The Biosynthesis of Transfer Ribonucleic Acid in the Developing Rat Brain and in Cultured Glial Cells

The biosynthesis of tRNA was investigated in cultured astroglial cells and the 3-day-old rat brain in vivo . In the culture system astrocytes were grown for 19 days and were then exposed to [ 3 H]guanosine for 1.5–7.5 h; 3-day-old rats were injected with [ 3 H]guanosine and were killed 5–45 min later. [ 3 H]tRNA was extracted, partially purified, and hydrolyzed to yield [ 3 H]-guanine and [ 3 H]methyl guanines. The latter were separated from the former by high performance liquid chromatography and their radioactivity determined as a function of the time of exposure to [ 3 H]guanosine. The findings indicate that labeling of astrocyte tRNA continued for 7.5 h and was maximal, relative to total RNA labeling, at 3 h, while in the immature brain tRNAs were maximally labeled at 20 min after [ 3 H]guanosine administration. The labeling pattern of the individual methyl guanines differed considerably between astrocyte and brain tRNAs. Thus, [ 3 H]1-methylguanine represented up to 35% of the total [ 3 H]methyl guanine radioactivity in astrocyte [ 3 H]tRNA, while it became only negligibly labeled in brain [ 3 H]tRNA. Conversely, brain [ 3 H]tRNA contained more [ 3 H]N 2 -methylguanine than did astrocyte [ 3 H]tRNA. Approximately equal proportions of [ 3 H]7-methylguanine were found in the [ 3 H]tRNAs of both neural systems. The [ 3 H]methylguanine composition of brain [ 3 H]tRNA was followed through several stages of tRNA purification, including benzoylated DEAE-cellulose and reverse phase chromatography (RPC-5), and differences were found between the [ 3 H]methylguanine composition of RPC-5 fractions containing, respectively, tRNA lys and tRNA phe . The overall results of this study suggest that developing brain cells biosynthesize their particular complement of tRNAs actively and in a cell-specific manner, as attested by the significant differences in the labeling rates of their methylated guanines. The notion is advanced that cell-specific tRNA modifications may be a prerequisite for the successful synthesis of cell-specific neural proteins.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65406/1/j.1471-4159.1980.tb09020.x.pd

A REGIONAL STUDY OF SOME OSMOTIC, IONIC AND AGE FACTORS AFFECTING THE STABILITY OF CEREBRAL LYSOSOMES 1

An examination was made of the effect of changes in the osmolarity and the ionic composition of the homogenizing medium on the partition of lysosomal arylsulphatase and N -acetylglucosaminidase of cerebral cortex, hypothalamus and thalamus of the rat. Sulphatase appeared to be more sensitive to hypotonicity than glucosaminidase, since a higher proportion of the sulphatase was released from the lysosomes into the soluble fraction of the cells from all three neuroanatomical areas examined. In the presence of 250 mM-sucrose, supplementation with 10 mM-Mg led to clumping of the lysosomes and their translocation into the heavy-particulate fraction; no such effect of 10 mM-Mg was noted in the absence of 250 mM-sucrose. The intracellular distribution of bound N -acetylneuraminic acid (bound-NANA) was also examined. The shifts observed in its intracellular localization as a result of changes in the ionic composition of the homogenizing medium rule out bound-NANA as a structural component of the membrane of the cortical lysosome. However regional differences in the response of bound-NANA to ionic factors were observed. Lysosomes from cerebral cortex of adult and 12-day-old rats were also compared. Differences in the pattern of distribution of lysosomes in linear sucrose gradients and in response to ionic factors were uncovered. The results support the previously enunciated concept (Sellinger and Hiatt, 1968) of a regional microheterogeneity of lysosomes and add a new, age-related dimension to it.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65807/1/j.1471-4159.1969.tb05969.x.pd

