Diminished cerebroside-sulfotransferase activity in the Jimpy mouse mutant due to altered lipid composition in microsomal membranes.

The mouse mutant Jimpy shows a deficient myelination. In the microsomes of the Jimpy brain, the cerebroside-sulfotransferase (EC 2.8.2.11) activity is low. The cerebroside-sulfotransferase activity of Jimpy microsomes could be normalised by delipidating the microsomes with cold acetone and adding to them acetone-extracted lipids from normal microsomes. The lipids extracted from Jimpy membranes did not influence the cerebroside-sulfotransferase activity of neither normal nor Jimpy microsomes. The same results were obtained if artificial cholesterol-phospholipid mixtures in ratios corresponding to the ones found in normal and Jimpy membranes were used for recombination experiments. Therefore the diminished enzyme activities in Jimpy microsomes may be related to the lower cholesterol-phospholipid ratio found in the microsomal membranes of the Jimpy mutant.

Influence of reduced cholesterol synthesis on the activity of cerebroside sulfotransferase in cultured glioblastoma cells treated with estradiol.

Cultured glioblastoma cells were inoculated with estradiol in concentrations of 0.5--10 microliter/ml medium in order to check the effect of this hormone on the activity of cerebroside sulfotransferase, an enzyme whose activity is strongly related to myelination. Thereby we could show that the cerebroside-sulfotransferase activity increases to a value of 200% of normal. Concomitant to this effect, the cholesterol content of the membrane bearing cerebroside sulfotransferase activity decreases to 60% of normal. The effect is fully reversible: after 48 h, cholesterol synthesis as well as cerebroside sulfotransferase activity reach normal values again. We suggest that cerebroside sulfotransferase activity is modulated by the changing cholesterol/phospholipid ratio in the cells during the inoculation period.

3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase. An optimized assay in homogenates of developing brain.

An optimized in vitro assay of 3'-phosphoadenylysulfate:galactosylceramide 3'-sulfotransferase (EC 2.8.2.11, galactosylceramide sulfotransferase, formerly known as galactocerebroside sulfotransferase) activity is presented, that can be used in crude homogenate of brain tissue of various developmental stages. The enzyme activity is determined by measuring the [35S]sulfatides formed by the enzymic transfer of [35S]sulfate from 3'-phosphoadenoside 5'-phospho [35S]sulfate to galactosylceramides. The sulfatide formation at 30 degrees C is linear up to 30 min and up to a protein concentration of 1 mg per 0.5 ml assay volume. The presence of 0.4% Triton X-100 and 50 micrometer exogenous bovine cerebrosides are optimal for enzyme activity. The pH optimum of the reaction is at pH 6.5 using 0.1 M imidazole buffer. The enzyme reaction is stimulated by NaCl, KCl, MgCl2, CaCl2, MnCl2, ATP and inhibited by ADP. The developmental enzyme activity pattern of mouse brain is the same, if derived from homogenates and microsomes, respectively, under our assay conditions.

Lipid-protein interactions with native and modified myelin basic protein.

The basic protein of central nervous system myelin has been shown to form complexes with acidic lipids in vitro. We measured the interaction of myelin basic protein with several charged and neutral lipids in a biphasic chloroform/methanol/water system and investigated the effect of decreasing the electrical charge of the basic amino groups of the myelin basic protein by acetylation. The modified myelin basic protein, which has an average of eight acetyl residues incorporated, was characterised by gel electrophoresis and circular dichroism. Complexes formed between the acetylated myelin basic protein and acidic lipids exhibited a reduction in the amount of lipids bound, a value that could be correlated with the number of modified amino groups. The significance of these experiments with reference to protein-lipid interaction in the myelin membrane is discussed.

Net sulfatide synthesis, galactosylceramide sulfotransferase and arylsulfatase A activity in the developing cerebrum and cerebellum of normal mice and myelin-deficient jimpy mice.

