Protein Kinase Inhibitor Staurosporine Research Articles

Neuroprotection against staurosporine by metalloporphyrins independent of antioxidant capability

Metalloporphyrin catalytic antioxidants are remarkably useful in protecting cells and tissues in a wide array of disease models, attributed primarily to functioning as superoxide dismutase (SOD) mimetics or by scavenging other reactive oxygen species (ROS). However, we recently showed that neuroprotection against Ca 2+-dependent excitotoxic insults did not correlate with antioxidant strength or capability [25], raising the question of whether scavenging of ROS underlies neuroprotection in other types of neuronal injury. The protein kinase inhibitor staurosporine causes neuronal demise primarily by apoptosis. Neuroprotection from staurosporine by a limited number of metalloporphyrin antioxidants has previously been attributed to antioxidant action. In the current study, a wide array of anionic and cationic metalloporphyrins and porphyrins, ranging in antioxidant strength or capability, provided protection against staurosporine in cortical neuron and cerebellar granule neuron (CGN) culture. Neuroprotection did not correlate with antioxidant strength or capability. In CGN but not cortical neuron cultures, NMDA receptor antagonists also prevented neurotoxicity, so metalloporphyrins may also target this secondary mode of death induced by staurosporine. Neuroprotection observed with antioxidant-inactive controls raises the possibility of an additional, or perhaps alternative, mechanism by antioxidant analogs not involving ROS scavenging.

Molecular characterization of a novel pathogen-responsive receptor kinase-like in Citrus limon

Infection by pathogenic microorganisms is a source of biological stress in plants. Understanding the interaction between plants and microbial infection at molecular level might contribute to our understanding for the effective control of the disease. Here, we isolated a novel putative receptor kinase-like protein (Citrus limon P5) that is the first Lec-receptor kinase-like protein isolated in lemon during a pathogen infection. C. limon P5 cDNA fragment was detected by differential display assay in C. limon during Capnodium citri (sooty mold) infection. The deduced amino acid sequence of P5 full-length cDNA revealed a 83% sequence homology with a receptor kinase-like protein from Arabidopsis thaliana characterized by a N-terminal lectin domain and C-terminal serine/threonine conserved domain. The inhibition of the pathogen-responsive P5 by the protein kinase inhibitor staurosporine and by a mitogen-activated protein kinase inhibitor (PD 98059) indicated a defense mechanism in C. limon against pathogens mediated via signal transduction pathway. To our knowledge, this is the first evidence that C. limon uses this putative defense mechanism against pathogens. The role of this protein is discussed as a starting point to understanding the molecular mechanisms in the C. limon in response to C. citri infection.

Brain-derived neurotrophic factor released from engineered mesenchymal stem cells attenuates glutamate- and hydrogen peroxide-mediated death of staurosporine-differentiated RGC-5 cells

The purpose of this study was to determine the viability of cell-based delivery of brain-derived neurotrophic factor (BDNF) from genetically modified mesenchymal stem cells (MSCs) for neuroprotection of RGC-5 cells. RGC-5 cells were differentiated with the protein kinase inhibitor staurosporine (SS) and exposed to the cellular stressors glutamate or H2O2. As a neuroprotective strategy, these cells were then co-cultured across a membrane insert with mesenchymal stem cells (MSCs) engineered with a lentiviral vector for production of BDNF (BDNF-MSCs). As a positive control, recombinant human BDNF (rhBDNF) was added to stressed RGC-5 cells. After SS-differentiation RGC-5s developed neuronal-like morphologies, and a significant increase in the proportion of RGC-5s immunoreactive for TuJ-1 and Brn3a was observed. Differentiated RGC-5s also had prominent TrkB staining, demonstrating expression of the high-affinity BDNF receptor. Treatment of SS-differentiated RGC-5s with glutamate or H2O2, produced significant cell death (56.0 ± 7.02 and 48.90 ± 4.58% of control cells, respectively) compared to carrier-solution treated cells. BDNF-delivery from MSCs preserved more RGC-5 cells after treatment with glutamate (80.0 ± 5.40% cells remaining) than control GFP expressing MSCs (GFP-MSCs, 57.29 ± 1.89%, p < 0.01). BDNF-MSCs also protected more RGC-5s after treatment with H2O2 (65.6 ± 3.47%) than GFP-MSCs (46.0 ± 4.20%, p < 0.01). We have shown survival of differentiated RGC-5s is reduced by the cellular stressors glutamate and H2O2. Additionally, our results demonstrate that genetically modified BDNF-producing MSCs can enhance survival of stressed RGC-5 cells and therefore, may be effective vehicles to deliver BDNF to retinal ganglion cells affected by disease.

