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Owing to their favorable design flexibility and eminent signal amplification ability, DNA nanomachine-supported biosensors have provided an attractive avenue for intracellular fluorescence imaging, especially for DNA walkers. However, this promising option not only suffers from poor controllability but also needs to be supplied with additional driving forces on account of the frequent employment of metal ion-dependent DNAzymes. Aiming at overcoming these obstacles, we introduce some fruitful solutions. On one hand, innovative light-activated walking behavior induced by a photocleavage mode is established on the surfaces of gold nanoparticles, and such a photoselective sensing system can be perfectly prevented from pre-activating during the intracellular delivery process and made to achieve target identification only under irradiation using a moderate ultraviolet light source. On the other hand, this light-switchable sensing frame is encapsulated within a dissociable metal-organic framework (ZIF-8) to facilitate endocytosis and ensure sufficient internal cofactors (Zn2+) to realize a self-driven pattern in the acidic environment of the cell lysosome. Based on the abovementioned efforts, the newly constructed autonomous three-dimensional DNA walkers present satisfactory sensitivity (a limit of detection of down to 19.4 pM) and specificity (even distinguishing single-base changes) toward a model biomarker (microRNA-21). More importantly, the sensing method allows determination of the variations in targets in living cancer cells with exceptional precision and efficiency, offering a powerful assay platform for intracellular imaging.

For the study on microbial contamination of reclaimed water from reverse osmosis process during storage, multiple controlled experiments were designed to simulate actual storage conditions and three influencing factors (storage materials, time and concentration of organic acids) which had an effect on the growth of microorganisms were investigated. The results showed that the stainless steel material was superior to PVC and glass material, but different materials of storage containers didn’t make a big difference in microorganism growth. In 30 days, all the total numbers of bacteria in three container materials were under 100cfu/mL which reached the Chinese Pharmacopoeia standards for purified water and also there is no clear law between organic acids concentration and microbial growth. Analyzed by DNA sequencing and checked by microscopic and electron microscopy, the pollution bacteria was identified as Bacillus. Bacteriostatic test results showed that the concentration 0-1000 mg/L of acetic acid had no significant inhibitory effect on the growth of Bacillus.

The production of hydrogen from hydrogen compounds for fuel cell or internal combustion engine applications is a potential method for responding to the energy crisis and environmental problems. In this work carbon dioxide reforming of methane and decomposition of ammonia using a Laval nozzle arc discharge (LNAD) reactor has been exploited at atmospheric pressure without external heating or catalysts. CH4 (or NH3) conversion and H2 selectivity were observed to be negatively correlated with the concentration of CH4 (or NH3) and the flux of CO2 (N2) and positively correlated with voltage and the Laval nozzle throat radius. Power consumption increased with the concentration of methane at the same CO2 flow rate, and the conversion of methane gradually increased with the content of water vapor in the gas mixture. A high conversion rate and fair H2 selectivity were achieved, 51% and 37.5%, respectively, when the methane and carbon dioxide flow rates were 4 L/min and 14 L/min, respectively, and the minimum distance between the two electrodes was 2.5 mm. The LNAD reactor used in this study exhibited a good conversion rate and low energy consumption, which should be suitable for the industrial scale-up of the system.

The wideband energy reflectance (ER) technique has become popular as a tool for evaluating middle ear function. Negative middle ear pressure (MEP) is a prevalent form of middle ear dysfunction, which may impact application of ER measurements in differential diagnosis. A negative MEP may be countervailed by application of an equivalent negative ear canal pressure. The present study examined ER in the same ears under normal and experimentally induced negative MEP conditions. Thirty-five subjects produced at least one negative MEP each (-40 to -225 daPa). Negative MEP significantly altered ER in a frequency-specific manner that varied with MEP magnitude. ER increased for low- to mid-frequencies with the largest change (~0.20 to 0.40) occurring between 1 and 1.5 kHz. ER decreased for frequencies above 3 kHz with the largest change (~-0.10 to -0.25) observed between 4.5 and 5.5 kHz. Magnitude of changes increased as MEP became more negative, as did the frequencies at which maximum changes occurred, and the frequency at which enhancement transitioned to reduction. Ear canal pressure compensation restored ER to near baseline values. This suggests that the compensation procedure adequately mitigates the effects of negative MEP on ER. Theoretical issues and clinical implications are discussed.

Four types of reverse osmosis membranes (LP, ULP, XLP and FR) were used for the treatment and reuse of the pharmaceutical condensates. The desalination rate and removal of TOC with pH 6.8 is significantly higher than pH 3.2 for all of the four membranes. When the pH is 6.8 the FR reverse osmosis membrane has the highest desalination rate, removal of TOC, permeate flux and the lowest transmembrane pressure, and it is energy conservation and not easy to be contaminated. So the FR reverse osmosis membrane is Suitable for the treatment of pharmaceutical condensate.

Four influencing factors (pH, pressure, temperature and feed water conductivity) in the separation of organic matter by the reverse osmosis were studied for the treatment and reuse of the pharmaceutical condensate. The pH of feed water had an obvious effect on the desalination rate and TOC removal rate. The temperature and pressure can obviously affect the recovery rate. In order to get a better treatment result the pH of the feed water should be maintained at above 6, the temperature should not exceed 35 °C during the operation, the pressure should be controlled at 1 to 1.5MPa.

Acetylferrocene was synthesized by acetyl chloride and ferrocene as raw materials, dichloromethane as the solvent, and ZnO as catalyst. Response surface methodology based on three-level, three-variable central composite rotable design was used to evaluate the interactive effects of the ratio of acetyl chloride and ferrocene(2-4), the amount of ZnO(1.0-1.3mol), reaction time(30-60 min)on the percentage yield of acylferrocene. The optimal raw material ratio, amount catalyst, and reaction time was 3:1, 1.19mol, 40min. Under the optimum conditions, the actual experimental yield can reach 86.72%.

