Search

The internal structure of aerobic granules has a significant impact on the hydrodynamic performance and mass transfer process, and severely affects the efficiency and stability of granules-based reactors for wastewater treatment. In this study, for the first time the granule complex structure was correlated with the hydrodynamic performance and substrates reactions process. First, a series of multiple fluorescence stained confocal laser scanning microscopy images of aerobic granules were obtained. Then, the form and structure of the entire granule was reconstructed. A three-dimensional computational fluid dynamics study was carried out for the hydrodynamic analysis. Two different models were developed on the basis of different fluorescence stained confocal laser scanning microscopy images to elucidate the roles of the granule structure in the hydrodynamic and mass transfer processes of aerobic granules. The fluid flow behavior, such as the velocity profiles, the pathlines and hence the hydrodynamic drag force, exerted on the granule in a Newtonian fluid, was studied by varying the Reynolds number. Furthermore, the spatial distribution of dissolved nutrients (e.g., oxygen) was acquired by solving the convection-diffusion equations on the basis of the reconstructed granule structure. This study demonstrates that the reconstructed granule model could offer a better understanding to the mass transfer process of aerobic granules than simply considering the granule structure to be homogeneous.

The molecular genetic diagnosis of inherited metabolic disorders is challenging. The diseases are rare, and most show locus heterogeneity. Hence, testing of the genes associated with IMDs is time consuming and often not easily available. We report a resequencing array that allows the simultaneous resequencing of up to 92 genes associated with IMDs. To validate the array, DNA samples from 51 patients with 52 different known variants (including point variants, small insertion, and deletions [indels]) in seven genes (C14ORF133, GAA, NPC1, NPC2, VPS33B, WFS1, and SLC19A2) were amplified by PCR and hybridized to the array. A further patient cohort with 48 different mutations in NPC1 were analyzed blind. Out of 76 point variants, 73 were identified using automated software analysis followed by manual review. Ten insertion and deletion variants were detected in the extra tiling using mutation specific probes, with 11 heterozygous deletions and 3 heterozygous insertions. In summary, we identified 96% (95% confidence interval [CI] 89-99%) of point variants added to the array, but the pickup rate reduced to 83% (95% CI 75-89%) when insertions/deletions were included. Although the methodology has strengths and weaknesses, application of this technique could expedite diagnosis in most patients with multilocus IMDs., ((c) 2010 Wiley-Liss, Inc.)

The method of liquid fermentation culture was used to study the influence of two synthetic surfactants, cetyl trimethylammonium bromide (CTAB) and Triton X-100, and a biosurfactant, dirhamnlipid (diRL), on phenol degradation by Candida tropicalis CICC 1463. The results showed that at the beginning of degradation the yeast population decayed, phenol metabolization and bacterial growth did not occur simultaneously, which indicated the toxicity of phenol and formation of intermediate product. CTAB was toxic to C. tropicalis, and it restrained phenol removal. The phenol degradation was accelerated by Triton X-100 of low concentrations of 0.1 and 0.3 CMC, and the complete degradation was achieved at 24 h and 36 h, respectively, compared to 48 h of control. When Triton X-100 concentration was increased to 1.0 CMC or higher concentration, decay of yeast in the initial phase was weakened, but phenol removal and bacterial growth were lagged. The biosurfactant diRL enhanced phenol degradation and growth of the C. tropicalis markedly, and the effect increased with increasing of diRL concentration. Complete degradation was achieved at 24 h in the presence of 1.0 and 3.0 CMC diRL. The diRL concentration also decreased gradually during the fermentation. These results indicated that diRL could reduce phenol toxicity to a great extent and favor the bacterial growth as co-substrate.

Objective: To investigate the changes in expression level of human leucocyte antigen-DR (HLA-DR) on CD14(+) monocyte (CD14(+)/HLA-DR) in the patients after orthotopic liver transplantation, and its role in monitoring postoperative infection., Methods: Sixty-three patients with liver transplantation were divided into three groups, non-infection group with 47 cases, infection group with 10 cases and septic shock group with 6 cases [according to the definition of septic shock of American College of Chest Physicians/Society for Critical Care Medicine (ACCP/SCCM)]. CD14(+)/HLA-DR expression ratio was assessed with flow cytometer, and its clinical implication was evaluated by receiver operating characteristic (ROC) curve assay., Results: CD14(+)/HLA-DR expression ratio in infection group [(29.6+/-7.2)%] and septic shock group [(16.3+/-10.5)%] were significantly lower than that in non-infection group [(62.3+/-18.3)%, both P<0.01], but no significant difference of CD14(+)/HLA-DR expression ratio was found between infection group and septic shock group (P=0.128). Total area under ROC curve of CD14(+)/HLA-DR expression ratio for the infection was 0.965, its sensitivity and specificity at 36.35% cut off were 100.0% and 93.6%, respectively. Total area under ROC curve of CD14(+)/HLA-DR expression ratio to predict septic shock was 0.968, its sensitivity and specificity at 31.97% cut off were 100.0% and 87.7%, respectively. Comparing the change of CD14(+)/HLA-DR expression, it was lower in the infection group and septic shock group (P<0.05 and P<0.01), and the expression rate was lowest during period of serious infection in the two groups [infection group: (29.6+/-7.2)%, septic shock group: (16.3+/-0.5)%, all P<0.01]., Conclusion: For the patients with possible infection after liver transplantation, sequential assessment of CD14(+)/HLA-DR expression ratio would be a good marker for the judgment of patient's conditions and outcome. CD14(+)/HLA-DR expression ratio below 36.35% could be used as the prewarning value for the diagnosis of postoperative infection, and 31.97% could be used as the critical value for the diagnosis of septic shock.

