Search

Tui Na or massage therapy alleviates symptoms related to intervertebral disc degeneration (IDD). However, precise, repeatable, standardized instructions for Tuina manipulation are lacking. This study establishes IDD model rabbits induced by fibrous ring puncture, creates targeted Tuina stimulation protocols at the acupuncture points in the lumbar region, and describes in detail the operation methods and requirements of kneading, pointing, and flicking. New Zealand male white rabbits (n = 15) were selected and randomly divided into a blank group, a model group, and a Tuina group. The rabbits in the model group and the Tuina group were molded by fibrous ring puncture; the rabbits in the model group were only immobilized on the operating table without treatment. In contrast, the Tuina group used the "8N/10N, 30 cycles/min" prescription for kneading, pointing, and flicking to perform the intervention, using tactile sensory aids to monitor and regulate the intensity of the Tuina operation. Imaging diagnosis and pathological tests were used to assess the effect of Tuina in rabbits, and the results showed improved imaging features and significantly lowered pathology scores of lumbar disc degeneration in the Tuina group compared to the model group (P < 0.01). Targeted Tuina in the lumbar region may be beneficial in the alleviation of lumbar disc degeneration, but further verification is needed. By regularly performing Tuina and recording the mechanical information involved enables reproducible manipulation prescriptions and helps to observe the basic features of the underlying mechanism of Tuina for IDD.

Objective: To observe the effect of acupuncture serum on the number of apoptosis of the cultured hippocampal neurons with seizure-like discharges and the expression levels of endoplasmic reticulum stress-inducible molecular chaperones glucose-regulated protein 78 (GRP 78), C/EBP homologous protein (CHOP) and cysteine protease protein-12(Caspase-12), so as to reveal its protective mechanism on seizure-induced injury of hippocampal neurons., Methods: A regular primary culture of neurons derived from the hippocampus of the newly-born SD rats was conducted for 10 days, then these cultured neurons that displayed seizure-like discharges in Mg 2+ -free medium were divided into normal extracellular fluid (medium) group (normal), Mg 2+ -free medium group, acupuncture serum group and non-acupuncture serum group ( n =30). Blood examples were taken from pentylenetetrazol (i.p.i.) -induced acute convulsion adult SD rats underwent manual acupuncture stimulation of "Baihui" (GV 20) and "Dazhui" (GV 14, once daily for 7 days) to prepare acupuncture serum. Hippocampal neuronal cultures were prepared from hip-pocampal tissue isolated from 24 h-old SD rats. The isolated neurons were incubated normally in Dulbecco's Modified Eagle Me-dium (DMEM)/Nutrient Mixture F 12(1:1) for 10 days, followed by exposure to the extracellular fluid {composed of NaCl, KCl, CaCl 2 , MgCl 2 , HEPES[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], D-glucose and aminoethanoic acid (pH 7.3)} for 3 h and returned to normal culture for normal group, by exposure to Mg 2+ -free extracellular fluid (to induce seizure-like discharges) for 3 h and returned to normal culture for Mg 2+ -free medium group, by exposure to Mg 2+ -free extracellular fluid for 3 h and returned to a regular culture medium[DMEM/F 12(1:1)] plus acupuncture serum (9:1) for acupuncture serum group, and by exposure to Mg 2+ -free extracellular fluid for 3 h and returned to DMEM/F 12(1:1) plus non-acupuncture serum (9:1) for non-acupuncture serum group. The cell apoptosis was assessed at 2, 12 and 48 h after application of acupuncture serum or non-acupuncture serum by using TUNEL method and the expression levels of GRP 78, CHOP and Caspase-12 proteins of the cultured cells at the 3 time-points detected using Western blot., Results: The number of apoptotic hippocampal neurons was significantly higher in the Mg 2+ -free medium group than in the normal medium group at 2, 12 and 48 h ( P <0.01), and considerably decreased in the acupuncture serum group (but not in the non-acupuncture serum group) relevant to the Mg 2+ -free medium group at the same time-points after application of acupuncture serum ( P <0.05, P <0.01). The expression levels of GRP 78 protein at 2 h, CHOP and Caspase-12 proteins at 2, 12 and 48 h were significantly up-regulated in the Mg 2+ -free medium group relavant to the normal me-dium group ( P <0.05, P <0.01). After application of acupuncture serum to the culture medium, the expression levels of GRP 78 protein at the three time-points were significantly increased in comparison with the Mg 2 + -free medium group ( P <0.01, P <0.05), while those of CHOP at 12 and 48 h, and Caspase-12 at the three time-points were notably down-regulated ( P <0.05, P <0.01). No significant differences were found between the non-acupuncture serum and Mg 2 + -free medium groups in the expression levels of GRP 78, CHOP and Caspase-12 proteins at the three time-points ( P >0.05)., Conclusions: Acupuncture serum can significantly reduce apoptosis of the cultured hippocampal neurons, which may be related to its effects in increasing the expression of GRP 78 protein and down-regulating the expression of CHOP and Caspase-12 proteins, suggesting an important role of acupuncture serum in maintaining the stability of the neuronal endoplasmic reticulum.

Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Type III secretion systems (T3SSs) are central virulence devices for many Gram-negative bacterial pathogens of humans, animals & plants. Upon physical contact with eukaryotic host cells, they translocate virulence-mediating proteins, known as effectors, into them during infection. T3SSs are gated from the outside by host-cell contact and from the inside via two cytoplasmic negative regulators, MxiC and IpaD in Shigella flexneri, which together control the effector secretion hierarchy. Their absence leads to premature and increased secretion of effectors. Here, we investigated where and how these regulators act. We demonstrate that the T3SS inner membrane export apparatus protein MxiA plays a role in substrate selection. Indeed, using a genetic screen, we identified two amino acids located on the surface of MxiA's cytoplasmic region (MxiAC) which, when mutated, upregulate late effector expression and, in the case of MxiAI674V, also secretion. The cytoplasmic region of MxiA, but not MxiAN373D and MxiAI674V, interacts directly with the C-terminus of MxiC in a two-hybrid assay. Efficient T3S requires a cytoplasmic ATPase and the proton motive force (PMF), which is composed of the ΔΨ and the ΔpH. MxiA family proteins and their regulators are implicated in utilization of the PMF for protein export. However, our MxiA point mutants show similar PMF utilisation to wild-type, requiring primarily the ΔΨ. On the other hand, lack of MxiC or IpaD, renders the faster T3S seen increasingly dependent on the ΔpH. Therefore, MxiA, MxiC and IpaD act together to regulate substrate selection and secretion mode in the T3SS of Shigella flexneri.

T3SSs are essential virulence determinants of many Gram-negative bacteria, used to inject bacterial effectors of virulence into eukaryotic host cells. Their major extracellular portion, a ∼50 nm hollow, needle-like structure, is essential to host cell sensing and the conduit for effector secretion. It is formed of a small, conserved subunit arranged as a helical polymer. The structure of the subunit has been studied by electron cryomicroscopy within native polymers and by solid-state NMR in recombinant polymers, yielding two incompatible atomic models. To resolve this controversy, we re-examined the native polymer used for electron cryomicroscopy via surface labelling and solid-state NMR. Our data show the orientation and overall fold of the subunit within this polymer is as established by solid-state NMR for recombinant polymers., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼ 20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies., (© 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Objective: To observe the protective effect of acupuncture stimulation on pyramidal cells in hippocampal CA 1 and CA 3 regions and to analyze the involvement of phosphatidy linositol-3-kinase (PI 3 K)/protein kinase B(PKB or Akt) signaling pathway in the acupuncture effect in epilepsy rats., Methods: A total of 120 SD rats were randomly divided into normal control group, model group, LY 294002 (a specific antagonist for PI 3 K/Akt signaling) group, acupuncture+ LY 294002 group and acupuncture group (n = 24 in each group, 12 for H. E. staining, and 12 for electron microscope observation). Epilepsy model was established by intraperitoneal injection of pentylenetetrazol (PTZ, 5 microL). Manual acupuncture stimulation was applied to "Baihui" (GV 20) and "Dazhui" (GV 14) once daily for 5 days. Dimethyl Sulfoxide (DMSO, 5 microL, a control solvent) was given to rats of the normal, model and acupuncture groups, and LY294002 (5 microL, dissolved in DMSO) given to rats of the LY 294002 and acupuncture+ LY 294002 groups by lateral ventricular injection. Four hours and 24 h after modeling, the hippocampus tissues were sampled for observing pathological changes of CA 1 and CA 3 regions after H. E. staining under light microscope and for checkin ultrastructural changes of the pyramidal cells under transmission electron microscope., Results: In comparison with the normal control group, the numbers of pyramidal cells of hippocampal CA 3 region in the model group were decreased significantly 4 h and 24 h after epileptic seizure (P < 0.01). While compared to the model group, the pyramidal cells of hippocampal CA 3 region in the acupuncture group were increased considerably in the number at both 4 h and 24 h after seizure (P < 0.01). No significant differences were found between the LY 294002 and model groups, and between the acupuncture+ LY 294002 and model groups in the numbers of pyramidal cells at 4 h and 24 h after seizure (P > 0.05). Findings of the light microscope and electron microscope showed that the injury severity of pyramidal cells of hippocampal CA 1 and CA 3 regions was moderate 4 h after epileptic seizure and even worse 24 h after seizure in the model group, LY 294002 group and acupuncture+ LY 294002 group, but relatively lighter in the acupuncture group. These results suggested an elimination of the acupuncture effect after blocking the PI 3 K/Akt signaling pathway by lateral ventricular injection of LY 294002 in epilepsy rats., Conclusion: Acupuncture intervention has a protective effect on pyramidal cells of hippocampal CA 1 and CA 3 regions in epilepsy rats, which is associated with the normal function of intracellular PI 3 K/Akt signaling pathway.

