Noradrenergic ß-Adrenoceptor-Mediated Intracellular Molecular Mechanism of Na-K ATPase Subunit Expression in C6 Cells.

Rapid eye movement sleep deprivation-associated elevated noradrenaline increases and decreases neuronal and glial Na-K ATPase activity, respectively. In this study, using C6 cell-line as a model, we investigated the possible intracellular molecular mechanism of noradrenaline-induced decreased glial Na-K ATPase activity. The cells were treated with noradrenaline in the presence or absence of adrenoceptor antagonists, modulators of extra- and intracellular Ca++ and modulators of intracellular signalling pathways. We observed that noradrenaline acting on ß-adrenoceptor decreased Na-K ATPase activity and mRNA expression of the catalytic α2-Na-K ATPase subunit in the C6 cells. Further, cAMP and protein kinase-A mediated release of intracellular Ca++ played a critical role in such decreased α2-Na-K ATPase expression. In contrast, noradrenaline acting on ß-adrenoceptor up-regulated the expression of regulatory ß2-Na-K ATPase subunit, which although was cAMP and Ca++ dependent, was independent of protein kinase-A and protein kinase-C. Combining these with previous findings (including ours) we have proposed a working model for noradrenaline-induced suppression of glial Na-K ATPase activity and alteration in its subunit expression. The findings help understanding noradrenaline-associated maintenance of brain excitability during health and altered states, particularly in relation to rapid eye movement sleep and its deprivation when the noradrenaline level is naturally altered.

The V-ATPase is expressed in the choroid plexus and mediates cAMP-induced intracellular pH alterations.

The cerebrospinal fluid (CSF) pH influences brain interstitial pH and, therefore, brain function. We hypothesized that the choroid plexus epithelium (CPE) expresses the vacuolar H+-ATPase (V-ATPase) as an acid extrusion mechanism in the luminal membrane to counteract detrimental elevations in CSF pH. The expression of mRNA corresponding to several V-ATPase subunits was demonstrated by RT-PCR analysis of CPE cells (CPECs) isolated by fluorescence-activated cell sorting. Immunofluorescence and electron microscopy localized the V-ATPase primarily in intracellular vesicles with only a minor fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na+-independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7.4, the cells failed to display significant concanamycin A-sensitive pHi recovery (i.e., V-ATPase activity). The recovery rate in the absence of Na+ amounted to <10% of the pHi recovery rate observed in the presence of Na+ Recovery of pHi was faster at pHo 7.8 and was abolished at pHo 7.0. The concanamycin A-sensitive pHi recovery was stimulated by cAMP at pH 7.4 in vitro, but intraventricular infusion of the membrane-permeant cAMP analog 8-CPT-cAMP did not result in trafficking of the V-ATPase. In conclusion, we find evidence for the expression of a minor fraction of V-ATPase in the luminal membrane of CPECs. This fraction does not contribute to enhanced acid extrusion at high extracellular pH, but seems to be activated by cAMP in a trafficking-independent manner.

Sensitive detection of intracellular RNA of human telomerase by using graphene oxide as a carrier to deliver the assembly element of hybridization chain reaction.

Since the level of human telomerase RNA (hTR) in tumor cells is higher than that in normal somatic cells, the quantitative assay of hTR is of significant importance in tumor diagnosis. Herein, graphene oxide (GO) was simultaneously exploited as a fluorescence quencher and a carrier of nucleic acid to successfully deliver two hairpin DNA probes of hybridization chain reaction (HCR) into the cancer cell for detecting telomerase RNA based on DNA nanoassembly of HCR. The sticky end of HCR probes could tightly absorb on the surface of GO, resulting in fluorescence quenching of the dye which was tagged at the sticky end of two hairpin probes. When faced with hTR, the fluorescence of DNA probes is subsequently recovered because hTR could trigger HCR to autonomous assembly of a DNA polymer which released from the GO and led to fluorescence recovery. Taking advantage of nucleic acid nanoassembly of HCR, this intracellular HCR strategy creates enormous signal amplification, and enables ultra-sensitive fluorescence imaging of hTR expression. By monitoring fluorescence change, human telomerase RNA could be specifically studied and this method can also be used for detecting single-base mutation. The GO-aided HCR strategy allowed us to sensitively detect hTR in a living cell, which holds great potential for analyzing other low-abundance biomolecules in living cells via HCR.

Mild mitochondrial metabolic deficits by α-ketoglutarate dehydrogenase inhibition cause prominent changes in intracellular autophagic signaling: Potential role in the pathobiology of Alzheimer's disease.

