Safety Assessment of Zinc Salts as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) assessed the safety of 27 inorganic and organometallic zinc salts as used in cosmetic formulations; these salts are specifically of the 2+ (II) oxidation state cation of zinc. These ingredients included in this report have various reported functions in cosmetics, including hair conditioning agents, skin conditioning agents, cosmetic astringents, cosmetic biocides, preservatives, oral care agents, buffering agents, bulking agents, chelating agents, and viscosity increasing agents. The Panel reviewed the relevant data for these ingredients, and concluded that these 27 ingredients are safe in cosmetics in the present practices of use and concentration described in this safety assessment when formulated to be non-irritating.

Safety Assessment of Polyaminopropyl Biguanide (Polyhexamethylene Biguanide Hydrochloride) as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of Polyaminopropyl Biguanide (polyhexamethylene biguanide hydrochloride), which functions as a preservative in cosmetic products. The Panel reviewed relevant data relating to the safety of this ingredient and concluded that Polyaminopropyl Biguanide is safe in cosmetics in the present practices of use and concentration described in the safety assessment, when formulated to be nonirritating and nonsensitizing, which may be based on a quantitative risk assessment or other accepted methodologies. The Panel also concluded that the data are insufficient to determine the safety of Polyaminopropyl Biguanide in products that may be incidentally inhaled.

Amended Safety Assessment of Parabens as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) assessed the safety of 21 parabens as preservatives in cosmetic products. All of these ingredients are reported to function in cosmetics as preservatives; however, 5 are reported to also function as fragrance ingredients. The Panel reviewed relevant data relating to the safety of these ingredients under the reported conditions of use in cosmetic formulations. The Panel concluded that 20 of the 21 parabens included in this report are safe in cosmetics in the present practices of use and concentration described in this safety assessment when the sum of the total parabens in any given formulation does not exceed 0.8%. However, the available data are insufficient to support a conclusion of safety for benzylparaben in cosmetics.

GMDTC Chelating Agent Attenuates Cisplatin-Induced Systemic Toxicity without Affecting Antitumor Efficacy.

Cisplatin is a platinum-based chemotherapeutic drug widely used in the treatment of various cancers such as testicular, ovarian, lung, bladder, and cervical cancers. However, its use and the dosage range applied have been limited by severe side effects (e.g., nephrotoxicity and ototoxicity) and by the development of resistance to cisplatin in patients during treatment. Metal chelators have shown promising potential in overcoming these problems often associated with platinum drugs. Previously, a new chelating agent, sodium (S)-2-(dithiocarboxylato((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)-4(methylthio)butanoate (GMDTC), was developed. In this study, we examined the effect of GMDTC in modifying cisplatin-induced toxicities following in vitro and in vivo exposures. GMDTC treatment dramatically reduced cisplatin-induced apoptosis and cytotoxicity in HK2 cells by decreasing the amount of intracellular platinum. In the 4T1 breast cancer mouse model, GMDTC reduced cisplatin-induced nephrotoxicity by reducing cisplatin deposition in the kidney. GMDTC attenuated cisplatin-induced elevations in blood urea nitrogen and plasma creatinine, ameliorated renal tubular dilation and vacuolation, and prevented necrosis of glomeruli and renal tubular cells. GMDTC also inhibited cisplatin-induced ototoxicity as shown by improved hearing loss which was assessed using the auditory brainstem response test. Furthermore, GMDTC attenuated cisplatin-induced hematotoxicity and hepatotoxicity. Importantly, co-treatment of cisplatin with GMDTC did not affect cisplatin antitumor efficacy. Tumor growth, size, and metastasis were all comparable between the cisplatin only and cisplatin-GMDTC co-treatment groups. In conclusion, the current study suggests that GMDTC reduces cisplatin-induced systemic toxicity by preventing the accumulation and assisting in the removal of intracellular cisplatin, without compromising cisplatin therapeutic activity. These results support the development of GMDTC as a chemotherapy protector and rescue agent to overcome the toxicity of and resistance to platinum-based antineoplastic drugs.

Mapping dynamic histone modification patterns during arsenic-induced malignant transformation of human bladder cells.

