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Long-term diabetic patients exhibit major clinical gastrointestinal problems, such as diarrhea and constipation. In recent years, water channel protein, aquaporin 1 (AQP1) has been identified in the enteric nervous system (ENS). We have examined the pathological changes in AQP1 immunoreactive (IR) neurons in streptozotocin-induced (STZ) diabetic rats. Eight-week-old Wistar rats were injected with streptozotocin, and artificial diabetes was induced. Sixteen-week-old STZ rats were then examined with double immunofluorescence staining and ABC immunohistochemical staining. AQP1-IR neurons in STZ rats were significantly increased compared with control rats (p

Most neurons in the central nervous system and peripheral nervous system do not express water transporting protein, aquaporin (AQP). In the present study, we have demonstrated the presence of AQP1 immunoreactivity in a particular neuronal subtype in the enteric nervous system (ENS) of the rat ileum. AQP1-immunoreactive (IR) neurons simultaneously expressed a neuronal marker HuC/D. Moderate numbers of AQP1-IR neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. AQP1-IR neurons can be classified as Dogiel type I cells, which have several short processes and a single long process. Many AQP1-IR fibers were found both in the myenteric and submucosal plexi. Many AQP1-IR varicose fibers were closely associated with neuronal somata in the ganglia, whereas other AQP1-IR fibers penetrated into the muscle layers. These results suggest that AQP1-IR neurons probably play a significant role within the ENS to control gut functions.

Background/Aims Nucleic acid damage by reactive nitrogen and oxygen species may contribute to inflammation-related carcinogenesis. To investigate the extent of nucleic acid damage in hepatitis C virus infection and its change after interferon treatment, we measured 8-nitroguanine and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the liver of patients with chronic hepatitis C (CHC) before and after interferon therapy. Methods Hepatic accumulation of 8-nitroguanine and 8-OHdG was immunohistochemically evaluated in 20 CHC patients and 7 control patients with non-alcoholic fatty liver. Results Immunoreactivities of 8-nitroguanine and 8-OHdG were strongly detected in the liver from patients with CHC, but not in control livers. 8-Nitroguanine accumulation was found not only in infiltrating inflammatory cells, but also hepatocytes particularly in the periportal area. The accumulation of 8-nitroguanine and 8-OHdG increased with inflammatory grade (8-nitroguanine; P =0.0019, 8-OHdG; P =0.0009). In the sustained virological responder group after interferon therapy, 8-nitroguanine and 8-OHdG accumulation were markedly decreased in the liver (8-nitroguanine; P =0.018, 8-OHdG; P =0.018). Conclusions In this study, we demonstrated for the first time that 8-nitroguanine accumulated in the liver of patients with CHC. 8-Nitroguanine is a useful biomarker to evaluate the severity of HCV-induced chronic inflammation in relation to hepatocellular carcinoma.

Inflammation mediated by infection is an important factor causing carcinogenesis. Opisthorchis viverrini (OV) infection is a risk factor of cholangiocarcinoma (CHCA), probably through chronic inflammation. Formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) were assessed in the liver of hamsters infected with OV. We newly produced specific anti-8-nitroguanine antibody without cross-reaction. Double immunofluorescence staining revealed that 8-oxodG and 8-nitroguanine were formed mainly in the same inflammatory cells and epithelium of bile ducts from day 7 and showed the strongest immunoreactivity on days 21 and 30, respectively. It is noteworthy that 8-oxodG and 8-nitroguanine still remained in epithelium of bile ducts on day 180, although amount of alanine aminotransferase activity returned to normal level. A time course of 8-nitroguanine was associated with iNOS expression. Furthermore, this study demonstrated that HO-1 expression and subsequent iron accumulation may be involved in enhancement of oxidative DNA damage in epithelium of small bile ducts. In conclusion, nitrative and oxidative DNA damage via iNOS expression in hamsters infected with OV may participate in CHCA carcinogenesis.

