Guard Cells Possess a Calcium-Dependent Protein Kinase That Phosphorylates the KAT1 Potassium Channel1

Increasing evidence suggests that changes in cytosolic Ca2+ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca2+ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca2+-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca2+ dependent. CDPK exhibits a Ca2+-induced electrophoretic mobility shift and its Ca2+-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPKα cross-react with CDPK. Micromolar Ca2+ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca2+-dependent protein phosphatase calcineurin enhances the Ca2+-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K+ channel KAT1 protein in a Ca2+-dependent manner. These results suggest that CDPK may be an important component of Ca2+ signaling in guard cells.

Protection from superoxide damage associated with an increased level of the YggX protein in Salmonella enterica

The deleterious effect of superoxide radicals on cell growth and survival is predominately caused by rapid oxidation of labile [Fe-S] clusters in proteins. Oxidation of these clusters releases Fe(II) ions, which participate in Fenton chemistry that damages DNA. Here it is shown that elevated levels of the YggX protein increase the resistance of Salmonella enterica to superoxide stress, reverse enzymatic defects attributed to oxidized [Fe-S] clusters, and decrease the spontaneous mutation frequency. The data are consistent with a model in which YggX protects protein [Fe-S] clusters from oxidation.

Functional cell surface expression of the anion transport domain of human red cell band 3 (AE1) in the yeast Saccharomyces cerevisiae.

We expressed the 52-kDa integral membrane domain (B3mem) of the human erythrocyte anion transporter (band 3; AE1) in a protease-deficient strain of the yeast Saccharomyces cerevisiae under the control of the inducible GAL10-CYC1 promoter. Immunoblots of total protein from transformed yeast cells confirmed that the B3mem polypeptide was overexpressed shortly after induction with galactose. Cell surface expression of the functional anion transporter was detected by using a simple transport assay to measure stilbene disulfonate-inhibitable chloride influx into intact yeast cells. The B3mem polypeptide was recycled and degraded by the cells with a half-life of approximately 1-3 hr, which led to a steady-state level of expression in exponentially growing cultures. Our data suggest that 5-10% of total B3mem is functionally active at the cell surface at any one time and that overexpression of this anion transport protein does not interfere with cell growth or survival. This is one of only a few reports of the functional expression of a plasma membrane transport protein in the plasma membrane of yeast cells and to our knowledge is the first report of red cell band 3-mediated anion transport at the plasma membrane of cDNA-transformed cells. The cell surface expression system we describe will provide a simple means for future study of the functional properties of band 3 by using site-directed mutagenesis.

Hydrogen peroxide-mediated neuronal cell death induced by an endogenous neurotoxin, 3-hydroxykynurenine.

3-Hydroxykynurenine (3-HK) is a tryptophan metabolite whose level in the brain is markedly elevated under several pathological conditions, including Huntington disease and human immunodeficiency virus infection. Here we demonstrate that micromolar concentrations (1-100 microM) of 3-HK cause cell death in primary neuronal cultures prepared from rat striatum. The neurotoxicity of 3-HK was blocked by catalase and desferrioxamine but not by superoxide dismutase, indicating that the generation of hydrogen peroxide and hydroxyl radical is involved in the toxicity. Measurement of peroxide levels revealed that 3-HK caused intracellular accumulation of peroxide, which was largely attenuated by application of catalase. The peroxide accumulation and cell death caused by 1-10 microM 3-HK were also blocked by pretreatment with allopurinol or oxypurinol, suggesting that endogenous xanthine oxidase activity is involved in exacerbation of 3-HK neurotoxicity. Furthermore, NADPH diaphorase-containing neurons were spared from toxicity of these concentrations of 3-HK, a finding reminiscent of the pathological characteristics of several neurodegenerative disorders such as Huntington disease. These results suggest that 3-HK at pathologically relevant concentrations renders neuronal cells subject to oxidative stress leading to cell death, and therefore that this endogenous compound should be regarded as an important factor in pathogenesis of neurodegenerative disorders.

Improved retroviral gene transfer into murine and Rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor.

