Phosphate groups modifying myelin basic proteins are metabolically labile; methyl groups are stable.

Young and adult rats received intracranial injections of [33P]orthophosphoric acid. The time course of the appearance and decay of the radioactive label on basic proteins in isolated myelin was followed for 1 mo. Incorporation was maximal by 1 h, followed by a decay phase with a half-life of approximately 2 wk. However, radioactivity in the acid-soluble precursor pool (which always constituted at least half of the total radioactivity) decayed with a similar half-life, suggesting that the true turnover time of basic protein phosphates might be masked by continued exchange with a long-lived radioactive precursor pool. Calculations based on the rate of incorporation were made to more closely determine the true turnover time; it was found that most of the phosphate groups of basic protein turned over in a matter of minutes. Incorporation was independent of the rate of myelin synthesis but was proportional to the amount of myelin present. Experiments in which myelin was subfractionated to yield fractions differing in degree of compaction suggested that even the basic protein phosphate groups of primarily compacted myelin participated in this rapid exchange. Similar studies were carried out on the metabolism of radioactive amino acids incorporated into the peptide backbone of myelin basic proteins. The metabolism of the methyl groups of methylarginines also was monitored using [methyl-3H]methionine as a precursor. In contrast to the basic protein phosphate groups, both the peptide backbone and the modifying methyl groups had a metabolic half-life of months, which cannot be accounted for by reutilization from a pool of soluble precursor. The demonstration that the phosphate groups of myelin basic protein turn over rapidly suggests that, in contrast to the static morphological picture, basic proteins may be readily accessible to cytoplasm in vivo.

Axonal transport of the mitochondria-specific lipid, diphosphatidylglycerol, in the rat visual system.

Rats 24 d old were injected intraocularly with [2-3H]glycerol and [35S]methionine and killed 1 h-60 d later. 35S label in protein and 3H label in total phospholipid and a mitochondria-specific lipid, diphosphatidylglycerol(DPG), were determined in optic pathway structures (retinas, optic nerves, optic tracts, lateral geniculate bodies, and superior colliculi). Incorporation of label into retinal protein and phospholipid was nearly maximal 1 h postinjection, after which the label appeared in successive optic pathway structures. Based on the time difference between the arrival of label in the optic tract and superior colliculus, it was calculated that protein and phospholipid were transported at a rate of about 400 mm/d, and DPG at about half this rate. Transported labeled phospholipid and DPG, which initially comprised 3-5% of the lipid label, continued to accumulate in the visual structures for 6-8 d postinjection. The distribution of transported material among the optic pathway structures as a function of time differed markedly for different labeled macromolecules. Rapidly transported proteins distributed preferentially to the nerve endings (superior colliculus and lateral geniculate). Total phospholipid quickly established a pattern of comparable labeling of axon (optic nerve and tract) and nerve endings. In contrast, the distribution of transported labeled DPG gradually shifted toward the nerve ending and stabilized by 2-4 d. A model is proposed in which apparent "transport" of mitochondria is actually the result of random bidirectional saltatory movements of individual mitochondria which equilibrate them among cell body, axon, and nerve ending pools.

How is an ideal satiating yogurt described? A case study with added-protein yogurts.

Protein is recognized as the macronutrient with the highest satiating ability. Yogurt can be an excellent basis for designing satiating food as it is protein-based food product. Five different set-type yogurts were formulated by adding extra skim milk powder (MP), whey protein concentrate (WPC), calcium caseinate (CAS) or a blend of whey protein concentrate with calcium caseinate (CAS-WPC). A control yogurt without extra protein content was also prepared. Differences in sensory perceptions (through CATA questions) were related to the consumers' expected satiating ability and liking scores (of several modalities). In addition, an "Ideal satiating yogurt" was included in the CATA question to perform a penalty analysis to show potential directions for yogurt reformulation and to relate sensory and non-sensory yogurt characteristics to satiating capacity.

The neurotoxicant, cuprizone, as a model to study demyelination and remyelination in the central nervous system.

