Protein Kinase Signaling Molecules Research Articles

Norcantharidin inhibits the malignant progression of cervical cancer by inducing endoplasmic reticulum stress.

The antitumor effect of norcantharidin (NCTD) has been widely reported. However, whether NCTD can inhibit cervical cancer remains unknown. In the present study, it was shown that NCTD inhibited the viability of cervical cancer cells and caused cell cycle arrest in a concentration‑dependent manner. Further analysis revealed that the NCTD‑induced reduction in cell viability could be reversed by the inhibitor of apoptosis z‑VAD‑FMK and by the inhibitor of endoplasmic reticulum (ER) stress, 4‑phenylbutyric acid (4‑PBA). Additionally, NCTD led to the accumulation of reactive oxygen species as well as a decrease in the mitochondrial membrane potential in cervical cancer cells, whereas 4‑PBA pre‑treatment attenuated these alterations. In addition, NCTD increased the expression of the apoptosis‑related proteins Bip, activating transcription factor (ATF) 4 and C/EBP homologous protein in a concentration‑dependent manner. Moreover, NCTD significantly increased the expression of the ER stress‑related signaling molecules protein kinase R‑like ER kinase, inositol‑requiring enzyme 1 and ATF6, but 4‑PBA abolished these effects. Invivo experiments showed that NCTD significantly inhibited the growth of subcutaneous tumors in mice. Additionally, the expression of ER stress‑related molecules and apoptosis‑related proteins increased significantly after NCTD treatment. In conclusion, NCTD induces apoptosis by activating ER stress and ultimately curtails the progression of cervical cancer.

Vitamin K2 Suppresses Proliferation and Inflammatory Cytokine Production in Mitogen-Activated Lymphocytes of Atopic Dermatitis Patients through the Inhibition of Mitogen-Activated Protein Kinases.

Vitamin K2 is suggested to have a suppressive effect on the peripheral blood mononuclear cells (PBMCs) of pediatric atopic dermatitis patients. We examined the molecular targets of vitamin K2 to suppress proliferation and cytokine production in T-cell mitogen-activated PBMCs of atopic dermatitis patients from the viewpoint of mitogen-activated protein kinase signaling molecules. The study population included 16 pediatric vitamin K2 patients and 21 healthy subjects. The effect of vitamin K2 on concanavalin A-activated PBMC proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell counting assays. T-helper (Th)1/Th2/Th17 cytokine profiles in plasma and PBMC-culture supernatants were analyzed by a cytometric beads array assay. Mitogen-activated protein kinase signaling molecules in concanavalin A-activated PBMCs were examined by enzyme-linked immunosorbent assay (ELISA) assays. At 10-100 µM, vitamin K2 significantly suppressed the proliferation of mitogen-activated PBMCs derived from atopic dermatitis patients and healthy subjects (p < 0.05). The interleukin (IL)-10 concentrations in plasma and the PBMC culture supernatants of atopic dermatitis patients were significantly higher than those of healthy subjects (p < 0.05). The IL-2 concentrations in the culture supernatants of atopic dermatitis PBMCs were significantly lower than those of healthy PBMCs (p < 0.05). Vitamin K2 significantly inhibited the IL-17A, IL-10, and tumor necrosis factor α (TNF-α) production (p < 0.05), and increased the IL-2 production (p < 0.01) in the culture supernatant of atopic dermatitis PBMCs. At 10-100 µM, vitamin K2 markedly decreased the of Mek1, extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase, and SAPK/c-Jun N-terminal kinase (JNK) expression in atopic dermatitis PBMCs (p < 0.05). Vitamin K2 is suggested to attenuate activated T-cell immunity in atopic dermatitis patients through the inhibition of mitogen-activated protein kinase-Mek1-ERK1/2 and SAPK/JNK signaling pathways.

