A case of combined laparoscopic reduction and open preperitoneal mesh repair for incarcerated small bowel in a retroperitoneal hernia between the external and internal iliac vessels.

We report a rare case of an incarcerated retroperitoneal hernia with or involving the small bowel through the orifice between the right external and internal iliac vessels. A 39-year-old woman was admitted to our hospital because of vomiting and abdominal pain. She had a history of right oophorocystectomy and appendectomy. Abdominal computed tomography revealed small bowel obstruction resulting from an incarcerated retroperitoneal hernia. The small bowel herniated into the retroperitoneal fossa through the orifice between the right external and internal iliac vessels. Laparoscopic reduction of the small bowel was performed, followed by ligation of the sac and placement of a mesh prosthesis through the preperitoneal approach, using a lower midline incision along the previous laparotomy scar. Her postoperative course was uneventful and no recurrence has been observed after surgery.

Arsenic speciation for the phytoremediation by the Chinese brake fern, Pteris vittata.

Arsenic (As) speciation for the phytoremediation by the Chinese brake fern was studied. In particular, the mechanism of how plants induce compounds containing thiol (SH) and proteins by As exposure in terms of the relationship between As and phosphate uptaken into plant cells was examined. Pteris vittata callus could efficiently reduce As(V) to As(III) by the rapid introduction of reductase and synthesize thiols leading to phytochelatins production. Furthermore, Pteris vittata could control phosphate concentration in the cells corresponding to the concentration of arsenite and arsenate. To our best knowledge, this is the first report to show the mechanisms of such high As tolerance of Pteris vittata using their callus in terms of in vitro approach for the analysis of As speciation and metabolism route.

Preservation of human cell bioassay device for the on-site evaluation of environmental waters.

We have already developed a novel disposable bioassay device based on the low-density lipoprotein (LDL) uptaking activity of human hepatoblastoma Hep G2 cells in our previous work. However, this device is not readily applicable to evaluate river water toxicity on-site because it cannot be preserved for more than one week. In this work, we developed the method for preservation of the device to enable it to be preserved for at least one month. The device can be supplied to individual environmental sites without any facilities for cell culture. We can evaluate river water toxicity by 2 hours of exposure after thawing. Therefore, this kind of device could be a promising tool for daily water quality management.

Cultured human-cell-based bioassay for environmental risk management.

Among bioassays for evaluating various impacts of chemicals on humans and ecosystems, those based on cultured mammalian-cells can best predict acute lethal toxicity to humans. We expect them to be employed in the future in environmental risk management alongside mutagenicity tests and endocrine-disrupting activity tests. We recently developed a disposable bioassay device that immobilizes human hepatocarcinoma cells in a small micropipette tip. This enables very quick (within 2 h) evaluation of acute lethal toxicity to humans. For bioassay-based environmental management, 2 promising approaches have been demonstrated by the US-EPA: toxicity identification evaluation (TIE) and toxicity reduction evaluation (TRE). The Japanese Ministry of Environment has been supporting a multi-center validation project, aimed at assembling a bioassay database. To make full use of these resources, we present a numerical model that describes contribution of individual chemical to observed toxicity. This will allow the selection of the most effective countermeasure to reduce the toxicity. Bioassay-based environmental risk management works retrospectively, whereas impact assessment using substance flow models and toxicity databases works prospective. We expect that these 2 approaches will exchange information, act complementarily, and work effectively in keeping our environment healthy in the 21 st century.

Development of a rapid and sensitive bioassay device using human cells immobilized in macroporous microcarriers for the on-site evaluation of environmental waters.

We developed a novel disposable bioassay device based on the fluorescein isothiocyanate-labelled low-density lipoprotein-uptake activity of human hepatoblastoma Hep G2 cells. The cells were cultured in porous microcarriers at a high cell density and packed in a filter tip that has a hydrophobic membrane. Upon evaluation of water samples, the culture medium was decanted by pipetting it down with a micropipet, and the samples were then introduced to the cell-immobilizing part of the tip only by pipetting them up after mixing them with x10 concentrated culture medium. The new device enabled us to detect almost the same toxicity levels of river water within 2 h of exposure as those detected by a conventional 48-h cell-survival assay. This is the first bioassay device for the rapid on-site evaluation of environmental waters using cultured human cells, and therefore promising for water-quality management based on risk to humans.