Myelin of the adult CNS is vulnerable to a variety of metabolic, toxic, and autoimmune insults. That remyelination can ensue, following demyelinating insult, has been well demonstrated. Details of the process of remyelination are, however difficult to ascertain since in most experimental models of demyelination/remyelination the severity, localization of lesion site, or time course of the pathophysiology is variable from animal to animal. In contrast, an experimental model in which massive demyelination can be reproducibly induced in large areas of mouse brain is exposure to the copper chelator, cuprizone, in the diet. We review work from several laboratories over the past 3 decades, with emphasis on our own recent studies, which suggest an overall picture of cellular events involved in demyelination/remyelination. When 8 week old C57BL/6 mice are fed 0.2% cuprizone in the diet, mature olidgodendroglia are specifically insulted (cannot fulfill the metabolic demand of support of vast amounts of myelin) and go through apoptosis. This is closely followed by recruitment of microglia and phagoctytosis of myelin. Studies of myelin gene expression, coordinated with morphological studies, indicate that even in the face of continued metabolic challenge, oligodendroglial progenitor cells proliferate and invade demyelinated areas. If the cuprizone challenge is terminated, an almost complete remyelination takes place in a matter of weeks. Communication between different cell types by soluble factors may be inferred. This material is presented in the context of a model compatible with present data -- and which can be tested more rigorously with the cuprizone model. The reproducibility of the model indicates that it may allow for testing of manipulations (e.g. available knockouts or transgenics on the common genetic background, or pharmacological treatments) which may accelerate or repress the process of demyelination and or remyelination.

Primary demyelination induced by exposure to tellurium alters mRNA levels for nerve growth factor receptor, SCIP, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin proteolipid protein in rat sciatic nerve.

Weanling rats fed a diet containing tellurium develop a peripheral neuropathy characterized by a highly synchronous primary demyelination; this demyelination is followed closely by a period of rapid remyelination. The demyelination is related to the inhibition of squalene epoxidase activity, which results in a block in cholesterol synthesis. Expression of mRNA for the major structural proteins of PNS myelin, myelin basic protein and P0, is coordinately down-regulated during the demyelinating phase and then up-regulated during the remyelinating phase (Toews et al., J. Neurosci. Res., 26 (1990) 501-507). We now report tellurium-induced alterations in gene expression for several proteins which are not major structural components of myelin in the peripheral nervous system. Expression of mRNA for nerve growth factor receptor in sciatic nerve was very low in control animals, but was markedly up-regulated after 3-5 days of exposure to tellurium, a time corresponding to the beginning of demyelination. Levels remained elevated during the subsequent period of remyelination. Expression of mRNA for SCIP (a presumptive transcription factor) was also up-regulated in sciatic nerve following tellurium exposure, with a time course similar to that for nerve growth factor receptor. When examined as a fraction of total RNA, steady-state mRNA levels for 2',3'-cyclic nucleotide 3'-phosphodiesterase and the myelin proteolipid protein were decreased during the demyelinating phase; however, this decrease could be largely accounted for by increased levels of total RNA. When analyzed on a 'per nerve' basis, steady-state mRNA levels for these two proteins were actually increased about 2-fold by 9 days after beginning tellurium exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

NGFR-mRNA expression in sciatic nerve: a sensitive indicator of early stages of axonopathy.

Expression of the low-affinity nerve growth factor receptor (NGFR) in the sciatic nerve (particularly Schwann cells) is high during development but is downregulated upon establishment of the mature axon-Schwann cell relationship. NGFR is re-expressed by Schwann cells if this relationship is altered by degeneration of axons (axotomy) or myelin (tellurium intoxication). To determine the sensitivity of NGFR expression to axonal injury, we have assayed NGFR-mRNA levels in proximal and distal regions of nerves exposed to the axonopathic agents acrylamide and isoniazid, as well as in proximal and distal stumps of axotomized nerves. NGFR-mRNA was elevated in all three models and correlated regionally with sites of axonal perturbation. In distal regions of acrylamide- and isoniazid-intoxicated nerves, NGFR-mRNA was elevated at least 2 days prior to visible signs of axonal degeneration as assayed by morphological techniques utilizing light microscopy. NGFR-mRNA was also elevated in proximal regions of axotomized and acrylamide-intoxicated nerves prior to signs of axonal degeneration. In these models, increased mRNA expression correlated with alterations in the size distribution of axonal cross sections. The common response in all of these situations indicates that NGFR expression, in addition to being a marker for axonal degeneration, is also a sensitive indicator of less profound perturbations in normal axon-Schwann cell interactions, including early stages of axonopathy. We suggest that assay for NGFR-mRNA may be utilized as a rapid and simple method (relative to more labor-intensive morphological methods) to screen for peripheral neurotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Tellurium causes dose-dependent coordinate down-regulation of myelin gene expression.

