Real-time monitoring of histamine released from rat basophilic leukemia (RBL-2H3) cells with a histamine microsensor using recombinant histamine oxidase

To detect low levels of histamine, we developed a histamine microsensor using recombinant histamine oxidase. Histamine oxidase with a histidine tag was readily purified using a histidine affinity column. The enzyme showed higher catalytic activity on histamine than diamines (e.g., putrescine and cadaverine) or N(tau)-methylhistamine. The sensor had three carbon film electrodes modified with osmium-polyvinylpyridine-based gel containing horseradish peroxidase, histamine oxidase, and Ag. When a standard solution of histamine was aspirated at a flow rate of 2 microl/min, the detected current was proportional to the histamine concentration and the lower detection limit was 11.3 nM. When rat basophilic leukemia cells (1 x 10(6)) were stimulated by various concentrations of antigen (2, 20, and 200 ng/ml), the histamine concentrations were 0.32, 2.7, and 1.3 microM, respectively, and 20 ng/ml of antigen was found to be the optimal concentration for the antigen-antibody reaction. In contrast, when thapsigargin, an inhibitor of Ca-ATPase in the endoplasmic reticulum, was added (50, 100, and 500 nM), the detected current increased with thapsigargin concentrations and the measured histamine concentrations were 28 nM, 1.3 microM, and 2.7 microM, respectively. These results indicate that the microsensor is useful for the analysis of histamine release from mast cells.     

Involvement of Rab27 in antigen-induced histamine release from rat basophilic leukemia 2H3 cells

The Rab family small G proteins regulate discrete steps in vesicular transport pathways. Recent studies indicate that one member of the Rab family, Rab27A, regulates the transport of lysosome-related organelles, such as melanosome distribution in melanocytes, lytic granule release in cytotoxic T cells, and dense granule release in platelets. Here, we have examined the involvement of Rab27A in the exocytic transport of another lysosome-related organelle, the basophilic secretory granule, in basophils. We have found that Rab27A locates on basophilic secretory granules containing histamine in rat basophilic leukemia (RBL) 2H3 cells. In addition, exogenous expression of dominant active Rab27A reduces antigen-induced histamine release from the cells. We have moreover identified Munc13-4 as a Rab27A target using a CytoTrap system and found that exogenous expression of Munc13-4 affects antigen-induced histamine release from RBL-2H3 cells. These results demonstrate that Rab27A plays a crucial role in antigen-induced histamine release from RBL-2H3 cells.     

Stereoselectivity in the placental transfer and kinetic disposition of racemic bupivacaine administered to parturients with or without a vasoconstrictor

The aim of the present study was to investigate the stereoselectivity in the kinetic disposition and the transplacental distribution of bupivacaine in term parturients during labor. Maternal age ranged from 18-37 years and fetal gestational age from 37.6-41.5 weeks. Healthy parturients (n = 23) received epidural 0.5% racemic bupivacaine alone (group A) or combined with epinephrine (group B). Maternal venous blood was sampled at regular intervals until 8 h after drug administration and umbilical venous blood was obtained at delivery. Bupivacaine enantiomers were determined in plasma samples by HPLC using a Chiralcel(R) OD-R column and a UV detector. One- or two-compartment models were fitted to data and differences between the (+)-(R) and (-)-(S) enantiomers were compared with the paired Wilcoxon test (P< 0.05). The influence of epinephrine was evaluated using the unpaired Mann-Whitney test (P< 0.05). The disposition of bupivacaine in maternal plasma was stereoselective, with higher V(d/f) (140.60 vs. 132.81 L for group A and 197.86 vs. 169.46 L for group B) and C(l/f) (29.00 vs. 25.43 L/h for group A and 33.15 vs. 26.39 L/h for group B) and lower t(1/2)beta (3.24 vs. 3.30 h for group A and 4.36 vs. 4.45 h for group B) being observed for (+)-(R)-bupivacaine. The combined administration of epinephrine resulted in higher V(d/f) (197.86 vs. 140.60 L for (+)-(R) and 169.46 vs. 132.81 L for (-)-(S)) and t(1/2)beta values (4.36 vs. 3.24 h for (+)-(R) and 4.45 vs. 3.30 h for (-)-(S)). The transplacental distribution of bupivacaine was stereoselective only when bupivacaine was administered without epinephrine (group B), with a higher cord blood/maternal blood ratio being observed for (-)-(S)-bupivacaine (0.40 vs. 0.35). Chirality 16:65-71, 2004.     