TASK channels: channelopathies, trafficking, and receptor-mediated inhibition

TWIK-related acid-sensitive K+ (TASK) channels contribute to the resting membrane potential in various kinds of cells, such as brain neurons, smooth muscle cells, and endocrine cells. Loss-of-function mutations at multiple sites in the KCNK3 gene encoding for TASK1 channels are one of the causes of pulmonary arterial hypertension in humans, whereas a mutation at only one site is reported for TASK3 channels, resulting in a syndrome of mental retardation, hypotonia, and facial dysmorphism. TASK channels are subject to regulation by G protein-coupled receptors (GPCRs). Two mechanisms have been proposed for the GPCR-mediated inhibition of TASK channels: a change in gating and channel endocytosis. The most feasible mechanism for altered gating is diacylglycerol binding to a site in the C-terminus, which is shared by TASK1 and TASK3. The inhibition of channel function by endocytosis requires the presence of a tyrosine residue subjected to phosphorylation by the non-receptor tyrosine kinase Src and a dileucine motif in the C-terminus of TASK1. Therefore, homomeric TASK1 and heteromeric TASK1-TASK3 channels, but not homomeric TASK3, are internalized by GPCR stimulation. Tyrosine phosphorylation by Src is expected to result in a conformational change in the C-terminus, allowing for AP-2, an adaptor protein for clathrin, to bind to the dileucine motif. It is likely that a raft membrane domain is a platform where TASK1 is located and the signaling molecules protein kinase C, Pyk2, and Src are recruited in sequence in response to GPCR stimulation.

Organic barn dust inhibits surfactant protein D production through protein kinase-c alpha dependent increase of GPR116.

Prolonged exposure to organic barn dusts can lead to chronic inflammation and a broad range of lung problems over time, mediated by innate immune mechanisms. The immune surfactant or collectin surfactant protein D (SP-D) is a crucial multifunctional innate immune receptor. Little work to date has examined the effect of such collectins in response to organic dusts. We provide evidence here that agricultural organic dusts can inhibit mRNA and protein expression of SP-D in a human alveolar epithelial cell line, and an in vivo mouse model. This inhibition was not a result of lipopolysaccharide (LPS) or peptidoglycans, the two most commonly cited immune active components of these dusts. We further show that inhibition of the signaling molecule protein kinase C alpha (PKCα) can reverse this inhibition implicating it as a mechanism of SP-D inhibition. Examination of the SP-D regulatory receptor GPR116 showed that its mRNA expression was increased in response to dust and inhibited by blocking PKCα, implicating it as a means of inhibiting SP-D in the lungs in response to organic dusts. This reduction shows that organic barn dust can reduce lung SP-D, thus leaving workers potentially at risk for a host of pathogens.

Therapeutic Chemical Screen Identifies Phosphatase Inhibitors to Reconstitute PKB Phosphorylation and Cardiac Contractility in ILK-Deficient Zebrafish.

Patients with inherited dilated cardiomyopathy (DCM) often suffer from severe heart failure based on impaired cardiac contractility leading to increased morbidity and mortality. Integrin-linked kinase (ILK) as a part of the cardiac mechanical stretch sensor was found to be an essential genetic regulator of cardiac contractility. Integrin-linked kinase localizes to z-disks and costameres in vertebrate hearts and regulates the activity of the signaling molecule protein kinase B (PKB/Akt) by controlling its phosphorylation. Despite identification of several potential drug targets in the ILK signaling pathway, pharmacological treatment strategies to restore contractile function in ILK-dependent cardiomyopathies have not been established yet. In recent years, the zebrafish has emerged as a valuable experimental system to model human cardiomyopathies as well as a powerful tool for the straightforward high-throughput in vivo small compound screening of therapeutically active substances. Using the ILK deficient zebrafish heart failure mutant main squeeze (msq), which shows reduced PKB phosphorylation and thereby impaired cardiac contractile force, we identified here, in an automated small compound screen, the protein phosphatase inhibitors calyculin A and okadaic acid significantly restoring myocardial contractile function by reconstituting PKB phosphorylation in msq ILK-deficient zebrafish embryos.

Protective effects of honokiol against oxidative stress-induced apoptotic signaling in mouse podocytes treated with H2O2.

