Optimized multilateral crop trade patterns can effectively mitigate phosphorus imbalance among the involved countries.

Phosphorus imbalance for cropland can greatly influence environmental quality and productivity of agricultural systems. Resolving cropland phosphorus imbalance may be possible with more efficient multilateral crop trade within the involved trading countries; however, the driving mechanisms are unclear. This study calculates phosphorus budgets in China and five central Asian countries and proposes two optimal multilateral crop trade models to mitigate the phosphorus imbalance. Results show that the current trading pattern between China and Central Asia is causing a phosphorus imbalance intensification. Phosphorus surpluses in China and Uzbekistan are 41.7 and 8.9 kg/ha, while Kazakhstan, Kyrgyzstan, Tajikistan, and Turkmenistan exhibit phosphorus deficits with the negative value of -0.7, -1.2, -0.8, and -0.8 kg/ha, respectively. However, under the optimal multilateral crop trade patterns, phosphorus budget of China and Central Asia will become balanced. Phosphorus imbalance intensification for China is reduced to -2525 and -2472 kt under the single- and bilevel-objective-based crop trades. In Kyrgyzstan, it will drop 61.5 % and 50.0 % and change to 321 and 417 kt under the two optimal crop trades. Moreover, changes of phosphorus imbalance mitigations for other central Asian countries range from 11.9 % to 28.2 %. This provides a scientific basis when establishing policies for strengthening optimal multilateral crop trading across the world to promote global phosphorus management.

Splenectomy attenuates severe thermal trauma-induced intestinal barrier breakdown in rats.

The severe local thermal trauma activates a number of systemic inflammatory mediators, such as TNF-α, NF-κB, resulting in a disruption of gut barrier. The gastrointestinal tight junction (TJ) is highly regulated by membrane-associated proteins including zonula occludens protein-1 (ZO-1) and occludin, which can be modulated by inflammatory cytokines. As splenectomy has been shown to reduce secretion of cytokines, we hypothesized that (1) severe scald injury up-regulates TNF-α and NF-κB, meanwhile down-regulates expression of ZO-1 and occludin, leading to the increased intestinal permeability, and (2) splenectomy can prevent the burn-induced decrease in ZO-1 and occludin expression, resulting in improved intestinal barrier. Wistar rats undergoing a 30% total body surface area (TBSA) thermal trauma were randomized to receive an accessorial splenectomy meanwhile or not. Intestinal injury was assessed by histological morphological analysis, and serum endotoxin levels, TNF-α, NF-κB, ZO-1 and occludin levels were detected by Western blotting in the terminal ileum mucosal tissue. 30% TBSA burn caused a significant increase in serum endotoxin levels, but NF-κB, and TNF-α, and the average intestinal villus height and mucosal thickness were decreased significantly. Burn injury could also markedly decrease the levels of ZO-1 and occludin in terminal ileum mucosal tissue (all P<0.01). Splenectomy at 7th day after burn significantly reversed the burn-induced breakdown of ZO-1 and occludin (all P<0.01). The results of this study suggest that severe thermal injury damages the intestinal mucosal barrier. Splenectomy may provide a therapeutic benefit in restoring burn-induced intestinal barrier by decreasing the release of inflammatory cytokines and recovering TJ proteins.

Hydrophosphination of vinyl-boranes with phosphinoamines.

Phosphinoamine ligands PhN(H)PR(2) (R = iPr 1, tBu 2) react with R(1)CH=CR(2)B(C(6)F(5))(2) (R(1) = Ph, p-FC(6)H(4), nPr, Et, R(2) = H, Ph, Et, C(6)F(5)) to effect uncatalyzed hydrophosphination of the olefinic bond, affording the C(2)BNP five-membered cyclization compounds 3-14. All the compounds are fully characterized and compounds 4, 7, 8 and 9 are studied by X-ray diffraction analysis.

Coexistence of Lewis acid and base functions: a generalized view of the frustrated Lewis pair concept with novel implications for reactivity.

