Influence of atropine and carbachol on the fluorescence of catecholaminergic structures in selected areas of the rat brain.

Using the formaldehyde-fluorescence technique, the authors studied the influence of atropine and carbachol, administered intraventricularly to Wistar rats, on the fluorescence of catecholaminergic structures in 20 areas of the CNS, situated within the range of the 10th-46th frontal plane according to KONIG and KLIPPEL. 1. A confirmation of the antagonistic action of atropine and carbachol was obtained. It was expressed by mutually opposed occurrence of the specific fluorescence of the catecholaminergic structures. 2. In 16 out of 20 studied areas of the CNS, carbachol abolished or considerably weakened the specific fluorescence. In 3 areas it was increased by this drug, and one area proved insensitive. 3. Atropine increased the specific fluorescence in the DA (dopaminergic system) areas, while it had varying effects in the NA (noradrenergic system areas. In some areas of the CNS it increased and in others reduced the specific fluorescence of the catecholaminergic structures. 4. An interference between atropine and carbachol is observed, but it seems that the results of the present experiment speak in favour of an interaction between the catecholamine transmitters and ACh in the particular areas of the CNS under the influence of atropine and carbachol. 5. The authors discuss in detail the reactions of the catecholaminergic structures in the particular areas of the CNS, in which, as compared with the control, an increase or a decrease of the specific fluorescence under the influence of the administered drugs was observed.     

Fluorescence microscopy with antisera against specific cellular structure: double photography method for cell identification in populations of multiple cell types

Immunofluorescent staining techniques using antitubulin antibody have been difficult to apply to meiotic tissue (testis) because of the large number of cell types present. Such techniques customarily use a fluorescent dye to counterstain nuclei, and this counterstain is hard to distinguish because of the fluorescence of the antitubulin. By counterstaining with dilute hematoxylin, we can photograph the same field using UV and then conventional illumination. This double photography allows us to identify precisely the many types of cells present, and it will be a useful tool for reexamining the staging of spermatogenesis.     

Cerebroside galactosidase: a method for determination and a comparison with other lysosomal enzymes in developing rat brain

• 1 A method is described for assaying brain for cerebroside galactosidase activity. The enzyme was liberated by sonication and addition of sodium taurocholate, then by digestion with pancreatic enzymes. It was further purified by precipitation at pH 3. The enzyme was then incubated with an emulsion of galactose-labelled cerebroside in taurocholate and oleate at pH 4·5, and the liberated galactose was determined by scintillation counting.
• 2 The content of cerebroside galactosidase in rat brain at various ages has been determined. The enzyme was present before cerebroside appears in noticeable amounts (4 days) and the amount rose considerably during the period of active cerebroside deposition and myelination. The amount then remained at a high concentration even in the adult.
• 3 Comparison with other lysosomal brain enzymes was made in the age study. Nitrophenyl galactoside hydrolase also increased during myelination but levelled off earlier; its activity paralleled the amount of ganglioside. Nitrophenyl glucoside hydrolase started at a lower level and decreased with age. Sulphatase activity rose during myelination, then decreased somewhat after 15 days. Ceramidase followed a pattern similar to that of nitrophenyl galactoside hydrolase; it is suggested that both of these enzymes reflect ganglioside metabolism.
• 4 The relative amounts of brain enzymes in different states were determined as a function of age in the case of cerebrosidase, nitrophenyl galactoside hydrolase and sulphatase. The proportion found in the high speed supernatant fraction was low but increased after myelination. The proportion that could be ‘solubilized’ by sonication decreased after myelination but the values differed greatly for the three enzymes. This treatment solubilized one-seventh of the cerebrosidase, half the nitrophenyl galactosidase and three-quarters of the sulphatase.