Anticoagulation factor II (ACF II), a coagulation factor X- binding protein from the venom of Agkistrodon acutus has both anticoagulant and hypotensive activities. Previous studies show that ACF II binds specifically with activated factor X (FXa) in a Ca(2+) -dependent manner and inhibits intrinsic coagulation pathway. In this study, the inhibition of extrinsic coagulation pathway by ACF II was measured in vivo by prothrombin time assay and the binding of ACF II to factor IX (FIX) was investigated by native polyacrylamide gel electrophoresis and surface plasmon resonance (SPR). The results indicate that ACF II also inhibits extrinsic coagulation pathway, but does not inhibit thrombin activity. ACF II also binds with FIX with high binding affinity in a Ca(2+) -dependent manner and their maximal binding occurs at about 0.1 mM Ca(2+) . ACF II has similar binding affinity to FIX and FX as determined by SPR. Ca(2+) has a slight effect on the secondary structure of FIX as determined by circular dichroism spectroscopy. Ca(2+) ions are required to maintain in vivo function of FIX Gla domain for its recognition of ACF II. However, Ca(2+) at high concentrations (>0.1 mM) inhibits the binding of ACF II to FIX. Ca(2+) functions as a switch for the binding between ACF II and FIX. ACF II extends activated partial thromboplastin time more strongly than prothrombin time, suggesting that the binding of ACF II with FIX may play a dominant role in the anticoagulation of ACF II in vivo., (Copyright © 2012 Wiley Periodicals, Inc.)

The type III secretion apparatus (T3SA), which is evolutionarily and structurally related to the bacterial flagellar hook basal body, is a key virulence factor used by many gram-negative bacteria to inject effector proteins into host cells. A hollow extracellular needle forms the injection conduit of the T3SA. Its length is tightly controlled to match specific structures at the bacterial and host-cell surfaces but how this occurs remains incompletely understood. The needle is topped by a tip complex, which senses the host cell and inserts as a translocation pore in the host membrane when secretion is activated. The interaction of two conserved proteins, inner-membrane Spa40 and secreted Spa32, respectively, in Shigella, is proposed to regulate needle length and to flick a type III secretion substrate specificity switch from needle components/Spa32 to translocator/effector substrates. We found that, as in T3SAs from other species, substitution N257A within the conserved cytoplasmic NPTH region in Spa40 prevented its autocleavage and substrate specificity switching. Yet, the spa40(N257A) mutant made only slightly longer needles with a few needle tip complexes, although it could not form translocation pores. On the other hand, Δspa32, which makes extremely long needles and also formed only few tip complexes, could still form some translocation pores, indicating that it could switch substrate specificity to some extent. Therefore, loss of needle length control and defects in secretion specificity switching are not tightly coupled in either a Δspa32 mutant or a spa40(N257A) mutant.

