[Time-dependent Protein Expressions of MMP-2 and MMP-9 in Liver Contusion Rats after Impact].

OBJECTIVES: To observe the protein expression patterns of matrix metalloproteinase （MMP）-2 and MMP-9 in the liver tissue of liver contusion rats at different time after impact. METHODS: Fifty healthy adult male SD rats were randomly and evenly divided into control group and experimental groups （1 h, 3 h, 6 h, 12 h, 18 h, 24 h, 3 d, 5 d, 7 d after liver contusion）. A rat liver contusion model was established by a free-falling device. The rats were killed at corresponding time, and the contused hepatic lobes were extracted. The protein expressions of MMP-2 and MMP-9 in contused liver tissue of the rats in each group were observed by immunohistochemical staining （SP method） and Western blotting. RESULTS: After the liver contusion, the expression of positive cell and the protein semiquantitative result showed that the protein expression of MMP-2 enhanced at 6 h and peaked at 24 h, then decreased gradually at 3-5 d, and returned to normal levels at 7 d. The difference of expression between group and its previous adjacent group after 6 h （except 18 h） had statistical significance （P<0.05）. The protein expression of MMP-9 rose obviously at 1 h after liver contusion and peaked at 18 h, then decreased gradually at 3-7 d which still higher than control group. The expression difference between group and its previous adjacent group （except 12 h and 24 h） had statistical significance （P<0.05）. CONCLUSIONS: The protein expressions of MMP-2 and MMP-9 in contused liver tissue after impact show good time-dependent patterns, which may provide important reference indicators for the time estimation of liver contusion.

CARMA3 regulates the invasion, migration, and apoptosis of non-small cell lung cancer cells by activating NF-кB and suppressing the P38 MAPK signaling pathway.

In our previous study, CARMA3 overexpression in lung cancer cells promoted cell proliferation and invasion; however, the mechanism underlying the role of CARMA3 in cancer cell invasion remained unclear. In the present study, knockdown of CARMA3 in A549 and H1299 cells suppressed cell invasion and migration, and downregulated matrix metalloprotease 9 expression at the protein and mRNA levels, as shown by Western blotting and real-time PCR. CARMA3 knockdown increased cell apoptosis, as shown by flow cytometry, increased the mRNA and protein expression levels of Bax and Caspase3, and downregulated Bcl-2 in A549 and H1299 cells. Phosphorylated P38 levels increased and NF-кB activation decreased following knockdown of CARMA3. SB203580, a P38 MAPK inhibitor, activated NF-кB, increased cell migration, and inhibited cell apoptosis after knockdown of CARMA3 compared to knockdown of CARMA3 without SB203580. These findings indicate that CARMA3 may suppress the activation of the P38 MAPK signaling pathway to regulate invasion, migration and apoptosis of lung cancer cells by activating NF-кB (P65) in the nucleus.

Improve oxidation resistance at high temperature by nanocrystalline surface layer.

An interesting change of scale sequence occurred during oxidation of nanocrystalline surface layer by means of a surface mechanical attrition treatment. The three-layer oxide structure from the surface towards the matrix is Fe3O4, spinel FeCr2O4 and corundum (Fe,Cr)2O3, which is different from the typical two-layer scale consisted of an Fe3O4 outer layer and an FeCr2O4 inner layer in conventional P91 steel. The diffusivity of Cr, Fe and O is enhanced concurrently in the nanocrystalline surface layer, which causes the fast oxidation in the initial oxidation stage. The formation of (Fe,Cr)2O3 inner layer would inhabit fast diffusion of alloy elements in the nanocrystalline surface layer of P91 steel in the later oxidation stage, and it causes a decrease in the parabolic oxidation rate compared with conventional specimens. This study provides a novel approach to improve the oxidation resistance of heat resistant steel without changing its Cr content.

Laparoscopic intersphincteric resection for low rectal cancer: comparison of stapled and manual coloanal anastomosis.