Arsenic is a known potent risk factor for bladder cancer. Increasing evidence suggests that epigenetic alterations, e.g., DNA methylation and histones posttranslational modifications (PTMs), contribute to arsenic carcinogenesis. Our previous studies have demonstrated that exposure of human urothelial cells (UROtsa cells) to monomethylarsonous acid (MMAIII), one of arsenic active metabolites, changes the histone acetylation marks across the genome that are correlated with MMAIII-induced UROtsa cell malignant transformation. In the current study, we employed a high-resolution and high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify and quantitatively measure various PTM patterns during the MMAIII-induced malignant transformation. Our data showed that MMAIII exposure caused a time-dependent increase in histone H3 acetylation on lysine K4, K9, K14, K18, K23, and K27, but a decrease in acetylation on lysine K5, K8, K12, and K16 of histone H4. Consistent with this observation, H3K18ac was increased while H4K8ac was decreased in the leukocytes collected from people exposed to high concentrations of arsenic compared to those exposed to low concentrations. MMAIII was also able to alter histone methylation patterns: MMAIII transformed cells experienced a loss of H3K4me1, and an increase in H3K9me1 and H3K27me1. Collectively, our data shows that arsenic exposure causes dynamic changes in histone acetylation and methylation patterns during arsenic-induced cancer development. Exploring the genomic location of the altered histone marks and the resulting aberrant expression of genes will be of importance in deciphering the mechanism of arsenic-induced carcinogenesis.

A time-series analysis of altered histone H3 acetylation and gene expression during the course of MMAIII-induced malignant transformation of urinary bladder cells.

Our previous studies have shown that chronic exposure to low doses of monomethylarsonous acid (MMAIII) causes global histone acetylation dysregulation in urothelial cells (UROtsa cells) during the course of malignant transformation. To reveal the relationship between altered histone acetylation patterns and aberrant gene expression, more specifically, the carcinogenic relevance of these alterations, we performed a time-course analysis of the binding patterns of histone 3 lysine 18 acetylation (H3K18ac) across the genome and generated global gene-expression profiles from this UROtsa cell malignant transformation model. We showed that H3K18ac, one of the most significantly upregulated histone acetylation sites following MMAIII exposure, was enriched at gene promoter-specific regions across the genome and that MMAIII-induced upregulation of H3K18ac led to an altered binding pattern in a large number of genes that was most significant during the critical window for MMAIII-induced UROtsa cells' malignant transformation. Some genes identified as having a differential binding pattern with H3K18ac, acted as upstream regulators of critical gene networks with known functions in tumor development and progression. The altered H3K18ac binding patterns not only led to changes in expression of these directly affected upstream regulators but also resulted in gene-expression changes in their regulated networks. Collectively, our data suggest that MMAIII-induced alteration of histone acetylation patterns in UROtsa cells led to a time- and malignant stage-dependent aberrant gene-expression pattern, and that some gene regulatory networks were altered in accordance with their roles in carcinogenesis, probably contributing to MMAIII-induced urothelial cell malignant transformation and carcinogenesis.

Mobilization and removing of cadmium from kidney by GMDTC utilizing renal glucose reabsorption pathway.

Chronic exposure to cadmium compounds (Cd(2+)) is one of the major public health problems facing humans in the 21st century. Cd(2+) in the human body accumulates primarily in the kidneys which leads to renal dysfunction and other adverse health effects. Efforts to find a safe and effective drug for removing Cd(2+) from the kidneys have largely failed. We developed and synthesized a new chemical, sodium (S)-2-(dithiocarboxylato((2S,3R,4R,5R)-2,3,4,5,6 pentahydroxyhexyl)amino)-4-(methylthio) butanoate (GMDTC). Here we report that GMDTC has a very low toxicity with an acute lethal dose (LD50) of more than 10,000mg/kg or 5000mg/kg body weight, respectively, via oral or intraperitoneal injection in mice and rats. In in vivo settings, up to 94% of Cd(2+) deposited in the kidneys of Cd(2+)-laden rabbits was removed and excreted via urine following a safe dose of GMDTC treatment for four weeks, and renal Cd(2+) level was reduced from 12.9µg/g to 1.3µg/g kidney weight. We observed similar results in the mouse and rat studies. Further, we demonstrated both in in vitro and in animal studies that the mechanism of transporting GMDTC and GMDTC-Cd complex into and out of renal tubular cells is likely assisted by two glucose transporters, sodium glucose cotransporter 2 (SGLT2) and glucose transporter 2 (GLUT2). Collectively, our study reports that GMDTC is safe and highly efficient in removing deposited Cd(2+) from kidneys assisted by renal glucose reabsorption system, suggesting that GMDTC may be the long-pursued agent used for preventive and therapeutic purposes for both acute and chronic Cd(2+) exposure.

Activation of transcription factor MEF2D by bis(3)-cognitin protects dopaminergic neurons and ameliorates Parkinsonian motor defects.

Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP(+)-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3ß (GSK3ß) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP(+)-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP(+)-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.

Cloning and expression analysis of carboxyltransferase of acetyl-coA carboxylase from Jatropha curcas.

A full-length cDNA of the carboxyltransferase (accA) gene of acetyl-coenzym A (acetyl-CoA) carboxylase from Jatropha curcas was cloned and sequenced. The gene with an open reading frame (ORF) of 1149 bp encodes a polypeptide of 383 amino acids, with a molecular mass of 41.9 kDa. Utilizing fluorogenic real-time polymerase chain reaction (RT-PCR), the expression levels of the accA gene in leaves and fruits at early, middle and late stages under pH 7.0/8.0 and light/darkness stress were investigated. The expression levels of the accA gene in leaves at early, middle and late stages increased significantly under pH 8.0 stress compared to pH 7.0. Similarly, the expression levels in fruits showed a significant increase under darkness condition compared to the control. Under light stress, the expression levels in the fruits at early, middle and late stages showed the largest fluctuations compared to those of the control. These findings suggested that the expression levels of the accA gene are closely related to the growth conditions and developmental stages in the leaves and fruits of Jatropha curcas.

Corrigendum to "Astilbin from Smilax glabra Roxb. Attenuates Inflammatory Responses in Complete Freund's Adjuvant-Induced Arthritis Rats".

[This corrects the article DOI: 10.1155/2017/8246420.].

Astilbin from Smilax glabra Roxb. Attenuates Inflammatory Responses in Complete Freund's Adjuvant-Induced Arthritis Rats.

Astilbin, a flavonoid compound, was isolated from the rhizome of Smilax glabra Roxb. (with red cross-section) grown in Guizhou Province, China. We accessed its effect and potential mechanism on attenuation of the inflammatory response in CFA-induced AA rats. Our results showed that daily oral administration of astilbin at 5.3 mg/kg reduced joint damage in the hind paw of AA rats. Accordingly, astilbin exhibited remarkable inhibitory effects on TNF-α, IL-1ß, and IL-6 mRNA expression. Significant decrease of serum cytokine levels of TNF-α, IL-1ß, and IL-6 was also observed in astilbin-treated AA rats compared to the vehicle-treated AA rats. The reduced expression of these cytokines was associated with protein activity suppression of three key molecular targets in the pathogenesis of RA, including IKKß, NF-κB p65 subunit, and TLR adaptor MyD88. Furthermore, the therapeutic effects of astilbin on the inhibition of cytokines production as well as the reduction of inflammatory response in AA rats are close to a commonly used antirheumatic drug, leflunomide. Collectively, our data suggest that the action mechanism of astilbin, as an anti-inflammatory agent for RA treatment, is associated with modulating the production of proinflammatory cytokines and inhibiting the expression of key elements in NF-κB signaling pathway mediated by TLR.

Phosphorylation of LAMP2A by p38 MAPK couples ER stress to chaperone-mediated autophagy.

Endoplasmic reticulum (ER) and lysosomes coordinate a network of key cellular processes including unfolded protein response (UPR) and autophagy in response to stress. How ER stress is signaled to lysosomes remains elusive. Here we find that ER disturbance activates chaperone-mediated autophagy (CMA). ER stressors lead to a PERK-dependent activation and recruitment of MKK4 to lysosomes, activating p38 MAPK at lysosomes. Lysosomal p38 MAPK directly phosphorylates the CMA receptor LAMP2A at T211 and T213, which causes its membrane accumulation and active conformational change, activating CMA. Loss of ER stress-induced CMA activation sensitizes cells to ER stress-induced death. Neurotoxins associated with Parkinson's disease fully engages ER-p38 MAPK-CMA pathway in the mouse brain and uncoupling it results in a greater loss of SNc dopaminergic neurons. This work identifies the coupling of ER and CMA as a critical regulatory axis fundamental for physiological and pathological stress response.

Interactive Influence of N6AMT1 and As3MT Genetic Variations on Arsenic Metabolism in the Population of Inner Mongolia, China.

