Effects of the ginkgo biloba extract on the superoxide dismutase activity and apoptosis of endothelial progenitor cells from diabetic peripheral blood.

Although ginkgo biloba extract (GBE) was shown to have antioxidant effects, little has been reported on the ability to GBE to help endothelial progenitor cells (EPCs) resist oxidative stress. The present study evaluated the influence of different concentrations of GBE on superoxide dismutase (SOD) and apoptosis of diabetic peripheral blood EPCs. Twenty-five diabetic patients without any vascular complications were included in the experimental group, while 15 healthy adults made up the control group. Peripheral blood mononuclear cells were isolated with density gradient centrifugation, and, after in vitro differentiation, were determined to be EPCs using FITC-UEA-I and Dil-Ac-LDL dual staining. After the colony and fusiform adherent cells were observed, on day 7, various concentrations of ginkgo biloba extract (0, 10, 25, 50 mg/L) were added to the culture medium for a 24-h incubation. EPC-SOD activity and apoptosis were subsequently detected. We found that within the experimental group, GBE significantly improved SOD activity within EPCs and reduced the rate of apoptosis. These effects became more obvious with increasing GBE concentrations (25 mg/L, P < 0.05; 50 mg/L, P < 0.01). GBE also improved SOD activity and reduced the rate of apoptosis within EPCs of the control group; however, the changes were not statistically significant. We conclude that GBE can improve SOD activity and reduce the rate of apoptosis of EPCs within the peripheral blood of diabetic patients, effects that are dose-dependent.

The use of a wound protector to prevent port site recurrence may not be totally logical.

The wound protector has been used widely to prevent port site metastases (PSM). Although port site metastases ties in with poor survival, it is not because of PSM itself, but rather because PSM is a sign of more widespread metastatic disease. Whilst being touted as a method of preventing PSM, it fails to address the bigger issue of preventing intra-abdominal recurrence. Proper surgical technique in tumour handling following rigorous oncological principles, and not just putting in a wound protector is the key to good surgery with low recurrences and excellent survival rates.

Optimization of submerged culture requirements for the production of mycelial growth and exopolysaccharide by Cordyceps jiangxiensis JXPJ 0109.

AIMS: The objective of the present study was to investigate the optimal culture requirements for mycelial growth and exopolysaccharide production by Cordyceps jiangxiensis JXPJ 0109 in submerged culture. METHODS AND RESULTS: The effects of medium ingredients (i.e. carbon and nitrogen sources, and growth factor) and other culture requirements (i.e. initial pH, temperature, etc.) on the production of mycelia and exopolysaccharide were observed using a one-factor-at-a-time method. More suitable culture requirements for mycelial growth and exopolysaccharide production were proved to be maltose, glycerol, tryptone, soya bean steep powder, yeast extract, medium capacity 200 ml in a 500-ml flask, agitation rate 180 rev min(-1), seed age 4-8 days, inoculum size 2.5-7.5% (v/v), etc. The optimal temperatures and initial pHs for mycelial growth and exopolysaccharide production were at 26 degrees C and pH 5 and at 28 degrees C and pH 7, respectively, and corresponding optimal culture age were observed to be 8 and 10 days respectively. According to the primary results of the one-factor-at-a-time experiments, the optimal medium for the mycelial growth and exopolysaccharide production were obtained using an orthogonal layout method to optimize further. Herein the effects of medium ingredients on the mycelial growth of C. jiangxiensis JXPJ 0109 were in the order of yeast extract > tryptone > maltose > CaCl2 > glycerol > MgSO4 > KH2PO4 and the optimal concentration of each composition was 15 g maltose (food-grade), 10 g glycerol, 10 g tryptone, 10 g yeast extract, 1 g KH2PO4, 0.2 g MgSO4, and 0.5 g CaCl2 in 1 l of distilled water, while the order of effects of those components on exopolysaccharide production was yeast extract > maltose > tryptone > glycerol > KH2PO4 > CaCl2 > MgSO4, corresponding to the optimal concentration of medium was as follows: 20 g maltose (food-grade), 8 g glycerol, 5 g tryptone, 10 g yeast extract, 1 g KH2PO4, and 0.5 g CaCl2 in 1 l of distilled water. CONCLUSIONS: Under the optimal culture requirements, the maximum exopolysaccharide production reached 3.5 g l(-1) after 10 days of fermentation, while the maximum production of mycelial growth achieved 14.5 g l(-1) after 8 days of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the submerged culture requirements for mycelial growth and exopolysaccharide in C. jiangxiensis, and this two-step optimization strategy in this study can be widely applied to other microbial fermentation processes.

[A bispecific monoclonal antibody with versatile adaptor mediating different cytotoxicities].