AIM: The study aim was to analyse the safety and feasibility of laparoscopic intersphincteric resection with stapled coloanal anastomosis for low rectal cancer. METHOD: Between March 2009 and August 2010, 22 patients underwent laparoscopic intersphincteric resection with a stapled coloanal anastomosis without a diverting ileostomy. The results were compared retrospectively with hand-sewn coloanal anastomoses performed between January 2001 and May 2009, which included 55 open and 38 laparoscopic intersphincteric resections. The morbidity comparison only included data relevant to the anastomosis. Function was compared using the Saito function questionnaire and the Wexner score and only involved data relevant to the laparoscopy. RESULTS: The anastomotic complication rates were similar for fistula, bleeding and neorectal mucosal prolapse (P = 0.526, P = 0.653 and P = 0.411, respectively). Anastomotic leakage and stricture formation of the stapled coloanal anastomosis were significantly lower than those of the hand-sewn coloanal anastomosis (P = 0.037 and P = 0.028, respectively). There were no significant differences in the Saito function questionnaire and the Wexner score between the stapled and hand-sewn coloanal anastomotic groups (all P > 0.05). CONCLUSION: Laparoscopic intersphincteric resection with a stapled coloanal anastomosis is technically feasible and is less likely to result in anastomotic leakage and stricture formation than a hand-sewn anastomosis.

Protective effect of Gui Qi mixture on the progression of diabetic nephropathy in rats.

Huang Qi (root of Astragalus membranaceus) and Dang Gui ( Angelica sinensis), two of the most widely used herbs in traditional Chinese medicine, have been proven to be effective in the treatment of diabetes mellitus (DM) although the underlying molecular mechanisms are not fully elucidated. This study was designed to investigate the protective effect of Dang Gui and Huang Qi mixture (GQM) on the development of diabetic nephropathy in rats with streptozotocin (STZ)-induced DM and the possible underlying molecular mechanism. The diabetic animal model was made by a single intraperitoneal injection of STZ and then treated with GQM or benazepril. Blood glucose, triglyceride (TG), cholesterol (CHO), high density lipoprotein (HDL), serum creatinine (Scr), creatinine clearance rate (Ccr), blood urea nitrogen (BUN), urine beta (2)-microglobin (beta (2)-MG), kidney/body weight (K/B) ratio, glomerular area (GA), renal transforming growth factor-beta (1) (TGF-beta (1)) mRNA expression and blood and renal angiotensin II (AngII) expression were determined 8 weeks after the treatment. The blood glucose, CHO and TG levels, BUN, SCr, Ccr. K/B ratio, GA, the excretion of beta (2)-MG, renal TGF-beta (1) mRNA expression and blood and renal AngII expression were significantly increased while the HDL level was decreased 8 week after STZ injection. The changes in blood glucose, TG, CHO and HDL were reversed by GQM, not by benazepril, whereas the changes in other variables were reversed by both GQM and benazepril. Our results suggest that GQM alleviates the disorder in blood glucose and lipids, protects against the progression of renal nephropathy in diabetic rats, probably by inhibiting the expression of AngII and TGF-beta (1) mRNA.

Induction of accommodation model by combined RNA interference targeting 1,3-galactosyltransferase gene and low-dose GS-IB4 lectin in vitro.

OBJECTIVE: This study sought to mimic the interaction of xenograft endothelial cells and human serum in vitro after successfully silencing the expression of porcine alpha1,3-galactosyltransferase (alpha1,3GT) gene by RNA interference (RNAi), and to investigate the possibility of inducing accommodation in vitro by stimulation of alpha-Gal-specific binding lectin, Griffonia simplicifolia isolectin B4 (GS-IB4) and RNAi. MATERIALS AND METHODS: Various alpha-Gal expression patterns on a pig endothelial cell immortalized line (PED) was achieved by serial doses of small interfering RNA (siRNA) targeting porcinc alpha1,3GT gene. alpha1,3GT-siRNA transfected PEDs were exposed to increasing doses of GS-IB4 lectin (0.5, 2, and 8 microg/mL) for 4 hours before incubation with normal human serum (NHS). Accommodation phenomenon of PEDs in NHS was observed by 51Cr release and antibody/complement binding assays. RESULTS: With combined RNAi and low-dose GS-IB4 stimulation, PEDs remarkably inhibited complement-mediated cytotoxicity, which showed a better protective effect than using RNAi alone. At a concentration of 2 mug/mL, GS-IB4 exhibited the maximum protective effect. The expression of E-selectin on alpha1,3GT-siRNA transfected PEDs did not differ from that on parental PEDs with heat-inactivated NHS (HINHS) stimulation. Combined with GS-IB4 stimulation, however, it inhibited expression of E-selectin, which was GS-IB4 dose dependent, resulting in mean fluorescence intensity values of 98.5, 42.0, and 36.3 at 0.5, 2, and 8 microg/mL. The mRNA expression of the protective gene HO-1 was significantly up-regulated after treatment with RNAi and low-dose of GS-IB4. CONCLUSIONS: Combined RNAi and low-dose GS-IB4 induced pig endothelial cell accommodation in vitro. The level of alpha-Gal expression played an important role in the induction of accommodation.