Decomposition of toluene as a typical aromatic volatile organic compound was experimentally investigated in a rotating gliding arc discharge nonthermal plasma reactor. The experiments were conducted at a gas flow rate of 5.4-10.8 m3h-1. Important parameters, including applied voltage, gas flow rate, initial concentration, and water vapor concentration, were investigated. The results showed that a higher applied voltage results in higher toluene removal and higher power deposited into a discharge system; the decomposition achieved was highly dependent on the total gas flow rate. Under the fixed specific energy density (SED), both toluene removal efficiency and energy efficiency decrease with the increase in gas flow rate. The removal efficiency of toluene decreased with an increase in the initial concentration, whereas the absolute removal amount and energy efficiency increased. For example, the removal efficiency decreased from 77% to 56% with the initial toluene concentration ranging from 200 to 2000 m g-3 as the SED was 266 J L-1; the toluene removal efficiency evidently increased as the water vapor concentration increased from 0.5945 to 1708.3435 mg m-3. A dynamics model was developed to describe the relation of the removal efficiency with SED and initial concentration.

High-performance liquid chromatography (HPLC) was employed in a fingerprint analysis of Radix astragali. A characteristic HPLC profile of known Radix astragali samples collected from five primary habitats in China was established by evaluating 15 samples with chemometric methods, including similarity evaluation, hierarchical cluster analysis (HCA), principal component analysis (PCA) and discriminant analysis (DA). The feasibility and advantages of employing chromatographic fingerprinting combined with chemometric methods were investigated and demonstrated in the evaluation of Radix astragali for the first time. The results showed that the chromatographic fingerprint combined with chemometric methods could efficiently distinguish Radix astragali from various areas and provide quality control in the quantitative analysis.

A series of ferrocene schiff bases derived from Thiadiazole have been synthesized and characterized by IR, 1H NMR, and elemental analysis, the results conformed well with expected structures. The synthesized compounds were screened in vitro for their antibacterial activity against three Gram-negative (Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi) and two Gram-positive (Bacillus subtilis and Staphylococcus aureus) bacterial strains. The results showed that these compounds are a series potential new drugs in antibacterial activity against one or more species.

A number of novel 13-membered chalcone-imidazole derivatives were prepared and have been synthesized and characterized by IR, 1H NMR, 13C NMR and elemental analysis, the results conformed well to expected structures. Substituted acetophenones and benzaldehydes were condensed using the Claisen–Schmidt base-catalyzed aldol condensation. Methyl on the aromatic ring of chalcones was brominated by NBS, and then the resulting mixture was reacted with imidazole to get the target compound. Several chalcones showed in vitro antibacterial activity against Gram-bacterial. The results showed that these are potential antibacterial compounds.

Layered double hydroxides (LDHs) are potential scavengers for anionic contaminants due to their “memory effect”. Here we report the removal of thiosulfate anion from contaminated water by calcined MgAl–CO3 LDH (CLDH). The results indicate that CLDH exhibits a good performance in adsorption of thiosulfate.We studied the kinetics and thermodynamics of adsorption of thiosulfate from water. The equilibrium isotherm showed that the uptake of thiosulfate anion well agrees with the Langmuir equation, the positive value of △HӨ demonstrates the endothermic feature of the adsorption, the pseudo-second order kinetics model can be uItalic textsed to describe the uptake process and the value of Ea was calculated to be 75.898 kJ/mol. Our results suggest that the process of thiosulfate uptaking was controlled by the reaction rate of thiosulfate with the CLDH rather than diffusion.

Electrolyte containing F–was employed to conduct two-step anodic oxidation. Through adjustment of the composition of electrolyte, TiO2nanotube array with high aspect ratio was grown in situ on Ti matrix, and meanwhile the binary doping with non-metallic elements, namely, F and S, was achieved. Using FSEM, XRD and EDS, the characterization on the topography, structure and doped elements of TiO2nanotubes were performed. Taking methyl orange as the target degradation substance and using xenon lamp to simulate natural lighting, the comparison between the doped non-high-aspect-ratio nanotube and the doped high-aspect-ratio nanotube was made to investigate the related photoelectrocatalytic property. The results showed that the photoelectrocatalytic property of doped nanotube with high aspect ratio was significantly superior to that with non-high aspect ratio. The mixed crystal ratio of anatase and rutile obtained after calcination for 2h at 500°C enabled higher photoelectrocatalytic activity of TiO2nanotube array.

Using metallurgical grade aluminum hydroxide as the raw material self-dispersed g-AlOOH nanopowders were made by sol-hydrothermal crystallization and the charge process. The AlOOH/polyimide nanocomposite membrane was prepared by compounding soluble polyamide with AlOOH using sol dispersed quasi-homogeneous blending and compounding method. TEM, XRD, IR and other techniques were employed to characterize AlOOH nanopowders and AlOOH/polyimide nanocomposite membranes, respectively. The results showed that: the particle size of AlOOH crystal was about 70nm, with good self-dispersion in water and some organic solvents; in the polyimide matrix, the AlOOH was dispersed at nanoscale, without agglomeration; when its content in composite membrane reached 15 %, it still had a good dispersion and transmission property.

A series of Ferrocene Schiff bases derived from glycine and their cobalt (II), copper (II), nickel (II), and zinc (II) metal complexes have been synthesized and characterized by IR, 1H NMR, MS and elemental analysis, the results conformed well with expected structures. The synthesized ligands, along with their metal (II) complexes, were screened in vitro for their antibacterial activity against three Gram-negative (Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi) and two Gram-positive (Bacillus subtilis and Staphylococcus aureus) bacterial strains. The results showed that the metal (II) complexes are more potent in antibacterial activity against one or more species in comparison with those of uncomplexed ligands.

