Effects of mutation and growth rates on patterns of microsatellite instability.

The detection of somatic microsatellite (MS) alterations in tumors is often interpreted as a sign of underlying genomic instability. However, it is unclear why the proportions of altered MS loci vary between different mutator phenotype tumors. We present a simple mathematical analysis that can account for some of these differences, recognizing that the mutations accumulated in a tumor reflect both its mutation rate and number of cell divisions. Only a small proportion of mutated MS loci are expected in tumors with normal or low mutation rates. In contrast, tumors with high mutation rates may or may not acquire mutations depending on the numbers of divisions that proceed the onset of the mutator phenotype. The majority of MS loci should accumulate mutations if high mutation rates are acquired early in tumor progression. Somatic MS mutations provide clues to both the mode and tempo of tumori-genesis.     

A new liver metastatic and peritoneal dissemination model established from the same human pancreatic cancer cell line: analysis using cDNA macroarray

To elucidate the mechanisms of metastasis, we established two sublines HPC-1H5 with a highly liver metastatic cell line and HPC-1P5a with a highly peritoneal disseminating cell line, which were sequentially selected from the parental pancreatic cancer cell line HPC-1. Using these three cell lines, we investigated several biological properties and mRNA levels of differentially-expressed genes involved in cancer metastasis by cDNA macroarray. Microscopic findings for the three cell lines were the same. The tumorigenicity, in vitro growth ability, motile activity, adhesive activity and the production of IL-8 of metastatic sublines were higher than those of parental HPC-1 cells. Particularly, HPC-1H5 cells showed clearly higher levels of IL-8 expression and tumors of HPC-1H5 cells grew faster and bigger than those of HPC-1P5a cells. In cDNA macroarray analysis of HPC-1H5 cells, 22 genes were up-regulated and 44 genes were down-regulated compared with parental HPC-1 cells. In HPC-1P5a cells, 9 genes were up-regulated and 28 genes were down-regulated compared with parental HPC-1 cells. This study provides a demonstration of global gene expression analysis of pancreatic cancer cells with liver metastasis and peritoneal dissemination. Furthermore, our results provide a new insight into the study of liver metastasis and peritoneal dissemination of human pancreatic cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Importance of a biofouling-resistant phospholipid polymer to create a heparinized blood-compatible surface

Heparinization is believed to be one of the methods to suppress thrombus formation on blood-contacting surfaces. However, this study hypothesizes that heparinization alone might not be sufficient to provide a blood-compatible surface; that is, a surface property that resists biofouling is necessary to obtain an effective heparin-modified surface. 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymers with 2-aminoethyl methacrylate (AEMA) were synthesized to immobilize heparin through ionic bonding. The primary amino groups of AEMA were considered to be the polymer surface because the zeta-potential of the surface was positive when the mole fraction of the AEMA units was above 0.2. The antithrombogenic character of the polymer surface modified with heparin was evaluated by both Lee-White and microsphere column methods. The coagulation period of human whole blood in the absence of anticoagulant in glass tubing coated with the MPC polymer was longer than that in the original glass tube. Cell adhesion was completely inhibited on the MPC polymer surface after contact with human whole blood without anticoagulant. However, many adherent blood cells were observed on poly(2-ethylhexyl methacrylate-co-AEMA) (no MPC unit) even after heparinization. These results strongly indicate that the MPC polymer is a useful substrate where the heparin works well and that the heparin-immobilized MPC polymer has superior blood compatibility to the simple MPC polymer.     

Structural identification of an epitope of antigenic factor 5 in mannans of Candida albicans NIH B-792 (serotype B) and J-1012 (serotype A) as beta-1,2-linked oligomannosyl residues.