Objective: To study the effects of high mobile group box-1 protein (HMGB1) and lipopolysaccharide (LPS) singly or in combination on release of cytokines from human liver carcinoma cell line (HepG2)., Methods: HepG2 cells were cultured, and purified HMGB1 protein was prepared by chromatography on Ni(2+)-NTA Sepharose column under natural conditions with recombinant expression plasmid pET14b-HMGB1. Different concentrations of HMGB1 (0, 0.01, 0.1, 1, 10 mg/L) and LPS (0, 0.1, 1, 10, 100 mg/L) were added into the cultured cells for 24 hours, respectively. Then the supernatant were collected to detect the levels of granulocyte/macrophage colony stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, IL-8, IL-10, and IL-12 by using LiquiChip system. Lastly, HepG2 cells were co-stimulated with 10 mg/L LPS and 1 mg/L HMGB1 for 24 hours. The supernatant were collected to determine the levels of above ten of cytokines ., Results: The expression and release of IL-6 and IL-8 increased from HepG2 cells after being stimulated by LPS in a dose-dependent manner, but there were no changes in other eight kinds of cytokines (P<0.05 or P<0.01). Low concentration of HMGB1 could up-regulate the expression of IL-6 and IL-8 in HepG2 cells (both P<0.01). But the extent of induction decreased with higher concentration of HMGB1. Similar to LPS, there was no effect of HMGB1 on the expression of other eight kinds of cytokines from cultured HepG2 cells. Furthermore, high concentration of HMGB1 could obviously inhibit the upregulation of IL-6 and IL-8 by high concentration of LPS when the HepG2 cells were co-stimulated with LPS and HMGB1 (both P<0.01)., Conclusion: HepG2 cells could only express and release a few kinds of cytokines when the cells were stimulated with pro-inflammatory agents, such as LPS or HMGB1. Two kinds of cytokines, IL-6 and IL-8 could be up-regulated by LPS and low concentration of HMGB1, and HMGB1 acted as an inhibitor of LPS to down-regulate the expression and release of IL-6 and IL-8 from HepG2 cells.

Objective: This purpose of this study was to determine if serum procalcitonin (PCT) concentration at the time of admission to the ICU is a predictor of all-cause short-term mortality., Design and Methods: This prospective cross-sectional study was conducted over a 16-month period with 86 consecutive critically ill patients. The semi-quantitative PCT-Q test was performed and APACHE II scores and C-reactive protein (CRP) concentrations were determined within 24 h of admission., Results: PCT-Q test value was a better predictor of all-cause short-term mortality than CRP value or APACHE II score. PCT > or = 10 ng/mL was highly and independently correlated with mortality. Use of PCT-Q > or = 10 ng/mL was superior to use of APACHE II > or = 25 or CRP > or = 10 mg/dL as a predictor of poor outcome., Conclusions: A PCT-Q value > or = 10 ng/mL obtained at the time of admission to the ICU is a strong predictor of short-term mortality.

The methanol and ammonia solvated Ca (+) or Mg (+) clusters are known to go through intracluster H or CH 3 eliminations which are typically switched on just below n = 6. By first principles calculations at the B3LYP/6-311+G** level, we have identified the transition structures, activation barriers, and energy changes in these reactions for clusters with 2-6 solvent molecules. The activation barrier is crucial to explain the previously reported experimental results. While increasing number of solvent molecules stabilizes a transition structure, the increasing presence of solvent molecules in the first solvation shell makes it difficult for the metal ion to assist the bond breaking through its interaction with the departing H atom or CH 3 group. The balance of these two factors determines whether a particular elimination channel could be switched on.

The intracluster elimination reactions in solvated alkaline earth metal monocation clusters, M (+)L n , are known to be size-dependent, indicating links between chemical reactivity and the solvation environment controlled by the cluster size. For the methanol and ammonia clusters, there are a number of competing elimination channels involving the breaking of O-H, C-H, O-CH 3, or N-H bond. In this report, we focus on the four clusters with only one solvent molecule and systematically map out the reaction paths and intermediates. The interaction between the metal ion and the departing H atom or CH 3 group varies considerably, depending on the interaction between the metal ion and the remaining group. The understanding of the nature of these interactions and the evaluation of various theoretical levels in treating these reactions provide a solid base for the investigation of the solvation effects on the chemical reactivity of the larger clusters.

To characterize quantitatively the viscoelastic behaviors of cryopreserved rabbit carotid arteries, five mechanical models (a standard linear model, and four generalized Maxwell models) were comparatively applied. Specific aim of this study was to test the validity of these models to simulate the stress relaxation behaviors of the cryopreserved arteries and to find a best-fit model for identification of viscoelastic parameters. Non-linear curve fittings were applied to the stress-relaxation data measured with dynamic mechanical thermal analyzer (DMTA IV; Rheometric. Scientific Inc.; Piscataway, NJ, USA), it was found that 5-element model (generalized Maxwell body composed of an elastic element and two Kelvin body) could provide satisfactory description of the cryopreserved rabbit carotid arteries.