Exposure of developing rats to a diet containing elemental tellurium systemically inhibits cholesterol synthesis at the level of squalene epoxidase. At high tellurium exposure levels (> 0.1% in the diet), there is an associated segmental demyelination of the PNS. Low levels of dietary tellurium (0.0001%) led to in vivo inhibition of squalene epoxidase activity in sciatic nerve, and inhibition increased with increasing exposure levels. With increasing dose and increasing exposure times, there was an increasing degree of demyelination and increasing down-regulation of mRNA levels for myelin P0 protein, ceramide galactosyltransferase (rate-limiting enzyme in cerebroside synthesis), and HMG-CoA reductase (rate-limiting enzyme in cholesterol synthesis). Because these were all down-regulated in parallel, we conclude there is coordinate regulation of the entire program for myelin synthesis in Schwann cells. An anomaly was that at early time points and low tellurium levels, mRNA levels for HMG-CoA reductase were slightly elevated, presumably in response to tellurium-induced sterol deficits. We suggest the eventual down-regulation relates to a separate mechanism by which Schwann cells regulate cholesterol synthesis, related to the need for coordinate synthesis of myelin components. Levels of mRNA for the low-affinity nerve growth factor receptor (indicator of alterations in axon-Schwann cell interactions) and for lysozyme (marker for phagocytic macrophages) were both up-regulated in a dose- and time-dependent manner which correlated with the presence of segmental demyelination. Levels of mRNA coding for myelin-related proteins were down-regulated at low tellurium exposure levels, without demyelination or up-regulation of nerve growth factor receptor. This suggests the down-regulation is related to the tellurium-induced cholesterol deficit, and not to the loss of axonal contact associated with early stages of demyelination or to the entry of activated macrophages.

Axonal transport of proteins and glycoproteins in the rat nigro-striatal pathway and the effects of 6-hydroxydopamine.

Following stereotaxic injection of [35S]methionine into the substantia nigra of adult rats, there was rapid local incorporation of radioactivity into acid-insoluble material. Incorporation peaked by 4 h and then decreased. In contrast, acid-precipitable radioactivity in the corpus striatum (the major projection site of the substantia nigra) rose markedly between 1 and 8 h followed by a plateau period and another even more marked increase between 24 h and 6 days. Experiments involving injection of [3H]fucose gave similar results except that most of the acid-precipitable radioactivity in the striatum appeared in an early wave. In each case radioactivity in the contralateral striatum was less than 11% of that on the ipsilateral side. Stereotaxic injection of colchicine (20 microgram) into the nigrostriatal pathway (within the median forebrain bundle) blocked transport of [35S]protein and [3H]glycoprotein by 90% and 50%, respectively. In animals treated with 6-hydroxydopamine (6-OHDA; treated neonatally or as adults) the accumulation of striatal [35S]protein was reduced to 7 to 26% of control levels; striatal [3H]glycoprotein was also reduced, but not as much (29% to 73% of control). In control experiments, [3H]DOPA wa injected into the substantia nigra, and [3H]dopamine was measured in corpus striatum; 6-OHDA treatment reduced the amounts of striatal [3H]dopamine recovered to 3% of control values. The failure of colchicine or 6-OHDA to block transport of incorporated fucose as effectively as the transport of incorporated methionine is possible due to greater diffusion of fucose away from the injection site to non-dopaminergic nuclei projecting to the striatum. The molecular weight distribution of radioactive proteins at the substantia nigra and corpus striatum was analyzed by polyacrylamide gel electrophoresis. For both [35S]methionine and [3H]fucose, the gel electrophoretic pattern of radioactive proteins in the injection site (substantia nigra) was complex and did not change greatly between 2 h and 6 days. At the projection site (striatum) the electrophoretic distribution pattern was initially different from that of the substantia nigra, and changed markedly over the course of several days. In 6-OHDA-treated animals (treated neonatally or as adults), the bulk of proteins transported in nigro-striatal non-dopaminergic neurons appears to be very similar to that transported in the intact pathway in control rats. However, in striata of 6-OHDA-treated animals, a consistent reduction in striatal 35S- and 3H-radioactivitiy was observed in proteins with molecular weight from about 67,000 to 77,000. Assuming that the 6-OHDA treatment did not substantially affect the non-dopaminergic neurons, we interpret this to mean that some of the proteins in this molecular weight range are transported primarily by dopaminergic neurons.