Mechanical dynamics of single cells during early apoptosis

Dynamic mechanical properties of cells are becoming recognized as indicators and regulators of physiological processes such as differentiation, malignant phenotypes and mitosis. A key process in development and homeostasis is apoptosis and whilst the molecular control over this pathway is well studied, little is known about the mechanical consequences of cell death. Here, we study the caspase-dependent mechanical kinetics of single cells during early apoptosis initiated with the general protein-kinase inhibitor staurosporine. This results in internal remodelling of the cytoskeleton and nucleus which is reflected in dynamic changes in the mechanical properties of the cell. Utilizing simultaneous confocal and atomic force microscopy (AFM), we measured distinct mechanical dynamics in the instantaneous cellular Young's Modulus and longer timescale viscous deformation. This allowed us to visualize time-dependent nuclear and cytoskeletal control of force dissipation with fluorescent fusion proteins throughout the cell. This work reveals that the cell death program not only orchestrates biochemical dynamics but also controls the mechanical breakdown of the cell. Importantly, the consequences of mechanical disregulation during apoptosis may be a contributing factor to several human pathologies through the poorly timed release of dead cells and cell debris.

PINOID Kinase Regulates Root Gravitropism through Modulation of PIN2-Dependent Basipetal Auxin Transport in Arabidopsis

Reversible protein phosphorylation is a key regulatory mechanism governing polar auxin transport. We characterized the auxin transport and gravitropic phenotypes of the pinoid-9 (pid-9) mutant of Arabidopsis (Arabidopsis thaliana) and tested the hypothesis that phosphorylation mediated by PID kinase and dephosphorylation regulated by the ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1) protein might antagonistically regulate root auxin transport and gravity response. Basipetal indole-3-acetic acid transport and gravitropism are reduced in pid-9 seedlings, while acropetal transport and lateral root development are unchanged. Treatment of wild-type seedlings with the protein kinase inhibitor staurosporine phenocopies the reduced auxin transport and gravity response of pid-9, while pid-9 is resistant to inhibition by staurosporine. Staurosporine and the phosphatase inhibitor, cantharidin, delay the asymmetric expression of DR5revGFP (green fluorescent protein) at the root tip after gravistimulation. Gravity response defects of rcn1 and pid-9 are partially rescued by treatment with staurosporine and cantharidin, respectively. The pid-9 rcn1 double mutant has a more rapid gravitropic response than rcn1. These data are consistent with a reciprocal regulation of gravitropism by RCN1 and PID. Furthermore, the effect of staurosporine is lost in pinformed2 (pin2). Our data suggest that reduced PID kinase function inhibits gravitropism and basipetal indole-3-acetic acid transport. However, in contrast to PID overexpression studies, we observed wild-type asymmetric membrane distribution of the PIN2 protein in both pid-9 and wild-type root tips, although PIN2 accumulates in endomembrane structures in pid-9 roots. Similarly, staurosporine-treated plants expressing a PIN2GFP fusion exhibit endomembrane accumulation of PIN2GFP, but no changes in membrane asymmetries were detected. Our data suggest that PID plays a limited role in root development; loss of PID activity alters auxin transport and gravitropism without causing an obvious change in cellular polarity.