Honokiol (HNK), an important bioactive compound purified from Magnolia officinalis Cortex, has been demonstrated to have manifold beneficial anti-oxidative, anti-inflammatory, anti-bacterial and antitumor pharmacological effects. In the present study, the association of HNK in the signaling mechanism associated with hydrogen peroxide (H2O2)-induced apoptosis of cultured mouse podocytes was investigated. HNK did not cause significant changes in podocyte viability when its concentration remained below 20 µM. MTS assay and flow cytometry confirmed that H2O2 significantly enhanced the rates of apoptosis while produce significant reduction in viability of podocytes. Following 24 h of pre-treatment with different concentrations of HNK, the viability of adherent podocytes increased and apoptosis significantly decreased in a dose-dependent manner below 20 µM. Reverse transcription-polymerase chain reaction and western blot results indicated that HNK significantly decreased the expression of mRNA and cleaved protein of caspase-3 and caspase-9 in podocytes pre-treated with H2O2. Furthermore, phosphorylation of the signaling molecules protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) 1/2 appeared to increase following HNK treatment. In conclusion, HNK largely eliminated the role of promoting podocyte apoptosis in an oxidative stress environment, which was a protective factor on podocytes cultured with H2O2. The anti-oxidative stress mechanisms of HNK are partly due to suppressing the expression of caspase-3 and caspase-9 and upregulating phosphorylated-Akt and -Erk 1/2.

PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer.

Placenta-specific 1 (PLAC1), a novel cancer-testis antigen (CTA), is expressed in a number of different human malignancies. It is frequently produced in breast cancer, serving a function in tumorigenesis. Adoptive immunotherapy using T cell receptor (TCR)-engineered T cells against CTA mediates objective tumor regression; however, to the best of our knowledge, targeting PLAC1 using engineered T cells has not yet been attempted. In the present study, the cDNAs encoding TCRα- and β-chains specific for human leukocyte antigen (HLA)-A*0201-restricted PLAC1 were cloned from a cytotoxic T-lymphocyte, generated by in vitro by the stimulation of CD8+ T cells using autologous HLA-A2+ dendritic cells loaded with a PLAC1-specific peptide (p28-36, VLCSIDWFM). The TCRα/β-chains were linked by a 2A peptide linker (TCRα-Thosea asigna virus-TCRβ), and the constructs were cloned into the lentiviral vector, followed by transduction into human cytotoxic (CD8+) T cells. The efficiency of transduction was up to 25.16%, as detected by PLAC1 multimers. TCR-transduced CD8+ T cells, co-cultured with human non-metastatic breast cancer MCF-7 cells (PLAC1+, HLA-A2+) and triple-negative breast cancer MDAMB-231 cells (PLAC1+, HLA-A2+), produced interferon γ and tumor necrosis factor α, suggesting TCR activation. Furthermore, the PLAC1 TCR-transduced CD8+ T cells efficiently and specifically identified and annihilated the HLA-A2+/PLAC1+ breast cancer cell lines in a lactate dehydrogenase activity assay. Western blot analysis demonstrated that TCR transduction stimulated the production of mitogen-activated protein kinase signaling molecules, extracellular signal-regulated kinases 1/2 and nuclear factor-κB, through phosphoinositide 3-kinase γ-mediated phosphorylation of protein kinase B in CD8+ T cells. Xenograft mouse assays revealed that PLAC1 TCR-transduced CD8+T cells significantly delayed the tumor progression in mice-bearing breast cancer compared with normal saline or negative control-transduced groups. In conclusion, a novel HLA-A2-restricted and PLAC1-specific TCR was identified. The present study demonstrated PLAC1 to be a potential target for breast cancer treatment; and the usage of PLAC1-specific TCR-engineered T cells may be a novel strategy for PLAC1-positive breast cancer treatment.

