Overexpression of CDC25B overrides radiation-induced G2-M arrest and results in increased apoptosis in esophageal cancer cells.

CDC25B phosphatase plays a key role in controlling G2-M progression by dephosphorylating two inhibitory residues of CDC2 and also has been suggested to have an oncogenic property. In this study, we investigated the effect of CDC25B overexpression on radiation-induced G2-M arrest and radiation sensitivity in esophageal cancer cells. TE8-CDC25B, in which CDC25B was overexpressed under an inducible system, was more radiosensitive than the vector control (TE8-neo) in a clonogenic survival assay. Without radiation, CDC25B overexpression had little effect on cell cycle fractions or growth rate. After 10-Gy radiation, TE8-CDC25B showed decreased G2-M arrest and increased apoptosis, whereas TE8-neo displayed prolonged G2-M arrest and less apoptosis. During this period, there were no differences in the protein amounts of CDC2 and cyclin B1 between the two cell lines. However, more CDC25B expression, which was reduced immediately by radiation, was sustained in TE8-CDC25B than in TE8-neo. Moreover, induction of tyrosine phosphorylation of CDC2 and reduction of CDC2 kinase activity after irradiation was less significant in TE8-CDC25B than in TE8-neo. These results indicate that cancer cells that overexpress CDC25B override G2-M arrest by retaining CDC2 kinase activity and undergo apoptosis after radiation. This may point to an effective approach toward improving radiotherapy outcomes of various cancers.

Analytical approach to the temporal changes in NIRS signals to separate hemodynamic change from brain activity.

In brain function measurements by near-infrared spectroscopy (NIRS), we focus on the temporal changes in the oxygenated and deoxygenated hemoglobin concentrations and propose an analytical approach to the temporal changes of the hemoglobin concentrations' cross-correlation coefficient in the brain functional signals. The cross-correlation coefficient between the oxygenated and deoxygenated hemoglobin concentrations shows negative values for brain activity and positive values for hemodynamic changes. Experiments with a 16-channel functional NIRS system show that our proposed approach clarifies the interpretation of the measured NIRS signals and it can be used to estimate the influence of hemodynamic change on brain activity.

Region-of-interest cone beam computed tomography (ROI CBCT) with a high resolution CMOS detector.

Cone beam computed tomography (CBCT) systems with rotational gantries that have standard flat panel detectors (FPD) are widely used for the 3D rendering of vascular structures using Feldkamp cone beam reconstruction algorithms. One of the inherent limitations of these systems is limited resolution (<3 lp/mm). There are systems available with higher resolution but their small FOV limits them to small animal imaging only. In this work, we report on region-of-interest (ROI) CBCT with a high resolution CMOS detector (75 µm pixels, 600 µm HR-CsI) mounted with motorized detector changer on a commercial FPD-based C-arm angiography gantry (194 µm pixels, 600 µm HL-CsI). A cylindrical CT phantom and neuro stents were imaged with both detectors. For each detector a total of 209 images were acquired in a rotational protocol. The technique parameters chosen for the FPD by the imaging system were used for the CMOS detector. The anti-scatter grid was removed and the incident scatter was kept the same for both detectors with identical collimator settings. The FPD images were reconstructed for the 10 cm x10 cm FOV and the CMOS images were reconstructed for a 3.84 cm × 3.84 cm FOV. Although the reconstructed images from the CMOS detector demonstrated comparable contrast to the FPD images, the reconstructed 3D images of the neuro stent clearly showed that the CMOS detector improved delineation of smaller objects such as the stent struts (~70 µm) compared to the FPD. Further development and the potential for substantial clinical impact are suggested.

Murine interleukin-2 receptor subunits differentially detected with anti-interleukin-2 monoclonal antibodies.

Cross-linking of radioiodinated interleukin-2 to murine CTLL-2 cells enabled detection of 70 kDa, 85 kDa and 105 kDa complexes of IL-2 and its binding proteins under the high-affinity binding condition. A series of anti-interleukin-2 monoclonal antibodies (L15, L20, L23, L34, and L61) were tested for their activity to immunoprecipitate these cross-linked complexes. L61, which had strong neutralizing activity, precipitated only the 70 kDa complex. L15, L20, and L34, which also had neutralizing activity, precipitated not only the 70 kDa complex but also the 85 kDa complex. L23, which had practically no neutralizing activity, precipitated the 105 kDa complex as well as the 85 kDa complex. These results suggest that there are at least three distinct receptor binding sites for each receptor subunit on the interleukin-2 molecule, which are discernible by these monoclonal antibodies and that the 105 kDa complex may play a significant role in the formation of the high-affinity receptor complex and the signal transduction.