Glycine Receptor in Rat Hippocampal and Spinal Cord Neurons as a Molecular Target for Rapid Actions of 17-β-Estradiol

Glycine receptors (GlyRs) play important roles in regulating hippocampal neural network activity and spinal nociception. Here we show that, in cultured rat hippocampal (HIP) and spinal dorsal horn (SDH) neurons, 17-β-estradiol (E2) rapidly and reversibly reduced the peak amplitude of whole-cell glycine-activated currents (IGly). In outside-out membrane patches from HIP neurons devoid of nuclei, E2 similarly inhibited IGly, suggesting a non-genomic characteristic. Moreover, the E2 effect on IGly persisted in the presence of the calcium chelator BAPTA, the protein kinase inhibitor staurosporine, the classical ER (i.e. ERα and ERβ) antagonist tamoxifen, or the G-protein modulators, favoring a direct action of E2 on GlyRs. In HEK293 cells expressing various combinations of GlyR subunits, E2 only affected the IGly in cells expressing α2, α2β or α3β subunits, suggesting that either α2-containing or α3β-GlyRs mediate the E2 effect observed in neurons. Furthermore, E2 inhibited the GlyR-mediated tonic current in pyramidal neurons of HIP CA1 region, where abundant GlyR α2 subunit is expressed. We suggest that the neuronal GlyR is a novel molecular target of E2 which directly inhibits the function of GlyRs in the HIP and SDH regions. This finding may shed new light on premenstrual dysphoric disorder and the gender differences in pain sensation at the CNS level.

CFTR is activated through stimulation of purinergic P2Y2 receptors

It has been reported that the cystic fibrosis transmembrane conductance regulator (CFTR) can be activated through cAMP- and protein kinase A-independent pathways involving GTP-binding proteins and an unknown kinase. In this study, we further examined how G protein-coupled pathways regulate CFTR. We demonstrate that stimulation of purinergic P2Y(2) receptors in CFTR-expressing oocytes and in airway epithelial cells activates CFTR Cl(-) currents. Activation of CFTR Cl(-) currents via P2Y(2) was inhibited by CFTR(inh)-172 and was independent of intracellular Ca(2+), protein kinase C, or calmodulin-dependent kinase (CAMK). However, activation of CFTR was suppressed by inhibition of phospholipase C and by the nonselective protein kinase inhibitor staurosporine. Activation of CFTR through P2Y(2) receptors was enhanced when G(i) proteins were inhibited by pertussis toxin. Inhibition of protein kinase A and of protein kinases downstream of P2Y(2) receptors such as mitogen-activated protein kinases, tyrosine kinase, or c-src kinase did not interfere with activation of CFTR. The present results demonstrate an antagonistic regulation of CFTR by P2Y(2) receptors: CFTR is inhibited by stimulation of G(i) proteins and is activated by stimulation of G(q/11)/PLC and an unknown downstream protein kinase.

Proteinase-activated Receptor 2 Stimulates Na,K-ATPase and Sodium Reabsorption in Native Kidney Epithelium

Proteinase-activated receptors 2 (PAR2) are expressed in kidney, but their function is mostly unknown. Since PAR2 control ion transport in several epithelia, we searched for an effect on sodium transport in the cortical thick ascending limb of Henle's loop, a nephron segment that avidly reabsorbs NaCl, and for its signaling. Activation of PAR2, by either trypsin or a specific agonist peptide, increased the maximal activity of Na,K-ATPase, its apparent affinity for sodium, the sodium permeability of the paracellular pathway, and the lumen-positive transepithelial voltage, featuring increased NaCl reabsorption. PAR2 activation induced calcium signaling and phosphorylation of ERK1,2. PAR2-induced stimulation of Na,K-ATPase Vmax was fully prevented by inhibition of phospholipase C, of changes in intracellular concentration of calcium, of classical protein kinases C, and of ERK1,2 phosphorylation. PAR2-induced increase in paracellular sodium permeability was mediated by the same signaling cascade. In contrast, increase in the apparent affinity of Na,K-ATPase for sodium, although dependent on phospholipase C, was independent of calcium signaling, was insensitive to inhibitors of classical protein kinases C and of ERK1,2 phosphorylation, but was fully prevented by the nonspecific protein kinase inhibitor staurosporine, as was the increase in transepithelial voltage. In conclusion, PAR2 increases sodium reabsorption in rat thick ascending limb of Henle's loop along both the transcellular and the paracellular pathway. PAR2 effects are mediated in part by a phospholipase C/protein kinase C/ERK1,2 cascade, which increases Na,K-ATPase maximal activity and the paracellular sodium permeability, and by a different phospholipase C-dependent, staurosporine-sensitive cascade that controls the sodium affinity of Na,K-ATPase.

