B-RAF mutation and accumulated gene methylation in aberrant crypt foci (ACF), sessile serrated adenoma/polyp (SSA/P) and cancer in SSA/P.

BACKGROUND: Sessile serrated adenomas/polyps (SSA/Ps) are a putative precursor of colon cancer with microsatellite instability (MSI). However, the developmental mechanism of SSA/P remains unknown. We performed genetic analysis and genome-wide DNA methylation analysis in aberrant crypt foci (ACF), SSA/P, and cancer in SSA/P specimens to show a close association between ACF and the SSA/P-cancer sequence. We also evaluated the prevalence and number of ACF in SSA/P patients. METHODS: ACF in the right-side colon were observed in 36 patients with SSA/Ps alone, 2 with cancers in SSA/P, and 20 normal subjects and biopsied under magnifying endoscopy. B-RAF mutation and MSI were analysed by PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP, respectively, in 15 ACF, 20 SSA/P, and 2 cancer specimens. DNA methylation array analysis of seven ACF, seven SSA/P, and two cancer in SSA/P specimens was performed using the microarray-based integrated analysis of methylation by isochizomers (MIAMI) method. RESULTS: B-RAF mutations were frequently detected in ACF, SSA/P, and cancer in SSA/P tissues. The number of methylated genes increased significantly in the order of ACF

The first probe of a FLASH proton beam by PET.

The recently observed FLASH effect related to high doses delivered with high rates has the potential to revolutionize radiation cancer therapy if promising results are confirmed and an underlying mechanism understood. Comprehensive measurements are essential to elucidate the phenomenon. We report the first-ever demonstration of measurements of successive in-spill and post-spill emissions of gammas arising from irradiations by a FLASH proton beam. A small positron emission tomography (PET) system was exposed in an ocular beam of the Proton Therapy Center at MD Anderson Cancer Center to view phantoms irradiated by 3.5 × 1010protons with a kinetic energy of 75.8 MeV delivered in 101.5 ms-long spills yielding a dose rate of 164 Gy s-1. Most in-spill events were due to prompt gammas. Reconstructed post-spill tomographic events, recorded for up to 20 min, yielded quantitative imaging and dosimetric information. These findings open a new and novel modality for imaging and monitoring of FLASH proton therapy exploiting in-spill prompt gamma imaging followed by post-spill PET imaging.

The first PET glimpse of a proton FLASH beam.

We demonstrate the first ever recorded positron-emission tomography (PET) imaging and dosimetry of a FLASH proton beam at the Proton Center of the MD Anderson Cancer Center. Two scintillating LYSO crystal arrays, read out by silicon photomultipliers, were configured with a partial field of view of a cylindrical poly-methyl methacrylate (PMMA) phantom irradiated by a FLASH proton beam. The proton beam had a kinetic energy of 75.8 MeV and an intensity of about 3.5 × 1010protons that were extracted over 101.5 ms-long spills. The radiation environment was characterized by cadmium-zinc-telluride and plastic scintillator counters. Preliminary results indicate that the PET technology used in our tests can efficiently record FLASH beam events. The instrument yielded informative and quantitative imaging and dosimetry of beam-activated isotopes in a PMMA phantom, as supported by Monte Carlo simulations. These studies open a new PET modality that can lead to improved imaging and monitoring of FLASH proton therapy.

Purification and Characterization of Membrane-Bound Inositol Phospholipid-Specific Phospholipase C from Suspension-Cultured Rice (Oryza sativa L.) Cells (Identification of a Regulatory Factor).

A membrane-bound inositol phospholipid-specific phospholipase C was solubilized from rice (Oryza sativa L.) microsomal membranes and purified to apparent homogeneity using a series of chromatographic separations. The apparent molecular mass of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000 D, and the isoelectric point was 5.1. The optimum pH for the enzyme activity was approximately 6.5, and the enzyme was activated by both Ca2+ and Sr2+. The chemical and catalytic properties of the purified membrane-bound phospholipase C differed from those of the soluble enzyme reported previously (K. Yotsushima, K. Nakamura, T. Mitsui, I. Igaue [1992] Biosci Biotech Biochem 56: 1247-1251). In addition, we found a regulatory factor for the phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolyzing activity of phospholipase C from rice cells. The regulatory factor was dissociated from the catalytic subunit of phospholipase C during the purification. The regulatory factor was necessary to induce PIP2-hydrolyzing activity of both membrane-bound and -soluble phospholipase C; these purified enzymes had no activity alone. Because the plasma membranes isolated from rice cells could also act as a regulatory factor, the regulatory factor seems to be localized in the plasma membranes. Regulation of inositol phospholipid turnover in rice cells is discussed.