Apoptosis in megakaryocytes results in the formation of platelets. The role of apoptotic pathways in platelet turnover and in the apoptotic-like changes seen after platelet activation is poorly understood. ABT-263 (Navitoclax), a specific inhibitor of antiapoptotic BCL2 proteins, which is currently being evaluated in clinical trials for the treatment of leukemia and other malignancies, induces a dose-limiting thrombocytopenia. In this study, the relationship between BCL2/BCL-XL inhibition, apoptosis, and platelet activation was investigated. Exposure to ABT-263 induced apoptosis but repressed platelet activation by physiologic agonists. Notably, ABT-263 induced an immediate calcium response in platelets and the depletion of intracellular calcium stores, indicating that on BCL2/BCL-XL inhibition platelet activation is abrogated because of a diminished calcium signaling. By comparing the effects of ABT-263 and its analog ABT-737 on platelets and leukemia cells from the same donor, we show, for the first time, that these BCL2/BCL-XL inhibitors do not offer any selective toxicity but induce apoptosis at similar concentrations in leukemia cells and platelets. However, reticulated platelets are less sensitive to apoptosis, supporting the hypothesis that treatment with ABT-263 induces a selective loss of older platelets and providing an explanation for the transient thrombocytopenia observed on ABT-263 treatment.

A biodegradable tumor targeting nano-probe based on poly(ɛ-caprolactone)-b-poly(ethylene glycol) block copolymer (PCL-b-PEG) micelle functionalized with a magnetic resonance imaging (MRI) contrast agent diethylenetriaminepentaacetic acid-gadolinium (DTPA-Gd3+) on the shell and a near-infrared (NIR) dye in the core for magnetic resonance and optical dual-modality imaging was prepared. The longitudinal relaxivity (r1) of the PCL-b-PEGDTPA-Gd3+ micelle was 13.4 (mmol/L)−1s−1, three folds of that of DTPA-Gd3+, and higher than that of many polymeric contrast agents with similar structures. The in vivo optical imaging of a nude mouse bearing xenografted breast tumor showed that the dual-modality micelle preferentially accumulated in the tumor via the folic acid-mediated active targeting and the passive accumulation by the enhanced permeability and retention (EPR) effect. The results indicated that the dualmodality micelle is a promising nano-probe for cancer detection and diagnosis.

Caofeidian is a large harbor being constructed in north of China. It is greatly meaningful to investigate the pollution situation of this area. In this paper, firstly, a tidal flow and pollutant transport numerical model based on Delft 3D-FLOW was established and verified by measured data to simulate and analyze phosphate distribution in Caofeidian sea area. Then, phosphate concentration under six hydrological conditions (spring, middle and neap tides of flood season; and spring, middle and neap tides of dry season) was computed by this numerical model, and the comparisons were made between the computed phosphate concentration at the boundary of the mixing zones and corresponding water quality standards. At last, the allowances discharge amount and cut rate of phosphate of each outfall was calculated. The results show that the high phosphate concentration appears the sea area around the river outlets of Douhe and Shahe, and the phosphate concentration of Douhe and Shuanglonghe rivers’ mixing zones exceed their allowed standard values, the total phosphate discharge from these rivers must be cut down.

The enhanced reactivity exhibited by six pseudo-α-bases, RC≡CZ− (R = H and Cl; Z = O, S, and Se) in gas-phase E2 reactions with ethyl chloride was examined at the G2(+) level. It is found that anomalous reactivity is observed despite the fact that these chalcogen bases do not possess adjacent lone-pair electrons. The influence of the halide leaving groups on the α-effect and the origin of the α-effect in the E2 reactions of ethyl halides are investigated and discussed.

The results of a theoretical study of the reaction mechanism for the neutral hydration of ketene, H2C=C=O + (n + 1) H2O → CH3COOH + nH2O (n = 0–4), in solution are presented. All structures were optimized and characterized at the MP2(fc)/6-31 + G* level of theory, and then re-optimized by MP2(fc)/6-311 ++G**, and the effect of the bulk solvent is taken into account according to the conductor-like polarized continuum model (CPCM) using the gas MP2(fc)/6-311 ++G** geometries. Energies were refined for five-water hydration at higher level of theory, QCISD(T)(fc)/6-311 ++G**//MP2(fc)/6-311 ++G**. In the combined supermolecular/continuum model, one water molecule directly attacks the central C-atom, and the other four explicit water molecules are divided into two groups, one acting as catalyst(s) by participating in the proton transfer to reduce the tension of proton transfer ring, and the other being placed near the non-reactive oxygen or carbon atom in order to catalyze the hydration by engaging in hydrogen-bonding to the substrate (the so-called cooperative effect). Between the two possible nucleophilic addition reactions of water molecule, across the C=O bond or the C=C bond, the former one is preferred. Our calculations suggest that the favorable hydrolysis mechanism of ketene involves a sort of eight-membered ring transition structure formed by a three-water proton transfer loop, and a cooperative dimeric water near the non-reactive carbon-atom. The best-estimated in the present paper for the rate-determining barrier in solution, $$ \Updelta G_{\text{sol}}^{ \ne } $$ (298 K), is about 58 kJ/mol, reasonably close to the available experimental result.

