Influence of Trendelenburg position and pneumoperitoneum treatment on gastroesophageal reflux in dogs.

Gastroesophageal reflux is a latent factor that may cause esophagitis, esophageal stenosis, and aspiration pneumonia through the regurgitation of the gastric fluid contents. For laparoscopic surgery, posture-changing and pneumoperitoneum operations are conducted to develop the visual field. However, few studies have examined the influence of these operations on gastroesophageal reflux. In this experiment using 10 Beagles, 10 mL of contrast medium was administered into the stomach, and the dogs were placed in the Trendelenburg position with 10-degree tilting. Pneumoperitoneum treatment with carbon dioxide was performed, with an intraperitoneal pressure of 10 mmHg. The presence or absence of gastroesophageal reflux was evaluated using computed tomography (CT). In horizontal and Trendelenburg positions, there was no reflux of Contrast medium. However, reflux was observed in the Trendelenburg position under pneumoperitoneum (p<0.05). These results suggest that the risk of gastroesophageal reflux increases during laparoscopic surgery in the Trendelenburg position with 10-degree tilting under an intraperitoneal pressure of 10 mmHg.

Development of new glass composite membranes and their properties for low temperature H2/O2 fuel cells.

A new glass electrolyte formed by constant amounts of titanium oxide (TiO2) and various amount of phosphotungstic acid (PWA) doped P2O5-SiO2 is prepared using the sol-gel process. The structural formation is confirmed by Fourier infrared spectroscopy (FTIR) and from thermogravimetric and differential thermal analysis (TG/DTA) measurements, the glasses display good thermal stability. Further characterisation is undertaken by N2 adsorption/desorption measurements, proton conductivity and hydrogen permeability analyses and a H2/O2 fuel cell test is also performed. The glass materials with large pores and specific surface area are suitable for use as the electrolyte in H2/O2 fuel cells. The effect of TiO2 processing with constant amount of PWA in phosphosilicate glasses, is investigated and discussed. The hydrogen permeability is 1.57x10(-11) mol cm(-1) s(-1) Pa(-1) at 110 degrees C for 0.8 mm thick glass; a power density of 46.3 mW cm(-2) at 125 mA cm(-2) and a current density of 175 mA cm(-2) is obtained (T=28 degrees C, relative humidity).

Carbonate-containing hydroxyapatite derived from calcium tripolyphosphate gel with urea.

Carbonate containing hydroxyapatite (CO3HAp) is one of the candidate materials as a bioresorbable bone substitute. In the present work, CO3HAp was efficiently prepared by a hydrothermal treatment of calcium tripolyphosphate gel with urea at 140 degrees C for 24 h. Chemical potential plots of the CO3HAp for estimation of its dissolution behavior suggested that the CO3HAp is more soluble than hydroxyapatite (HAp) and is as soluble as octacalcium phosphate (OCP) and/or beta -tricalcium phosphate (TCP). This material is expected to be applied to bioresorbable materials such as bone fillers.

Immunohistochemistry of atrial natriuretic peptide in brain infarction.

Atrial natriuretic peptide (ANP) was originally isolated from cardiac atria, and has potent natriuretic, diuretic, and vasorelaxant properties. It has been localized in neurons and astrocytes in the cerebral cortex and the white matter. We hypothesize that glial ANP may contribute to the regulation of cerebral blood flow in brain infarction. In order to elucidate this possible role, the immunohistochemistry of ANP was studied in cases of brain infarction and in other cases of brain trauma for comparison. A statistically significant increase in the number of ANP-immunoreactive glial cells (mainly astrocytes) was observed in the white matter surrounding the brain infarction compared with the intact area. No statistically significant increase in ANP-immunoreactive glial cell number was observed in the cerebral white matter from brain haemorrhage, contusion and control cases. Our results indicate that glial ANP may increase in number in brain infarction, and that it may be involved in the regulation of the cerebral blood flow in the infarcted area.

Genomic anatomy of a premier major histocompatibility complex paralogous region on chromosome 1q21-q22.