A study of HLA-G1 protection of porcine endothelial cells against human NK cell cytotoxicity.

UNLABELLED: Human natural killer (NK) cells, which can directly lyse porcine endothelial cells, play an important role in xenotransplantation. HLA-G is a nonclassical major histocompatibility complex (MHC) class I molecules that has been implicated in protecting susceptible target cells from lysis by NK cells. The objective was to study the effect of protecting porcine endothelial cells transfected with HLA-G1 from human NK cell lysis. METHODS: The recombinant expression vector pcDNA3-HLA-G1 was transfected into primary cultured porcine aortic endothelial cells (PAECs) by lipofection. Surface expression of HLA-G1 in transected PAECs was confirmed by an immunofluoresence technique. Peripheral blood mononuclear cells (PBMC) and NK cell line (NK92) were used as NK effects cells with pcDNA3-HLA-G1-transfected PAECs as targets in a MTT method using pcDNA3 transfection as a negative control. RESULTS: Expression of HLA-G1 on PAECs conferred significant protection against NK-mediated lysis. The rate of NK92 cytotoxicity was reduced to 41.5% +/- 14.0% from 75.3% +/- 10.5% in the control group (P < .01). Similarly the rate of the PBMC cytotoxicity among different donors (n = 7) was reduced to 45.4% +/- 12.1% in contrast to 74.6% +/- 11.2% in the control group (P < .05). CONCLUSIONS: HLA-G1 molecules can directly protect xenogeneic PAECs against attack by human NK cells. These results indicate that the expression of HLA-G1 on the porcine cell surface may provide a new approach to overcome NK-mediated immunity to xenografts.

Crystallization and preliminary crystallographic studies of trichomaglin, a novel ribosome-inactivating protein.

Trichomaglin, a novel ribosome-inactivating protein, has been crystallized in two crystal forms using the hanging-drop vapour-diffusion method. The form A and form B crystals belong to the orthorhombic space group P2(1)2(1)2(1) and the hexagonal space group P6(1) (or P6(5)), respectively. X-ray data have been collected to 3.3 and 2.2 A resolution for the form A and B crystals, respectively.

Crystal structure of recombinant trypsin-solubilized fragment of cytochrome b(5) and the structural comparison with Val61His mutant.

The crystal structure of the recombinant trypsin-solubilized fragment of the microsomal cytochrome b(5) from bovine liver has been determined at 1.9 A resolution and compared with the reported crystal structure of the lipase-solubilized fragment of the membrane protein cytochrome b(5). The two structures are similar to each other. However, some detailed structural differences are observed: the conformation of the segment Asn16-Ser20 is quite different, some helices around the heme and some segments between the helices are shifted slightly, the heme is rotated about the normal of the mean plane of heme, one of the propionates of the heme exhibits a different conformation. The average coordination distances between the iron and the two nitrogen atoms of the imidazole ligands are the same in the two structures. Most of the structural differences can be attributed to the different intermolecular interactions which result from the crystal packing. The wild-type protein structure is also compared with its Val61His mutant, showing that the heme binding and the main chain conformations are basically identical with each other except for the local area of the mutation site. However, when Val61 is mutated to histidine, the large side chain of His61 is forced to point away from the heme pocket toward the solvent region, disturbing the micro-environment of the heme pocket and influencing the stability and the redox potential of the protein.