The water-catalyzed hydrolysis reaction of carbon disulfide (CS(2)) has been investigated at the levels of HF and MP2 with the basis set of 6-311++G(d,p) using the combined supramolecular/continuum models, in which up to six water molecules are involved in the hydrolysis and the effect of water bulk solvent is taken into account according to the polarizable continuum model (PCM). The activation Gibbs free energies in water solution, DeltaG(sol) (not equal) (298 K), for the rate-determining steps of one up to six water hydrolyses are 247.9, 184.2, 152.3, 141.8, 134.4, and 118.9 kJ/mol, respectively. The most favorable hydrolysis path of CS(2) involves a sort of eight-membered ring transition structure formed by six water molecules, among which three water molecules are not involved in the proton transfer, two near to the nonreactive sulfur atom, and one below the parent carbon disulfide. This suggests that the hydrolysis of CS(2) can be mediated with the water molecule(s) and be significantly facilitated by the cooperative effects of the water molecule(s) in the nonreactive region. The catalytic effects of water molecule(s) due to the alleviation of ring strain in the proton transfer process may result from the synergistic effects of rehybridization and charge reorganization from the prereaction complex to the rate-determining transition state structure induced by water molecule(s). PCM solvation models could significantly lower the rate-determining activation Gibbs free energies by 20-38 kJ/mol when two up to six explicit water molecules involved in the neutral hydrolysis of CS(2).

Distortion product otoacoustic emissions (DPOAEs) were measured in the absence and presence of contralateral noise at five levels--below, equal to, and above the middle-ear muscle (MEM) reflex threshold. The resultant changes in DPOAE level and phase were dependent on stimulus frequency and noise level. Both low-level noise, believed to elicit the medial olivocochlear (MOC) reflex, and high-level noise, thought to activate both MOC and MEM reflexes, significantly decreased the DPOAE level. However, the shift from sole MOC effect to mixed MOC and MEM effects was not as dramatic as we thought. While low-level noise resulted in a minimum DPOAE phase change, high-level noise caused a substantial phase lead for 1 and 2kHz. With increasing frequency, phase lag became more notable. The present study suggests the following: (1) DPOAE contralateral suppression by low-level sound most likely does not involve the effect of the MEM reflex and signal crossover; and (2) combined analysis of DPOAE level and phase changes warrants further investigations to overcome the difficulty in separating the effects of MOC efferents and MEM contraction. The results also imply that OAE measurement has the potential for being used to investigate the effect of the MEM reflex on sound transmission.

We have used chromosome microdissection and microcloning to construct a DNA library of the entire B chromosome (B) of rye. New rye B-specific sequences have been screened from this pool, blasted with other sequences and analyzed to elucidate the characters of DNA constitution and the possible pathway of the origin of the rye B chromosome. We report the discovery of a new sequence that is specific to the rye B centromere.

Artemisinin and its derivatives are currently recommended as first-line antimalarials in regions where Plasmodium falciparum is resistant to traditional drugs. The cytotoxic activity of these endoperoxides toward rapidly dividing human carcinoma cells and cell lines has been reported, and it is hypothesized that activation of the endoperoxide bridge by an iron(II) species, to form C-centered radicals, is essential for cytotoxicity. The studies described here have utilized artemisinin derivatives, dihydroartemisinin, 10beta-(p-bromophenoxy)dihydroartemisinin, and 10beta-(p-fluorophenoxy)dihydroartemisinin, to determine the chemistry of endoperoxide bridge activation to reactive intermediates responsible for initiating cell death and to elucidate the molecular mechanism of cell death. These studies have demonstrated the selective cytotoxic activity of the endoperoxides toward leukemia cell lines (HL-60 and Jurkat) over quiescent peripheral blood mononuclear cells. Deoxy-10beta-(p-fluorophenoxy)dihydroartemisinin, which lacks the endoperoxide bridge, was 50- and 130-fold less active in HL-60 and Jurkat cells, respectively, confirming the importance of this functional group for cytotoxicity. We have shown that chemical activation is responsible for cytotoxicity by using liquid chromatography-mass spectrometry analysis to monitor endoperoxide activation by measurement of a stable rearrangement product of endoperoxide-derived radicals, which was formed in sensitive HL-60 cells but not in insensitive peripheral blood mononuclear cells. In HL-60 cells the endoperoxides induce caspase-dependent apoptotic cell death characterized by concentration- and time-dependent mitochondrial membrane depolarization, activation of caspases-3 and -7, sub-G(0)/G(1) DNA formation, and attenuation by benzyloxycarbonyl-VAD-fluoromethyl ketone, a caspase inhibitor. Overall, these results indicate that endoperoxide-induced cell death is a consequence of activation of the endoperoxide bridge to radical species, which triggers caspase-dependent apoptosis.

The reactivity order of 12 anions toward ethyl chloride has been investigated by using the G2(+) method, and the competitive E2 and SN2 reactions are discussed and compared. The reactions studied are X(-) + CH3CH2Cl → HX + CH2═CH2 + Cl(-) and X(-) + CH3CH2Cl → CH3CH2X + Cl(-), with X = F, Cl, Br, HO, HS, HSe, NH2 PH2, AsH2, CH3, SiH3, and GeH3. Our results indicate that there is no general and straightforward relationship between the overall barriers and the proton affinity (PA) of X(-); instead, discernible linear correlations only exist for the X's within the same group of the periodic table. Similar correlations are also found with the electronegativity of central atoms in X, deformation energy of the E2 transition state (TS), and the overall enthalpy of reaction. It is revealed that the electronegativity will significantly affect the barrier height, and a more electronegative X will stabilize the E2 and SN2 transition states. Multiple linear regression analysis shows that there is a reasonable linear correlation between E2 (or SN2) overall barriers and the linear combination of PA of X(-) and electronegativity of the central atom.

Raf‐1 protein kinase has been identified as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf‐1 is achieved by Ras.GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. We have used gene targeting to generate a ‘knockout’ of the raf‐1 gene in mice as well as a rafFF mutant version of endogenous Raf‐1 with Y340FY341F mutations. Raf‐1 −/− mice die in embryogenesis and show vascular defects in the yolk sac and placenta as well as increased apoptosis of embryonic tissues. Cell proliferation is not affected. Raf‐1 from cells derived from raf‐1 FF/FF mice has no detectable activity towards MEK in vitro , and yet raf‐1 FF/FF mice survive to adulthood, are fertile and have an apparently normal phenotype. In cells derived from both the raf‐1 −/− and raf‐1 FF/FF mice, ERK activation is normal. These results strongly argue that MEK kinase activity of Raf‐1 is not essential for normal mouse development and that Raf‐1 plays a key role in preventing apoptosis.