Human chromosomes 1q21-q25, 6p21.3-22.2, 9q33-q34, and 19p13.1-p13.4 carry clusters of paralogous loci, to date best defined by the flagship 6p MHC region. They have presumably been created by two rounds of large-scale genomic duplications around the time of vertebrate emergence. Phylogenetically, the 1q21-25 region seems most closely related to the 6p21.3 MHC region, as it is only the MHC paralogous region that includes bona fide MHC class I genes, the CD1 and MR1 loci. Here, to clarify the genomic structure of this model MHC paralogous region as well as to gain insight into the evolutionary dynamics of the entire quadriplication process, a detailed analysis of a critical 1.7 megabase (Mb) region was performed. To this end, a composite, deep, YAC, BAC, and PAC contig encompassing all five CD1 genes and linking the centromeric +P5 locus to the telomeric KRTC7 locus was constructed. Within this contig a 1.1-Mb BAC and PAC core segment joining CD1D to FCER1A was fully sequenced and thoroughly analyzed. This led to the mapping of a total of 41 genes (12 expressed genes, 12 possibly expressed genes, and 17 pseudogenes), among which 31 were novel. The latter include 20 olfactory receptor (OR) genes, 9 of which are potentially expressed. Importantly, CD1, SPTA1, OR, and FCERIA belong to multigene families, which have paralogues in the other three regions. Furthermore, it is noteworthy that 12 of the 13 expressed genes in the 1q21-q22 region around the CD1 loci are immunologically relevant. In addition to CD1A-E, these include SPTA1, MNDA, IFI-16, AIM2, BL1A, FY and FCERIA. This functional convergence of structurally unrelated genes is reminiscent of the 6p MHC region, and perhaps represents the emergence of yet another antigen presentation gene cluster, in this case dedicated to lipid/glycolipid antigens rather than antigen-derived peptides.

Calcium phosphate invert glass-ceramic coatings joined by self-development of compositionally gradient layers on a titanium alloy.

A glass-ceramic layer containing beta-Ca3(PO4)2 crystals could be joined easily with a new type of titanium alloy (Ti-29Nb-13Ta-4.6Zr) consisting of a beta-titanium phase by heating the metal, on which glass powders with a composition of 60CaO x 30P2O5 7Na2O x 3TiO2 were placed, at 800 degrees C in air. Measurement of tensile bonding strength revealed that the joining between the coating layer and the substrate is very strong. Even after the large deformation (e.g., approximately 90 degrees in bending angle) of the titanium alloy, the coating layer was not peeled off from the substrate. A compositionally gradient layer in the TiO2-P2O5-Na2O-CaO system is developed automatically on the titanium alloy during the heating, resulting in the formation of the strong joining. By soaking in simulated body fluid at 37 degrees C, hydroxyapatite phase was formed newly on the surface of the coating layer.

Preparation and mechanical properties of polylactic acid composites containing hydroxyapatite fibers.

Ceramic-polymer composite biomaterials were prepared by hot-pressing a mixture consisting of poly-L-lactic acid (PLA) and hydroxyapatite fibers (HAF) with dimensions of 40-150 microm length and 2-10 microm diameter, which were converted from beta-Ca(PO3)2 fibers. After PLA dissolved with methylene chloride was mixed with the fibers, the mixture was dried completely and subsequently hot-pressed uniaxially under a pressure of 40 MPa at 180 degrees C, resulting in the fabrication of the PLA/HAF composite. The modulus of elasticity was improved effectively even by introducing a small amount of HAF; almost no degradation in the bending strength was observed and the modulus of elasticity showed high values of 5-10 GPa when the fibers of 20-60 wt% were introduced. With increasing HAF content, the maximum strain decreases and the specimen is apt to show a brittle fracture; this result implies that HAF in the composites can share the applied load efficiently due to the formation of a bond between HAF and PLA.

Optical bistability of stimulated-emission lines in Sm(3+)-doped glass microspheres.

