Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse liver

Phenobarbital-induced coumarin 7-hydroxylase is high in DBA/2J and low in C57BL/6N inbred mice; this genetic difference is encoded by the Coh locus on chromosome 7. The aim of this study was to develop an antibody specific for this cytochrome P-450 polymorphism. P-450 fractions, highly specific for phenobarbital-inducible coumarin 7-hydroxylase activity, were purified from DBA/2J and C57BL/6N mouse liver microsomes. Both proteins are 49 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soret peaks of the reduced cytochrome . CO complexes are 451 nm. Reconstituted DBA/2J coumarin 7-hydroxylase activity exhibits a V twice as high as, and a Km value 10-fold less than, the reconstituted C57BL/6N activity. Antibodies were raised in rabbit. By Ouchterlony immunodiffusion, both antibodies show 100% cross-reactivity with DBA/2J and C57BL/6N microsomes and purified antigens. Yet, DBA/2J but not C57BL/6N 7-hydroxylase activity is inhibited by the antibody to DBA/2J P-450. Both DBA/2J and C57BL/6N activities are blocked by the antibody to C57BL/6N P-450. Neither antibody has any effect on liver microsomal d-benzphetamine N-demethylase, ethylmorphine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, acetanilide 4-hydroxylase, or aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. The DBA/2J protein most specific for phenobarbital-induced coumarin 7-hydroxylation is designated 'P-450Coh'. Anti-(P-450Coh) precipitates a relatively minor 49-kDa protein from detergent-solubilized microsomes and from in vitro translation of poly(A+)-enriched total RNA of phenobarbital-treated DBA/2J mouse liver, whereas the major phenobarbital-induced P-450 proteins exhibit a molecular mass of about 51 kDa. The immunoprecipitated translation products correspond to a messenger RNA of 2100 +/- 100 nucleotides.     

Myelin development in infant brain

Myelin was isolated from subcortical areas of ten human brains, with ages ranging from 24 days to 350 days-of-age; samples were subsequently analyzed for lipid composition. Eight infants were victims of Sudden Infant Death Syndrome, and two infants were accident cases. Gray and white matter samples from each brain were also dissected and analyzed. Galactolipids were only 12% of the total lipids in white matter from brains of infants that were 24 days-of-age, a time when myelination was just starting in the subcortical areas. At 175 and 350 days of age, myelination was well underway and galactolipids measured 22% of the total lipids. Total phospholipids decreased (65% to 54%) in white matter during development, with the decrease mostly in phosphatidylcholine (23% to 15%). Even though there was little white matter present at early ages, myelin could be isolated. Surprisingly, the lipid composition of myelin, from the 24-day-infant brain was similar to that from adult brain. Galactolipids were 22% of the total lipids, cholesterol, 23%, and phospholipids, 52%. These results suggest that only subtle remodeling of myelin occurs in humans once myelination commences. All four major gangliosides were present in myelin during this first year of development. Interestingly, the yield of myelin from the 350-day-old infant subcortical white matter was similar to that from an adult. Thus major tracts in this area may have acquired most of the myelin by one year-after-birth. Since the control samples blend quite well into the developmental pattern obtained, it is believed there are no abnormalities in myelin lipids from SIDS infants.     

ISOLATION AND IDENTIFICATION OF γ-AMINOBUTYRYL-CYSTATHIONINE FROM HUMAN BRAIN AND CSF

Abstract— A new dipeptide, γ-aminobutyryl-cystathionine, has been identified in human brain and CSF. The compound was isolated from peptide concentrates which were prepared by removing free a-amino acids from deproteinized brain extracts on a copper Sephadex column. The isolated peptide was shown to be GABA-Cysta by standard chemical methods, and its identification was confirmed by mass spectrometry. GABA-Cysta is present in both biopsy and autopsy specimens of adult human brain, its content in some brain areas being as high as 0.090 μrnol/g wet weight. Its concentration in CSF is much lower. What physiologic role this unusual peptide plays in brain remains to be determined.     

Glutathione contents of tissues in the aging mouse.

1. Previous results from this laboratory demonstrated that the erythrocyte content of reduced glutathione (GSH) decreased as a function of both increasing cell age and mouse age [Abraham, Taylor & Lang (1978) Biochem. J. 174, 819-825]. In the present investigation glutathione concentrations were determined in other tissues of the C57BL/6J mouse of different ages (6--31 months) throughout the life-span. 2. At all ages the total glutathione and the GSH concentrations in liver were 3 times that in kidney and 10 times that in heart. In the old (31 months) mouse the GSH contents were lower by 30% in the liver, 34% in the kidney and 20% in the heart than in the mature (17--23 months) animals. 3. The oxidized glutathione (GSSG) concentrations of the tissues did not vary with age and constituted less than 3% of the total glutathione. 4. The decreased in GSH concentrations were not due to changes in organ weights, which were constant from 10 to 36 months of age. 5. These findings extend our previous results and indicate that a general characteristic of aging tissues may be a decrease in GSH concentrations. Further, this is consistent with our hypothesis that the reducing potential of tissues decreases in senescence.     

Sexual development of patients with isochromosomes for the long arm of the X chromosome

Summary The sexual development of 14 girls with non-mosaic monocentric 46,X,iXq karyotype was studied. Seven out of eight girls were found to have immature secondary sexual characteristics and amenorrhoea, a finding greatly contrasting with that in Triplo-X girls. The relative ineffectiveness of the isochromosome Xq in maintaining fertility may be due to the absence of one short arm, which probably also carries a gonadal determinant. Alternatively, the presence of two inactivation sites on one isochromosome may render the gonadal determinants inactive at an important stage in gonadal development.     

BIOCHEMICAL EFFECTS IN MAN and RAT OF THREE DRUGS WHICH CAN INCREASE BRAIN GABA CONTENT

Abstract— Brain amino acids were measured in rats given aminooxyacetic acid (AOAA) by mouth, and in rats given sodium dipropylacetate (DPA) both orally and by intraperitoneal injection. Brain GABA content was significantly elevated by AOAA doses of 10mg/kg/day, but not by 5mg/kg/day. Approximately 4 times as much AOAA is required by mouth as by parenteral injection to raise brain GABA content in the rat. DPA (400mg/kg) increased brain GABA and lowered brain aspartate content significantly 1 h after a single injection. However, DPA given orally (350 mg/kg/day) produced no alterations of any amino acids in rat brain.
Amino acids were measured in plasma and urine from patients treated orally with isonicotinic acid hydrazide (INH) or DPA, and from a volunteer who took AOAA. INH (10–21 mg/kg/day) increased concentrations of β -alanine and ornithine in plasma, as well as urinary excretion of β -alanine. DPA had no such effect. AOAA in oral doses ranging from 1.25 to 5.0 mg/kg/day increased plasma concentrations of β -alanine, ornithine, β -aminoisobutyric acid, proline and hydroxyproline, and produced massive urinary excretion of β -alanine, β -aminoisobutyric acid, and taurine.
Both INH and AOAA, given in doses practical for human use, inhibit the transamination of β -alanine and ornithine in liver, and may also inhibit the transamination of GABA in brain. In addition, AOAA interferes with the catabolism of β -aminoisobutyric acid, proline, and hydroxyproline. AOAA, in the lowest dose employed, appeared more effective than INH as an inhibitor of GABA aminotransferase in man, and might therefore be useful in the treatment of neurological diseases in which brain GABA is deficient.