Synthesis and pharmacological evaluation of some dual-acting amino-alcohol ester derivatives of flurbiprofen and 2-[1,1'-biphenyl-4-yl]acetic acid: a potential approach to reduce local gastrointestinal toxicity

The search for safer non-steroidal anti-inflammatory drugs (NSAIDs) continues with the failure of anticipated 'ideal' anti-inflammatory agents, the coxibs, on long-term usage. Increased gastric motility and acidity due to the free carboxy group are involved in the etiology of gastric toxicity, common to conventional NSAIDs. Keeping this fact in mind, it was planned to modify some of the conventional NSAIDs to amino-alcohol ester derivatives, which satisfied the structural requirements for these compounds to possess anticholinergic activity in the intact form. Besides blocking the acidic carboxylic group, incorporation of anticholinergic acivity in these molecules was expected to reduce the gastric toxicity by decreasing gastric acid secretion and motility. Synthesis and pharmacological evaluation of six different N,N-disubstituted amino-ethyl ester derivatives, structurally resembling the amino-alcohol ester class of anticholinergic agents, each for [1,1'-biphenyl]-4-acetic acid (3) and flurbiprofen (10), have been reported as potential substitutes for these NSAIDs, with improved therapeutic profile. All the ester derivatives were found to have sufficient chemical stability in buffers (pH 2.0 and 7.4), ensuring them to be absorbed as intact moieties from the gastrointestinal tract. A significant reduction in ulcerogenic potency in comparison to the parent drugs with a slightly higher anti-inflammatory potency suggests that the majority of these candidates have an improved therapeutic profile over their parent drugs. Hence, a promising novel approach, different from the conventional prodrug concept, has been successfully worked out to overcome the local gastric toxicity, yielding therapeutically better compounds for long-term oral anti-inflammatory therapy.     

HPLC with electrochemical detection to examine the pharmacokinetics of baicalin and baicalein in rat plasma after oral administration of a Kampo medicine

A highly sensitive method for determining baicalin and baicalein in rat plasma was developed using micro HPLC with electrochemical detection (muHPLC-ED). Peak heights for baicalin and baicalein were found to be linearly related to the amounts injected, ranging from 2.2 pg to 4.5 ng (r = 0.997) and from 1.4 pg to 2.7 ng (r = 0.997), respectively. The detection limits (signal/noise ratio = 3) of the current method for baicalin and baicalein were 0.89 and 0.54 pg, respectively. The concentrations of baicalin, baicalein, and their conjugates in rat plasma after oral administration of 2.0 mg/kg of Saiko-keishi-to (TJ-10) were determined. The glucuronide and sulfate forms of baicalin and baicalein in plasma were hydrolyzed enzymatically using beta-glucuronidase and sulfatase, respectively, and the hydrolyzed solutions were extracted with a 0.1-mol/L phosphoric acid-ethyl acetate mixture (1:1, v/v). Based on the time courses of the concentrations of the free and conjugated forms of baicalin and baicalein in rat plasma after oral administration of Saiko-keishi-to, the pharmacokinetic parameters of C(max), t(max), and AUC(0-6 h) were obtained.     

Characterization of MgATP-Driven H+ uptake in to a microsomal vesicle franction from rat pancreatic acinar cells