Previously, adsorption feature of a dirhamnolipid biosurfactant on diverse microbial cells was studied and the effect of the adsorption on cell surface hydrophobicity was compared. In this paper, the adsorption behavior of a monorhamnolipid and a dirhamnolipid on cells of two Pseudomonas aeruginosa strains was investigated in order to further reveal the influence of biosurfactant structure and cell property on the adsorption and the relation between the adsorption and cell surface hydrophobicity. Experimental results showed that the adsorption capacity of all the cells to monorhamnolipid was much stronger than to dirhamnolipid, and the rhamnolipid-sourced P. aeruginosa cells, no matter grown on glucose or hexadecane, released extra dirhamnolipid when aqueous concentration of dirhamnolipid was too high. Length of surfactant alkyl chain as well as the type of carbon source used to cultivate the cell adsorbents had only minor influence on the adsorption. The adsorption was assumed to be driven by polar interaction between the rhamnolipid molecules and the cell surface chemical groups. The directional orientation of the rhamnolipid molecules with hydrophobic moiety extending to the environment may account for the rapid increase of cell surface hydrophobicity at low aqueous concentrations of the surfactant, while the stable or decreased cell hydrophobicity was probably the consequence of multiple surfactant layer formation or hemimicelle accumulation.

Objective: To establish a new method for the study of DNA-protein interaction., Methods: Six SPF BALB/c mice were divided into two groups, the lipopolysaccharide (LPS)-treated group (n=3) and the normal control group (n =3). The LPS-treated mice were subjected to tail vein-injection of LPS (25 mg/kg) to reproduce an endotoxic shock model. The biotin-labeled DNA probe for the LPS inducible gene (LIG) promoter was amplified by polymerase chain reaction (PCR). The cell nuclear extracts from liver of mice were prepared and mixed together. DNA pull-down assays were performed by using magnetic beads conjugated with streptavidin. Proteins binding on the beads were eluted by salts with different concentrations. The samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and displayed by silver-stain to compare differential bands between two groups., Results: Fifteen proteins in the nuclear extracts were found to be different between the mice of two groups. Analysis of the differential bands of SDS-PAGE displayed that, compared with normal control group, there were four proteins increased and one protein declined in the mice treated with LPS when eluted with low salt. While under the condition of high affinity elution, there were nine proteins increased and one disappeared after LPS treatment., Conclusion: In this study, a novel method was set up by combining the biotin-streptavidin system and magnetic beads isolation technique, providing a new way to study DNA-protein interaction. This strategy is helpful for further understanding the regulation of gene expression with a view of "omics".

The measured vibrational OH-stretch spectra of size-selected Na(H2O)n clusters for n=8, 10, 16, and 20 are compared with first-principle calculations, which account for the interaction of the sodium cation, the electron, and the water molecules with the hydrogen-bonded network. The calculated harmonic frequencies are corrected by comparing similar results obtained for pure water clusters with experiment. The experimental spectra are dominated by intensity peaks between 3350 and 3550 cm(-1), which result from the interaction of the H atoms with the delocalized electron cloud. The calculations, which are all based upon the average spectra of the four lowest-energy isomers, indicate that most of the peaks at the lower end of this range (3217 cm(-1) for n=8) originate from the interaction of one H atom with the electron distribution in a configuration with a single hydrogen-bonding acceptor. Those at the upper end (3563 cm(-1) for n=8) come from similar interactions with two acceptors. The doublets, which arise from the interaction of both H atoms with the electron, appear in the red-shifted part of the spectrum. They are with 3369/3443 cm(-1) quite pronounced for n=8 but slowly vanish for the larger clusters where they mix with the other spectral interactions of the hydrogen-bonded network, namely, the fingerprints of the free, the double, and the single donor OH positions known from pure water cluster spectroscopy. For all investigated sizes, the electron is sitting at the surface of the clusters.

The constant ionization potential for hydrated sodium clusters Na(H2O)n just beyond n=4, as observed in photoionization experiments, has long been a puzzle in violation of the well-known (n+1)(-1/3) rule that governs the gradual transition in properties from clusters to the bulk. Based on first principles calculations, a link is identified between this puzzle and an important process in solution: the reorganization of the solvation structure after the removal of a charged particle. Na(H2O)n is a prototypical system with a solvated electron coexisting with a solvated sodium ion, and the cluster structure is determined by a balance among three factors: solute-solvent (Na+-H2O), solvent-solvent (H2O-H2O), and electron-solvent (OH{e}HO) interactions. Upon the removal of an electron by photoionization, extensive structural reorganization is induced to reorient OH{e}HO features in the neutral Na(H2O)n for better Na+-H2O and H2O-H2O interactions in the cationic Na+(H2O)n. The large amount of energy released, often reaching 1 eV or more, indicates that experimentally measured ion signals actually come from autoionization via vertical excitation to high Rydberg states below the vertical ionization potential, which induces extensive structural reorganization and the loss of a few solvent molecules. It provides a coherent explanation for all the peculiar features in the ionization experiments, not only for Na(H2O)n but also for Li(H2O)n and Cs(H2O)n. In addition, the contrast between Na(H2O)n and Na(NH3)n experiments is accounted for by the much smaller relaxation energy for Na(NH3)n, for which the structures and energetics are also elucidated.