Objective To examine the role of constitutive and inducible nitric oxide synthases (cNOS and iNOS) in platelet-activating factor (PAF)-induced shock and intestinal injury. Design Prospective, randomized, controlled experimental study. Setting Hospital research laboratory. Subjects Young adult male Sprague-Dawley rats were anesthetized and studied. Interventions Rats were injected with PAF, either alone or after the following pretreatments: a) selective iNOS inhibitors aminoguanidine or S-methylisothiourea; b) 3-morpholinosydnonimine, a NO donor; c) S-methylisothiourea + 3-morpholinosydnonimine; and d) antineutrophil antibody (to deplete neutrophils). Measurements and main results Blood pressure, hematocrit, white blood cell counts, intestinal injury, and intestinal cNOS and iNOS activities were assessed. We found that: a) cNOS is the predominant NOS in the intestine and its activity is inversely correlated to the level of tissue injury; b) there is a time-dependent increase in cNOS activity in sham-operated animals, which was abolished by PAF; c) Western blotting and immunohistochemistry showed iNOS present in the normal intestine, localizing mainly in crypt cells; d) iNOS inhibitors attenuated PAF-induced injury in animals with high cNOS activity, but had no protective effect in animals with low cNOS activity; e) 3-morpholinosydnonimine, alone or together with S-methylisothiourea, alleviated PAF-induced injury; and f) neutrophil depletion blocked the suppressive effect of PAF on cNOS and prevented injury. Conclusions We conclude that cNOS and iNOS play different roles in PAF-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of iNOS inhibitors.

In a previous study, we showed that anti-CD11b or anti-CD18 antibody markedly attenuated platelet-activating factor (PAF)-induced shock and intestinal necrosis in rats, whereas anti-P-selectin anti-body was ineffective. Here we used genetically altered mice to study the mechanism of PAF in mice. We found that P-selectin-deficient mice are completely protected from the adverse effects of PAF with no mortality or intestinal injury and only mild hemoconcentration and transient hypotension. In contrast, CD18- or intercellular adhesion molecule 1 (ICAM-1)-deficient mice were not protected from PAF-induced tissue injury and death. However, when ICAM-1-, but not CD18-, deficient mice were pretreated with fucoidin, the adverse effects of PAF were markedly reduced; survival was 100%, although hypotension still developed. Neutrophil-depleted mice were protected from PAF-induced intestinal injury but still developed hypotension and hemoconcentration. PAF increases peripheral blood neutrophil counts, probably by inducing granulopoiesis, since neutrophil-depleted mice still showed granulocytosis 60 min after PAF. Thus P-selectin plays an important role in PAF-induced injury in mice, and the selectins and the integrin-ICAM-1 system work in concert to mediate the inflammatory response to PAF in vivo.

Leukocyte adhesion and diapedesis, critical steps in the inflammatory process, depend on the expression of integrin CD11b/CD18. In this study, we examined the preventive effect of monoclonal antibodies (MAb) against CD11b (1B6), CD11a, or CD18 (CL26) on platelet-activating factor (PAF)-induced bowel injury. Young male Sprague-Dawley rats were anesthetized and injected with either of two doses of PAF (2.5 or 3 micrograms/kg iv) to induce transient hypotension and irreversible shock. Some rats wee also injected intravenously with 1B6 (anti-CD11b), anti-CD11a, CL26 (anti-CD18), or combined anti-CD11a and 1B6, 30 min before PAF. Animals receiving a low dose of PAF developed mild hypotension, hemoconcentration, increased intestinal myeloperoxidase, and bowel injury after 1 h. These effects were completely prevented by pretreatment with 1B6. A high dose of PAF induced irreversible shock and gross intestinal necrosis. Both CL26 and 1B6 were partially effective in attenuating PAF-induced bowel injury. Addition of anti-CD11a to 1B6 in the treatment further ameliorated the systemic adverse effects of PAF and intestinal injury. However, focal minor injury still developed. Anti-CD11a alone, fucoidin, or anti-P-selectin was ineffective. Rats depleted of neutrophils were also largely protected from the adverse effects of PAF at high doses, although minor intestinal injury often persisted. We conclude that leukocyte beta 2-integrins play an important role in PAF-induced hypotension, leukopenia, hemoconcentration, and intestinal necrosis, and that CD11b/CD18 is the main adhesion molecule involved in the pathogenesis of injury. However, CD11/CD18- and neutrophil-independent pathways exist for mediating PAF-induced bowel injury, although their role is probably a minor one.

Background & Aims: Platelet-activating factor (PAF), an endogenous mediator for experimental sepsis, has been shown to induce shock and intestinal necrosis in vivo. However, it is unclear whether PAF exerts its injurious effects on the intestinal tissue directly or via synergism with other endogenous products. The aim of this study was to examine the role of endogenous bacterial products, such as endotoxin, in PAF-induced intestinal injury. Methods: PAF (3 μg/kg) was injected intravenously into normally colonized rats, germfree rats, and normal rats pretreated with a combination of antibiotics, and the systemic response and intestinal injury were assessed. Results: PAF did not cause prolonged shock, leukopenia, hemoconcentration, and bowel necrosis in germfree rats. When germfree rats were primed with a low dose (0.5 mg/kg) of endotoxin, the protection was lost. Combined treatment of the normally colonized rats with neomycin, polymyxin B, and metronidazole for 7 days largely protected the animal from PAF-induced shock and intestinal necrosis. Conclusions: PAF does not directly induce prolonged hypotension, hemoconcentration, persistent leukopenia, and gross intestinal necrosis but causes these changes via a synergism with endogenous bacterial toxins, presumably from the gut flora.