Sequence and electrophysiological characterization of two insect-selective excitatory toxins from the venom of the Chinese scorpion Buthus martensi.

The two insecticidal peptides Bm32-VI and Bm33-I, isolated from the venom of the Chinese scorpion Buthus martensi induce paralytical symptoms typical of insect contractive toxins. They show, respectively, 74% and 77% homology with AaIT from Androctonus australis, comparable insecticidal activity and no vertebrate toxicity. Under voltage-clamp conditions, both toxins induced (1) an increased fast Na(+) current, (2) a shift in voltage dependence of Na(+) current activation, (3) the occurrence of a delayed current, and (4) a slow development of a holding current. Increased Na(+) conductance at negative potential values is responsible for axonal hyperexcitability and the contractive paralysis of insect prey.

Marked, sustained expression of a novel 150-kDa oxygen-regulated stress protein, in severely ischemic mouse neurons.

The 150-kDa oxygen-regulated protein (ORP150) first was described with reference to the central nervous system in cultured astrocytes subjected to dense hypoxia. Subsequently its transcript was found in macrophages within human aortic atherosclerotic plaques, suggesting a role in protecting cells under hypoxic stress. In a mouse model of permanent focal brain ischemia, we aimed to elucidate the constitutive cellular localization in vivo of ORP150 in the central nervous system as well as the sequential alteration in its mRNA and protein expression during this severe ischemic insult. Immunohistochemical study demonstrated that ORP150 protein normally is present predominantly in neurons. The 78-kDa glucose-regulated protein, which is another well-known stress protein retained in the endoplasmic reticulum, also was stained in neurons. During the first 3 h after ischemia, ORP150 antigenicity was markedly enhanced in severely damaged neurons, while the amount of the glucose-regulated protein was decreased. Preceding this change, orp150 mRNA was selectively induced in neurons undergoing postischemic cytoskeletal proteolysis, as early as 1 h after middle cerebral artery occlusion. These results indicated that ORP150 might be regulated by transcriptional level as for many stress proteins, but unlike previously described other stress proteins it was translated in the center of ischemic lesions despite nearly complete energy depletion. In this paper, the biological potentials of ORP150 protein in the setting of brain ischemia in vivo will also be discussed.

Histological and biochemical evaluation of osteogenic response in porous hydroxyapatite coated alumina ceramics.

We compared the osteogenic response in porous alumni (Al) and hydroxyapatite coated porous alumni ceramic (HA/Al) by grafting rat bone marrow cells with these implants subcutaneously in the back of syngeneic rats. The ceramics did not show any bone formation without marrow, but did when combined with marrow cells. The osteogenesis in HA/Al began directly on the ceramic surface, but in Al began away from the ceramic surface, and fibrous tissue interposition was seen between the de novo bone and its surface. Alkaline phosphatase activity and bone specific bone gla protein content in HA/Al with marrow were both about three times higher than in Al with marrow. These results indicate that HA/Al has excellent osteoconductive properties and that a composite of marrow and HA/Al is clinically applicable osteogenic biomaterial.

Ouabain-resistant cells cultured in a synthetic medium.

Several strains of kidney and liver cells cultured in a synthetic medium were found to be resistant to ouabain. These cell strains were characterized because this resistance may serve as a good marker in genetic studies on somatic cells in chemically defined conditions in the absence of Na+ related growth factors and hormones. The phenotype was stable in the absence of selection for at least two years, and the original strains before adaptation to the synthetic medium were found to have ouabain sensitivity equal to the corresponding cells in the synthetic medium. The resting membrane potential, Na+,K+-ATPase activity, and growth rate of the resistant cells were similar to those of ouabain-sensitive cells. The resistance of the cells was not affected by serum or antibodies against some cytoskeletal proteins and the sensitivity of the Na+,K+-ATPase was not restored by partial purification of the membranes. Western blotting of the Na+,K+-ATPase of the ouabain-resistant cells showed that the molecular weights of its two subunits and its immunoreactivity were similar to those of the enzyme from the ouabain-sensitive strain. Thus the ouabain resistance is caused not by ouabain-like hormone produced by the cells or change in the cytoskeletal system, but by a mutation resulting in expression of an ouabain-resistant ATPase gene.