Carbon monoxide (CO), an activator of soluble guanylate cyclase (SGC) and generated enzymatically by heme oxygenases (HO), is considered to function as an intra- and intercellular neuromodulator or neurotransmitter in the central and peripheral nervous systems. HO-2 is the constitutive isoform of HO and is more prevalent in nervous tissues than in the other peripheral tissues. Because previous studies have demonstrated different distributions of HO-2 in the retina depending on the species of animals, the aim of this study was to identify which cell types of the monkey retina express HO-2. The expression of HO-2 protein was examined in monkey retina by Western blot analysis. Immunoblottings from monkey homogenates revealed a single clear protein band with a molecular mass of 36 kDa that is corresponding to rat HO-2. Immunoreactivity of HO-2 was found in the perikarya of ganglion cells. Density of immunoreactive ganglion cells was higher in the central area of retina than in the peripheral retina, and somata of larger ganglion cells were stained more densely than smaller ones. In electron microscopy, immunoreactivity of HO-2 was localized on the membrane of the endoplasmic reticulum and the nuclear outer membrane of the ganglion cells. By contrast, inner plexiform layer, inner nuclear layer and outer nuclear layer were devoid of HO-2 immunoreactivity. cGMP were strongly localized in all of ganglion cells. Some cells contributed to the relatively faint cGMP staining were seen in the inner nuclear layer. In combination of HO-2 and cGMP immunocytochemistry, the overlap of co-localization of HO-2 and cGMP would suggest that HO-2 in the ganglion cells would serve as a source for CO generation and CO could serve as a gaseous signaling molecule modulator of neural activity in the retina of monkey.

Helicobacter pylori infection causes chronic inflammation, which can lead to gastric carcinoma. A double immunofluorescence labeling study demonstrated that the level of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) apparent in gastric gland epithelium was significantly higher in gastritis patients with H. pylori infection than in those without infection. A significant accumulation of proliferating cell nuclear antigen, a prognostic factor for gastric cancer, was observed in gastric gland epithelial cells in patients with H. pylori infection as compared to those without infection, and its accumulation was closely correlated with the formation of 8-nitroguanine and 8-oxodG. These results suggest that nitrosative and oxidative DNA damage in gastric epithelial cells and their proliferation by H. pylori infection may lead to gastric carcinoma. 8-Nitroguanine could be not only a promising biomarker for inflammation but also a useful indicator of the risk of gastric cancer development in response to chronic H. pylori infection.

The Goto-Kakizaki (GK) rat is a spontaneous model of non-insulin-dependent diabetes mellitus without obesity and diabetic retinopathy. We examined the retinal distribution of L-glutamate, gamma aminobutyric acid (GABA), glycine, and L-aspartate as neurotransmitters in the GK rat retina, using an immunohistochemical method with high-affinity antibodies. The retinal structures in the GK rats were the same as the controls. However, in the GK rats, immunoreactivity of L-glutamate and GABA was observed in the Müller and photoreceptor cells in addition to the immunoreactivity in normal rats. There was no change in glycine distribution between GK rats and controls. In the GK rats, L-aspartate accumulated in the inner segment of the photoreceptor cells in addition to the normal distribution. We consider that these immunoreactivity patterns in the GK rat retina might be induced by ischemia associated with diabetes mellitus.

