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A 17,000 g supernatant of mouse brain microsomes was subfractionated on a continuous sucrose gradient in order to localise ceramide galactosyltransferase (CGalt, EC 2.4.1.47) and cerebroside sulphotransferase (CST, EC 2.8.2.1 l), both enzymes involved in the synthesis of myelin lipids. The submicrosomal fractions were analysed for marker enzymes of myelin, plasma membranes, Golgi membranes, endoplasmic reticulum and lysosomes, and their protein distribution was studied. The results and EM studies give evidence that CGalT and CST are located in the Golgi membranes of the brain.

The association of NK cell counts and beta2-microglobulin or sIL-2r serum levels with prognostically favorable factors such as ER positive tumor and older age supports the assumption that the immune system plays a role in the course of early breast cancer. The exact nature of this role requires further study.

SummaryThe experiments reported herein compare growth kinetics and biochemical properties of cultured skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) and matched normal controls.On day 7 after plating (6000 cells/cm2) cell number and DNA per dish are significantly reduced ( P < 0.001) in the cultures from DMD patients (n = 14)' compared to those from controls (n = 10). Moreover DMD cells contain less lipids and proteins per dish but more per cell than normal fibroblasts (not significant). Variations of media (McCoy's medium instead of Eagle's minimum essential medium) and sera (human cord serum instead of fetal calf serum) resulted in the same differences between DMD and control cells.Cell kinetic experiments (plating density: 2000 cells/cm2) show increased doubling times of DMD fibroblasts ( P < 0.001; ~D M D = 5; n,o,t,o~s = 4) whereas plating efficiency is equal for both DMD and controls.On day 7 the activity of the membrane bound enzyme 9-nucleotidase either per mg protein or per pg DNA is significantly elevated in cells from DMD patients ( P < 0.0005; nDMD = 8; nCont,,ls = 9) independent of cell density. Thus all findings in cultured DMD fibroblasts: increased doubling time, tendency to more voluminous cells, and elevated 5'-nucleotidase activity per cell suggest, that the DMD cells behave similar to prematurely aging cells. Until now we were not able to check whether any alterations of the plasma membrane are inducing early senescence or, reversely, premature aging is the cause of the postulated membrane alterations.If these findings were to be confirmed in cultured amniotic cells 13, 27, 40). All these findings are supposed to be secondary or tertiary alterations. The underlying metabolic defect is not yet known. Cultured fibroblasts obtained from explants of human skin biopsies have proved to be very useful for the investigations of numerous inborn errors of metabolism. Studies of cultured fibroblasts from DMD patients have been initiated only very recently. There are reports of altered morphology (43), reduced cell aggregation (17), decreased protein synthesis (5, 29), and decreased lysosomal dipeptidyl aminopeptidase I activity (1 1).Herein we report the results of investigations on cultured fibroblasts from patients with DMD: plating efficiency, doubling time, effects of different media and sera, 14C-thymidine incorporation in DNA, and activity of the membrane bound enzyme 5'-nucleotidase (EC 3.1.3.5.). MATERIALS AND METHODS MATERIALWe studied fibroblasts growing from skin biopsies of 14 patients with DMD, aged 4 to 14 yr, and of 10 normal boys matched by age, who were undergoing surgical treatment. The cells were usually cultured in Eagle's minimum essential medium (MEM), supplemented with nonessential amino acids and 10% fetal calf serum (FCS). To test the influence of different growth conditions, McCoy's 5A modified medium (26) was used instead of MEM, and MEM was supplemented with 10% human cord serum instead of FCS. METHODS Dfrom DMD fetuses, they could serve as a potential ...

Summaryuted exclusively to hypoglycemia could not be defined. Hypoglycemia in the newborn is usually accompanied by secondary pheThe present work was designed to a mode' for studying nomena, such as hypoxia, circulatory failure, or toxic influences the effect of substrates on brain cell metabolism. The synthesis of due to infections, all of which may lead to brain damage per se, sulfatide, a myelin lipid, which takes place predominantly in oliBrain changes in animals induced by expenmental hypoglycegodendrocytes was measured in vitro in dissociated cell cultures of mia showed a decrease of brain weight with decreased total protein newborn mouse brains a t 13 days in culture. This corresponds to and DNA in young rats (8), swelling of neurones and oligodenan active stage of in vivo myelination. The effect of increasing droglia with only mild swelling of astrocytes in rat brains durations glucose (below in the and cortical changes in rhesus monkeys (19,30). Metabolic conwas assessed 0' ""'r~ho'og~~ ce'"lar protein, DNA, sequences hypoglycemia in animals comprise alterations in sulfatide synthesis, cerebroside sulfotransferase activity, and mu-brain aminoacids (25. 41). glucose and glycogen (8, 25) and copolysaccharide synthesis.glycolytic as well as citric acid cycle intermediates (25). The brain was by the "Itured above a high-energy phosphates were depleted only at marked hypoglyconcentration of 0.4 mM in the medium. At high and intermed~ate cemia (26). The membrane lipids cholesterol and phospholipid glucose concentrations. between 43.8 and 63.6% of the glucose we_ diminished (*), Brain cerebroside as well as sulfate incorpoconsumed was accumulated a s Iactate on a molar ratio, indicating ration into the myelin lipid sulfatide was also reduced, whereas that the pyruvate deh~drobenaw com~lex was rate limitin& In lipid N-acelyl-neuraminic acid as a measure of gangliosides losevere glucose deprivation. Iactate consumption of the cultures cated in neurones was not by hypoglycemia (*).exceeded accumulation. The purpose of the present work was to characterize the effect With increasing duration of severe glucose deprivation (below of glucose deprivation on a specific step in structural brain metabmM)* the fOllowing major changes wereThe number olism during early development. Sulfatide synthesis (SFS) was of phase dark cells identified by immunofluorescence a s oligoden-chosen as a specific function of oligodendrocytes which plays a drocytes progressively decreased. Immunologically identified as-central role in myelination, A of cultured trocytes were unaffected glucose Sul-brain cells from newborn mice was utilized which shows an ageIatide 'ynthesis ~~~b~~~~~~~ to 3*8% * and dependenl SFS to the ; , , ,,jvo developmenl ( 4 2 ) This cerebroside sulfotransferase dropped to 10.3% of control with system makes it possible to study directly the effects of glucose prolonged glucose Thus, Oligodendroc~tes and One on brain cell metabolism, avoiding changes at the blood their specific functions were damaged by glucose deprivati...

Several metabolic activities in dissociated cultures of newborn mouse brain were compared to the situation in vivo. The developmental activity pattern of cerebroside-sulfotransferase, cyclic nucleotide phosphohydrolase, and beta-hydroxy-beta-methyl glutaryl-coenzyme A-reductase and the synthesis and deposition of sulfatide and cholesterol in culture were estimated. The enzyme activity patterns in vivo and in culture are the same. Since the cultures show very little myelin formation, the parallel increase of enzyme activities necessary for myelination in vivo and in culture suggest the existence of intrinsic factors regulating the biochemical differentiation. In addition, the formation of the products, determined in culture, follows the patterns of the enzyme activities. Dissociated brain cell cultures are therefore a valid model for the study of biochemical parameters related to the synthesis of brain lipids during development.

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