In previous studies we showed that 5 days of treatment with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) mobilized murine repopulating cells to the peripheral blood (PB) and that these cells could be efficiently transduced with retroviral vectors. We also found that, 7-14 days after cytokine treatment, the repopulating ability of murine bone marrow (BM) increased 10-fold. In this study we examined the efficiency of gene transfer into cytokine-primed murine BM cells and extended our observations to a nonhuman primate autologous transplantation model. G-CSF/SCF-primed murine BM cells collected 7-14 days after cytokine treatment were equivalent to post-5-fluorouracil BM or G-CSF/SCF-mobilized PB cells as targets for retroviral gene transfer. In nonhuman primates, CD34-enriched PB cells collected after 5 days of G-CSF/SCF treatment and CD34-enriched BM cells collected 14 days later were superior targets for retroviral gene transfer. When a clinically approved supernatant infection protocol with low-titer vector preparations was used, monkeys had up to 5% of circulating cells containing the vector for up to a year after transplantation. This relatively high level of gene transfer was confirmed by Southern blot analysis. Engraftment after transplantation using primed BM cells was more rapid than that using steady-state bone marrow, and the fraction of BM cells saving the most primitive CD34+/CD38- or CD34+/CD38dim phenotype increased 3-fold. We conclude that cytokine priming with G-CSF/SCF may allow collection of increased numbers of primitive cells from both the PB and BM that have improved susceptibility to retroviral transduction, with many potential applications in hematopoietic stem cell-directed gene therapy.

The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol.

The antimycobacterial compound ethambutol [Emb; dextro-2,2'-(ethylenediimino)-di-1-butanol] is used to treat tuberculosis as well as disseminated infections caused by Mycobacterium avium. The critical target for Emb lies in the pathway for the biosynthesis of cell wall arabinogalactan, but the molecular mechanisms for drug action and resistance are unknown. The cellular target for Emb was sought using drug resistance, via target overexpression by a plasmid vector, as a selection tool. This strategy led to the cloning of the M. avium emb region which rendered the otherwise susceptible Mycobacterium smegmatis host resistant to Emb. This region contains three complete open reading frames (ORFs), embR, embA, and embB. The translationally coupled embA and embB genes are necessary and sufficient for an Emb-resistant phenotype which depends on gene copy number, and their putative novel membrane proteins are homologous to each other. The predicted protein encoded by embR, which is related to known transcriptional activators from Streptomyces, is expendable for the phenotypic expression of Emb resistance, but an intact divergent promoter region between embR and embAB is required. An Emb-sensitive cell-free assay for arabinan biosynthesis shows that overexpression of embAB is associated with high-level Emb-resistant arabinosyl transferase activity, and that embR appears to modulate the in vitro level of this activity. These data suggest that embAB encode the drug target of Emb, the arabinosyl transferase responsible for the polymerization of arabinose into the arabinan of arabinogalactan, and that overproduction of this Emb-sensitive target leads to Emb resistance.

Depletion of intracellular polyamines relieves inward rectification of potassium channels.

Two different approaches were used to examine the in vivo role of polyamines in causing inward rectification of potassium channels. In two-microelectrode voltage-clamp experiments, 24-hr incubation of Xenopus oocytes injected with 50 nl of difluoromethylornithine (5 mM) and methylglyoxal bis(guanylhydrazone) (1 mM) caused an approximate doubling of expressed Kir2.1 currents and relieved rectification by causing an approximately +10-mV shift of the voltage at which currents are half-maximally inhibited. Second, a putrescine auxotrophic, ornithine decarboxylase-deficient Chinese hamster ovary (O-CHO) cell line was stably transfected with the cDNA encoding Kir2.3. Withdrawal of putrescine from the medium led to rapid (1-day) loss of the instantaneous phase of Kir2.3 channel activation, consistent with a decline of intracellular putrescine levels. Four days after putrescine withdrawal, macroscopic conductance, assessed using an 86Rb+ flux assay, was approximately doubled, and this corresponded to a +30-mV shift of V1/2 of rectification. With increasing time after putrescine withdrawal, there was an increase in the slowest phase of current activation, corresponding to an increase in the spermine-to-spermidine ratio over time. These results provide direct evidence for a role of each polyamine in induction of rectification, and they further demonstrate that in vivo modulation of rectification is possible by manipulation of polyamine levels using genetic and pharmacological approaches.

Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress.

