Effects of Monensin and Colchicine on Myelin Galactolipids

Monensin and colchicine have been used in a variety of systems to disrupt functioning of the Golgi apparatus and transport of Golgi-derived vesicles to the plasma membrane. In this study the effects of monensin and colchicine on the synthesis of cerebroside and sulfatide and their appearance in myelin were examined to determine whether these myelin components are processed through the Golgi apparatus. Brain slices from rats 17 days old were incubated with [3H]galactose and [35S]-sulfate to label cerebroside and sulfatide. Myelin was isolated on sucrose density gradients. Fractions highly enriched in cerebroside and sulfatide were prepared from homogenates and myelin fractions by lipid extraction, alkaline methanolysis, and in some cases TLC. Monensin at 0.1 microM had no significant effect on synthesis of these galactolipids as measured by incorporation of [3H]-galactose into cerebroside or [35S]sulfate into sulfatide in homogenates. However, appearance of [35S]sulfatide in the myelin fraction was reduced to 49% of control, while appearance of [3H]cerebroside was not significantly reduced. Colchicine from 1 mM to 0.1 microM had effects similar to monensin, that is, appearance of [35S]sulfatide in myelin was depressed, but again [3H]cerebroside was not affected. Incorporation of [35S]sulfate into sulfatide in homogenate was 93% of control, while appearance of [35S]sulfatide in the myelin fraction was depressed to 58% of control. The inhibition of appearance of sulfatide in myelin by colchicine and monensin is consistent with the view that sulfation of cerebroside occurs in the Golgi and that sulfatide is transported via Golgi-derived vesicles to the forming myelin membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myelin Galactolipid Synthesis in Different Strains of Mice

Previous studies have indicated that the brains of DBA/2J (D2) mice have a more heavily myelinated CNS than those of C57BL/6J (B6) at postnatal days 17-21. However, the amount of myelin in the brains of F1 (B6 X D2) hybrids is even higher than in their parental strains. To investigate further factors involved in regulating myelinogenesis in these mice, we have focused on the synthesis of cerebrosides and sulfatides, galactolipids enriched in myelin. Brain slices from 14-, 17-, and 21-day-old D2, B6, and F1 mice were incubated with [3H]galactose and [35S]sulfate. After incubation, microsomes, myelin, and oligodendroglial cells were isolated, and the galactolipids were analyzed. At 21 days of age, the labeling of cerebrosides in F1 mice was higher than in D2 and B6 mice when the results were expressed as microsomal or myelin radioactivity per gram wet weight. At 14 and 17 days of age, the labeling of cerebrosides in F1 animals was similar to that in D2 mice and was considerably higher than that in B6 mice. The labeling of sulfatides in F1 animals was significantly higher than in the B6 parent at all ages studied, whereas it remained higher than that in the D2 parent only at 17 days of age. A similar relationship among the strains was observed when the synthesis of myelin galactolipids was estimated by measuring the in vitro activity of UDP-galactose:ceramide galactosyltransferase and 3'-phosphoadenylyl sulfate:galactosylceramide 3'-sulfotransferase. The results indicate that the increased accumulation of myelin galactolipids previously reported in the F1 mice is partially due to enhanced synthetic activity.     

Inhalation carcinogenesis of high-fired 241AmO2 in rats

Wistar rats were given a single inhalation exposure to high-fired 241AmO2 particles and examined over their life span. A total of 310 rats were used: 259 exposed to 241Am for life-span study, 30 exposed to 241Am for early metabolism study, and 21 unexposed life-span controls. The activity median aerodynamic diameter of the aerosols was 0.75-1.39 microns. About 55% of alveolarly deposited 241Am was cleared from the lung with a half-life of 0.5 days, 37% with a half-life of 7 days, and 8% with a half-life of 580 days. Group mean lung doses ranged from less than 5.7 rad up to 1500 rad. Significant early mortality due to radiation pneumonitis was seen only in the highest exposure group. The percentage of rats with lung tumors was 0% for controls (21 rats), 1% at lifetime lung doses less than 10 rad (139 rats), 7% at 10-50 rad (86 rats), 0% at 50-100 rad (9 rats), 60% at 100-500 rad (10 rats), and 7% at 500 rad (15 rats). Only one liver and one bone tumor were found in all exposed rats, both at lifetime tissue doses less than 10 rad. The fate and carcinogenicity of inhaled 241AmO2 in the lung of rats were similar to what has previously been described for inhaled 244CmO2.     