The value of LYM-1 cells for examining vacuole formation and loss of cell viability induced by culture supernates of Helicobacter pylori.

Some strains of Helicobacter pylori are known to produce an extracellular cytotoxin that causes vacuolation in cultured mammalian cells. Screening for such strains makes use of HeLa cells which may not be sensitive enough to detect minimal changes. The aim of this study was to develop a more sensitive cell line. Vacuole formation was examined in HeLa cells, as well as four other cell lines established in this laboratory by ammonium chloride induction. Among five cell lines tested, LYM-1 cells were most sensitive for the detection of intracellular vacuolation with this agent. Loss of cell viability of LYM-1 and HeLa cells induced by H. pylori culture supernates was also examined: LYM-1 were more sensitive than HeLa cells. Cell death was not always accompanied by vacuole formation. This suggests that the mechanism whereby cell death occurs must be different from that for vacuole formation. LYM-1 cells may be useful when measuring vacuole formation and cell death of the cultured cells induced by culture supernates of clinical isolates of H. pylori.

A sensitive new method for measurement of guanase with 8-azaguanine in bicine bis-hydroxy ethyl glycine buffer as substrate.

Serum guanase activity has been considered as a possible specific indicator of hepatocellular diseases. However, no suitable method is available for routine clinical determination of serum guanase activity. Conventional assay methods are troublesome and inaccurate, since guanine and 8-azaguanine, the substrates of the enzyme, are scarcely soluble in water so that it is not possible to prepare a stable substrate solution of sufficient concentration for use in assays. A new method was developed for assay of guanase activity by direct colorimetric determination of ammonia. In this method, bicine bis-hydroxy ethyl glycine (dotite bicine) buffer is used for preparation of a stable substrate solution and with a fixed concentration of substrate of sufficient strength serum guanase can be measured sensitively and reproducibly. This assay system could be used as a routine clinical laboratory test in the diagnosis of liver damage.

Liposomal transfection of human gamma-interferon gene into human glioma cells and adoptive immunotherapy using lymphokine-activated killer cells.

The authors evaluated the effect of liposomal transfection of human gamma-interferon (HuIFN-gamma) gene into human glioma cells and lymphokine-activated killer (LAK) cells, alone and in combination. An HuIFN-gamma gene inserted in a eukaryotic expression vector was entrapped in liposomes bearing positive surface charges. Liposomal gene transfection induced production of HuIFN-gamma and its secretion in culture medium of human glioma cell lines (SK-MG-1 and U-251 MG). At 4 days after transfection, the cells produced 10 to 50 U/ml of HuIFN-gamma in the medium, whereby the major histocompatibility complex (MHC) class I and II antigens, as well as intercellular adhesion molecule-1 (ICAM-1), were induced on the glioma cell surface. The growth-inhibiting effect of transfection-induced HuIFN-gamma was much stronger in comparison with control cultures exposed to 500 U/ml of exogenously added HuIFN-gamma. In addition, 20% to 40% growth inhibition was obtained in the glioma cells when they were treated with LAK cells alone at a 5:1 ratio of effector to target cells. Liposomal transfection of HuIFN-gamma gene into human glioma cells combined with immunotherapy using LAK cells was more effective than either technique alone. The reinforcement of growth inhibition in the case of combined therapy was quenched by anti-ICAM-1 monoclonal antibody, but not by anti-MHC class I or II monoclonal antibodies. These results suggest that the combined effect of liposomal transfection of HuIFN-gamma gene plus LAK cells into human glioma cells is a potentially useful therapy for malignant glioma, and that the mechanisms of the reinforcement of growth inhibition are closely related to the expression of ICAM-1 on the glioma cell surface.

Stimulation of prostaglandin E2 production by phorbol esters and epidermal growth factor in porcine thyroid cells.

Effects of phorbol esters and epidermal growth factor (EGF) on prostaglandin E2 production by cultured porcine thyroid cells were examined. Both phorbol 12-myristate 13-acetate (PMA) and EGF stimulated prostaglandin E2 production by the cells in dose related fashion. PMA stimulated prostaglandin E2 production over fifty-fold with the dose of 10(-7) M compared with control. EGF (10(-7) M) also stimulated it about ten-fold. The ED50 values of PMA and EGF were respectively around 1 X 10(-9) M and 5 X 10(-10) M. Thyroid stimulating hormone (TSH), however, did not stimulate prostaglandin E2 production from 1 to 24-h incubation. The release of radioactivity from [3H]-arachidonic acid prelabeled cells was also stimulated by PMA and EGF, but not by TSH. These results indicate that both PMA and EGF are potent stimulators of prostaglandin E2 production, associated with the activity to stimulate arachidonic acid release in porcine thyroid cells.