Crystal structures of the complexes of trichosanthin with four substrate analogs and catalytic mechanism of RNA N-glycosidase.

Four substrate analogs-nicotinamide adenine dinucleotide, adenylyl (3', 5') guanosine, guanylyl (3',5') adenosine, and adenosine 2', 5'-diphosphate-have been used to prepare the complexes with trichosanthin (TCS), a type I ribosome-inactivating protein that possesses the activity of N-glycosidase. The crystal structures of the complexes have been determined and refined at high resolution. The refined structures show that the N-glycosidic bonds of all the four substrate analogues are hydrolyzed and a common structure is shared by the four complexes, in which only adenine, the product of the enzymatic reaction, is bound in the active center. The structure is compared with those of native trichosanthin and a previously reported trichosanthin-NADPH complex in which the N-glycosidic bond is uncleaved. The structural comparison shows that the conformation of Tyr70 obviously differs from those in the latter two structures, i.e., the side chain of Tyr70 is rotated along its Cbeta-Cgamma bond by approximately 70 degrees. The water molecule found to be preassociated with the N-glycosidic bond in the TCS-NADPH complex structure and proposed to be the water candidate responsible for hydrolyzing the N-glycosidic bond disappears in the trichosanthin-product complex structure. Based on the comparison of the three structures representing the different stages of the enzymatic reaction, the catalytic mechanism of RNA N-glycosidase has been further elucidated. Proteins 2000;39:37-46.

Effect of mutation at valine 61 on the three-dimensional structure, stability, and redox potential of cytochrome b5.

To elucidate the role played by Val61 of cytochrome b(5), this residue of the tryptic fragment of bovine liver cytochrome b(5) was chosen for replacement with tyrosine (Val61Tyr), histidine (Val61His), glutamic acid (Val61Glu), and lysine (Val61Lys) by means of site-directed mutagenesis. The mutants Val61Tyr, Val61Glu, Val61His, and Val61Lys exhibit electronic spectra identical to that of the wild type, suggesting that mutation at Val61 did not affect the overall protein structure significantly. The redox potentials determined by differential pulse voltammetry were -10 (wild type), -25 (Val61Glu), -33 (Val61Tyr), 12 (Val61His), and 17 mV (Val61Lys) versus NHE. The thermal stabilities and urea-mediated denaturation of wild-type cytochrome b(5) and its mutants were in the following order: wild type > Val61Glu > Val61Tyr > Val61His > Val61Lys. The kinetics of denaturation of cytochrome b(5) by urea was also analyzed. The first-order rate constants of heme transfer between cytochrome b(5) and apomyoglobin at 20 +/- 0.2 degrees C were 0.25 +/- 0.01 (wild type), 0.42 +/- 0.02 (Val61Tyr), 0.93 +/- 0.04 (Val61Glu), 2.88 +/- 0.01 (Val61His), and 3.88 +/- 0.02 h(-)(1) (Val61Lys). The crystal structure of Val61His was determined using the molecular replacement method and refined at 2.1 A resolution, showing that the imidazole side chain of His61 points away from the heme-binding pocket and extends into the solvent, the coordination distances from Fe to NE2 atoms of two axial ligands are approximately 0.6 A longer than the reported value, and the hydrogen bond network involving Val61, the heme propionates, and three water molecules no longer exists. We conclude that the conserved residue Val61 is located at one of the key positions, the "electrostatic potential" around the heme-exposed area and the hydrophobicity of the heme pocket are determinant factors modulating the redox potential of cytochrome b(5), and the hydrogen bond network around the exposed heme edge is also an important factor affecting the heme stability.

Three-dimensional structure of beta-momorcharin at 2.55 A resolution.

