Poplar leaf abscission through induced chlorophyll breakdown by Mg-dechelatase.

Chlorophyll breakdown is observed during senescence. The first step in chlorophyll breakdown is the removal of central Mg by Mg-dechelatase. This reaction is the rate-limiting step in the chlorophyll breakdown pathway. We evaluated the effect of induced chlorophyll breakdown on abscission through the removal of Mg by Mg-dechelatase. Poplar transformants carrying the dexamethasone-inducible Mg-dechelatase gene were prepared using the Arabidopsis Stay-Green1 cDNA. When leaves were treated with dexamethasone, chlorophyll was degraded, photosynthetic capacity was reduced, and an abscission zone was formed, resulting in leaf abscission. In addition, ethylene, which plays an important role during senescence, was produced in this process. Thus, chlorophyll breakdown induces the phenotype in the same way as commonly observed during leaf senescence. This study suggests a physiological role of chlorophyll breakdown in the leaf abscission of deciduous trees. Furthermore, this study shows that the dexamethasone-inducible gene expression system is an available option for deciduous tree studies.

Antisense vimentin cDNA combined with chondroitinase ABC promotes axon regeneration and functional recovery following spinal cord injury in rats.

The formation of glial scar restricts axon regeneration after spinal cord injury (SCI) in adult mammalian. Chondroitin sulfate proteoglycans (CSPGs) are mostly secreted by reactive astrocytes, which form dense scar tissues after SCI. Chondroitinase ABC (ChABC), which can digest CSPGs, is a promising therapeutic strategy for SCI. However, to date ChABC has exhibited only limited success in the treatment of chronic SCI. The intermediate filament protein vimentin underpins the cytoskeleton of reactive astrocytes. We targeted glial scar in injured spinal cord by sustained infusion of ChABC and antisense vimentin cDNA. Using anterograde tracing, BBB scoring and hind limb placing response, we found that this combined treatment promoted axon regeneration and functional recovery after SCI in rats. Our results indicate that axon regeneration may be promoted by modified physical and biochemical characteristics of intra- and extracellular architecture in glial scar tissues. Theses findings could potentially help us to understand better the composition of glial scar in central nervous system injury.

Gene delivery of bone morphogenetic protein-2 plasmid DNA promotes bone formation in a large animal model.

In the field of bone regeneration, BMP-2 is considered one of the most important growth factors because of its strong osteogenic activity, and is therefore extensively used in clinical practice. However, the short half-life of BMP-2 protein necessitates the use of supraphysiological doses, leading to severe side-effects. This study investigated the efficiency of bone formation at ectopic and orthotopic sites as a result of a low-cost, prolonged presence of BMP-2 in a large animal model. Constructs consisting of alginate hydrogel and BMP-2 cDNA, together acting as a non-viral gene-activated matrix, were combined with goat multipotent stromal cells (gMSCs) and implanted in spinal cassettes or, together with ceramic granules, intramuscularly in goats, both for 16 weeks. Bone formation occurred in all cell-seeded ectopic constructs, but the constructs containing both gMSCs and BMP-2 plasmid DNA showed higher collagen I and bone levels, indicating an osteogenic effect of the BMP-2 plasmid DNA. This was not seen in unseeded constructs, even though transfected, BMP-2-producing cells were detected in all constructs containing plasmid DNA. Orthotopic constructs showed mainly bone formation in the unseeded groups. Besides bone, calcified alginate was present in these groups, acting as a surface for new bone formation. In conclusion, transfection of seeded or resident cells from this DNA delivery system led to stable expression of BMP-2 during 16 weeks, and promoted osteogenic differentiation and subsequent bone formation in cell-seeded constructs at an ectopic location and in cell-free constructs at an orthotopic location in a large animal model.

cDNA cloning, overexpression, purification and pharmacologic evaluation for anticancer activity of ribosomal protein L23A gene (RPL23A) from the Giant Panda.