Sterically congested Lewis pairs cannot form Lewis adducts; instead they establish encounter complexes of "frustrated" Lewis pairs (FLPs). These encounter complexes have recently been recognized to be capable of activating, i.e., splitting, homopolar and polar single and double bonds, which rendered a new reactivity principle. With the help of qualitative orbital considerations this chapter reviews and explains the reactivity of FLPs toward homopolar Z-Z or Z-Z' single bonded molecules, such as H-H and C-H single bonds, assuming in the encounter complexes the action of strongly polarizing Coulombic fields originating from the FLP constituents. This reactivity principle has been extended in its view to the activating potential for homopolar Z-Z or Z-Z' single bonds of strongly polarized [X-Y ↔ X(+)-Y|(-)] σ and [X=Y ↔ X(+)-Y|(-)] π bonded molecules (X,Y = atoms or molecular fragments; electronegativity of X < electronegativity of Y). A striking analogy in the reaction behavior of FLPs and strongly polarized σ and π bonded molecules could be revealed based on the analyses of selected examples of "metal-free" (main group element reactions) or metal-based (containing transition metals) σ bond metathesis reactions and σ bond additions of H2 and alkanes to polarized main group element and metal to ligand π bonds. Related to the described polar reaction types are Z,Z' double atom or group transfers between highly polarized double bonds of XY and X'Y' molecules combining a Z,Z' elimination with an addition process. Multiple consecutive Z,Z' double atom or group transfers are denoted as double H transfer cascade reactions. Analyzed by examples are concerted or stepwise double H transfers and double H transfer cascades with Z,Z'=H,H from the "metal-free" and metal-based realms: the Meerwein- Pondorf-Verley reduction, H,H exchanges between amine boranes and between amine boranes and unsaturated organic compounds, and the crucial H,H transfer steps of Noyori's bifunctional and Shvo type transfer hydrogenation catalyses.

Bulky derivatives of boranes, boronic acids and boronate esters via reaction with diazomethanes.

Reactions of the perfluoroarylboranes RB(C(6)F(5))(2) (R = C(6)F(5), Ph, Cl, OC(6)F(5)) with Me(3)SiCH(N(2)), (C(6)F(5))CH(N(2)) or Ph(2)C(N(2)) yield (C(6)F(5))(2)B(Me(3)SiCH(C(6)F(5))) 1, (C(6)F(5))B(Me(3)SiCH(C(6)F(5)))(2) 2, (C(6)F(5))B(Me(3)SiCH(C(6)F(5)))(Me(3)SiCH(C(6)H(5))) 3, (C(6)F(5))(2)B(CH(C(6)F(5))(2)) 4, ClB(C(6)F(5))(Ph(2)C(C(6)F(5))) 5 and (C(6)F(5)O)B(C(6)F(5))(Me(3)SiCH(C(6)F(5))) 6 as a result of single or double insertion of a Me(3)SiCH, C(6)F(5)CH or Ph(2)C fragment into a B-C bond of the respective borane. Reactions of one or two equivalents of ethyl α-diazomethylacetate with B(C(6)F(5))(3) yielded (Me)(C(6)F(5))(C=C)(OC(2)H(5))(OB(C(6)F(5))(2)) 8 and [(Me)(C(6)F(5))(C=C)(OC(2)H(5))](2)(O(2)B(C(6)F(5))) 9, in addition to the corresponding pyridine adducts (Me)(C(6)F(5))(C=C)(OC(2)H(5))(OB(C(6)F(5))(2))(py) 10 and [(Me)(C(6)F(5))(C=C)(OC(2)H(5))](2)(O(2)B(C(6)F(5)))(py) 11. Similarly, reaction of α-diazomethylacetate with BPh(3) yielded analogous products of borane reorganization, (Me)(C(6)H(5))(C=C)(OC(2)H(5))(OBPh(2)) 12 and was isolated as a mixture of E and Z-isomers whereas BPh(3) reacts with Me(3)SiCH(N(2)) and pyridine yielding (py)B(Ph(2)(Me(3)SiCH(Ph)) 7. Reactions of Ph(2)C(N(2)) with RB(OH)(2) (R = C(6)F(5), p-F-C(6)H(4), C(6)H(5)) yielded cyclic boroxines of the form [Ph(2)C(R)BO](3) (R = C(6)F(5) 13, p-FC(6)H(4) 14, C(6)H(5) 15) while reactions of the boronate esters (C(6)H(4)O(2))BR (R = C(6)F(5), p-F-C(6)H(4)) with three or five equivalents of Me(3)SiCH(N(2)) yielded (C(6)H(4)O(2))B(Me(3)SiCH(Ar)) (Ar = C(6)F(5) 16, p-F-C(6)H(4) 17) and [(Py)B(C(6)H(4)O(2))(Me(3)SiCH(Ar))] (Ar = C(6)F(5) 18, p-F-C(6)H(4), 19) upon complexation with pyridine. Reaction of HBCat and ClBCat with Ph(2)C(N(2)) yielded the products of B-H and B-Cl bond derivatization (C(6)H(4)O(2))B(Ph(2)CR) (R = H 20, Cl 21), while the triethylphosphine oxide adduct (Et(3)PO)B(C(6)H(4)O(2))(CPh(2)Cl) 22, is readily isolable.