SRC1 promotes Th17 differentiation by overriding Foxp3 suppression to stimulate RORγt activity in a PKC-θ–dependent manner

Th17 cells are major players in multiple autoimmune diseases and are developmentally contingent on reciprocal functionality between the transcription factor Retineic acid receptor-related orphan nuclear receptor gamma (RORγt) and Forkhead box protein P3 (Foxp3). Here we deciphered a previously unappreciated role of Steroid receptor coactivator 1 (SRC1) in defining the lineage decision for the development of Th17 versus induced T-regulatory (iTreg) cells. We demonstrate that SRC1 functions as a critical coactivator for RORγt in vivo to promote the functional dominance of RORγt over Foxp3 and thus establishing an unopposed Th17 differentiation program. In the absence of SRC1, T cell polarization resulted in decreased IL-17+ and increased Foxp3+ cells during both in vitro differentiation and in vivo development of experimental autoimmune encephalomyelitis. Mechanistically, T cell receptor (TCR) signaling molecule protein kinase C theta (PKC-θ)-mediated phosphorylation of SRC1 is important for inducing enhanced RORγt-SRC1 interaction, stable DNA binding, and resultant IL-17A transcription. Furthermore, phospho-SRC1-mediated recruitment of CARM1 induced prominent asymmetric dimethylation of H3R17 while preventing repressive H3K9 trimethylation and hence further modifying the IL-17 locus for optimal transcription. Moreover, binding of phospho-SRC1 to RORγt displaced bound Foxp3, leading to prompt degradation of the dissociated Foxp3 via a ubiquitin-proteosomal pathway and hence reversing the inhibitory action of Foxp3 on RORγt activity. Thus, SRC1 acts as a crucial molecular mediator to integrate positive PKC-θ-dependent TCR signals to induce peak RORγt activity and establish phenotypic dominance of Th17 over the iTreg pathway.

A Model of Induction of Cerebellar Long-Term Depression Including RKIP Inactivation of Raf and MEK.

We report an updated stochastic model of cerebellar Long Term Depression (LTD) with improved realism. Firstly, we verify experimentally that dissociation of Raf kinase inhibitor protein (RKIP) from Mitogen-activated protein kinase kinase (MEK) is required for cerebellar LTD and add this interaction to an earlier published model, along with the known requirement of dissociation of RKIP from Raf kinase. We update Ca2+ dynamics as a constant-rate influx, which captures experimental input profiles accurately. We improve α-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA) receptor interactions by adding phosphorylation and dephosphorylation of AMPA receptors when bound to glutamate receptor interacting protein (GRIP). The updated model is tuned to reproduce experimental Ca2+ peak vs. LTD amplitude curves at four different Ca2+ pulse durations as closely as possible. We find that the updated model is generally more robust with these changes, yet we still observe some sensitivity of LTD induction to copy number of the key signaling molecule Protein kinase C (PKC). We predict natural variability in this number by stochastic diffusion may influence the probabilistic LTD response to Ca2+ input in Purkinje cell spines and propose this as an extra source of stochasticity that may be important also in other signaling systems.

Protein kinase C theta is required for efficient induction of IL-10-secreting T cells.

Secretion of interleukin-10 (IL-10) by CD4+ T cells is an essential immunoregulatory mechanism. The work presented here assesses the role of the signaling molecule protein kinase C theta (PKCθ) in the induction of IL-10 expression in CD4+ T cells. Using wildtype and PKCθ-deficient Tg4 T cell receptor transgenic mice, we implemented a well-described protocol of repeated doses of myelin basic protein (MBP)Ac1-9[4Y] antigen to induce Tr1-like IL-10+ T cells. We find that PKCθ is required for the efficient induction of IL-10 following antigen administration. Both serum concentrations of IL-10 and the proportion of IL-10+ T cells were reduced in PKCθ-deficient mice relative to wildtype mice following [4Y] treatment. We further characterized the T cells of [4Y] treated PKCθ-deficient Tg4 mice and found reduced expression of the transcription factors cMaf, Nfil3 and FoxP3 and the surface receptors PD-1 and Tim3, all of which have been associated with the differentiation or function of IL-10+ T cells. Finally, we demonstrated that, unlike [4Y] treated wildtype Tg4 T cells, cells from PKCθ-deficient mice were unable to suppress the priming of naïve T cells in vitro and in vivo. In summary, we present data demonstrating a role for PKCθ in the induction of suppressive, IL-10-secreting T cells induced in TCR-transgenic mice following chronic antigen administration. This should be considered when contemplating PKCθ as a suitable drug target for inducing immune suppression and graft tolerance.