A third-order optical nonlinear effect in a spherical glass resonator doped with Sm(3+) ions has been observed on stimulated emission lines of the (4)G(5/2)-(6)H(7/2) transition, in which the excitation beam of an Ar(+) laser in a 488-nm single line is coupled with the glass resonator through a prism. A spherical form is produced during a free fall of fractional glasses in a longitudinal electric furnace configured vertically. The prism-coupled glass microsphere exhibits sufficient performance to confine photon energy as a resonance of light inside the sphere and offers the prospect of new applications to functional photonic circuits.

A glucose biosensor based on electrodeposited biocomposites of gold nanoparticles and glucose oxidase enzyme.

Electrodeposition was used for the codeposition of glucose oxidase enzyme and a gold nanoparticle-silicate network onto an indium tin oxide (ITO) glass electrode. This co-entrapment of glucose oxidase enzyme in a gold nanoparticle-silicate network imparts biocatalytic activity to the film. The gold nanoparticles in the network catalyse the oxidation and reduction of H2O2, the by-product of the enzymatic reaction. The low operating potential of the sensor eliminates the interference from common interferents, such as acetaminophen, ascorbic acid, dopamine, etc.

An immunohistochemical study on cathepsin D in human hippocampus.

Cathepsin D is a lysosomal enzyme involved in neuronal degeneration. In this study, the immunohistochemistry of cathepsin D was studied in hippocampal CA1 neurons that are vulnerable to ischemia, and parahippocampal glial cells. CA1 neurons from the majority of cases showed cathepsin D immunoreactivity in the cytoplasm, whereas shrunk neurons were unstained in only one case. There was no statistically significant correlation between the postmortem interval between death and autopsy, and cathepsin D immunoreactivity in CA1 neurons. These observations indicate that cathepsin D immunoreactivity is not a sensitive marker for neuronal degeneration or postmortem changes. On the other hand, there was a statistically significant correlation between age and cathepsin D immunoreactivity in the cytoplasm of parahippocampal glial cells. This shows that senescence is correlated with cathepsin D expression in humans as has been reported previously in an animal study.

Newly identified repeat sequences, derived from human chromosome 21qter, are also localized in the subtelomeric region of particular chromosomes and 2q13, and are conserved in the chimpanzee genome.

Subtelomeric regions have been a target of structural and functional studies of human chromosomes. Markers having a defined structure are especially useful to such studies. Here, we report 93 bp tandem repeat sequences found in the subtelomeric region of human chromosome 21q. They were also detected in the telomeric region of several other chromosomes. Interestingly, the repeat was also found in the 2q13 region which is known to be a position of chromosomal fusion, a major difference between the human and chimpanzee karyotypes. To the best of our knowledge, this repetitive sequence is a new member of human subtelomeric interspersed repeats.

Intranuclear arrangement of human chromosome 12 correlates to large-scale replication domains.

The intranuclear arrangement of human chromosome 12 in G0(G1) nuclei from human myeloid leukemia HL60 cells was analyzed by multicolor fluorescence in situ hybridization (FISH) using band-specific cosmid clones as probes. Pairs of differently colored cosmids were detected on paraformaldehyde-fixed HL60 nuclei, and their relative positions, internal or peripheral, in individual nuclei were scored. Our results suggest that the intranuclear arrangement of human chromosome 12 is not random. Some chromosomal domains, including the centromere, were located in the periphery of the nucleus, while other domains, including the telomeres, were positioned in the internal areas of the nucleus in GO(G1) cells. Based on the replication banding patterns of metaphase spreads, human chromosome 12 was divided roughly into five large domains. Interestingly, the clones in late replicating domains were preferentially localized in the nuclear periphery, whereas clones in early replicating domains were arranged in the internal areas of the nuclei. The DNA replication timing of each cosmid determined by FISH-based assay did not reflect the replication bands, but an overall profile of the replication timing was relatively correlated with these domains on chromosome 12. These results suggest that the intranuclear arrangement of a human chromosome is correlated with the large-scale replication domains, even before DNA replication.