PROTEIN SYNTHESIS IN NEURONS AND GLIAL CELLS OF THE DEVELOPING RAT BRAIN: AN IN VIVO STUDY 1

A technique for the isolation of pure neuronal perikarya and intact glial cells from cerebral cortex has been developed for routine use. The yield of neuronal perikarya and glial cells was greater from highly immature (5–10 days) rat cerebral cortex than from the cortex of older rats (18–43 days). The perikarya/glia yield ratio decreased with age indicating that, as the glial population matured, the procedure succeeded in isolating a gradually smaller proportion of the existing neurons. The perikarya/glia ratio was highest for the 5-day-old cortex in which no mature glial cells could be identified. After a 10-min pulse in vivo of intrathecally injected [ 14 C]phenylalanine, the specific radioactivity of the neuronal proteins was higher than that of the glial proteins in the 5-, 10- and 18-day-old rat but was lower in the 43-day-old rat. The values for absolute specific radioactivity of the 14 C-labelled proteins in both cell types were greater, the younger the brain. The 14 C-labelling of neuronal and glial proteins in the 18-day-old rat was assessed in vivo as a function of time by determining the incorporation of [ 14 C]phenylalanine into such proteins at 5, 10, 20 and 45 min after administration of the amino acid. The rate of incorporation of [ 14 C]phenylalanine into the glial cells was faster than into the neurons since higher specific radioactivities of the glial proteins could be achieved at earlier times. Also, a biphasic pattern of 14 C-labelling of the glial proteins was noted, suggesting, perhaps, a sequential involvement of the oligodendrocytes and astrocytes. Homogenates of prelabelled neuronal perikarya were fractionated into the nuclear, mitochondrial microsomal and soluble cell sap fractions. In the 18-day-old cerebral cortex, the proteins of the microsomal fraction exhibited the highest specific radioactivity at the end of 10 min, whereas by 20 min proteins of the mitochondrial fraction were most highly labelled. The specific radioactivity of the nuclear proteins increased over the entire 45-min experimental period. On the contrary, the proteins of the soluble cell sap, in which the specific radioactivity was at all times by far the lowest, were maximally labelled by 5 min. Examination of the labelling of the neuronal subcellular fractions as a function of age revealed that at 10 min after administration of [ 14 C]phenylalanine, the specific radioactivities of all 14 C-labelled proteins were highest in the youngest (5-day-old) neurons. The proteins of the microsomal fraction were most rapidly labelled at all ages. During this interval the proteins of the soluble cell sap were only moderately labelled in the 5-day-old neurons and were totally unlabelled in the 43-day-old neurons, indicating age-dependent differences in the rate of utilization of the amino acid precursor by the neurons.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65968/1/j.1471-4159.1971.tb00007.x.pd

METHYLATION OF E. COLI TRANSFER RIBONUCLEIC ACIDS BY A tRNA ADENINE-l-METHYLTRANSFERASE FROM RAT BRAIN CORTEX AND BULK-ISOLATED NEURONS

Brain cortices or bulk-isolated neuronal cell bodies prepared from cortices of 8-day old male rats were used as the source of a l-methyl adenine-specific tRNA methyltransferase (tRNA-AMT). Ammonium sulfate fractionation and chromatography on spheroidal hydroxylapatite and Sephadex G-200 yielded an 80-fold purified enzyme, as determined by using E. coli bulk tRNA as substrate. The kinetic parameters of tRNA-AMT for the substrate S -adenosyl-l-methionine (SAM) ( K m = 6 ΜM) and the inhibitor, S -adenosyl-l-homocysteine (SAH) ( K i = 3.4 Μm) were determined and several SAH analogs tested as inhibitors. S -Adenosyl-l-cysteine (SAC) ( 10 -4 m) and S -adenosyl-d-homocysteine (SADH) (10 -4 m) produced a 35 and a 21% reduction in enzyme activity, respectively. The effects of Mg 2+ , NH 4 + acetate and of the polyamines spermine, putrescine and spermidine on the brain tRNA-AMT mimicked the effects of these agents on hepatic tRNA-AMT (Glick et al , 1975). Comparing the ability of cerebral tRNA-AMT to methylate E. coli tRNA glu2 , tRNA val , tRNA phe and bulk tRNA revealed tRNA glu2 as the best and tRNA phe as the least effective substrate. tRNA-AMT prepared from neuronal cell bodies showed closely similar characteristics to the cortical enzyme. A comparison of the activities of tRNA-AMT in neurons and glial cells revealed higher values in the former.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66287/1/j.1471-4159.1978.tb12436.x.pd