Cleavage of DNA into oligonucleosomal fragments, recognizable as a DNA ladder on agarose gel electrophoresis, is usually considered as the biochemical hallmark of apoptosis. Recently, it has been shown that this internucleosomal cleavage is preceded by the formation of large fragments of DNA of > or = 700, 200-250, and 30-50 kilobase pairs (kbp) in length. Using isolated thymocyte nuclei, we now demonstrate that the formation of these large fragments is Mg(2+)- but not Ca(2+)-dependent. Further degradation of > or = 460- and 200-250-kbp fragments to 30-50-kbp fragments but not oligonucleosomal cleavage is also Mg(2+)-dependent but is facilitated by Ca2+. In contrast, formation of oligonucleosomal fragments does not occur in the presence of either Ca2+ or Mg2+ alone but requires the presence of both cations. These results support the hypothesis that the formation of these large fragments and the subsequent internucleosomal cleavage are distinct steps in the degradation of DNA in apoptosis in thymocytes.

Etoposide, a DNA topoisomerase II inhibitor, caused a concentration-dependent induction of apoptosis in immature thymocytes. Using a flow cytometric method to separate and quantify normal and apoptotic cells, etoposide-induced apoptosis was inhibited by cycloheximide and actinomycin D but not by zinc. Etoposide induced a marked cleavage of DNA into nucleosomal length fragments or multiples thereof, which was completely inhibited if the thymocytes were also incubated in the presence of zinc. Etoposide, alone, induced the classical ultrastructural features of apoptosis, but in the presence of zinc, the morphological pattern was markedly different and dominated by discrete clumps of condensed chromatin abutting the nuclear membrane. These latter changes resemble those described as the earliest changes in apoptosis. These results support the hypothesis that, in the induction of apoptosis, critical alterations in nuclear chromatin occur prior to endonuclease cleavage of DNA into nucleosomal fragments.

We have recently developed a method for the separation and quantification of viable apoptotic cells without the need for permeabilisation or fixation of the cells. The method is based on the observation that apoptotic rat thymocytes fluoresce more brightly than normal cells after a brief incubation with the DNA binding dye, Hoechst 33342. In order to understand these differences, we have investigated the uptake of Hoechst 33342 by normal and apoptotic thymocytes. By staining with fluorescein diacetate, we have shown that the efflux of fluorescein from apoptotic cells is more rapid than that from normal thymocytes. This result demonstrated an increase in the permeability of the plasma membrane of the apoptotic thymocytes and it is this change which probably results in the more rapid uptake of Hoechst 33342. The data also revealed two populations of apoptotic thymocytes. © 1993 Wiley-Liss, Inc.

Using flow cytometry, we describe a method for separating and quantifying normal and apoptotic thymocytes. Apoptosis was induced in isolated thymocytes from immature rats by treatment with the glucocorticoid dexamethasone or the antitumor agent etoposide. Subsequent incubation with the vital bisbenzimidazole dye Hoechst 33342 and the DNA intercalating agent propidium iodide enabled three distinct populations of cells to be identified and sorted by flow cytometry. Dead cells fluoresced red due to propidium iodide whereas normal and apoptotic cells fluoresced blue due to Hoechst 33342. Apoptotic cells were distinguished from normal thymocytes both by their higher intensity of blue fluorescence and by their smaller size as determined by a reduction in forward light scatter. The larger cells, with low blue fluorescence, showed normal thymocyte morphology by electron microscopy and the absence of any DNA fragmentation as measured by agarose gel electrophoresis. In contrast, the smaller cells showed both the morphological characteristics of apoptosis and extensive internucleosomal fragmentation of DNA to multiples of approximately 180 bp. Using this method, a time-dependent induction of apoptosis by dexamethasone, which was inhibited by cycloheximide, actinomycin D, and aurin tricarboxylate, was observed. The method should facilitate mechanistic studies on the induction of apoptosis in thymocytes.

The detailed reaction pathways for the hydration of ketenimine by water and water clusters containing up to five explicit water molecules (CH(2) =C=NH + (n + 1)H(2)O --CH(3)CONH(2) + nH(2)O, n = 0-4) in the gas phase have been investigated by the MP2 method in conjunction with the 6-31+G* and 6-311++G** basis sets, and the effects of bulk solvent are taken into account according to the conductor-like polarizable continuum model (COSMO). In the hybrid cluster/continuum model, apart from one directly attacking water molecule, four explicit water molecules participating in the water-assisted hydrolysis of ketenimine are divided into two groups, one involving in the proton relay and the other near the nonreactive nitrogen or carbon atom. Two possible reaction channels, water addition across the C=C bond or across the C horizontal lineN bond of ketenimine, are discussed. Our results indicate that the kinetically favorable mechanism involves an eight-membered ring transition state structure formed by a proton transfer chain of three water molecules. Meanwhile, two additional cooperative water molecules near the nonreactive region assist the hydration by engaging in hydrogen bonding to the substrate; such an interaction is found to be important in the hydration of ketenimine and other cumulenes. The lowest rate-determining activation barriers of C=C and C=N addition are 98.9 and 95.1 kJ/mol, respectively, suggesting that the two channels are competitive when more water molecules take part in the hydration. COSMO solution models do not modify the calculated energy barriers in a significant way.

Necrotizing enterocolitis (NEC) is an important neonatal disease with a high mortality rate. The pathophysiology is unclear but epidemiologic studies suggest that hypoxia and infection are important risk factors. In this review we discuss the effect of hypoxia and platelet-activating factor (PAF) on intestinal blood flow and intestinal necrosis, and implicate PAF as an important mediator in hypoxia-induced intestinal injury. Finally we provide evidence that PAF may be important in neonatal NEC.