The pyrolytic behavior of wood is investigated under inert and oxidative conditions. The TGA experiment is given a temperature variation from 323 to 1173 K by setting the heating rate between 5 and 40 K/min. The results of DTG curves show that the hemicellulose shoulder peak for birch is more visible under inert atmosphere due to the higher content of reactive xylan-based hemicellulose (mannan-based for pine). When oxygen presents, thermal reactivity of biomass (especially the cellulose) is greatly enhanced due to the acceleration of mass loss in the first stage, and complex reactions occur simultaneously in the second stage when char and lignin oxidize. A new kinetic model is employed for biomass pyrolysis, namely the distributed activation energy model (DAEM). Under inert atmosphere, the distributed activation energy for the two species is found to be increased from 180 to 220 kJ/mol at the solid conversion of 10-85% with the high correlation coefficient. Under oxidative atmosphere, the distributed activation energy is about 175-235 kJ/mol at the solid conversion of 10-65% and 300-770 kJ/mol at the solid conversion of 70-95% with the low correlation coefficient (below 0.90). Comparatively, the activation energy obtained from established global kinetic model is correspondingly lower than that from DAEM under both inert and oxidative environments, giving relatively higher correlation coefficient (more than 0.96). The results imply that the DAEM is not suitable for oxidative pyrolysis of biomass (especially for the second mass loss stage in air), but it could represent the intrinsic mechanism of thermal decomposition of wood under nitrogen better than global kinetic model when it is applicable., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Type III secretion systems (T3SSs) are key determinants of virulence in many Gram-negative bacterial pathogens. Upon cell contact, they inject effector proteins directly into eukaryotic cells through a needle protruding from the bacterial surface. Host cell sensing occurs through a distal needle "tip complex," but how this occurs is not understood. The tip complex of quiescent needles is composed of IpaD, which is topped by IpaB. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which other virulence effector proteins may be translocated. IpaB is required for regulation of secretion and may be the host cell sensor. It binds needles via its extreme C-terminal coiled coil, thereby likely positioning a large domain containing its hydrophobic regions at the distal tips of needles. In this study, we used short deletion mutants within this domain to search for regions of IpaB involved in secretion regulation. This identified two regions, amino acids 227 to 236 and 297 to 306, the presence of which are required for maintenance of IpaB at the needle tip, secretion regulation, and normal pore formation but not invasion. We therefore propose that removal of either of these regions leads to an inability to block secretion prior to reception of the activation signal and/or a defect in host cell sensing.

Lignin is a key component in the biomass with a complex polymeric structure of the phenyl-C(3) alkyl units. The kraft lignin from the wood pulping process is tested in TG-FTIR and Py-GC-MS. The samples are pyrolyzed in TGA coupled with FTIR from 30 to 900 degrees C at the heating rate of 20 and 40K/min. The evolution of phenolic compounds in the initial pyrolysis stage of lignin is determined by FTIR, while the second stage is mainly attributed to the production of the low molecular weight species. A bench-scale fast pyrolysis unit is employed to investigate the effect of temperature on the product yield and composition. It is found that the guaiacol-type and syringol-type compounds as the primary products of lignin pyrolysis are predominant in bio-oil, acting as the significant precursors for the formation of the derivatives such as the phenol-, cresol- and catechol-types. A series of free-radical chain-reactions, concerning the cracking of different side-chain structures and the methoxy groups on aromatic ring, are proposed to demonstrate the formation pathways for the typical compounds in bio-oil by closely relating lignin structure to the pyrolytic mechanisms. The methoxy group (-OCH(3)) is suggested to work as an important source for the formation of the small volatile species (CO, CO(2) and CH(4)) through the relevant free radical coupling reactions., ((c) 2010 Elsevier Ltd. All rights reserved.)

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(II) and has disulfide-bond linkages between two peptide chains. The effects of the reduction of the disulfide-bonds and Cu(II) in AA-NADase by small-molecule reductants on its NADase and ADPase activities have been investigated by polyacrylamide gel electrophoresis, high performance liquid chromatography, electron paramagnetic resonance spectroscopy and isothermal titration calorimetry. The results show that AA-NADase has six disulfide-bonds and fifteen free cysteine residues. L-ascorbate inhibits AA-NADase on both NADase and ADPase activities through the reduction of Cu(II) in AA-NADase to Cu(I), while other reductants, dithiothreitol, glutathione and tris(2-carboxyethyl)phosphine inhibit both NADase and ADPase activities through the reduction of Cu(II) to Cu(I) and the cleavage of disulfide-bonds in AA-NADase. Apo-AA-NADase can recover its NADase and ADPase activities in the presence of 1 mM Zn(II). However, apo-AA-NADase does not recover any NADase or ADPase activity in the presence of 1 mM Zn(II) and 2 mM TCEP. The multicatalytic activity relies on both disulfide-bonds and Cu(II), while Cu(I) can not activate the enzyme activities. AA-NADase is probably only active as a dimer. The inhibition curves for both ADPase and NADase activities by each reductant share a similar trend, suggesting both ADPase and NADase activities probably occur at the same site. In addition, we also find that glutathione and L-ascorbate are endogenous inhibitors to the multicatalytic activity of AA-NADase., (Copyright 2009 Wiley Periodicals, Inc.)