THE ELEVATION OF CEREBRAL HISTAMINE- N -AND CATECHOL- O -METHYL TRANSFERASE ACTIVITIES BY l-METHIONINE-dl- SULFOXIMINE 1

The administration of the convulsant, l-methionine-dl-sulfoximine (MSO), increased histamine N -methyl transferase (E.C. 2.1.1.8) (HMT) activity in rat and mouse brain and, to a lesser extent, catechol- O -methyl transferase (E.C. 2.1.1.6) (COMT) activity in rat brain. The duration of this effect was shortened by co-administration of l-methionine. The increased HMT activity was seen in 5 or 7 rat brain regions tested. l-Methionine administration had no effect on the activity of either enzyme. Partially purified HMT preparations from rat or guinea-pig brain exhibited no alterations in activity after the in vitro addition of MSO or l-methionine over a wide range of histamine and S -adenosyl-l-methionine concentrations. Rat brain COMT was equally unaffected by MSO and l-methionine. The in vitro inhibition of HMT and COMT by S -adenosyl-l-homocysteine was the same whether tested on preparations derived from MSO-treated or control animals. The data are discussed with respect to the possible involvement of aberrant methylation processes in the MSO-induced seizure.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65751/1/j.1471-4159.1975.tb07696.x.pd

Decreased Transmethylation of Biogenic Amines After In Vivo Elevation of Brain S -Adenosyl-l-Homocysteine

The ability of S -adenosyl-l-homocysteine (AdoHcy) to inhibit biologic transmethylation reactions in vitro has led us to explore the possibility of pharmacologically manipulating AdoHcy levels in vivo and examining the consequences of these alterations on the transmethylation of some biogenic amines. Swiss-Webster mice were injected intraperitoneally with different doses of adenosine (Ado) and d,l-homocysteine thiolactone (Hcy) and were killed at various times thereafter. S -Adenosyl-l-methionine (AdoMet) and AdoHcy concentrations were determined by using a modified isotope dilution-ion exchange chromatography-high pressure liquid chromatography technique sensitive to less than 10 pmol. Increasing doses of Ado + Hcy (50-1000 mg/kg of each) produced a dose-related increase in blood, liver, and brain AdoHcy levels. At a dose level of 200 mg/kg Ado + Hcy, AdoHcy levels were markedly elevated, with minimal concomitant perturbations of AdoMet. This elevation was maximal 40 min after giving Ado + Hcy, returning to control values within 6 h. Ado + Hcy treatment resulted in decreased activities of catechol- O -methyltransferase, histamine- N -methyltransferase, and AdoHcy hydrolase in vitro. The cerebral catabolism of intraventricularly administered [ 3 H]histamine (HA) was decreased in a dose-related manner by Ado + Hcy treatment as evidenced by higher amounts of nonutilized [ 3 H]HA in brain, concurrent decreases in [ 3 H]methylhistamine formation, and decreases in the transmethylation conversion index. Steady state levels of HA also showed dose-related increases after Ado + Hcy treatment. It is concluded that injections of Ado + Hcy can markedly elevate AdoHcy levels in vivo , which can, in turn, decrease the rate of transmethylation reactions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66442/1/j.1471-4159.1981.tb00426.x.pd

RAT AND MOUSE BRAIN HISTAMINE N -METHYLTRANSFERASE: MODULATION BY METHYLATED INDOLEAMINES 1

A purification procedure for rat and mouse brain histamine N -methyltransferase (HMT, EC 2.1.1.8) is described which achieves the preparation of 87-fold purified rat brain and 166-fold purified mouse brain enzyme. The purified HMT (MW 29,000) is inhibited by a number of physiologically and pharmacologically active amines, among them several methylated indoleamines, at concentrations above 5 ± 10 -6 M. At concentrations below 1 ± 10 -7 M, most of the methylated indoleamines stimulate HMT , provided histamine is maintained at, or close to, its optimal concentration as an HMT substrate, namely 1 ± 10 -5 M. A study of the nature of the inhibitory process revealed a non-competitive inhibition of HMT by dopamine as against a competitive inhibition of the enzyme by most methylated indoleamines. Increasing the concentration of histamine beyond the optimal value, i.e. to inhibitory levels, resulted in less stimulation. The findings support the notion that MSO elicits the formation in selected brain cells of supranormal amounts of several methylated indoleamines which are able to stimulate HMT (and possibly other methyltransferases, see Salas et al. , 1977), thereby causing the depletion of the cerebral levels of S-adenosyl-L-methionine, reported previously (Schatz & Sellinger, 1975b).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65930/1/j.1471-4159.1978.tb06548.x.pd