Systematic genetic array analysis links the Saccharomyces cerevisiae SAGA/SLIK and NuA4 component Tra1 to multiple cellular processes

BackgroundTra1 is an essential 437-kDa component of the Saccharomyces cerevisiae SAGA/SLIK and NuA4 histone acetyltransferase complexes. It is a member of a group of key signaling molecules that share a carboxyl-terminal domain related to phosphatidylinositol-3-kinase but unlike many family members, it lacks kinase activity. To identify genetic interactions for TRA1 and provide insight into its function we have performed a systematic genetic array analysis (SGA) on tra1SRR3413, an allele that is defective in transcriptional regulation.ResultsThe SGA analysis revealed 114 synthetic slow growth/lethal (SSL) interactions for tra1SRR3413. The interacting genes are involved in a range of cellular processes including gene expression, mitochondrial function, and membrane sorting/protein trafficking. In addition many of the genes have roles in the cellular response to stress. A hierarchal cluster analysis revealed that the pattern of SSL interactions for tra1SRR3413 most closely resembles deletions of a group of regulatory GTPases required for membrane sorting/protein trafficking. Consistent with a role for Tra1 in cellular stress, the tra1SRR3413 strain was sensitive to rapamycin. In addition, calcofluor white sensitivity of the strain was enhanced by the protein kinase inhibitor staurosporine, a phenotype shared with the Ada components of the SAGA/SLIK complex. Through analysis of a GFP-Tra1 fusion we show that Tra1 is principally localized to the nucleus.ConclusionWe have demonstrated a genetic association of Tra1 with nuclear, mitochondrial and membrane processes. The identity of the SSL genes also connects Tra1 with cellular stress, a result confirmed by the sensitivity of the tra1SRR3413 strain to a variety of stress conditions. Based upon the nuclear localization of GFP-Tra1 and the finding that deletion of the Ada components of the SAGA complex result in similar phenotypes as tra1SRR3413, we suggest that the effects of tra1SRR3413 are mediated, at least in part, through its role in the SAGA complex.

B7-H1 is a ubiquitous antiapoptotic receptor on cancer cells

AbstractB7-H1 is an immunoglobulin-like immune suppressive molecule broadly detectable on the majority of human and rodent cancers, and its functions have been attributed to delivering an inhibitory signal to its counter-receptor programmed death-1 (PD-1) on T cells. Here we report that B7-H1 on cancer cells receives a signal from PD-1 to rapidly induce resistance against T cell–mediated killing because crippling signaling capacity of B7-H1 but not PD-1 ablates this resistance. Importantly, loss of B7-H1 signaling is accompanied by increased susceptibility to immune-mediated tumoricidal activity. In addition to resistance against T-cell destruction, B7-H1+ cancer cells also become refractory to apoptosis induced by Fas ligation or the protein kinase inhibitor Staurosporine. Our study reveals a new mechanism by which cancer cells use a receptor on immune cells as a ligand to induce resistance to therapy.

Cloning and characterization of a novel LpWRKY1 transcription factor in tomato

The initiation of defence responses in plants is accompanied by fundamental changes in gene expression: the expression of pathogenesis-related genes is co-ordinately regulated with metabolic changes such as down regulation of photosynthesis and induction of sink metabolism. To identify candidate regulators of this co-ordinated regulatory mechanism, the role of WRKY transcription factors in the initiation of defence response was analysed in tomato. A WRKY-type transcription factor (LpWRKY1) from tomato was cloned by a reverse Northern approach. The corresponding mRNA is rapidly and transiently induced after challenging the cells with an elicitor-preparation derived from the wilt inducing fungus Fusarium oxysporum lycopersici (E-FOL) and the fungal elicitor chitosan, whereas the endogenous signals systemin and salicylic acid are inactive. Inhibition of protein biosynthesis by cycloheximide results in sustained induction of mRNA for LpWRKY1. In contrast, the transient induction of the gene encoding LpWRKY1 in response to elicitation by E-FOL is inhibited by the protein-kinase inhibitor staurosporine and may be mimicked by the phosphatase inhibitors endothall and cantharidine indicating the involvement of protein phosphorylation in the regulation of WRKY-type transcription factors. Direct proof of this postranslational modification of LpWRKY1 was obtained by demonstrating in-gel kinase assays using recombinant LpWRKY1 as substrate. A 44kDa and a 67 kDa protein kinase were shown to be transiently activated to phosphorylate LpWRKY1 protein in response to elicitation with E-FOL.