Brain activities of the mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) are reduced in Alzheimer's disease and other age-related neurodegenerative disorders. The goal of the present study was to test the consequences of mild impairment of KGDHC on the structure, protein signaling and dynamics (mitophagy, fusion, fission, biogenesis) of the mitochondria. Inhibition of KGDHC reduced its in situ activity by 23-53% in human neuroblastoma SH-SY5Y cells, but neither altered the mitochondrial membrane potential nor the ATP levels at any tested time-points. The attenuated KGDHC activity increased translocation of dynamin-related protein-1 (Drp1) and microtubule-associated protein 1A/1B-light chain 3 (LC3) from the cytosol to the mitochondria, and promoted mitochondrial cytochrome c release. Inhibition of KGDHC also increased the negative surface charges (anionic phospholipids as assessed by Annexin V binding) on the mitochondria. Morphological assessments of the mitochondria revealed increased fission and mitophagy. Taken together, our results suggest the existence of the regulation of the mitochondrial dynamism including fission and fusion by the mitochondrial KGDHC activity via the involvement of the cytosolic and mitochondrial protein signaling molecules. A better understanding of the link among mild impairment of metabolism, induction of mitophagy/autophagy and altered protein signaling will help to identify new mechanisms of neurodegeneration and reveal potential new therapeutic approaches.

Intracellular ß-glucosidases CEL1a and CEL1b are essential for cellulase induction on lactose in Trichoderma reesei.

Lactose (1,4-O-ß-d-galacto-pyranosyl-d-glucose) induces cellulolytic enzymes in Trichoderma reesei and is in fact one of the most important soluble carbon sources used to produce cellulases on an industrial level. The mechanism underlying the induction is, however, not fully understood. In this study, we investigated the cellular functions of the intracellular ß-glucosidases CEL1a and CEL1b in the induction of cellulase genes by lactose in T. reesei. We demonstrated that while CEL1a and CEL1b were functionally equivalent in mediating the induction, the simultaneous absence of these intracellular ß-glucosidases abolished cbh1 gene expression on lactose. d-Galactose restored the efficient cellulase gene induction in the Δcel1a strain independently of its reductive metabolism, but not in the Δcel1a Δcel1b strain. A further comparison of the transcriptional responses of the Δcel1a Δcel1b strain complemented with wild-type CEL1a or a catalytically inactive CEL1a version and the Δcel1a strain constitutively expressing CEL1a or the Kluyveromyces lactis ß-galactosidase LAC4 showed that both the CEL1a protein and its glycoside hydrolytic activity were indispensable for cellulase induction by lactose. We also present evidence that intracellular ß-glucosidase-mediated lactose induction is further conveyed to XYR1 to ensure the efficiently induced expression of cellulase genes.

Function, therapeutic potential and cell biology of BACE proteases: current status and future prospects.

The ß-site APP cleaving enzymes 1 and 2 (BACE1 and BACE2) were initially identified as transmembrane aspartyl proteases cleaving the amyloid precursor protein (APP). BACE1 is a major drug target for Alzheimer's disease because BACE1-mediated cleavage of APP is the first step in the generation of the pathogenic amyloid-ß peptides. BACE1, which is highly expressed in the nervous system, is also required for myelination by cleaving neuregulin 1. Several recent proteomic and in vivo studies using BACE1- and BACE2-deficient mice demonstrate a much wider range of physiological substrates and functions for both proteases within and outside of the nervous system. For BACE1 this includes axon guidance, neurogenesis, muscle spindle formation, and neuronal network functions, whereas BACE2 was shown to be involved in pigmentation and pancreatic ß-cell function. This review highlights the recent progress in understanding cell biology, substrates, and functions of BACE proteases and discusses the therapeutic options and potential mechanism-based liabilities, in particular for BACE inhibitors in Alzheimer's disease. The protease BACE1 is a major drug target in Alzheimer disease. Together with its homolog BACE2, both proteases have an increasing number of functions within and outside of the nervous system. This review highlights recent progress in understanding cell biology, substrates, and functions of BACE proteases and discusses the therapeutic options and potential mechanism-based liabilities, in particular for BACE inhibitors in Alzheimer disease.

Differential expression of neuroblastoma cellular proteome due to AICD overexpression.