Components of the multidrug resistance-associated protein (mrp) family mediate the adenosine triphosphate (ATP)-dependent transport of conjugated organic anions in the liver. Of these, mrp1 and mrp2 have been shown to have similar substrate specificity and nucleotide sequence. The intracellular localization and distribution of mrp1 under normal condition and cholestasis have not been as yet completely elucidated. To clarify this point, in the present study we evaluated the intracellular localization of mrp1 in rat liver and kidney after bile duct ligation (BDL). Bile duct was ligated in Wistar rats. Sequential staining of mrp1 by immunofluorescence was carried out in rat liver and kidneys 1, 3, and 5 days after bile duct ligation using confocal laser scanning microscopy. Weak granular staining of mrp1 was observed in cytoplasm of control rat hepatocytes. In addition to increased cytoplasm staining of mrp1, belt-and granule-like staining of mrp1 in basolateral membrane of hepatocytes was also shown after BDL. Furthermore, mrp1 immunofluorescence increased over time after BDL. No specific immunoflurescence of mrp1 was detected in control rat kidney. However, mrp1-positive staining was observed in epithelia of some renal tubules after BDL. This study showed that mrp1 immunofluorescence increased in hepatocyte basolateral membrane and cytoplasm and epithelia of some renal tubules after BDL. This increased mrp1 expression may be an adaptive response to impairment of hepato-biliary organic anion transport during obstructive cholestasis.

The congenital aganglionosis rat is considered to be an animal model of Hirschsprung's disease. The mutants had a long constricted segment (from distal ileum to rectum) below the dilated distal ileum. In the dilated region, synaptophysin-immunoreactivity (IR) was almost preserved in all layers of the intestinal wall. In the constricted distal ileum and oral proximal colon, synaptophysin-IR was scarce in all layers, including the circular and longitudinal muscle layers. In the anal proximal and distal colon, synaptophysin-IR was almost scarce in the circular muscle layer (CML). An ultrastructural study confirmed that almost no terminals were found in the CML of any regions of constricted intestine. Therefore, the CML in any region of a constricted segment, is presumed to be poor innervation. However, a few synaptophysin-IR were found in the longitudinal muscle layer (LML) of an anal part of a constricted segment. An ultrastructural study also confirmed that some terminals were observed in the LML of this segment. The present study suggested that denervated CML is related to the production of constricted segment, irrespective of the presence or absence of terminals in the LML.

L-Arginine is a precursor of nitric oxide (NO) that may be involved in neuronal activity in the gastrointestinal tract. It is known that NO is formed from L-arginine by NO synthase which is localized in neurons in the enteric nervous system. The present study demonstrated that significant L-arginine immunoreactivity was present in the enteric ganglia. Ultrastructural examination showed that L-arginine immunoreactivity was present in the ganglionic glial cells but not in neurons. These findings suggest that enteric glial cells may represent the main reservoir of L-arginine, which may possibly be transferred to neurons when used.

Recent studies have shown that carbon monoxide (CO) may function as a gaseous signaling molecule in a similar way to nitric oxide. In the gastrointestinal tract, immunoreactivity against a CO-producing enzyme, heme oxygenase-2 (HO-2), was reported in epithelial cells and neurons of submucosal and myenteric plexus. However, details of the epithelial cells in the gastric mucosa remain unknown. The aim of this study was to clarify if mRNA for HO-2 is expressed in the rat stomach, if HO-2 protein is present in the mucosa, and to define the cell types of the HO-2-immunoreactive cells. HO-2 mRNA and protein were detected in fundic and pyloric mucosa of rat stomach using an RNA protection assay and western blot analysis. Immunohistochemical study showed that HO-2 was localized in parietal cells of the fundic glands and gastrin cells of the pyloric glands of both rat and monkey. The results suggest that HO-2 enzyme is produced in the gastric mucosa, and that CO is released from parietal cells and gastrin cells.

An investigation was made of age-related changes in levels of the beta-subunit of nerve growth factor (beta-NGF) in selected tissues and of testosterone in serum in senescence-accelerated mice (SAM-P/8) and in the control mice (senesence-resistant mice; SAM-R/1). The concentrations of testosterone in serum were higher in SAM-P/8 than in SAM-R/1 at ages 2 and 4 mo. The level of beta-NGF in the thymus from SAM-R/1 increased with age, resulting in a statistically significant difference in its level between mice at ages 2 and 12 mo. By contrast, there was a transient increase in SAM-P/8 at around age 4 mo with a subsequent decrease. Consequently, significant differences were apparent in levels of beta-NGF between the two types of mouse at ages 2 and 4 mo. Similar results were obtained in the adrenal gland and testis. Compared to SAM-R/1 at age 2 mo, the average concentrations of beta-NGF in the hypophysis were higher in SAM-R/1 at ages 4 and 8 mo and in SAM-P/8 at all ages. In other tissues tested, no remarkable differences were detected. Our present results indicate that, in SAM-P/8, the elevation in levels of beta-NGF in the thymus, adrenal gland, testis, and hypophysis occurs in the early period of life compared to the control mice. Possible dysfunction of the disorder of hypophysis is discussed.