Potential role of NADPH-oxidase in early steps of lead-induced oxidative burst in Vicia faba roots

The mechanism of oxidative burst induced by lead in Vicia faba excised roots was investigated by luminol-dependent chemiluminescence. Results showed that lead triggered a rapid and dose-dependent increase in chemiluminescence production. In this study, specific inhibitors of putative reactive oxygen species (ROS) sources were used to determine the mechanism of lead-induced ROS generation. This generation was sensitive to dephenylene iodonium (DPI), quinacrine and imidazole, some inhibitors of the NADPH-oxidase and not inhibited by other putative ROS sources inhibitors. Data reported in this work clearly demonstrated the pivotal role of NADPH-oxidase-like enzyme in early steps of lead-induced oxidative burst. To investigate the respective implication of calmodulin and protein kinase (PK) in lead-induced NADPH-oxidase activation, excised roots were treated with the calmodulin inhibitor W7 or with the PK inhibitor staurosporine. The chemiluminescence generation inhibition by these inhibitors illustrated the role of PK in lead-induced NADPH-oxidase activation and revealed a calmodulin-dependent step. Using the calcium entry blocker La(3+) or different concentrations of calcium in the extra-cellular medium, our data highlighted the implication of Ca(2+) channel in lead-induced oxidative burst.

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H +-dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of d- and l-[U- 14C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3- O-methyl- d-glucose/ d-[U- 14C]glucose and 3- O-methyl- d-glucose/3- O-methyl- d-[U- 14C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH 4Cl inhibited neither the linear component of d- and l-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3- O-methyl- d-[U- 14C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1–43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol − 1 , respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol − 1 ). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.

Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells

Protein phosphorylation/dephosphorylation and cytoskeletal reorganization regulate the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) during osmotic shrinkage; however, the mechanisms involved are unclear. We show that in cytoplasts, plasma membrane vesicles detached from Ehrlich ascites tumor cells (EATC) by cytochalasin treatment, NKCC1 activity evaluated as bumetanide-sensitive (86)Rb influx was increased compared with the basal level in intact cells yet could not be further increased by osmotic shrinkage. Accordingly, cytoplasts exhibited no regulatory volume increase after shrinkage. In cytoplasts, cortical F-actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the myosin light chain kinase (MLCK) inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact EATC. Cytoplast NKCC1 activity was potentiated by the Ser/Thr protein phosphatase inhibitor calyculin A, partially inhibited by the protein kinase A inhibitor H89, and blocked by the broad protein kinase inhibitor staurosporine. Cytoplasts exhibited increased protein levels of NKCC1, Ste20-related proline- and alanine-rich kinase (SPAK), and oxidative stress response kinase 1, yet they lacked the shrinkage-induced plasma membrane translocation of SPAK observed in intact cells. The basal phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in cytoplasts compared with intact cells, yet in contrast to the substantial activation in shrunken intact cells, p38 MAPK could not be further activated by shrinkage of the cytoplasts. Together these findings indicate that shrinkage activation of NKCC1 in EATC is dependent on the cortical F-actin network, myosin II, and MLCK.