Evidence for a mate-attracting chemosignal in the dwarf African clawed frog Hymenochirus.

Many male frogs and toads possess sexually dimorphic skin glands (breeding glands). However, in most anuran species, the functional significance of the glands is unknown. Here we show that the breeding glands of male dwarf African clawed frogs (Hymenochirus sp. ) release a mate-attractant chemosignal. The mate-attractant activity was assessed using a two-choice aquatic Y-maze. Female Hymenochirus were allowed to choose between different treatment waters (e.g., plain water and water housing males) in the upstream arms of the maze, and the females' movements were monitored by computer-linked motion sensors. Females showed a positive chemotaxis to water housing males and to water containing homogenized breeding glands. Females showed no reaction to water housing conspecific females or to water housing breeding gland-ablated males. Additional choice tests demonstrated that females were more attracted to water housing males than to water housing females and to water containing homogenized breeding glands than to water housing breeding gland-ablated males. Males in the maze showed no response to water housing either females or other males, indicating that the attractant is specific for females and is therefore neither a species aggregation signal nor a food-related attractant. These results represent the first experimental demonstration of a mate-attractant function for anuran breeding glands. Because many anuran species possess breeding glands, these results suggest that pheromonal communication could be more widespread among frogs and toads than previously believed.

Rapid and sensitive neurotoxicity test based on the morphological changes of PC12 cells with simple computer-assisted image analysis.

In order to develop a rapid and sensitive bioassay for the screening of chemicals with possible neurotoxicity, a computer-assisted simple image-analysis system was developed to quantify small changes in the specific morphology of the cultured pheochromocytoma cell line, PC12. This cell forms a neuron-like microfibril network (neurites) in response to a nerve growth factor (NGF) stimulation in vitro. Dichrolvos (DDVP) and methylmercury chloride (MMC) were employed as model neurotoxicants. In DDVP treatment, there was no large difference in the ED50s (effective dose that reduces the morphological index by 50%) among the toxicities determined from various morphological indices, but they were significantly lower than those observed by whole-cell-area-based toxicity assay using the hepatoblastoma cell line, Hep G2. In contrast, in MMC treatment, neurite-length-based toxicity was observed as early as 2 h, and at 48 h this was lower by over three orders of magnitude compared with whole-cell-area-based one (2.06 x 10(-7) mM vs. 6.42 x 10(-4) mM). These results demonstrate that the developed bioassay using image analysis of nerve-tissue-derived cell morphology allows us to screen possible neurotoxic chemicals very rapidly with highly enhanced sensitivity, particularly for some chemicals that preferentially act on nerve fibers.

Preservation of microplate-attached human hepatoma cells and their use in cytotoxicity tests.

We investigated the feasibility of hypothermic- orcryogenically-preserved human hepatoma Hep G2 cell preculturedin 96-well plates in cytotoxicity testings. First, we observedthat microplates precoated with both collagen (CN) and pronectin (PN) showed significantly improved living cell adhesion (71.0 +/- 5.5%) after 48 hr of cryopreservation with 10%-DMSO containing culture medium, whereas non-coated surfaces gave very low living cell adhesion (33.5 +/- 2.1%). Hypothermic preservation was most suitable for short-term storage, and cryogenic preservation at -20 degrees C allowed cells to be used within a week of the storage period. Only cryopreservation in a deep freezer (-85 degrees C) gave satisfactory results in much longer period of storage. Second, we evaluated the cytotoxicity of ten chemicals during 48 hr of exposure using hypothermically - (4 degrees C for 2 days) or cryogenically - (-85 degrees C for 7 days) preserved cells cultured inCN/PN-precoated microplates in comparison with results fromfreshly inoculated cells. Although almost the same LD(50)values were obtained, LD(10) values of relatively hydrophilic chemicals obtained with cryopreserved cell were significantly lowered. These results shown that CN/PN-precoating is effective in keeping cells attached even in recultivation of preserved cells and that the toxicities of relatively hydrophilic chemicals tend to be overestimated when we use preserved cells in that manner.