Beta-Momorcharin (Mr approximately 29 kDa) is a single-chained ribosome-inactivating protein (RIP) with a branched hexasaccharide bound to Asn51. The crystal structure of beta-momorcharin has been determined using the molecular-replacement method and refined to 2. 55 A resolution. The final structural model gave an R factor of 17. 2% and root-mean-square deviations of 0.016 A and 1.76 degrees from ideal bond lengths and bond angles, respectively. beta-Momorcharin contains nine alpha-helices, two 310 helices and three beta-sheets, and its overall structure is similar to those of other single-chained RIPs. Residues Tyr70, Tyr109, Glu158 and Arg161 are expected to define the active site of beta-momorcharin as an rRNA N-glycosidase. The oligosaccharide is linked to the protein through an N-glycosidic bond, beta-GlcNAc-(1-N)-Asn51, and stretches from the surface of the N-terminal domain far from the active site, which suggests that it should not play a role in enzymatic function. The oligosaccharide of each beta-momorcharin molecule interacts with the protein through hydrogen bonds, although in the crystals most of these are intermolecular interactions with the protein atoms in an adjacent unit cell. This is the first example of an RIP structure which provides information about the three-dimensional structure and binding site of the oligosaccharide in the active chains of RIPs.

Purification and characterization of trichomaglin--a novel ribosome-inactivating protein with abortifacient activity.

Trichomaglin, a novel ribosome-inactivating protein, has been isolated from root tuber of a plant Maganlin (Trichosanthes Lepiniate, Cucurbitaceae). The isolation and purification procedure included ammonium sulfate precipitation, Sephadex G-75 chromatography and CM-Sephadex C-50 chromatography. The protein was identified to be homogeneous by SDS-PAGE and FPLC analysis. Its molecular weight is 24,673 dalton and isoelectric point is 5.8, determined by electrospray ionization mass spectroscopy and isoelectric focusing gel electrophoresis respectively. Trichomaglin can inhibit protein synthesis in rabbit reticulocyte lysate with ID50 of 10.1 nM. When rat ribosome was incubated with trichomaglin, a diagnostic RNA fragment appeared on polyacrylamide gel after ribosomal RNAs were treated with acidic aniline. It was concluded that trichomaglin is an RNA N-glycosidase. In addition, it has been verified to be an abortifacient protein.

Detailed active site configuration of a new crystal form of methanol dehydrogenase from Methylophilus W3A1 at 1.9 A resolution.

The three-dimensional structure of a new crystal form of methanol dehydrogenase from Methylophilus W3A1 has been obtained in the presence of substrate using data recorded at a synchrotron. The structure of this approximately 140 kDa heterotetramer, refined at 1. 9 A resolution, reveals the detailed configuration of its redox cofactor, pyrroloquinoline quinone (PQQ). C4, one of the oxygen-bearing atoms of this orthoquinone is in a planar configuration while C5, which bears the other quinone oxygen, is tetrahedral, suggesting that the PQQ is in the semiquinone redox state. The substrate binding site has been identified close to PQQ and to the side chain of Asp297, the putative active site base. The proximity of the hydroxyl of methanol to C5 of PQQ compared to the greater separation of the substrate methyl group from C5 supports the addition-elimination reaction mechanism involving a hemiketal intermediate.

A possible hydrolysis mechanism of beta-naphthyl acetate catalyzed by antibodies.

The mechanism of ester hydrolysis has been extensively studied; however, the precise function of active-site residues in promoting catalysis is unclear. We describe here the structural models for the complex of a catalytic antibody Fv fragment with a phosphonate transition-state analogue, constructed by using gene cloning, sequencing and molecular modeling, mainly based on a known X-ray structure of a catalytic antibody. Hydrophobic and electrostatic analyses of the Fv/analog and Fv/substrate interaction suggest the hydrolysis mechanism: Tyr L91 and Tyr H97 play important roles to stabilize the beta-naphthyl group of hapten through pi-stack; His H35 donates a pair of free electrons at the atom NE2 to an active water and let it to be a partial hydroxide, which attacks the carbon atom of the carbonyl group of the substrate. Both His H35 and Arg L96 can form hydrogen bonds and stabilize the anionic tetrahedral intermediate formed during turnover. This mechanism emphasizes that an active water bridge may be formed during hydrolysis process.