Monoclonal antibodies which differentiate high- and low-affinity binding sites of interleukin-2.

Five monoclonal antibodies (L15, L20, L23, L34, and L61) against human recombinant interleukin-2 were tested for their effects on the interleukin-2 bioactivity and binding. Four of these monoclonal antibodies, L15, L20, L34, and L61, which had neutralizing activity, completely blocked interleukin-2 binding to the high-affinity receptor. On the other hand, L23, which had a very weak neutralizing activity, blocked interleukin-2 binding to the low-affinity receptor. These results suggest that there are at least two distinct binding sites on the interleukin-2 molecule; those for the high-affinity receptor and those for the low-affinity receptor. These monoclonal antibodies should be useful tools in the study of the interaction between interleukin-2 and interleukin-2 receptor.

Conditioned media of glial cell lines induce alkaline phosphatase activity in cultured artery endothelial cells. Identification of interleukin-6 as an induction factor.

Conditioned media of human glial cell lines induced alkaline phosphatase activity in cultured calf artery endothelial cells. The maximal alkaline phosphatase activity in the culture was comparable to the level in isolated brain capillary endothelial cells. An induction factor in the conditioned media was purified and identified as interleukin-6 from its amino-terminal sequence, molecular weight, amino acid composition and immunoreactivity. Recombinant interleukin-6 had similar induction activity. Our findings raise the possibility that interleukin-6 induces and modulates alkaline phosphatase activity in endothelial cells during normal development of the blood-brain barrier and under certain pathological conditions.

High throughput screening for human interferon-gamma production inhibitor using homogenous time-resolved fluorescence.

An immunoassay for interferon-gamma (IFN-gamma) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IFN-gamma can be detected by simply adding a mixture of three reagents-biotinylated polyclonal antibody, europium cryptate (fluorescence donor, EuK)-labeled monoclonal antibody, and crosslinked allophycocyanin (fluorescence acceptor, XL665) conjugated with streptavidin-and then measuring the time-resolved fluorescence. The detection limit of IFN-gamma by the proposed method is about 625 pg/ml. We applied the method to the detection of IFN-gamma secreted from NK3.3 cells and employed it in high throughput screening for IFN-gamma production inhibitors. With this screening format, IFN-gamma can be measured by directly adding the above reagents to microplate wells where NK3.3 cells are being cultured and stimulated with interleukin-12. This "in situ" immunoassay requires only pipetting reagents, with no need to transfer the culture supernatant to another microplate or wash the plate. Therefore, this screening format makes possible full automation of cell-based immunoassay, thus reducing cost and experimental time while increasing accuracy and throughput.

A homogeneous caspase-3 activity assay using HTRF technology.

Caspases are cysteine proteases presenting a conserved active site that cleaves protein substrates at a highly specific position. They are involved in different aspects of the active cell death pathway. Most of them act through proteolytic degradations of cellular components. This paper describes the assay development, assay validation, and screening for inhibitors of this enzyme, which could be potential drug candidates. The assay uses homogeneous time-resolved fluorescence based on energy transfer from europium cryptate as donor to cross-linked allophycocyanin as acceptor (XL665). A double-tagged substrate, biotinyl-epsilon-aminocaproyl-L-aspartyl-L-glutamyl-L-valyl-Laspartyl-L-alanyl-L-propyl-N(epsilon)-(2,4-dinitrophenyl)-L-lysine-amide (biotin-X-DEVDAPK(dnp)-NH(2)), is conjugated with streptavidin cryptate and anti-dnp-XL665 monoclonal antibody. The close proximity between donor and acceptor induces a specific time-resolved fluorescence signal. In the presence of enzyme activity, the substrate cleavage induces an unlinking of the two fluorescent probes and, subsequently, the disappearance of the specific signal as a result of loss of proximity. Experiments to optimize the reagent concentration, incubation times, precision, reproducibility, and robustness are discussed in comparison with a fluorometric method.

Cycloprodigiosin hydrochloride, a H+/Cl- symporter, induces apoptosis in human breast cancer cell lines.

The effect of cycloprodigiosin hydrochloride (cPrG.HCl), a H+/Cl- symporter, on five human breast cancer cell lines (KPL-1, T-47D, MCF-7, MKL-F, and MDA-MB-231), a human breast epithelial cell line (HBL-100), and a human fibroblast cell line (WI-38-40) was examined. cPrG.HCl inhibited the growth of all five breast cancer cell lines (IC50: 0.46-0.62 microM) and slightly inhibited HBL-100 and WI-38-40 cell growth (IC50: 1.75 microM and 2.26 microM respectively). cPrG.HCl treatment in KPL-1 cells increased the pH of acidic organelles, decreased intracellular pH, and caused apoptosis, which was confirmed by the appearance of a sub-G1 population by flow cytometry and DNA fragmentation. In addition, cPrG.HCl-induced apoptosis was strongly suppressed by imidazole, a cell-permeable base, suggesting that intracellular acidification was essential for the apoptosis. Further, cPrG.HCl treatment up-regulated Bax and Bak expression, down-regulated Bcl-2 expression, and activated caspase-3. Therefore, the intracellular acidification by cPrG.HCl treatment suppressed the growth of human breast cancer cell lines by inducing apoptosis.