Phosphinimine-borane combinations in frustrated Lewis pair chemistry.

The phosphinimines Ph(3)PNR (R = Ph 1, C(6)F(5) 2, tBu 3) are combined with B(C(6)F(5))(3) in an effort to explore the frustrated Lewis pair (FLP) chemistry. While compound 1 is shown to form an adduct with the borane, compounds 2 and 3 exhibit no apparent interaction. Nonetheless exposure of each of the three combinations to H(2) resulted in the formation of the corresponding salts [Ph(3)PN(H)R][HB(C(6)F(5))(3)] (R = Ph 5, C(6)F(5) 6, tBu 7). Reaction of 1 or 2 with B(C(6)F(5))(3) and carbon dioxide afforded Ph(3)PN(R)COOB(C(6)F(5))(3) (R = Ph 8, C(6)F(5) 9) while the corresponding reaction with 3 gave rise only to the tBuNCO and (Ph(3)PO)B(C(6)F(5))(3). Reactions of 1-3 and B(C(6)F(5))(3) with PhC≡CH proceeds to give either deprotonation or addition affording products of the form [Ph(3)PN(H)R][PhC≡CB(C(6)F(5))(3)] or (Ph(3)PNR)(Ph)C=CH(B(C(6)F(5))(3)). The factors governing the nature of the dominant products are considered.

Reversible, metal-free hydrogen activation by frustrated Lewis pairs.

The Lewis acid cyclohexylbis(pentafluorophenyl)boron 1, which exhibits about 15% lower Lewis acidity in comparison with B(C(6)F(5))(3), activates H(2) in the presence of the bulky Lewis bases 2,2,6,6-tetramethylpiperidine (TMP), 1,2,2,6,6-pentamethylpiperidine (PMP), tri-tert-butylphosphine (t-Bu(3)P) leading in facile reactions at room temperature to heterolytic splitting of dihydrogen and formation of the salts [TMPH][CyBH(C(6)F(5))(2)] 2, [PMPH][CyBH(C(6)F(5))(2)] 3 and [t-Bu(3)PH][CyBH(C(6)F(5))(2)] 4, which could be dehydrogenated at higher temperatures. The related Lewis acid 1-phenyl-2-[bis(pentafluorophenyl)boryl]ethane 5 exhibiting about 10% lower Lewis acidity than B(C(6)F(5))(3) is also capable of splitting H(2) in a heterolytic fashion in the presence of TMP, PMP and t-Bu(3)P yielding [TMPH][PhC(2)H(4)BH(C(6)F(5))(2)] 6, [PMPH][PhC(2)H(4)BH(C(6)F(5))(2)] 7 and [t-Bu(3)PH][PhC(2)H(4)BH(C(6)F(5))(2)] 8. Under comparable conditions as for 2-4, the dehydrogenations of 6-8 were much slower. 4b and 6 were characterized by single crystal X-ray diffraction studies.

Reversal of P-glycoprotein-mediated multidrug resistance in SGC7901/VCR cells by PPARgamma activation by troglitazone.