Summary In microsomal vesicles, as isolated from exocrine pancreas cells, MgATP-driven H⁺ transport was evaluated by measuring H⁺-dependent accumulation of acridine orange (AO). Active H⁺ uptake showed an absolute requirement for ATP with simple Michaelis-Menten kinetics (K _m for ATP 0.43 mmol/liter) with a Hill coefficient of 0.99. H⁺ transport was maximal at an external pH of 6.7, generating an intravesicular pH of 4.8. MgATP-dependent H⁺ accumulatioin was abolished by protonophores. such as nigericin (10^–6 mol/liter) or CCCP (10^–5 mol/liter), and by inhibitors of nonmitochondria H⁺ ATPase, such as NEM or NBD-Cl, at a concentration of 10^–5 mol/liter. Inhibitors of both mitochondrial and nonmitochondrial H⁺ pumps, such as DCCD (10^–5 mol/liter) or Dio 9 (0.25 mg/ml), reduced microsomal H⁺ transport by about 90%. Vanadate (2×10^–3 mol/liter). a blocker of those ATPases, which form a phosphorylated intermediate, did not inhibit H⁺ transport. The stilbene derivative DIDS (10^–4 mol/liter), which inhibits anion transport systems, abolished H⁺ transport completely. MgATP-dependent H⁺ transport was found to be anion dependernt in the sequence Cl^–>Br^–>gluconate^–; in the presence of SO ₄ ^–2 . CH₃COO^– or No ₃ ^– , no H⁺ transport was observed. MgATP-dependent H⁺ accumulation was also cation dependent in the sequence K⁺>Li⁺>Na⁺=choline⁺, As shown by dissipation experiments in the presence of different ion gradients and ionophores, both a Cl^– and a K⁺ conductance, as well as a small H⁺ conductance. were found in the microsomal membranes. When membranes containing the H⁺ pump wer further purified by Percoll gradient centrifugatin (ninefold enrichment comparad to homogenate), no correlation with markers for endoplasmic reticulum., mitochondria, plasma membranes, zymogen graules or Golgi membranes was found.The present data indicate that the H⁺ pump located in microsomes from rat exocrine pancreas is a vacuolar-or V-type H⁺ ATPase and has most similarities to that described in endoplasmic reticulum. Golgi apparatus or endosomes.     

Lafutidine,a novel histamine H2-receptor antagonist,vs lansoprazole in combination with amoxicillin and clarithromycin for eradication of Helicobacter pylori

Background. In contrast to the growing amount of data concerning proton pump inhibitor‐based triple therapy for Helicobacter pylori infection, it is still controversial whether proton pump inhibitor can be replaced by H₂ receptor antagonist without compromising efficacy. Lafutidine is a novel potent H₂ receptor antagonist with gastroprotective activities such as enhancement of gastric mucosal blood flow. Methods. 122 outpatients with positive cultures and subsequent successful cultivation of H. pylori for antimicrobial susceptibility tests were randomized to receive a 7‐day course of either lafutidine (20 mg twice daily) or lansoprazole (30 mg twice daily), plus clarithromycin (200 mg twice daily) and amoxicillin (750 mg twice daily). Eradication was considered successful if the rapid urease test, culture, histology and [ 13 ]C‐urea breath test were all negative at least 4 weeks after cessation of therapy. Cytochrome p450 2C19 genotype status using polymerase chain reaction‐restriction fragment length polymorphism was also studied. Results. On intention‐to‐treat basis, H. pylori cure was achieved in 52 of 61 (85.2%) patients and 49 of 61 (80.3%) patients for the lafutidine‐ and lansoprazole‐based therapies, respectively. The predicted 95% confidential intervals for the 4.9% of the difference were ?1.8–11.6%. Using per protocol analysis, the eradication rates were 88.2% (52/59) and 84.5% (49/58), respectively. The predicted 95% confidential intervals for the 3.7% of the difference were ?2.6–10.0%. Adverse events were observed in five and six patients, from the lafutidine and lansoprazole groups, respectively, but they were generally mild. Genetic predisposition of cytochrome p450 2C19 had no significant influence on treatment outcome in both regimens. Conclusions. The lafutidine‐clarithromycin‐amoxicillin therapy yielded satisfactory results for eradicating H. pylori, which was comparable with those of the lansoprazole‐based regimen with the same drug combination.     