Taspine derivative TAS9 regulates cell growth and metastasis of human hepatocellular carcinoma.

Taspine has been indicated to be a potential anti‑carcinogenic agent. The present study investigated the effects of TAS9, a modified taspine derivative, on the proliferation and migration of the SMMC‑7721 human liver cancer cell line. First, the effects of TAS9 on SMMC‑7721 cell growth were examined using MTT and colony formation assaya. In vivo Transwell and wound healing assays were then performed to assess the inhibitory effects of TAS9 on cell invasion and migration, respectively. The expression of cell proliferation‑ and migration‑associated signaling molecules was investigated by western blot analysis. The results indicated that TAS9 inhibited SMMC‑7721 cell growth by downregulating the signaling molecules protein kinase Cβ (PKCβ), Akt, mammalian target of rapamycin, mitogen‑activated protein kinase kinase 2, RAF and c‑Jun N‑terminal kinase‑1, and inhibiting SMMC‑7721 cell migration by suppressing the expression of matrix metalloproteinase (MMP)‑2, MMP‑9, chemokine (C‑X‑C motif) receptor 4, nuclear factor κB, p38 and p53. Small interfering RNA‑mediated knockdown of PKCβ in the SMMC‑7721 cells significantly attenuated the tumor inhibitory effects of TAS9. In conclusion, the results of the present study suggested that TAS9 may have inhibitory effects on the proliferation and migration of SMMC‑7721 cells, and may serve as a potential candidate for cancer treatment.

The Association of Lipoprotein Lipase and Phosphatidylinositol 3‐Kinase with Macrophage Lipid Accumulation

Lipoprotein lipase (LPL) is an extracellular enzyme that hydrolyzes triglycerides and phospholipids within lipoproteins. LPL is expressed in several tissues, including macrophages within atherosclerotic lesions, where it has been shown to promote foam cell formation. Our laboratory previously showed that the hydrolysis products generated by LPL from total lipoproteins, and specifically the free fatty acids (FFA), increased the phosphorylation of the signaling molecule protein kinase B (or Akt). Thus, we hypothesized that the FFA liberated from total lipoproteins by LPL promote lipid accumulation in macrophages in part through a mechanism associated with the phosphatidylinositol 3-kinase (PI3K) signaling pathway. To test this hypothesis, THP-1 macrophages were incubated with a mixture of purified FFA (which matched the concentrations of the FFA species liberated by LPL lipoprotein hydrolysis) for 18 hours in the absence or presence of the PI3K inhibitor LY294002. As expected, Oil Red O staining showed an increased cellular accumulation of lipids following the FFA mixture treatment. THP-1 macrophages in the presence of both the FFA mixture and LY294002 exhibited a 20% decrease of Oil Red O staining (n=9, p<0.0001). However, no differences in Oil Red O staining were observed between THP-1 macrophages incubated with individual species of FFA in the absence or presence of LY294002, thus suggesting that multiple FFA species together are necessary to modulate the PI3K signaling pathway. Overall, our data show that the FFA mixture indeed promotes macrophage lipid accumulation partially through a PI3K pathway. Funded by the Natural Sciences and Engineering Research Council of Canada (grant #402185/2011).

Protein kinase C inhibition rescues manic-like behaviors and hippocampal cell proliferation deficits in the sleep deprivation model of mania.