Objective: To construct a recombinant adenovirus vector for expressing interferon-gamma-inducible protein 10 (IP-10) by homogenous bacterial recombination., Methods: IP-10 gene was cloned into the shuttle plasmid pAdTrack-CMV that contained the coding sequence of enhanced green fluorescent protein (EGFP). The shuttle plasmid was then transformed into E. coli BJ5183 with pAdEasy-1 vector by chemical transformation. The recombinant adenovirus vector pAd/IP-10 was identified by enzyme digestion with Pac I and the linearized plasmid was transfected into HEK293 cells., Results: The positive clones were identified with enzyme digestion and polymerase chain reaction (PCR) and were further verified by DNA sequencing. The recombinant adenovirus of high titration was obtained after transfection and packaging in HEK293 cells., Conclusion: A recombinant adenovirus vector for expression of IP-10 has been constructed successfully and high-titer active adenovirus is obtained for functional study of IP-10 protein.

Objective: To construct an cell penetrating peptide-based expression vector capable of targeted delivery of proteins into the cell nuclei and study its function of protein transduction., Methods: The fusion protein expression vector pET14b-HC(L)NE (pET14-b-His-CPP-Linker-NLS-EGFP) incorporating cell penetrating peptide (CPP), nuclear localization signal(NLS), linker and enhanced green fluorescent protein (EGFP) was constructed based on His-tagged pET14b-HE (pET14b-His-EGFP) by site-directed mutagenesis PCR method. After identification by enzyme digestion and DNA sequencing, the recombinant plasmid was transformed into BL21(DE(3)) strain. The HC(L)NE fusion protein was expressed following IPTG induction and purified with Ni(2+)-NTA affinity chromatography. After dialysis and filtration, the HC(L)NE fusion protein was added into cultured eukaryotic cells. The protein transduction in the living cells was observed under fluorescence microscope and analyzed by Western blotting., Results: Enzyme digestion and DNA sequencing confirmed successful construction of the pET14b-HC(L)NE vector, and the fusion protein efficiently expressed in E. coli. Protein transduction experiments in eukaryotic cells revealed that the fusion protein could rapidly penetrate the cell membrane and reach the cell nucleus, and this internalization was time- and concentration-dependent., Conclusion: The cell penetrating peptide-based expression vector for targeted protein delivery to the cell nucleus has been successfully constructed, and a transport system that can delivery exogenous proteins or polypeptides into the cytoplasm and cell nucleus is established, which provides an economical and efficient means for functional study of the proteins and polypeptide in cells and targeted drug delivery.

Objective: To establish human integrin-alpha v adenovirus expressed in human liver cancer SMMC-7721 cells and analyze the characteristics of integrin-alpha adhesion to liver cancer cells., Methods: Human integrin-alpha v cDNA was cloned into the plasmid pAdTrack-CMV. Then, pAdTrack-integrin-alpha v was cotransformed with adenoviral backbone vector into E. coli strain BJ5183. The recombinant adenoviral plasmid was identified by restriction analysis with Pac I and transfected into HEK-293A cells to package and amplify recombinant adenovirus particles. Western blot showed that integrin-alpha v gene was exactly transcript and expressed in SMMC-7721 cells. Twenty four hours after transfection, the effect of integrin-alpha on adhesion of SMMC-7721 cells was detected by adhesion experiment., Results: Recombinant adenovirus vector of integrin-alpha v gene was successfully constructed and high titers of recombinant adenovirus was obtained. Adhesion cell count was significantly higher in integrin-alpha v transfected cells than in untransfected and Ad-null-transfected cells (P<0.01)., Conclusion: Over expression of integrin-alpha v promotes adhesion of SMMC-7721 cells, is an important molecular mechanism of tumor metastasis.

Objective: To observe the localization and translocation of human high mobility group protein B1 (HMGB1) protein in eukaryotic cells., Methods: The vector that expressed the fusion protein of human HMGB1 and enhanced green fluorescent protein (EGFP) in mammalian cells were constructed. HMGB1 and EGFP were subcloned into hytohemagglutinin (HA) tagged vector pcDNA3-HA by two steps, the construct of which was then transfected into 293A cells and observed with fluorescence microscope., Results: The recombinant plasmid was verified by enzyme digestion, polymerase chain reaction (PCR) and sequence analysis, and the fusion protein was highly expressed in 293A cells. With fluorescence microscopy the green fluorescence was found to be distributed in the nuclei. Eighteen hours after the stimulation with tumor necrosis factor-alpha (TNF-alpha), it was found to have translocated into the cytoplasm in some of the cells., Conclusion: The expression vector for HMGB1-EGFP fusion protein is successfully constructed and effectively expressed in mammalian cells, and it can serve as an important tool in the study of the signal pathway of HMGB1 in mammalian cells.

Objective: To explore the reasons for the low intracellular transduction efficiency of a previously constructed His-TAT-Flag recombinant protein and establish a more efficient transduction system., Methods: The Flag tag of pET14b-His- Tat-Flag vector was deleted with PCR mutant kit, and enhanced green fluorescent protein (EGFP) coding sequence was inserted into the new pET14b-His-TAT recombinant vector. Enzyme digestion and DNA sequencing were performed for identification of pET14b-His-TAT-EGFP vector, which was then transformed into E. coli BL21(DE(3)). After IPTG induction, the recombinant protein of His-TAT-EGFP was isolated and analyzed with SDS-PAGE. Purified His-TAT-EGFP recombinant protein was added to ECV304 cells and the fluorescence was observed to evaluate the transduction efficiency., Results: pET14b-His-TAT vector and pET14b-His-TAT- EGFP vector were successfully constructed, which was identified with enzyme digestion and DNA sequencing. His-TAT-EGFP fusion protein was expressed and purified successfully and showed cellular transduction activity., Conclusion: The prokaryotic expression vector has been successfully constructed by modifying pET14b-His-TAT-Flag, and the expressed and purified recombinant protein of His-TAT-EGFP possesses high efficiency of intracellular transduction activity.

