Quantitation of proenkephalin A messenger RNA in bovine brain, pituitary and adrenal medulla: correlation between mRNA and peptide levels.

Concentrations of mRNA coding for the opioid peptide precursor proenkephalin A were measured in bovine brain areas, pituitary and adrenal medulla. In all tissues, a single hybridizable species of 1400 bases in size was found by Northern blot analysis using as a probe a single-stranded (ss) cDNA complementary to bovine proenkephalin A mRNA. In solution hybridization experiments the distribution of the mRNA was quantified. Considerable differences were found for the abundance of proenkephalin A mRNA in the various tissues: from 0.023% in the adrenal medulla to 0.00002% in the adenohypophysis. Relative abundance in the various brain areas varied greater than 20-fold, being highest in the caudate nucleus (0.0025%) and lowest in the thalamus and substantia nigra (0.0001%). Comparison with immunoreactive peptide concentrations in these tissues showed a close correlation between the levels of proenkephalin A mRNA and the immunoreactive peptides.     

Proenkephalin B Messenger RNA in Porcine Tissues: Characterization, Quantification, and Correlation with Opioid Peptides

Concentrations of proenkephalin B (PENK B) mRNA in porcine brain, pituitary, spinal cord, and peripheral tissues were measured using RNA blotting and solution hybridization. A single hybridizing species of approximately 2,800 bases in size was present in the CNS, with the highest concentration in the caudate nucleus, followed by hypothalamus and hippocampus. The abundance of PENK B mRNA ranged from 22 pg/micrograms of poly(A)-rich RNA in caudate nucleus to less than 0.1 pg/microgram in cerebellum. Concentrations of immunoreactive PENK B-derived peptides showed a similar distribution, with the exception of the hypothalamus, which had lower PENK B mRNA levels than expected from peptide concentrations. PENK B mRNA of the same size as in the brain was also found in the anterior lobe of the pituitary and in the heart ventricle, whereas in intestine, lung, and kidney, smaller mRNA species of 1,800 bases became apparent by RNA blot analysis. An intermediate size of 2,200 bases was found in heart atrium. As revealed by S1 mapping, however, these smaller mRNAs are not completely homologous with PENK B mRNA, but rather may represent closely related mRNAs from a different gene(s).     

THE EFFECTS OF PENTOBARBITONE-SODIUM ANAESTHESIA, SHOCK AVOIDANCE CONDITIONING AND ELECTRICAL SHOCK ON ACETYLCHOLINESTERASE ACTIVITY AND PROTEIN CONTENT IN REGIONS OF THE RAT BRAIN

Abstract— Pentobarbitone sodium anaesthesia was found to produce an increase in protein content in some regions of the rat brain, i.e. posterior cortex, caudate nucleus, and a decrease in protein content in the ventral cortex.
Acetylcholinesterase expressed in terms of wet weight was found to increase in the cerebellum, medulla, and to decrease in the medial cortex, hippocampus, thalamus and caudate nucleus. The changes in activity were not explicable in terms of a direct effect of the anaesthetic on the enzyme. A decrease in protein content of rat brain was observed in the frontal cortex, ventral cortex, hippocampus and caudate nucleus after electrical shocks. Following shock avoidance conditioning procedure (shuttle-box), decreases in protein content were observed in the medial cortex, posterior cortex, cerebellum and ventral cortex; in the thalamus an increase in protein content was observed.
Changes in AChE activity were observed following footshock in the frontal cortex and medulla where there was an increase in activity and in the caudate nucleus, hypothalamus, thalamus, and olfactory tubercle where there was a decrease in activity.
Following shock avoidance conditioning the activity of the AChE increased in posterior cortex, hippocampus, thalamus and hypothalamus and the activity of the enzyme decreased in the ventral cortex.     

Reduced Neuropeptide Y Concentrations in Suicide Brain

Neuropeptide Y (NPY) was measured in postmortem brain tissue from victims of suicide and from individuals dying a sudden natural or accidental death (controls). Concentrations of NPY-immunoreactivity were measured by radioimmunoassay in frontal cortex (BA 10), temporal cortex (BA 22), caudate nucleus, and cerebellum. Concentrations of NPY-immunoreactivity were significantly lower in postmortem frontal cortex (-14%) and caudate nucleus (-27%) from suicide victims compared with age-matched controls. A subgroup of suicides with evidence of a history of depression revealed more robust reductions in concentrations of NPY-immunoreactivity in frontal cortex and caudate nucleus, as did four individuals who died from natural causes and also were described as having a possible history of depression. Concentrations of NPY-immunoreactivity in temporal cortex and cerebellum from victims of suicide or from the subgroup of subjects with a possible history of depression were not significantly different from those of age-matched controls. We suggest there is a deficit in the brain NPY system leading to region-specific reductions in peptide concentrations in subjects who have a history of depression.     