Over-expression of P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, represents one of the major mechanisms that contribute to multidrug resistance (MDR) in cancer cells. This study examined the effects of troglitazone, a ligand of peroxisome proliferator-activated receptor gamma (PPARgamma), on P-gp-mediated MDR in SGC7901/VCR cells (a vincristine-resistant human gastric cancer cell line). The expression of P-gp was detected by RT-PCR and Western blotting, respectively. The SGC7901/VCR cells were treated with 0.1 mg/L vincristine (VCR) alone or in combination with 1, 5, 10 micromol/L troglitazone for 24 h. PPARgamma was measured by electrophoretic mobility shift assay (EMSA). The intracellular concentration of Rhodamine123 (Rh123, a fluorescent P-gp substrate) was assayed to evaluate the activity of P-gp. The cell cycle and apoptosis were measured by flow cytometry. The results showed that the P-gp was increasingly expressed in SGC7901, BGC823 and SGC7901/VCR cells in turn, suggesting that MDR in the SGC7901/VCR cells was mediated by the increased expression of P-gp. In the SGC7901/VCR cells, the expression level of total PPARgamma was increased, however, the protein level and activity of PPARgamma in the nuclei of cells decreased significantly. Troglitazone elevated the PPARgamma activity in SGC7901/VCR cells in a dose-dependent manner. Troglitazone decreased the P-gp expression and markedly enhanced the accumulation of Rh123 in SGC7901/VCR cells in a dose-dependent manner. We also found that troglitazone significantly increased the percentage of SGC7901/VCR cells in the G2/M phase and decreased the cell percentage in G1 and S phase in a dose-dependent manner. Troglitazone significantly increased the apoptotic rate of SGC7901/VCR cells treated by VCR or ADR in a dose-dependent manner. It was concluded that P-gp-overexpressed SGC7901/VCR cells have minor endogenous PPARgamma activity. Elevation of the PPARgamma activity by troglitazone can reverse P-gp-mediated MDR via down-regulating the expression and activity of P-gp in SGC7901/VCR cells. It was suggested that troglitazone can dramatically enhance the sensitivity of P-gp-mediated MDR cancer cells to chemotherapeutic agents.

Metal-free hydrogen activation and hydrogenation of imines by 1,8-bis(dipentafluorophenylboryl)naphthalene.

In the presence of 2,2,6,6-tetramethylpiperidine (TMP) 1,8-bis(dipentafluorophenylboryl)naphthalene has been found to activate H2 and to hydrogenate various imines under mild conditions.

Metal complexes with N-(2-pyridylmethyl)iminodiacetate: from discrete polynuclear compounds to 1D coordination polymers.

The reactions of M(ClO(4))(2).6H(2)O (M = Mn(2+), Co(2+) and Ni(2+)) with N-(2-pyridylmethyl)-iminodiacetic acid (H(2)pmida) gave three discrete polynuclear complexes {Na[Mn(3)(pmida)(3)(H(2)O)(3)]}ClO(4) (), {Na[Co(3)(pmida)(3)(H(2)O)(3)](H(2)O)}ClO(4) () and [Co(H(2)O)(6)][Co(3)(pmida)(3)(H(2)O)(3)](2)(ClO(4))(2).4H(2)O () and two 1D polymers {[Co(pmida)(H(2)O)].H(2)O}(n) () and {[Ni(pmida)(H(2)O)].H(2)O}(n) (). All complexes were characterized by elemental analysis, infrared (IR) spectroscopy and single-crystal X-ray diffraction analysis. Complexes and show similar structures with an unprecedented trinuclear unit [M(3)(pmida)(3)(H(2)O)(3)] in which the three six-coordinated divalent metal ions are joined by three coplanar syn-anti bridging carboxylate groups of three ligands into a closed planar twelve-membered ring with one Na(+) ion located on the C(3) axis. Complex possesses two kinds of coordination units: mononuclear cation [Co(H(2)O)(6)](2+) and trinuclear unit [Co(3)(pmida)(3)(H(2)O)(3)]. The Co(2+) and Ni(2+) ions in complexes and , respectively, present distorted octahedral geometries and are bridged by anti-anti bridging carboxylate groups of the ligands to form 1D chains. Compounds display a structure variation from discrete polynuclear to 1D coordination polymer along Mn(ii), Co(ii) to Ni(ii), and a dependence of the formation of discrete compound or coordination polymer on the identity of the metal ion. The magnetic investigations of were also carried out. The negative Weiss constants and coupling constants obtained from two kinds of fitting models indicate the presence of dominant antiferromagnetic exchanges mediated by the syn-anti bridging carboxylate groups between the metal centers of three compounds.

Three-dimensional metal azide coordination polymers with amino carboxylate coligands: synthesis, structure, and magnetic properties.