[Aldehyde dehydrogenase 2(ALDH2) gene polymorphism in NIDDM patients with chronic renal failure].

In order to investigate the influence of aldehyde dehydrogenase 2(ALDH2) genotype in the pathogenesis of nephropathy due to non-insulin dependent diabetes mellitus (NIDDM), genotyping of ALDH2 was measured using the PCR-RFLP method in patients with NIDDM on chronic hemodialysis (HD). The results were as follows; 1) The frequency of active ALDH2 was 63% and that of inactive ALDH2 was 37%. 2) The percentage of active ALDH2 was significantly higher in patients with alcohol tolerance than that in those without it (38%). 3) The estimated amount of alcohol consumption in the past was 506 +/- 720 g/week in the active ALDH2 group, and 156 +/- 288 g/week in the inactive ALDH2 group, showing a significant difference between the two groups. 4) Interdialytic body weight gain was larger in patients with active ALDH2 than in those with inactive ALDH2. Since the frequency of active ALDH2 was similar to that in patients without nephropathy, these results do not support the hypothesis that ALDH2 gene polymorphism is involved in the development and persistence of chronic renal failure due to NIDDM. However, salt and water craving in dialysis patients may be influenced partially by an active ALDH2 gene.

Embryogenesis-promoting factors in rat serum.

Regarding whole rat embryo cultures in vitro, rat serum as a culture medium is known to support the normal growth of rat embryos in the organogenesis phase. The purpose of the present study was to isolate the embryogenesis-promoting factors from rat serum as a first step in the development of a defined serum-free medium for a whole embryo culture system. Pooled rat serum after heat inactivation was fractionated into three major peaks (frA, containing a region of void volume, frB, and frC) by gel filtration. The 9.5-day rat embryos that were cultivated for 48 hr in essential salt medium containing frB (with a molecular size range of 100-500 kDa) revealed normal growth. Three proteins (27 kDa, 76 kDa, and 190 kDa) that had the embryogenesis-promoting effects were isolated from 3-hr delayed centrifuged rat serum by the ion exchange chromatography. The 76-kDa protein was found to be rat transferrin by immunoblotting. The 27-kDa protein was identified as apo-AI (the major apoprotein of high-density lipoprotein) by immunoblotting. High-density lipoprotein obtained from pooled rat serum by a NaBr density gradient ultracentrifugation was found to have a positive effect on embryogenesis. The 10-kDa protein was also identified as alpha 1-inhibitor 3 by immunoblotting. In addition, the embryogenesis-promoting effect of the fraction containing 27-kDa and 190-kDa proteins declined within a short period of storage at -20 degrees C. This decrease was countered by supplementing its fraction (D-2) with albumin isolated from rat serum. These results in the present study suggest that transferrin, high-density lipoprotein, and alpha 1-inhibitor 3 in rat serum may be embryogenesis-promoting factors, and that albumin appeared to play a role in the embryogenesis of rat embryos in whole embryo cultures.

Cochlear histopathology of the mutant bustling mouse, BUS/Idr.

The inner ear of mutant bustling mice, BUS/Idr, was examined histopathologically. LM examinations revealed an age-dependent degeneration of the auditory organ of Corti in BUS homozygotes, but not heterozygotes. Cochlear base-to-apex gradient in severity of the degeneration was noted. First signs of degeneration were found in the outer hair cells of the cochlear basal turn at about 3 weeks of age, followed by degeneration of the spiral ganglion cells which occurred slowly. As examined by SEM, stereociliary derangements of both the inner and outer hair cells were apparent in homozygotes as early as after 10 days. No normal arrays of stereocilia were found in homozygotes examined at 10 days through 6 months. The results of immunohistochemical examinations suggest that the sensory cells of the Corti's organ of homozygotes are structurally once normally innervated. No significant difference was found in the expression of protooncogene c-mos in the CNS between BUS homozygotes and control mice. We propose that BUS mice be categorized as a member of the so-called "waltzer-shaker" mutants group.