Effect of Chronic Manganese Treatment on Adenosine Tissue Levels and Adenosine A2a Receptor Binding in Diverse Regions of Mouse Brain

In the present study the effects of chronic manganese (Mn) treatment on adenosine A_2a receptor binding in mouse brain have been assessed. Male albino mice were divided in two groups: In the Mn-treated group, the animals were injected intraperitoneally (i.p.) with MnCl₂ (5 mg/kg/day) five days per week during 9 weeks; in the control group, they were injected likewise with a saline solution. A significant decrease of the Kd without alteration of Bmax in the cerebellum and, an increase of the Kd and Bmax in hippocampus of mice treated with Mn were found. Also, an increase of Kd in frontal cortex was observed. The binding parameters in caudate nucleus, olfactory bulb and hypothalamus were not altered by Mn. A significant decrease in the adenosine concentration in caudate nucleus, olfactory bulb and hypothalamus, without significant changes in hippocampus, frontal cortex and cerebellum was also detected. These findings suggest that chronic administration of Mn could affect adenosine receptor function and turnover, depending on the brain region analyzed.     

Preferential affinity of 3H-2-oxo-quazepam for type I benzodiazepine recognition sites in the human brain

The hypnotic drug quazepam and its active metabolite 2-oxo-quazepam (2-oxo-quaz) are two benzodiazepines (BZ) containing a trifluoroethyl moiety on the ring nitrogen at position 1, characterized by their preferential affinity for Type I BZ recognition sites. In the present study we characterized the binding of 3H-2-oxo-quaz in discrete areas of the human brain. Saturation analysis demonstrated specific and saturable binding of 3H-2-oxo-quaz to membrane preparations from human cerebellum. Hill plot analysis of displacement curves of 3H-flunitrazepam (3H-FNT) binding by 2-oxo-quaz yielded Hill coefficients of approximately 1 in the cerebellum and significantly less than 1 in the cerebral cortex, hippocampus, caudate nucleus, thalamus and pons. Self and cross displacement curves for 3H-FNT and 3H-2-oxo-quaz binding in these brain areas indicated that 2-oxo-quaz binds with different affinities to two populations of binding sites. High affinity binding sites were more abundant in the cerebellum (95% of total sites), cerebral cortex, hippocampus and thalamus, whereas low affinity sites were predominant in the caudate nucleus and pons. Competition studies of 3H-2-oxo-quaz (2 nM) and 3H-FNT (0.5 nM) using unlabelled ligands indicated that compounds which preferentially bind to Type I sites are more potent at displacing 3H-2-oxo-quaz than 3H-FNT from cerebral cortex membrane preparations. The results suggest that 3H-2-oxo-quaz may be used for selectively studying Type I BZ recognition sites in the human brain.     

Distribution of testican expression in human brain

Testican is a putative extracellular heparan/ chondroitin sulfate proteoglycan of unknown function that is expressed in a variety of human tissues at widely different levels but is most abundant in the brain. In mice, testican mRNA has been detected only in brain and it is therefore likely to have an important function in the central nervous system. RNA blot analysis reveals the relative intensity of testican in various regions of the human brain. Levels of testican message are most pronounced in the thalamus, hippocampus, occipital lobe, nucleus accumbens, temporal lobe, and caudate nucleus, with somewhat lower levels in the cerebral cortex, medulla oblongata, frontal lobe, amygdala, putamen, spinal cord, substantia nigra, and cerebellum. In situ hybridization reveals the cellular distribution of the mRNA within these areas to be highest in neurons and in choroid plexus epithelium, and moderately lower in ependymal cells lining the ventricles and in vascular endothelial cells. Testican mRNA is not detected in oligodendrocytes or in most astrocytes. However, astrocytes in regions of reactive gliosis do express testican mRNA. These findings, along with a cysteine-rich pattern similarity to neurocan, brevican, versican, and other proteoglycans found in brain, suggest that testican may be a part of the specialized extracellular matrix of the brain.     