Bispecific monoclonal antibody (bsMAb), secreted by hybrid-hybridoma, has an intact 1g molecule construction with dual antigen binding specificities. bsMAb has several advantages over conventional conjugate in tumor targeting therapy: (1) The damage to both antibody and tumoricidal agents due to chemical crosslinking can be avoided. (2) The antigen modulation can be decreased due to monovalence binding of bsMAb with target antigen. (3) The binding affinity between Fc fragment of bsMAb with heterogenous heavy chains and Fc receptor is decreased, so that nonspecific distribution and side effects can be reduced. (4) The bsMAb can pretarget to tumor site, increasing the T/NT ratio and cytotoxicity. In the present study, A bsMAb with versatile adaptor was designed. In this bsMAb, one arm could react with gastric cancer-associated antigen, and the other with a hapten TNP. The bsMAb could mediate different tumoricidal agents crosslinked to TNP. A variant hybridoma 3H11-HAT(s) secreting McAb against gastric cancer was fused with spleen cells of mice immunized with KLH-TNP. After screening and subcloning, 10 hybrid-hybridomas were obtained, which secreted antibodies against both gastric cancer target cells BGC823 and BSA-TNP. By using special bridge method, only hybrid-hybridomas 6A3 (gamma 2b, mu), and 1G7 (gamma 2b, gamma 2b) were confirmed to secrete bsMAb. Further experiments showed that the bsMAb 6A3 and 1G7 could mediate different cytotoxicities, for example, Ricin-TNP, MMC-HSA-TNP and ADM-BSA-TNP. This system is useful for evaluating different tumoricidal agents in bsMAb targeting therapy, and has potential value in clinics.(ABSTRACT TRUNCATED AT 250 WORDS)

[3H]ryanodine binding sites in rat brain demonstrated by membrane binding and autoradiography.

High affinity [3H]ryanodine binding sites were characterized in P1 (crude nuclear), P2 (mitochondrial/synaptosomal) and P3 (microsomal) subcellular fractions of rat brain. Binding in each of the fractions was highest at 37 degrees C and pH 8-9, optimal in the presence of 100 microM Ca2+, 550 microM ATP and 1.0 M KCl, and increased linearly as a function of protein. Saturation analyses revealed a single class of binding sites with mean KD values (nM) of 8.9, 1.6 and 5.7 and Bmax values (fmol/mg protein) of 122, 69 and 106 for the P1, P2 and P3 fractions, respectively. The levels of [3H]ryanodine binding in P1 and P2 fractions of 4 brain regions were fairly uniform while those in P3 fractions were 5-fold greater in cerebral cortex than in the other areas examined. By autoradiography, a high concentration of [3H]ryanodine binding sites was seen in the dentate gyrus and CA3 subregions of the hippocampus. The results suggest that [3H]ryanodine binding sites, perhaps similar to [3H]ryanodine receptors in muscle, are associated with various subcellular structures and are heterogeneously distributed in the CNS.

A rapid and efficient method for testing immunohistochemical reactivity of monoclonal antibodies against multiple tissue samples simultaneously.

A flexible, efficient and rapid method was developed whereby a small volume of monoclonal antibody could be used to immunohistochemically stain many different tissues, simultaneously, on one standard glass slide. This method is based on the preparation of 'cores' of paraffin-embedded tissue from standard histology blocks. The paraffin cores are straightened, inserted into a casing cut from an ordinary drinking straw, mounted in a paraffin block and sectioned. Over 120 individual tissue samples can be organized on a slide and stained for screening or characterization with 0.25 ml of diluted primary antibody. Advantages of this paraffin core method include: great economies in time, reagents, tissue specimens and antibodies; ease of producing multiple, regular, stable, easily handled tissue core samples; direct identification of intratissue regions of interest for inclusion; efficient use of rare tissue samples; versatility for rapid construction of multiple tissue slides containing any combination of relevant tissues from a 'library' of tissue cores; and, no need for deparaffinization and reembedding of tissues.

Stereochemistry of the hydrolysis of the endo isomer of uridine 2',3'-cyclic phosphorothioate catalyzed by the nonspecific phosphohydrolase from Enterobacter aerogenes.

The nonspecific phosphohydrolase from Enterobacter aerogenes (ATCC 13048) requires divalent metal ions for activity, since zinc present in the isolated enzyme can be removed by extensive dialysis against 8-hydroxyquinoline-5-sulfonate at pH 7.5 to yield an inactive enzyme which can be reactivated by addition of Zn2+, Cd2+, Co2+, Mn2+, or Ni2+; six ions of either zinc or cadmium can be incorporated into the inactive enzyme, and this incorporation of metal ion can be correlated with the regaining of activity (J. A. Gerlt, R. Dhesi, and H. C. Hemmings, unpublished experiments). The cadmium-reactivated phosphohydrolase catalyzes the hydrolysis of the endo isomer of uridine 2',3'-cyclic phosphorothioate (U greater than pS) to yield uridine 3'-monophosphorothioate as the major product. After enzymatic hydrolysis of the cyclic phosphorothioate in 19.8% H218O and chemical recyclization of the 18O-labeled acyclic phosphorothioates to yield a mixture of the endo and exo isomers of U greater than pS, 18O is found primarily in the exo isomer, as judged by examination of the 145.7-MHz phosphorus-31 nuclear magnetic resonance spectrum of the mixture. This observation indicates that the cadmium phosphosphohydrolase catalyzes hydrolysis of endo-U greater than pS with inversion of configuration, implying that the hydrolysis reaction proceeds by an in-line attack of water on the phosphorus.