Mouse Brain Samples Research Articles

Age-related changes in neural cell adhesion molecule (NCAM) isoforms in the mouse telencephalon

The concentrations of different polypeptide isoforms of the neural cell adhesion molecule NCAM were examined in telencephalic and brainstem-cerebellar tissue from groups of young (3 months) and old (25 months) mice. Antibodies against chick brain NCAM were used in immunoblot analyses to quantify 180 (NCAM 180) and 140 (NCAM 140) kDa NCAM forms in mouse brain samples containing equal amounts of protein. Telencephalic homogenates from the older group exhibited 37% and 31% less NCAM 180 and NCAM 140 immunoreactivity, respectively, when compared with homogenates from the younger animals. Brainstem-cerebellar homogenates, however, did not express such age-related changes in the two NCAM isoforms. Age-related changes in isoforms labeled by the anti-NCAM antibodies were not evident in synaptic plasma membranes. NCAM 180:NCAM 140 ratios were 2- to 3-fold greater in the synaptic membranes vs. homogenates for both age groups. These data suggest that expression levels of NCAM 180 and NCAM 140 are selectively impaired with aging in the telencephalon, whereas the synaptic contents of these molecules appear to be stably regulated.

Ca 2+/calmodulin-dependent protein kinase II from hen brain : Purification and characterization

Ca 2+/calmodulin-dependent protein kinase II (CaM-kinase II) has been purified from hen whole brain. The enzyme was purified 3000-fold using phosphocellulose and calmodulin-Agarose column chromatography. The specific activity was 200 nmol/min/mg protein. Microtubule associated protein-2 (MAP-2) was used as a substrate to assess the activity of the enzyme during purification and for its characterization. CaM-kinase II consisted of α and β/β′ subunits of molecular weights 46,000 and 55,000/ 52,000, respectively. The ratio of α to β/β′ subunits was 3:1 in the enzyme purified from the whole brain. The enzyme exhibited broad substrate specificity and phosphorylated myelin basic protein, MAP-2, histone II, histone VIII, casein, tubulin, myosin light chains, glycogen synthase, and phosvitin in decreasing order. Phosphorylase b was phosphorylated at a negligible rate. Autophosphorylation of CaM-kinase II for 10 min in the presence of calcium and calmodulin decreased its total activity to 33%, and calcium/calmodulin-independent activity reached 30% after 1 min and then dropped to 14% after 10 min of autophosphorylation. The K m value of ATP was 19 ± 1.3 μM, and the K 0.5 values of calcium and calmodulin were 4.4±0.5 and 3.0±0.5 μM, respectively. The latter were determined using myelin basic protein as the substrate. CaM-kinase II exhibited great differences in the calmodulin requirement for phosphorylation of MAP-2, histone II and myelin basic protein. MAP-2 required the least amount of calmodulin for its phosphorylation. Autophosphorylation of CaM-kinase II resulted in decreased mobility of the α-subunit but apparently not of the β/β′ subunits in sodium dodecyl/sulfate-polyacrylamide gel. Antiserum was raised against the CaM-kinase II α subunit and used for testing cross-reactivity of hen brain enzyme with that of other species. The antiserum which reacted with both α and β subunits of hen brain CaM-kinase II cross-reacted with only the α subunit of rat, mouse, rabbit, cat, dog, pig and human brain samples. The purified hen brain CaM-kinase II is a multifunctional enzyme and resembled rat brain CaM-kinase II several properties. Immunocross-reactivity suggested that there was similarity in the α but not the β/β′ subunits of the hen brain enzyme and the brain enzyme of other species.

Changes in the distribution of anionic sites in brain micro-blood vessels with and without amyloid deposits in scrapie-infected mice

Cationic colloidal gold (CCG) and scrapie-infected mouse brain samples embedded in Lowicryl K4M were used for ultrastructural localization of negatively charged microdomains (anionic sites) in the cerebral microvasculature. The distribution of anionic sites on both fronts (luminal and abluminal) of endothelial cells and in the basement membrane (BM) in the majority of micro-blood vessels (MBVs) located outside the plaque area and in the remaining cerebral cortex was similar to that which has been previously observed in non-infected animals. Some MBVs (especially capillaries), however, located inside the plaque areas and surrounded directly by amyloid fibers contained attenuated endothelium, the luminal surface of which showed a segmental lack or diminution of anionic sites. In these vessels the BM was frequently infiltrated and replaced by the amyloid fibers. In some vessels located mainly in the areas of the neuropil vacuolization deposits of homogenous material causing the thickening of the BM were noted. These changes were accompanied by irregular labeling of the BM with gold particles. At the sites of bifurcation of some MBVs, predominantly in the area of the venular estuary at the mouth of capillary (at capillary-venular connections), a discontinuity in the distribution of anionic sites was noted. The observed disturbances in the distribution of anionic sites can be associated with a previously noted increased permeability of some MBVs in the brains of scrapie-infected mice.

Ultrastructural studies of glycoconjugates in brain micro-blood vessels and amyloid plaques of scrapie-infected mice.