Tumor necrosis factor-alpha (TNF-alpha), a key inflammatory cytokine, plays an important role in atherosclerosis. However, its precise characters in primary stage of the disease remain unclear. To assess the influence of TNF-alpha on inflammatory factors in aorta and liver in apoE and TNF-alpha double mutant (AT) mice, a comparative study on early fatty-streak lesion, the mRNA level of target gene in aorta and liver of adolescent AT and apoE-null (apoE(-/-)) mice were achieved. The characteristics of expression of inflammatory factors, and early fatty-streak lesion relevance were analyzed. The plasma cytokines in 6-week-old AT and apoE(-/-) mice were also measured. Lipid accumulation in the intima of the aorta existed as early as 3 weeks of age in apoE(-/-) mice. Fatty-streak lesion was mild in AT mice but prominent in apoE(-/-) mice, at age of 6 weeks. Furthermore, most interesting findings indicate that mRNA levels of pro-atherosclerotic factors, i.e. IL-1beta, IFN-gamma, ICAM-1, VCAM-1, MCP-1, GM-CSF and NF-kappaB (p65) were significantly downregulated in AT mice. Whereas IL-2 and IkappaB-alpha were upregulated in aorta of AT mice versus those in apoE(-/-) mice (p

Platelet activating factor (PAF) is an endogenous phospholipid mediator that causes pulmonary hypertension and thrombocytopenia in experimental animal models. To investigate circulating PAF in persistent pulmonary hypertension of the newborn (PPHN), we studied PAF and its degradative enzyme, acetylhydrolase. Thirteen neonates with PPHN, diagnosed by routine clinical methods including echocardiography, were compared to six age-matched control patients with respiratory distress. Overall, plasma PAF levels were elevated in patients with PPHN compared to control patients (20.1 +/- 3.9 versus 1.6 +/- 0.7 ng/ml, p less than 0.01). In addition, plasma PAF concentrations in patients with PPHN correlated with the severity of disease as defined by the delta AaPO2 (r = 0.65, p = 0.015). In three patients with elevated PAF levels, as the clinical status improved, the plasma PAF values decreased. Acetylhydrolase activity was similar in both groups (3.96 +/- 0.90 versus 3.78 +/- 1.44 nmol/ml/min, p = NS). We conclude that PAF production is increased in PPHN and that abnormal production of PAF may be associated with pulmonary hypertension.

We have previously shown that injection of plateletactivating factor causes necrotizing enterocolitis in the rat and that platelet-activating factor is an endogenous mediator in lipopolysaccharide-induced bowel necrosis. Because hypoxia is a known predisposing factor for neonatal necrotizing enterocolitis, we investigated the effect of hypoxia on platelet-activating factor formation and intestinal necrosis. Young male Sprague-Dawley rats were made severely hypoxic by placing them in a 100% N 2 chamber for 2 minutes; moderate hypoxia was accomplished using 10% O 2 for 15 or 30 minutes. To evaluate the role of platelet-activating factor on intestinal perfusion and injury, two platelet-activating factor antagonists, SRI 63-441 and WEB 2086, were injected 10 minutes before the hypoxic exposure. We found that plasma platelet-activating factor levels were significantly elevated after 2 minutes of severe hypoxia (13.8 ± 2.9 ng/mL vs. control 2.1 ± 0.8 ng/mL) and after 30 minutes of moderate hypoxia (41.1 ± 11.7 ng/mL). This increase in platelet-activating factor level was not caused by decreased degradation, because neither plasma nor intestinal platelet-activating factor acetylhydrolase was decreased in the hypoxic rats. (Intestinal acetylhydrolase activity was actually increased.) Intestinal perfusion was markedly decreased at 30 minutes in hypoxic animals. In contrast, all platelet-activating factor antagonist-treated animals had normal intestinal perfusion. Histological examination of affected bowel from hypoxic animals showed early intestinal necrosis which was completely prevented by pretreatment with SRI 63–441 and WEB 2086. Because 30 minutes of hypoxia also resulted in metabolic acidosis, we further investigated if acidosis alone could induce platelet-activating factor release and bowel injury. We found that acidosis alone resulted in moderate increase of plasma platelet-activating factor but did not produce bowel injury. We conclude that platelet-activating factor plays a central role in mediating hypoxia-induced intestinal necrosis. Acidosis may enhance the effect of hypoxia on platelet-activating factor production.

Apoptosis is a major form of cell death, characterized morphologically by chromatin condensation and biochemically by endonuclease cleavage of DNA into oligonucleosomal fragments. Recently, we reported that zinc arrested dexamethasone-induced apoptosis in thymocytes at an early stage, as characterized morphologically by condensation of heterochromatin into clumps abutting the nuclear membrane. In this study, we show that zinc completely inhibits endonuclease cleavage of DNA into oligonucleosomal fragments but does not prevent the cleavage of DNA into high molecular weight fragments. These results indicate that the formation of these high molecular weight fragments, which correlates with the very early morphological features of apoptosis, is a critical event in glucocorticoid-induced apoptosis. The formation of these high molecular weight fragments, despite the inhibition by zinc of the endonuclease cleavage of DNA, suggests that key enzyme(s), other than the Ca2+/Mg(2+)-dependent endonuclease, are involved at the earliest stages of induction of apoptosis.