Net sulfatide synthesis, galactosylceramide sulfotransferase (EC 2.8.2.11) and arylsulfatase A (EC 3.1.6.1) activities were measured in two brain regions, cerebrum and cerebellum, of normal and jimpy mice during postnatal development. In normally myelinating mice, two phases of increasing rates of net sulfatide synthesis were observed, the first coinciding with oligodendrocyte proliferation and the second with myelination. Net sulfatide synthesis was quantitatively higher in the cerebellum than in the cerebrum. In both brain regions, the developmental patterns of net sulfatide synthesis were related to the activity patterns of both galactosylceramide sulfotransferase and arylsulfatase A. In jimpy mice, a neurological mutant showing hypomyelination in brain, the first phase of net sulfatide synthesis was preserved in both brain regions and galactosylceramide sulfotransferase and arylsulfatase A activities were normal up to 12 days. However, during the phase in which myelination occurred in controls, the net sulfatide synthesis in both brain regions of jimpy mice was zero or even negative. The sulfatide deficit was larger in the cerebellum than in the cerebrum. In both mutant brain parts, galactosylceramide sulfotransferase activity increased up to 12 days showing about 50% of the maximal activities observed in normal brain regions. Thereafter up to 15 days, enzyme activity decreased to about 25% of that of controls and remained low in both brain regions. The developmental patterns and the activities of arylsulfatase A were, however, normal in the cerebrum and cerebellum of jimpy mice. These results suggest that the enzyme activities and the developmental patterns of galactosylceramide sulfotransferase and arylsulfatase A as measured in vitro reflect to a high degree their functional activity in vivo. Furthermore, sulfatide degradation by arylsulfatase A seems to be important in regulating net sulfatide synthesis during normal and impaired myelination.

Age-dependent modulation of 3'-phosphoadenosine-5'-phosphosulfate-galactosylceramide sulfotransferase by lipids extracted from the microsomal membranes and artificial lipid mixtures.

The 3'-phosphoadenosine-5'-phosphosulfate-galactosylceramide-sulfotransferase (cerebroside sulfotransferase) is microsomal enzyme, which shows a definite developmental activity pattern. This report gives evidence that the enzyme activity of partially delipidated microsomes is modulated by the cholesterol:phospholipid ratio of the extracted microsomal lipids in an age-dependent manner. These findings suggest that in vivo the enzyme activity is modulated by the lipid surrounding.

Progressive somatofugal activation of cerebroside sulfotransferase in the developing chick optic fibers.

A myelination parameter of the chick optic pathway has been investigated: the activity of cerebroside sulfotransferase. Evidence is presented for a somatofugal progression of the enzyme activity peak along the optic nerve, chiasm, optic tract, tectum anterior, and tectum posterior. The enzyme activity appears on the fifteenth embryonic day and reaches a maximum in the nerve two days before hatching. One day after hatching, the peak is found in the chiasm, and three days after that in the optic tract. The peak is found in the tectum anterior on the tenth day. The results suggest a somatofugal progression of myelination.

Abnormal metabolism of 35SO4-sulfatide in jimpy brains expressed in brain organotypic cultures.

Cerebroside-sulfotransferase (CST), creatine-phosphokinase (CPK), and 3-hydroxy-3-methylglutaroyl CoA (HMG CoA) reductase activity, protein, and DNA content were measured in an easy-to-perform organotypic culture system of newborn normal and jimpy brains. The defective sulfatide synthesis which has been shown in vivo in jimpy brains could also be demonstrated in organ cultures of jimpy mice in the form of lowered CST activity in the homogenate as well as reduced 35SO4 incorporation into 35SO4-sulfatide. HMG CoA reductase was reduced to 60% of that found in 16-day-old normal cultures, similar to the findings in vivo. DNA of jimpy cultures was significantly lower than that in normal cultures, suggesting the possibility of an arrest in the differentiation or increased cellular death of presumptive oligodendrocytes, as was found in vivo. Organ cultures of jimpy mouse brain can serve as an appropriate model for further study of the primary defect in this animal mutant.

Expression of antigenic markers during the development of oligodendrocytes in mouse brain cell cultures.

The expression of myelin basic protein (MBP) and galactocerebroside (GC), two antigenic markers for oligodendrocytes, was checked on 7-, 14-, 21- and 28-day-old dissociated mouse brain cell cultures (BCC) by using the indirect immunofluorescence method with double staining. The number of GC positive cells increased between the 7th and the 14th day of culture before a steady state was reached. In contrast to this, the MBP-positive cells appeared only on the 14th day of culture, and their number increased with the age of the culture. In double staining, the serum produced against isolated oligodendrocytes shows the same picture as the anti-GC serum, while only a part of GC-positive cells showed also the presence of MBP. Our data suggest that the GC appears very early on the membrane of the oligodendrocytes during development while cells exhibiting both GC and MBP probably represent a more differentiated oligodendrocyte population.