The reaction of M(II) ions with azido ligands in the presence of different amino carboxylic acids gave four three-dimensional metal-azido coordination polymers, [Mn(3,5-daba)(N(3))](n) (1), [Cd(3,5-daba)(N(3))](n) (2; 3,5-daba = 3,5-diaminobenzoate), [Mn(4-aba)(N(3))](n) (3; 4-aba = 4-aminobenzoate), and [Cu(2)(gly)(2)(N(3))(2)](n) (4; gly = glycinate), which display different topological structures. Polymers 1 and 2 present 4,6-connected 3D networks with different Schlafli symbols. However, 3 and 4 feature an unprecedented trinodal 3,6-connected network with the Schlafli symbol (4(2).6)(4.6(2))(4(3).6(6).8(6)) and an unusual 4-connected 3D net with the Lonsdaleite (hexagonal diamond) topology, respectively. Magnetic susceptibility measurements revealed dominant antiferromagnetic couplings for 1 and 3 and an overall dominant ferromagnetic coupling for 4, which presents metamagnetic behavior with a magnetic phase transition at a critical temperature of 6 K and a transition field of ca. 6030 Oe. The results demonstrate that the EE azido and syn-anti carboxylato bridges in our cases induce an antiferomagnetic interaction, and the anti-anti carboxylato bridge in 4 mediates a ferromagnetic interaction. The magnetic interaction through the EO azido bridge in 3 and 4 has a dependence on the value of the M-N-M bond angle.

Hyaluronic acid fragments evoke Kupffer cells via TLR4 signaling pathway.

Kupffer cells, expressing toll-like receptor 4 (TLR4), play a central role in hepatic ischemia/reperfusion (I/R) injury. Hyaluronic acid (HA) fragments, degradative products of high-molecular-weight HA (HMW-HA), acquire the ability to activate immune cells under inflammatory conditions. Here we investigated whether HA fragments could activate Kupffer cells and analyzed the underlying mechanism. Kupffer cells were isolated from wild-type mice (WT, C3H/HeN) and TLR4 mutant mice (C3H/HeJ) and HA fragments were produced by the methods of enzyme digestion and chromatography. Then Kupffer cells were stimulated by HA fragments or other control stimuli. The activation of Kupffer cells was estimated as the release of pro-inflammatory cytokines. The activation of p38 MAPK pathway of Kupffer cells was checked and blocking experiments were done as well. The results indicated that HA fragments acquired the ability to activate Kupffer cells in vitro, which was TLR4 dependent and not due to contamination of lipopolysaccharide. Experiments of p38 MAPK kinase inhibition by SB-203580 verified p38 MAPK was required in HA fragments induced Kupffer cells activation. This suggests that HA fragments, degradative products of one of the major glycosaminoglycans of the extracellular matrix, play critical roles in Kupffer cell activation mediated by TLR4 signaling pathway, which is, at least partially, dependent on p38 MAPK activation.

Relationship between carbachol hyperstimulation-induced pancreatic intracellular trypsinogen and NF-kappa B activation in rats in vitro.

The relationship between intracellular trypsinogen activation and NF-kappa B activation in rat pancreatic acinar cells induced by M3 cholinergic receptor agonist (carbachol) hyperstimulation was studied. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc) and NF-kappa B inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The activity of NF-kappa B was monitored by using electrophoretic mobility shift assay. The results showed that after pretreatment with 2 mmol/L pefabloc, the activities of trypsin and NF-kappa B in pancreatic acinar cells treated with high concentrations of carbachol (10(-3) mol/L) in vitro was significantly decreased as compared with control group (P<0.01). The addition of 10(-2) mol/L PDTC resulted in a significant decrease of NF-kappa B activities in pancreatic acinar cells after treated with high concentrations of carbachol (10(-3) mol/L) in vitro, but the intracellular trypsinogen activity was not obviously inhibited (P>0.05). It was concluded that intracellular trypsinogen activation is likely involved in the regulation of high concentrations of carbachol-induced NF-kappa B activation in pancreatic acinar cells in vitro. NF-kappa B activation is likely not necessary for high concentrations of carbachol-induced trypsinogen activation in pancreatic acinar cells in vitro.

Endogenous danger signals trigger hepatic ischemia/reperfusion injury through toll-like receptor 4/nuclear factor-kappa B pathway.