Class III β-Tubulin and γ-Tubulin are Co-expressed and Form Complexes in Human Glioblastoma Cells

We have previously shown that the neuronal-associated class III beta-tubulin isotype and the centrosome-associated gamma-tubulin are aberrantly expressed in astrocytic gliomas (Cell Motil Cytoskeleton 2003, 55:77-96; J Neuropathol Exp Neurol 2006, 65:455-467). Here we determined the expression, distribution and interaction of betaIII-tubulin and gamma-tubulin in diffuse-type astrocytic gliomas (grades II-IV) (n = 17) and the human glioblastoma cell line T98G. By immunohistochemistry and immunofluorescence microscopy, betaIII-tubulin and gamma-tubulin were co-distributed in anaplastic astrocytomas and glioblastomas and to a lesser extent, in low-grade diffuse astrocytomas (P < 0.05). In T98G glioblastoma cells betaIII-tubulin was associated with microtubules whereas gamma-tubulin exhibited striking diffuse cytoplasmic staining in addition to its expectant centrosome-associated pericentriolar distribution. Treatment with different anti-microtubule drugs revealed that betaIII-tubulin was not associated with insoluble gamma-tubulin aggregates. On the other hand, immunoprecipitation experiments unveiled that both tubulins formed complexes in soluble cytoplasmic pools, where substantial amounts of these proteins were located. We suggest that aberrant expression and interactions of betaIII-tubulin and gamma-tubulin may be linked to malignant changes in glial cells.     

A Halothane-Related Effect on Rat Brain Myelination: A Comparison of Chronic Prenatal or Postnatal Exposure

Rats were exposed to 0.5% halothane in air for 8 h per day during the intervals (1) 5 days postconception to birth, (2) birth to 5 days postnatal age, or (3) birth to 10 days postnatal age. Controls were exposed to an equivalent flow of air. Prenatal exposure had no significant effect on body or brain weight and no subsequent effect on the relative synthesis of brain subcellular membranes. Five days of postnatal exposure caused a 10% reduction in body and brain weight and a 10% relative reduction in the synthesis of brain myelin. The effect persisted throughout the period of rapid postnatal brain myelination. Ten days of postnatal exposure produced equivalent, more severe effects on body and brain weights and a more severe effect on myelin synthesis. Postnatal exposure had no apparent effect on the relative synthesis of non-myelin particulate proteins.     

Effects of hypoxia and hyperoxia on proteoglycan production by bovine pulmonary artery endothelial cells

Bovine pulmonary artery endothelial cells in culture were used to assess the influence of oxygen tension on proteoglycan synthesis. Cells exposed to 3% O2 (hypoxia) for 72 h and then labeled with [35S]sulfate for 5 h accumulated significantly less [35S]proteoglycan in medium than cells exposed to 20% O2 (control). This decrease was due primarily to a reduction in heparan sulfate. Cells exposed to 80% O2 (hyperoxia) for 72 h secreted slightly more [35S]proteoglycan into medium than controls. Greater accumulation of chondroitin sulfate was responsible for the increase. The amount of cell-associated proteoglycan did not change significantly in cells cultured in 3% or 80% O2 as compared with control cells cultured in 20% O2. Proteoglycans produced by hypoxia- or hyperoxia-treated cells were found to be similar in size to proteoglycans produced by cells cultured at 20% O2. Glycosaminoglycan sulfation, as measured by ion-exchange chromatography, did not appear to change with varying oxygen tensions. Our results demonstrate that production of proteoglycans secreted by endothelial cells in culture is sensitive to oxygen tension.     