Purification from brain of synenkephalin, the N-terminal fragment of proenkephalin

Abstract: The primary sequence of adrenal proenkephalin was recently deduced from the structure of the cloned cDNA that codes for this protein. Several enkephalin-containing proteins with molecular weights between 8,000 and 20,000 daltons were purified from the bovine adrenal medulla. These proteins appear to represent intermediates in the processing of proenkephalin into physiologically active opioid peptides. While the concentrations of these large processing intermediates in the adrenal medulla are quite high, similar proteins have not yet been shown to be present in brain, and there is some question as to whether the brain synthesizes an enkephalin precursor similar to adrenal proenkephalin. We report here the purification from bovine caudate nucleus of synenkephalin, the N-terminal fragment of adrenal proenkephalin. The amino acid composition of synenkephalin indicates that the protein represents residues 1–70 of adrenal proenkephalin. Thus the brain and adrenal glands appear to utilize a similar precursor for enkephalin biosynthesis.     

Postnatal development of the scratch reflex in rabbits]

The development of the scratch reflex was studied in newborn (up to 2 months old) rabbits in norm and after elimination or activation of some parts of their nervous system (reticular formation, cerebellum, caudate nucleus, cerebral cortex, superior cervical sympathetic ganglia). The experiments with the section of the brain stem at the border between the medulla and the midbrain showed that in very young (5-10 days old) rabbits in norm the scratch reflex is controlled by the spinal cord with no influences of structures situated above the section's level. Later on the spinal mechanism of the scratch reflex becomes subject to supraspinal influences, among which in 2-3 weeks old animals facilitatory effects are predominant produced, in particular, by the reticular formation and the cerebellum, whereas in older age prevail inhibitory influences of the cerebral cortex, cerebellum, caudate nucleus and the sympathetic nervous system.     

FREE AMINO ACIDS AND RELATED COMPOUNDS IN FIVE REGIONS OF BIOPSIED CAT BRAIN

Abstract— Contents (μmol/g wet wt.) of 34 free amino acids and related compounds were measured in grey matter from three areas of cerebral cortex, from the cerebellum, and from the caudate nucleus in unanaesthetized cats with classical cerveau isolé preparations. Brain specimens were frozen in liquid nitrogen within 10 s of removal; thus, the values found were expected to approximate those which occur in living cat brain. Levels of most of the compounds measured were lower than those previously reported for the cat. In the case of GABA, alanine, and ethanolamine, the lower values found seemed attributable to the rapid freezing of brain tissue, and may more closely approximate levels occurring in living cat brain. On the other hand, the relatively low levels of aspartic and glutamic acids found may have resulted from use of the cerveau isolé preparation. Little difference in levels of amino compounds was found among the three cerebral cortical areas examined. However, there were significant differences in the contents of a number of amino acids between cerebral cortex and the cerebellum or caudate nucleus. These differences resembled those previously observed in autopsied human brain. The content of GABA was two-fold higher in biopsied cat cortex than in biopsied human cortex, whereas the content of cystathionine was only 10 per cent of that in human cortex. Homocarnosine and α-(γ-aminobutyryl)-lysine, two GABA-containing dipeptides found in relatively large amounts in human brain, were not detectable in cat brain. Living cat brain contained two amino acids not previously reported for this species:putreanine and ɛ- N -methyllysine.     

Regional Distribution of the GABAA/Benzodiazepine Receptor (α Subunit) mRNA in Rat Brain

A human cDNA clone containing the 5' coding region of the GABAA/benzodiazepine receptor alpha subunit was used to quantify and visualize receptor mRNA in various regions of the rat brain. Using a [32P]CTP-labelled antisense RNA probe (860 bases) prepared from the alpha subunit cDNA, multiple mRNA species were detected in Northern blots using total and poly A rat brain RNA. In all brain regions, mRNAs of 4.4 and 4.8 kb were observed, and an additional mRNA of 3.0 kb was detected in the cerebellum and hippocampus. The level of GABAA/benzodiazepine receptor mRNA was highest in the cerebellum followed by the thalamus = frontal cortex = hippocampus = parietal cortex = hypothalamus much greater than pons = striatum = medulla. In situ hybridization revealed high levels of alpha subunit mRNA in cerebellar gray matter, olfactory bulb, thalamus, hippocampus/dentate gyrus, and the arcuate nucleus of the hypothalamus. These data suggest the presence of multiple GABAA/benzodiazepine receptor alpha subunit mRNAs in rat brain and demonstrate the feasibility of studying the expression of genes encoding the GABAA/benzodiazepine receptor after pharmacological and/or environmental manipulation.     

