Aminopeptidase activity is asymmetrically distributed in selected zones of rat brain

Levels of soluble aminopeptidase (AP), measured as arylamidase activity using L-Leucine-2-Naphthylamide (Leu-2-NA) as substrate, were determined in the soluble fraction of eleven zones of rat brain. Results showed that AP activity is asymmetrically distributed in frontal cortex and hypothalamus with both left sides having significantly higher levels of AP activity, respectively, than the right sides. Simultaneously, the activities of lactate dehydrogenase (LDH) and glutamate-oxalacetate aminotransferase (GOT) were measured in the same cerebral regions; no significant difference was recorded in these activities between either side of the rat brain in any of the zones studied. Provided that aminopeptidases are involved in the degradation of some endogenously released neuropeptides, the results suggest a new mode of expression of cerebral lateralization.     

Aminopeptidase activity in the postmortem brain of human heroin addicts

Several studies have reported that the chronic administration of opioids induces changes in the biosynthesis of endogenous opioid peptides or their precursors in specific brain regions of the adult central nervous system. However, little is known about the catabolic regulation of opioid peptides and its contribution to neuroadaptative changes underlying drug addiction. In the present study, we have analyzed the activity of two enkephalin-degrading enzymes (puromycin-sensitive aminopeptidase or PSA and aminopeptidase N or APN) and two functionally different, soluble aminopeptidases (aminopeptidase B and aspartyl-aminopeptidase) in postmortem samples of prefrontal cortex and caudate nucleus of eight human heroin addict brains and eight matched-controls. Enzyme activities were fluorimetrically measured using beta-naphthylamide derivatives. An increase in the activity of soluble PSA in the prefrontal cortex of heroin abusers was observed (heroin addict group: 51,452+/-3892 UAP/mg protein versus control group: 42,003+/-2597 UAP/mg protein; P<0.05), while the activity of the other peptidases in both brain regions remained unaltered. This result agrees with previous findings in morphine-tolerant rats, and indicates that soluble PSA may be involved in neurobiological processes which underlie heroin addiction.     

Aminopeptidase P from rat brain. Purification and action on bioactive peptides.

Aminopeptidase P (EC 3.4.11.9) was purified from rat brain cytosol. A subunit Mr of 71,000 was determined for the reduced, denaturated protein whereas an Mr of 143,000 was determined for the native enzyme. The purified aminopeptidase P selectively liberated all unblocked, preferentially basic or hydrophobic ultimate amino acids from di-, tri- and oligopeptides with N-terminal Xaa-Pro- sequences. Corresponding peptides with penultimate Ala instead of Pro were cleaved with much lower rates; oligopeptides with residues other than Pro or Ala in the penultimate position appeared not to be substrates for the enzyme. Several bioactive peptides with Xaa-Pro sequences, especially bradykinin, substance P, corticortropin-like intermediate lobe peptide, casomorphin and [Tyr]melanostatin were shortened by the N-terminal amino acid by aminopeptidase P action. Rat brain aminopeptidase P was optimally active at pH 7.6-8.0 in the presence of Mn2+. Chelating agents and SH-reacting reagents inhibited the enzyme, but common inhibitors of aminopeptidases, like amastatin or bestatin, of prolidase or of dipeptidyl peptidases II and IV, like N-benzoyloxycarbonyl-proline or epsilon-benzyl-oxycarbonyl-lysyl-proline, as well as antibiotics like beta-lactam ones, bacitracin or puromycin, had little or no effect.     

Aminopeptidase P activity in rat organs and human serum

The substrate Lys(epsilon-Dnp)-Pro-Pro-NH-CH2-CH2-NH-ABz in which the fluorescent 2-aminobenzoyl (ABz) group (lambda ex = 320, lambda em = 410 nm) is intramolecularly quenched by the 2,4-dinitrophenyl (Dnp) chromophore was synthesized and used for the development of a sensitive assay for aminopeptidase P (EC 3.4.11.9). The emission of the intact compound was 160 times less than that of an equimolar concentration of Pro-Pro-NH-CH2-CH2-NH-ABz under the same conditions. The efficient resonance energy transfer permits an increased assay sensitivity as compared to the previously reported Phe(p-NO2)-Pro-Pro-NH-CH2-CH2-NH-ABz in which the p-nitrophenylalanyl [Phe(p-NO2)] residue caused only a 3.4-fold collisonal quenching. The kinetic constants Km were determined as 100 +/- 3.0 and 38 +/- 1.0 microM (mean of four experiments) for the human serum and the rat-lung enzymes, respectively. Both enzymes were inhibited by metal chelating agents and were not affected by 2.8 microM diisopropyl fluorophosphate. The mean activity in the sera of 53 healthy adults was 37.4 +/- 2.7 (standard error) with a standard deviation of 19.2 units/ml of serum. Only 10 microliters of serum was required for a reliable assay of the enzyme. The specific activity in rat-organ extracts was determined. High aminopeptidase P activity was observed in the testis, lung, kidney, and ovary and lower activity was observed in the serum.     