BACKGROUND: Restoration of blood flow to the ischemic liver lobes may paradoxically exacerbate tissue injury, which is called hepatic ischemia/reperfusion injury (IRI). Toll-like receptor 4 (TLR4), expressed on several liver cell types, and the nuclear factor-kappa B (NF-kappaB) signaling pathway are crucial to mediating hepatic inflammatory response. Because IRI is essentially a kind of profound acute inflammatory reaction evoked by many kinds of danger signals, we investigated TLR4/NF-kappaB signaling pathway activation in a murine model of partial hepatic IRI. METHODS: Wild-type mice (WT, C3H/HeN) or TLR4 mutant mice (C3H/HeJ) were subjected to 45 minutes of partial hepatic ischemia followed by 1 hour, 3 hours of reperfusion. Sham group accepted the same procedure without the obstruction of blood supply. At the end of reperfusion, the compromise of liver function and the histological change of liver sections were measured as the severity of liver injury. The level of endotoxin in the portal vein was measured by limulus assay. NF-kappaB activation was determined by electrophoretic mobility shift assay (EMSA). The levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in systemic blood after hepatic IRI were assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The compromise of liver function and the morphological injuries in mutant mice were relieved more markedly than those in WT mice after partial hepatic IRI. NF-kappaB activation in WT mice was stronger than that in TLR4 mutant mice, and both were stronger than those in the sham operated mice (P < 0.01). Endotoxin in each group was undetectable. The levels of TNF-alpha and IL-1beta in systemic blood were elevated in both strains, but lower in the sham operated group. These mediators were significantly decreased in TLR4 mutant mice compared with those in WT mice (P < 0.01). CONCLUSIONS: The TLR4/NF-kappaB signaling pathway may mediate hepatic IRI triggered by endogenous danger signals. Inhibition of the TLR4/NF-kappaB pathway may be a potential therapeutic target for attenuating ischemia/reperfusion-induced tissue damage in some clinical settings.

Exogenous PTEN gene induces apoptosis in breast carcinoma cell line MDA468.

The effects and mechanisms of exogenous phosphatase and tensin homolog deleted from chromosome ten (PTEN) gene on phosphatase activity-dependent apoptosis of breast cancer cell line MDA468 were investigated. PTEN gene packaged with lipofectin was transferred into breast cancer cell line MDA468 and parental MDA468 cells served as controls. RT-PCR and Western blot were done to detect the expression of target genes. The expression of phosphospecific protein kinase B (PKB/Akt) and focal adhesion kinase (FAK) protein stimulated by epidermal growth factor (EGF) was also detected. Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI. MDA468 cells transfected with PTEN gene could express PTEN mRNA and protein. PTEN decreased the phosphorylation level of AKT protein and down-regulated FAK protein expression in MDA468 stimulated by EGF. The apoptosis rate was 21.68%. PTEN induced breast cancer apoptosis phosphatase activity-dependently. The mechanism is possibly related with phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB)/AKT signaling pathway. Those results may provide new clues on the gene therapy in breast cancer.

Construction and expression of human PTEN tumor suppressor gene recombinant adenovirus vector.

The recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector Ad-PTEN containing green fluorescent protein (GFP) was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2. 5 X 10(10) pfu/mL, and about 70% breast cancer cells were infected with Ad-PTEN when multiplicity of infection (MOI) reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer.

[Adult intestinal heterotopic gastric mucosa resulting in alimentary tract hemorrhage: a report of 11 cases].

OBJECTIVE: To investigate the clinical characteristics,diagnosis and treatment of intestinal heterotopic gastric mucosa resulting in alimentary tract hemorrhage. METHODS: Eleven cases of intestinal heterotopic gastric mucosa with alimentary tract hemorrhage during the past 24 years in our hospital were reviewed and the clinical data were analyzed retrospectively. RESULTS: The median age was 29 years old. Nine cases had abdominal pain, and radionuclide (99m)Tc-pertechnetate scan revealed bleeding lesion in 6 cases preoperatively. Segmental resection of the intestine with bleeding lesion were performed in all patients, postoperative pathology confirmed heterotopic gastric mucosa. The lesion was located in the jejunum in five cases and in the ileum in six cases. All lesions were complicated with diverticulum, or inflammatory mass on the intestinal wall, or abnormity of intestinal duplication. CONCLUSIONS: Intestinal heterotopic gastric mucosa is difficult to be diagnosed preoperatively, and radionuclide (99m)Tc-pertechnetate scan plays a role in preoperative diagnosis.

