Reiji Semba

Distribution of brain-derived neurotrophic factor in rats and its changes with development in the brain.

Abstract: A newly established, sensitive, two‐site enzyme‐immunoassay system for brain‐derived neurotrophic factor (BDNF) is described. Using this system, we investigated the tissue distribution of BDNF and developmental changes in tissue levels of BDNF in rats. The minimal limit of detection of the assay was 3 pg/0.2 ml of assay mixture. BDNF was successfully solubilized from tissues in the presence of guanidine hydrochloride but not in any of the other buffers examined. In the rat brain at 1 month of age, the highest level of BDNF was detected in the hippocampus (5.41 ng/g of wet weight), followed by the hypothalamus (4.23 ng/g) and the septum (1.68 ng/g). In other regions, levels of BDNF ranged between 0.9 and 1.7 ng/g. The level of BDNF in the posterior lobes of the cerebellum from rats at 30 days of age was slightly higher than that in the anterior lobes. The concentration of BDNF increased in all regions of the brain with postnatal development. In peripheral tissues, BDNF was found at very low concentrations (0.65 ng/g in the spleen, 0.21 ng/g in the thymus, and 0.06 ng/g in the liver). The subfractionation of the hippocampal homogenate indicated that ∼50% of BDNF was contained in the crude nuclear fraction. Immunoblots of BDNF‐immunoreactive proteins extracted from the hippocampus, hypothalamus, and cerebellum contained doublet bands of protein of ∼14 kDa, a value close to the molecular mass of recombinant human BDNF. Immunocytochemical investigations showed that, in the hippocampus, BDNF was localized in the nucleus of the granule cells in the dentate gyrus and of the cells in the pyramidal cell layer. The frequency of cells that were stained in the dentate gyrus was greater than that of cells in the pyramidal cell layer.

AGE-RELATED CHANGES IN LEVELS OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN SELECTED BRAIN REGIONS OF RATS, NORMAL MICE AND SENESCENCE-ACCELERATED MICE : A COMPARISON TO THOSE OF NERVE GROWTH FACTOR AND NEUROTROPHIN-3

Age-related changes in the levels of brain-derived neurotrophic factor (BDNF) in selected regions of brains from rats, normal mice and senescence-accelerated mice were compared to those of nerve growth factor (NGF) and neurotrophin-3 (NT-3). The concentration of BDNF increased with age in the rat hippocampus while it decreased in the rat cerebral cortex. The level of BDNF in the hippocampus from aged rats was about 260%, of that in the same region from young adult rats. A strong staining with antibodies specific for BDNF was observed in the hilus of the dentate gyrus in the hippocampus from aged rats. By contrast, BDNF levels were significantly lower in four brain regions from aged rats as compared to young adult rats (30, 56, 52 and 52%, lower in the septum, cerebral cortex, cerebellum and striatum, respectively). Patterns of age-related changes in the level of BDNF in the mouse hippocampus. cerebral cortex, cerebellum and olfactory bulb were similar to those in the respective regions from rats. In rats, the concentration of NGF decreased with age in the cerebral cortex but remained unchanged in the hippocampus, cerebellum and olfactory bulb. In mice, levels of NGF increased in all four brain regions from 1 to 18 months after birth. The concentrations of NT-3 increased and decreased with age in the rat cerebral cortex and cerebellum, respectively, while minimal changes were observed in the rat hippocampus and olfactory bulb as was also true in mice. In senescence-accelerated mice with memory disturbances, no marked increases in levels of NGF and BDNF in the hippocampus and in the level of NT-3 in the cerebral cortex were found. Thus, increases in levels of BDNF and NT-3 occurred in the murine hippocampus and cerebral cortex, respectively, during normal aging, but not during aging of mice with pathological changes.

Predominant localization in glial cells of freel-arginine. Immunocytochemical evidence

Nitric oxide has been recently identified as an endogenous activator of the soluble guanylate cyclase in the brain as well as in vascular endothelial cells and macrophages. In the present study, we determined the localization of free arginine in the brain because nitric oxide was formed from the terminal guanido group of L-arginine. Anti-arginine antiserum was raised in guinea pigs by repeated injection of L-arginine covalently conjugated to guinea pig serum albumin via glutaraldehyde. Specific anti-arginine antibody was purified from the antiserum by using an affinity gel coupled with L-arginine. Arginine-like immunoreactivity in the rat brain and spinal cord was found concentrated mainly in astrocytes including Bergmann glial cells in the cerebellum and processes of astrocytes around blood vessels. The present results suggest that glial cells, particularly astrocytes, are the main locus of L-arginine, a nitric oxide precursor, in the brain.