Objective: To construct a p38 MAPK protein delivery system based on TAT protein and study its functions in eukaryotic cells., Methods: Recombinant vectors pHis-TAT-p38 and pHis-TAT-p38(AF) were constructed, and two recombinant proteins, His-TAT-p38 and His-TAT-p38(AF), were expressed and purified in E. coli. The two fusion proteins were then incubated with ECV304 cells, respectively. The phosphorylation of ATF2 was detected to assay the effect of His-TAT-p38 on endogeneious p38 activity after the cells were stimulated by sorbitol., Results: The results of restriction enzyme digestion and DNA sequencing showed that the two recombinant vectors were correctly constructed. The recombinant proteins of His-TAT-p38 and His-TAT-p38(AF) were isolated and purified by SDS-PAGE, and Western blotting suggested that His-TAT-p38 and its mutant with dual phosphorylation sites could enter the cells efficiently in a time- and concentration-dependent manner. His-TAT-p38 was found capable of increasing the activity of endogenous p38 in ECV304 cells, but His-TAT-p38(AF) inhibited the phosphorylation of ATF2 so as to block the transduction of p38 signal pathway when the cells were stimulated with sorbitol., Conclusion: p38 MAPK protein delivery system based on TAT protein has been constructed successfully. It is confirmed that TAT can transfer the proteins into the cells in a time- and dose-depended manner. TAT-p38 and its dominant negative form possess high biological activity after transduction into ECV304 cells by TAT protein delivery system, and the former can increase the activity of endogenous ATF2, but the latter inhibits the transduction of endogeneious p38 signal pathway in ECV304 cells with high osmotic stress.

The decrease in the reaction rate for the charge separation in SO(4) (2-)(H(2)O)(n) with increasing cluster size is examined by first-principles calculations of the energetics, activation barriers, and thermal stability for n=3-7. The key factor governing the charge separation is the difference in the strength of solvation interaction: while interaction with water is strong for the reactant SO(4) (2-) and the product OH(-), it is relatively weak for HSO(4) (-). It gives rise to a barrier for charge separation as SO(4) (2-) is transformed into HSO(4) (-) and OH(-), although the overall reaction energy is exothermic. The barrier is high when more than two H(2)O are left to solvate HSO(4) (-), as in the case of symmetric solvation structure and in the case of large clusters. The entropy is another important factor since the potential surface is floppy and the thermal motion facilitates the symmetric distribution of H(2)O around SO(4) (2-), which leads to the gradual reduction in reaction rate and the eventual switch-off of charge separation as cluster size increases. The experimentally observed products for n=3-5 are explained by the thermally most favorable isomer at each size, obtained by ab initio molecular-dynamics simulations rather than by the isomer with the lowest energy.

The structures and energies of hydrated oxalate clusters, C2O4(2-)(H2O)n, n = 6-12, are obtained by density functional theory (DFT) calculations and compared to SO4(2-)(H2O)n. Although the evolution of the cluster structure with size is similar to that of SO4(2-)(H2O)n, there are a number of important and distinctive futures in C2O4(2-)(H2O)n, including the separation of the two charges due to the C-C bond in C2O4(2-), the lower symmetry around C2O4(2-), and the torsion along the C-C bond, that affect both the structure and the solvation energy. The solvation dynamics for the isomers of C2O4(2-)(H2O)12 are also examined by DFT based ab initio molecular dynamics.

By a systematic examination on Na(H2O)n, with n = 4-7, 9, 10, and 15, we demonstrate that a hydrogen loss reaction can be initiated by a single sodium atom with water molecules. This reaction is similar to the well-known size-dependent intracluster hydrogen loss in Mg+(H2O)n, which is isoelectronic to Na(H2O)n. However, with one less charge on Na(H2O)n than that on Mg+(H2O)n, the hydrogen loss for Na(H2O)n is characterized by a higher barrier and a more flexible solvation shell around the metal ion, although the reaction should be accessible, as the lowest barrier is around 8 kcal/mol. Interestingly, the hydroxide ion OH- produced in the process is stabilized by the solvation of H2O molecules and the formation of an ion pair Na+(H2O)4(H2O)n-l-4[OH-(H2O)l]. The activation barrier is reduced as the unpaired electron in Na(H2O)n moves to higher solvation shells with increasing cluster size, and the reaction is not switched off for larger clusters. This is in sharp contrast to the reaction for Mg+(H2O)n, in which the OH- ion is stabilized by direct coordination with Mg2+ and the reaction is switched off for n > 17, as the unpaired electron moved to higher solvation shells. Such a contrast illustrates the important link between microsolvation environment and chemical reactivity in solvation clusters.