Experiment is performed to investigate the mechanism of the cellulose pyrolysis and the formation of the main products. The evolution of the gaseous products is examined by the 3-D FTIR spectrogram at the heating rate of 5-60 K/min. A pyrolysis unit, composed of fluidized bed reactor, carbon filter, vapour condensing system and gas storage, is employed to investigate the products of the cellulose pyrolysis under different temperatures (430-730 degrees C) and residence time (0.44-1.32 s). The composition in the bio-oil is characterized by GC-MS while the gases sample is analyzed by GC. The effects of temperature and residence time on the main products in bio-oil (LG, 5-HMF, FF, HAA, HA and PA) are examined thoroughly. Furthermore the possible routes for the formation of the products are developed from the direct conversion of cellulose molecules and the secondary reactions of the fragments. It is found that the formation of CO is enhanced with elevated temperature and residence time, while slight change is observed for the yield of CO(2).

Objective: To study the underlying mechanism of electroacupuncture (EA) in relieving epilepsy in pentylenetetrazole (PTZ)-induced epilepsy rats., Methods: Twenty SD rats were randomly divided into normal control, model, EA, Nimodipine groups, with 5 cases in each. Epilepsy model was established by intraperitoneal injection of PTZ (32.0 mg/kg), once daily for 28 days. EA (100 Hz, 0.6 mA) was applied to "Baihui" (GV 20) and "Dazhui" (GV 14), once daily for 7 days. For Nimodipine group, the rats were given with nimodipine (0.25 mg/kg, i.p.), once daily for 7 days. Electroencephalogram (EEG) was recorded and the fluorescence intensity of Ca2+ of the hippocampus tissue sections was detected by laser scanning confocal microscope (LSCM) after incubation in artificial cerebrospinal fluid containing Flou-3/AM (10 micromol/L) and pluronic F-127 (5 microl)., Results: Compared with model group, the latencies of epileptic EEG seizure prolonged obviously (P < 0.05), and epileptic EEG seizure frequencies decreased significantly (P < 0.05) in EA and Nimodipine groups. The fluorescence intensity of intracellular Ca2+ in hippocampus tissue in model group was obviously higher than that in control group (P < 0.01). In comparison to model group, Ca2+ levels in EA and Nimodipine groups lowered considerably (P < 0.05, P < 0.01). No significant differences were found between EA and Nimodipine groups in the aforementioned 3 indexes (P > 0.05)., Conclusion: EA has an obvious anti-epileptic effect, which may be closely related to its effect in downregulating the increased hippocampal Ca2+ level in PTZ-kindled epilepsy rats.

The Pseudomonas aeruginosa type III secretion system (T3SS) is known to be a very important virulence factor in acute human infections, but it is less important in maintaining chronic infections in which T3SS genes are downregulated. In vitro, the activation of T3SS expression involves a positive activating loop that acts on the transcriptional regulator ExsA. We have observed that in vivo T3SS expression is cell density-dependent in a manner that does not need known quorum-sensing (QS) signals. In addition, stationary-phase culture supernatants added to exponential-phase growing strains can inhibit T3SS expression. The analysis of transposon insertion mutants showed that the production of such T3SS-inhibiting signals might depend on tryptophan synthase and hence tryptophan, which is the precursor of signalling molecules such as indole-3-acetic acid (IAA), kynurenine and Pseudomonas quinolone signal (PQS). Commercially available tryptophan-derived molecules were tested for their role in the regulation of T3SS expression. At millimolar concentrations, IAA, 1-naphthalacetic acid (NAA) and 3-hydroxykynurenine inhibited T3SS expression. Inactivation of the tryptophan dioxygenase-encoding kynA gene resulted in a decrease in the T3SS-inhibiting activity of supernatants. These observations suggest that tryptophan catabolites are involved in the downregulation of T3SS expression in the transition from a low- to a high-cell-density state.