Acetylcholinesterase activity in developing rat brain during undernutrition

The effect of low protein diet on rat brain AChE activity has been studied during gestation, lactation and postweaning periods. There was decrease in enzyme activity of pups undernourished either during gestation and lactation or lactation alone, the decrease being maximum in 18-day-old pups. In postweaning rats, a significant decrease was observed after 2 and 4 weeks of undernutrition compared to the control. However, the effect of undernutrition was annuled by 2-week rehabilitation, thereby indicating that imposed undernutrition only delays the normal level of the enzyme. Moreover, it appears that the enzyme activity depends both on the nutritional status and the development age.     

Glutatmine synthetase activity in the chick brain during postnatal growth. Effect of MSO

Glutamine synthetase activity was estimated in the chick cerebral hemispheres, optic lobes and cerebellum between the 1st and the 30th day of postnatal growth. Glutamine synthetase activity is higher in the cerebellum than in the cerebral hemispheres and lowest in the optic lobes at 1 day after hatching; at 30 days after hatching, it is the same in the optic lobes and in the cerebellum and lowest in the cerebral hemispheres. The great increase of glutamine synthetase activity between the 1st and the 4th day after hatching corresponds to the appearance of the heterogeneity of the chick brain glutamate metabolism. The glutamine synthetase activity is inhibited by MSO $\text{in vivo}$ at a concentration of 100 mg kg ^?1 at values of 87, 90 and 89 % in cerebral hemispheres, optic lobes and cerebellum of 1, 2 and 4-day-old chicks. The enzyme inhibition is less pronounced $\text{in vitro}$ and reaches values of about 25 and 75 % for 1 and 10 mM MSO concentrations respectively in the three brain areas of the 1 to 4-day-old chick and values slightly lower in the 30-day-old chick brain.     

Changes of hippocampal protein levels during postnatal brain development in the rat

Information on postnatal brain protein expression is very limited, and we therefore compared hippocampal protein levels in rat hippocampus at different developmental time points using two-dimensional gel electrophoresis followed by mass spectrometrical protein identification and specific software for quantification. Proteins from several cascades as e.g., antioxidant, metabolic, cytoskeleton, proteasomal, and chaperone pathways were developmentally regulated, which is relevant for design and interpretation of protein chemical studies in the mammalian brain.     

Free choline levels in the rat brain

C holine levels in nervous tissue have been investigated by a number of workers in recent years. The values have varied widely from 39.3 nmol/g (E wetz , S parf and S öbo , 1970) to 700 nmol/g (S mith and S aelens , 1967). Many of the values published may have been too high for one of the following reasons: (1) post mortem formation of choline, (2) hydrolysis of phospholipids (PL) by extractants and (3) inadequate assay systems. In the past we too have obtained values which we can now with confidence say were too high due to the post mortem formation of choline. In a method which employed bioassay as an end-step after extraction of choline by acid-ethanol the values we obtained were 138 ±27 nmol/g. Despite criticism of this method by E wetz et al. (1970) and S chuberth , S parf and S undwall (1970a) we were reasonably sure that the assay system was both sensitive and specific, and that extraction with acid-ethanol did not lead to liberation of choline from PL, especially since values for plasma choline by this method were in a number of trial extractions as low as 8 nmol/ml. In view of these results we decided to re-investigate free choline levels in the brain using a method similar to that of E wetz et al. (1970) in that the living animal (in this case anaesthetized) was frozen in liquid nitrogen before removing the brain, and comparing the results of three different methods of analysis applied to brain extracts prepared in this way.     

Mitochondrial glycerol kinase activity in rat brain.

Glycerol kinase activity was found in the particulate fraction of rat brain homogenates predominantly associated with mitochondria. The enzyme remained bound to the particulate fraction after treatment with a variety of solubilizing agents.     

Aminopeptidase and lysozyme activity levels and serum protein concentrations in Biomphalaria glabrata (Mollusca) challenged with bacteria.