In previous articles, we reported the presence of phosphate-bound beta-1,2-linked oligomannosyl residues in the mannans of strains of Candida albicans serotypes A and B and Candida stellatoidea. To identify the antigenic factor corresponding to this type of oligomannosyl residue, a relationship between chemical structure and antigenic specificity in the mannans of C. albicans NIH B-792 (serotype B, B-strain) and C. albicans J-1012 (serotype A, J-strain) was investigated by using a combination of two-dimensional 1H nuclear magnetic resonance spectroscopy of H-1, H-2, and H-5 regions in the mannans and an enzyme-linked immunosorbent assay that employed concanavalin A-coated microtiter plates. It was shown in the present 1H nuclear magnetic resonance study that an examination of chemical shifts not only in the H-1 region but also in the H-5 region was useful for the quantitative determination of the phosphate-bound beta-1,2-linked oligomannosyl residues. In the enzyme-linked immunosorbent assay using concanavalin A-coated plates, it was revealed that, of factor sera 1, 4, and 5, only factor serum 5 showed a reactivity proportional to the densities of the beta-1,2-linked oligomannosyl residues of the mannan subfractions of different phosphate contents that had been prepared from the bulk B-strain mannan by DEAE-Sephadex chromatography. The above results indicate that the phosphate-bound beta-1,2-linked oligomannosyl residues, Manp beta 1----(2Manp beta 1----)n2Man (n = 0-5), correspond to antigenic factor 5.     

Biological effects of static magnetic fields on the microcirculatory blood flowin vivo: a preliminary report

There have been few studies of the effect of static magnetic fields on microcirculatory haemodynamics in vivo. The rat skinfold transparent chamber technique was used, which provides an excellent means of observing and quantifying direct in vivo microvascular haemodynamic responses to static magnetic fields up to 8 T. An intravital videomicroscope was used to measure the changes in blood flow before and after exposure to a magnetic field for 20 min in a horizontal type superconducting magnet with a bore 100 mm in diameter and 700 mm long. After exposure, microcirculatory blood flow showed an initial increase for about 5 min followed by a gradual decrease and a return to the control value. It is hypothesised that these changes represent rebound hyperaemia following reduced blood flow during exposure.     

The effect of epidermal growth factor on production of vascular endothelial growth factor by amnion-derived (WISH) cells

Our objective was to clarify the physiological role of vascular endothelial growth factor (VEGF) by amnion-derived (WISH) cells. WISH cells were cultured, and the effect of epidermal growth factor (EGF), mitogen-activated protein (MAP) kinase kinase or extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors (U0126) or phosphatidylinositol (PI) 3-kinase on the production of VEGF was examined. VEGF was assayed by ELISA. The activation of MAP kinase and akt, which is phosphorylated by PI 3-kinase, were detected by Western blot analysis using anti-phosphorylated MAP kinase antibody and anti-phosphorylated akt antibody. In the time course of VEGF production following EGF treatment, VEGF production showed a significant increase only after 16 (p < 0.01)-32h (p < 0.01). EGF increased the production of VEGF by WISH cells in a dose-dependent manner. The MAP kinase and akt activity were determined by treatment with EGF. VEGF production was significantly decreased following pretreatment with U0126 or wortmannin for two hours before treatment with EGF (p < 0.01, p < 0.01). WISH cells appeared to produce VEGF via a mechanism involving tyrosine kinase activation of EGF receptor and MAP kinase or PI 3-kinase. It is suggested that VEGF may contribute to the neovascularization and proliferation of the placenta and gestational tissue, and EGF may play an important role in regulation of VEGF production in the placenta.     

Proventricular glands in fowl.