Objective: Pseudomonas aeruginosa is a ubiquitous and opportunistic pathogen that uses the type III secretion system (TTSS) to inject effector proteins directly into the cytosol of target cells to subvert the host cell's functions. Specialized bacterial chaperones are required for effective secretion of some effectors. To identify the chaperone of ExoS, the representative effector secreted by the TTSS of P. aeruginosa, we analyzed the role of a postulated chaperone termed Orf1., Methods: By allelic exchange, we constructed the mutant with the deletion of gene Orf1. Analysis of secreted and cell-associated fractions was performed by SDS-PAGE and Western blotting. Using strain expressing in trans Orf1, tagged by V5 polypeptide and histidine, protein-protein interaction was determined by affinity resin pull-down assay in combination with MALDI-TOF. The role of Orf1 in the expression of exoS was evaluated by gene reporter analysis., Results: Pull-down assay showed that Orf1 binds to ExoS and ExoT. Secretion profile analysis showed that Orf1 was necessary for the optimal secretion of ExoS and ExoT. However, Orf1 had no effect on the expression of exoS., Conclusion: Orf1 is important for the secretion of ExoS probably by maintaining ExoS in a secretion-competent conformation. We propose to name Orf1 as SpcS for "specific Pseudomonas chaperone for ExoS".

Objective: To observe the effect of Aconite cake-separated moxibustion on primary knee osteoarthritis of liver and kidney deficiency type., Methods: Fifty-six cases of such disease (80 knees) were randomly divided into a cake-separated moxibustion group (41 knees) with Neixiyan (EX-LE 5), Dubi (ST 35), Yinlingquan (SP 9) etc. selected, and a western medicine group (39 knees) were treated with oral administration of Sodium Diclofenate Slow-released Tablet., Results: The cumulative score for symptoms and signs was (37.41 +/- 6.61) points before treatment and (9.37 +/- 8.15) points after treatment in the cake-separated moxibustion group and (37.64 - 6.00) points before treatment and (12.05 +/- 8.83) points after treatment in the western medicine group, with a very significant difference before and after treatment in the two groups (P < 0.01); two months after treatment, it was (11.71 +/- 8.69) points in the cake-separated moxibustion group and (15.95 +/- 9.96) points in the western medicine group, the former being better than the latter (P < 0.05). After the end of treatment, there was no significant difference in the comprehensive therapeutic effect between the two groups (P > 0.05), the cured and markedly effective rate was 63.4% in the cake-separated moxibustion group and 48.7% in the western medicine group, but two months after treatment, the cured and markedly effective rate of 56.1% in the cake-separated moxibustion group was better than 33.3% in the western medicine group (P < 0.05)., Conclusion: Aconite cake-separated moxibustion has an ideal therapeutic effect on primary knee osteoarthritis of liver and kidney deficiency type, and the therapeutic effect at anaphase is better than that of Sodium Diclofenate Slow-released Tablet.

The type III secretion system (TTSS) of Pseudomonas aeruginosa is induced in vivo upon contact with eukaryotic cells and in vitro by calcium depletion in culture medium. We have observed a previously identified protein, PsrA, necessary for full activation of TTSS gene expression in P. aeruginosa. Electrophoretic mobility shift assays showed that recombinant PsrA could bind to the exsCEBA promoter region. A mutant with a deletion in the psrA gene was constructed. Using transcriptional fusions, we demonstrated that PsrA is required for the full activation of transcription of the TTSS regulatory operon exsCEBA and effector exoS, although the deletion mutant still responded to calcium depletion, to serum, and to host cell contact. The psrA mutant showed a marked decrease in the secretion of the type III effectors and weak resistance to phagocyte-like PLB-985 cells. The defect in TTSS transcription and secretion in the psrA mutant could be complemented by expression in trans of psrA. PsrA was previously identified as a transcriptional activator of RpoS, a central regulator during stationary phase. We confirmed with our strain that RpoS has a negative effect on TTSS gene expression. Taken altogether, these results suggest that PsrA is a newly identified activator that is involved in the expression of the TTSS by enhancing the exsCEBA transcriptional level.