Levels of neurotrophin-3 markedly decrease in the rat cerebellum after the first 10 days of life, suggesting an importance during early development. To further examine the effect of neurotrophin-3 on the developing cerebellum, we injected a monoclonal antibody against neurotrophin-3 into the lateral ventricle of 7.5-day-old rats. The resultant depletion of neurotrophin-3 caused a significant decrease in cerebellar wet weights noted at 7 and 23 days thereafter. Other changes noted 48 h after injection of monoclonal antibodies against neurotrophin-3 included reduced incorporation of bromodeoxyuridine into granule neurons in the external germinal layer, an elevated density of atrophic neurons that had just migrated under the Purkinje cell layer, and an increased number of apoptotic neurons in the internal granule cell layer. These changes were limited to the central lobe. The concentration of neurotrophin-3 protein in the posterior region, including the central lobe, was about four- and threefold higher than that in the anterior region of the cerebellum of 9.5- and 30-day-old rats, respectively. Immunocytochemical examination showed higher amounts of neurotrophin-3 protein in the central lobe than in the anterior lobe. Our results provide evidence that neurotrophin-3 regulates the proliferation of granule precursors and supports the survival of mature granule neurons in restricted lobules, suggesting an involvement in limited regions at a specific stage in development of the rat cerebellum.

For the quantitative analysis of vitamin D-dependent 28-kDa calcium-binding protein (calbindin-D) in the CNS, we have established a highly sensitive immunoassay method. The antisera were raised in rabbits with purified calbindin-D from rat kidneys, and the antibodies were purified with a calbindin-D-coupled Sepharose column. The purified antibodies were specific for calbindin-D, showing a single band on the immunoblot with the extract of rat kidney or cerebellum. The sandwich-type immunoassay system was prepared by the use of purified monospecific antibodies, and the minimum detection limit of the assay was 0.1 pg or 3.6 amol of calbindin-D, which was sufficiently sensitive for the measurement of calbindin-D content in isolated Purkinje cell bodies at the level of single cells. The average content of calbindin-D in a single Purkinje cell was 0.05 pg. Calbindin-D was detected in most of the rat tissues examined, but it was present predominantly in the kidney and CNS, especially in the cerebellum. Calbindin-D was detected at a similarly low level in the cerebral cortex, cerebellum, and brainstem of rat embryos of 15 gestational days, and it increased gradually but differently in these regions, reaching the respective adult levels by 4–5 weeks of postnatal age. In contrast, kidney calbindin-D increased sharply between 15 gestational days and 3 postnatal days, reaching the adult level by 6 days of age. Calbindin-D levels in the adult rat CNS were affected little by age, whereas the concentrations in human cerebral cortices were significantly low in the aged brain as compared with those in the young brain. However, the concentrations in various regions of cerebral cortex from patients with Alzheimer's disease showed values similar to those in the relevant regions of the age-matched control patients.