     

Distribution of mRNA for the calmodulin-sensitive adenylate cyclase in rat brain: expression in areas associated with learning and memory

The Drosophila learning mutant, rutabaga, is deficient in the calmodulin-sensitive adenylate cyclase, and studies of associative learning in Aplysia have implicated this enzyme in neuroplasticity. Therefore, the distribution of mRNA encoding the calmodulin-sensitive adenylate cyclase in rat brain was examined by in situ hybridization. mRNA for this enzyme is expressed in specific areas of brain that have been implicated in learning and memory, including the neocortex, the hippocampus, and the olfactory system. The presence of mRNA for this enzyme in the pyramidal and granule cells of the hippocampal formation provides evidence that it is found in neurons. These data are consistent with the proposal that the calmodulin-sensitive adenylate cyclase plays an important role in learning and memory.     

Identical immunoreactivity of afferents to the rat suprachiasmatic nucleus with antisera against avian pancreatic polypeptide,molluscan cardioexcitatory peptide and neuropeptide Y

Summary Avian pancreatic polypeptide (APP)-like, molluscan cardioexcitatory peptide (FMRF)-like and neuropeptide Y (NPY)-like immunoreactivities were studied in a secondary visual pathway in rat brain. The cell bodies of this pathway are located in the lateral geniculate nucleus and its terminal plexus is found in the suprachiasmatic hypothalamic nucleus (SCN). The neurons and terminal plexus demonstrated by antiserum to each peptide are identical, and immunoreactivity is blocked by preabsorption of each antiserum with a low concentration of the antigen against which it was raised. Immunoreactivity is also blocked by preabsorption of each antiserum with either NPY or APP. In contrast, APP- and NPY-like immunoreactivities are blocked only partially when these antisera are preabsorbed with concentrations of FMRF as high as 100 M. Since NPY is the only one of these peptides that has been isolated from mammalian brain, we conclude that NPY is the endogenous CNS peptide produced by neurons of the lateral geniculate-SCN projection.     

Purification and properties of malonyl-CoA decarboxylase from rat liver mitochondria and its immunological comparison with the enzymes from rat brain, heart, and mammary gland.

Malonyl-CoA decarboxylase (EC 4.1.1.9) was found to be localized in the mitochondria in rat liver. Low ionic strength (10 mm Na phosphate) buffer extracted the bulk (>85%) of the enzyme from the mitochondria. From this extract the enzyme was purified over 2,000-fold using a combination of (NH₄)₂SO₄ precipitation, gel filtration with Sepharose 4B and Sephadex G-150, ion exchange chromatography with QAE-Sephadex and CM-Sephadex, and finally chromatography on NADP-agarose. The purified enzyme, which had a specific activity of about 16 μmol/min/mg, appeared to be electrophoretically homogeneous and had a molecular weight of 160,000. The decarboxylase had a broad pH optimum between 8.5 and 10.0 and showed a typical Michaelis-Menten substrate saturation pattern from which K_m and V were calculated to be 54 μm and 18.8 μmol/min/mg, respectively. This enzyme decarboxylated neither malonic acid nor methylmalonyl-CoA and was severely inhibited by thiol-directed reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide but not by iodoacetamide. Acetyl-CoA, propionyl-CoA, and methylmalonyl-CoA also inhibited the enzyme. The purified decarboxylase was immunogenic in rabbits and Ouchterlony double diffusion analysis revealed a single precipitant line with the purified enzyme. The IgG fraction isolated from the antiserum inhibited the enzyme from not only liver mitochondria but also the mammary gland, heart, and kidney of the rat. However, malonyl-CoA decarboxylase from rat brain mitochondria was not inhibited by the antibody. Malonyl-CoA decarboxylase purified from the uropygial gland of a domestic goose neither cross reacted nor was it inhibited by the antiserum prepared against the rat liver mitochondrial enzyme and the antibody against the goose enzyme neither cross-reacted nor inhibited the enzyme from the rat. It is proposed that a role for mitochondrial malonyl-CoA decarboxylase is to decarboxylate malonyl-CoA generated by propionyl-CoA carboxylase and thus protect mitochondrial enzymes susceptible to inhibition by malonyl-CoA.     