The infinite long hollow cylinder was selected as the model for studying the thermal stress inside the vascular wall during the common cryopreservation process (i.e. cooling-holding at a certain temperature-warming-holding at a second temperature). Transient distributions of temperature and stress were obtained; and effects of cooling rates, warming rates, the holding temperatures on this two distributions were analyzed. Coupled with the experimental results of other researchers, our theoretical predictions indicated that the appearance of the axial compressive stress during the initial period of cooling process was not the governing role that may cause the circumferential crack of the inner and outer vascular wall. Instead, it was the axial tensile stress during the initial period of the warming process that may cause the cracks. Furthermore, the stress history in the multi-steps warming methods was studied. Contrary to the common opinion, the results revealed that the multi-steps method could not decrease the maximal stress while it can change the temperature range containing the maximal stress from low temperature stage to relatively high temperature stage. As the flexibility of the blood vessel was partially recovered in the relative high temperature stage, it can bear the maximal stress that it cannot bear when still left in the low temperature stage, and the intrinsic feature of multi-steps warming method was revealed by this study. This paper provides a theoretical supplement to the study of Pegg et al. (1997) and Buján et al. (2001) published in the journal of Cryobiology.

Objective: To express and purify the fusion protein of His-tagged mouse mitochondrial transcription factor A (mtTFA) in prokaryocytes and prepare rabbit anti-mtTFA polyclonal antibody., Methods: The total RNA was extracted from mouse liver cells, and the coding sequence of mtTFA without signal peptides was amplified with reverse transcriptional (RT)-PCR. The PCR product was then cloned into the prokaryotic expression vector pET14b. After enzyme digestion and DNA sequencing, the plasmid was transformed into BL21(DE3) competent cells, and IPTG was used to induce the expression of His-tagged mtTFA. The expressed fusion protein was purified with Ni(2+)-NTA His-bind resin and quantified with Bradford method., Results: The amplified product was confirmed as the coding sequence of mtTFA without signal peptides by DNA sequencing, and the recombinant expression vector could express His-tagged mtTFA in E.coil following the induction by IPTG. The fusion protein of high purity and rabbit anti-mtTFA serum with high specificity were obtained., Conclusions: The prokaryotic expression vector for mtTFA is successfully constructed and His-tagged mtTFA protein and specific polyclonal antibody are obtained, which can be helpful for further investigation of the biological function and signaling pathways involved in the regulation of functional cooperation between the nucleus and mitochondria.

Mg+(H2O)n clusters ions are known to undergo a hydrogen loss reaction to produce (MgOH)+(H2O)n-1, when the cluster size n falls between 5 and 15. It indicates that the microscopic solvation environment has a strong effect on the reaction mechanisms, which have now been elucidated. Above n = 6, Mg+(H2O)n exists in the form of an ion pair, Mg2+(H2O)l...e-(H2O)n-l, and H+ is produced by the autoionization of H20, enhanced by the hydrolysis of Mg2+. Production of the H atom is due to a reductive half reaction H(+) + e --> H, with the electron being the solvated electron in the ion pair. The reaction barrier is reduced when the electron is in the first or second solvation shell, promoting the H loss reaction. As the number of H2O molecules increases, the electron moves beyond the third solvation shell and the reaction barrier increases significantly, which is responsible for the subsequent switch-off of H loss. It has been one of the important goals in cluster sciences to understand the microscopic correlation between solvation and chemical reactivity by in-depth study on clusters, which has now been achieved in the case of Mg+(H2O)n, linking electron solvation with reaction paths for electron transfer.

Objective: To investigate the effect of allitridin injection on the expression of human cytomegalovirus (HCMV) immediate-early antigens (IEAs including IE72 and IE86) in human embryonic lung cells., Method: HCMV AD 169 Virus strain infected cell model (MOI = 2.5 and 0.25, respectively) were established, and then treated with ICm5 and MTC doses of allitridin. Western blot was used to analyze the of IE72 and IE86 expression after the treatment, ganciclovir(GCV, IC50 and 2.3 x IC50) treatment as control., Result: No matter what kind of MOI was used, both IE86 and IE72 antigens' expression was effectively suppressed by allitridin treatment, and the inhibitory rate of IE86 was almost twice of IE72's. Compared with GCV, allitridin had stronger inhibitory effect on IE86 expressing, although its efficacy on IE72 was weaker than GCV., Conclusion: Allitridin could suppress the expression of IE72 and IE86, especially for IE86 expressing, maybe it is ore of key role in the mechanism of allitridin against HCMV.

Objective: To observe the selective killing of MCF-7 human breast cancer cells and vascular endothelial ECV304 cells by adenovirus (Ad)-mediated double suicide gene driven by KDR promoter., Methods: The plasmid pAdEasy-KDR- CDglyTK was transfected into 293 packaging cells for amplification of the infectious Ad, which were then used to infect KDR-producing ECV304 and MCF-7 cells and LS174T cells that did not produce KDR. The infected cells were treated with 5-FC and/or GCV at different doses to observe the cell-killing and bystander effects of AdKDR-CdglyTK. The cell cycle changes were also detected by flow cytometry., Results: The Ad at the titer of 2.0 x 10(12) pfu/ml was obtained after the amplification, whose infection rates of the cells were similar, but could be increased gradually with the multiplicity of infection (MOI) till reaching 100% with the MOI of 200. The infected cells exhibited different sensitivities to the two prodrugs: ECV304 cells and MCF-7 cells infected with Ad-KDR-CDglyTK showed similar high sensitivity to the prodrugs (P=1.00), whereas the infected LS174T cells appeared to be less sensitive (P<0.001). The killing effect of CD/TK fusion gene on the target cells was much stranger than that of either suicide gene (P<0.001), but all exhbiting considerable bystander effect. In addition, the cell cycle of MCF-7 cells was arrested at G1 phase., Conclusions: CD/TK fusion gene system driven by KDR promoter can selectively kill KDR-expressing vascular endothelial cells and MCF-7 cells.