Early atherosclerotic lesions develop in a topographical pattern that strongly suggests involvement of hemodynamic forces in their pathogenesis. We hypothesized that certain endothelial genes, which exhibit differential responsiveness to distinct fluid mechanical stimuli, may participate in the atherogenic process by modulating, on a local level within the arterial wall, the effects of systemic risk factors. A differential display strategy using cultured human endothelial cells has identified two genes, manganese superoxide dismutase and cyclooxygenase-2, that exhibit selective and sustained up-regulation by steady laminar shear stress (LSS). Turbulent shear stress, a nonlaminar fluid mechanical stimulus, does not induce these genes. The endothelial form of nitric oxide synthase also demonstrates a similar LSS-selective pattern of induction. Thus, three genes with potential atheroprotective (antioxidant, antithrombotic, and antiadhesive) activities manifest a differential response to distinct fluid mechanical stimuli, providing a possible mechanistic link between endothelial gene expression and early events in atherogenesis. The activities of these and other LSS-responsive genes may have important implications for the pathogenesis and prevention of atherosclerosis.

Potassium homeostasis in vacuolate plant cells.

Plant cells contain two major pools of K+, one in the vacuole and one in the cytosol. The behavior of K+ concentrations in these pools is fundamental to understanding the way this nutrient affects plant growth. Triple-barreled microelectrodes have been used to obtain the first fully quantitative measurements of the changes in K+ activity (aK) in the vacuole and cytosol of barley (Hordeum vulgare L.) root cells grown in different K+ concentrations. The electrodes incorporate a pH-selective barrel allowing each measurement to be assigned to either the cytosol or vacuole. The measurements revealed that vacuolar aK declined linearly with decreases in tissue K+ concentration, whereas cytosolic aK initially remained constant in both epidermal and cortical cells but then declined at different rates in each cell type. An unexpected finding was that cytoplasmic pH declined in parallel with cytosolic aK, but acidification of the cytosol with butyrate did not reveal any short-term link between these two parameters. These measurements show the very different responses of the vacuolar and cytosolic K+ pools to changes in K+ availability and also show that cytosolic K+ homeostasis differs quantitatively in different cell types. The data have been used in thermodynamic calculations to predict the need for, and likely mechanisms of, active K+ transport into the vacuole and cytosol. The direction of active K+ transport at the vacuolar membrane changes with tissue K+ status.

Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

On the role of antigen in maintaining cytotoxic T-cell memory.

This study evaluated whether T-cell memory reflects increased precursor frequencies of specific long-lived T cells and/or a low-level immune response against some form of persistent antigen. Antivirally protective CD8+ T-cell memory was analyzed mostly in the original vaccinated host to assess the role of antigen in its maintenance. T-cell mediated resistance against reinfection was measured in the spleen and in peripheral solid organs with protocols that excluded protection by antibodies. In vivo protection was compared with detectable cytotoxic T-lymphocyte precursor frequencies determined in vitro. In the spleen, in vitro detectable cytotoxic T-lymphocyte precursor frequencies remained stable independently of antigen, conferring resistance against viral replication in the spleen during reinfection. In contrast, T-cell mediated resistance against reinfection of peripheral solid organs faded away in an antigen-dependent fashion within a few days or weeks. We show that only memory T cells persistently or freshly activated with antigen efficiently extravasate into peripheral organs, where cytotoxic T lymphocytes must be able to exert effector function immediately; both the capacity to extravasate and to rapidly exert effector function critically depend on restimulation by antigen. Our experiments document that the duration of T-cell memory protective against peripheral reinfection depended on the antigen dose used for immunization, was prolonged when additional antigen was provided, and was abrogated after removal of antigen. We conclude that T-cell mediated protective immunity against the usual peripheral routes of reinfection is antigen-dependent.

Elevation of intracellular calcium by muscarinic receptor activation induces a block of voltage-activated rat ether-à-go-go channels in a stably transfected cell line.

We have studied the properties of r-eag voltage-activated potassium channels in a stably transfected human embryonic kidney cell line. It was found that r-eag channels are rapidly and reversibly inhibited by a rise in intracellular calcium from 30 to 300 nM. The inhibition does not appear to depend on the activity of calcium-dependent kinases and phosphatases. The effect of calcium on r-eag channel activity was studied in inside-out membrane patches. Calcium inhibited r-eag channel activity with a mean IC50 of 67 nM. Activation of muscarinic receptors, generating calcium oscillations in the transfected cells, induced a synchronous inhibition of r-eag mediated outward currents. This shows that calcium can mediate r-eag current inhibition following muscarinic receptor activation. The data indicate that r-eag channels are calcium-inhibitable voltage-activated potassium channels.