Chronic arsenic exposure via drinking water has become a worldwide public health concern. In humans, inorganic arsenic (iAs) is metabolized to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) mainly mediated by arsenic (+3 oxidation state) methyltransferase (As3MT). We reported recently that N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) was involved in arsenic metabolism, and examined its interactive effect with As3MT on arsenic metabolism in vitro To further evaluate the interactive effect of N6AMT1 and As3MT on arsenic biomethylation in humans, we conducted a human population-based study including 289 subjects living in rural villages in Inner Mongolia, China, and assessed their urinary arsenic metabolites profiles in relation to genetic polymorphisms and haplotypes of N6AMT1 and As3MT Five N6AMT1 single nucleotide polymorphisms (SNPs; rs1003671, rs7282257, rs2065266, rs2738966, rs2248501) and the N6AMT1 haplotype 2_GGCCAT were significantly associated with the percentage of iAs (% iAs) in urine (e.g., for rs7282257, mean was 9.62% for TT, 6.73% for AA). Rs1003671 was also in a significant relationship with urinary MMA and DMA (the mean of %MMA was 24.95% for GA, 31.69% for GG; the mean of % DMA was 69.21% for GA, 59.82% for GG). The combined effect of N6AMT1 haplotype 2_GGCCAT and As3MT haplotype 2_GCAC showed consistence with the additive significance of each haplotype on % iAs: the mean was 5.47% and 9.36% for carriers with both and null haplotypes, respectively. Overall, we showed that N6AMT1 genetic polymorphisms were associated with arsenic biomethylation in the Chinese population, and its interaction with As3MT was observed in specific haplotype combinations.

Advanced glycation end product (AGE)-induced hepatic stellate cell activation via autophagy contributes to hepatitis C-related fibrosis.

AIMS: Advanced glycation end products (AGEs) have been implicated in pulmonary and renal fibrosis. Herein, we investigated whether AGEs are associated with liver fibrosis and examined the underlying mechanism by focusing on hepatic stellate cell (HSC) activation and autophagy induction. METHODS: Liver fibrosis was assessed by transient elastography (FibroScan). Serum AGE levels were determined by ELISA. Rat primary HSCs and HSC-T6 were treated with BSA-AGEs, cell proliferation was examined by WST-1 assay, and cell activation was evaluated by qPCR for transcripts of α-SMA and collagen type Iα1 and by Western blotting. Autophagy was measured by detection of LC3-II lipidation, p62 degradation, and puncta GFP-LC3 formation. Receptor of AGE (RAGE)-blocking antibodies and soluble RAGE were employed to inhibit AGE-RAGE signaling. RESULTS: First, elevated AGE levels were observed in CHC patients than patients with chronic hepatitis B, especially in those with insulin resistance. Second, compared to controls, AGE-treated rat primary HSCs displayed an enhanced cell proliferation (1.39-fold), increased transcripts of α-SMA (2.40-fold) and proCOL1A1 (1.76-fold), and a higher level of α-SMA protein (1.85-fold). Moreover, AGE-induced HSC activation improved autophagy flux, as evidenced by significantly more LC3-II lipidation, p62 degradation, as well as GFP-LC3 puncta formations. In addition, our results showed that AGE-induced HSC autophagy and HSC activation could be reduced by RAGEs. CONCLUSION: AGEs were found to induce autophagy and activation of HSCs, which subsequently contributes to the fibrosis in CHC patients. Blocking AGE-RAGE signaling may be a promising way to alleviate fibrosis.

Application of human haploid cell genetic screening model in identifying the genes required for resistance to environmental toxicants: Chlorpyrifos as a case study.

INTRODUCTION: High-throughput loss-of-function genetic screening tools in yeast or other model systems except in mammalian cells have been implemented to study human susceptibility to chemical toxicity. Here, we employed a newly developed human haploid cell (KBM7)-based mutagenic screening model (KBM7-mu cells) and examined its applicability in identifying genes whose absence allows cells to survive and proliferate in the presence of chemicals. METHODS: KBM7-mu cells were exposed to 200 µM Chlorpyrifos (CPF), a widely used organophosphate pesticide, a dose causing approximately 50% death of cells after 48h of treatment. After a 2-3 week period of continuous CPF exposure, survived single cell colonies were recovered and used for further analysis. DNA isolated from these cells was amplified using Splinkerette PCR with specific designed primers, and sequenced to determine the genomic locations with virus insertion and identify genes affected by the insertion. Quantitative realtime reverse transcription PCR (qRT-PCR) was used to confirm the knockdown of transcription of identified target genes. RESULTS: We identified total 9 human genes in which the cells carrying these genes conferred the resistance to CPF, including AGPAT6, AIG1, ATP8B2, BIK, DCAF12, FNBP4, LAT2, MZF1-AS1 and PPTC7. MZF1-AS1 is an antisense RNA and not included in the further analysis. qRT-PCR results showed that the expression of 6 genes was either significantly reduced or completely lost. There were no changes in the expression of DCAF12 and AGPAT6 genes between the KBM7-mu and the control KBM7 cells. DISCUSSION: The KBM7-mu genetic screening system can be modified and applied to identify novel susceptibility genes in response to environmental toxicants, which could provide valuable insights into potential mechanisms of toxicity.

