET assay to obtain the dissociation constant of the VCAM-1/VLA-4 interaction, and is also the first quantification of this interaction. Moreover, the value of K-d can serve as an indicator of abnormal protein-protein interactions; hence, this assay can potentially be further developed into a drug screening platform of VLA-4/VCAM-1 as well as other protein-ligand interactions.

These data collectively establish a novel role for the CD70-CD27 axis in human gamma delta T-cell activation and hence open new perspectives for its modulation in clinical settings.”
“In recent years, there has been a great deal of interest in proteasome inhibitors as a novel class of anticancer drugs. We report that fenbendazole (FZ) (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl)carbamate) exhibits a potent growth-inhibitory activity against cancer cell lines but not normal cells. We show here, using fluorogenic

substrates, that FZ treatment leads to the inhibition of proteasomal activity in the cells. Succinyl-Leu-Leu-Val-Tyr-methylcoumarinamide (MCA), benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-MCA, and t-butoxycarbonyl-Gln-Ala-Arg-7-amido-4-MCA click here fluorescent derivatives were used to assess chymotrypsin-like, post-glutamyl peptidyl-hydrolyzing, and trypsin-like protease activities, respectively. Non-small cell lung cancer cells transiently transfected with an expression plasmid encoding selleck products pd1EGFP and treated with FZ showed

an accumulation of the green fluorescent protein in the cells due to an increase in its half-life. A number of apoptosis regulatory proteins that are normally degraded by the ubiquitin-proteasome pathway like cyclins, p53, and I kappa B alpha were found to be accumulated in FZ-treated cells. In addition, FZ induced distinct ER stress-associated genes like GRP78, GADD153, ATF3, IRE1 alpha, and NOXA in these cells. Thus, treatment of human NSCLC cells with fenbendazole induced endoplasmic reticulum stress, reactive oxygen species production, decreased mitochondrial

membrane potential, and cytochrome c release that eventually led to cancer cell death. This is the first report to demonstrate the inhibition of proteasome function and induction of endoplasmic reticulum stress/reactive oxygen species-dependent apoptosis in human lung cancer cell lines by fenbendazole, which may represent a new class of anticancer agents showing selective toxicity against cancer cells.”
“A Merck molecular force field classical potential combined with Poisson-Boltzmann electrostatics (MMFF/PB) has been used to estimate the binding free energy of seven guest molecules (six tertiary amines and one primary amine) into a synthetic receptor (acyclic cucurbit[4]uril congener) www.selleckchem.com/products/BKM-120.html and two benzimidazoles into cyclic cucurbit[7]uril (CB[7]) and cucurbit[8]uril (CB[8]) hosts. In addition, binding enthalpies for the benzimidazoles were calculated with density functional theory (DFT) using the B3LYP functional and a polarizable continuum model (PCM). Although in most cases the MMFF/PB approach returned reasonable agreements with the experiment (+/- 2 kcal/mol), significant, much larger deviations were reported in the case of three host-guest pairs. All four binding enthalpy predictions with the DFT/PCM method suffered 70% or larger deviations from the calorimetry data.

“
“Clostridium difficile was

investigated as a possible cause of enteritis in calves. see more The organism and its toxins (TcdA and TcdB), respectively, were found in 25.3% and 22.9% of stool samples from diarrheic calves. Culture positive samples were more likely than culture negative samples to be toxin positive. However, toxin positive stools were more common among nondiarrheic calves, but diarrheic calves were nearly twice as likely to be culture positive. Ribotype 078 was dominant among isolates. Salmonella sp. was isolated from both diarrheic and nondiarrheic calves, but large numbers of E. coli were found more commonly in diarrheic calves than in nondiarrheic animals. Prevalence rates for coronavirus and Cryptosporidium sp. were substantially higher in nondiarrheic calves than in diarrheic, but rates of detection of rotavirus and Giardia sp. were more nearly equal between groups. Lesions in naturally infected calves included https://www.selleckchem.com/products/BEZ235.html superficial mucosal erosion with associated fibrinous exudates. Neutrophils and eosinophils

infiltrated lamina propria. Large Gram-positive rods morphologically compatible with C. difficile were abundant in the colonic lumen and the organism was isolated by bacteriologic culture. Toxins were found throughout the colon. Purified toxins A and B (individually and conjointly) caused comparable lesions, as well as fluid accumulation, in ligated intestinal loops. Our findings are Geneticin in substantial agreement with those of others [Rodriguez-Palacios, A., Stampfli, H.R., Duffield, T., Peregrine, A.S., Trotz-Williams, L.A., Arroyo, L.G., Brazier, J.S., Weese, J.S., 2006. Clostridium difficile PCR ribotypes in calves, Canada.