Background:Recent studies revealed that bipolar disorder may be associated with deficits of neuroplasticity. Additionally, accumulating evidence has implicated alterations of the intracellular signaling molecule protein kinase C (PKC) in mania.Methods:Using sleep deprivation (SD) as an animal model of mania, this study aimed to examine the possible relationship between PKC and neuroplasticity in mania. Rats were subjected to SD for 72h and tested behaviorally. In parallel, SD-induced changes in hippocampal cell proliferation were evaluated with bromodeoxyuridine (BrdU) labeling. We then examined the effects of the mood stabilizer lithium, the antipsychotic agent aripiprazole, and the PKC inhibitors chelerythrine and tamoxifen on both behavioral and cell proliferation impairments induced by SD. The antidepressant fluoxetine was used as a negative control.Results:We found that SD triggered the manic-like behaviors such as hyperlocomotion and increased sleep latency, and reduced hippocampal cell proliferation. These alterations were counteracted by an acute administration of lithium and aripiprazole but not of fluoxetine, and only a single administration of aripiprazole increased cell proliferation on its own. Importantly, SD rats exhibited increased levels of phosphorylated synaptosomal-associated protein 25 (SNAP-25) in the hippocampus and prefrontal cortex, suggesting PKC overactivity. Moreover, PKC inhibitors attenuated manic-like behaviors and rescued cell proliferation deficits induced by SD.Conclusions:Our findings confirm the relevance of SD as a model of mania, and provide evidence that antimanic agents are also able to prevent SD-induced decrease of hippocampal cell proliferation. Furthermore, they emphasize the therapeutic potential of PKC inhibitors, as revealed by their antimanic-like and pro-proliferative properties.

Functional characterization of zebrafish K2P18.1 (TRESK) two-pore-domain K+ channels

The human KCNK18 gene is predominantly expressed in brain, spinal cord, and dorsal root ganglion neurons. Encoded K2P18.1K(+) channels are functionally implicated in migraine, pain and anesthesia. Data delineating the in vivo significance of K2P18.1 are still limited owing to a lack of model systems allowing for rapid, whole organism phenotypic analyses. We hypothesized that zebrafish (Danio rerio) might close this scientific gap. This work was designed to characterize the zebrafish ortholog of K2P18.1 in comparison to human K2P18.1 channels. The complete coding sequence of zKCNK18 was amplified from zebrafish cDNA. Zebrafish KCNK18 expression was assessed by in situ hybridization. Human and zebrafish K2P18.1 currents were functionally analyzed using two-electrode voltage clamp electrophysiology and the Xenopus oocyte expression system. KCNK18 mRNA is expressed in zebrafish brain and eyes. Human and zebrafish K2P18.1 proteins share 32% identity. Zebrafish K2P18.1 channels mediate K(+)-selective background currents that stabilize the negative resting membrane potential. Functional similarities between human and zK2P18.1 currents include open rectification properties, inhibition by barium, and regulation by signaling molecules protein kinase (PK)C, PKA, and phospholipase C. In contrast to the human ortholog, zK2P18.1 exhibited reduced sensitivity to elevation of intracellular calcium levels by ionomycin and was virtually insensitive to inhibition by quinidine. Zebrafish and human K2P18.1 channels share functional and regulatory properties, indicating that the zebrafish may serve as model to assess K2P18.1 function in vivo. However, distinct differences in K2P18.1 current regulation require careful consideration when zebrafish data are extrapolated to human physiology.

Anthocyanins attenuate body weight gain via modulating neuropeptide Y and GABAB1 receptor in rats hypothalamus

Anthocyanins in a variety of plant species have been identified and are known for its hypolipidemic and anti-obesity effects. The effect of anthocyanins extracted from black soybean on body weight and daily food intake in adult rats raised on normal diet were studied. Male Sprague–Dawley rats were daily intra-gastric administered water or anthocyanins 6mg/kg and 24mg/kg for 40days. During this period daily food intake and body weight were measured prior to anthocyanins treatment. These findings showed that anthocyanins treatment resulted in significantly lowered body weight and food intake compared with water treated rats. In addition, anthocyanins dose dependently reduced the adipose tissue size compared with control group. Western blot analysis showed that high dose of anthocyanins treatment significantly reduced the expression of neuropeptide Y (NPY) and increased γ-amino butyric acid receptor (GABAB1R) in hypothalamus. Furthermore, these events were followed by a decreased in expression of GABAB1R downstream signaling molecules protein kinase A-α (PKA) and phosphorylated cAMP-response element binding protein (p-CREB) in hypothalamus. These data support the concept that anthocyanins even in normal circumstances have the capability to reduce body weight and food intake through its modulatory effect on NPY and GABAB1R in hypothalamus. These results suggest that anthocyanins from black soybean seed coat might have a novel role in preventing obesity in rats on normal diet.