Identification of a stable complex of trichosanthin with nicotinamide adenine dinucleotide phosphate.

Trichosanthin, a type I ribosome-inactivating protein with RNA N-glycosidase activity, forms a stable complex with nicotinamide adenine dinucleotide phosphate, a substrate analog. Difference UV spectroscopy, fluorescence spectroscopy, and 31P NMR are used to identify the formation of the complex, followed by a crystal structure analysis carried out to elucidate the active-site structure of trichosanthin. The determination of germinal vesicle breakdown indicates that the complex does not, at least for abortion-inducing activity, result in competitive inhibition to the protein.

Crystal structure of trichosanthin-NADPH complex at 1.7 A resolution reveals active-site architecture.

We describe here the crystal structure of the trichosanthin-NADPH complex determined at a resolution of 1.7 A. The adenine base stacks between Tyr 70 and Tyr 111. Arg 163, Glu 160 and Tyr 70 form hydrogen bonds to N(3), O(3') and, through a water molecule, to N(9) of adenosine, respectively. This is the first high resolution structure of a complex between a ribosome-inactivating protein and a substrate analogue, in which the electron density of the N-glycosidic bond is well defined and the preassociated water, thought to be responsible for hydrolyzing the N-C bond, is also explicitly elucidated.

Ionic strength dependence of the reaction between methanol dehydrogenase and cytochrome c-551i: evidence of conformationally coupled electron transfer.

The quinoprotein methanol dehydrogenase and cytochrome c-551i are two soluble acidic proteins that form a physiological complex in which electrons are transferred from pyrroloquinoline quinone to heme. The oxidation of methanol dehydrogenase by the cytochrome was studied as a function of ionic strength using stopped-flow spectroscopy. The dissociation constant (Kd) for complex formation decreased 2-fold with increasing ionic strength from 0.21 to 1.3 M and increased at higher ionic strengths. The rate constant for the electron transfer reaction (kET) increased 2-fold with increasing ionic strength from 0.21 to 1.3 M and decreased at higher ionic strengths. The variation of Kd and kET over this range of ionic strengths was described by Van Leeuwen theory, which takes into account monopole-dipole and dipole-dipole forces, in addition to the monopole-monopole force, to predict the interactions between large molecules. Analysis of the kinetic results in terms of these electrostatic interactions indicated the probable orientations for protein-protein binding and electron transfer. To explain the ionic strength dependence of the observed kET, a model is presented in which the true kET is reduced by a factor Kc, an equilibrium constant that describes some rearrangement of the proteins after a nonoptimal collision to produce the most efficient orientation for electron transfer. This model is consistent with the notion that the large reorganizational energy obtained from temperature-dependence studies of this electron transfer reaction [Harris, T. K., & Davidson, V. L. (1993) Biochemistry 32, 14145-14150] is due to such an intracomplex rearrangement.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallization and preliminary crystallographic study of beta-momorcharin.

Beta-Momorcharin from seeds of Momordica charantia, Cucurbitaceae Linn, has been crystallized using a vapor diffusion method. The crystals belong to space group P1 with unit cell parameters: a = 49.09 A, b = 50.58 A, c = 61.12 A, alpha = 72.98 degrees, beta = 78.39 degrees, gamma = 76.97 degrees. There are two molecules in the unit cell and the diffraction data up to 2.4 A resolution were collected on an X-200B area detector, giving an Rmerge of 7.8%.

The active site structure of the calcium-containing quinoprotein methanol dehydrogenase.

Pyrroloquinoline quinone (PQQ), widely found in nature, serves as the redox cofactor in bacterial methanol dehydrogenase (MEDH), a heterotetrameric enzyme that oxidizes methanol to formaldehyde. The refined structure of MEDH at 2.4-A resolution, based on recently obtained amino acid sequence data, reveals that the PQQ, located in a central channel of the disk-shaped protein, is sandwiched between a Trp side chain and a very unusual vicinal disulfide. A Ca2+ ion forms a bridge between PQQ and the protein molecule, very close to a putative substrate binding pocket. The vicinal disulfide may form during PQQ incorporation and possibly act to hold the latter in place.