Amyloid-ß protein precursor intracellular domain (AICD), which exerts intracellular effects by interacting with proteins involved in a plethora of biological processes, is a key player behind the pathophysiology of Alzheimer's disease (AD). Keeping in mind that overwhelming presence of AICD would mimic AD-like conditions in neuroblastoma cell lines, we hypothesized alteration in the proteomic expression pattern in these cells in the presence of AICD compared to their normal proteome. The rationale behind the study was to distinguish between symptomatic pathophysiological effects as opposed to any artifactual consequence due to protein overload in the cell lines. Using 2D-DIGE analysis and MALDI-MS identifications in neuro2A (mouse) and SHSY5Y (human) cell lines, we have identified several proteins belonging to different functional classes and involved in several biological pathways including protein folding, cytoskeletal dynamics, metabolism, and stress. Many of these were being upregulated or downregulated due to AICD effects and could be correlated directly with AD phenotypes.

Elevated [Ca2+]i levels occur with decreased calpain activity in aged fibroblasts and their reversal by energy-rich compounds: new paradigm for Alzheimer's disease prevention.

Elevated intracellular Ca2+ levels in the aging brain are widely thought to hyperactivate Ca2+ signaling and Ca2+-dependent enzymes, leading to neuronal death through an excitatory mechanism in Alzheimer's disease (AD). This "Ca2+ overload" hypothesis has been questioned by our theoretical analyses. To better understand the relationship between the "level" and functionality of Ca2+ in aging, in this study we simultaneously measured intracellular Ca2+ transients and calpain activity in cultured human fibroblasts. We found that Ca2+ transitions elicited by bradykinin were indeed overstayed or elevated in levels in old cells but, remarkably, calpain activity was decreased compared to young cells. Also, treating young cells with the energy inhibitor rotenone or with H2O2 recapitulated the Ca2+ overstay and calpain inactivation found in old cells. More importantly, treating old cells with high-energy compounds such as phosphoenol pyruvate or phosphocreatine, which boosted cellular ATP content, reduced the Ca2+ overstay and re-activated calpain. Moreover, Ca2+ levels and calpain activity were dramatically raised in the dying cells killed by detergent. Finally, Ca2+ oscillations induced by low dose of bradykinin in old cells exhibited lower spike frequency, but higher overall levels. Collectively, these results suggest that (a) Ca2+ overload in old cells arises from an inefficient Ca2+ handling system compromised by age-related energy depletion and oxidative stress; and (b) despite elevated levels, the functionality of Ca2+ signaling has diminished in old cells. Thus, the study reinforces the concept that tonic promotion of bioenergetics and Ca2+ signaling function is a reasonable and new paradigm to protect the aging brain cells to prevent AD.

Analysis of intracellular enzyme activity by surface enhanced Raman scattering.

Dysfunctional intracellular enzymatic activity is believed to be an underlying cause of a myriad of diseases. We present the first use of surface enhanced Raman scattering (SERS) as a detection technique capable of reporting intracellular activity of a specific enzyme. Careful choice of reagents allowed the preparation of high resolution cellular activity maps highlighting the specific conversion of the commonly used ELISA reagent 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-Gal), by wild type ß-galactosidase enzymes. Further, through co-addition of X-Gal substrate and inhibitors we were able to demonstrate that intracellular substrate conversion occurred predominantly through an enzymatically specific pathway. The data presented therefore supports the application of SERS probes as sensitive, specific sensors of biochemical activity and demonstrates the use of SERS probes for the first time as beacons capable of high resolution subcellular localisation of native enzymes.

Intracellular transactivation of epidermal growth factor receptor by α1A-adrenoceptor is mediated by phosphatidylinositol 3-kinase independently of activation of extracellular signal regulated kinases 1/2 and serine-threonine kinases in Chinese hamster ovary cells.