Glycine-like immunoreactivity in the rat auditory pathway

From neurophysiological and biochemical studies it has been suggested that glycine can function as a major inhibitory neurotransmitter in the central nervous system of mammals. In the present study, anti-glycine antiserum was obtained from rabbits immunized with glycine conjugated to rabbit serum albumin via glutaraldehyde and purified by affinity chromatography. The antibody thus obtained was found specific for glycine as determined by an enzyme immunoassay system. The immunocytochemical distribution of glycine in the auditory tract and internal ear was investigated with the antibody. In the central auditory pathway, glycine-like immunoreactivity was mainly located in the ventral and dorsal cochlear nuclei, trapezoid body, lateral lemniscus and inferior colliculus. In the labyrinth, immunoreactivity was detected in the vestibular ganglion and the supporting cells of the crista ampullaris and the organ of Corti, but not in the spiral ganglion. These findings suggest an important role of glycine in the auditory and vestibular pathways.

Distribution of nervous system-specific forms of enolase in peripheral tissues

The distribution of 3 forms of rat enolase (alpha alpha, alpha gamma and gamma gamma forms), including nervous system-specific forms (alpha gamma and gamma gamma), was determined in various tissues with a sensitive enzyme immunoassay system. The brain and spinal cord contained more than 100 pmol/mg protein of the alpha alpha, alpha gamma and gamma gamma enolases. Organs such as the lungs, heart, spleen, liver, and kidney contained similarly high levels of alpha alpha enolase, but these tissues contained alpha gamma and gamma gamma enolases at levels less than 1% of the central nervous tissues. High levels of the alpha gamma (greater than 10 pmol/mg) and the gamma gamma (greater than 1.5 pmol/mg) forms were found in rectum, bladder, and uterus. In gut, major portions of the nervous system-specific forms were localized in the muscle layers. Skeletal muscle and diaphragm, which are composed of striated muscle, contained low levels of 3 forms of enolase. Megakaryocytes separated from the suspension of bone marrow contained 11.3, and 0.53 amol/cell of the alpha alpha and gamma gamma enolases, respectively, with little, if any, of the alpha gamma form.

Determination of Brain Enolase Isozymes with an Enzyme Immunoassay at the Level of Single Neurons

Abstract Ultrasensitive enzyme immunoassay systems for the assay of rat brain enolase isozymes (αα, αγ, and γγ forms) were prepared by use of β‐d‐galactosidase from Escherichia coli as label and the purified rabbit antibodies to αα and γγ enolases. The antibodies were purified from the immunoglobulin G (IgG) fractions of antisera by immunoaffinity chromatography with a column of the corresponding antigen‐coupled Sepharose. Sandwich‐type immunoassay systems with the galactosidase‐labeled antibody Fab’fragments and the antibody F(abapos;)2‐immobilized polystyrene beads could determine amounts as small as 1 amol (10−18 mol) of each isozyme. Purkinje cell bodies picked up from the bulk‐separated fraction by means of a nylon loop were subjected to the assay at the level of single cells. In contrast to previous report, this neuron contained not only the γγ but also the αγ and αα enolases at a level of amol per cell body, although the concentration of γγ was the highest. Immunohistochemical experiments on the cerebellum with the peroxidase‐labeled antirabbit IgG antibody and the unlabeled antibody method confirmed the above results, and indicated that both α and γ subunits of the enolase were stained intensely in axons.