Relative locations of the centromere and imprinted SNRPN gene within chromosome 15 territories during the cell cycle in HL60 cells.

Investigations of imprinted regions provide clues that increase our understanding of the regulation of gene functions at higher order chromosomal domains. Here, the relative positions of the chromosome 15 centromere and the imprinted SNRPN gene in interphase nuclei of human myeloid leukemia HL60 cells were compared, because the homologous association of this imprinted chromosomal domain was previously observed in lymphocytes and lymphoblasts. Four targets including the chromosome 15 territory, its centromere, the SNRPN gene on this chromosome, and the nucleus, were visualized simultaneously in three-dimensionally preserved nuclei using multicolor fluorescence in situ hybridization, and the spatial distributions of these probes were analyzed with a cooled CCD camera deconvolution system. We found that preferential association of SNRPN interhomologues did not occur during the cell cycle in HL60 cells, although this gene exhibited asynchronous replication and monoallelic expression in this cells. SNRPN was found to localize at the periphery of the chromosome territories, and it preferentially faced the nuclear membrane, unlike the adjacent centromeric repeat. The SNRPN gene and the centromere were located close to each other late in S phase, reflecting that these DNA segments may be compacted into the same intranuclear subcompartments with the progress of S phase and in course of preparation for the following G(2) phase. Our results suggest that, although an imprinted gene has features similar to those observed with intranuclear localization of other gene coding sequences, the characteristic of mutual recognition of imprinted regions is determined by certain cellular regulation, and it is not necessary for the allele-specific features of an imprinted gene.

Surface modification of calcium metaphosphate fibers.

beta-calcium metaphosphate fibers having high aspect ratios of 10-120 with diameters of 2-10 microm show high strength and good biocompatibility. When the fibers are soaked in simulated body fluid at 37 degrees C, however, no calcium phosphate phase is newly formed on the fibers. In the present work, by treating the fibers at 70 degrees C with dilute NaOH aqueous solution, the surface phase was converted successfully into the orthophosphate phase that was in fine sizes and was adhered. After soaking the treated fibers in simulated body fluid at 37 degrees C for 30 days, a new calcium phosphate phase was precipitated. This was attributed to the surface phase modified using dilute NaOH. The treated fibers are expected to show bone-bonding ability, i.e. bioactivity.

Immunohistochemical localization of P-selectin in the glomeruli from forensic autopsies.

The expression of the adhesion molecule P-selectin is known to be up-regulated in several vital organs including the kidney after trauma in experimental animals. We examined the expression of P-selectin in the kidney by immunohistochemistry in 41 forensic autopsies mainly from trauma cases. P-selectin immunoreactivity was present in the glomerular capillary endothelial tufts and cortical interstitial vascular endothelial cells. The P-selectin immunoreactivity in the glomeruli was not co-localized with CD41 (platelet marker) immunoreactivity. The antemortem interval between the onset of injury and death (AMI) was statistically significantly longer in the cases with more P-selectin-positive capillary endothelial tufts in the glomeruli. Our results show that P-selectin immunoreactivity exists in the glomerular capillary endothelial cells rather than platelets. Our results also indicate that the P-selectin expression increases in the glomerular endothelial cells of the human kidney with the longer duration of the state under injury.

Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation.

Synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a signaling phospholipid, is primarily carried out by phosphatidylinositol 4-phosphate 5-kinase [PI(4)P5K], which has been reported to be regulated by RhoA and Rac1. Unexpectedly, we find that the GTPgammaS-dependent activator of PI(4)P5Kalpha is the small G protein ADP-ribosylation factor (ARF) and that the activation strictly requires phosphatidic acid, the product of phospholipase D (PLD). In vivo, ARF6, but not ARF1 or ARF5, spatially coincides with PI(4)P5Kalpha. This colocalization occurs in ruffling membranes formed upon AIF4 and EGF stimulation and is blocked by dominant-negative ARF6. PLD2 similarly translocates to the ruffles, as does the PH domain of phospholipase Cdelta1, indicating locally elevated PI(4,5)P2. Thus, PI(4)P5Kalpha is a downstream effector of ARF6 and when ARF6 is activated by agonist stimulation, it triggers recruitment of a diverse but interactive set of signaling molecules into sites of active cytoskeletal and membrane rearrangement.