In vitro formation of polyunsaturated fatty acids by desaturation in rat brain: some properties of the enzymes in developing brain and comparisons with liver

Abstract— In vitro desaturation of [1-C¹⁴]linolenic, linoleic, oleic, and icosatrienoic acids was determined using homogenates and subcellular fractions of developing rat brain and liver. Linolenic, linoleic, and oleic acids were desaturated in the δ6-position and activity was optimal in the presence of CoA, ATP, MgCl₂, and NADH in a citrate-phosphate buffer at pH 6.0. Icosatrienoic acid was desaturated in the δ5-position with a much broader pH optimum. The unstable desaturation systems of brain were protected by reduced glutathione and niacinamide and markedly inhibited by dithiothreitol, p-chloromercuribenzoate, sodium cyanide or bathophenanthroline sulfonate. With brain homogenate of neonatal rats, the relative rates of desaturation of these substrates were 18:3(n - 3) > 18:2(n - 6) > 20:3(n - 6) > 18:l (n - 9). Specific activity of brain enzymes was greatest in neonatal rats with fluctuations in activity between 3 and 6 days of age. During this period, liver enzyme appeared to alter in a reciprocal manner. Total desaturation capacity of brain was maximal and fairly constant between 4 and 20 days of age, whereas liver activity increased dramatically after weaning. The activity of crude microsomal preparations from neonatal brain, like that of liver microsomes, was stimulated by a heat-labile component of the cytosolic fraction. These results demonstrate that brain has a high capacity for desaturation of the essential fatty acids during crucial stages of brain development when liver activity is relatively low.     

Membrane receptors for estrogen,progesterone, and testosterone in the rat brain: Fantasy or reality

Summary 1. There are numerous circumstantial evidence supporting the concept that steroid hormones control cellular function by means other than the nuclear receptor steroid binding mechanism. It is the intent of this report to present evidence indicating that steroids bind to specific sites in neuronal membranes.2. Some of the criteria to define steroid membrane receptors using steroid-BSA conjugates that can be radioiodinated to desired specific activity have been fulfilled for each of the three sex steroids using crude synaptosomal membrane preparations (P2 fractions) from the CNS of female and male rats. Ligand binding for each of the three steroids indicate high-affinity and high-capacity sites with distinct brain selectivity and stereospecificity. For example, 17-E-6-[¹²⁵I]BSA binds hypothalamic P2 fractions (HYP-P2) with an estimatedK _d of about 3±0.7 nM (X ± SE;n=3), whereas the cerebellum P2 (CB-P2) fractions bind the ligand with aK _d of 34±7 nM and, aB _max of 3 and 42 pmol/mg protein, respectively. Estrogen and testosterone binding fit best a one-single site, while progesterone binding sites can be best represented by a two-binding site, one high-affinity (K _d=1–2 nM) and one low affinity (K _d=62 nM), in CB-P2 fractions from intact adult female rat brain. Kinetics studies for T-3-[¹²⁵I]BSA indicate that the estimatedK _d of 30±2 nM for the olfactory bulb P2 fractions (OB-P2) from male rats is in good agreement withK _d values computed from Scatchard-derived data using the LIGAND algorithm.3. 17-E-6-[¹²⁵I]BSA binding sites are stereospecific and appears to be present as early as 5 days of age in both the OB- and the CB-P2 fractions without changes during development. In contrast, P-6-[¹²⁵I]BSA binding sites are practically absent during days 5 and 12 and appear by day 22.4. Finally, membrane receptor molecules for estrogen and progesterone have been isolated and purified by affinity chromatography and characterized by PAGE and Western blot. Microsequencing of one of the membrane estrogen binding proteins indicates that the high-affinity site corresponds to the OSCP subunit of the proton ATP synthase.5. It remains to be determined if P and T also bind to this complex enzyme or if they bind to other subunits of the family of proton ATPases. Overall the data indicate that steroid hormones conjugated to BSA are important tools to study the reality of membrane steroid receptors.     