Protein kinase C inhibition attenuates hypochlorite-induced acute lung injury

Neutrophil-derived oxidative stress plays a crucial role in acute lung injury. Hypochlorite/hypochlorous acid (HOCl) is a major oxidant of neutrophils. Protein kinase C (PKC) may be an appropriate target for HOCl due to its functionally important thiols. This study investigates the role of PKC in HOCl-induced acute lung injury. Isolated lung preparations were from 30 rabbits. HOCl (1000 nmol min(-1)) or buffer (control) were infused into isolated rabbit lungs. Pulmonary artery pressure (PAP [Torr]) and lung weight were continuously measured. Capillary filtration coefficient (K(f,c)), was measured at baseline and at 30, 60, 90 min. Experiments were terminated at 105 min or when fluid retention exceeded 50 g. The non-selective protein kinase inhibitor staurosporin (100 nM) or the selective PKC inhibitor bisindolylmaleimide I (GF109203X, 10nM) were added to the perfusate 5 min prior to the start of the experiments. Staurosporin completely prevented the HOCl-induced increase in PAP (no change versus DeltaPAP(max) 5.2+/-0.78) but did not influence the increase in vascular permeability. GF109203X delayed the HOCl-induced increase in PAP and vascular permeability. PAP(max) was observed significantly later in the HOCl-GF109203X group (84.4+/-4.0 min) in comparison with the HOCl group (52.1+/-3.5 min). Termination of the experiments due to edema formation occurred significantly later in experiments with GF109203X (91.8+/-1.9 versus 79.2+/-4.1 min). Protein kinases are involved in HOCl-induced acute lung injury. Specifically PKC inhibition delayed HOCl-induced increases in PAP and vascular permeability.

PI3 Kinase Dependent Stimulation of Gastric Acid Secretion by Dexamethasone

Excessive gastric acid secretion plays an important role in the pathogenesis of peptic ulcers. Dexamethasone, a widely used drug, is known to stimulate gastric acid secretion and increase the incidence of peptic ulcers. However little is known about the mechanism of the dexamethasone’s effect on parietal cells. The present study was performed to investigate the contribution of the phosphatidylinositol-3-kinase (PI3 kinase) to dexamethasone induced stimulation of gastric acid secretion. In vivo pretreatment with dexamethasone injections (150µg/100g for 3 days) or in vitro exposure to (10 µM for > 20 minutes) significantly increased acid secretion in isolated gastric glands ñ 2-3 fold. The dexamethasone induced stimulation of gastric acid secretion was concentration dependent and significantly blunted by the H²⁺/K²⁺ ATPase inhibitor omeprazole (200 µM), the PI3 kinase inhibitor Wortmannin (500 nM), the protein kinase inhibitor staurosporine (2.5 µM) and the Cl^- channel blocker NPPB (100 µM); but not by the H₂ antagonist cimetidine (100 µM). In conclusion, it was observed that dexamethasone’s effect on proton extrusion requires the activity of a PI3 kinase pathway, an apical Cl^- channel and the H²⁺/K²⁺ ATPase.

Rapid and Drastic Induction of CYP3A4 mRNA Expression via Vitamin D Receptor in Human Intestinal LS180 Cells

The aim of this study was to evaluate the usefulness of human intestinal LS180 cells for studying the induction of CYP3A4 mRNA expression via vitamin D receptor (VDR). CYP3A4 mRNA expression in LS180 cells treated with 100 nM 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) for 6 and 24 h was approximately 80- and 500-fold higher than the control, respectively. A protein kinase (PK) inhibitor (staurosporine), c-jun N-terminal kinase (JNK) pathway inhibitor (curcumin), and JNK inhibitor (SP600125) attenuated 1alpha,25(OH)(2)D(3)-induced CYP3A4 mRNA expression, suggesting that the PK-JNK pathway contributed to the rapid and drastic induction of CYP3A4 expression via VDR in LS180 cells. The ability of CYP3A4 mRNA induction in LS180 cells was highly dependent on the site and number of vitamin D(3) and D(2) hydroxylation. In addition, short-time (6 h) treatment of LS180 cells with cytotoxic secondary bile acids, lithocholic acid (LCA) and 3-keto-LCA also significantly induced the mRNA expression of CYP3A4. LS180 cells may be useful to quickly investigate the CYP3A4-inducing effect of drugs, xenobiotics, and/or endogenous substrates in the intestinal epithelia.