The structures of hydrated sulfate clusters, SO(4) (2-)(H(2)O)(n) with n=6-12, are obtained by density functional theory calculations. For SO(4) (2-)(H(2)O)(12), two structures with symmetric distribution of H(2)O molecules around the sulfate group are favored in energy. The structures for the smaller clusters, SO(4) (2-)(H(2)O)(n) with n=6-11, are obtained by taking away one H(2)O molecule successively from the two symmetric SO(4) (2-)(H(2)O)(12) isomers. The hydrogen bonding between the sulfate O atoms and H(2)O molecules are strong. So are the hydrogen bonds among H(2)O molecules, which are facilitated by the structure of the polyatomic sulfate group. The solvation energy is quite large (often exceeding 15 kcal/mol). The patterns for structural and energy changes as the cluster size increases are very different from the well studied hydrated halide ions, although the competition between solute-solvent and solvent-solvent interactions is again an important factor. Ab initio molecular dynamics simulations also show "crowding" effects in the first solvation of SO(4) (2-)(H(2)O)(12) at raised temperature.

Objective: To study the mechanism of total Panax notoginseng saponin (tPNS) in regulating the transcription activity of human endothelial nitric oxide synthase (heNOS) gene promoter., Methods: With gene recombination technique, we subcloned the heNOS gene promoter sequence (from -1 to -1 600 bp) into the BglII/HindIII sites of the firefly luciferase reporter gene vector, pGL2-Basic (Promega), to yield the recombinant plasmid designated as peNOS-Luc. With lipofectamine- mediated co-transfection technique, peNOS-Luc, pGL2-Basic and pCMV-beta were cotransfected into NIH3T3 cells, which were treated with lipopolysaccharide (LPS), tPNS and transforming growth factor beta1 (TGFbeta1) respectively. The relative activities (Luc/beta-gal) were subsequently determined in the cell lysates to evaluate the effects of these 3 factors on the activity of heNOS gene promoter., Results: Double restriction enzyme digestion and sequencing both confirmed that the recombinant plasmid, peNOS-Luc, was constructed correctly, which could be effectively expressed in NIH3T3 cells. Upon LPS stimulation, the luciferase activity was obviously decreased, contrary to the results of tPNS and TGFbeta1 treatment, and between the latter two agents, TGFbeta1 produced higher transcription activity., Conclusions: A firefly luciferase reporter gene vector containing heNOS gene promoter sequence has been constructed correctly. tPNS can up-regulate the activity of heNOS gene promoter in NIH3T3 cells.

Aim: To explore the expressions and significance of heat shock protein 70 (Hsp70) and proliferating cell nuclear antigen (PCNA) in the development of forestomach carcinoma in NIH mice induced with N-Nitrososarcosineethylester(NSEE)., Methods: 144 mice were divided into 6 groups and their stomachs were infused with NSEE. Then 24 mice were killed every two weeks and their forestomachs were used for the study of the expressions of Hsp70 and PCNA by immunohistochemical staining., Results: With the progress of carcinogenesis, the expression pattern of Hsp70 was up-down-up, but the expression trend was still increasing. On day 56 and day 70 after NSEE treatment, the expression level of Hsp70 was higher than that of control (P<0.05). On day 84, the expression level of Hsp70 was markedly higher than that of control (P<0.01). The expression of PCNA increased gradually with the carcinogenesis. As compared with the control, stronger expression was found on day 42 and day 56 (P<0.05) and notably stronger expression on day 84 (P<0.01). The expression of Hsp70 was positively correlated to that of PCNA (r=0.98, P<0.01)., Conclusion: In the development of forestomach carcinoma of NIH mice, the expressions of both Hsp70 and PCNA increased and were positively correlated with each other.

The long disputed mechanism for the F(2) addition to ethene is elucidated by density functional theory calculations. With hybrid functionals and a large basis set, DFT provides an excellent description for the dissociation of ground state F(2), and a reasonable account for the F(2)...C(2)H(4) van der Waals complex, which makes it possible for the modeling of reactions between elemental fluorine and organic molecules. The attack of F(2) on ethene first produces a diradical intermediate, which then dissociates into two radicals CH(2)F-CH(2) and F. The first step is exothermic with a low barrier around 1.8 kcal/mol, and the exothermic energy is more than enough to overcome the barrier in the second step for the homolysis of the dangling F-F bond in the diradical, although the presence of solvents and matrices environment could stabilize the diradical. Our calculations provide a coherent framework to understand this reaction not only in the gas and solution phases, but also in the matrices environment where mode-specific enhancement is observed for the addition process induced by infrared radiation., ((c) 2004 American Institute of Physics.)