Isolation and Characterization of Opioid Peptides from Rabbit Cerebellum

The rabbit cerebellum has been shown to contain significant quantities of opioid receptors consisting of both mu- and kappa-subtypes. To determine the nature of the endogenous opioid ligands in this tissue, extracts from rabbit cerebellum were separated by various chromatography techniques and fractions were assayed initially for opioid peptides with a radioimmunoassay capable of detecting all peptides with an amino-terminal Tyr-Gly-Gly-Phe sequence. This sequence is common to all mammalian opioid peptides and is critical for recognition by all known opioid receptors. Each of the three immunoreactive opioid peptide peaks detected was purified to homogeneity and subjected to amino acid composition and sequence analysis. One peak was analyzed further by mass spectrometry. This identified the major opioid peptides in the cerebellum as [Met5]enkephalin, [Leu5]enkephalin, and heptapeptide [Met5]enkephalyl-Arg6-Phe7. The comprehensiveness of this initial detection scheme in identifying biologically active opioid peptides was substantiated through subsequent analysis. Using specific radioimmunoassays for representative opioid peptides of the three opioid systems currently known, no other peptides of either the proenkephalin, proopiomelanocortin, or prodynorphin series were detected in any appreciable amounts. Collectively, these results are consistent with the position that rabbit cerebellar opioids are derived from proenkephalin. However, given that no appreciable quantities of either [Met5]enkephalyl-Arg6-Arg7-Val8-NH2 (metorphamide) or [Met5]enkephalyl-Arg6-Gly7-Leu8 were detected suggests that rabbit proenkephalin may have a slightly altered sequence and/or is differentially processed relative to other mammalian species studied.     

Twenty-four hour daily rhythms of glycine in the rodent brain

Male Sprague Dawley rats weighing 200–250 grams each were adapted for a minimum period of three weeks to a 12-hour light: 12-hour dark programmed illumination cycle with a temperature of 23 ± 1°C before used in this study. Every four hours for a twenty-four hour period glycine levels were measured in specific brain areas. Glycine levels were determined in the caudate nucleus, cerebellum, cortex, midbrain and pons by the method of Aprison and Shank (16). The highest level of glycine was observed in the pons. Through concentrations appeared in the cerebellum, cortex, midbrain and pons at D-0400 hours and the peak concentrations occurred at L-1600 hours. The glycine levels were significantly lower during the dark period of the light-dark cycle for all brains except the caudate nucleus.     

Distribution and characterization of synenkephalin immunoreactivity in the bovine brain and pituitary

The distribution of synenkephalin, the N-terminal fragment of proenkephalin, was studied in various parts of the bovine brain (globus pallidus, caudate nucleus, hypothalamus) and in the posterior pituitary by the use of a radioimmunoassay. The distribution of synenkephalin-immunoreactivity (IR) was compared to the distribution of Met-enkephalin-IR. Gel exclusion chromatography was used to examine the molecular forms of the immunoreactivities present in the tissues. The distribution of synenkephalin-IR was similar to the distribution of Met-enkephalin-IR, with a molar ratio of Met-enkephalin/synenkephalin ranging between 2.7 and 5.9. In all regions tested except the hypothalamus the synenkephalin-IR was present as a single species. However, in the hypothalamus a small amount of IR material (3% of the total synenkephalin-IR) was detected in fractions where larger Met-enkephalin-containing peptides eluted. Based on the concordance between the molar ratio of Met-enkephalin to synenkephalin found in the tissues and the molar ratio present in the sequence of adrenal proenkephalin, it is concluded that the brain and adrenal glands utilize a similar precursor for enkephalin biosynthesis.     