Results of opening-wedge osteotomy for the treatment of a post-traumatic varus deformity of the ankle.

We performed a one-stage opening-wedge valgus osteotomy in nine patients to correct a post-traumatic progressive varus deformity of the ankle. The osteotomy site was stabilized with two, three, or four Kirschner wires or with a plate and screws (in one patient). The site of the osteotomy united within two months after the operation in eight patients and at six months in one patient who was fifty-nine years old. The average duration of follow-up was seven years and four months (range, two years and four months to thirteen years and two months). Postoperatively, the range of motion of the ankle was decreased in six patients and remained unchanged in three. However, none of the patients reported any limitation in the activities of daily living, and the four adolescent patients were able to participate in sports activities. The result was graded as excellent for four ankles, good for two, and fair for three.

Development of a Simple Culture Method for the Tissues Contaminated with Microorganisms and Application to Establishment of a Fish Cell line.

Cell cultures are good in vitro model systems in place of using living animals. In the present study, we developed a simple culture method in which tissues were pretreated with a low concentration of sodium hypochlorite solution (NaClO) to prevent not only bacteria but also fungi. Scales removed from a goldfish (Carassius auratus) body were treated with 70% ethanol and then with 0.3% of sodium hypochlorite solution, and cultured in vitro in an atmosphere containing 0.5% CO2. The doubling time of the established cells (GAKS) was 24 hr. The GAKS cells contained alkaline phosphatase activity (8.3+/-1.1 nmol/min/mg protein) and secreted 0.32+/-0.07 pg/ml endothelin during a 3 day culture of a full monolayer sheet.

Comparison of antibody titer to human and rat acetylcholine receptor in myasthenia gravis.

Antibody titer to acetylcholine receptor (AChR Ab) was measured in 34 patients with myasthenia gravis (MG) using an immunoprecipitation method with human and rat AChR as antigens, to study the reliability of these assay systems and the cross-reactivity between human and rat AChR. There was a good correlation in the titer of Ab to AChR between human and rat (P < 0.01) when Ab titers were expressed logarithmically. The titer of Ab to human AChR was higher in a group of patients with a high concentration of AChR Ab and lower in a group with a low concentration of AChR Ab than that to rat AChR. This indicates that there may be distinctive characteristics in AChR Ab between groups of patients with high and low concentrations of AChR Ab.

Extractive-spectrophotometric determination of trace iron(II) with di-2-pyridylmethanone 2-(5-nitro)pyridylhydrazone.

The optimum conditions for the extractive-spectrophotometric determination of trace iron(II) with di-2-pyridylmethanone 2-(5-nitro)pyridylhydrazone have been established. Iron(II) reacts with this reagent at pH 2.0-7.5 to form an uncharged 1:2 (metal-to-ligand) complex, which can be extracted with toluene. Beer's law is obeyed over the range up to 0.84 mug/ml of iron(II) at 505 nm. The molar absorptivity of the extracted species is 5.83 x 10(4) 1.mole(-1).cm(-1). The proposed method is extremely sensitive and reproducible, and has been satisfactorily applied to the determination of total iron in freshwater samples by adding ascorbic acid to reduce iron(III).

Tarsal tunnel syndrome caused by coalition associated with a ganglion.

We examined seven patients with tarsal tunnel syndrome in one foot caused by talocalcaneal coalition and a ganglion. We excised the coalition and the ganglion in six of them. All the patients had pain, sensory disturbance in the sole, and a positive Tinel's sign. Older patients with a long history showed atrophy and weakness of the plantar muscles. Talocalcaneal coalition can be diagnosed on a plain lateral radiograph and an anteroposterior radiograph externally rotated 20 degrees, and confirmed by CT. MRI is also useful for diagnosis. The coalitions were medial, and the ganglion had developed from the incomplete part of the coalition; it was multilocular in some patients. After resection, there was early pain relief but sensory disturbances and Tinel's sign persisted. The postoperative results were excellent in one patient, good in four and fair in one.