Developmental studies of the uptake of choline, GABA and dopamine by crude synaptosomal preparations after in vivo or in vitro lead treatment.

The kinetics of sodium dependent, high affinity uptake of choline and dopamine by striatal synaptosomal preparations and of GABA (gamma-aminobutyric acid) by cortical synaptosomal preparations have been examined during the development of Long-Evans control and lead-treated rats. Choline uptake was very low until 12 days postnatally, then the Vmax increased and approached adult values of 29 pmol/mg prot./min within a week. GABA uptake was somewhat elevated at birth and only after three weeks did it decrease to the adult value of 0.7 nmol/mg prot./min. Dopamine uptake was low at birth, developed linearly with age and by 30 days postnatally approached the adult value of 68 pmoles/mg prot./min. The high affinity uptake constants (choline, 0.66 microM; GABA, 4.4 muM; and dopamine, 0.31 muM) did not change markedly during development. Similar studies were conducted with rats treated at the highest lead dosage which did not result in weight loss (100 microgram lead as lead acetate/g body weight/day via intubation). Blood and brain lead determinations confirmed a substantial lead exposure. Such chronic exposure did not markedly affect the amount or developmental pattern of uptake of the putative neurotransmitters. The effect of 2.5 x 10(-5) M lead acetate in vitro on the kinetics of high affinity uptake of these compounds into preparations from 20-26-day-old rats was investigated. When uptake was assayed in the absence of calcium, lead caused a 20% increase in the Vmax for dopamine. This stimulation was reduced if samples were assayed in the presence of n mM CaCl2. The Km for high affinity uptake of these neurotransmitter-related compounds was not affected by lead. In other studies, crude synaptosomal preparations were preloaded with neurotransmitter by preincubation with radioactive choline, GABA, or dopamine. Release of radioactive neurotransmitter, either spontaneous or in response to potassium depolarization, was quantitated and correlated with the presence or absence of 2.5 x 10(-5) M lead and/or 10(-3) M calcium ions. Lead slightly inhibited calcium dependent spontaneous release of dopamine. Lead also appeared to partially substitute for calcium in the potassium depolarized release of dopamine and GABA, although subtraction of the spontaneous (potassium independent) component reduced the magnitude of the lead effect.

Appearance of myelin proteins in rat sciatic nerve during development.

Rats between 5 and 45 days of age were sacrificed and their sciatic nerves dissected. Myelin was prepared from these sciatic nerves by a procedure involving purification on discontinuous sucrose gradients. The proteins of whole sciatic nerves at different ages and the proteins derived from the myelin isolated from these sciatic nerves were examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate. Over half of the proteins of sciatic nerve myelin migrated in a single band on the gel (P0). There were only minor changes in the protein distribution of sciatic nerve mylein during development. In contrast, the polyacrylamide gel patterns of whole sciatic nerve homogenate changed markedly during development between 5 and 15 days of age. The amount of P0 protein as a proportion of the total sciatic nerve protein increased from 3% at 5 days of age to 13% at 15 days of age after which it remained constant. Several other proteins which were also characteristic of the isolated myelin increased in relative importance during this time period. Parallel experiments dealing with a metabolic parameter of myelinogenesis, incorporation of intraperitoneally injected [35S]sulfate into sulfatide, were conducted. The maximum synthesis of sulfatide occurred between 6 and 16 days of age, coincident with the marked accumulation of myelin proteins in sciatic nerve.