Some researchers have already described the fowl proventriculus. However, we believed there was a need for detailed carbohydrate histochemical investigations on the same glands. Moreover, some researchers had erred about the lamina muscularis mucosae. The results of these investigations are as follows. 1. The proventricular glands consist of both superficial and profound gastric glands. 2. The superficial glands are distributed in the lamina propria mucosae while the profound glands exist in the tela submucosa. 3. The superficial glands are simple, branched tubular glands. The columnar glandular cells are arranged in a simple layer and react strongly to PAS, AB (pH 2.5 and 0.5). These appear to be dark purple when they are stained with PAS-AB (pH 2.5). Some other methods have also been tried. 4. Judging from the data 3), the superficial gastric glands contain neutral, weak and strong acids, sulfuric and acid mucopolysaccharides, sialomucin, and II and III neutral mucus type. 5. Glandular cells in the body and basal portions of the superficial gastric glands contain a large number of fine pepsinogen granules. 6. Judging from the data of 3)-5), we believe that the superficial gastric glands are undifferentiated gastric glands and that they are same kinds of glands that are found in mammals. 7. A large number of profound gastric glands fill the tela submucosa. They are compound tubular glands, and are composed of many glandular alveoli. Their columnar glandular cells are arranged in a simple layer. 8. These glandular cells react moderately to PAS, negatively to AB (pH 2.5 and 0.5) and PAS-AB (pH 2.5). Moreover, we observed some other reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylcobalamin induces a long-lasting enhancement of the field potential in rat suprachiasmatic nucleus slices

Optic nerve stimulation has been reported to evoke a field potential (FP) in rat suprachiasmatic nucleus (SCN) slices. Methylcobalamin,δ-(5,6-dymethylbenzimidazoyl)-Co-methyl-cobamide (Me-B₁₂) enhanced this FP and the enhancement lasted more than 1 h after washing out. Maximal enhancement (143.6±9.8%) was achieved at a concentration of 10 μM. By contrast, cyanocobalamin containing CN- instead of CH₃-showed no enhancement of the amplitude in the FP. Me-B₁₂ induced enhancement of FP was strongly blocked by an N-methyl- -aspartate (NMDA) receptor antagonist, -2-amino-5-phosphonovaleric acid (APV). These results indicate that CH₃- in the Me-B₁₂ is required to modulate the FP amplitude and the NMDA receptor is involved in the long-lasting FP enhancement induced by Me-B₁₂. The present results suggest that Me-B₁₂ modifies the photic entrainment of the circadian clock in the suprachiasmatic nucleus via an activation of NMDA receptors.     

Apoptotic signaling pathway activated by Helicobacter pylori infection and increase of apoptosis-inducing activity under serum-starved conditions

The enhanced gastric epithelial cell apoptosis observed during infection with Helicobacter pylori has been suggested to be of significance in the etiology of gastritis, peptic ulcers, and neoplasia. To investigate the cell death signaling induced by H. pylori infection, human gastric epithelial cells were incubated with H. pylori for up to 72 h. H. pylori infection induced the activation of caspase -8, -9, and -3 and the expression of the proapoptotic Bcl-2 family proteins Bad and Bid. The peak of the activity of the caspases occurred at 24 h. At this time, the inhibition of caspase-8 or -9 almost completely suppressed H. pylori-induced apoptosis. Inhibition of caspase-8 suppressed the expression of Bad and Bid and the subsequent activation of caspase-9 and -3. These observations indicate that H. pylori induces apoptosis through a pathway involving the sequential induction of apical caspase-8 activity, the proapoptotic proteins Bad and Bid, caspase-9 activity, and effector caspase-3 activity. Activation of the pathway was independent of CagA or vacuolating toxin. A membrane fraction of H. pylori was sufficient to activate this pathway, and treatment with proteinase K eliminated the activity. Apoptotic activity of the membrane fraction was significantly increased by incubating the bacteria under serum-starved conditions for 24 h. These observations suggest that environmental conditions in the human stomach could induce H. pylori-mediated pathogenesis, leading to a variety of clinical outcomes.     

Inhibitory effect of antiserum to surface antigen P50 of Babesia gibsoni on growth of parasites in severe combined immunodeficiency mice given canine red blood cells

The inhibitory effect of an antiserum to surface protein P50 of Babesia gibsoni on the growth of the parasite was determined with severe combined immunodeficiency mice given canine red blood cells. The antiserum to the recombinant P50 protein significantly inhibited the parasite growth, indicating that P50 might be a useful vaccine candidate.