A functional polymorphism in interleukin-1α (IL1A) gene is associated with risk of alopecia areata in Chinese populations

Alopecia areata (AA) is an inflammatory hair loss disorder with a major genetic component, which may cause great psychosocial distress for those affected. Studies have shown that interleukin-1 (IL-1) is a very potent inducer of hair loss and a significant human hair growth inhibitor. The 4-bp insertion/deletion (Indel) polymorphism (rs3783553) within the 3′ untranslated regions of IL1A gene has been suggested to be associated with risk of various types of cancers, possibly through regulating expression of IL-1α levels. In the current study, we estimated the susceptibility to AA associated with rs3783553 in two independent case–control panels of Eastern and Southern Chinese populations, totally containing 313 AA cases and 626 healthy controls. Logistic regression analysis showed that the heterozygote and the homozygote 4-bp ins/ins confer a significantly lower risk of AA in both panels and total subjects [odds ratio (OR) = 0.55, 95% confidence interval (C.I.) = 0.41–0.75, P = 6.24 × 10^− 5; OR = 0.47, 95% C.I. = 0.28–0.76, P = 0.001, respectively]. Stratification analysis based on age onset showed that the protective roles of ins/del and ins/ins genotype against developing AA was more obvious in AA patients with early age onset (< 30 years) under dominant model (OR = 0.48, 95% C.I. = 0.29–0.77, P = 0.001). The results of luciferase assay showed that rs3783553 could influence expression of IL-1α in a miR-122 dependant manner. Taken together, our results suggested that the IL1A 4-bp indel polymorphism may be a marker for genetic susceptibility to patchy (mild) AA in Chinese populations, likely through miR-122 mediated regulation.     

Characterization of the N-methyltransferase CalM involved in calcimycin biosynthesis by Streptomyces chartreusis NRRL 3882

Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the calcimycin biosynthesis gene cluster is required for calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl calcimycin to calcimycin in vitro. The enzyme was determined to have a kinetic parameter of K_m 276 μM, k_cat 1.26 min⁻¹ and k_cat/K_m 76.2 M⁻¹ s⁻¹. These results proved that CalM is a N-methyltransferase that is required for calcimycin biosynthesis, and they set the stage for generating much desired novel calcimycin derivatives by rational genetic and chemical engineering.     

The association between CD14 gene C-260T polymorphism and coronary heart disease risk: a meta-analysis

Monocyte differentiation antigen CD14 is considered an important cell-activating mediator of inflammatory responses that may result in atherosclerosis, coronary heart disease (CHD), thrombus formation, and myocardial infarction (MI). A common C-260T polymorphism in the promoter of the CD14 gene, the trans-membrane receptor of lipopolysaccharides, has been inconsistently associated with CHD. To investigate this inconsistency, we performed a meta-analysis of 28 studies involving a total of 13,335 CHD cases and 7,979 controls for C-260T of the CD14 gene to evaluate the effect of CD14 on genetic susceptibility for CHD. An overall random effects odds ratio of 1.24 (95 % CI: 1.12–1.36, P < 10^?5) was found for T allele. Significant results were also observed using dominant (OR = 1.34, 95 % CI: 1.17–1.54, P < 10^?4) or recessive genetic model (OR = 1.25, 95 % CI: 1.10–1.41, P = 0.0004). There was strong evidence of heterogeneity (P < 10^?5), which largely disappeared after stratification by ethnicity. After stratified by ethnicity, significant results were found in East Asians; whereas no significant associations were found among Caucasians and other ethnic populations in all genetic models. In the stratified analysis according to sample size, CHD endpoints, and HWE status, significantly increased risks for the polymorphism were found in all genetic models. In conclusion, our results indicate that the CD14 C-260T polymorphism is a risk factor of CHD, especially in East Asians. However, additional very large-scale studies are warranted to confirm our results.     