Subtalar arthrography in acute injuries of the calcaneofibular ligament.

We treated 43 acute tears of the calcaneofibular ligament by operation in 43 patients after subtalar arthrography. There were 22 men and 21 women with a mean age of 22.3 years (14 to 61). Anteroposterior (AP), lateral and oblique views were obtained with the foot in 45 degrees of internal rotation and the ankle in the neutral position. Any communication or leakage to the ankle, tendon sheaths, subcutaneous tissue and sinus tarsi was recorded. We examined an oblique view of the microrecess along the interosseous ligament and an AP view of the lateral recess just under the distal end of the fibula. We also studied a control group of 27 patients with isolated injuries of the anterior talofibular ligament without rupture of the calcaneofibular ligament. The findings in the two groups were significantly different when examined for leakage to the ankle (p=0.0002), to the peroneal tendon sheaths (p=0.0347) and to the subcutaneous tissue (p=0.0222), absence of the microrecess (p=0.0055) and presence of the lateral recess (p=0.0012). Many ankle sprains which involve tearing of the calcaneofibular ligament are accompanied by injuries of the subtalar joint. Combined injuries of the anterior talofibular ligament and calcaneofibular ligament, and isolated injury of the anterior talofibular ligament should be differentiated.

Characterization of bacteriocin N15 produced by Enterococcus faecium N15 and cloning of the related genes.

Enterococcus faecium N15 was isolated from nuka (Japanese rice-bran paste), which is utilized as starter in the fermenting of vegetables, and was found to produce a bacteriocin that exhibited a broad spectrum of activity, including activity against Listeria monocytogenes and Bacillus circulans JCM2504. The bacteriocin was sensitive to proteases (alpha-chymotrypsin, proteinase K, trypsin, and pepsin) and alpha-amylase, but it was resistant to lipase. The bacteriocin was resistant to heat treatment at 100 degrees C for 2 h, but its activity was completely lost after autoclaving at 121 degrees C for 15 min. It was active over a wide pH range from 2.0 to 10.0. The bacteriocin showed bactericidal activity against Lactobacillus sake JCM1157 at a concentration of 40 AU/ml. Its molecular weight was estimated by SDS-PAGE to be about 3-5 kDa. PCR primers were designed based on the conserved amino acid sequences of class IIa bacteriocins. A 3-kb DNA fragment was amplified and three open reading frames (ORFs) were found. The first encodes a probable immunity protein of 103 amino acid residues and shows complete homology with the putative immunity protein of E. faecium DPC1146. The second and third ORFs respectively encode a probable transposase gene and an inducing factor. The upstream region of the immunity gene, in which the bacteriocin structural gene is located, was amplified. A homology search revealed that the bacteriocin produced by E. faecium N15 exhibits complete identity to enterocin A, a bacteriocin produced by E. faecium DPC1146. PCR using the primers designed in this study is a rapid and sufficient method of screening for bacteriocin-producing strains.

Inhibition of bacterial and glucan adherence to various light-cured fluoride-releasing restorative materials.

OBJECTIVES: This study investigated the potential plaque adhesion properties of various light-cured fluoride-releasing restorative materials by measuring the amount of adhering radiolabeled bacteria and glucan. METHODS: Three resin-modified glass ionomer cements (RMGI) and two polyacid-modified resin composites (compomer) were used in this study. As a control, one light-cured resin composite was added. Disk-shaped specimens were made following the manufacturers' recommendations and the respective surfaces were finished with a 600-grit abrasive paper. Streptococcus sobrinus B13 was selected as a cariogenic bacterial strain. The amount of bacteria and glucan adhered to these specimens were measured after 3, 8 and 24h incubations with radiolabeled cariogenic bacteria and sucrose. RESULTS: After 3 and 8h incubations, the amount of adhered bacteria and glucan was small and there were no significant differences among the restorative materials except in the resin composite. Although after 24h incubation the amounts of adhered bacteria and glucan, significantly increased on the RMGIs and compomers, these were still significantly less than the resin composite except one compomer. Although at 3h no good correlation was found between the contact angles and the amount of bacteria and glucans, the correlation coefficients were high at 8 or 24h. In addition, the coefficients for bacteria were always higher than those for glucan irrespective of the incubation times. CONCLUSIONS: After 24h resin-modified glass ionomer cements and compomers showed significantly smaller amounts of adhered bacteria and glucans compared to resin composite with an exception of glucan adherence on one compomer.