The structure of sugar chains of Japanese quail ovomucoid. The occurrence of oligosaccharides not expected from the classical biosynthetic pathway for N-glycans; a method for the assessment of the structure of glycans present in picomolar amounts.

While the structure of the major oligosaccharide of Japanese quail ovomucoid was reported earlier (Hase, S. et al. (1982) J. Biochem. 91, 735-737), the structures of the minor oligosaccharide units were investigated for the first time in the present studies. For this purpose, the glycans of the protein were liberated from the polypeptide chain by hydrazinolysis. After N-acetylation, the reducing ends of the oligosaccharides obtained were coupled with 2-aminopyridine, and then the resulting fluorescent derivatives were purified by Bio-Gel P-2 column chromatography and reversed-phase HPLC. The chemical structures of two minor oligosaccharide units were determined with the aid of exoglycosidases, and by methylation analysis and Smith degradation. The results demonstrated that the ovomucoid contains the following two monoantennary glycans: Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc and Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. The latter structure was not predicted by the classical metabolic pathway for the N-glycans to be formed. The structures of three additional minor heterosaccharides were deduced from their elution positions on HPLC together with the results of determination of their molecular sizes and the HPLC elution positions of their enzymatic degradation products. It is noteworthy that for the latter procedure for the estimation of the structures of oligosaccharides only minute quantities of glycans (several hundreds pmol) are required.

Structure of a sugar chain of a protease inhibitor isolated from barbados pride (Caesalpinia pulcherrima Sw.) seeds.

An asparagine-linked sugar chain of a protease inhibitor from barbados pride (Caesalpinia pulcherrima Sw.) was liberated by hydrazinolysis. After N-acetylation, the reducing end residue of this carbohydrate unit was coupled with 2-aminopyridine and the pyridylamino (PA-) derivative was purified by gel-filtration and reversed-phase HPLC. The structure of the resulting PA-sugar chain was determined mainly by stepwise exoglycosidase digestions and 500 MHz 1H-NMR spectroscopy and proved to be as follows: (formula; see text).

The effect of penfluridol and some psychotropic drugs on monoamine metabolism in central nervous system.

Five neuroleptics: penfluridol, haloperidol, chlorpromazine, pimozide and oxypertine, and two benzodiazepine minor tranquilizers; chlordiazepoxide and diazepam, were studied in mice and rats. When the drugs were injected i.p. in mice, all neuroleptics increased the brain homovanillic acid (HVA) concentration but two benzodiazepines did not change it. Of the drugs tested, only pimozide increased the brain level of 5-hydroxyindoleacetic acid. Penfluridol accelerated the turnover rate of brain dopamine (DA), wheras haloperidol accelerated both DA and noradrenaline turnover rates. In rats, oral administration of penfluridol caused an increase in the striatal HVA and 3,4-dihydroxyphenylacetic acid which persisted longer than that following haloperidol. The concentration of brain 3-methoxy-4-hydroxyphenylethylene glycol, however, was not affected by penfluridol even when the drug was administered at a high dose. Based on these and earlier findings, it is concluded that the effect of penfluridol on DA metabolism resembles that of typical neuroleptic compounds and penfluridol acts only on DA neurons as a long-acting DA receptor blocker.

Stereoselective reaction of alpha-sulfinyl carbanion derived from chiral 2-(Trialkylsilyl)ethyl sulfoxides: evidence for a novel silicon-oxygen interaction

Reactions of alpha-sulfinyl carbanions, derived from p-tolyl sulfoxides bearing various alkyl groups, with various electrophiles were examined. The reaction of alpha-sulfinyl carbanions, derived from the beta-silylethyl sulfoxides, with ketones or trimethyl phosphate, gave the syn products with high stereoselectivity. Interaction between the silicon in the trialkylsilyl group and the carbonyl oxygen in nucleophiles was postulated to stabilize the transition state, leading preferably to the syn diastereisomers. This novel silicon-oxygen interaction was supported by an MO calculation study using the MOPAC 93/PM3 and the Gaussian 94 Beche3LYP/3-21+G methods.