Axonal transport and metabolism of [3H]fucose- and [35S]-sulfate-labeled macromolecules in the rat visual system.

The axonal transport of labeled macromolecules in retinal ganglion cells of rats was investigated from 1 to 20 days following intraocular injection of [3H]fucose and [35S]sulfate. Maximal incorporation of [3H]fucose into acid insoluble material in the retina was at 8 h, followed by a biphasic decline. Transported [3H]fucose (98% as glycoprotein) was in the optic nerve at 1 h, the optic tract and lateral geniculate body by 2 h, and the superior colliculus by 3 h after injection, indicating a rate of transport of approximately 200 mm/day. Radioactivity continued to accumulate in the superior colliculus for at least 8 h and began to decline rapidly by 24 h. Between 3 and 6 days levels rose again in both optic tract and superior colliculus before starting a gradual decline, indicating that a wave of rapidly transported material was delayed in leaving the retina. When proteins in the superior colliculus were fractionated by gel electrophoresis, the composition of the two fucosylated protein transport phases could be partially resolved. Radioactivity in individual gel peaks represented primarily in the first phase decayed with an average half-life of one day, althouth that in one prominent protein of molecular weight 280,000 turned over with a half-life of the order of 12 h. Radioactive peaks primarily in the second phase decayed with an average half-life of more than a week. Incorporation of [35S]sulfate into acid insoluble material in the retina was maximal at 1-2 h, after which there was a rapid loss of label. The appearance of [35S]sulfate in the optic tract, lateral geniculate body and superior colliculus preceded by a short time that of the [3H]fucose; indicating a shorter retinal processing time for this label. The total transported [35S]sulfate in the superior colliculus peaked by 4-8 h and had fallen by 65% at one day; no prominent second wave of transport was observed as was the case for [3H]fucose. Acid insoluble [35S]sulfate in the superior colliculus was equally divided between glycopeptides and glycosaminoglycans at all times examined, indicating that these macromolecules are transported at the same rate. [35S]Sulfate incorporated into various proteins fractionated by gel electrophoresis had heterogeneous turnover rates, the average being around 12 h. Radioactivity in one group of proteins, of molecular weight around 90,000, decayed with a half-life of only a few hours.

Modulation by neuropeptides of bradykinin-stimulated second messenger release in dorsal root ganglion neurons.

Fetal rat dorsal root ganglion neurons (7-8 days in culture) were labeled with [3H]arachidonic acid for 24 h. Stimulation with 10 microM bradykinin (BK) for 30 s resulted in nearly 2-fold increases in levels of radioactive diglyceride and arachidonic acid. A similar result was obtained in the absence of receptor stimulation using the Ca2+ channel agonist BAY K 8644 (10 microM, in the presence of 100 mM potassium chloride) or the Ca2+ ionophore, ionomycin (2.5 microM). If Ca2+ influx was inhibited by adding 3 mM Co2+, a blocker of voltage-sensitive calcium channels, or 2.5 mM EDTA, then BK-stimulated accumulation of both arachidonate and diglyceride was inhibited. These data suggest Ca2+ influx is required for ligand-stimulated accumulation of both arachidonate (a product of diglyceride-lipase or phospholipase A2) and diglyceride (a product of phospholipase C). Two distinct populations of channels may be involved in these reactions since pretreatment with 10 microM nifedipine or 50 microM verapamil (agents which block a subset of voltage-sensitive Ca2+ channels) inhibited BK-stimulated accumulation of arachidonic acid, but did not inhibit diglyceride accumulation. Such functional discrimination appears to have physiological importance; the inhibitory effect of nifedipine and verapamil on BK-stimulated arachidonate release was mimicked by pretreatment with peptides which decrease Ca2+ channel conductance in dorsal root ganglion neurons. The three peptides used were 1 microM neuropeptide Y, 10 microM somatostatin, and 10 microM [N-MePhe3,D-Pro4]-morphiceptin. The effect of neuropeptide Y was blocked by pretreatment with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Axonal transport through nodes of Ranvier.