RPL23A gene encodes a ribosomal protein that is a component of the 60S subunit. The protein belongs to the L23P family of ribosomal proteins, which is located in the cytoplasm. The purpose of this paper was to explore the structure and anti-cancer function of ribosomal protein L23A (RPL23A) gene of the Giant Panda (Ailuropoda melanoleuca). The cDNA of RPL23A was cloned successfully from the Giant Panda using RT-PCR technology. We constructed a recombinant expression vector containing RPL23A cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified by using Ni chelating affinity chromatography. Recombinant protein of RPL23A obtained from the experiment acted on Hep-2 cells and human HepG-2 cells, then the growth inhibitory effect of these cells was observed by MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay. The result indicated that the length of the fragment cloned is 506 bp, and it contains an open-reading frame (ORF) of 471 bp encoding 156 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL23A protein is 17.719 kDa with a theoretical pI 11.16. The molecular weight of the recombinant protein RPL23A is 21.265 kDa with a theoretical pI 10.57. The RPL23A gene can be really expressed in E. coli and the RPL23A protein, fusioned with the N-terminally His-tagged protein, gave rise to the accumulation of an expected 22 KDa polypeptide. The data showed that the recombinant protein RPL23A had a time- and dose-dependency on the cell growth inhibition rate. The data also indicated that the effect at low concentrations was better than at high concentrations on Hep-2 cells, and that the concentration of 0.185 µg/mL had the best rate of growth inhibition of 36.31%. All results of the experiment revealed that the recombinant protein RPL23A exhibited anti-cancer function on the Hep-2 cells. The study provides a scientific basis and aids orientation for the research and development of cancer protein drugs as well as possible anti-cancer mechanisms. Further research is on going to determine the bioactive principle(s) of recombinant protein RPL23A responsible for its anticancer activity.

An automated system for intracellular and intranuclear injection.

The Xenopus oocyte expression system has played an important role in the study of cellular proteins, particularly in the field of membrane physiology; expression of transporters and ion channels has significantly advanced our knowledge of these membrane proteins and the rapid and easy expression of mutants has been crucial in many structure-function studies. Xenopus oocytes are an expression system in many ligand-binding assays and in functional screening for ion channel modulators. Several commercially available automated technologies use this system, generating a demand for large numbers of oocytes injected with ion channel genes. Injection of oocytes with genetic material is generally carried out manually. Here we describe an automated system capable of injecting up to 600 oocytes per hour. Oocytes are contained in microplates with conical wells, a simple calibration procedure by the operator is required and pipette filling and oocyte injection are carried out automatically. Following intracellular injection of mRNA coding for ligand-gated ion channels close to 100% of oocytes tested positive for expression, and intranuclear injection of cDNA gave a rate of expression >50%. Moreover, we demonstrate that this method can also be successfully applied to inject zebrafish embryos and could be extended to other cell types.

Alteration of epithelial structure and function associated with PtdIns(4,5)P2 degradation by a bacterial phosphatase.

Elucidation of the role of PtdIns(4,5)P(2) in epithelial function has been hampered by the inability to selectively manipulate the cellular content of this phosphoinositide. Here we report that SigD, a phosphatase derived from Salmonella, can effectively hydrolyze PtdIns(4,5)P(2), generating PtdIns(5)P. When expressed by microinjecting cDNA into epithelial cells forming confluent monolayers, wild-type SigD induced striking morphological and functional changes that were not mimicked by a phosphatase-deficient SigD mutant (C462S). Depletion of PtdIns(4,5)P(2) in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of phospholipase Cdelta, used as a probe for the phosphoinositide by conjugation to green fluorescent protein. Single-cell measurements of cytosolic pH indicated that the Na(+)/H(+) exchange activity of epithelia was markedly inhibited by depletion of PtdIns(4,5)P(2). Similarly, anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of PtdIns(4,5)P(2) was associated with marked alterations in the actin cytoskeleton and its association with the plasma membrane. The junctional complexes surrounding the injected cells gradually opened and the PtdIns(4,5)P(2)-depleted cells eventually detached from the monolayer, which underwent rapid restitution. Similar observations were made in intestinal and renal epithelial cultures. In addition to its effects on phosphoinositides, SigD has been shown to convert inositol 1,3,4,5,6-pentakisphosphate (IP(5)) into inositol 1,4,5,6-tetrakisphosphate (IP(4)), and the latter has been postulated to mediate the diarrhea caused by Salmonella. However, the effects of SigD on epithelial cells were not mimicked by microinjection of IP(4). In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing PtdIns(4,5)P(2) with a membrane-targeted 5-phosphatase or by occluding the inositide using high-avidity tandem PH domain constructs. We therefore suggest that opening of the tight junctions and inhibition of Na(+)/H(+) exchange caused by PtdIns(4,5)P(2) hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced diarrhea.