Characteristics and Regulatory Properties of the H+-ATPase in a Plasma Membrane Fraction Purified from Arabidopsis thaliana

The aqueous two-phase partitioning technique was utilized to isolate a plasma membrane (PM) fraction from etiolated seedlings of Arabidopsis thaliana. The purification procedure adopted yielded a fraction highly enriched in PM as compared to inner membranes, with a recovery of about 30%, as judged from the activities of PM markers such as vanadate-sensitive ATPase, FC binding and UDP-glucose sterol glucosyltransferase. The purified PM fraction displayed vanadate-sensitive H⁺ pumping activity. Its purity was confirmed by the biochemical characteristics of its ATPase activity assayed in the absence of Ca²⁺: sensitivity to vanadate (IC₅₀ ca. 1 μM), Mg²⁺-dependence, insensitivity to molybdate, oligomycin and nitrate, pH optimum at 6.6. The PM H⁺-ATPase activity was stimulated by fusicoccin and by a controlled treatment of the PM with trypsin. In both cases stimulation was much stronger on the activity assayed at pH 7.5 than on the activity at pH 6.6. Moreover, neither fusicoccin nor the treatment with trypsin stimulated the portion of activity (30 to 40% at pH 7.5) which decayed upon preincubation of the PM in assay medium without ATP.     

Spontaneous and evoked release of [3H]taurine from a P2 subcellular fraction of the rat retina

The effects of spontaneous and evoked [³H]taurine release from a P₂ fraction prepared from rat retinas were studied. The P₂ fraction was preloaded with [³H]taurine under conditions of high-affinity uptake and then examined for [³H]taurine efflux utilizing superfusion techniques. Exposure of the P₂ fraction to high K⁺ (56 mM) evoked a Ca²⁺-independent release of [³H]taurine. Li⁺ (56 mM) and veratridine (100 M) had significantly less effect (8–15% and 15–30%, respectively) on releasing [³H]taurine compared to the K⁺-evoked release. 4-Aminopyridine (1 mM) had no effect on the release of [³H]taurine. The spontaneous release of [³H]taurine was also Ca²⁺-independent. When Na⁺ was omitted from the incubation medium K⁺-evoked [³H]taurine release was inhibited by approximately 40% at the first 5 minute depolarization period but was not affected at a second subsequent 5 minute depolarization period. The spontaneous release of [³H]taurine was inhibited by 60% in the absence of Na⁺. Substitution of Br^– for Cl^– had no effect on the release of either spontaneous or K⁺-evoked [³H]taurine release. However, substitution of the Cl^– with acetate, isethionate, or gluconate decreased K⁺-evoked [³H]taurine release. Addition of taurine to the superfusion medium (homoexchange) resulted in no significant increase in [³H]taurine efflux. The taurine-transport inhibitor guanidinoethanesulfonic acid increased the spontaneous release of [³H]taurine by approximately 40%. These results suggest that the taurine release of [³H]taurine is not simply a reversal of the carrier-mediated uptake system. It also appears that taurine is not released from vesicles within the synaptosomes but does not rule out the possibility that taurine is a neurotransmitter. The data involving chloride substitution with permeant and impermeant anions support the concept that the major portion of [³H]taurine release is due to an osmoregulatory action of taurine while depolarization accounts for only a small portion of [³H]taurine release.     

Fusicoccin Binding to its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase. II. Stimulation of the H+-ATPase in a Plasma Membrane Fraction Purified by Phase-partitioning

The effect of fusicoccin (FC) on the activity of the PM H⁺-ATPase was investigated in a plasma membrane (PM) fraction from radish seedlings purified by the phase-partitioning procedure. FC stimulated the PM H⁺-ATPase activity by up to 100 %; the effect was essentially on V_max with only a slight decrease of the apparent K_M of the enzyme for ATP. FC-induced stimulation of the PM H⁺-ATPase was evident within the first minute and maximal within five minutes of membrane treatment with the toxin indicating that transmission of the signal from the activated receptor to the PM H⁺-ATPase is very rapid. Both FC-induced stimulation of the PM H⁺-ATPase and FC binding to its receptor decreased dramatically upon incubation of the membranes in ATPase assay medium at 33 °C in the absence of FC, due to the lability of the free FC receptor. FC-induced stimulation of the PM H⁺-ATPase was strongly pH dependent: absolute increase of activity was maximal at pH 7, while percent stimulation increased with the increase of pH up to pH 7.5; FC binding was scarcely influenced by pH in the pH range investigated. Taken as a whole, these results indicate that FC binding is a condition necessary, but not sufficient, for FC-induced stimulation of the PM H⁺-ATPase.     