Immunological properties of bulk isolated mouse oligodendrocytes.

Mouse oligodendrocytes were isolated in bulk. These cells were 95% galactocerebroside-positive. Rabbit anti-mouse oligodendrocyte antisera reacted in radioimmunoassay and in indirect immunofluorescence with mouse oligodendrocytes in suspension or in culture. The activity of the sera was not blocked by adsorption with galactocerebroside or by preincubation of cells with anti-galactocerebroside serum, although it cross-reacted with galactocerebroside in a serological test. The staining was not impaired by the pretreatment of cells with trypsin, neuraminidase or acetone, nor by adsorption of serum with myelin basis protein.

Relative changes in the amount of galactocerebroside during development as observed in mouse brain cell cultures.

Relative changes in the amount of galactocerebroside (GC) during development were measured in mouse brain cell cultures at different stages of development. For this purpose we used an 125I-labelled protein A indirect assay modified in the respect that the total amount of cellular proteins was evaluated before counting the radioactivity. The amount of GC greatly increased between the 10th and the 14th day of culture, then a steady state was reached between the 14th and the 20th day of culture. This change correlates well with the dynamics of the number of oligodendrocytes we observed earlier. These data suggests that the increase of the GC amount in culture during development corresponds to the increase in the number of GC-positive oligodendrocytes rather than to the increase in the number of GC molecules per cell.

Theophylline reduces the activity of cerebroside-sulfotransferase, a key enzyme in myelination, in cell cultures from newborn mouse brain.

Theophylline, a drug used in neonatology for the treatment of apnea, affects cholesterol synthesis if administered in concentrations of 10(-4) M (a concentration found in serum of treated patients) for 24 hr to dissociated brain cell cultures. The rate-limiting enzyme of cholesterol synthesis, beta-hydroxy-beta-methylglutaryl-coenzyme A reductase (EC 1.1.1.34), is lowered to 45% 48 hr after removal of theophylline. At the same time, cholesterol content of the cells is lowered to 73%. Inasmuch as the phospholipid content of the cells remains stable, the treatment changes the cholesterol phospholipid ratio. Concomitant to this effect, the activity of cerebroside-sulfotransferase (EC 2.8.2.11) is lowered to 60% of control values. We postulate that these two effects are linked to each other by means of modulation of the cerebroside-sulfotransferase activity by membrane lipids.

Automated and computer-controlled method for the measurement of the crystallization of calcium oxalate monohydrate in urine.

Monitoring of crystallization of calcium salts with ion-selective electrodes has turned out to be a very sensitive method. The difficulties of handling these electrodes in native whole urine and other biological fluids have been eliminated by new calcium analyzers, which clean and calibrate the electrodes after each measurement. To study crystallization kinetics, repeated calcium ion measurements have to be performed at regular intervals. For this purpose we have developed a special sampler and software. The sampler brings a thermostat-controlled crystallization chamber to the analyzer at preselected intervals. The computer directs and coordinates the sampler and the analyzer, stores the received results and prints out growth curves. Furthermore it calculates the half-time (h) and the maximum decrease of ionic calcium at infinite incubation time (delta Ca2+ infinity). Both values are shown to characterize the growth of calcium oxalate monohydrate in urine. Results are obtained within 40 min.

Measurement of metastability, growth and aggregation of calcium oxalate in native urine. A new approach for clinical and experimental stone research.

Nucleation, growth and aggregation are thought to be the most important crystallization processes in stone formation. Since crystallization properties change with urinary dilution, centrifugation and filtration, crystallization should always be studied in freshly voided and not pretreated urine. Recently we developed an automated method where calcium oxalate crystallization is induced in native urine by an exogenous oxalate load and nucleation and growth are monitored by an ion-selective calcium electrode. The method has now been supplemented with the spectrophotometric measurement of crystal aggregation. Repeated experiments in the same urine with different oxalate loads enable the determination of the critical oxalate additionable to induce crystallization (metastable limit) and the calculation of an oxalate load-independent growth rate constant. Preliminary results obtained in the native urine of healthy controls showed an extremely high limit of metastability and a complete absence of crystal aggregation. These findings may explain why, despite frequent urinary calcium oxalate supersaturation, healthy people do not form stones.