[Inhibition of rat RAW264.7 macrophage inflammatory cytokines release by small hairpin RNAi targeting Toll-like receptor].

OBJECTIVE: To construct a eukaryotic expression vector carrying the small hairpin RNA (shRNA) for Toll-like receptor 4 (TLR4) mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by rat RAW264.7 macrophages induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism. METHODS: The H1 promotor and double BbsIrestrict endoenzyme site from the plasmid psiRNA-hH1neo were cloned into the reporter gene plasmid pEGFP-C1 at the MluIrestrict endoenzymic site, thus forming the plasmid pEGFP-H1/siRNA containing Bbs site and reporter EGFP gene. Then an oligo nuclear hairpin sequence targeting TLR4 gene was designed by the internet tool siRNA Wizard and then inserted into the plasmid pEGFP-H1/siRNA so as to form the plasmid pEGFP-H1/TLR4-siRNA. Rat macrophages of the line RAW264.7 were cultured and transfected with pEGFP-H1/TLR4-siRNA mediated by lipofectamine 2000. Another RAW264.7 cells were transfected with pEGFP-H1/control sequence-siRNA or blank plasmid. Lipopolysaccharide was added into the 3 kinds of culture fluid for 2 and 68 hours respectively. ELSA was used to detect the levels of tumor necrosis factor-alpha (TNF-alpha) in the supernatants. RESULTS: Restriction endonuclease analysis showed that the construction pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene was successful. The expression of EGFP gene was 50% +/- 8%. The TNF-alpha level of the TLR4-siRNA transfection group 2 hours and after transfection was 825 pg/ml +/- 136 pg/ml, significantly lower than those of the pEGFP-H1/control sequence-siRNA and blank plasmid groups (2190 pg/ml +/- 359 pg/ml and 1265 pg/ml +/- 283 pg/ml respectively, both P < 0.01). The TNF-alpha level of the TLR4-siRNA transfection group 8 hours and after transfection was 1179 pg/ml +/- 240 pg/ml, significantly lower than those of the pEGFP-H1/control sequence-siRNA and blank plasmid groups (4720 pg/ml +/- 227 pg/ml and 4689 pg/ml +/- 310 pg/ml respectively, both P < 0.01). CONCLUSION: shRNA targeting TLR4 gene can inhibit the TNF-alpha release by RAW264.7 cells evoked by LPS.

Relationship between carbachol hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen or NF-kappaB activation in rats in vitro.

The relationship between M3 cholinergic receptor agonist (carbachol) hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen activation or NF-kappaB activation in rats was studied in vitro. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc), and NF-kappaB inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The cellular injury was evaluated by measuring the leakage of LDH from pancreatic acinar cells. The results showed that as compared with control group, 10(-3) mol/L carbachol induced a significant increase of the intracellular trypsin activity and the leakage of LDH from pancreatic acinar cells. Pretreatment with 2 mmol/L pefabloc could significantly decrease the activity of trypsin and the leakage of LDH from pancreatic acinar cells (P < 0.01) following the treatment with a high concentration of carbachol (10(-3) mol/L) in vitro. The addition of 10(-2) mol/L PDTC didn't result in a significant decrease in the activity of trypsin and the leakage of LDH from pancreatic acinar cells treated with a high concentration of carbachol (10(-3) mol/L) in vitro (P > 0.05). It was concluded that intracellular trypsinogen activation is likely involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro. NF-kappaB activation may not be involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro.

Inhibition of RAW264.7 macrophage inflammatory cytokines release by small hairpin RNAi targeting TLR4.

In order to construct an expression vector carrying small hairpin (sh) RNA (shRNA) for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism, the reporter gene plasmid pEGFP-C1 (4.7 kb) and psiRNA-hHlneo (2979 bp) were used. The H1 promotor and double Bbs I restrict endoenzyme site were cloned from plasmid psiRNA-hH1neo and reconstructed them into plasmid pEGFP-C1 in the Mlu I restrict endoenzymic site, forming plasmid pEGFP-H1/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H1/siRNA forming plasmid pEGFP-H1/TLR4-siRNA. After transfection of pEGFP-H1/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha (TNF-alpha) release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was (50.37+/-8.23) % and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA the level of TNF-alpha released by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-alpha release by RAW264.7 cells evoked by LPS.