Immunohistochemical localization of c-fos in the nuclei of the medulla oblongata in relation to asphyxia.

The immediately early gene product c-fos is known to be induced in neurons under noxious stimuli. Therefore, the immunohistochemistry of c-fos expression in human brains might offer information on the localization of stimulated neurons. In this study, the immunohistochemical localization of c-fos was studied in the neurons of the hypoglossal nucleus (XII), the dorsal motor nucleus of the vagal nerve (X), the nucleus solitarius (Sol), the accessory cuneate nucleus (Cun), the spinal trigeminal nucleus (V) and the inferior olive (Oli) of the human medulla oblongata from forensic autopsy cases. The neurons in the X nucleus showed the highest percentage of positive reactions for c-fos, followed in descending order by the Cun, V, Oli, XII and Sol. The c-fos immunoreactivity in the Cun and X was statistically significantly higher than in the Sol, XII and Oli. Although neurons in the Sol are known to be involved in respiration, there was no statistically significant difference in the c-fos immunoreactivity in the neurons in the Sol between asphyxia and non-asphyxia cases. On the other hand, the percentage of neurons positive for the c-fos immunoreactivity was statistically significantly higher in the Oli of asphyxia cases than of non-asphyxia cases. Our results indicate the difference in the immunoreactivity of c-fos among the nuclei of the human medulla oblongata and that the c-fos immunoreactivity in the Oli might assist the diagnosis of asphyxia.

A novel sperm-specific hypomethylation sequence is a demethylation hotspot in human hepatocellular carcinomas.

Certain human DNA regions are strikingly undermethylated at CpG sites in sperm compared to adult somatic tissues. These sperm-specific hypomethylation sequences are thought to function early in embryogenesis or gametogenesis. By using the restriction landmark genomic scanning (RLGS) cloning method, we have isolated a novel sperm-specific hypomethylation sequence, the status of which changes during spermatogenesis, embryonal growth and differentiation. This sequence is a part of a new 'NotI repeat' consisting of a 1.4 kb repetitive unit sequence named DE-1. The sequence is GC-rich and has high homology to a CpG DNA clone that was isolated by a methyl CpG protein binding column, indicating that it was normally highly methylated. We investigated the methylation status of this sequence. In the normal genome the sequence was methylated, but in the human hepatocellular carcinoma (HCC) genome, the target sequence was demethylated at the cytosine residue of the CpG dinucleotides with high frequency (75% in the previous study). These data suggest that this regional DNA hypomethylation may play a role in both cell differentiation and hepatocarcinogenesis.

Isolation of NotI clusters hypomethylated in HBV-integrated hepatocellular carcinomas by two-dimensional electrophoresis.

To examine genetic and epigenetic alterations associated with HBV integration in hepatocarcinogenesis, we compared genomic DNA profiles of primary hepatocellular carcinomas (HCCs) and cell lines that either contained or did not contain integrated HBV. To accomplish this, we carried out Restriction Landmark Genomic Scanning (RLGS), a two-dimensional system that displays 2000-3000 Not I landmark sites in a single gel electrophoresis experiment. We identified one Not I landmark spot that showed high signal intensity in HBV-integrated cell lines or in primary HCCs, but not in HCCs or tumor-cell lines free of HBV integration. Cloning of this spot revealed that it consisted of a Not I cluster sequence enriched with CpG dinucleotides. This sequence, hypomethylated in association with HBV integration, was found in the peri-centromeric region of human acrochromosomes. The results demonstrate that epigenetic changes at specific sequences in the genome occur in association with HBV integration during the process of hepatocarcinogenesis.