Effect of chemoreceptor modification on assembly and activity of the receptor-kinase complex in Escherichia coli

Bacterial chemoreceptors are embedded in the inner cell membrane in tight clusters. We show that changes in receptor methylation that generate large changes in kinase activity have relatively little effect on cluster morphology. Thus, changes in receptor activity do not appear to be mediated by changes in receptor-kinase assembly.     

Acute regulation of Na/H exchanger NHE3 by adenosine A(1) receptors is mediated by calcineurin homologous protein

Adenosine is an autacoid that regulates renal Na(+) transport. Activation of adenosine A(1) receptor (A(1)R) by N(6)-cyclopentidyladenosine (CPA) inhibits the Na(+)/H(+) exchanger 3 (NHE3) via phospholipase C/Ca(2+)/protein kinase C (PKC) signaling pathway. Mutation of PKC phosphorylation sites on NHE3 does not affected regulation of NHE3 by CPA, but amino acid residues 462 and 552 are essential for A(1)R-dependent control of NHE3 activity. One binding partner of the NHE family is calcineurin homologous protein (CHP). We tested the role of NHE3-CHP interaction in mediating CPA-induced inhibition of NHE3 in opossum kidney (OK) and Xenopus laevis uroepithelial (A6) cells. Both native and transfected NHE3 and CHP are present in the same immuno-complex by co-immunoprecipitation. CPA (10(-6) M) increases CHP-NHE3 interaction by 30 - 60% (native and transfected proteins). Direct CHP-NHE3 interaction is evident by yeast two-hybrid assay (bait, NHE3(C terminus); prey, CHP); the minimal interacting region is localized to the juxtamembrane region of NHE3(C terminus) (amino acids 462-552 of opossum NHE3). The yeast data were confirmed in OK cells where truncated NHE3 (NHE3(delta552)) still shows CPA-stimulated CHP interaction. Overexpression of the polypeptide from the CHP binding region (NHE3(462-552)) interferes with the ability of CPA to inhibit NHE3 activity and to increase CHPNHE3(Full-length) interaction. Reduction of native CHP expression by small interference RNA abolishes the ability of CPA to inhibit NHE3 activity. We conclude that CHPNHE3 interaction is regulated by A(1)R activation and this interaction is a necessary and integral part of the signaling pathway between adenosine and NHE3.     

Electrooptical analysis of the Escherichia coli-phage interaction

This article describes electrooptical (EO) characterization of biospecific binding between the bacterium Escherichia coli XL-1 and the phage M13K07. The electrooptical analyzer (ELUS EO), which has been developed at the State Research Center for Applied Microbiology, Obolensk, Russia, was used as the basic instrument for EO measurements. The operating principle of the analyzer is based on the polarizability of microorganisms, which depends strongly on their composition, morphology, and phenotype. The principle of analysis of the interaction of E. coli with the phage M13K07 is based on registration of changes of optical parameters of bacterial suspensions. The phage-cell interaction includes the following stages: phage adsorption on the cell surface, entry of viral DNA into the bacterial cell, amplification of phage within infected host, and phage ejection from the cell. In this work, we used M13K07, a filamentous phage of the family Inoviridae. Preliminary study had shown that combination of the EO approach with a phage as a recognition element has an excellent potential for mediator-less detection of phage-bacteria complex formation. The interaction of E. coli with phage M13K07 induces a strong and specific EO signal as a result of substantial changes of the EO properties of the E. coli XL-1 suspension infected by the phage M13K07. The signal was specific in the presence of foreign microflora (E. coli K-12 and Azospirillum brasilense Sp7). Integration of the EO approach with a phage has the following advantages: (1) bacteria from biological samples need not be purified, (2) the infection of phage to bacteria is specific, (3) exogenous substrates and mediators are not required for detection, and (4) it is suitable for any phage-bacterium system when bacteria-specific phages are available.     