The effect of long-term enrichment of diet with selenium, vitamin E and B15 on the activity of certain enzymes in rat brain

The aim of this study was tracing of changes in the activity of glutathione peroxidase (GSHPx), glutathione transferase (GSH S-Tr), aspartate aminotransferase (AspAT) and alanine aminotransferase (A1AT) in the brain as a result of diet enrichment with antioxidants: selenium (Se), vitamin E and vitamin B15 (pangamic acid). The experiment was carried out on Wistar rats with initial body weight 150 g. Following prolonged enrichment of diet with Se (0.1 ppm of sodium selenite), vitamin E (6 mg/100 g of diet) and vitamin B15 (2.5 mg/100 g of diet) the following results were obtained. The activity of GSHPx in brain microsomes was not changed after one year of vitamin E administration when it was measured against hydrogen hydroxide and against cumene hydrochloride; vitamin E administration increased the activity of GSH S-Tr in the cytoplasmic fraction of brain cells. Diet enrichment with selenium increased after 12 and 18 months the activity of GSHPx measured against both substrates, and GSH S-Tr activity increased considerably. Presence of vitamin B15 in diet reduced GSHPx activity after one-year or longer administration, after 18 months the activity of GSH S-Tr was reduced also. No changes were noted in the activity of AspAT and A1AT.     

Neuroprotective effect of rutin against colistin-induced oxidative stress,inflammation and apoptosis in rat brain associated with the CREB/BDNF expressions

The purpose of the current study was to examine the neuroprotective effect of rutin against colistin-induced neurotoxicity in rats. Thirty-five male Sprague Dawley rats were randomly divided into 5 groups. The control group (orally received physiological saline), the rutin group (orally administered 100 mg/kg body weight), the colistin group (i.p. administered 15 mg/kg body weight), the Col?+?Rut 50 group (i.p. administered 15 mg/kg body weight of colistin, and orally received 50 mg/kg body weight of rutin), the Col?+?Rut 100 group (i.p. administered 15 mg/kg body weight of colistin, and orally received 100 mg/kg body weight of rutin). Administration of colistin increased levels of glial fibrillary acidic protein and brain-derived neurotrophic factor and acetylcholinesterase and butyrylcholinesterase activities while decreasing level of cyclic AMP response element binding protein and extracellular signal regulated kinases 1 and 2 (ERK1/2) expressions. Colistin increased oxidative impairments as evidenced by a decrease in level of nuclear factor erythroid 2-related factor 2 (Nrf-2), glutathione, superoxide dismutase, glutathione peroxidase and catalase activities, and increased malondialdehyde content. Colistin also increased the levels of the apoptotic and inflammatoric parameters such as cysteine aspartate specific protease-3 (caspase-3), p53, B-cell lymphoma-2 (Bcl-2), nuclear factor kappa B (NF-κB), Bcl-2 associated X protein (Bax), tumor necrosis factor-α (TNF-α) and neuronal nitric oxide synthase (nNOS). Rutin treatment restored the brain function by attenuating colistin-induced oxidative stress, apoptosis, inflammation, histopathological and immunohistochemical alteration suggesting that rutin supplementation mitigated colistin-induced neurotoxicity in male rats.