Vestibulocochlear defects and effects of deuterium oxide in mutant bustling (BUS) mice.

Bustling mouse (BUS/Idr: bus) is a mutant mouse strain which exhibits bustling/hyperkinetic behavior and functional disorders related to the vestibulocochlear system such as rapid circling and loss of auditory startling response. In homozygous (bus/bus) mice this phenotype develops progressively after birth and has been shown from cross experiments to be inherited by a single autosomal recessive gene. Using light and electron microscopy, we examined the development of pathological changes in the inner ear of homozygous mice. Effects of deuterium oxide, which has been shown to influence vestibular function, on the abnormal behavior of homozygous mice of different ages were also examined. Pathological changes of the inner ear including impairment and/or loss of auditory hair cells, deformation and/or loss of the inner and outer tunnels of the organ of Corti, hypoplasia of the otoliths, and decrease in number of neurons of the spiral genglion were observed in the homozygous mice starting before the weaning stage and progressively thereafter, but not in the heterozygous mice. Although histological changes were also noted in the crista ampullaris and maculae, they were less evident than those in the cochlea at least until the mice were 6 weeks old. Administration of deuterated physiological saline (8-16 ml/kg, per os) tended to decrease the abnormal behavior of the homozygous mice, including rapid circling. These morphological and functional findings suggest that BUS mice may be a useful model for analysis of the pathogenesis of vestibulo-cochlear disorders.

Localization of nitric oxide-related substances in the peripheral nervous tissues.

Nitric oxide (NO) is now recognized as a transduction molecule in many biological systems, and is known to promote the synthesis of cGMP by activating the soluble guanylate cyclase. NO synthase which fully accounts for all the neuronal activity of NADPH diaphorase catalyzes L-arginine to NO and L-citrulline. In the present study, the localization of NO-related substances, L-arginine, NO synthase, L-citrulline and cGMP in the enteric plexus and dorsal root ganglia was demonstrated with immuno- or enzyme-histochemical methods. L-Arginine was proved accumulated in glial cells, while NO synthase and L-citrulline were found in neurons. Cyclic GMP was predominantly observed in glial cells. These results reveal L-arginine-NO-cGMP pathway may be present in the enteric plexus and dorsal root ganglion as in the brain, and provide visible evidence that NO mediates neuron-glia communications in this pathway.

Biochemical comparison of brain glycosaminoglycans between normal and reeler mutant mice.

Glycosaminoglycans (GAGs) were isolated from the brains of reeler and normal mice on postnatal days 13 and 20. The GAG content of the reeler mouse brain, based upon the amount of DNA, was about 150% that of the normal mouse brain on both days. The GAGs consisted of chondroitin sulfate (CS), heparan sulfate (HS), hyaluronic acid (HA) and polysialosyl glycopeptides. There was no significant difference in the composition of GAGs isolated from either reeler or normal brain. Repeating disaccharide compositions of CS and HS were also similar in reeler and normal brains. Core proteins of brain chondroitin sulfate proteoglycans (CSPGs), solubilized with phosphate buffered saline, were prepared by digesting purified CSPGs with chondroitinase ABC, and were analyzed by SDS-polyacrylamide slab gel electrophoresis. There was no difference in the composition of core proteins from either reeler or normal brain. These results indicate that, although the GAG content of the reeler mouse brain is higher than the normal, all structural parameters of GAGs/CSPGs so far examined were normal. The rate of synthesis and/or degradation of brain GAGs may be affected in the mutant mouse brain.

Defect of a fiber cell-specific 94-kDa protein in the lens of inherited microphthalmic mutant mouse Elo.