Lyophilized tea extracts of Brickellia cavanillesii (Asteraceae): in vitro characterization of biological activity.

UNLABELLED: Lyophilized Brickellia cavanillesii (LBC) tea extracts and identified chemical compounds of LBC were examined using in vitro human carcinoma liver (HepG2) cells with and without fetal bovine serum (FBS). Cells were incubated for 24 h with varying concentrations of FBS and LBC, respectively; cytotoxicity was determined spectrophotometrically using MTT (Formazan 3-(4,5-dimethyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide) assay. Furthermore, the potential hypoglycemic activity of LBC tea extracts was investigated using glucose transport and metabolism proteins biomarkers. FBS (0% to 10%) increased the viability of HepG2 cells steadily with increasing concentration. Possible therapeutic effects of LBC were concentration dependent with and without FBS. The cytotoxicity of 12 identified compounds from the LBC extract suggests that the individual compounds inhibited the proliferation of HepG2 cells differentially and do not reflect the inhibition of the whole aqueous LBC. Western blot analysis of glucose facilitated transporter protein 2 (GLUT 2) expression of HepG2 cells exposed to 0 mg/mL (Control) and 0.2 mg/mL LBC for 2, 4, 6, and 24 h suggests that GLUT 2 expression was increased. Increase in GLUT 2 expression in the absence of FBS was statistically significant with time of exposure. Significant difference was observed for GLUT 2 expression between 6 and 24 h and also between 4 and 24 h at 0.2 mg/mL LBC. Results obtained indicate that LBC may exhibit antidiabetic activity. However, further studies will be necessary to clearly delineate LBC potential therapeutic benefit and biological activities in animal studies as well as other in vitro models. PRACTICAL APPLICATION: Brickellia cavanillesii (Asteraceae) is a herbal plant widely used (Central America, Mexico and South-Western U.S.A.) in Type 2 diabetes mellitus therapy. Unfortunately, there is insufficient scientific data to validate its presumed pharmacological properties. This study examines the cytotoxic properties of whole, and certain identified chemical compounds in B. cavanillesii. It also investigates the potential hypoglycemic activity of lyophilized B. cavanillesii using glucose transport and metabolism proteins as biomarkers.

Chemical characterization of Brickellia cavanillesii (Asteraceae) using gas chromatographic methods.

A methanol extract of lyophilized Brickellia cavanillesii was quantitatively analyzed using gas chromatographic (GC) techniques. The chromatographic methods employed were (i) GC-flame ionization detector (GC-FID), (ii) GC-mass spectrometry (GC-MS), and (iii) purge and trap GC-MS (P&T GC-MS). Thirteen compounds were identified with a quality match of 90% and above using GC-MS. The compounds were (1) Cyclohexene, 6-ethenyl-6-methyl-1-(1-methylethyl)-3-(1-methylethylidene)-, (S)-; (2) Bicylo (2.2.1) heptan-2-one, 1, 7, 7-trimethyl-(1S, 4S)-; (3) Phenol, 2-methoxy-4-(1-propenyl)-; (4) Benzene, 1-(1, 5-dimethyl-4-hexenyl)-4-methyl-; (5) Naphthalene, 1, 2, 3, 5, 6, 8a-hexahydro4, 7-dimethyl-1-1-(1-methylethyl)-, (1S-cis)-; (6) Phenol, 2-methoxy-; (7) Benzaldehyde, 3-hydroxy-4-methoxy-; (8) 11, 13-Eicosadienoic acid, methyl ester; (9) 2-Furancarboxaldehyde, 5-methyl-; (10) Maltol; (11) Phenol; (12) Hydroquinone; (13) 1H-Indene, 1-ethylideneoctahydro-7a-methyl-, (1E, 3a.alpha, 7a.beta.). Other compounds (14) 3-methyl butanal; (15) (D)-Limonene; (16) 1-methyl-4-(1-methyl ethyl) benzene; (17) Butanoic acid methyl ester; (18) 2-methyl propanal; (19) 2-butanone; (20) 2-pentanone; and (21) 2-methyl butane were also identified when P&T GC-MS was performed. Of the 21 compounds identified, 12 were validated using chemical standards. The identified compounds were found to be terpenes, derivatives of terpenes, esters, ketones, aldehydes, and phenol-derived aromatic compounds; these are the primary constituents of the essential oils of many plants and flowers.

