Perioperative blood transfusion is associated with poorer survival in patients with gastric cancer

The putative prognostic significance of perioperative blood transfusions on gastric cancers is controversial and the published results are contradictory. The aim of this study was to evaluate the prognostic influence of transfusion on Chinese gastric cancer surgery. Six hundreds and seventy-six patients who underwent curative gastrectomy for gastric cancer from 2000 to 2004 were retrospectively reviewed. Uni- and multivariate analyses of the incidence and amount of transfusion, and a comparison of the clinicopathological features were performed. Subgroup analyses of prognosis according to stage, tumor size, and pretreatment anemia were carried out. Blood transfusion was significantly associated with older age (>60 year), larger tumor (>6 cm), upper and middle location, surgical margin status, and pretreatment anemia. In addition, tumors in the transfused group were more advanced in depth of invasion, nodal stage, and TNM stage. No significant relationship was found between the amount of transfused blood and prognosis. Subgroup analyses of prognosis according to stage showed significant differences in stages II and III, between the transfused and nontransfused groups. Significant difference between the transfused and nontransfused groups could be observed in two subgroups of tumor size. Patients with or without anemia in the nontransfused group both had a longer survival time than those in the transfused group. On multivariate analysis, transfusion was shown to be an independent risk factor for poor prognosis. This study suggests that perioperative blood transfusion is associated with a significantly worse prognosis following gastric cancer surgery. The parameters such as advanced stage, tumor size, and anemia do not affect its prognostic value.     

Expression, refolding, and characterization of a novel recombinant dual human stem cell factor

A novel recombinant dual human stem cell factor (rdhSCF) gene which consisted of a full-length hSCF(1-165aa) cDNA and a truncated hSCF (1-145aa) cDNA, linked by a peptide (GGGGSGGGGSGG) coding region, was constructed and cloned into Escherichia coli expression vector pET-22b. The rdhSCF was expressed at high level in E. coli BL21(DE3) and existed mainly as inclusion bodies. The inclusion bodies were solubilized in urea and refolded by ion-exchange chromatography. After renaturation, the purity of the yielded rdhSCF was up to 90%. Cell proliferation assay showed that the specific activity of the rdhSCF was 2.86x10(5) U/mg, about 1.66 times as high as that of monomer rhSCF expressed in E. coli.     

Expression of a novel recombinant stem cell factor/macrophage colony-stimulating factor fusion protein in baculovirus-infected insect cells

A novel human stem cell factor (SCF)/macrophage colony-stimulating factor (M-CSF) fusion protein gene was constructed, in which the coding regions of human SCF cDNA (1-165aa) and the truncated M-CSF cDNA (1-149aa) were connected by a linker sequence encoding a short peptide GGGGSGGGGSGG. The SCF/M-CSF gene was cloned into baculovirus transfer vector pVL1392 under the control of polyhedrin promoter and expressed in the Sf9 cells (Spodoptera frugiperda). SDS-PAGE and Western blot analysis showed that the purified fusion protein was a homodimer with a molecular weight about 84kDa under non-reducing conditions or a monomer about 42kDa under reducing conditions. The specific activity of rhSCF/M-CSF was 17 times as high as that of monomeric rhSCF to stimulate the proliferation of TF-1 cell. The results of macrophages colony-forming (CFU-M) assay performed with human bone marrow mononuclear cells demonstrated that rhSCF/M-CSF was more potent in promoting CFU-M than the equimolar of SCF, M-CSF or that of two cytokines mixture.     

Kinetic studies on the glutathione peroxidase activity of selenium-containing glutathione transferase

Selenium-containing glutathione transferase (seleno-GST) was generated by biologically incorporating selenocysteine into the active site of glutathione transferase (GST) from a blowfly Lucilia cuprina (Diptera: Calliphoridae). Seleno-GST mimicked the antioxidant enzyme glutathione peroxidase (GPx) and catalyzed the reduction of structurally different hydroperoxides by glutathione. Kinetic investigations reveal a ping-pong kinetic mechanism in analogy with that of the natural GPx cycle as opposed to the sequential one of the wild type GST. This difference of the mechanisms might result from the intrinsic chemical properties of the incorporated residue selenocysteine, and the selenium-dependent mechanism is suggested to contribute to enhancement of the enzymatic efficiency.     

Chemical characterization and radioprotective effect of polysaccharide from <Emphasis Type="Italic">Monostroma angicava</Emphasis> (Chlorophyta)

Polysaccharide from the green alga Monostroma angicava was extracted with boiling water and was purified by ion-exchange and size-exclusion column chromatography. The radioprotective effect of the polysaccharide was investigated in mice. The results show that polysaccharide from M. angicava has a different chemical composition to other Chlorophyta having a high rhamnose – containing sulfated polysaccharide. The sulfate ester content was estimated to be 21.8%. When the polysaccharide was applied to BALB/c mice following whole-body X-ray irradiation the counts of leukocytes, thrombocytes and erythrocytes recovered more rapidly in the polysaccharide treated mice after irradiation. In the irradiated mice, the polysaccharide significantly increased the spleen index, natural killer cytostatic activity and the transformation response of splenic lymphocytes. The present observations suggest that polysaccharide from M. angicava led to leukocytogensis and hematopoetic activation in mice after irradiation and that the biological response might be caused by immune activation.     