Lectin or glycoprotein-gold complexes and samples of scrapie-infected mouse brain embedded in Lowicryl K4M were used for ultrastructural localization of glycoconjugates. The lectins tested recognize the following residues: beta-D-galactosyl [RCA, Ricinus communis agglutinin (aggl.) 120], N-acetyl and N-glycolyl neuraminic acid (LFA, Limax flavus aggl.), N-acetyl-D-glucosaminyl and sialyl (WGA, Wheat germ aggl.), N-acetyl-D-galactosaminyl (HPA, Helix pomatia aggl., and DBA, Dolichos biflorus aggl.), alpha-D-mannosyl/alpha-D-glucosyl (Con A, Concanavalin A), alpha-D-galactosyl and alpha-D-galactopyranoside (BSA, Bandeirea simplicifolia aggl., izolectin B4). Labeling of the majority of micro-blood vessels (MBVs) located outside the plaque area and in the remaining cerebral cortex was similar to that which has been previously observed in non-infected animals. Some MBVs, however, located inside the plaque area and surrounded directly by amyloid fibers showed attenuation of the endothelium, the surface of which was scarcely and irregularly decorated with RCA, LFA, WGA and Con A. These abnormalities in the composition of glycoconjugates can be associated with previously noted increased permeability of some MBVs in the brains of scrapie-infected mice. Some vessels in the plaque area were encapsulated by perivascular deposits of homogeneous or flocculogranular material containing several glycoconjugates. A very intimate structural relation between reactive (microglial-like) cells and amyloid fibers suggests the participation of these cells in elaboration of plaque material. Labeling of the cell surface and adjacent amyloid fibers with the same lectins (RCA, WGA, DBA, Con A) suggests the possibility that the glycosylation of these fibers occurs extracellularly. Only WGA and DBA were occasionally labeling some Golgi elements of the reactive cells.

The determination of pterins in biological samples by liquid chromatography/electrochemistry

A method is presented for determining the concentrations of several oxidized pterins in a variety of biological samples. This methodology employs reverse-phase "ion-pair" liquid chromatography with electrochemical detection. Detection limits below a picomole have been achieved with responses linear over four orders of magnitude. This methodology was employed to study the excretion pattern of pterins in human urine. The concentration of several pterins in mouse brain and liver samples were also determined. The direct detection of tetrahydrobiopterin was demonstrated.

A New Technique for Simultaneous Assay of Biogenic Amines and Their Metabolites in Unpurified Mouse Brain

Abstract We developed a chromatographic system which separates cate-cholamines and indolamines as well as a large number of their metabolites (acids, aminoacids and glycols). Waters Radial Pak columns compressed radially to avoid formation of cavities in the packing, were used. These columns must be washed with methanol in order to obtain reproducible results when an ion-pairing mobile phase is employed. The conditioning of bhe columns was not affected by washing with triethylamine or variations in radial pressure. Good resolution of 13 samples was obtained in 28 minutes with a mobile phase composed of 0.1 M KH2PO4, 0.1 mM EDTA and 5 mM heptane sulfonic acid in water and Me OH (85:15), pH = 3.6. Products were identified with the use of a Metrohm 64l electrochemical detector. The technique was used for rapid, simultaneous determination of NA, DA, DOPAC, HVA, 5HT and 5HIAA in deproteinated mouse brain samples.

Histochemical demonstration of sialic acid-containing compounds in the CNS of mice and goldfish by means of the mPAT ("mild" periodic acid-thionine)-method (author's transl)

The "mild" periodic acid-thionine (mPAT)-method was examined for the quantitative research of histochemical localization of sialic acid (NeuAc)-containing compounds in brain samples of goldfish and albino mice in comparison to salivary gland (Glandula submandibularis) of the mouse. Biochemical determinations of NeuAc contents of the tissues showed that during histological treatment a significant decrease in the amount of lipid-bound NeuAc, especially in the brain had occurred, because of its high content of lipid-bound NeuAc. A previous KOH-treatment caused in an increase of the colour reaction, which indicated mainly the amount of O-acylated NeuAc just as possibly lactone containing NeuAc-compounds. The intensity of staining, especially in the optic tectum of goldfish, decreased to 45% after neuraminidase-treatment. The low concentration of NeuAc-compounds in the brain in comparison to the salivary glands, results in only a faint staining. Therefore, the use of the mPAT-method seems to be not very suitable for a specific and quantitative staining of all NeuAc-compounds in the CNS.

Post-column derivatization procedure for the colorimetric analysis of tissue cannabinoids separated by high performance liquid chromatography

A new method for quantitating cannabidiol (CBD) and Δ 9-tetrahyrocannabinol (THC) in mouse plasma and brain involves (1) the separation of CBD and THC from their majro metabolites by the use of isocratic, revrsed-phase high-performance liquid chromatography (HPLC), and (2) the on-line reaction of the cannabinoids with Fast Blue Salt B (FBB) as teh former elute from the column; the colored cannabinoid-FBB derivatives are then detected at 490 nm in a spectrophotometer with a sensitivity of less than 50 ng. In addition to this HPLC—FBB analytical procedure, a method for extracting CBD and THC from brain and plasma is described, and selected examples illustrate the procedure's application to the analysis of CBd and THC in mouse plasma and brain samples taken from animals injected with these two cannabinoids.

Amino acids of nervous tissue.

SummaryThe pattern of free amino acids in mouse brain, as shown by 2-dimensional paper chromatography, does not reflect the amino acid composition of the brain as a whole. The brains of mice of several strains and various parts of the nervous system of the rabbit were studied. Free glutamic and aspartic acids, γ-aminoibutyric acid, taurine, glutamine, cystine, serine, glycine, alanine, valine, and the leucines were detected consistently. The variations in these constituents with age, sex, and strain in mice are discussed. The sciatic nerve in the rabbit had considerably smaller quantities of detectable constituents than the various parts of the central nervous system. A transplantable neuroblastoma in mice had a pattern of free amino acids which was similar to that found in other malignant tissues and in brains of 15-day embryos and significantly different from that found in all of the other samples of mouse brain examined. The suitability of the chromatographic method for the study of changes in the free...