Cycloprodigiosin hydrochloride, a H+/Cl- symporter, induces apoptosis in human colon cancer cell lines in vitro.

Recently, we reported that cycloprodigiosin hydrochloride (cPrG.HCl), a novel H+/Cl- symporter, induces acidification of the cytosol and leads to apoptosis on rat and human liver cancer cells. In the present study, the effects of cPrG.HCl, a H+/Cl- symporter, were examined in colon cancer cell lines in vitro. In the MTT assay, cPrG.HCl inhibited the growth of two colon cancer cell lines (WiDr and SW480) in a dose- and time-dependent manner. The cPrG.HCl treatment of both types of cells induced apoptosis as confirmed by the appearance of a sub-G1 population and intranucleosomal DNA fragmentation. In addition, cPrG.HCl lowered pHi (below pH 6.8) respectively. Therefore, these results suggest that cPrG.HCl may be useful for the treatment of colon cancer cells.

Granulation at the porta hepatis following hepatic portoenterostomy for biliary atresia: the healing of experimental hepatoenterostomy.

After hepatic portoenterostomy for biliary atresia, granulation that formed at the porta hepatis caused biliary obstruction in seven out of 27 patients (26%). Six of the seven developed the complication during the first 6 weeks after surgery. The mortality rate was 29% (2/7). Among the same group, the incidence and mortality rate of ascending cholangitis was 19% (5/27) and 20% (1/5), respectively. The features characteristic of biliary obstruction caused by granulation, as compared with those of ascending cholangitis, were the absence of signs of infection and the lack of response to medical treatment. Quantitative analysis of the healing process of an experimental hepatoenterostomy in the rat showed that the mean thickness of the granulation formed at the anastomotic site (area, 5 x 20 mm) decreased almost constantly--from 1.49 mm on day 7 to 0.64 mm on day 42. Mucosa covered 20% of the granulation on day 7, 55% on day 14, 63% on day 21, 76% on day 28, and 92% on day 42. The increase in coverage was greatest during the second postoperative week. The results suggested that the healing of the hepatoenteostomy should be almost complete within 6 weeks. The operative method and postoperative management to prevent excessive granulation should be chosen so as to decrease the incidence of postoperative biliary obstruction.

Morphological analyses of the human tongue musculature for three-dimensional modeling.

Skilled movements of the tongue in speech articulation reflect complex formation of the tongue musculature, although its description in the anatomical literature is rather limited for developing a realistic computational model of the tongue. This study presents detailed descriptions of the muscular structure of the human tongue based on macroscopic and microscopic observations and provides three-dimensional schemata of the tongue musculature. Histologic examination revealed that the tongue consists of five strata, stacked along the courses of the fibers of the genioglossus muscle in proximal-distal directions. This stratum structure exists in the entire tongue tissue, indicating that the lingual musculature can be divided into the inner and outer regions. The former consisted of the "stem" and "core," and the latter of the "cover" and "fringe." In gross dissection, the tongue was cut into wedge-like blocks along the course of the genioglossus muscle to examine muscle fiber arrangement. Using this approach, it was determined that serial repetitions of "structural units" composed the inner musculature of the tongue. Each unit consisted of a pair of thin muscle fiber laminae; one was composed of the genioglossus and vertical muscles, and the other of the transverse muscle. In the apex, the laminae lacked the fibers of the genioglossus. These findings have been incorporated in three-dimensional schemata of the tongue musculature.

[Experimental and clinical studies on the intestinal anastomosis and reconstruction of the alimentary tract].

Healing process of anastomosis and its procedure were studied in following points: Four-interrupted sutures anastomosis in the Wistar rats revealed recanalization without leakage in 64 out of 69. Lymphangial recanalization through the anastomosis was completed within 21 days after operation by Gambee' layer-to-layer anastomosis. While 8 weeks were required by everted or inverted anastomosis. Serosal surface of invaginated intestinal segment of which length corresponded to x1-x2 luminal diameter in the telescoping anastomosis was covered within 8 weeks by the proliferated mucosae of the both proximal and distal segments. Telescoping anastomosis was found to be useful to make an intestinal valve which worked just the same as ileocoecal junction. IVH and elementary diet were effective on the healing of anastomosis. According to the above mentioned findings, following operative procedures were recommended: Esophago-jejunal conduit duodenostomy following total gastrectomy. Choledocho-jejunal conduit duodenostomy with the intestinal valve as bile duct reconstruction. Construction of the intestinal valve and an artificial sphincter using rectus abdominis muscle fibers for ileostomy or short bowel syndrome. Endorectal pull-through operation for anterior-resection of the rectum as well as for radical treatment of Hirschsprung' disease.