The roboocyte: automated electrophysiology based on Xenopus oocytes.

Automated electrophysiological assays are of great importance for modern drug discovery, and various approaches have been developed into practical devices. Here, we describe the automation of two-electrode voltage-clamp (TEVC) recording from Xenopus oocytes using the Roboocyte automated workstation, jointly developed by Multi Channel Systems and Bayer Technology Services. We briefly discuss the technology, including its advantages and limitations relative to patch clamp and other TEVC systems. We provide a step-by-step description of typical operating procedures and show that the Roboocyte represents a practical and highly effective way to perform automated electrophysiology in an industrial setting.

Activation of 5-HT3 receptors in the rat and mouse intestinal tract: a comparative study.

This study provides a comprehensive evaluation of 5-HT(3) receptor functional distribution in both the rat and mouse intestinal tract. 5-HT(3A-S) receptor splice variant mRNA was expressed throughout the intestine of the rat and mouse; the 5-HT(3A-L) variant being more common in the rat.5-HT, m-CPB, 1-PBG and 2-methyl-5-hydroxytryptamine (2m5-HT) induced contraction in the jejunum, ileum, proximal colon and distal colon of the rat (pEC(50) range: 2m5-HT, 5.86+/-0.40 to m-CPB, 7.47+/-0.27) and mouse (pEC(50) range: 1-PBG, 5.34+/-0.06 to m-CPB, 6.49+/-0.14) in the presence of nontarget 5-HT receptor antagonists, methysergide (1 muM) and GR125487 (0.1 microM). The rank orders of potency in the four regions of the rat and mouse intestine were concordant with the accepted order and the responses to 5-HT were inhibited by ondansetron (0.1 microM).5-HT(3)-induced contractions to 5-HT were reduced by tetrodotoxin (1 microM). Pargyline (10 muM) and fluoxetine (1 microM) potentiated responses in the rat jejunum. Atropine (0.1 microM) potentiated 5-HT(3)-induced responses in the rat jejunum (E(max) 49-65%), but attenuated responses in most other regions of the rat and mouse (e.g. mouse ileum: E(max) 57-26%). In the rat jejunum, L-NAME (100 microM) mimicked the effect of atropine, hexamethonium (100 microM) suppressed 5-HT(3)-induced responses, but tachykinin receptor antagonists were without effect. It is concluded that functional 5-HT(3) receptors are present in nerves along the length of the rat and mouse intestinal tract. The mouse proximal colon was found to discriminate 5-HT(3) receptor agonist profiles better than any other region in the rat or mouse. The rat jejunum shows evidence of 5-HT uptake and inactivation processes as well as inhibitory nitrergic and nontachykinin excitatory pathways associated with the 5-HT(3)-induced response.

In vivo inhibition of steroidogenic acute regulatory protein expression by dexamethasone parallels induction of the negative transcription factor DAX-1.

In this study, we investigated the effect of dexamethasone on the synthesis of steroidogenic acute regulatory protein (StAR) and the expression of DAX-1 (dosage sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1) and SF-1 (steroidogenic factor-1) in vivo. Male rats were treated with dexamethasone (0.4 and 4 mg/kg body wt per day) by intraperitoneal injections using phosphate-buffered saline as the vehicle for 7 d. At the end of 7 d, serum testosterone levels were decreased. Response to luteinizing hormone (LH) and 8-bromo-cyclic-AMP (8-Br-cAMP) in vitro was reduced in testicular cells isolated from dexamethasone-treated rat testes. Dexamethasone decreased LH-stimulated cAMP production. The conversion of 22(R)-hydroxycholesterol, pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone, and androstenedione to testosterone was not affected by dexamethasone. Dexamethasone increased DAX-1 expression and concordantly decreased StAR protein and mRNA in testicular cells. The increase in DAX-1 protein corresponded to a 57% reduction in StAR mRNA levels concomitant with a 79% reduction in serum testosterone levels. Dexamethasone had no effect on the level of SF-1, but increased the amount of complexed DAX-1-SF-1. Dexamethasone in vitro suppressed StAR promoter activity when an increasing amount of DAX-1 cDNA was transfected. These results demonstrate that dexamethasone increases expression of DAX-1, which results in increased amounts of complexed DAX-1-SF-1, in the absence of any change in the expression of SF-1. These observations strongly support the concept that dexamethasone suppresses rat testicular testosterone production, at least in part, by increasing the amount of complexed DAX-1-SF-1 in these cells, which leads directly to decreased availability of free SF-1 and, therefore, decreased activation of transcription of the rat StAR gene.