A mathematical model for brain tumor response to radiation therapy

Gliomas are highly invasive primary brain tumors, accounting for nearly 50% of all brain tumors (Alvord and Shaw in The pathology of the aging human nervous system. Lea & Febiger, Philadelphia, pp 210-281, 1991). Their aggressive growth leads to short life expectancies, as well as a fairly algorithmic approach to treatment: diagnostic magnetic resonance image (MRI) followed by biopsy or surgical resection with accompanying second MRI, external beam radiation therapy concurrent with and followed by chemotherapy, with MRIs conducted at various times during treatment as prescribed by the physician. Swanson et al. (Harpold et al. in J Neuropathol Exp Neurol 66:1-9, 2007) have shown that the defining and essential characteristics of gliomas in terms of net rates of proliferation (rho) and invasion (D) can be determined from serial MRIs of individual patients. We present an extension to Swanson's reaction-diffusion model to include the effects of radiation therapy using the classic linear-quadratic radiobiological model (Hall in Radiobiology for the radiologist. Lippincott, Philadelphia, pp 478-480, 1994) for radiation efficacy, along with an investigation of response to various therapy schedules and dose distributions on a virtual tumor (Swanson et al. in AACR annual meeting, Los Angeles, 2007).     

Novel inhibition of proteoglycan synthesis and exocytosis by diethylcarbamazine in the Swarm rat chondrocyte

Pretreatment of cultured chondrosarcoma chondrocytes at 37 degrees C for 15 min with 15 mM diethylcarbamazine (DEC) followed by a 60-min pulse with [35S] sulfate in the presence of DEC resulted in an approximate 40% inhibition of synthesis and a 75% inhibition of secretion of 35S-proteoglycan. The inhibition was dose-related and was not due to a decrease in protein synthesis. Chondrocytes exposed for 75 min to 15 mM DEC, washed, incubated for 17 h in DEC-free medium, and then pulsed with [35S]sulfate showed no inhibition in the rate of synthesis of proteoglycan or in the per cent of radiolabeled proteoglycans exocytosed into the culture medium, indicating full reversibility of the inhibitory effect. When chondrocytes were incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, secretion of beta-D-xyloside-bound 35S-glycosaminoglycan was inhibited by more than 70% despite an approximate 3-fold increase in intracellular 35S-macromolecules, as compared to cells exposed to beta-D-xyloside alone. Upon removal of DEC, the block in the secretion of beta-D-xyloside-bound 35S-glycosaminoglycans was reversed, although there was a 15-30-min lag in the initiation of exocytosis. Light and electron microscopic examination of chondrocytes after 75 min of incubation with 15 mM DEC revealed large vacuoles, a distended Golgi apparatus, and a distended endoplasmic reticulum which contained electron dense material. Upon removal of DEC, the vacuoles disappeared and distended organelles returned to their normal appearance between 15 and 30 min, coincident with the start of exocytosis of 35S-proteoglycan and beta-D-xyloside-bound 35S-glycosaminoglycan. These biochemical and morphological studies indicate that DEC treatment of chondrosarcoma chondrocytes alters the transport of molecules from the endoplasmic reticulum to the Golgi and the transport of molecules from the Golgi to the cell surface.     

Decreased susceptibility of Malassezia furfur to UV light by synthesis of tryptophane derivatives

Recently, tryptophane (Trp)-dependent synthesis of pigments and fluorochromes in Malassezia furfur was described. The possible significance of this metabolic pathway for the microorganism remains to be explored. Since the upper parts of the human epidermis are a natural habitat of M. furfur, increased exposure to UV light may be hazardous. Five reference strains and one wild type strain of M. furfur were grown on m-Dixon agar, in which the nitrogen source peptone had been substituted either by pigment-inducing tryptophane or arginine. The yeast cells thus obtained were harvested after 6 days, washed with physiological saline and inoculated on to the modified Dixon medium. Immediately after inoculation, the yeast cells were irradiated with UVA (100, 150 and 200 Jcm^-2, single dose) or UVB (100, 500, 1000, 1500, 2000 mJcm^-2, single dose; 500, 1500, 2500 mJcm^-2cumulative dose). Irrespective of the primary nitrogen source (Trp or Arg), unexposed controls showed nearly identical cell yield after 5 days. In the case of irradiation, however, growth reduction of cells cultured on Trp was lesser than that of cells fed with arginine. High significance (p<0.0001) was found especially with the upper UVA and UVB doses. Differences were also found among the individual test strains, the wild strain being most sensitive. One strain (CBS 6094) failed to produce pigment on Trp medium, and there were no differences in the growth behavior of subcultures of this strain fed with either arginine or tryptophane under irradiation. In conclusion, synthesis of pigments and fluorochromes by M. furfur implies the generation of potent UV filters in the UVA and UVB spectrum.     