ASTROGLIAL UPTAKE IS MODULATED BY EXTRACELLULAR K +

Primary cultures of rat brain astrocytes were used to examine the uptake of the glucose analogue, 2-deoxy- d -glucose (2-DOG). 2-DOG competes with glucose for uptake, indicating that both are transported by the same carrier system. Extracellular K + at 11.9 mM increased the uptake of 2-DOG at 2-DOG concentrations greater than 100 ΜM. Uptake. appears Na + -dependent only at high concentrations of 2-DOG. This suggests that the extracellular concentrations of Na + and K + may regulate the astrocytic uptake of 2-DOG.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65513/1/j.1471-4159.1979.tb05224.x.pd

Biosynthesis of Polyamines in Mouse Brain: Effects of Methionine Sulfoximine and Adenosylhomocysteine

This study examines the consequences on cerebral polyamine biosynthesis of increases and decreases in cerebral methylation. Increases were elicited by administering the convulsant agent methionine sulfoximine (MSO) and decreases by elevating in vivo the cerebral levels of the methylation inhibitor S -adenosyl-homocysteine. Following the intraventricular (i.vt.) administration of one of the two possible polyamine precursors, [1,4- 14 C]putrescine, the specific radioactivity (sra) of the newly formed [ 14 C]spermidine remained unchanged. Conversely, after i.vt. l-[3,4- 14 C]methionine, the other polyamine precursor, significantly higher sra values for [ 14 C]spermidine and [ 14 C]spermine were recorded in the brains of the MSO-treated animals. [ 14 C] S - adenosylmethionine in the brain of the MSO-treated animals was also more highly labeled following [1- 14 C]-methionine, indicating its accelerated formation relative to controls. We also investigated the effect of the administration of adenosine + homocysteine, a treatment that results in elevated brain adenosylhomocysteine levels, on polyamine biosynthesis from [3,4- 14 C]-methionine. The results of these experiments show both significantly lower sra values for [ 14 C]spermidine and [ 14 C]spermine and significantly higher than control endogenous methionine levels, a clear sign of the existence of a retardation in the conversion of methionine to polyamines under these conditions. In conclusion, the present study demonstrates that while interference with cerebral methylation results in significant alterations of the rate of formation of the methionine moiety of spermidine and spermine, it has no effect on the entry of the putrescine moiety into the two polyamine molecules.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66159/1/j.1471-4159.1983.tb08055.x.pd

Kinetics of Carboxylmethylation of the Charge Isoforms of Myelin Basic Protein by Protein Methyltransferase II

The charge isoforms (C1-C5) of bovine myelin basic protein (MBP) were used as substrates for the rat brain enzyme protein carboxylmethyltransferase (PM II). The objective of these experiments was to ascertain whether the kinetic behavior of the MBP isoforms reflected differences in the structures of this molecular family. Initial velocity plots as a function of the MBP-isoform concentration showed significnt differences ( p > 0.05) among the assayed isoforms except for isoforms C2 and C4. Under the conditions of our experiment all the curves exhibited a consistent sigmoidicity. The kinetic data were best fitted by a model, previously described for the enzyme D-Β-hydroxybutyrate dehydrogenase, in which two independent sites must be randomly occupied before any catalytic activity can occur. This mechanism is substantially different from that proposed by other investigators for similar PM II enzymes and other substrates. The differences in the rates of isoform carboxylmethylation are largely accounted for by the different apparent dissociation constants K s and is explained on the basis of inherent structural differences among the charge isoforms.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65821/1/j.1471-4159.1989.tb09257.x.pd