Inflammation, a process necessary for destroying invading organisms, may cause tissue damage in the host. The intestinal mucosa is especially at risk due to its constant exposure to abundant bacteria flora (1012 organisms/g). Invasion by bacteria or their products, such as endotoxin (LPS), may elicit the release of inflammatory mediators that synergize with each other and induce one another’s production, thereby leading to eventual tissue destruction. Mild inflammatory reactions may be self-limiting, due to protective mechanisms intrinsic to the intestinal mucosa. Severe insults overwhelm the protective mechanism resulting in irreversible intestinal necrosis. This paper will discuss some interactions between pro-inflammatory lipid mediators and protective mediators, e. g., PAF, eicosanoids, phospholipase A2 (PLA2), and nitric oxide (NO), in an experimental model of intestinal injury. PAF (platelet-activating factor, paf-acether), an endogenous phospholipid mediator (for review, see Refs. 1–3), is used to elicit experimental injury because PAF has been shown to be the endogenous mediator for LPS-induced bowel injury (4), and has been implicated in necrotizing enterocolitis (reviewed in ref. 5), and other intestinal inflammatory lesions (reviewed in ref. 6). PAF is synthesized by intestinal epithelial cells (7), as well as various inflammatory cells and endothelial cells (for review, see Refs. 1–3) which are resident cells in the lamina propria of the intestinal mucosa. Systemic administration of PAF induces potent pathophysiological changes mimicking circulatory shock (reviewed in Refs. 1–3), and intestinal necrosis (8). LPS and PAF act synergistically to induce shock and bowel necrosis (8). Combined LPS and TNF-a administration also cause bowel necrosis (9), which is probably mediated by endogenous PAF (9).

A novel radioiodinated ligand with a high specific activity (2,200 Ci mmol ), 3-[4-(4-iodophenyl)piperazin-1-yl]methyl-1H-pyrrolo(2,3-b)pyridine([125I]IPMPP), was successfully prepared. Binding characteristics of [125I]IPMPP were evaluated using human dopamine D4 (D4.2 variant) receptors expressed in Chinese hamster ovary (CHO) cells. Saturation analysis revealed high-affinity binding sites for [125I]IPMPP (Kd = 0.39 ± 0.18 nM). The number of D4 receptors labeled with [125I]IPMPP at room temperature was four times higher than that labeled with [125I]S(−)5-OH-PIPAT, a radioiodinated agonist ligand (572 fmol mg protein vs. 125 fmol mg protein). A significant decrease in the number of binding sites was observed with [125I]S(−)5-OH-PIPAT when assays were carried out at a higher temperature (37 °C vs. 25 °C). In contrast to [125I]S(−)5-OH-PIPAT, [125I]IPMPP labeled more D4 sites at 37 °C. Neither magnesium ion nor guanyl-imidodiphosphate (Gpp(NH)p) affected [125I]IPMPP binding. These data support the conclusion that [125I]IPMPP is an antagonist ligand. The potency of various compounds, including clozapine, to inhibit [125I]IPMPP binding is consistent with the rank order measured with other radioligands for D4 receptors. In addition, measuring D4 receptor stimulation of [35S]GTPγS binding further demonstrated the antagonist property of IPMPP.

C3H/HeJ (endotoxin-resistant) mice have been used widely in biological research. However, the mechanism of endotoxin (LPS) resistance is only partially understood. In this study, we first investigated the differences in response to PAF, a mediator of endotoxin shock, between normal and C3H/HeJ mice. We found that in control mice, PAF (2.5 micrograms/kg) caused shock, hemoconcentration, complement activation, intestinal hypoperfusion, and necrosis with 75% mortality, whereas all C3H/HeJ mice survived, without complement activation or intestinal injury, and manifesting only mild hypotension and hemoconcentration. PAF also caused elevated serum TNF-alpha in some control mice but not in C3H/HeJ mice. We also observed that PAF induces endogenous PAF production and intestinal phospholipase A2 activation in normal mice, whereas PAF production and phospholipase A2 are suppressed in C3H/HeJ mice. The low endogenous PAF production may account, at least in part, for the resistance to LPS and PAF of C3H/HeJ mice.

A time- and dose-dependent thymic atrophy was observed in young male Fischer 344 rats dosed intraperitoneally with etoposide (10, 30, or 100 mg/kg). Histopathological examination of the thymus revealed that the pattern of cell death in the majority of thymocytes had a characteristic apoptotic morphology typified by nuclear condensation. This observation was supported by the formation of internucleosomal fragments of DNA in thymocytes isolated from animals dosed with etoposide. Administration of the protein synthesis inhibitor, cycloheximide (1.5 mg/kg, ip), 1 hr prior to etoposide inhibited the induction of apoptosis in thymocytes, assessed by both biochemical and histological criteria. Flow cytometric analysis of thymocytes from animals dosed with etoposide in vivo revealed the formation of both apoptotic cells and apoptotic bodies in contrast to previous in vitro studies which showed the formation of only apoptotic cells. Our data indicate that the induction of apoptosis in thymocytes is a major mechanism involved in etoposide-induced thymic atrophy.

The pathogenesis of necrotizing enterocolitis (NEC) is poorly understood. We have established several animal models of NEC by using a combination of various stimuli and stress, including endotoxin, PAF, TNF, and hypoxia. We discuss the mechanism of their actions and the possible roles of these factors in the pathogenesis of human NEC.

Apoptosis and necrosis are two important mechanisms of cell death. Several methods have recently been described for quantifying apoptotic cells by flow cytometry. We report a novel method for the quantification and separation of viable normal and apoptotic cells. We have applied this method both to immature rat thymocytes treated with a variety of agents and to a murine haemopoetic cell line after withdrawal of a growth factor. The cells were incubated with two dyes which give fluorescent complexes when bound to DNA, the bis-benzimidazole, Hoechst 33342, and propidium iodide. Three populations were identified and characterized. On excitation with UV radiation, dead cells fluoresced red due to the uptake of propidium iodide whereas apoptotic cells fluoresced bright blue; normal cells showed low blue, low red fluorescence. In this paper, we demonstrate how this method may be used to help to distinguish between cell death by apoptosis and necrosis.