Transactivation of epidermal growth factor receptor (EGFR) by α1-adrenoceptor (α1-AR) is implicated in contraction and hypertrophy of vascular smooth muscle (VSM). We examine whether all α1-AR subtypes transactivate EGFR and explore the mechanism of transactivation. Chinese hamster ovary (CHO) cells stably expressing one subtype of α1-AR were transiently transfected with EGFR. The transactivation mechanism was examined both by coexpression of a chimeric erythropoietin (EPO)-EGFR with an extracellular EPO and intracellular EGFR domain, and by pharmacologic inhibition of external and internal signaling routes. All three α1-AR subtypes transactivated EGFR, which was dependent on the increase in intracellular calcium. The EGFR kinase inhibitor AG1478 [4-(3'-chloroanilino)-6,7-dimethoxyquinazoline] abrogated α1A-AR and α1D-AR induced phosphorylation of EGFR, but both the inhibition of matrix metalloproteinases by GM6001 [(R)-N4-hydroxy-N(1)-[(S)-2-(1H-indol-3-yl)-1-methylcarbamoyl-ethyl]-2-isobutyl-succinamide] or blockade of EGFR by cetuximab did not. Stimulation of α1A-AR and α1D-AR also induced phosphorylation of EPO-EGFR chimeric receptors. Moreover, α1A-AR stimulation enhanced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 and serine-threonine kinases (Akt), which were both unaffected by AG1478, indicating that ERK1/2 and Akt phosphorylation is independent of EGFR transactivation. Accordingly, inhibitors of ERK1/2 or Akt did not influence the α1A-AR-mediated EGFR transactivation. Inhibition of calcium/calmodulin-dependent kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3K), and Src, however, did block EGFR transactivation by α1A-AR and α1D-AR. These findings demonstrate that all α1-AR subtypes transactivate EGFR, which is dependent on an intracellular signaling route involving an increase in calcium and activation of CaMKII, PI3K, and Src, but not the of ERK1/2 and Akt pathways.

[Biodegradation of decabromodiphenyl ether by intracellular enzyme obtained from Pseudomonas aeruginosa].

The degradation characteristics of decabromodiphenyl ether (BDE-209) by crude enzyme from Pseudomonas aeruginosa were investigated. The results revealed that the degradation efficiency of the intracellular enzyme excreted from this bacterial strain reached 69.22% after incubation with 1 mg x L(-1) BDE-209 for 12 h. Temperature, pH, enzyme concentration and BDE-209 concentration all influenced the ability of crude enzyme to degrade BDE-209. When the BDE-209 concentration was 1 mg x L(-1), the optimal condition for enzymatic degradation was temperature 30 degrees C and pH 7.5, and the degradation rate increased with increasing enzyme concentration. The degradation process of BDE-209 by intracellular enzyme of the strain conformed to the first-order kinetic model. The highest reaction rate was achieved when the initial concentration of BDE-209 was 1 mg x L(-1) and the half-life of this substrate was 6.9 h. In addition, the biodegradation of BDE-209 can be well described by enzymatic reaction of high concentration substrate inhibition, with a maximum substrate utilization rate of 0.133 mg x (L x h)(-1), a Michaelis-Menten constant of 0.642 mg x L(-1), and an inhibitory constant of 1.558 mg x L(-1), respectively.

GPI-anchored carbonic anhydrase IV displays both intra- and extracellular activity in cRNA-injected oocytes and in mouse neurons.

Soluble cytosolic carbonic anhydrases (CAs) are well known to participate in pH regulation of the cytoplasm of mammalian cells. Membrane-bound CA isoforms--such as isoforms IV, IX, XII, XIV, and XV--also catalyze the reversible conversion of carbon dioxide to protons and bicarbonate, but at the extracellular face of the cell membrane. When human CA isoform IV was heterologously expressed in Xenopus oocytes, we observed, by measuring H(+) at the outer face of the cell membrane and in the cytosol with ion-selective microelectrodes, not only extracellular catalytic CA activity but also robust intracellular activity. CA IV expression in oocytes was confirmed by immunocytochemistry, and CA IV activity measured by mass spectrometry. Extra- and intracellular catalytic activity of CA IV could be pharmacologically dissected using benzolamide, the CA inhibitor, which is relatively slowly membrane-permeable. In acute cerebellar slices of mutant mice lacking CA IV, cytosolic H(+) shifts of granule cells following CO(2) removal/addition were significantly slower than in wild-type mice. Our results suggest that membrane-associated CA IV contributes robust catalytic activity intracellularly, and that this activity participates in regulating H(+) dynamics in the cytosol, both in injected oocytes and in mouse neurons.

Participation of 47C>T SNP (Ala-9Val polymorphism) of the SOD2 gene in the intracellular environment of human peripheral blood mononuclear cells with and without lipopolysaccharides.