Inhibition of nephrin activation by c-mip through Csk–Cbp–Fyn axis plays a critical role in Angiotensin II-induced podocyte damage

It has been demonstrated that nephrin inactivation plays a critical role in Angiotensin II (AngII)-induced podocyte damage both in in vitro and in vivo, but the underlying molecular mechanisms are still unclear. Recently, c-maf inducing protein (c-mip) has been identified as a key component in the molecular pathogenesis of acquired podocyte diseases. In this study, the role of c-mip on AngII-induced nephrin inactivation and podocyte damage was explored in a mouse podocyte cell line. AngII stimulation caused podocyte damage, presenting with a time and dose dependent cell apoptosis increment, and obvious reorganization of actin cytoskeleton, both of which was remarkably prevented by knockdown of c-mip (siCmip). In AngII stimulated podocyte, c-mip and Csk expressions increased obviously at protein level, and nephrin phosphorylation decreased while Cbp phosphorylation increased. AngII-induced Csk increment and nephrin inactivation was remarkably inhibited by siCmip treatment. AngII stimulation increased the interaction of c-mip and Csk, as well as Csk and Cbp. Notably, the binding of Csk to active form pY418 decreased while the binding of Csk to inactive form pY530 of Src kinase Fyn increased in AngII-stimulated podocyte. Nevertheless, c-mip knockdown prevented AngII-induced reduction of pY418 and increase of pY530. In addition, AngII stimulation significantly decreased the expression of phosphor-Akt (Ser473) and antiapoptotic protein Bcl-2, whereas increased the expression of apoptotic proteins caspase-3 and BAD, all of which were prevented by siCmip treatment. Taken together, our results demonstrated that AngII induced nephrin inactivation and podocyte damage by the novel podocyte protein c-mip through Csk–Cbp–Fyn signaling pathway.     

Zinc Transporter 7 Induced by High Glucose Attenuates Epithelial-to-Mesenchymal Transition of Peritoneal Mesothelial Cells

Zinc (Zn) is an essential micronutrient and cytoprotectant involved in preventing many types of epithelial-to-mesenchymal transition (EMT)-driven fibrosis in vivo. The zinc-transporter family SLC30A (ZnT) is a pivotal factor in the regulation of Zn homeostasis. However, its function in EMT in peritoneal mesothelial cells (PMCs) remains unknown. This study explored the regulation of zinc transporters and the role they play in cell EMT, particularly in rat peritoneal mesothelial cells (RPMCs), surrounding glucose concentrations and the molecular mechanism involved. The effects of high glucose (HG) on zinc transporter gene expression were measured in RPMCs by real-time PCR. We explored ZnT7 (Slc30A7): the effect of ZnT7 over-expression and siRNA-mediated knock-down on HG-induced EMT was investigated as well as the underlying molecular mechanisms. Over-expression of ZnT7 resulted in significantly inhibited HG-induced EMT in RPMCs, while inhibition of ZnT7 expression using a considerable siRNA-mediated knock-down of RPMCs increased the levels of EMT. Furthermore, over-expression of ZnT7 is accompanied by down-regulation of TGF-β/Smad pathway, phospho-Smad3,4 expression levels. The finding suggests that the zinc-transporting system in RPMCs is influenced by the exposure to HG. The ZnT7 may account for the inhibition of HG-induced EMT in RPMCs, likely through targeting TGF-β/Smad signaling.     

IL‐1β and TNF‐α induce neurotoxicity through glutamate production: a potential role for neuronal glutaminase

Glutaminase 1 is the main enzyme responsible for glutamate production in mammalian cells. The roles of macrophage and microglia glutaminases in brain injury, infection, and inflammation are well documented. However, little is known about the regulation of neuronal glutaminase, despite neurons being a predominant cell type of glutaminase expression. Using primary rat and human neuronal cultures, we confirmed that interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), two pro‐inflammatory cytokines that are typically elevated in neurodegenerative disease states, induced neuronal death and apoptosis in vitro. Furthermore, both intracellular and extracellular glutamate levels were significantly elevated following IL‐1β and/or TNF‐α treatment. Pre‐treatment with N‐Methyl‐d ‐aspartate (NMDA) receptor antagonist MK‐801 blocked cytokine‐induced glutamate production and alleviated the neurotoxicity, indicating that IL‐1β and/or TNF‐α induce neurotoxicity through glutamate. To determine the potential source of excess glutamate production in the culture during inflammation, we investigated the neuronal glutaminase and found that treatment with IL‐1β or TNF‐α significantly upregulated the kidney‐type glutaminase (KGA), a glutaminase 1 isoform, in primary human neurons. The up‐regulation of neuronal glutaminase was also demonstrated in situ in a murine model of HIV‐1 encephalitis. In addition, IL‐1β or TNF‐α treatment increased the levels of KGA in cytosol and TNF‐α specifically increased KGA levels in the extracellular fluid, away from its main residence in mitochondria. Together, these findings support neuronal glutaminase as a potential component of neurotoxicity during inflammation and that modulation of glutaminase may provide therapeutic avenues for neurodegenerative diseases.     