Vesicular transport of sulfatide in the myelinating mouse brain. Functional association with lysosomes?

Sulfatide synthesis and its subcellular distribution kinetics was followed in the myelinating brain of 17-day-old mice. Pulse-labeling-chasing conditions were achieved by an intraperitoneal injection of (35S)sulfate followed 2 h later by a second injection of a high dose of unlabeled sulfate. At 1, 2, 3, 4, and 6 h after the (35S)sulfate injection, the brains were removed, homogenized, and subcellular fractions were obtained by differential and discontinuous sucrose gradient centrifugation (Eichberg, J., Whittaker, V. P., and Dawson, R. M. (1964) Biochem. J. 92, 91-100). The microsomal membranes were further subfractionated (Siegrist, H. P., Burkart, T., Wiesmann, U. N., Herschkowitz, N. N., and Spycher, M. A. (1979) J. Neurochem. 33, 497-504) into light myelin, plasma membranes, Golgi vesicles, endoplasmic reticulum membranes, and heavy vesicles associated with acid hydrolase activities. The [35S]sulfatide-labeling kinetics was measured in all subcellular fractions. The results indicate that sulfatides are synthesized in the Golgi-endoplasmic reticulum complex and transferred in vesicles at least partially associated with lysosomes to the myelin membranes. The association of sulfatides with lysosomes could explain the existence of the previously described labile pool of newly synthesized sulfatides (Burkart, T., Hofmann, K., Siegrist, H. P., Herschkowitz, N. N., and Wiesmann, U. N. (1981) Dev. Biol. 83, 42-48) and also could be a form of vesicular transport to the myelin.

Synthesis of lipids in mouse brain cell cultures during development.

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.

Abnormal growth kinetics and 5'-nucleotidase activities in cultured skin fibroblasts from patients with Duchenne muscular dystrophy.

The 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 less than 0.0001) 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) 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 less than 0.001; nDMD = 5; ncontrols = 4) whereas plating efficiency is equal for both DMD and controls. On day 7 activity of the membrane bound enzyme 5'-nucleotidase either per mg protein or per microgram DNA is significantly elevated in cells from DMD patients (P less than 0.0005; nDMD = 8; ncontrols = 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 from DMD fetuses, thay could serve as a potential prenatal diagnosis of the disease.

Utilization of galactose in cultured brain cells of neonatal mice.

Metabolism of galactose was examined in dissociated brain cells from neonatal mice after 10-13 days in culture. Consumption of galactose at levels up to 26 mM was much less than consumption of glucose at corresponding concentrations. Lactate was consumed from the media at all galactose levels, in contrast to experiments with glucose in which lactate was formed and released into the media. Generation of CO2 from 4 mM glucose was 9-fold greater than from an equimolar level of galactose. Relatively low concentrations of glucose could reduce uptake of galactose, whereas galactose at levels up to 11.6 mM failed to inhibit consumption of glucose or formation of lactate. In glucose-deficient states, galactose supplementation of the media led to a marked increase in sulfatide synthesis by oligodendrocytes in the culture with a maximum effect at 2.3 mM. Under these conditions, [1-14C]galactose was incorporated directly into the carbohydrate portion of sulfatide, although most of the label was found in phospholipids and in the nonlipid fraction of the cellular homogenate. These data suggest that galactose is poorly metabolized by brain cells, but does not exhibit toxic effects.

Nucleation and aggregation of calcium oxalate in whole urine; spectrophotometric sedimentation analysis: a new approach to study the aggregation of calcium oxalate dihydrate.

Spectrophotometric and scanning electron microscopic (SEM) studies of oxalate-induced crystallization have been performed in whole urine with and without continuous magnetic stirring and before and after millipore filtration of urine. With continuous stirring, preferential nucleation was observed and this followed second order kinetics. Important crystal aggregation only occurred after an oxalate load above 1 mmol/l and without stirring. Under these conditions and at an ionic calcium concentration of 2 mmol/l, single crystals and aggregates of calcium oxalate dihydrate and monohydrate of well defined sizes were produced. Single dehydrates, their aggregates and the other particles could be distinguished by their significantly different sedimentation rates. From sedimentation curves an aggregation ratio for calcium oxalate dihydrate (aggregated/total dihydrate particles) was extrapolated. Millipore filtration removing important urinary macromolecules increased this aggregation ratio as well as the size of the aggregates on SEM pictures.