Stereoselective construction of tetra-substituted tetrahydrofuran compounds from benzylic hemiacetal in the presence of H2 and a Pd catalyst: stereoselective synthesis of a stereoisomer of (-)-virgatusin and its antimicrobiological activity

Tetra-substituted tetrahydrofuran compounds were stereoselectively prepared from benzylic hemiacetal in the neutral condition by employing the simple reagent, H(2), and a Pd catalyst. The stereoselective conversion of benzylic hemiacetal to two different stereoisomers of the tetrasubstituted tetrahydrofuran compound was observed. One of these tetrahydrofuran compounds was converted to the virgatusin stereoisomer to estimate its antimicrobiological activity.     

Effect of CoCl2 on the content of different metals and a relative activity of DNA‐hydrolyzing abzymes in the blood plasma of mice

Cobalt is a transition metal and an essential trace element that is required for vitamin B₁₂ biosynthesis, enzyme activation, and so on but is toxic in high concentrations. It was shown that the content of different elements in the plasma of 2‐month‐old BALB/c mice (control group) decreased in the following order: Ca > Mg > Si > Fe > Zn > Cu ≥ Al ≥ B. The treatment of mice with CoCl₂ did not appreciably change the relative content of Ca, Cu, and Zn, but a significant increase in the content of B (2.3‐fold), Mg (1.5‐fold), Al and Fe (2.1‐fold), and Si (3.4‐fold) was found. The treatment of mice led to a 2.2‐fold decrease in the concentration of the total blood protein and a 1.7 ± 0.2‐fold decrease of total immunoglobulin Gs (IgGs). Deoxyribonuclease IgGs corresponding to mice treated (t‐IgGs) and non‐treated (nt‐IgGs) with CoCl₂ contained intrinsically bound metal ions; these IgGs hydrolyzed DNA with very low activity but were not active in the presence of ethylenediaminetetraacetic acid or after Ab dialysis against ethylenediaminetetraacetic acid. The average RAs of deoxyribonuclease nt‐IgGs increased after addition of external metal ions in the following order: Zn²⁺ < Ca²⁺ < Cu²⁺ < Fe²⁺ < Mn²⁺ < Mg²⁺ < Co²⁺ < Ni²⁺. Interestingly, t‐IgGs demonstrated lower activities than those for nt‐IgGs either in the absence of external metal ions (2.7‐fold) or in the presence of Cu²⁺ (9.5‐fold) > Co²⁺ (5.6‐fold) > Zn²⁺ (5.1‐fold) > Mg²⁺ (4.1‐fold) > Ca²⁺ (3.0‐fold) > Fe²⁺ (1.3‐fold). However, the RAs of t‐IgGs were remarkably more active than nt‐IgGs in the presence of best activators of t‐IgGs Ni²⁺ (1.4‐fold) and especially Mn²⁺ (2.2‐fold). The data may be useful for an understanding of Co toxicity, its effect on the concentration of other metal ions, and a change of metal‐dependent specificity of Abzs. Copyright © 2012 John Wiley & Sons, Ltd.     

Sensitivity of ATPase-ADPase activities from synaptic plasma membranes of rat forebrain to lipid peroxidation in vitro and the protective effect of vitamin E

The in vitro effects of membrane lipid peroxidation on ATPase-ADPase activities in synaptic plasma membranes from rat forebrain were investigated. Treatment of synaptic plasma membranes with an oxidant generating system (H₂O₂/Fe²⁺/ascorbate) resulted in lipid peroxidation and inhibition of the enzyme activity. Besides, trolox as a water soluble vitamin E analogue totally prevented lipid peroxidation and the inhibition of enzyme activity. These results demonstrate the susceptibility of ATPase-ADPase activities of synaptic plasma membranes to free radicals and suggest that the protective effect against lipid peroxidation by trolox prevents the inhibition of enzyme activity. Thus, inhibition of ATPase-ADPase activities of synaptic plasma membranes in cerebral oxidative stress probably is related to lipid peroxidation in the brain.     