Myelinogenesis in optic nerve. A morphological, autoradiographic, and biochemical analysis

Morphological, autoradiographic, and biochemical methods were used to study the time of appearance, distribution, and nature of sulfated constituents in the developing rat optic nerve. Electron microscope studies showed that myelination begins (6 days postnatal) shortly after the appearance of oligodendroglia (5 days postnatal). Over the ensuing 3 wk, myelination increased rapidly. During the 1st postnatal wk, mucopolysaccharides and glycoproteins were labeled with 35S and autoradiographs showed grains over arachnoidal cells, astroglia, and the glia limitans. These results indicated that astroglia synthesize sulfated mucopolysaccharides of the glia limitans. After the onset of myelination, however, the major portion of [35S]sulfate was incorporated into sulfatide. Autoradiographs showed a shift of radioactive grains from astroglia and arachnoidal cells to myelin, indicating that actively myelinating oligodendroglia incorporate [35S]sulfate into myelin sulfatide; there was a concomitant increase in the activity of cerebroside sulfotransferase. In addition, the increasing amounts of proteolipid protein and myelin basic protein corresponded with the morphological appearance of myelin. These results point to a strict correlation between the structural and biochemical changes occurring during myelination. This system provides a useful model for studies designed to evaluate the effects of various perturbations on the process of myelination.     

Effects of insulin-like growth factor-I, corticosterone, and 3,3', 5-tri-iodo-L-thyronine on glycosaminoglycan synthesis in vertebral cartilage of the clearnose skate, Raja eglanteria.

Effects of insulin-like growth factor-I (IGF-I), corticosterone, and triiodothyronine (T(3)) on in vitro growth of vertebral cartilage of the clearnose skate, Raja eglanteria, were investigated. Uptake of [(35)S]sulfate in cultured vertebrae was used to characterize glycosaminoglycan (GAG) synthesis and cartilage growth. IGF-I significantly enhanced cartilage growth when concentrations of 1.28 and 12.8 nM were present in the culture system. Corticosterone significantly inhibited vertebral GAG synthesis at concentrations of 1, 10, and 100 nM. This effect was markedly pronounced in cartilage exposed to 1 and 10 nM corticosterone, in which GAG synthesis was virtually ceased. In contrast, T(3) (0.75, 7.5, and 75.0 nM) had no significant effect on sulfate uptake. These data suggest that IGF-I and corticosteroids may play important roles in regulating skeletal growth of elasmobranchs, as they appear to do in other vertebrates. While T(3) does not appear to exert an immediate, direct effect on vertebral growth, it may still influence elasmobranch chondrogenesis over longer culture periods or indirectly through other regulatory pathways. Thus, further information is necessary to characterize the role of thyroid hormones in the skeletal growth of these fishes. The present study is the first in vitro investigation on the hormonal regulation of elasmobranch cartilage growth. As such, the methods described herein provide a useful technique for examining these physiological processes. J. Exp. Zool. 284:549-556, 1999.     

Mitogen-stimulated activation of the Na+/H+ antiporter does not regulate S6 phosphorylation or protein synthesis in murine thymocytes or Swiss 3T3 fibroblasts

The activation of protein synthesis by mitogens in quiescent (G0) mammalian cells is obligatory for progression from G0 through G1 to DNA synthesis in S phase. When the activation of the Na+/H+ antiporter which occurs in mitogen-stimulated Swiss 3T3 fibroblasts or murine fibroblasts is completely blocked by dimethylamiloride, there is little or no effect on the phosphorylation of the ribosomal protein S6 or the activation of protein synthesis assayed by [35S]methionine incorporation. Furthermore, the accumulation of the protein product of the activated c-myc gene is unaffected by dimethylamiloride in 3T3 fibroblasts. The data show that there is no requirement for activation of the Na+/H+ antiporter for the activation of S6 phosphorylation or protein synthesis by mitogens but do not preclude the possibility that activation of the antiporter is necessary for some other response(s) obligatory for DNA synthesis. These data are contrasted with previous reports for Chinese hamster lung fibroblasts that the increase in intracellular pH which results from activation of the Na+/H+ antiporter in bicarbonate-free media is necessary for S6 phosphorylation, protein synthesis, and hence, for subsequent DNA synthesis (Pouyssegur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3935-3939; Chambard, J.C., and Pouyssegur, J. (1986) Exp. Cell Res. 164, 282-294).     