Nitric oxide regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells.

Decreased nitric oxide (NO) activity, the formation of reactive oxygen species, and increased endothelial expression of the redox-sensitive vascular cell adhesion molecule 1 (VCAM-1) gene in the vessel wall are early and characteristic features of atherosclerosis. To explore whether these phenomena are functionally interrelated, we tested the hypothesis that redox-sensitive VCAM-1 gene expression is regulated by a NO-sensitive mechanism. In early passaged human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the NO donor diethylamine-NO (DETA-NO, 100 microM) reduced VCAM-1 gene expression induced by the cytokine tumor necrosis factor alpha (TNF-alpha, 100 units/ml) at the cell surface level by 65% and intracellular adhesion molecule 1 (ICAM-1) gene expression by 35%. E-selectin gene expression was not affected. No effect on expression of cell adhesion molecules was observed with DETA alone. Moreover, DETA-NO suppressed TNF-alpha-induced mRNA accumulation of VCAM-1 and TNF-alpha-mediated transcriptional activation of the human VCAM-1 promoter. Conversely, treatment with NG-monomethyl-L-arginine (L-NMMA, 1 mM), an inhibitor of NO synthesis, augmented cytokine induction of VCAM-1 and ICAM-1 mRNA accumulation. By gel mobility shift analysis, DETA-NO inhibited TNF-alpha activation of DNA binding protein activity to the VCAM-1 NF-kappa B like binding sites. Peroxy-fatty acids such as 13-hydroperoxydodecanoeic acid (linoleyl hydroperoxide) may serve as an intracellular signal for NF-kappa B activation. Using thin layer chromatography, DETA-NO (100 microM) suppressed formation of this metabolite, suggesting that DETA-NO modifies the reactivity of oxygen intermediates in the vascular endothelium. Through this mechanism, NO may function as an immunomodulator of the vessel wall and thus mediate inflammatory events involved in the pathogenesis of atherosclerosis.

Expression and analysis of presenilin 1 in a human neuronal system: localization in cell bodies and dendrites.

Mutations in the recently identified presenilin 1 gene on chromosome 14 cause early onset familial Alzheimer disease (FAD). Herein we describe the expression and analysis of the protein coded by presenilin 1 (PS1) in NT2N neurons, a human neuronal model system. PS1 was expressed using recombinant Semliki Forest virions and detected by introduced antigenic tags or antisera to PS1-derived peptides. Immunoprecipitation revealed two major PS1 bands of approximately 43 and 50 kDa, neither of which were N-glycosylated or O-glycosylated. Immunoreactive PS1 was detected in cell bodies and dendrites of NT2N neurons but not in axons or on the cell surface. PS1 was also detected in BHK cells, where it was also intracellular and colocalized with calnexin, a marker for the rough endoplasmic reticulum. A mutant form of PS1 linked to FAD did not differ from the wild-type protein at the light microscopic level. The model system described here will enable studies of the function of PS1 in human neurons and the role of mutant PS1 in FAD.

Deletion of the mouse T-cell receptor beta gene enhancer blocks alphabeta T-cell development.

Intrathymic T-cell development requires temporally regulated rearrangement and expression of T-cell receptor (TCR) genes. To assess the role of the TCR beta gene transcriptional enhancer (Ebeta) in this process, mouse strains in which Ebeta is deleted were generated using homologous recombination techniques. We report that mice homozygous for the Ebeta deletion, whether a selectable marker gene is present or not, show a block in alphabeta T-cell development at the CD4-CD8- double-negative cell stage, whereas the number of gammadelta+ T cells is normal, few CD4+CD8+ double-positive thymocytes and no alphabeta+ T cells are produced. DNA-PCR and RNA-PCR analyses of thymic cells from homozygous mutants showed no evidence of TCR beta gene rearrangement although germ-line Vbeta transcripts were detected at a low level, in heterozygous T cells, the targeted allele is not rearranged. Thus, deletion of Ebeta totally prevents rearrangement, but not transcription, of the targeted beta locus. These data formally establish the critical role played by Ebeta in cis-activation of the TCR beta locus for V(D)J recombination during alphabeta T-cell development.