Deferoxamine inhibits iron induced hippocampal tau phosphorylation in the Alzheimer transgenic mouse brain

Prior work has shown that iron interacts with hyperphosphorylated tau, which contributes to the formation of neurofibrillary tangles (NFTs) in Alzheimer’s disease (AD), whereas iron chelator desferrioxamine (DFO) slows down the clinical progression of the cognitive decline associated with this disease. However, the effects of DFO on tau phosphorylation in the presence or absence of iron have yet to be determined. Using amyloid precursor protein (APP) and presenilin 1 (PS1) double transgenic mouse brain as a model system, we investigated the effects and potential mechanisms of intranasal administration of DFO on iron induced abnormal tau phosphorylation. High-dose iron treatment markedly increased the levels of tau phosphorylation at the sites of Thr205, Thr231 and Ser396, whereas highly induced tau phosphorylation was abolished by intranasal administration of DFO in APP/PS1 transgenic mice. Moreover, DFO intranasal administration also decreases Fe-induced the activities of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3β (GSK3β), which in turn suppressing tau phosphorylation. Cumulatively, our data show that intranasal DFO treatment exerts its suppressive effects on iron induced tau phosphorylation via CDK5 and GSK3β pathways. More importantly, elucidation of DFO mechanism in suppressing tau phosphorylation may provide insights for developing therapeutic strategies to combat AD.     

Downregulation of multiple CDK inhibitor ICK/KRP genes upregulates the E2F pathway and increases cell proliferation,and organ and seed sizes in Arabidopsis

The ICK/KRP cyclin‐dependent kinase (CDK) inhibitors are important plant cell cycle factors sharing only limited similarity with the metazoan CIP/KIP family of CDK inhibitors. Little is known about the specific functions of different ICK/KRP genes in planta. In this study, we created double and multiple mutants from five single Arabidopsis ICK/KRP T‐DNA mutants, and used a set of 20 lines for the functional investigation of the important gene family. There were gradual increases in CDK activity from single to multiple mutants, indicating that ICK/KRPs act as CDK inhibitors under normal physiological conditions in plants. Whereas lower‐order mutants showed no morphological phenotypes, the ick1 ick2 ick6 ick7 and ick1 ick2 ick5 ick6 ick7 mutants had a slightly altered leaf shape. The quintuple mutant had larger cotyledons, leaves, petals and seeds than the wild‐type control. At the cellular level, the ICK/KRP mutants had more but smaller cells in all the organs examined. These phenotypic effects became more apparent as more ICK/KRPs were downregulated, suggesting that to a large extent ICK/KRPs function in plants redundantly in a dosage‐dependent manner. Analyses also revealed increased expression of E2F‐dependent genes, and elevated RBR1 as well as an increased level of phospho‐RBB1 protein in the quintuple mutant. Thus, downregulation of multiple ICK/KRP genes increases CDK activity, upregulates the E2F pathway and stimulates cell proliferation, resulting in increased cell numbers, and larger organs and seeds.     

Fused thiazolyl alkynes as potent mGlu5 receptor positive allosteric modulators

A new series of potent fused thiazole mGlu₅ receptor positive allosteric modulators (PAMs) (10, 11 and 27–31) are disclosed and details of the SAR and optimization are described. Optimization of alkynyl thiazole 9 (Lu AF11205) led to the identification of potent fused thiazole analogs 10b, 27a, 28j and 31d. In general, substituted cycloalkyl, aryl and heteroaryl carboxamides, and carbamate analogs are mGlu₅ PAMs, whereas smaller alkyl carboxamide, sulfonamide and sulfamide analogs tend to be mGlu₅ negative allosteric modulators (NAMs).