A change in the oxygen effect throughout the cell-cycle of human cells of the line NHIK 3025 cultivated in vitro.

NHIK 3025 cells were synchronized by repeated mitotic selection. The S-phase was determined by 3H-thymidine incorporation and scintillation counting. By comparing the age-response surves of aerobic cells irradiated with 500 rad with those of extremely hypoxic (less than4 p.p.m. O2) cells irradiatedwith 1500 rad, it was found that the sensitizing effect of oxygen was not constant throuhgout the cycle. It was significantly higher in S, G2 and mitosis than in G1. No significant sensitizing effect of 120 p.p.m. O2 (compared with less than4 p.p.m.O2) was found on cells in G1 when the cells were irradiated with 1500 rad. In S, G2 and mitosis, however, the sensitizing effect of oxygen at 120 p.p.m. is considered to be significant. Experiments performed with cells irradiated with 2000 rad incontact with either less than4 p.p.m. O2 or 80 p.p.m. O2 showed the same trend, little sensitizing effect in G1 and higher in S, G2 andmitosis. Dose-response curves for cells in mid-G1 and mid-S under aerobic and extremely hypoxic conditions were well fitted by the formula S=exp (-alphaD-betaD2). From the dose-response curves it was conculded that the change in the sensitizing effect of oxygen throughout the cell-cycle only appeared for low doses (in the dose region where alpha dominates). The sensitizing effect of oxygen on cells in mid-G1 was found to be increasing with increasing dose.     

Photoperiodic alteration of postpartum reproductive function in suckled cows

Three experiments were conducted using a total of 41 cows to determine if photoperiod modulates the establishment of postpartum estrous cycles and conception. Cows calving in the autumn and winter were exposed to either 18 hr light/day (18L:6D) or natural photoperiods. In Exp. 1, cows receiving 18L:6D had shorter (P<0.025) intervals from calving to estrus (61 +/- 3.8 days) than cows not receiving supplemental light (154 +/- 23.9 days). The same was true for primiparous cows in Exp. 2 (76 +/- 5.5 days vs 153 +/- 38.0 days; P<0.06) but not for the multiparous cows in Exp. 2 (56 +/- 5.2 days vs 40 +/- 7.4 days) or for all cows in Exp. 3 (60 +/- 10.1 days vs 70 +/- 13.5 days). Because conception rate was higher for cows exposed to 18L:6D for the multiparous cows in Exp. 2 and all cows in Exp. 3, interval to conception was significantly shorter for animals exposed to 18L:6D in all experiments. Generally, interval from calving to uterine involution was also reduced by exposure to 18L:6D. No effects of photoperiod were observed on body weight changes, serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) or estradiol or on estradiol-induced release of gonadotropins. It was concluded that in certain situations day length can hasten estrus and conception in postpartum cows. The effect of photoperiod interacts with other conditions, one of which is parity. The endocrine basis for these effects are not known.     

[3 5S]Sulfate incorporation into myelin glycoproteins II. Peripheral nervous tissue

The in vivo incorporation of [^{3 5}S]sulfate, [³H]fucose and [³H]leucine into sciatic nerve myelin was investigated. Polyacrylamide gel electrophoresis of the proteins indicated that the ^{3 5}S-labeling of proteins occurred almost exclusively in the major myelin protein. A smaller myelin glycoprotein migrating just ahead of the major one was labeled with [³H]fucose but did not incorporate ^{3 5}S to a detectable extent. There was little or no ^{3 5}S associated with basic proteins on polyacrylamide gels when the proteins were extracted with chloroform/methanol. Fucose-labeled myelin glycoproteins were converted to glycopeptides by pronase digestion. The glycopeptides gave a single peak on Sephadex G-50 in which the ³H and ^{3 5}S coincided. The association of ^{3 5}S with glycopeptides was not caused by binding of sulfatide or free inorganic sulfate. This study shows that the major myelin protein in the sciatic nerve of the rat is glycosylated and sulfated.     

Chromosome aberration yields in human lymphocytes induced by fractionated doses of x-radiation.

