Hymenolepis diminuta: Partial characterization of the membrane-bound and solubilized alkaline phosphohydrolase activities of the isolated brush border plasma membrane

The membrane-bound and solubilized (using Triton ×-100 or sodium dodecyl sulfate (SDS)) alkaline phosphohydrolase (APase) activities of the isolated brush border membrane of Hymenolepis diminuta require a divalent cation for maximum activity. Highest rates of substrate (p-nitrophenyl phosphate) hydrolysis are obtained with low concentrations of Mg²⁺ (1 mM), although low concentrations of Mn²⁺, Ca²⁺, or Zn²⁺ will also partially satisfy this requirement; higher concentrations of Mg²⁺ and Mn²⁺, and other divalent cations (Cu²⁺, Fe²⁺, and Pb²⁺), inhibit the membrane-bound APase activity. Solubilization of the membrane-bound enzyme in either Triton or SDS results in an increase in specific activity and K_m, but has little effect on thermal stability of the APase activity. Phosphate, pyrophosphate, adenosine 5′-triphosphate, adenosine 5′-monophosphate, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-diphosphate inhibit substrate hydrolysis, and the relative affinities of these inhibitors for the APase enzyme are altered only slightly upon solubilization. Graphic analyses of data from inhibitor studies indicate that all eight inhibitors will inhibit membrane-bound and solubilized APase activities 100% at high inhibitonsubstrate ratios. Molybdate, F^?, 2-mercaptoethanol, cysteine, and p-chloromercuribenzoate inhibit membrane-bound APase activity. Inhibitor data indicate that if more than one enzyme is responsible for the APase activity of the brush border membrane of H. diminuta, the enzymes cannot be differentiated on the basis of substrate specificity.     

Restraint stress augments postprandial gastric contractions but impairs antropyloric coordination in conscious rats

Central corticotropin-releasing factor (CRF) plays an important role in mediating restraint stress-induced delayed gastric emptying. However, it is unclear how restraint stress modulates gastric motility to delay gastric emptying. Inasmuch as solid gastric emptying is regulated via antropyloric coordination, we hypothesized that restraint stress impairs antropyloric coordination, resulting in delayed solid gastric emptying in conscious rats. Two strain gauge transducers were sutured onto the serosal surface of the antrum and pylorus, and postprandial gastric motility was monitored before, during, and after restraint stress. Antropyloric coordination, defined as a propagated single contraction from the antrum to the pylorus within 10 s, was followed by > or = 20 s of quiescence. Restraint stress enhanced postprandial gastric motility in the antrum and pylorus to 140 +/- 9% and 134 +/- 9% of basal, respectively (n = 6). The number of episodes of antropyloric coordination before restraint stress, 2.4 +/- 0.4/10 min, was significantly reduced to 0.6 +/- 0.3/10 min by restraint stress. Intracisternal injection of the CRF type 2 receptor antagonist astressin 2B (60 microg) or guanethidine partially restored restraint stress-induced impairment of antropyloric coordination (1.6 +/- 0.3/10 min, n = 6). The restraint stress-induced augmentation of antral and pyloric contractions was increased by astressin 2B and guanethidine but abolished by atropine, hexamethonium, and vagotomy. Restraint stress enhanced postprandial gastric motility via a vagal cholinergic pathway. Restraint stress-induced delay of solid gastric emptying is due to impairment of antropyloric coordination. Restraint stress-induced impairment of antropyloric coordination might be mediated via a central CRF pathway.     

Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS

The newly established hyphenated instrumentation of LC/DAD/SPE/NMR and LC/UV/(ESI)MS techniques have been applied for separation and structure verification of the major known constituents present in Greek Hypericum perforatum extracts. The chromatographic separation was performed on a C18 column. Acetonitrile-water was used as a mobile phase. For the on-line NMR detection, the analytes eluted from column were trapped one by one onto separate SPE cartridges, and hereafter transported into the NMR flow-cell. LC/DAD/SPE/NMR and LC/UV/MS allowed the characterization of constituents of Greek H. perforatum, mainly naphtodianthrones (hypericin, pseudohypericin, protohypericin, protopseudohypericin), phloroglucinols (hyperforin, adhyperforin), flavonoids (quercetin, quercitrin, isoquercitrin, hyperoside, astilbin, miquelianin, I3,II8-biapigenin) and phenolic acids (chlorogenic acid, 3-O-coumaroylquinic acid). Two phloroglucinols (hyperfirin and adhyperfirin) were detected for the first time, which have been previously reported to be precursors in the biosynthesis of hyperforin and adhyperforin.     