Purpose: To evaluate histopathologic retinal changes in rabbit eyes after injection of pure perfluoropropane (C3F8) gas, pure sulfur hexafluoride (SF6) gas, or air into the vitreous cavity. Methods: Air, C3F8 gas, or SF6 gas (0.4 mL each) were injected into rabbit vitreous cavities. Two, 4, and 6 weeks later, light and electron microscopic examinations were conducted, and the immunohistochemical localization of glutamate in the retina was studied. Noninjected eyes served as controls. Results: At all time points, thinning or disappearance of the outer plexiform layer in the superior retina in eyes that received C3F8 gas was found; the inferior retina was the same as in controls. In eyes that received SF6 gas or air, light and electron microscopy showed that the superior and inferior retina were the same as in controls at all time points. Immunohistochemical examination showed abnormal glutamate distribution of the superior retina in eyes injected with C3F8 gas, SF6 gas, or air. However, glutamate distribution was the same as in controls in the inferior retina in eyes injected with C3F8 gas, SF6 gas, or air. Conclusions: Retinal tamponade using intraocular gases induces histopathologic retinal changes in the superior retina of the rabbit eye, where the gases are in continuous contact with the eye.

A Chinese hamster with a single X chromosome was found in 23 off-spring from crosses between a male heterozygous for T(1;3)8Idr translocation and karyologically normal females. All karyotypes of cells from various tissues of this animal were 21, X. The animal had an external genitalia generally characteristic to the female but no typical proestrous and estrous figure was found in the vaginal smear, and the animal showed no indication of pregnancy by any crosses. On autopsy, two gonads were found. Microscopically, there were some large fluid-filled cysts and many follicle-like bodies in the cortex of both gonads, but no mature follicle containing an oocyte was observed. The XO conditions in the Chinese hamster are compared with those of other mammals.

The behavior of marker proteins of glial cells [alpha-enolase, beta-S100 protein, and glial fibrillary acidic protein (GFAP)] was investigated quantitatively by using enzyme immunoassay systems during the development of cerebellar hypoplasia in jaundiced Gunn rats. A neuronal marker protein, gamma-enolase, was also measured as a reference. At postnatal day 8 corresponding to the early stage of cerebellar damage, the amount of beta-S100 on a protein basis was significantly higher in jaundiced homozygotes (jj) than in control nonjaundiced heterozygotes (j+), whereas no differences in alpha- and gamma-enolases and GFAP were observed between the two groups of rats. At days 15 and 30, which correspond, respectively, to the advanced and late stages of cerebellar damage, the three glial proteins, especially GFAP, were higher and the neuronal protein was lower in the jj rat cerebellum than in the control. These results are consistent with the reported histological observations that neuronal cells are vulnerable and damaged by bilirubin, whereas glial cells seem to be less sensitive. On the other hand, the amounts of beta-S100 and alpha-enolase per cerebellum were significantly lower in jj rats at days 15 and 30, as in the case of gamma-enolase, whereas that of GFAP remained at the same level as the control at day 15 and showed a slight but significant decrease at day 30. The possibility is suggested that beta-S100 and GFAP may be available as biochemical indicators of glial cells, especially in the early and advanced stages of cerebellar damage, respectively, but that alpha-enolase is less available.

Postnatal changes in cerebellar wet weight and the effect of phototherapy were studied using a new strain of Gunn rats with a genetic background of the Sprague-Dawley strain (SDGR). The cerebellar wet weight of homozygous rats reached a maximum at postnatal day 10 and then it gradually decreased up to day 20, suggesting that the cerebellar hypoplasia of the rats was a consequence both of cellular underdevelopment and degeneration. Histological examinations of the 10 cerebellar lobules showed that lobules I-VIII of homozygotes were severely damaged while lobules IX and X were less affected, coinciding with previous reports of Wistar strain Gunn rats. Photoirradiation with blue light of homozygous rats from day 1 through 15 markedly improved their cerebellar development, and the cerebellar wet weight of these rats was almost comparable to that of heterozygous rats as examined at day 30. Photoirradiation from day 1 to 5 protected, though not fully, lobules I-V and VIII against damage, but not lobules VI and VII at all. Photoirradiation from day 6 to 10 prevented all the lobules from hypoplasia. Irradiation from day 11 to 15 was effective only at the tips of lobules VI and VII. These results indicate that the cerebellar hypoplasia of jaundiced Gunn rats can be effectively prevented by a series of photoirradiation during a particular period after birth and also that the most effective period is different among the lobules.