Hepatocyte growth factor enhances protein phosphatase Cdc25A inhibitor compound 5-induced hepatoma cell growth inhibition via Akt-mediated MAPK pathway.

We have previously shown that Compound 5 (Cpd 5), an inhibitor of protein phosphatase Cdc25A, inhibits Hep3B human hepatoma cell growth. We now show that hepatocyte growth factor (HGF), a hepatocyte growth stimulant, can strongly enhance Cpd 5-induced growth inhibition in Hep3B cells, and this enhancement in cell growth inhibition is correlated with a much stronger ERK phosphorylation when compared to cells treated with Cpd 5 or HGF separately. We found that HGF/Cpd 5-induced ERK phosphorylation and cell growth inhibition were mediated by Akt (protein kinase B) pathway, since combination HGF/Cpd 5 treatment of Hep3B cells inhibited Akt phosphorylation at Ser-473 and its kinase activity, which led to the suppression of Raf-1 phosphorylation at Ser-259. The suppression of Raf-1 Ser-259 phosphorylation caused the induction of Raf-1 kinase activity, as well as hyper-ERK phosphorylation. Transient transfection of Hep3B cells with dominant negative Akt c-DNA further enhanced both Cpd 5- and HGF/Cpd 5-induced ERK phosphorylation, while over-expression of wild-type Akt c-DNA diminished their effects. In contrast, HGF antagonized the growth inhibitory actions of Cpd 5 on normal rat hepatocytes, thus showing a selective effect on tumor cells compared to normal cells. Our data suggest that Akt kinase negatively regulates MAPK activity at the Akt-Raf level. Suppression of Akt activity by either combination HGF/Cpd 5 treatment or by dominant negative Akt c-DNA transfection antagonizes the Akt inhibitory effect on Raf-1, resulting in an enhancement of Cpd 5-induced MAPK activation and cell growth inhibition.

Intracorporal injection of hSlo cDNA restores erectile capacity in STZ-diabetic F-344 rats in vivo.

The ability of gene transfer with the pore-forming subunit of the human maxi-K channel (hSlo) to ameliorate the decline in erectile capacity commensurate with 12-24 wk of streptozotocin (STZ)-diabetes was examined in 181 Fischer-344 rats. A 2-mo period of STZ-diabetes was induced before gene transfer, and erectile capacity was evaluated by measuring the intracavernous pressure response (ICP) to cavernous nerve (CN) stimulation (ranging from 0.5 to 10 mA). In the first series of experiments, ANOVA revealed increased CN-stimulated ICP responses at 1 and 2 mo postinjection of 100 microg pcDNA-hSlo compared with control values. A second series of experiments further examined the dose dependence and duration of gene transfer. The ICP response to submaximal (0.5 mA) and maximal (10 mA) nerve stimulation was evaluated 3 or 4 mo postinjection of a single dose of pcDNA-hSlo ranging from 10 to 1,000 microg. ANOVA again revealed that hSlo overexpression was associated with increased CN-stimulated ICP responses compared with responses in corresponding control animals. Histological studies revealed no immune response to the presence of hSlo. PCR analysis documented that expression of both plasmid and transcript were largely confined to the corporal tissue. In the third series of pharmacological experiments, hSlo gene transfer in vivo was associated with iberiotoxin-sensitive relaxation responses to sodium nitroprusside in corporal tissue strips in vitro. The latter data indicate that gene transfer produces functional maxi-K channels that participate in the modulation of corporal smooth muscle cell tone. Taken together, these observations suggest a fundamental diabetes-related change in corporal myocyte maxi-K channel regulation, expression, or function that may be corrected by expression of recombinant hSlo.

Serum response factor promotes re-epithelialization and muscular structure restoration during gastric ulcer healing.