大白鼠脚内核的传入性联系

众所周知,肉食动物和大白鼠的脚内核,相当于灵长类的内侧苍白球(Nagy et al.1978;Fox and Schmitz 1944);它们的细胞形态、传入及传出均相同。早期以及近年来的一些研究工作者,虽然在研究其他核团的投射时,联系到一些本核团的传入,但是尚缺乏对本核团传人的系统研究。本实验即是应用辣根过氧化物酶的逆行传递法来研究大白鼠脚内核的传入性联系。     

Gangliosides in various brain areas of three inbred strains of mice

The ganglioside patterns of cerebellum, cortex, pons-medulla, hypothalamus, hippocampus and caudate nucleus of three inbred strains of mice (C57BL/6J, DBA/2J and BALB/cJ) have been analysed. All brain areas contained both the simple and complex species of gangliosides. GD1a was the major ganglioside in cortex, hippocampus and caudate nucleus whereas GT1b was the major species in cerebellum, hypothalamus and pons-medulla. In hippocampus, the percentages of GT1b and GD1a were quite similar. Pons and medulla exhibited the highest levels of GM1 (which approaches the value of GT1b) and the lowest values of GD1a. A ganglioside, termed here GT1L, was located between GD1b and GT1b. This ganglioside, which was present in highest amounts in cerebellum disappeared after alkali treatment. Highly significant differences were observed in the amounts and patterns of gangliosides among brain areas of the three strains. Highly significant differences (p<0.001) were also found in the ganglioside distribution of various brain areas among the strains, especially for tri-and tetrasialogangliosides between Balb and DBA. A significant difference of GM1 was observed in the cerebellum when comparing DBA with the two other strains. It is likely that the differences might be related to their relative abundances in certain cell types and for defining synaptic circuits in brain areas of some strains.     

Proenkephalin is a nuclear protein responsive to growth arrest and differentiation signals

Neuropeptide precursors are traditionally viewed as molecules destined to be cleaved into bioactive peptides, which are then released from the cell to act on target cell surface receptors. In this report we demonstrate nuclear localization of the enkephalin precursor, proenkephalin, in rodent and human embryonic fibroblasts (Swiss 3T3 and MRC-5 cells) and in rodent myoblasts (C2C12 cells). Nuclear proenkephalin, detected by immunofluorescence with a panel of antiproenkephalin monoclonal antibodies, is distributed predominantly in three patterns. Selective abolition of these patterns with salt, nuclease, or methanol is associated with liberation of immunoprecipitable proenkephalin into the extraction supernatant. Proenkephalin antigenic domains, mapped using phage display libraries and synthetic peptides, are differentially revealed in the three distribution patterns. Selective epitope revelation may reflect different conformational forms of proenkephalin or its existence in complexes with other nuclear proteins, forms which therefore have different biochemical associations with the nuclear substructure. In fibroblast cell populations in transition to growth arrest, nuclear proenkephalin responds promptly to mitogen withdrawal and cell-cell contact by transient, virtually synchronous unmasking of multiple antigenic domains in a fine punctate distribution. A similar phenomenon is observed in myoblasts undergoing differentiation. The acknowledgment of growth arrest and differentiation signals by nuclear proenkephalin suggests its integration with transduction pathways mediating these signals. To begin to address the mechanism of nuclear targeting, we have transfected mutated and nonmutated proenkephalin into COS (African green monkey kidney) cells. Nonmutated proenkephalin is localized exclusively in the cytoplasm; however, proenkephalin mutated at the first ATG codon, or devoid of its signal peptide sequence, is targeted to the nucleus as well as to the cytoplasm. From this we speculate that nuclear proenkephalin arises from a primary translation product that lacks a signal peptide sequence because of initiation at a different site.     

Studies on genomic DNA topology and stability in brain regions of Parkinson's disease

DNA damage has been postulated as a mechanism of neuronal death in Parkinson's disease (PD). In the present study, genomic DNA was isolated from eight brain regions (frontal, temporal, and occipital cortex, hippocampus, caudate/putamen, thalamus, cerebellum, and midbrain) from five neuropathologically confirmed cases of Parkinson's disease and six control brains and analyzed for the presence of single and double strand breaks, melting temperature, EtBr intercalation, DNAse digestion pattern, and DNA conformations. The results showed that DNA from midbrain in PD accumulated significantly higher number of strand breaks than age-matched controls. Caudate nucleus/putamen, thalamus, and hippocampus also showed more DNA fragmentation compared to control brains. Circular dichroism studies showed that DNA conformation was altered with imprecise base stacking in midbrain, caudate nucleus/putamen, thalamus, and hippocampus in PD. However, DNA from frontal, temporal, and occipital cortex, and cerebellum was not affected significantly in PD group as compared to controls. This study provides a comprehensive database on stability, damage, and conformations of DNA in different regions in brains of PD patients.