Deficiency in a 94,000-dalton protein in the non-crystallin fraction from the Elo mouse lens was shown. To perform further investigations, we raised an antibody against the 94,000-dalton protein isolated from normal mouse lens. Western blot analysis with the antibody indicated that the protein was only present in the lens and not in the brain, lung, heart, liver, and kidney. In the lens, it was unique to the cortex and nucleus fractions, not being present in the epithelial cells. Furthermore, it was observed in the water-soluble fraction as well as in the urea-soluble fraction. The antibody weakly but clearly reacted with the chick CP97 lens peptide, a fiber cell-specific protein, and anti-CP97 antibody also reacted with the 94,000-dalton protein. From these results, we concluded that the protein corresponds to CP97 cytoskeletal protein in the mouse lens. The protein was deficient in the lenses from Elo mice, but microphthalmic lenses from CTA mice contained a normal level.

Three distinct molecular species of proteoglycan synthesized by the rat limb bud at the prechondrogenic stage.

To characterize proteoglycans in the prechondrogenic limb bud, proteoglycans were extracted with 4 M guanidine HCl containing a detergent and protease inhibitors from Day 13 fetal rat limb buds which had been labeled with [35S]sulfate for 3 h in vitro. About 90% of 35S-labeled proteoglycans was solubilized under the conditions used. The proteoglycan preparation was separated by DEAE-Sephacel column chromatography into three peaks; peak I eluted at 0.45 M NaCl concentration, peak II at 0.52 M, and peak III at 1.4 M. Peaks I and III were identified as proteoglycans bearing heparan sulfate side chains. The heparan sulfate proteoglycan in peak III was larger in hydrodynamic size than the proteoglycan in peak I. The heparan sulfate side chains of peak III proteoglycan were smaller in the size and more abundant in N-sulfated glucosamine than those of peak I proteoglycan. Peak II contained a chondroitin sulfate proteoglycan with a core protein of a doublet of Mr 550,000 and 500,000. The chondroitin sulfate proteoglycan was easily solubilized with a physiological salt solution and the heparan sulfate proteoglycan in peak I was partially solubilized with the physiological salt solution. The remainder of the proteoglycan in peak I and the heparan sulfate proteoglycan in peak III could be solubilized effectively only with a solution containing a detergent, such as nonanoyl-N-methylglucamide. This observation indicates the difference in the localization among these three proteoglycans in the developing rat limb bud.

Dysplasia of subcommissural organ in congenital hydrocephalus spontaneously occurring in CWS/Idr rats.

The subcommissural organ (SCO) of the congenital hydrocephalus spontaneously occurring in CWS/Idr rats was severely reduced in size and displaced at some distance from the anterior end of the cerebral aqueduct. The cerebral aqueduct of the hydrocephalic rats was open throughout its total length during postnatal days 1-20, though it was somewhat narrower at its middle region than in the normal brain.

Developmental change in the amount of polysialosyl glycopeptides isolated from the rat brain.

Polysialosyl glycopeptides were coisolated with glycosaminoglycans by Pronase digestion of the whole brains of perinatal rats and could be separated from known glycosaminoglycans by two-dimensional electrophoresis on cellulose acetate film. The polysialosyl glycopeptides could not be obtained from fetal rat brain on day 13 of gestation, but began to be detected on day 14. The amount of polysialosyl glycopeptides was estimated from the dye concentration of the Alcian blue-stained spot in the electrophoretogram. The glycopeptide content increased almost linearly, on the basis of brain DNA, up to 10 days after birth. Thereafter, the content decreased rapidly, and hardly any polysialosyl glycopeptides could be isolated from the brain at approximately 30 days. This developmental change may be involved in morphogenesis and maturation of the brain. The polysialosyl glycopeptides could be isolated from the cerebellum, from the cerebrum, or from the brainstem of the neonatal rat. However, each region of the brain had a postnatal developmental change in glycopeptide content different from those of the other regions.

Absence of subcommissural organ in the cerebral aqueduct of congenital hydrocephalus spontaneously occurring in MT/HokIdr mice.

The midbrains of pups with congenital hydrocephalus spontaneously occurring in MT/HokIdr mice were histologically examined. The subcommissural organ (SCO) and the posterior commissure were completely absent in the hydrocephalic brain. The cerebral aqueduct in the hydrocephalic brain was never completely stenosed, though it was somewhat narrowed in its middle region as compared with that in the normal brain. A possible interrelationship between an absence of SCO and a cause of congenital hydrocephalus is discussed.