The phospholipase B family (PLB) are enzymes sharing phospholipase (PL), lysophospholipase (LPL) and lysophospholipase-transacylase (LPTA) activities. They have been shown to be important virulence factors in several human fungal pathogens including Candida albicans and Cryptococcus neoformans. Aspergillus fumigatus, a human opportunistic fungal pathogen leading to a high rate of mortality in immunosuppressed patients is known to possess an extracellular phospholipase B activity. In this paper, we report the molecular characterisation of three PLB genes from A. fumigatus (afplb) using degenerate primers in PCR amplification and data from the A. fumigatus genome project. They are expressed at 37 degrees C, and two of them (afplb1 and afplb3) are induced by lecithin. They encode proteins of 633, 588 and 630 amino acids, respectively, presenting together a T-Coffee score of 81. They also possess the amino acid triad responsible for enzymatic activity in the mammalian cytosolic PLA2 and other fungal PLBs. AfPLB1 and afPLB3 are secreted with a cleaved signal peptide. The complete cDNA sequences were obtained by RACE-PCR for the two secreted afPLBs and probably account for the extracellular phospholipase activity previously reported in the culture media of A. fumigatus.

The structural characteristics and optical and electrical properties of molecular-beam-epitaxy (MBE) grown ZnS(0.8)Se(0.2) thin films on indium-tin-oxide (ITO) glass substrates were investigated in this work. The X-ray diffraction (XRD) results indicated that high quality polycrystalline ZnS(0.8)Se(0.2) thin film grown at the optimized temperature had a preferred orientation along the (111) planes. The transmission electron microscopy (TEM) cross-sectional micrograph of the sample showed a well defined columnar structure with lateral crystal dimension in the order of a few hundred angstroms. Ultraviolet (UV) photoresponsivity as high as 0.01 A/W had been demonstrated and for wavelengths longer than 450 nm, the response was down from the peak response by more than 3 orders of magnitude. The thin ZnS(0.8)Se(0.2) photosensor layer, with a wide energy gap and anisotropic electrical property, makes a transmission UV liquid crystal light valve (LCLV) with high resolution feasible.

Schistosoma japonicum causes schistosomiasis in humans and livestock in the Asia-Pacific region. Knowledge of the genome of this parasite should improve understanding of schistosome-host interactions, biomedical aspects of schistosomiasis and invertebrate evolution. We assigned 43,707 expressed sequence tags (ESTs) derived from adult S. japonicum and their eggs to 13,131 gene clusters. Of these, 35% shared no similarity with known genes and 75% had not been reported previously in schistosomes. Notably, S. japonicum encoded mammalian-like receptors for insulin, progesterone, cytokines and neuropeptides, suggesting that host hormones, or endogenous parasite homologs, could orchestrate schistosome development and maturation and that schistosomes modulate anti-parasite immune responses through inhibitors, molecular mimicry and other evasion strategies.

MBE growth of ZnS(x)Se(1 - x) thin films on ITO coated glass substrates were carried out using ZnS and Se sources with the substrate temperature ranging from 270 degrees C to 330 degrees C . The XRD theta/2theta spectra resulted from these films indicated that the as-grown polycrystalline ZnS(x)Se(1 - x) thin films had a preferred orientation along the (111) planes. The evaluated crystal sizes as deduced from the FWHM of the XRD layer peaks showed strong growth temperature dependence, with the optimized temperature being about 290 degrees C. Both AFM and TEM measurements of these thin films also indicated a similar growth temperature dependence. High quality ZnS(x)Se(1 - x) thin film grown at the optimized temperature had the smoothest surface with lowest RMS value of 1.2 nm and TEM cross-sectional micrograph showing a well defined columnar structure.

Objective: To sub-clone and express the gene encoding Schistosoma japonicum calcium-binding protein (SjE16) and study its immunological response., Methods: The specific primers were designed according to the expressed sequence tags (ESTs) sequence, which was used for amplification of the encoding sequence from the cDNA clone containing SjE16. The gene was subcloned into pGEX4T-1 plasmid and expressed. The rSjE16 was tested for its immunological response by ELISA., Results: The gene encoding Schistosoma japonicum SjE16 was cloned and expressed successfully. The immunogenicity and diagnostic potential of rSjE16 were investigated. It was demonstrated by immunoassay in rabbits that the specificity and sensitivity of the test were 94.1% (16/17) and 88.2% (15/17), respectively, and the level of antibody titer of the untreated group reached a peak at 9-11 wk post infection and maintained high at least for 21 wk post infection, while the antibody level in the treated group rapidly decreased to pre-infection level in 11 wk after treatment. In human, the specificity of the test was 98.3% (57/58); the sensitivity of acute and chronic patient serum assay was 85.5% (53/62) and 70.2% (40/57), respectively., Conclusion: The recombinant protein of SjE16 (rSjE16) was acquired. It can be recognized by the sera from schistosomiasis patients, and the level of antibodies decreased quickly after treatment in experimental rabbits, which implicates the potential value for the evaluation of chemotherapy and detection of active infection.