Emerg. Infect. Dis. 12, 1730-1736; Porter, M.C., Reggiardo, C., Bueschel, D.M., Keel, M.K., Songer, J.G., 2002. Association of Clostridium difficile with bovine neonatal diarrhea. Proc. 45th Ann. Mtg. Amer. Assoc. Vet. Lab. Diagn., St. Louis, MO, U.S.A.] and add strength to a working hypothesis that C. difficile infection and the accompanying intoxication can manifest as diarrhea in calves. It seems clear that calves serve as multiplying hosts for this organism. (C) 2007 Elsevier B.V. All rights reserved.”
“Background: Age, Injury severity score (ISS), hyperglycemia (HGL) at admission, and morbid obesity are known risk factors of poor outcome in trauma patients. Our aim was to which risk factors had the highest risk of death in the critically ill trauma patient.\n\nMethods: A Trauma Registry of the American College of Surgeons database retrospective study was performed at our Level I trauma center from January 2000 to October 2004. Inclusion criteria were age >15 years and >= 3 days hospital stay. Data collected included age, gender, and ISS.

Within Armillaria mellea and

Amanita citrina f. lavendula, we found evidence of interbreeding and recombination. Within G. dichrous and H. flavescens/ chlorophana, hybrids were identified but there was no evidence for F-2 or PARP inhibitor cancer higher progeny in natural populations suggesting that the hybrid fruitbodies might be an evolutionary dead end and that the genetically divergent Mendelian populations from which they were derived are, in fact, different species. The association between ITS haplotype divergence of less than 5% (Armillaria mellea = 2.6% excluding gaps; Amanita citrina f. lavendula = 3.3%) with the presence of putative recombinants and greater than 5% (Gymnopus dichrous LY2835219 in vivo = 5.7%; Hygrogbe flavescens/ chlorophana = 14.1%) with apparent failure of F-1 hybrids to produce F-2 or higher progeny in populations may suggest a correlation between genetic distance and reproductive isolation.to determine the outcome of hybridization events. Within Armillaria mellea and Amanita citrina f. lavendula, we found evidence of interbreeding and recombination. Within G. dichrous and

H. flavescens/ chlorophana, hybrids were identified but there was no evidence for F-2 or higher progeny in natural populations suggesting that the hybrid fruitbodies might be an evolutionary dead end and that the genetically divergent Mendelian populations from which they were derived are, Selleckchem PD-1/PD-L1 Inhibitor 3 in fact, different species. The association between ITS haplotype divergence of less than 5% (Armillaria mellea = 2.6% excluding gaps; Amanita citrina f. lavendula = 3.3%) with the presence of putative recombinants and greater than 5% (Gymnopus dichrous = 5.7%; Hygrogbe flavescens/ chlorophana = 14.1%) with apparent failure of F-1 hybrids to produce F-2 or higher progeny in populations may suggest a correlation between genetic distance and reproductive isolation.”
“The acidosis that accompanies many diseases and pathological conditions can promote osteoclast formation and activation. Acidosis mainly acts on the last phase of osteoclast formation to generate large osteoclasts and promote

bone resorption. There are several acid-sensing mechanisms, among which transient receptor potential (TRP) channels and G protein-related receptors have been focused on. TRPV4 channels appear to be, at least partly, implicated in acidosis-promoted large osteoclast formation. Other TRP channels including TRPV1 and TRPV2 might be components of the acid-sensing machinery. Several reports suggest the involvement of ovarian cancer G protein-coupled receptor 1 (OGR1), a G-protein-related acid sensor, in receptor activator of nuclear factor kappa-B ligand (RANKL) expression via cyclooxygenase-2 (COX-2). On the other hand, acidosis impairs osteoblast differentiation, which is further impeded in the presence of inflammatory cytokines.

All patients required re-interventions in adulthood. Tricuspid valve (TV) (n = 5), pulmonary valve (PV)/conduit (n = 6), and mitral valve (n = 2) replacements were the most frequent re-intervention in the biventricular repair subset. Atrial arrhythmias were present in 80% of the total cohort, the highest