Increased seizure susceptibility and cortical malformation in beta-catenin mutant mice

Beta-catenin has been implicated in epilepsy because of its altered post seizure expression and the role of Wnt2 signaling in autism. To determine beta-catenin's role in seizure susceptibility, we injected penetylenetetrazol intraperitoneally in beta-catenin cerebral cortex- and hippocampus-specific knockout mice. We then analyzed the latency, number, and duration of four phases of seizure behaviors: (I) non-seizure activity, (II) myoclonic jerks, (III) generalized clonic seizures, and (IV) tonic seizures. The latencies to both death and Phase IV were significantly reduced in mutant mice. Mutant mice also spent significantly more time in Phases III and IV and showed significantly less time in the non-convulsive state (Phase I). Nissl and gold chloride staining indicated that the knockout mice had underdeveloped cortices, lacked a corpus callosum, and were missing hippocampal structures. This suggests that dysfunction of beta-catenin-mediated signaling pathways in mice leads to cortical malformation and increased seizure susceptibility.     

Augmented heme oxygenase-1 induces prostaglandin uptake via the prostaglandin transporter in micro-vascular endothelial cells

Previous studies show that expression of heme oxygenase-1 (HO-1) in endothelial cells results in decreased cyclooxygenase expression and prostaglandin (PG) levels through limiting heme availability. Regulation of PGs, important inflammatory mediators, may contribute to the anti-inflammatory potential of HO-1. Here we examine the effects of HO-1 expression on PG clearance via the prostaglandin transporter (PGT). Endothelial cells expressing human HO-1 via retroviral transfer exhibit approximately 7-fold higher levels of PGT RNA and equivalently elevated uptake of [(3)H]PGE(2). The pattern and extent of uptake and the substrate inhibitory constants of PGE(2), PGF(2alpha), and thromboxane B(2) are similar to those of cloned PGT. Treatment of cells with stannous chloride, an inducer of HO-1, results in increased expression of PGT while incubation of cells expressing human HO-1 with stannic mesophorphyrin, a substrate inhibitor of HO-1, decreases PG uptake. Therefore, PG clearance via PGT may contribute to the cellular regulation of PG levels by HO-1.     

Bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase. Comparison of pre-steady state and single turnover methylation of 40-mer duplexes containing two (un)modified target sites

We analyzed pre-steady state and single turnover kinetics of bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to 40-mer duplexes containing native recognition sites (5'-GATC/5'-GATC) or some modified variant(s). The results extend a model from studies with single-site 20-mer duplexes. Under pre-steady state conditions, monomeric T4Dam methyltransferase-AdoMet complexes were capable of rapid methylation of adenine residues in 40-mer duplexes containing two sites. During processive movement of T4Dam to the next site, the rate-limiting step was the exchange of the product S-adenosyl-l-homocysteine (AdoHcy) for AdoMet without T4Dam dissociating from the duplex. Consequently, instead of a single exponential rate dependence, complex methylation curves were obtained with at least two pre-steady state steps. With 40-mer duplexes containing a single target site, the kinetics were simpler, fitting a single exponential followed by a linear steady state phase. Single turnover methylation of 40-mer duplexes also proceeded in two stages. First, two dimeric T4Dam-AdoMet molecules bound, and each catalyzed a two-step methylation. Instead of processive movement of T4Dam, a conformational adaptation occurred. We propose that following methyl transfer to one strand, dimeric (T4Dam-AdoMet)-(T4Dam-AdoHcy) was capable of rapidly reorienting itself and catalyzing methyl transfer to the target adenine on the complementary, unmethylated strand. This second stage methyl transfer occurred at a rate about 25-fold slower than in the first step; it was rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. Under single turnover conditions, there was complete methylation of all target adenine residues with each of the two-site 40-mer duplexes.     