Selective use of TRAM in lipopolysaccharide (LPS) and lipoteichoic acid (LTA) induced NF-kappaB activation and cytokine production in primary human cells: TRAM is an adaptor for LPS and LTA signaling

TLR signal via Toll-IL-1R (TIR) homology domain-containing adaptor proteins. One of these adaptors, Toll-IL-1R domain-containing adaptor inducing IFN-beta-related adaptor molecule (TRAM), has been shown to be essential for TLR4 signaling in TRAM(-/-) mice and cell lines. Previously, we showed that MyD88 or Mal dominant-negative constructs did not inhibit LPS induction of cytokines in primary human M-CSF-derived macrophages. A possible explanation was redundancy of the adaptors during LPS signaling. TRAM is a suitable candidate to compensate for these adaptors. To investigate a potential role for TRAM in LPS signaling in human M-CSF-derived macrophages, we engineered an adenoviral construct expressing dominant-negative TRAM-C117H (AdTRAMdn). Synovial fibroblasts (SF) and human umbilical endothelial cells (HUVECs) were used as a nonmyeloid comparison. AdTRAMdn inhibited LPS-induced signaling in SFs and HUVECs, reducing NF-kappaB activation and cytokine production, but did not inhibit LPS signaling in M-CSF-derived human macrophages. Further investigation of other TLR ligands showed that AdTRAMdn was also able to inhibit signaling initiated by lipoteichoic acid, a TLR2 ligand, in SFs and HUVECs and lipoteichoic acid and macrophage-activating lipopeptide 2 signaling was also inhibited in TRAM(-/-) murine embryonic fibroblasts. We conclude that TRAM is an adaptor protein for both TLR4 and TLR2/6 signaling in SFs, HUVECs, and murine embryonic fibroblasts, but cannot demonstrate a role in human macrophages.     

Hydrogen peroxide inhibits caspase-dependent apoptosis by inactivating procaspase-9 in an iron-dependent manner

Apoptosis represents a physiological form of cell death, the perturbation of which may contribute to the development of several diseases connected with accumulation of unwanted cells or excessive cell loss. We have previously shown that the continuous presence of low concentrations of H2O2 (generated by the action of glucose oxidase) was able to inhibit caspase-mediated apoptosis in Jurkat cells. The main purpose of the present study was to elucidate the exact molecular mechanism(s) underlying this inhibitory action of H2O2. The results presented show that events like outer mitochondrial membrane permeabilization, release of cytochrome c from mitochondria, oligomerization of Apaf-1, and recruitment of procaspase-9 to apoptosomes were taking place normally, but further advancement toward activation of the execution caspases was interrupted when H2O2 was present during the apoptotic process. From the results presented in this work, it emerges that the inhibition of procaspase-9 autoactivation was probably due to the reversible oxidation of sensitive cysteine residues in this molecule. Remarkably, caspase-9 activation and the ensuing caspase cascade proceeded normally in the presence of H2O2 under conditions of iron deprivation, indicating that the inhibition of procaspase-9 activation was an iron-dependent process. Collectively, these results highlighted the potential role of available intracellular iron ions in signaling mechanisms related to apoptotic cell death.     

A Replicative Plasmid Vector Allows Efficient Complementation of Pathogenic Leptospira Strains

Leptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including the parA, parB, and rep genes into pMAT, a plasmid that cannot replicate in Leptospira spp. and contains a backbone consisting of an aadA cassette, ori R6K, and oriT RK2/RP4. The inserted DNA segment was isolated from a 52-kb region within Leptospira mayottensis strain 200901116 that is not found in the closely related strain L. mayottensis 200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use of trans complementation in the pathogen Leptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of the perR gene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells. In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel of Leptospira strains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation of Leptospira spp.     

Proteomics reveals that redox regulation is disrupted in patients with ethylmalonic encephalopathy

Deficiency of the sulfide metabolizing protein ETHE1 is the cause of ethylmalonic encephalopathy (EE), an inherited and severe metabolic disorder. To study the molecular effects of EE, we performed a proteomics study on mitochondria from cultured patient fibroblast cells. Samples from six patients were analyzed and revealed seven differentially regulated proteins compared with healthy controls. Two proteins involved in pathways of detoxification and oxidative/reductive stress were underrepresented in EE patient samples: mitochondrial superoxide dismutase (SOD2) and aldehyde dehydrogenase X (ALDH1B). Sulfide:quinone oxidoreductase (SQRDL), which takes part in the same sulfide pathway as ETHE1, was also underrepresented in EE patients. The other differentially regulated proteins were apoptosis inducing factor (AIFM1), lactate dehydrogenase (LDHB), chloride intracellular channel (CLIC4) and dimethylarginine dimethylaminohydrolase 1 (DDAH1). These proteins have been reported to be involved in encephalopathy, energy metabolism, ion transport, and nitric oxide regulation, respectively. Interestingly, oxidoreductase activity was overrepresented among the regulated proteins indicating that redox perturbation plays an important role in the molecular mechanism of EE. This observation may explain the wide range of symptoms associated with the disease, and highlights the potency of the novel gaseous mediator sulfide.