rate among Fontan repairs (n = 7) and biventricular repairs (n = 7). Ventricular arrhythmias occurred in 15% of the cohort.\n\nConclusions: Although limited in number, the adult PA/IVS patients in this series continue to have high rates of morbidity and mortality, with arrhythmias and need for re-operations as the major causes. Patients with biventricular repairs had the highest re-intervention rate in adulthood. While this subset of patients might not be representative of all adult AZD4547 in vitro PA/IVS survivors, continued follow-up at centers with expertise in adult congenital cardiology is recommended for all patients. AZD6094 (c) 2011 Elsevier Ireland Ltd. All rights reserved.”
“Abnormal c-Src expression and activation has been observed in a number of tumors. To determine the therapeutic potential of Src inhibitors for ovarian cancer patients, this study aimed to explore the expression patterns of c-Src and phospho-Src in epithelial ovarian cancer. A total of 82 patients with epithelial ovarian cancer

treated at Sun Yat-sen University Cancer Center from January 1999 to December 2005 were enrolled along with 25 patients with benign ovarian lesions; 20 normal ovarian tissues served as controls. Expression of c-Src and phospho-Src (Tyr416) was examined using immunohistochemistry. Survival analyses were performed using Kaplan-Meier curves. As compared to the control group, a significantly greater proportion of ovarian cancer

tissues were positive for c-Src and phospho-Src expression (P < 0.001). c-Src expression was associated with age, while phospho-Src expression was significantly associated with age, FIGO stage, histology grade, and residual tumor size after surgery (all P < 0.05). BLZ945 solubility dmso The mean survival time was associated with phospho-Src expression, but not with c-Src expression. The mean survival times of patients with phospho-Src-positive tumors were significantly greater than those with phospho-Src-negative tumors (87.4 months, 95 % CI = 74.3-100.5 months and 91.5 months, 95 % CI = 84.7-98.2 months, respectively; P = 0.013). The increased c-Src expression and activation in epithelial ovarian cancer suggests that ovarian cancer patients may benefit from tyrosine kinase inhibitors such as Dasatinib. Activation of c-Src through phosphorylation at Tyr416 may play a role in the early stages of ovarian cancer development, and evaluation of its expression may be a useful prognostic marker of epithelial ovarian cancer.

21) to 65 (FIO(2) = 0.5) to 140 s (FIO(2) = 1.0).\n\nConclusions: Findings from this swine hemorrhagic shock model confirm that FIO(2) and the level of hemorrhagic shock, but not fluid resuscitation, influence the rate of apneic desaturation. A five-fold increase in time until critical oxygen desaturation occurs can be achieved when preoxygenating with 100% oxygen compared with room air, underscoring the importance of adequate preoxygenation

before emergent airway management.”
“Treatment with sigma1 receptor (Sigma1) ligands can inhibit cell proliferation in vitro and tumor growth in vivo. However, the cellular pathways engaged in response to Sigma1 ligand treatment that contribute to these outcomes remain largely undefined. Here, we show that treatment with putative antagonists this website of Sigma1 decreases cell mass. This effect corresponds with repressed 4EGI-1 cap-dependent translation initiation in multiple breast and prostate cancer cell lines. Sigma1 antagonist treatment suppresses phosphorylation of translational regulator proteins p70S6K, 56, and 4E-BP1. RNAi-mediated knockdown of Sigma1 also results in translational repression, consistent with the effects of antagonist treatment. Sigma1 antagonist mediated

translational repression and decreased cell size are both reversible. Together, these data reveal a role for Sigma1 in tumor cell protein synthesis, and demonstrate that small molecule Sigma1 ligands can be used as modulators of protein translation. (c) 2012 Elsevier Torin 2 Inc. All rights reserved.”
“Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) phosphorylate and activate specific downstream protein kinases, including CaMKI, CaMKIV, and 5′-AMP-activated protein kinase, which mediates a variety of Ca2+ signaling cascades. CaMKKs have been shown to undergo autophosphorylation, although their role in enzymatic regulation remains unclear. Here, we found that CaMKK alpha and beta isoforms expressed in nonstimulated transfected COS-7 cells, as well as recombinant CaMKKs expressed in and purified

from Escherichia coli, were phosphorylated at Thr residues. Introduction of a kinase-dead mutation completely impaired the Thr phosphorylation of these recombinant CaMKK isoforms. In addition, wild-type recombinant CaMKKs were unable to transphosphorylate the kinase-dead mutants, suggesting that CaMKK isoforms undergo Ca2+/CaM-independent autophosphorylation in an intramolecular manner. Liquid chromatography tandem mass spectrometry analysis identified Thr(482) in the autoinhibitory domain as one of the autophosphorylation sites in GaMKK beta, but phosphorylation of the equivalent Thr residue (Thr(446)) in the alpha isoforrn was not observed. Unlike CaMKK alpha that has high Ca2+/CaM-dependent activity, wild-type CaMKK beta displays enhanced autonomous activity (Ca2+/CaM-independent activity, 71% of total activity).