Activation of the Ano1 (TMEM16A) chloride channel by calcium is not mediated by calmodulin.

The Ca(2+)-activated Cl channel anoctamin-1 (Ano1; Tmem16A) plays a variety of physiological roles, including epithelial fluid secretion. Ano1 is activated by increases in intracellular Ca(2+), but there is uncertainty whether Ca(2+) binds directly to Ano1 or whether phosphorylation or additional Ca(2+)-binding subunits like calmodulin (CaM) are required. Here we show that CaM is not necessary for activation of Ano1 by Ca(2+) for the following reasons. (a) Exogenous CaM has no effect on Ano1 currents in inside-out excised patches. (b) Overexpression of Ca(2+)-insensitive mutants of CaM have no effect on Ano1 currents, whereas they eliminate the current mediated by the small-conductance Ca(2+)-activated K(+) (SK2) channel. (c) Ano1 does not coimmunoprecipitate with CaM, whereas SK2 does. Furthermore, Ano1 binds very weakly to CaM in pull-down assays. (d) Ano1 is activated in excised patches by low concentrations of Ba(2+), which does not activate CaM. In addition, we conclude that reversible phosphorylation/dephosphorylation is not required for current activation by Ca(2+) because the current can be repeatedly activated in excised patches in the absence of ATP or other high-energy compounds. Although Ano1 is blocked by the CaM inhibitor trifluoperazine (TFP), we propose that TFP inhibits the channel in a CaM-independent manner because TFP does not inhibit Ano1 when applied to the cytoplasmic side of excised patches. These experiments lead us to conclude that CaM is not required for activation of Ano1 by Ca(2+). Although CaM is not required for channel opening by Ca(2+), work of other investigators suggests that CaM may have effects in modulating the biophysical properties of the channel.

Oxidation of survival factor MEF2D in neuronal death and Parkinson's disease.

AIMS: Dysfunction of myocyte enhancer factor 2D (MEF2D), a key survival protein and transcription factor, underlies the pathogenic loss of dopaminergic (DA) neurons in Parkinson's disease (PD). Both genetic factors and neurotoxins associated with PD impair MEF2D function in vitro and in animal models of PD. We investigated whether distinct stress conditions target MEF2D via converging mechanisms. RESULTS: We showed that exposure of a DA neuronal cell line to 6-hyroxydopamine (6-OHDA), which causes PD in animals models, led to direct oxidative modifications of MEF2D. Oxidized MEF2D bound to heat-shock cognate protein 70 kDa, the key regulator for chaperone-mediated autophagy (CMA), at a higher affinity. Oxidative stress also increased the level of lysosomal-associated membrane protein 2A (LAMP2A), the rate-limiting receptor for CMA substrate flux, and stimulated CMA activity. These changes resulted in accelerated degradation of MEF2D. Importantly, 6-OHDA induced MEF2D oxidation and increased LAMP2A in the substantia nigra pars compacta region of the mouse brain. Consistently, the levels of oxidized MEF2D were much higher in postmortem PD brains compared with the controls. Functionally, reducing the levels of either MEF2D or LAMP2A exacerbated 6-OHDA-induced death of the DA neuronal cell line. Expression of an MEF2D mutant that is resistant to oxidative modification protected cells from 6-OHDA-induced death. INNOVATION: This study showed that oxidization of survival protein MEF2D is one of the pathogenic mechanisms involved in oxidative stress-induced DA neuronal death. CONCLUSION: Oxidation of survival factor MEF2D inhibits its function, underlies oxidative stress-induced neurotoxicity, and may be a part of the PD pathogenic process.

Cdk5: mediator of neuronal development, death and the response to DNA damage.

In the central nervous system, cyclin-dependent kinase 5 (Cdk5), an unusual member of the Cdk family, is implicated in the regulation of various physiological processes ranging from neuronal survival, migration and differentiation, to synaptogenesis, synaptic plasticity and neurotransmission. Dysregulation of this kinase has been demonstrated to play a critical role in the pathogenic process of neurodegenerative disorders. DNA damage is emerging as an important pathological component in various neurodegenerative conditions. In this review, we discuss the recent progress regarding the regulation and roles of Cdk5 under physiological conditions, and its dysregulation under pathological conditions, especially in neuronal death mediated by DNA damage.