     

Short peptide tools for monitoring caspase and proteasome activities in embryonal and adult rat brain lysates: an approach for the differential identification of proteases

The numerous caspase-like activities present in nervous tissue can be investigated with labelled peptides. However, the cross-reactivities of peptides with both proteasomes and caspases complicate the analysis of protease activity. The pharmacological features of substrates and inhibitors specific for either caspases or proteasome caspase-like proteases in rat brain lysates were similar or identical to the profiles of commercially purified proteasome preparations. Caspase inhibitors bind directly to active proteasome centres, thus competing with selective antagonists of proteasomes. Separation of lysates by molecular weight does not separate active caspases from proteasomes because these enzymes co-localize under native electrophoresis. The addition of ATP or its analogues is associated with the differential modulation of proteasomal activity, which also leads to ambiguity in the data. However, induced caspase activity could be successfully differentiated from proteasome activity in embryonal brain lysates with the non-selective caspase inhibitors Z-VAD-FMK and Q-VD-OPh and the proteasome inhibitor AdaAhx(3)L(3)VS that are not cross-reactive. This strategy is proposed for the simultaneous examination of caspases and proteasomes using proteolysis experiments. The present study reveals that all of the caspase-like activities in the tissue lysates of non-injured adult rat brains were related to proteasomal caspase-like activities.     

Argininosuccinase from bovine brain: isolation and comparison of catalytic, physical, and chemical properties with the enzymes from liver and kidney.

Homogeneous argininosuccinase has been isolated from bovine brain: compared to liver and kidney argininosuccinases from the same species, the catalytic activity (1400 U/mg). molecular weight of the fully active form (202,000 by gel filtration), and the minimum molecular weight (50, 000 in sodium dodecyl sulfate and mercaptoethanol) were in agreement with published liver and kidney enzyme values from this laboratory. That the brain enzyme is composed of four identical, or closely similar, polypeptide chains is supported by peptide maps analyzed after tryptic or cyanogen bromide cleavage. One-fourth the number of peptide fragments were produced as compared to the total number of susceptible residues per mole. The number of peptides containing other specific residues, or methionyl residues, were consistently one-fourth of the total considered. As maps of peptide fragments prepared from the brain enzyme were also superimposable, or nearly so, on liver enzyme maps, the four polypeptide chains from both sources were closely similar to each other in amino acid sequence. Distribution of the 16 sulfhydryl groups, as based on titration with Ellman's reagent, was in accord with the liver enzyme: Four sulfhydryl groups reacted without affecting catalytic activity, a second group of 4 became accessible on cold dissociation of the tetramers to catalytically inactive dimers, and the final 8 became accessible in strong dissociating agents. On analysis, K_m values and negative homotropic interactions with substrate were in accord with liver enzyme kinetics. Immunological studies indicated a ciose resemblance in antigenic properties. The brain enzyme, as antigen, was fully crossreactive in the formation of precipitin bands with rabbit antibody to either liver or kidney enzymes already known to be mutually cross-reactive. The antibody to the liver enzyme was an effective inhibitor of brain enzyme activity comparable to inhibition of the homologous liver and kidney antigen.     

Expression and electrophysiological identification of the receptor for bombesin and gastrin-releasing peptide in Xenopus laevis oocytes injected with polyA+ RNA from rat brain

The receptor for bombesin and the related peptide, gastrin-releasing peptide (GRP) has been induced in frog oocytes by injection of polyA+ RNA from rat brain. The primed oocytes responded to peptides of the bombesin family (GRP, neuromedin C of bombesin) by showing dose-dependent oscillations in membrane currents as recorded by the voltage-clamp method. The induced membrane changes were suppressed when oocytes were pretreated with a bombesin-receptor antagonist.     

Gene expression of alpha-endosulfine in the rat brain: correlative changes with aging, learning and stress

Endosulfine (EDSF) belongs to a highly conserved cAMP-regulated phosphoprotein (ARPP) family and was first isolated from ovine brain as a possible endogenous ligand for sulfonylurea receptors. To explore its involvement in brain functions, we investigated regional distribution of alpha-EDSF gene expression in the rat brain, and its regulation under physiological and pathological conditions. The majority of alpha-EDSF gene was expressed in the pyramidal neurons, which represent the principal excitatory neurons in various brain regions. Down-regulation of alpha-EDSF mRNA was detected in the rat hippocampus during long-term memory consolidation following a spatial learning experience, whereas swimming-related stress caused persistent up-regulation of alpha-EDSF gene expression in several brain regions. These changes, however, were absent from brains of diabetic rats that were subjected to the same behavioral treatments. Intracerebroventricular injection of streptozocin with a toxic dose induced severe learning deficits and brain structure alteration accompanied by a massive increase of alpha-EDSF mRNA in the somatosensory cortex. These results suggest that alpha-EDSF gene expression is differentially regulated by distinct brain processes involving excitatory neuronal activities.     