Unstimulated (G0) human peripheral blood lymphocytes were exposed at 37 degrees C to doses of 200 or 500 rad of X-rays delivered in two equal fractions. The dose fractions were separated by intervals of up to 7 h in the 200 rad study and up to 48 h for 500 rad. In both studies the mean levels of dicentrics and total unstable aberrations began to decline when fractions were delivered with intervals of greater than 2 h. With 200 rad the yield had decreased to an additive baseline (i.e. equal to only twice the yield of a single 100-rad fraction) by an interval of 4 h. Following 500 rad the yield declined until 8 h and then remained 20% above the additive baseline even when 48 h separated the fractions. Possible explanations for this discrepancy are discussed. In a second experiment PHA stimulated lymphocyte cultures were exposed to 2 doses of 125 rad of X-rays up to 7 h apart in an attempt to demonstrate the late peak in aberration yields originally reported by Lane [5]. Control cultures received unsplit doses of 250 rad at the time of the corresponding second 125-rad fraction. No evidence of a late peak in dicentric yield was observed. The yield remained approximately the same irrespective of the time interval between fractions but these split dose yields were significantly different from the accompanying unsplit controls.     

Fibrillogenic nuclei composed of P301L mutant tau induce elongation of P301L tau but not wild-type tau

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an autosomal, dominantly inherited neurodegenerative disorder caused by tau gene mutations, is neuropathologically characterized by intraneuronal filamentous inclusions of hyperphosphorylated tau protein. Biochemical and immunocytochemical analyses have shown that only mutant tau is deposited in patients harboring P301L missense mutation, whereas both wild-type and mutant tau are deposited in patients harboring R406W mutation (Miyasaka, T., Morishima-Kawashima, M., Ravid, R., Kamphorst, W., Nagashima, K., and Ihara, Y. (2001) J. Neuropathol. Exp. Neurol. 60, 872- 884 and Miyasaka, T., Morishima-Kawashima, M., Ravid, R., Heutink, P., van Swieten, J. C., Nagashima, K., and Ihara, Y. (2001) Am. J. Pathol. 158, 373-379). Here we have tested the nucleation ability of monomeric tau and the seeding ability of fibrillogenic nuclei obtained from bacterially expressed human tau. P301L mutant tau showed a higher nucleation ability than wild-type tau, whereas R406W mutant tau shows similar ability to wild-type tau. Surprisingly, fibrillogenic nuclei composed of P301L mutant tau enhanced the assembly of P301L mutant tau into filaments but did not promote filament formation from wild-type tau. In contrast, nuclei composed of R406W mutant tau supported filament formation from both wild-type tau and R406W mutant tau, as did nuclei composed of wild-type tau. Proteolytic analyses indicated that the substructure of nuclei composed of P301L mutant tau was different from that of nuclei composed of wild-type or R406W mutant tau. Thus, the interaction between fibrillogenic nuclei and monomeric protein appears to play an important role in the mechanism of tau filament assembly.     

Metabolism of Functional Groups Modifying the CNS Myelin-Associated Glycoprotein

We have examined the metabolic turnover of the peptide backbone of the CNS myelin-associated glycoprotein (MAG) and of the fucose and sulfate groups modifying this protein. Rats (20 or 90 days old) were injected intracranially with mixtures of [3H]fucose and [14C]glycine, [3H]glycine and [35S]sulfuric acid, or [3H]fucose and [35S]sulfuric acid. At times ranging from 30 min to 4 weeks later, myelin was isolated, and radioactivity in MAG was determined following electrophoretic separation. Following the peak of incorporation, glycine-derived radioactivity in the MAG peptide backbone declined several-fold during the first week and was then metabolically stable (half-life much greater than 1 month). Declines with time in [3H]fucose- and [35S]sulfate-derived radioactivity in MAG were similar to that of [3H]glycine, an observation indicating that the fucose and sulfate groups modifying MAG are metabolized together with the peptide backbone as a single metabolic entity. These results were confirmed by experiments involving selective immunoprecipitation of MAG. The rates of incorporation of labeled glycine, fucose, and sulfate into MAG all decreased approximately 12-fold between 20 days of age and adulthood, a finding providing further evidence for concerted turnover of the entire molecule. Because of this concerted turnover, we suggest that functional groups modifying MAG serve some permanent structural role in protein configuration.