Go, a GTP-binding protein : Immunochemical and immunohistochemical localization in the rat

Abstract: The tissue and cellular distribution of a GTP‐binding protein, Go, was investigated in the rat by immunochemical and immunohistochemical methods. Because the specific antibody for the α subunit of bovine Go (Goα) cross‐reacted with rat Goα, an enzyme immunoassay method developed for bovine Goα was applied for measuring the tissue concentration of Goα in the rat. Goα was detected in all tissues examined except blood cells. The concentration of Goα was highest in the CNS (∼7.7 and 4.4 nmol/g in the cerebrum and cerebellum, respectively), followed by the pituitary gland and sciatic nerve. Among the other peripheral tissues, relatively high concentrations of Goα were observed in the urinary bladder, stomach, and intestines; however, these values were <2% of the concentration in the cerebrum. Goα in the intestine was located mostly in the muscle layer. Immunohistochemical study showed that Goα was associated mostly with the neural elements but not with cells particular to each peripheral organ. Goα was also present in the membranes of neuroen‐docrine cells, including glandular cells in the anterior lobe of the pituitary gland, chromaffin cells in the medulla of the adrenal gland, islets cells in the pancreas, and parafollicular cells in the thyroid. These results indicate that Go is localized exclusively in the nervous tissues and neuroendocrine cells.

Highly Sensitive Immunoassay for the α Subunit of the GTP-Binding Protein Go and Its Regional Distribution in Bovine Brain

Abstract: Antisera were raised in rabbits against the α sub‐unit of a GTP‐binding protein, Go. Because the antisera cross‐reacted weakly with the α subunit of inhibitory GTP‐binding protein of adenylate cyclase (Gi), they were purified with a Goα‐coupled Sepharose column. Purified antibodies reacted only with Goα and did not cross‐react with the Giα subunit or βγ subunits in an immunoblot assay. Using these purified antibodies, a highly sensitive enzyme immunoassay method for the quantification of bovine brain Goα was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab′)2 fragments and the same antibody Fab’ fragments labeled with β‐D‐galactosidase from Escherichia coli. The minimal detection limit of the assay was 0.1 fmol, or 4 pg. The assay was specific for Goa, and it did not cross‐react with Giα or βγ. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Goα were determined. Goα was detected in all the regions, and the highest concentration was observed in the cerebral cortex. The immunohistochemical study showed that the neu‐ropil was rich in Goα.

Purification of specific antibody against aspartate and immunocytochemical localization of aspartergic neurons in the rat brain

The distribution of L-aspartate known as a putative excitatory neurotransmitter in the central nervous system was investigated immunocytochemically in the rat brain. Anti-aspartate antiserum was raised in rabbits using L-aspartate covalently conjugated to rabbit serum albumin with glutaraldehyde as the immunogen and was found to be cross-reactive with an L-glutamate conjugate. Monospecific anti-L-aspartate antibody was successfully purified using affinity gels coupled with several amino acids including L-aspartate and L-glutamate and with the L-glutamate conjugate. Putative aspartergic neurons were generally immunoreactive to the purified antibody, but epithelia of the choroid plexus were also stained. These results show that the antibody is a useful tool for the immunocytochemical demonstration of possible aspartergic neurons in the central nervous system, although the immunochemical expression of L-aspartate not used as a neurotransmitter must be taken into consideration.

Inducible nitric oxide synthase-dependent DNA damage in mouse model of inflammatory bowel disease

Increased cancer risk occurs in inflammatory bowel disease (IBD) undergoing long‐term chronic inflammation. To evaluate whether inducible nitric oxide synthase (iNOS)‐dependent DNA damage plays a role in the carcinogenic process triggered by IBD, we prepared a mouse model of IBD induced by transfer of CD45RBhighCD4+ T cells lacking regulatory T cells to female severe combined immunodeficiency (SCID) mice. CD45RBhighCD4+ T cells were isolated from mouse spleen after staining with fluorescein isothiocyanate (FITC)‐conjugated anti‐CD45RB monoclonal antibody, followed by anti‐FITC‐conjugated microbeads. This IBD mouse model showed that the bodyweight increased with aging to a lesser extent than non‐treated controls, and that the intestine was shortened. Pathological findings of this mouse model, which showed severe inflammation in colon tissues, were similar to IBD patients. Double immunofluorescence technique revealed that both 8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) were formed mainly in epithelial cells of the IBD mouse model. 8‐Nitroguanine was formed in most of 8‐oxodG‐immunoreactive nuclei of epithelial cells. iNOS, proliferating cell nuclear antigen and p53 protein were also expressed in the colon epithelium. These results indicate that nitrative DNA damage, as well as oxidative DNA damage, is induced in colon epithelial cells of the IBD mouse model followed by proliferation of these cells, which may contribute to colon carcinogenesis. (Cancer Sci 2005; 96: 157–163)