The outcome of sepsis occurs due to influence of environmental and genetic factors besides genes variants whose expression support its outcome or not. Oxidative stress is associated to the pathogenicity of sepsis, occurring when there is a reactive species overproduction associated with inflammation. The aim of this study was to characterize the cellular redox status of human peripheral blood mononuclear cells (PBMCs) with either -9Ala (AA) or -9Val (VV) SOD2 genotypes and evaluate their response to oxidative stress induced by lipopolysaccharide (LPS). The PBMCs were isolated from the blood of 30 healthy human volunteers (15 volunteers for each allele) and the following assays were performed: antioxidant enzyme activities (superoxide dismutase; catalase; glutathione peroxidase), total radical-trapping antioxidant parameter, non-enzymatic antioxidant capacity (total antioxidant reactivity), and quantification of conjugated dienes (lipid peroxidation). At basal conditions (i.e., not stimulated by LPS), cells from 47C allele carriers showed higher activities of CAT and SOD, as well as higher TAR compared to 47T allele. However, when 47CC cells were challenged with LPS, we observed a higher shift toward a pro-oxidant state compared to 47TT cells. The CAT activity and lipid peroxidation were increased in cells with both alleles, but SOD activity increased significantly only in 47TT cells. These results demonstrate that SOD2 polymorphisms are associated with different cellular redox environments at both basal and LPS-stimulated states, and identification of this polymorphism may be important for a better understanding of pro-inflammatory conditions.

Cutting edge: NADPH oxidase modulates MHC class II antigen presentation by B cells.

Phagocyte NADPH oxidase plays a key role in pathogen clearance via reactive oxygen species (ROS) production. Defects in oxidase function result in chronic granulomatous disease with hallmark recurrent microbial infections and inflammation. The oxidase's role in the adaptive immune response is not well understood. Class II presentation of cytoplasmic and exogenous Ag to CD4(+) T cells was impaired in human B cells with reduced oxidase p40(phox) subunit expression. Naturally arising mutations, which compromise p40(phox) function in a chronic granulomatous disease patient, also perturbed class II Ag presentation and intracellular ROS production. Reconstitution of patient B cells with a wild-type, but not a mutant, p40(phox) allele restored exogenous Ag presentation and intracellular ROS generation. Remarkably, class II presentation of epitopes from membrane Ag was robust in p40(phox)-deficient B cells. These studies reveal a role for NADPH oxidase and p40(phox) in skewing epitope selection and T cell recognition of self Ag.

Adaptor protein APPL1 couples synaptic NMDA receptor with neuronal prosurvival phosphatidylinositol 3-kinase/Akt pathway.

It is well known that NMDA receptors (NMDARs) can both induce neurotoxicity and promote neuronal survival under different circumstances. Recent studies show that such paradoxical responses are related to the receptor location: the former to the extrasynaptic and the latter to the synaptic. The phosphoinositide 3-kinase (PI3K)/Akt kinase cascade is a key pathway responsible for the synaptic NMDAR-dependent neuroprotection. However, it is still unknown how synaptic NMDARs are coupled with the PI3K/Akt pathway. Here, we explored the role of an adaptor protein-adaptor protein containing pH domain, PTB domain, and leucine zipper motif (APPL1)-in this signal coupling using rat cortical neurons. We found that APPL1 existed in postsynaptic densities and associated with the NMDAR complex through binding to PSD95 at its C-terminal PDZ-binding motif. NMDARs, APPL1, and the PI3K/Akt cascade formed a complex in rat cortical neurons. Synaptic NMDAR activity increased the association of this complex, induced activation of the PI3K/Akt pathway, and consequently protected neurons against starvation-induced apoptosis. Perturbing APPL1 interaction with PSD95 by a peptide comprising the APPL1 C-terminal PDZ-binding motif dissociated the PI3K/Akt pathway from NMDARs. Either the peptide or lentiviral knockdown of APPL1 blocked synaptic NMDAR-dependent recruitment and activation of PI3K/Akt pathway, and consequently blocked synaptic NMDAR-dependent neuroprotection. These results suggest that APPL1 contributes to connecting synaptic NMDARs with the intracellular PI3K/Akt cascade and the downstream prosurvival signaling pathway in rat cortical neurons.

Metformin reverses hexokinase and phosphofructokinase downregulation and intracellular distribution in the heart of diabetic mice.