H3 Cys-110 is in close proximity to the C-terminal regions of H2B and H4 in a nucleosome core with an altered internal arrangement of histones

A particle obtained by nuclease digestion of nucleohistone complexes prepared by direct mixing of histones with DNA in 0.15 M NaCl was indistinguishable by composition and physical properties from nucleosome cores prepared under the same conditions from nucleohistone preannealed in 0.6 M NaCl. We show here that different photo-cross-links form when these particles are prepared from H3 labeled with photoaffinity reagents on the unique histone H3 cysteine. H3-H3 histone dimers were dominant when the particles were prepared by dilution of the nucleohistone from 0.6 M NaCl while H3-H2B and H3-H4 histone dimers were prominent if the nucleohistone complex was prepared directly in 0.15 M NaCl. Peptide mapping of the novel H3-H4 and H3-H2B dimers showed that Cys-110 of histone H3 is cross-linked to the 18 amino acid C-terminal end of H4 or to the 66 amino acid C-terminal half of H2B.     

Extrapolating leaf CO2 exchange to the canopy: a generalized model of forest photosynthesis compared with measurements by eddy correlation

Over the last 4 years, two data sets have emerged which allow increased accuracy and resolution in the definition and validation of a photosynthesis model for whole forest canopies. The first is a greatly expanded set of data on the nitrogen-photosynthesis relationship for temperate and tropical woody species. The second is a unique set of long-term (4 year) daily carbon balance measurements at the Harvard Forest, Petersham, Massachusetts, collected by the eddy-correlation technique. A model (PhET-Day) is presented which is derived directly from, and validated against, these data sets. The PnET-Day model uses foliar nitrogen concentration to calculate maximum instantaneous rates of gross and net photosynthesis which are then reduced for suboptimal temperature, photosynthetically active radiation (PAR), and vapor pressure deficit (VPD). Predicted daily gross photosynthesis is closely related to gross carbon exchange at the Harvard Forest as determined by eddy-correlation measurements. Predictions made by the full canopy model were significantly better than those produced by a multiple linear regression model. Sensitivity analyses for this model for a deciduous broad-leaved forest showed results to be much more sensitive to parameters related to maximum leaf-level photosynthetic rate (A _max) than to those related to light, temperature, VPD or total foliar mass. Aggregation analyses suggest that using monthly mean climatic data to drive the canopy model will give results similar to those achieved by averaging daily eddy correlation measurements of gross carbon exchange (GCE).     

Identification of a high molecular weight presumptive precursor to albumin mRNA in the nucleus of rat liver and hepatoma cell line H4AZC2.

Nuclear RNAs prepared from rat liver and rat hepatoma cell line H4AZC2 have been fractionated and examined for albumin mRNA sequences by annealing to specific albumin [3H]cDNA. In both instances, sucrose gradient analysis revealed nuclear RNA molecules containing albumin RNA sequences which sedimented at 26 S (26 S albumin RNA). In contrast, cytoplasmic albumin messenger RNA sediments exclusively at 17 S. 26 S albumin RNA is resistant to both heat denaturation (65 degrees C X 5 min) and denaturation in 85% formamide (v/v), and 75% of these molecules are polyadenylated. These results provide evidence for the existence of an intact, high molecular weight, polyadenylated nuclear RNA which contains albumin mRNA sequences.     

Pretreatment with the probiotic VSL#3 delays transition from inflammation to dysplasia in a rat model of colitis-associated cancer