Two cerebellar proteins with apparent molecular weights of 250,000 (GR-250) and 50,000 (GR-50) are closely associated with cerebellar hypoplasia in jaundiced homozygous Gunn rats. These proteins, found in Gunn rat cerebellum (4-60 days of age) and cerebrum as well as staggerer mouse cerebellum, were studied with electrophoretic techniques. After 8 days of life, GR-250 decreased and GR-50 increased in the homozygous Gunn rat cerebellum. The pI's of GR-250 and GR-50 were 4.7-5.8 and 4.6-4.9, respectively, and the former protein was shown to bind to Concanavalin A. A comparative study between cerebella of Gunn rats and staggerer mice revealed that GR-250 and P400, a protein generally thought to be characteristic of the Purkinje cells, were identical. Evidence was also obtained showing that GR-250 was present in the Gunn rat cerebrum. GR-50 was not detectable in the staggerer mouse cerebellum but instead, a protein (MW 47,000) was found to be increased in the mutant mouse cerebellum.

Three forms of enolase isozymes (alpha alpha, alpha gamma, gamma gamma), including nervous system-specific forms, were measured in the cerebrospinal fluid and the blood plasma of jaundiced or nonjaundiced infant rats by means of enzyme immunoassay systems capable of detecting each form of enolase at the 1 amol (10(-18) mol) level. Average enolase levels in cerebrospinal fluid in normal rat were 2.0, 0.2 and 0.1 pmol/ml for alpha alpha, alpha gamma, gamma gamma forms, respectively. Levels of alpha gamma and gamma gamma forms (nervous system-specific enolases; NSE) in jaundiced rats, which suffer Purkinje cell degeneration due to the inborn hyperbilirubinemia, were three to four times as high as the normal values. When kernicterus was induced in jaundiced rats by an injection of bucolome, the NSE level in cerebrospinal fluid was elevated up to more than 30-fold the control, together with a significantly higher level of alpha gamma form in blood plasma are helpful in detecting neuronal damage in the central nervous system.

On the basis of our previous findings that a 50,000-dalton protein (GR-50) shows a marked increase in the hypoplastic cerebellum of jaundiced homozygous Gunn rats and its electrophoretic behavior is similar to that of glial fibrillary acidic protein (GFAP), we tried to identify GR-50 as GFAP by two-dimensional electropho-resis of rat cerebellar membrane proteins using an improved immunoblotting method. In this method a blot immunostained for a specific antigen was also visualized for other proteins, thereby enabling us to determine the location of the antigen on the blot more precisely. With the methodology it was found that GFAP antigen occupied exactly the same position as GR-50 on the blot, suggesting strongly the identity of both proteins. Immunohistochemical studies revealed that in the cerebellum of homozygotes compared with that of heterozygotes GFAP antigen was greatly increased and that it was especially rich in the homozygous rat cerebellar lobules with a high degree of hypoplasia. Further, it was shown that not only the fibers of the Bergmann glial cells but also their somata were intensely immunostained in the affected lobules. A 47,000-dalton protein (SG-47), which has been reported to be increased in staggerer mutant mice with cerebellar hypoplasia, also immunoreacted with the antiserum to GFAP.

Photoirradiation of globin, obtained from human hemoglobin, in the presence of oxygen and protoporphyrin produced a series of polymers of globin subunits ranging from dimer to dodecamer. These polymers are useful as molecular weight markers because they cover a relatively wide range of molecular weights (Mr 15,500-186,000) with a constant and narrow interval of Mr 15,500.

A simple method for the drying, on filter paper, of sodium dodecyl sulfate polyacrylamide disc gels is described. This method has been successfully applied to disc gels with polyacrylamide concentrations of 5, 10, and 15%, and also with a linear gradient of 5–15%. The gels dehydrated without any longitudinal shrinkage are suitable for longtime storage and may be used for molecular weight estimation of proteins.