Diabetes mellitus is characterized by hyperglycemia and its associated complications, including cardiomyopathy. Metformin, in addition to lowering blood glucose levels, provides cardioprotection for diabetic subjects. Glycolysis is essential to cardiac metabolism and its reduction may contribute to diabetic cardiomyopathy. Hexokinase (HK) and phosphofructokinase (PFK), rate-limiting enzymes of glycolysis, are downregulated in cardiac muscle from diabetic subjects, playing a central role on the decreased glucose utilization in the heart of diabetic subjects. Thus, the aim of this study was to determine whether metformin modulates heart HK and PFK from diabetic mice. Diabetes was induced by streptozotocin injection on male Swiss mice, which were treated for three consecutive days with 250 mg/kg metformin before evaluating HK and PFK activity, expression, and intracellular distribution on the heart of these subjects. We show that metformin abrogates the downregulation of HK and PFK in the heart of streptozotocin-induced diabetic mice. This effect is not correlated to alteration on the enzymes' transcription and expression. However, the intracellular distribution of both enzymes is altered in diabetic hearts that show increased activity of the soluble fraction when compared to the particulate fraction. Moreover, this pattern is reversed upon the treatment with metformin, which is correlated with the effects of the drug on the enzymes activity. Altogether, our results support evidences that metformin alter the intracellular localization of HK and PFK augmenting glucose utilization by diabetic hearts and, thus, conferring cardiac protection to diabetic subjects.

Doxorubicin induces the persistent activation of intracellular transglutaminase 2 that protects from cell death.

The activation of transglutaminase 2 (TG2), an enzyme that catalyzes post-translational modifications of proteins, has been implicated in apoptosis, cell adhesion and inflammatory responses. We previously reported that intracellular TG2 is activated under oxidative stress conditions, such as ultraviolet irradiation, ischemia-reperfusion, and hypoxia. In this study, we examined the effect of genotoxic stress on the intracellular activity of TG2 using doxorubicin which generates reactive oxygen species that lead to double-strand breakage of DNA. We demonstrated that doxorubicin elicits the persistent activation of TG2. Doxorubicin-induced TG2 activity was suppressed by treatment with caffeine at the early phase, N-acetylcysteine at the mid-phase, and EGTA at the late phase. However, treatment with a blocking antibody against TGFß or toll-like receptor 2 showed no effect on TG2 activity, indicating that at least three different signaling pathways may be involved in the process of TG2 activation. In addition, using MEF cells defective for TG2 and cells overexpressing an activesite mutant of TG2, we revealed that doxorubicin-induced cell death is inversely correlated with TG2 activity. Our findings indicate that the persistent activation of TG2 by doxorubicin contributes to cell survival, suggesting that the mechanism-based inhibition of TG2 may be a novel strategy to prevent drug-resistance in doxorubicin treatment.

Mutation of a positively charged cytoplasmic motif within CD1d results in multiple defects in antigen presentation to NKT cells.

CD1d is an MHC class I-like molecule that presents glycolipid Ags to types I and II NKT cells. The YxxI motif in the cytoplasmic tail of CD1d contributes to its intracellular localization to the endolysosomal compartment and is important for Ag presentation to type I NKT cells. In this study, we identified the (327-329)RRR motif in CD1d and showed that it is critical for the control of CD1d intracellular trafficking and Ag presentation. The replacement of the arginines in this motif with alanines resulted in the extensive accumulation of CD1d in lysosomes but did not affect the cell surface expression. The defect in its cellular localization was accompanied by defects in Ag presentation to both type I and type II NKT cells. These results demonstrated that the (327-329)RRR motif of CD1d is required for proper cellular distribution of CD1d and optimal Ag presentation to both type I and type II NKT cells.

Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus.

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5mM dopachrome the oxygen consumption rate of TrT on 8mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.

CaMKII is involved in cadmium activation of MAPK and mTOR pathways leading to neuronal cell death.

Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative diseases. Recently, we have shown that Cd elevates intracellular free calcium ion ([Ca(2+) ](i) ) level, leading to neuronal apoptosis partly by activating mitogen-activated protein kinases (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains to be elucidated. In this study, we show that the effects of Cd-elevated [Ca(2+) ](i) on MAPK and mTOR network as well as neuronal cell death are through stimulating phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). This is supported by the findings that chelating intracellular Ca(2+) with 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester or preventing Cd-induced [Ca(2+) ](i) elevation using 2-aminoethoxydiphenyl borate blocked Cd activation of CaMKII. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of MAPK/mTOR pathways and cell death. Furthermore, inhibitors of mTOR (rapamycin), c-Jun N-terminal kinase (SP600125) and extracellular signal-regulated kinase 1/2 (U0126), but not of p38 (PD169316), prevented Cd-induced neuronal cell death in part through inhibition of [Ca(2+) ](i) elevation and CaMKII phosphorylation. The results indicate that Cd activates MAPK/mTOR network triggering neuronal cell death, by stimulating CaMKII. Our findings underscore a central role of CaMKII in the neurotoxicology of Cd, and suggest that manipulation of intracellular Ca(2+) level or CaMKII activity may be exploited for prevention of Cd-induced neurodegenerative disorders.