Diabetic nephropathy: Progression and pathophysiology

Diabetic nephropathy is the leading cause of kidney disease in patients starting renal replacement therapy and affects ~30% of type 1 and type 2 diabetic patients. This review focuses on the progression and pathophysiological aspects of the condition. The natural history of diabetic nephropathy is characterized by specific renal morphological and functional alterations. Features of early diabetic renal changes are microalbuminuria (30-300mg/day), glomerular hyperfiltration, glomerular and renal hypertrophy, increased basement membrane thickness, and mesangial expansion with the accumulation of extracellular matrix proteins such as collagen, fibronectin, and laminin. Advanced diabetic nephropathy is characterized by macroalbuminuria ( >300mg/day), a progressive decline in glomerular filtration rate, decreasing creatinine clearance, glomerulosclerosis, and interstitial fibrosis. Although poor glycemic control is an important risk factor, glycemia does not fully explain why only a subset of diabetic patients progress to end stage renal disease. Several decades of extensive research has elucidated various pathways to be implicated in the development of diabetic kidney disease such as systemic and glomerular hypertension, advanced glycation endproducts and the aldose reductase system. Furthermore, hemodynamic factors, the reninangiotensin system, the endothelin system, the intracellular signaling molecule protein kinase C, transforming growth factor-s, growth hormone, insulin like growth factors, vascular endothelial growth factor, and platelet-derived growth factor are believed to be involved in the pathogenesis. Thus, there are clearly many points at which therapeutic approaches could be tried to provide renoprotection in diabetes. It is likely that due to its complexity, targeting multiple points in altered metabolism in the diabetic kidney will be more successful in attenuating the development of diabetic nephropathy, rather than a single approach.

Transcript and protein profiling analysis of OTA-induced cell death reveals the regulation of the toxicity response process in Arabidopsis thaliana

Ochratoxin A (OTA) is a toxic isocoumarin derivative produced by various species of mould which mainly grow on grain, coffee, and nuts. Recent studies have suggested that OTA induces cell death in plants. To investigate possible mechanisms of OTA phytotoxicity, both digital gene expression (DGE) transcriptomic and two-dimensional electrophoresis proteomic analyses were used, through which 3118 genes and 23 proteins were identified as being up- or down-regulated at least 2-fold in Arabidopsis leaf in response to OTA treatment. First, exposure of excised Arabidopsis thaliana leaves to OTA rapidly causes the hypersensitive reponse, significantly accelerates the increase of reactive oxygen species and malondialdehyde, and enhances antioxidant enzyme defence responses and xenobiotic detoxification. Secondly, OTA stimulation causes dynamic changes in transcription factors and activates the membrane transport system dramatically. Thirdly, a concomitant persistence of compromised photosynthesis and photorespiration is indicative of a metabolic shift from a highly active to a weak state. Finally, the data revealed that ethylene, salicylic acid, jasmonic acid, and mitogen-activated protein kinase signalling molecules mediate the process of toxicity caused by OTA. Profiling analyses on Arabidopsis in response to OTA will provide new insights into signalling transduction that modulates the OTA phytotoxicity mechanism, facilitate mapping of regulatory networks, and extend the ability to improve OTA tolerance in Arabidopsis.

Are periodontal fibroblasts target cells for lipoxins and resolvin D1?