BACKGROUND & AIMS: Serum response factor (SRF) regulates transcription of immediate early genes and muscle genes. In this study, we examined the role of SRF in gastric ulcer healing and the mechanisms involved. METHODS: Gastric ulcers were induced in rats by serosal application of acetic acid. Gastric specimens were obtained sequentially after ulcer induction for analyses of SRF messenger RNA (mRNA), protein expression, and for immunohistochemistry. We examined the role of SRF in ulcer healing by local injection of an SRF expression plasmid into ulcers (gene therapy). To elucidate the cellular mechanisms of the action of SRF, we examined the effect of SRF overexpression on actin dynamics, cell migration, and proliferation in rat gastric epithelial cell (RGM1) and smooth muscle cell (A7R5). To determine the clinical relevance, we examined SRF expression in human gastric ulcer specimens. RESULTS: Gastric ulceration activated SRF expression in epithelial cells lining regenerating glands and in myofibroblasts and smooth muscle cells of granulation tissue. SRF up-regulation in human gastric ulcers was similar to that found in rat gastric ulcers. Gene therapy with SRF significantly accelerated experimental gastric ulcer healing and promoted re-epithelialization and muscle restoration. Overexpression of SRF in RGM1 and A7R5 cells accelerates migration and proliferation of these cells by promoting actin polymerization and activation of immediately early genes. CONCLUSIONS: Activation of SRF is an important component of ulcer healing. SRF promotes migration and proliferation of gastric epithelial and smooth muscle cells, which are essential for re-epithelialization and restoration of muscular structures.

Calmodulin regulates synaptic plasticity in the anterior cingulate cortex and behavioral responses: a microelectroporation study in adult rodents.

We developed a microelectroporation method for the transfer of genes into neurons in the cerebral cortex of adult rodents, both rats and mice. We selectively expressed either green-fluorescent protein (GFP) or a Ca2+-binding deficient calmodulin (CaM) mutant in the anterior cingulate cortex (ACC). In mice that expressed GFP, positive neuronal cell bodies were found specifically at the injection site in the ACC. Mice that expressed CaM12, a mutant CaM with two impaired Ca2+ binding sites in the N-terminal lobe, exhibited significant changes in vocalization, locomotion, and sensory functions. Long-term potentiation and long-term depression, two major forms of central plasticity, were completely abolished by expression of CaM12. Mice that expressed CaM34, a mutant CaM with two impaired Ca2+ binding sites in the C-terminal lobe, did not show any significant behavioral or electrophysiological alterations. These findings provide strong evidence that CaM is critical for bidirectional synaptic plasticity. This new method will be useful for investigating gene function in specific brain regions of freely moving animals. Furthermore, this approach also may facilitate gene therapy in adult human brains.

Genetic and cellular basis for acetylcholine inhibition of Caenorhabditis elegans egg-laying behavior.

Egg-laying behavior in Caenorhabditis elegans is activated by signaling through the G-protein G(rho)q and inhibited by signaling through a second G-protein, G(rho)o. Activation of egg laying depends on the serotonergic hermaphrodite-specific neurons (HSNs), but the neurotransmitter(s) and cell(s) that signal to inhibit egg laying are not known. Mutants for G-protein signaling genes have well characterized defects in egg laying. Here we present an analysis of mutants for other genes reported to lack inhibition of egg laying. Of the nine strongest, six have morphological defects in the ventral-type C (VC) neurons, which synapse onto both the HSNs and the egg-laying muscles and are thus the third cell type comprising the egg-laying system. Laser-ablating VC neurons could also disrupt the inhibition of egg laying. The remaining three mutants (unc-4, cha-1, and unc-17) are defective for synthesis or packaging of acetylcholine in the VCs. The egg-laying defects of unc-4, cha-1, and unc-17 were rescued by VC-specific expression of the corresponding cDNAs. In addition, increasing synaptic acetylcholine by reducing acetylcholinesterase activity, with either mutations or the inhibitor aldicarb, decreased egg laying. Finally, we found that a knock-out for the HSN-expressed receptor G-protein-coupled acetylcholine receptor 2 (GAR-2) shows a partial defect in the inhibition of egg laying and fails to respond to aldicarb. Our results show that acetylcholine released from the VC neurons inhibits egg-laying behavior. This inhibition may be caused, in part, by acetylcholine signaling onto the HSN presynaptic terminals, via GAR-2, to inhibit neurotransmitter release.

Thyroid hormone transport by the rat fatty acid translocase.