Activation of the pro-survival phosphatidylinositol 3-kinase/AKT pathway by transforming growth factor-beta1 in mesenchymal cells is mediated by p38 MAPK-dependent induction of an autocrine growth factor

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine involved in differentiation, growth, and survival of mesenchymal cells while inhibiting growth/survival of most other cell types. The mechanism(s) of pro-survival signaling by TGF-beta1 in mesenchymal cells is unclear. In this report, we demonstrate that TGF-beta1 protects against serum deprivation-induced apoptosis of mesenchymal cells isolated from patients with acute lung injury and of normal human fetal lung fibroblasts (IMR-90). TGF-beta receptor(s)-activated signaling in these cells involves rapid activation of the Smad and p38 MAPK pathways within minutes of TGF-beta1 treatment followed by a more delayed activation of the pro-survival phosphatidylinositol 3-kinase-protein kinase B (PKB)/Akt pathway. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a p38 kinase-deficient mutant protein inhibits TGF-beta1-induced PKB/Akt phosphorylation. Conditioned medium from TGF-beta1-treated cells rapidly induces PKB/Akt activation in an SB203580- and suramin-sensitive manner, suggesting p38 MAPK-dependent production of a secreted growth factor that activates this pro-survival pathway by an autocrine/paracrine mechanism. Inhibition of the phosphatidylinositol 3-kinase-PKB/Akt pathway blocks TGF-beta1-induced resistance to apoptosis. These results demonstrate the activation of a novel TGF-beta1-activated pro-survival/anti-apoptotic signaling pathway in mesenchymal cells/fibroblasts that may explain cell-specific actions of TGF-beta1 and provide mechanistic insights into its pro-fibrotic and tumor-promoting effects.     

Irradiation of mangoes as a postharvest quarantine treatment for fruit flies (Diptera: Tephritidae)

Mangoes infested with third instar larvae were irradiated using Co-60 gamma rays and a dose interval of 2-250 Gy to assess the irradiation dose required to prevent adult emergence of the Mexican fruit fly (Anastrepha ludens), the West Indies fruit fly (A. obliqua), the sapote fruit fly (A. serpentina), and the Mediterranean fruit fly (Ceratitis capitata). Doses of 76.9, 87.3, 91.4 and 112.7 Gy, were estimated to inhibit 99.9968% (probit 9) of adult emergence forA. obliqua, A. serpentina, A. ludens, and C. capitata, respectively. Using mangoes infested with a total of 100,000 larvae of each species, the results obtained in the laboratory were confirmed using a dose of 100 Gy for the Anastrepha species and 150 Gy for C. capitata. No adult emergence was observed for any of the four species compared with approximately 80% emergence in the controls. A dose of 150 Gy is recommended as a generic quarantine treatment against potential infestation of these species in exported mangoes. A minor decrease in the ascorbic acid content was the only adverse effects observed in irradiated mangoes.     

A dual role of IFN-alpha in the balance between proliferation and death of human CD4+ T lymphocytes during primary response

Type I IFNs (IFN-alphabeta) enhance immune responses, notably T cell-mediated responses, in part by promoting the functional activities of dendritic cells. In this study, we analyzed the direct impact of IFN-alpha on proliferative and apoptotic signals upon in vitro activation of human naive CD4+ T lymphocytes. We demonstrate that IFN-alpha protects T cells from the intrinsic mitochondrial-dependent apoptosis early upon TCR/CD28 activation. IFN-alpha acts by delaying entry of cells into the G1 phase of the cell cycle, as well as by increasing Bcl-2 and limiting Bax activation. Later, upon activation, T cells that were exposed to IFN-alpha showed increased levels of surface Fas associated with partially processed caspase-8, a key component of the extrinsic apoptotic pathway. Caspase-8 processing was augmented furthermore by Fas ligation. Overall, these findings support a model whereby IFN-alpha favors an enhanced clonal expansion, yet it sensitizes cells to the Ag-induced cell death occurring at the end of an immune response. These observations point to a complex role of type I IFN in regulating the magnitude of proliferation and survival of naive CD4+ T cells during primary response and underline how crucial could be the timing of exposure to this cytokine.