The total serum protein concentrations and levels of aminopeptidase and lysozyme activities in the sera of the gastropod Biomphalaria glabrata have been determined. The groups of snails from which hemolymph samples were taken for study included (1) untampered controls, (2) sham-injected snails, (3) heat-killed Bacillus megaterium-injected, and (4) live B. megaterium-injected ones. Our results indicate that there are significant elevations in the levels of aminopeptidase activity in 2 hr in the sera of snails that had been sham-, dead bacteria-, and liver bacteria-injected. The levels of lysozyme activity were not altered in sham-, dead bacteria-, and live bacteria-injected snails. This is contrary to an earlier finding (T. C. Cheng, M. J. Chorney, and T. P. Yoshino, 1977. J. Invertebr. Pathol., 29, 170–174), and the difference is believed to be due to the age of the snails employed. Comparisons of total serum proteins have revealed that the concentration in snails injected with live B. megaterium is significantly higher than in sham-injected ones. This may be due to increase of some yet undetermined serum protein fraction.     

Recovery of brain noradrenaline after 5,7-dihydroxytryptamine-induced axonal lesions in the rat

Time-dependent changes in regional CNS noradrenaline (NA) concentration, 3H-NA uptake and fluorescence morphology of CNS NA neurons were analysed in the adult rat up to 6 months after intraventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), and compared with the time-course of changes in brain and spinal cord indolamine neurons. Following a substantial depletion of both amines in all CNS regions (telodiencephalon, brainstem and spinal cord) at 10 days after 150 mug 5,7-DHT, brain NA--but not 5-HT--levels recovered to near-normal values in brainstem and forebrain (35% below the age-matched controls) within 4 months. This was accompanied by a total restoration of the initially decreased capacity of the brain tissue to accumulate 3H-NA in vitro. Within 10 days after 5,7-DHT, there was a disappearance of NA terminals from many telencephalic, diencephalic and lower brain stem nuclei, from the cerebral and cerebellar cortices, and the grey matter of the spinal cord, concomitant with the appearance of numerous distorted, highly fluorescent swellings along the non-terminal axons of the major noradrenergic projection pathways. The recovery of the NA levels was paralleled by a re-appearance of fluorescent fibres, signifying an intense sprouting and regrowth of the drug-lesioned axons, which eventually re-innervated some of the previously denervated telodiencephalic regions. Except for a permanent loss of some surface-near perikarya in group A1 (the main source of the bulbospinal projections) there was no evidence of a retrograde degeneration of noradrenergic cell bodies in the rat CNS. The results are compatible with the idea that 5,7-DHT mainly causes a lesion of NA axons at a distance from the cell bodies, and this is followed by sprouting and regrowth of axons from the lisioned neurites, and formation of new terminal-like fibres in some previously denervated telodiencephalic regions. These findings indicate that chemical axotomy of central NA neurons induced by 5,7-DHT is--in contrast to that induced by 6-hydroxydopamine--followed by extensive axonal regeneration.     

Patterns of growth, axonal extension and axonal arborization of neuronal lineages in the developing Drosophila brain

The Drosophila central brain is composed of approximately 100 paired lineages, with most lineages comprising 100-150 neurons. Most lineages have a number of important characteristics in common. Typically, neurons of a lineage stay together as a coherent cluster and project their axons into a coherent bundle visible from late embryo to adult. Neurons born during the embryonic period form the primary axon tracts (PATs) that follow stereotyped pathways in the neuropile. Apoptotic cell death removes an average of 30-40% of primary neurons around the time of hatching. Secondary neurons generated during the larval period form secondary axon tracts (SATs) that typically fasciculate with their corresponding primary axon tract. SATs develop into the long fascicles that interconnect the different compartments of the adult brain. Structurally, we distinguish between three types of lineages: PD lineages, characterized by distinct, spatially separate proximal and distal arborizations; C lineages with arborizations distributed continuously along the entire length of their tract; D lineages that lack proximal arborizations. Arborizations of many lineages, in particular those of the PD type, are restricted to distinct neuropile compartments. We propose that compartments are “scaffolded” by individual lineages, or small groups thereof. Thereby, the relatively small number of primary neurons of each primary lineage set up the compartment map in the late embryo. Compartments grow during the larval period simply by an increase in arbor volume of primary neurons. Arbors of secondary neurons form within or adjacent to the larval compartments, resulting in smaller compartment subdivisions and additional, adult specific compartments.     

Superoxide dismutase activity in rat brain during acute and chronic alcohol intoxication

The effect of acute and chronic ethanol administration on rat brain superoxide dismutase (SOD) activity was studied. Intraperitoneal injections of ethanol led to an inhibition of SOD activity. When ethanol was fed as the sole fluid, the SOD activity decreased progressively, reaching a plateau after 6 weeks of treatment. Withdrawal of ethanol produced a recovery of control values within 48 hr. SOD activity was also decreased in rats born from ethanol-drinking mothers. Inhibition of SOD activity by ethanol may allow an accumulation of cytotoxic O₂ ^– radicals; this may account for some nervous system disorders during alcohol intoxication.