We examined the hypothesis that rat fatty acid translocase (rFAT) mediates the cellular uptake of T(3) and other iodothyronines. Uninjected Xenopus laevis oocytes and oocytes injected 4 d previously with rFAT cRNA were incubated for 60 min at 25 C in medium containing 0.01-10 micro M [(125)I]T(3) and 0.1% BSA, or 1-100 micro M [(3)H]oleic acid and 0.5% BSA. Injection of rFAT cRNA resulted in a 1.9-fold increase in uptake of T(3) (10 nM) and a 1.4-fold increase in uptake of oleic acid (100 micro M). Total T(3) uptake was lower in the presence than in the absence of BSA, but relative to the free T(3) concentration, uptake was increased by BSA. The fold induction of T(3) uptake by rFAT was not influenced by BSA. By analyzing uptake as a function of the ligand concentration, we estimated a K(m) value of 3.6 micro M for (total) T(3) and 56 micro M for (total) oleic acid. In addition to T(3), rFAT mediates the uptake of T(4), rT(3), 3,3'-diiodothyronine, and T(3) sulfate. The injection of human type III deiodinase cRNA with or without rFAT cRNA resulted in the complete deiodination of T(3) taken up by the oocytes, indicating that T(3) is indeed transported to the cytoplasm. In conclusion, our results demonstrate transport of T(3) and other iodothyronines by rFAT.

The proximal and distal C-terminal tail domains of the CB1 cannabinoid receptor mediate G protein coupling.

The human CB1 cannabinoid receptor couples to G(i/o) proteins and inhibits neuronal voltage-gated Ca2+ channels. The role of the C-terminal tail of the CB1 cannabinoid receptor in G(i/o) protein coupling was examined using the superior cervical ganglion neuronal expression system. Deletion of the distal intracellular C-terminal tail (amino acids 418-472) slowed the kinetics and reduced the magnitude of Ca2+ channel inhibition. Deletion of the entire intracellular C-terminal tail (amino acids 401-472) abolished Ca2+ channel inhibition demonstrating the critical role of the proximal amino acids 401-417 of the C-terminal tail in G protein signaling. Expression of the C-terminal truncated receptors on the cell surface was examined using an N-terminal CB1 antibody. Both the C-terminal truncated receptors were expressed on the cell surface and were no different from wild type CB1 cannabinoid receptors. This study establishes that the proximal CB1 cannabinoid receptor intracellular C-terminal tail domain (amino acids 401-417) is critical for G(i/o) protein coupling and that the distal C-terminal tail domain (amino acids 418-472) profoundly modulates both the magnitude and kinetics of signal transduction. Thus, the C-terminal tail of the CB1 cannabinoid receptor has a wider role in G protein coupling than was previously thought.

Electrophysiological characteristics of rat gustatory cyclic nucleotide--gated channel expressed in Xenopus oocytes.

The complementary DNA encoding gustatory cyclic nucleotide--gated ion channel (or gustCNG channel) cloned from rat tongue epithelial tissue was expressed in Xenopus oocytes, and its electrophysiological characteristics were investigated using tight-seal patch-clamp recordings of single and macroscopic channel currents. Both cGMP and cAMP directly activated gustCNG channels but with markedly different affinities. No desensitization or inactivation of gustCNG channel currents was observed even in the prolonged application of the cyclic nucleotides. Single-channel conductance of gustCNG channel was estimated as 28 pS in 130 mM of symmetric Na(+). Single-channel current recordings revealed fast open-close transitions and longer lasting closure states. The distribution of both open and closed events could be well fitted with two exponential components and intracellular cGMP increased the open probability (P(o)) of gustCNG channels mainly by increasing the slower opening rate. Under bi-ionic conditions, the selectivity order of gustCNG channel among divalent cations was determined as Na(+) approximately K(+) > Rb(+) > Li(+) > Cs(+) with the permeability ratio of 1:0.95:0.74:0.63:0.49. Magnesium ion blocked Na(+) currents through gustCNG channels from both intracellular and extracellular sides in voltage-dependent manners. The inhibition constants (K(i)s) of intracellular Mg(2+) were determined as 360 +/- 40 microM at 70 mV and 8.2 +/- 1.5 mM at -70 mV with z delta value of 1.04, while K(i)s of extracellular Mg(2+) were as 1.1 +/- 0.3 mM at 70 mV and 20.0 +/- 0.1 microM at -70 mV with z delta of 0.94. Although 100 microM l-cis-diltiazem blocked significant portions of outward Na(+) currents through both bovine rod and rat olfactory CNG channels, the gustCNG channel currents were minimally affected by the same concentration of the drug.

Beta-amyloid peptide expression is sufficient for myotube death: implications for human inclusion body myopathy.