Lipoxins and resolvins are endogenous eicosanoids which participate in the resolution of inflammation. The connective tissue, including the fibroblasts of the periodontal ligament and the gingiva, is inflamed in periodontal disease. However, it is unknown if periodontal fibroblasts are targeted by lipoxins and resolvins. This study has investigated the impacts of lipoxin A4, lipoxin B4, and resolvin D1 on the response of periodontal ligament fibroblasts and gingival fibroblasts to the inflammatory mediators interleukin (IL)-1b and tumor necrosis factor-a (TNF-a) and platelet-derived growth factor BB (PDGF-BB). It was found that up to a concentration of 1 mM neither of the eicosanoids changed the expression of IL-6 and IL-8 by periodontal fibroblasts previously exposed to IL-1b and TNF-a. The eicosanoids also failed to modulate IL-1b-induced phosphorylation of p38 and c-Jun N-terminal protein kinase signaling molecules. Furthermore, lipoxins and resolvin D1 did not change proliferation rates or protein synthesis by periodontal fibroblasts exposed to PDGF-BB. Together, these results suggest that the response of periodontal fibroblasts to inflammatory mediators and a potent mitogen is not modulated by lipoxins and resolvin D1 under the in vitro conditions studied.

Lactobacillus pentosus strain b240 suppresses pneumonia induced by Streptococcus pneumoniae in mice

Oral administration of probiotics has been known to improve inflammatory responses against infectious diseases. Here, we describe the inhibitory effect of oral intake of heat-killed Lactobacillus pentosus strain b240 (b240) on pneumococcal pneumonia in a murine experimental model. The mice treated with oral b240 for 21 days before Streptococcus pneumoniae infection exhibited prolonged survival time and less body weight loss, compared with saline-treated control mice. Mild pneumonia with significantly reduced secretion of inflammatory cytokines/chemokines according to related mitogen-activated protein kinase signalling molecules (phosphorylated c-Jun N-terminal kinase) was found in b240-treated mice, whereas severe pneumonia with hypercytokinemia was evident in control mice. Prominent reduction in the number of pneumococci and elevated expression of Toll-like receptor 2 and 4 in the lung tissues was concomitantly noted in b240-treated mice. These findings indicate that b240 has inhibitory effects on pneumococcal pneumonia induced by Strep. pneumoniae infection and improves inflammatory tissue responses, resulting in reduced damages to the respiratory tissues. These results demonstrate that oral administration of b240 might protect host animals from Strep. pneumoniae infection by augmentation of innate immune response.

Proteomic Signatures of Epidermal Growth Factor Receptor and Survival Signal Pathways Correspond to Gefitinib Sensitivity in Head and Neck Cancer

Gefitinib targeting of the epidermal growth factor receptor (EGFR) has shown limited activity in clinical trials of head and neck squamous cell carcinoma (HNSCC). To investigate the underlying molecular mechanism, the proteomic signatures and responses of EGFR and downstream signals have been studied in a panel of HNSCC cell lines and tumor specimens pre- and post-gefitinib treatment. The IC(50) of gefitinib for HNSCC cell lines were determined using 3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay. The effects of gefitinib on activation of EGFR and downstream signaling molecules were determined by Western blot, ELISA, and reverse-phase protein microarray (RPMA). The biomarkers involved in the signaling pathways were examined in HNSCC tumor specimens from patients in a phase I gefitinib trial. In vitro, gefitinib inhibited cell proliferation with differing IC(50), and suppressed activation of EGFR and downstream signaling molecules protein kinase B (AKT), extracellular signal-regulated kinase 1/2, signal transducer and activator of transcription 3 (STAT3), and nuclear factor kappaB. The drug sensitivity was statistically correlated with activation of phosphorylated AKT (p-AKT) and phosphorylated STAT3 (p-STAT3) detected by ELISA, and consistent with results measured by RPMA. In patient samples, a broad suppression of activation of EGFR and downstream signaling molecules was observed in a molecular responder patient, in contrast to a lack of inhibition or increased activation of biomarkers in different pathways in nonresponder patients. Gefitinib sensitivity is correlated with p-AKT and p-STAT3 activation in HNSCC cell lines and tumor specimens. p-AKT and p-STAT3 could serve as potentially useful biomarkers and drug targets for further development of novel therapeutic agents for HNSCC.