Identification of a subversive substrate of Trichomonas vaginalis purine nucleoside phosphorylase and the crystal structure of the enzyme-substrate complex

Trichomonas vaginalis is an anaerobic protozoan parasite that causes trichomoniasis, a common sexually transmitted disease with worldwide impact. One of the pivotal enzymes in its purine salvage pathway, purine nucleoside phosphorylase (PNP), shows physical properties and substrate specificities similar to those of the high molecular mass bacterial PNPs but differing from those of human PNP. While carrying out studies to identify inhibitors of T. vaginalis PNP (TvPNP), we discovered that the nontoxic nucleoside analogue 2-fluoro-2'-deoxyadenosine (F-dAdo) is a "subversive substrate." Phosphorolysis by TvPNP of F-dAdo, which is not a substrate for human PNP, releases highly cytotoxic 2-fluoroadenine (F-Ade). In vitro studies showed that both F-dAdo and F-Ade exert strong inhibition of T. vaginalis growth with estimated IC(50) values of 106 and 84 nm, respectively, suggesting that F-dAdo might be useful as a potential chemotherapeutic agent against T. vaginalis. To understand the basis of TvPNP specificity, the structures of TvPNP complexed with F-dAdo, 2-fluoroadenosine, formycin A, adenosine, inosine, or 2'-deoxyinosine were determined by x-ray crystallography with resolutions ranging from 2.4 to 2.9 A. These studies showed that the quaternary structure, monomer fold, and active site are similar to those of Escherichia coli PNP. The principal active site difference is at Thr-156, which is alanine in E. coli PNP. In the complex of TvPNP with F-dAdo, Thr-156 causes the purine base to tilt and shift by 0.5 A as compared with the binding scheme of F-dAdo in E. coli PNP. The structures of the TvPNP complexes suggest opportunities for further improved subversive substrates beyond F-dAdo.     

Matrix-induced inhibition of membrane binding contributes to human immunodeficiency virus type 1 particle assembly defects in murine cells

Defective human immunodeficiency virus type 1 (HIV-1) assembly in murine cells is accompanied by poor plasma membrane binding and proteolytic processing of the HIV-1 Gag precursor. Here, we show that such defects are induced by the propensity of the HIV-1 MA globular head to inhibit membrane binding and particle assembly, particularly at the low expression levels observed in murine cells. Simple additions to or deletion of the MA globular head can improve the yield of infectious virions from murine cells by >50-fold. Expression level and autoinhibition can be important confounding variables in studies of HIV-1 assembly and contribute to defects encountered in murine cells.     

Burn injury promotes antigen-driven Th2-type responses in vivo

Severe injury induces detrimental changes in immune function, often leaving the host highly susceptible to developing life-threatening opportunistic infections. Advances in our understanding of how injury influences host immune responses suggest that injury causes a phenotypic imbalance in the regulation of Th1- and Th2-type immune responses. We report in this study, using a TCR transgenic CD4(+) T cell adoptive transfer approach, that injury skews T cell responses toward increased Th2-type reactivity in vivo without substantially limiting Ag-driven CD4(+) T cell expansion. The increased Th2-type response did not occur unless injured mice were immunized with specific Ag, suggesting that the phenotypic switch is Ag dependent. These findings establish that severe injury induces fundamental changes in the induction of Ag-specific CD4(+) Th cell responses favoring the development of Th2-type immune reactivity in vivo.     

Spatial approximation between two residues in the mid-region of secretin and the amino terminus of its receptor. Incorporation of seven sets of such constraints into a three-dimensional model of the agonist-bound secretin receptor

Photoaffinity labeling of receptors by bound agonists can provide important spatial constraints for molecular modeling of activated receptor complexes. Secretin is a 27-residue peptide hormone with a diffuse pharmacophoric domain that binds to the secretin receptor, a prototypic member of the Class B family of G protein-coupled receptors. In this work, we have developed, characterized, and applied two new photolabile probes for this receptor, with sites for covalent attachment in peptide positions 12 and 14, surrounding the previously most informative site of affinity labeling of this receptor. The [Tyr10,(BzBz)Lys12]rat secretin-27 probe covalently labeled receptor residue Val6, whereas the [Tyr10,(BzBz)Lys14]rat secretin-27 probe labeled receptor residue Pro38. When combined with previous photoaffinity labeling data, there are now seven independent sets of constraints distributed throughout the peptide and receptor amino-terminal domain that can be used together to generate a new molecular model of the ligand-occupied secretin receptor. The amino-terminal domain of this receptor presented a stable platform for peptide ligand interaction, with the amino terminus of the peptide hormone extended toward the transmembrane helix domain of the receptor. This provides clear insights into the molecular basis of natural ligand binding and supplies testable hypotheses regarding the molecular basis of activation of this receptor.