Immunocytochemical finding of the amidating enzymes in mouse pancreatic A-, B-, and D-cells: a comparison with human and rat.

alpha-Amidation is catalyzed by two enzymatic activities, peptidyl-glycine alpha-hydroxylating mono-oxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), denoted collectively as peptidyl-glycine alpha-amidating mono-oxygenase (PAM), which also may include transmembrane and cytoplasmic domains. PAM is present in mammalian pancreas, where it appears to be abundant in the perinatal period. Nevertheless, there is no agreement on the cell type(s) that produces PAM or even on its presence in adults. In the present study we found PAM (PHM and cytoplasmic domain) immunoreactivity (IR) in A-, B-, and D-cells of adult mouse pancreas. In contrast to previous reports, PAM IR was found in B-cells of human and rat. Most of the B/D-cells were PAM immunoreactive, although with variable intensity, whereas less than half of A-cells displayed IR. Immunocytochemistry and Western blotting suggested the existence of different PAM molecules. Differences in the cellular distribution of IR for PAM domains were also observed. Whereas PHM-IR was extended throughout the cytoplasm in the three cell types, presumably in the secretory granules, IR for the cytoplasmic domain in A/D-cells was restricted to a juxtanuclear region, perhaps indicating its cleavage in Golgi areas. Although glucagon, insulin, and somatostatin are non-amidated, amidated peptides (glucagon-like peptide 1, adrenomedullin, proadrenomedullin N-terminal 20 peptide) were found in the three cell types.     

Anti-catechol-O-methyltransferase: Demonstration of specificity and immunological cross-reactivity with the enzyme from rat liver,kidney, brain,and choroid plexuses

An antiserum to rat liver catechol-O-methyltransferase (COMT) was utilized in the immunological characterization of COMT from rat kidney, brain, and choroid plexuses, in addition to rat liver. The presence of anti-COMT activity was confirmed by the direct inhibition of the activity of the enzyme from rat liver by small quantities of the antiserum and by the inhibition of the activity of the enzyme from rat brain. The specificity of the antiserum was demonstrated both by immunoelectrophoresis of rat liver COMT, and by a partial purification of rat liver COMT in which changes in COMT specific activity were correlated with the appearance of a precipitin line in double-immunodiffusion experiments. The antigenic similarity of the enzyme derived from rat liver, kidney, brain, and choroid plexuses was demonstrated by the formation of a precipitin line of identity when preparations from these four tissues were diffused against the antiserum.     

Biochemical criteria for the evaluation of drug efficiency on adjuvant arthritis and nephrotoxic serum nephritis in the rat: studies with phenylbutazone, L-Asparaginase, colchicine, lysine acetylsalicylate, and pyridinol carbamate.

The levels of serum orosomucoid, haptoglobin, and seromucoid were evaluated as possible quantitative criteria for the estimation of drug efficiency in adjuvant arthritis and nephrotoxic serum nephritis. In adjuvant arthritis, haptoglobin, seromucoid, and chiefly orosomucoid serum levels were generally very sensitive to anti-inflammatory agents such as phenylbutazone and pyridinol carbamate, and to immunosuppressive agents such as L-asparaginase. There was a significant correlation between the serum levels of these glycoproteins and the arthritis scores. In nephrotoxic serum nephritis, seromucoid levels were correlated with the proteinuria of the autologous phase and were found to be a good complementary criterion for the analysis of the efficiency of pyridinol carbamate, colchicine, iysine acetylsalicylate, and L-asparaginase.