Allitridin (diallyl trisulfide), a main effective compound of Allium sativum (garlic), was previously shown to inhibit the expression of immediate-early antigens and viral proliferation of human cytomegalovirus (HCMV) in vitro. Here we have examined the prophylactic and therapeutic efficacy of allitridin in a non-lethal murine cytomegalovirus (MCMV) hepatitis in methylprednisolone-immunosuppressed BALB/c mice. Allitridin was administered at 25mg/kg per day (equal to the mean human dose) and 75 mg/kg per day in two regimens: prophylaxis plus therapy beginning at 2 days before infection and lasting for 18 days, and therapy lasting for 14 days initiated at 2 days after infection. Ganciclovir (GCV)-treated, infected, and non-infected mice served as controls. MCMV DNA load in the liver, plasma alanine aminotransferase (ALT) level and Knodell's histological activity index (HAI) score of liver section were evaluated. We found that MCMV DNA load was significantly decreased in all allitridin- and GCV-treated mice, compared with infected controls. Concomitantly, histopathological lesions in the liver and plasma ALT levels were reduced. Statistically, no significant differences were detected between the combined allitridin prophylaxis plus therapeutic and therapeutic groups regardless of dose and the GCV groups. Our results demonstrate the therapeutic efficacy of allitridin in mouse models with MCMV hepatitis.

Chemisorption of singlet (1)Delta(g) O2 on single-walled carbon nanotubes is reexamined by first principles calculations, and the reaction barrier is substantially lower than previously reported when the spin on O2 is correctly treated. The process is initiated by the cycloaddition of a singlet O2 on top of a C-C bond and ended with an epoxy structure with each of the two oxygen atoms occupying a bridge position. The overall process is exothermic, with an activation barrier as low as 0.61 eV for the (8, 0) tube. Our results raise the possibility that carbon nanotubes with small diameters could be degraded after exposure to air and sunlight, similar to the degradation of natural rubber and synthetic plastics.

We report computational studies on Al(+)(H(2)O)(n), and HAlOH(+)(H(2)O)(n-1), n = 6-14, by the density functional theory based ab initio molecular dynamics method, employing a planewave basis set with pseudopotentials, and also by conventional methods with Gaussian basis sets. The mechanism for the intracluster H(2) elimination reaction is explored. First, a new size-dependent insertion reaction for the transformation of Al(+)(H(2)O)(n), into HAlOH(+)(H(2)O)(n-1) is discovered for n > or = 8. This is because of the presence of a fairly stable six-water-ring structure in Al(+)(H(2)O)(n) with 12 members, including the Al(+). This structure promotes acidic dissociation and, for n > or = 8, leads to the insertion reaction. Gaussian based BPW91 and MP2 calculations with 6-31G* and 6-31G** basis sets confirmed the existence of such structures and located the transition structures for the insertion reaction. The calculated transition barrier is 10.0 kcal/mol for n = 9 and 7.1 kcal/mol for n = 8 at the MP2/6-31G** level, with zero-point energy corrections. Second, the experimentally observed size-dependent H(2) elimination reaction is related to the conformation of HAlOH(+)(H(2)O)(n-1), instead of Al(+)(H(2)O)(n). As n increases from 6 to 14, the structure of the HAlOH(+)(H(2)O)(n-1) cluster changes into a caged structure, with the Al-H bond buried inside, and protons produced in acidic dissociation could then travel through the H(2)O network to the vicinity of the Al-H bond and react with the hydride H to produce H(2). The structural transformation is completed at n = 13, coincident approximately with the onset of the H(2) elimination reaction. From constrained ab initio MD simulations, we estimated the free energy barrier for the H(2) elimination reaction to be 0.7 eV (16 kcal/mol) at n = 13, 1.5 eV (35 kcal/mol) at n = 12, and 4.5 eV (100 kcal/mol) at n = 8. The existence of transition structures for the H(2) elimination has also been verified by ab initio calculations at the MP2/6-31G** level. Finally, the switch-off of the H(2) elimination for n > 24 is explored and attributed to the diffusion of protons through enlarged hydrogen bonded H(2)O networks, which reduces the probability of finding a proton near the Al-H bond.

The mechanism of size-dependent intracluster hydrogen loss in the cluster ions Mg(+)(H(2)O)(n), which is switched on around n=6, and off around n=14, was studied by ab initio calculations at the MP2/6-31G* and MP2/6-31G** levels for n=1-6. The reaction proceeds by Mg(+)-assisted breaking of an H-O bond in one of the H(2)O molecules. The reaction barrier is dependent on both the cluster size and the solvation structure. As n increases from 1 to 6, there is a dramatic drop in the reaction barrier, from greater than 70 kcal mol(-1) for n=1 to less than 10 kcal mol(-1) for n=6. In the transition structures, the Mg atom is close to the oxidation state of +2, and H(2)O molecules in the first solvation shell are much more effective in stabilizing the transition structures and lowering the reaction barriers than H(2)O molecules in the other solvation shells. While the reaction barrier for trimer core structures with only three H(2)O molecules in the first shell is greater than 24 kcal mol(-1), even for Mg(+)(H(2)O)(6), it drops considerably for clusters with four-six H(2)O molecules in the first shell. The more highly coordinated complexes have comparable or slightly higher energy than the trimer core structures, and the presence of such high coordination number complexes is the underlying kinetic factor for the switching on of the hydrogen-loss reaction around n=6. For clusters with trimer core structures, the hydrogen loss reaction is much easier when it is preceded by an isomerization step that increases the coordination number around Mg(+). Delocalization of the electron on the singly occupied molecular orbital (SOMO) away from the Mg(+) ion is observed for the hexamer core structure, while at the same time this isomer is the most reactive for the hydrogen-loss reaction, with an energy barrier of only 2.7 kcal mol(-1) at the MP2/6-31G** level.