Evidence supports involvement of microflora in the transition of chronic inflammation to neoplasia. We investigated the protective efficacy of the probiotic VSL#3 in a model of colitis-associated colorectal cancer. Chronic colitis was induced in Sprague-Dawley rats by administration of trinitrobenzene sulfonic acid (TNBS), followed 6 wk later by systemic reactivation. To induce colitis-associated dysplasia and cancer, the animals received TNBS (intravenously) twice a week for 10 wk. One group received VSL#3 in drinking water from 1 wk before colitis induction until death. The colons were examined for damage and presence of dysplasia or cancer. Samples were analyzed for cell proliferation and apoptosis, vitamin D receptor (VDR) expression, angiogenic factors, and presence of alkaline sphingomyelinase or phosphatase. Microbial community composition was evaluated by terminal restriction fragment-length polymorphism analysis of the bacterial 16S rRNA gene. None of the probiotic-treated animals developed carcinoma, and no high-grade dysplasia was found in either the proximal or mid colon. In contrast, 29% of the animals in the control group developed carcinoma in one or more regions of the colon. VSL#3-treated animals had significantly less damage than the vehicle treated-controls in all areas of the colon, and this correlated with decreased richness and diversity of the mucosally adherent microbiota. Treatment with the probiotic increased the antiangiogenic factor angiostatin, VDR expression, and alkaline sphingomyelinase. We concluded that pretreatment with the probiotic VSL#3 can attenuate various inflammatory-associated parameters, delaying transition to dysplasia and cancer, thus offering its potential therapeutic use in patients with long-standing colitis.     

Isolation from the microsomal fraction of rat liver of a subfraction highly enriched in uncoated endocytic vesicles with high H+-ATPase activity and a 50 kDa phosphoprotein

A subcellular fraction, highly enriched in uncoated vesicles (UCV) with high H+-ATPase (EC 3.6.1.34) activity, was isolated from the crude microsomal fraction of rat liver homogenates by discontinuous sucrose gradient centrifugation. The UCV fraction, recovered at the interface of sucrose density 1.08 and 1.10 g/ml, was shown morphologically to be a mixture of small, smooth-surfaced univesicular and a few multivesicular structures. A permeable anion (e.g. chloride) was required for internal acidification, indicating an electroneutral proton pump. Specific inhibitors of anion transport (pyridoxal 5'-phosphate and 4-acetamide-4'-isothiocyanostilbene-2,2'-disulfonic acid) totally inhibit proton translocation. The proton pump activity was insensitive to oligomycin, but was completely inhibited by about 5 microM of the tridentate bathophenanthroline chelate of Fe(II). The activity was also inhibited 100% by low concentrations of the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the proton conduction inhibitor N,N'-dicyclohexylcarbodiimide and the ionophore monensin. The UCV fraction contained 2 proteins of Mr 50000 (major) and 54000 (minor) which were phosphorylated by an endogenous cyclic nucleotide- and Ca2+-independent protein kinase.     

Response of NAD(P)H dehydrogenase complex to the alteration of CO2 concentration in the cyanobacterium Synechocystis PCC6803

An NADPH-specific NDH-1 sub-complex was separated by native-polyacrylamide gel electrophoresis and detected by activity staining from the whole cell extracts of Synechocystis PCC6803. Low CO2 caused an increase in the activity of this sub-complex quickly, accompanied by an evident increase in the expression of NdhK and PSI-driven NADPH oxidation activity that can reflect the activity of NDH-1-mediated cyclic electron transport. During incubation with high CO2, the activities of NDH-1 sub-complex and PSI-driven NADPH oxidation as well as the protein level of NdhK slightly increased at the beginning, but decreased evidently in various degrees along with incubation time. These results suggest that CO2 concentration in vitro as a signal can control the activity of NDH-1 complex, and NDH-1 complex may in turn function in the regulation of CO2 uptake.     

Cu(II) complexes with a sulfonamide derived from benzoguanamine. Oxidative cleavage of DNA in the presence of H2O2 and ascorbate

Reaction between benzoguanamine (2,4-diamino-6-phenyl-1,3,5-triazine) and 2-mesitylenesulfonyl chloride leads to formation of a sulfonamide able to form two mononuclear Cu(II) complexes with a CuL(2) stoichiometry. The local environment of the metal cation is a distorted octahedron, with two ligands and two solvent molecules; both complexes crystallize in the monoclinic structure, space group P2(1), with Z=2. In the presence of ascorbate/H(2)O(2,) the two complexes significantly cleavage double-strand pUC18 DNA plasmid. Both complexes exhibit more nuclease efficiency that the copper phenantroline complex. From scavenging reactive oxygen studies we conclude that the hydroxyl radical and a singlet oxygen-like entity, such a peroxide copper complex, are the radical species involved in the DNA damage.