Elicitin-responsive lectin-like receptor kinase genes in BY-2 cells

The inhibition of elicitor-induced plant defense responses by the protein kinase inhibitors K252a and staurosporine indicates that defense responses require protein phosphorylation. We isolated a cDNA clone encoding Nicotiana tabacum lectin-like receptor protein kinase 1 (NtlecRK1), an elicitor-responsive gene; in tobacco bright yellow (BY-2) cells by a differential display method. NtlecRK forms a gene family with at least three members in tobacco. All three NtlecRK genes potentially encode the N-terminal legume lectin domain, transmembrane domain and C-terminal Ser/Thr-type protein kinase domain. Green fluorescent protein (GFP) fusion showed that the NtlecRK1 protein was located on the plasma membrane. In addition, NtlecRK1 and 3 were responsive to INF1 elicitin and the bacterial elicitor harpin. These results indicate that NtlecRKs are membrane-located protein kinases that are induced during defense responses in BY-2 cells.

Cell death, caspase activation, and HMGB1 release of porcine choroid plexus epithelial cells during Streptococcus suis infection in vitro

The choroid plexus epithelium constitutes the structural basis of the blood–cerebrospinal fluid barrier. We previously demonstrated that Streptococcus suis ( S. suis), a relevant cause of bacterial meningitis in pigs and humans, affects porcine choroid plexus epithelial cell (PCPEC) barrier function and integrity. We now characterized PCPEC cell death and investigated whether apoptosis or necrosis is responsible for the cytotoxicity after infection with different S. suis isolates. We found S. suis strain-dependent histone associated DNA-fragments quantified by ELISA. This response could partially be inhibited by cylcoheximide, cytochalasin D, dexamethasone, herbimycin A, but most effectively by the pan-caspase inhibitor zVAD-fmk. We further detected caspase-3 and -9 activation after infection with all tested S. suis isolates that could also be blocked by zVAD-fmk. However, we found a significantly stronger caspase activity with the protein kinase inhibitor staurosporine. All tested S. suis isolates induced loss of cell viability in PCPEC as shown with the Live/Dead assay (R), but strain dependent lactate dehydrogenase-release. Both parameters could not be influenced by zVAD-fmk. Immunostaining showed release of high-mobility group box 1 (HMGB1) protein from the nucleus, indicative of necrosis. Transmission electron microscopy showed cell swelling, cytoplasmic vacuolization, loss of membrane integrity, nuclear fermentation but no nuclear condensation, indices for a primarily necrotic cell morphology. Taken together, our findings indicate that S. suis causes cell death in PCPEC by different mechanisms. Although apoptosis may be involved in the process of PCPEC cell death, necrosis seems to be the predominant mechanism. Through inflammation in the choroid plexus during bacterial meningitis, the blood–cerebrospinal fluid barrier function will be compromised.

Cross‐inhibitory interactions between GABAA and P2X channels in myenteric neurones

Inhibitory interactions between GABA(A)[induced by gamma-aminobutyric acid (GABA)] and P2X [activated by adenosine 5'-triphosphate (ATP)] receptors of myenteric neurones from the guinea pig small intestine were characterized using whole-cell recordings. Currents induced by GABA (I(GABA)) or ATP (I(ATP)) were inhibited by picrotoxin or pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, respectively. Currents induced by GABA + ATP (I(GABA+ATP)) were only as large as the current induced by the most effective transmitter, revealing current occlusion. This occlusion requires maximal activation of at least one of these receptors. Sequential applications of neurotransmitters, and kinetic and pharmacological properties of I(GABA+ATP) indicate that they are carried through both GABA(A) and P2X channels. ATP did not affect I(GABA) in neurones: (i) in which P2X channels were not present; (ii) after inhibiting P2X channels with Ca2+ (iii) in the presence of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, a P2X receptor antagonist; (iv) after P2X receptor desensitization or (v) at I(ATP) reversal potential. Similarly, GABA did not affect P2X-mediated currents in neurones: (i) in which GABA(A) channels were not present; (ii) in the presence of picrotoxin, a GABA(A) channel blocker; (iii) after GABA(A) receptor desensitization or (iv) at the I(GABA) reversal potential. Current occlusion occurred as fast as current activation and it was still present in the absence of Ca2+, at 11 degrees C, after adding to the pipette solution a cocktail of protein kinase inhibitors (staurosporine + genistein + K-252a), after substituting the GTP in the pipette with GDP-beta-S and after treating the cells with N-ethylmaleimide. Taken together, all of these results are consistent with a model of cross-inhibition between GABA(A) and P2X.