Rationale and Objectives: The authors evaluated gas-filled liposomes as echocardiographic contrast agents in rabbits with myocardial infarcts., Methods: Ten rabbits underwent ligation of the left anterior descending coronary artery. Five animals underwent echocardiography before and after production of myocardial infarct (MI) and four animals had post-MI imaging only. In either case, images were obtained before and after injection of a single dose of 1 mL of gas-filed liposomes. Three radiologists blinded to clinical information reviewed the pre- and postcontrast images and assessed endomyocardial border definition, wall motion, confidence levels for normal versus abnormal wall motion and visualization of papillary muscle and mitral valve., Results: Postcontrast scans showed significant improvement (P < .05) in endomyocardial border definition, visualization of wall motion, papillary muscle and mitral valve as well as increased reader confidence level., Conclusions: These results are encouraging and suggest that gas-filled liposomes may be a useful contrast agent for echocardiography.

Rationale and Objectives: The purpose of this study was to further develop and compare manganese-based liposomes prepared by two different approaches wherein a manganese ion was entrapped within the internal aqueous space of the vesicles or into the bilayer surface via membrane bound complexes., Methods: Small unilamellar liposomes (SUVs) were prepared entrapping manganese chloride. Alkylated complexes of manganese were prepared and also incorporated into SUVs. The two different manganese-based liposomes were compared for in-vitro relaxivity, stability, toxicity, and in-vivo imaging in rats with liver tumors., Results: Liposomes entrapping manganese had a concentration-dependent change in relaxivity that was maximal at a several-fold molar excess of phospholipid relative to manganese ion. Liposomes bearing membrane-bound complexes showed relaxivity inversely proportional to vesicle size. In-vivo imaging showed greater and more specific hepatic enhancement with manganese liposomes bearing alkylated complexes than those entrapping manganese ion., Conclusions: Correlation effects likely explain the increased relaxivity of manganese entrapped in phospholipid vesicles. Greater efficacy, however, is afforded by liposomes bearing alkylated complexes.

Recent work on the development of liposomal magnetic resonance (MR) contrast agents has yielded structures with higher overall relaxivity than that of other nanoparticles of similar diameter. Liposomes incorporating membrane-bound complexes of manganase ("memsomes") produce greater hepatic enhancement per micromole of metal ion than either ferrite particles or paramagnetic chelates. Memsomes also hold promise for targeting of sites outside the liver. Work is in progress to take these agents into clinical trials.

Liposomes with a mean diameter of 1-2 microns were made to entrap nitrogen gas and tested as an ultrasound (US) contrast agent. The gas-filled liposomes, or Aerosomes (ImaRx Pharmaceutical, Tucson) were tested in vitro for size, stability, reflectivity, and acoustic characterization, and were tested in vivo for acute toxicity in mice and for cardiac imaging in rabbits after intravenous injection. Aerosomes have much greater reflectivity and higher attenuation than do standard liposomes and retain their acoustic properties after storage in aqueous media for several months. The interpolated median lethal dose of Aerosomes is approximately 2.5 mmol of lipid per kilogram, and the imaging dose is under 5 mumol of lipid per kilogram, yielding a potential therapeutic index of over 500 to 1. Postcontrast US images showed sustained enhancement of all four cardiac chambers as well as enhancement in the aorta, vena cava, and hepatic veins. Aerosomes hold promise as a contrast agent for cardiac and blood-pool imaging. Further work is in progress to characterize and develop this novel US contrast agent.

Liposomes are generally thought of as being useful for entrapping drugs within their internal aqueous space. When used with MR contrast agents, this has the drawback that water flux across the membrane bilayer is limiting to contrast enhancement. This can be partially overcome by making the liposomes very small, such that surface area is relatively great compared to internal volume, thereby facilitating water exchange. Alternatively the membranes can be designed to be permeable to water but this may render the vesicles unstable in serum. Another approach is to incorporate the contrast agents into the lipid bilayer. By designing novel complexes of manganese with the ligands incorporated onto dual acyl chains, we have achieved R1 and R2 values of over 20/mmol sec-1 or more than five times higher than Gd-DTPA. Hepatic metastasis detection is significantly improved in rats at doses of only 10 mumol/kg manganese. Membrane-bound manganese complexes function as highly effective liver imaging agents and merit further study for development as agents to undergo human clinical trials.