Inclusion body myositis (sIBM) is the most common disorder of skeletal muscle in aged humans. It shares biochemical features with Alzheimer's disease, including congophilic deposits, which are immunoreactive for beta-amyloid peptide (Abeta) and C'-terminal betaAPP epitopes. However, the etiology of myofiber loss and the role of intracellular Abeta in IBM is unknown. Here we report correlative evidence for apoptotic cell death in myofibers of IBM patients that exhibit pronounced Abeta deposition. HSV-1-mediated gene transfer of Abeta(42) into cultured C2C12 myotubes resulted in a 12.6-fold increase in dUTP-labeled and condensed nuclei over nonexpressing myotubes (P < 0.05). The C'-terminal betaAPP domain C99 also induced myotube apoptosis, but to a significantly lesser extent than Abeta. Apoptosis specific to Abeta-expressing myotubes was also demonstrated through DNA fragmentation, decreased mitochondrial function and the loss of membrane phospholipid polarity. Myotubes laden with Abeta(42), but not other transgene products, developed cytoplasmic inclusions consisting of fibrillar material. Furthermore, injection of normal mouse gastrocnemius muscle with HSV-encoding Abeta cDNA resulted in TUNEL-positive myofibers with pyknotic nuclei. We conclude that Abeta is sufficient to induce apoptosis in myofibers both in vivo and in vitro and suggest it may contribute to myofiber loss and muscle dysfunction in patients with IBM.

Acidosis induces necrosis and apoptosis of cultured hippocampal neurons.

Acidosis, hypoxia, and hypoglycemia rapidly and transiently appear after reduction of cerebral blood flow. Acidosis also accompanies head trauma and subarachnoid hemorrhage. These insults result in necrotic and apoptotic loss of neurons. We previously demonstrated that transient acidification of intracellular pH from 7.3 to 6.5 induces delayed neuronal loss in cultured hippocampal slices (49). We now report that acidosis induced both necrotic and apoptotic loss of neurons. Necrosis and apoptosis were distinguished temporally and pharmacologically. Necrosis appeared rapidly and was dose dependent with the duration of the acidosis treatment. Apoptosis was delayed with maximal number of apoptotic cells seen with a 30-min acidosis treatment. Apoptotic neuronal loss was accompanied by DNA fragmentation and was blocked by inhibitors of protein and RNA synthesis, ectopic expression of the anti-apoptotic gene bcl-2, or an inhibitor of caspases, proteases known to be activated during apoptosis. Necrotic neuronal loss was unaffected by these treatments. Hypothermia, a treatment known to attenuate neuronal loss following a variety of insults, blocked both acidosis-induced necrosis and apoptosis. These results indicate that acidosis is neurotoxic in vitro and suggest that acidosis contributes to both necrotic and apoptotic neuronal loss in vivo.

Overexpression of protein kinase C-beta1 isoenzyme suppresses indomethacin-induced apoptosis in gastric epithelial cells.

BACKGROUND & AIMS: We have previously reported that nonsteroidal anti-inflammatory drugs (NSAIDs) could induce apoptosis of gastric epithelial cells both in vivo and in vitro. This study investigated the role of protein kinase C (PKC) isoforms in the regulation of NSAID-induced apoptosis. METHODS: Protein levels of 12 PKC isoforms in AGS cells, in the presence or absence of indomethacin, were determined by Western blot. The effect of PKC-beta1 overexpression by transfection with its complementary DNA (cDNA) on indomethacin-induced apoptosis and apoptosis-related genes, including p53, p21(waf1/cip1), and c-myc, was further investigated. RESULTS: Treatment with indomethacin decreased the abundance of PKC-beta1 and increased that of PKC-beta2, eta, and epsilon, but did not alter the expression of PKC alpha, gamma, zeta, delta, iota, and micro. Overexpression of PKC-beta1 attenuated the apoptotic response of AGS cells to indomethacin, associated with overexpression of p21(waf1/cip1) in both messenger RNA and protein levels. Inhibition of PKC-beta1-mediated overexpression of p21(waf1/cip1) by its antisense cDNA partially reduced the antiapoptotic effect of PKC-beta1. CONCLUSIONS: Indomethacin-induced apoptosis in gastric cancer cells is partly mediated by differential regulation of PKC isoform expression. Enhanced expression of exogenous PKC-beta1 protects against indomethacin-induced apoptosis through up-regulation of p21(waf1/cip1).