Axonally transported glycoproteins are shown to accumulate at nodes of Ranvier. We hypothesize that the increased labeling in nodal regions results from the rheological effects of axonal constriction as well as from selective deposition of some transported labeled molecules.

The use of Synapsin I as a biochemical marker for neuronal damage by trimethyltin.

The content of Synapsin I (Protein I) was examined in brain regions of adult rats exposed to trimethyltin (TMT), and in control animals. Long Evans hooded rats were intragastrically dosed with 4 mg TMT hydroxide/kg body weight for 4 days. No perturbations in Synapsin I levels were evident by 24 h following the fourth dose; however, by 36 h, a significant decrease of 28% in Synapsin I level was present in the hippocampus of TMT treated animals. This decrease was selective, no other brain region examined was affected. As determined by regional analysis of inorganic tin, this specificity was not due to a profound preferential accumulation of tin in the hippocampus. Despite the absence of an alteration in Synapsin I levels at 24 h, morphological examination revealed perturbation in the normal uniform arrangement of granule cell neurons, with dead neurons diffusely distributed throughout the facia dentata. At 36 h, these changes were only slightly more extensive. In contrast, examination of the terminal projection area of these cells, the mossy boutons, showed to be unaffected at 24 h after the 4th dose of TMT. However, by 36 h, many of the mossy boutons contained dense bodies and showed signs of degeneration. This result suggested that the loss of Synapsin I coincides with degeneration of the nerve terminal region. In order to better establish the temporal correlation, a less severe dosing regimen (only 3 days of exposure to 4 mg TMT/kg body wt) was utilized to attenuate the time course of necrosis. Again, necrotic changes were visible in the perikaryon by 1 day after termination of toxicant dosing.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental lead encephalopathy in the suckling rat: concentration of lead in cellular fractions enriched in brain capillaries.

Five-day old rats subjected to short-term (2-day) lead exposure by gastric gavage of aqueous lead acetate at the highest non-lethal dosage (1mgPb/g body weight/day) developed a hemorrhagic encephalopathy. Capillaries and microvessels isolated from brains of these rats showed abnormal morphology consisting of an increased number of irregularly dispersed endothelial nuclei and swollen, vacuolated endothelial cells. Lead was concentrated in isolated brain capillary-microvessel fractions, as demonstrated by both atomic absorption and 210Pb tracer methods. When lead exposure was continued for 20 days (at the maximal dosage regime compatible with a 60% survival rate), the rats recovered from the initial encephalopathy and capillaries and microvessels isolated from brains of these rats appeared morphologically normal. This recovery occurred despite continued high levels of lead in the blood and in the isolated capillary-microvessel fractions, suggesting that, as capillary endothelial cells mature, they are able to adapt to the presence of large amounts of lead.

Diacylglycerol modulates action potential frequency in GH3 pituitary cells: correlative biochemical and electrophysiological studies.

We have investigated the involvement of enhanced phosphoinositide metabolism in mediating TRH-induced alteration of electrophysiological events related to prolactin secretion by GH3 cells (a line of pituitary origin). Patch-clamp recording (in the current clamp, whole-cell configuration) showed that a few seconds after TRH application there was a brief period (about 30 s) of membrane hyperpolarization followed by several minutes of increased calcium-dependent action potential frequency. In parallel experiments cells were labeled for 24 h with either [3H]myo-inositol or [3H]arachidonate. Application of TRH resulted in rapid increases in levels of inositol phosphates and diacylglycerol. The time course of elevation of inositol 1,4,5-triphosphate (maximal by 5 s) is compatible with an initial burst of intracellular calcium mobilization associated with a transient phase of TRH-induced prolactin release. Application of TRH was also followed by a rapid but more sustained (several minutes) period of elevated diglyceride accumulation; a time course corresponding to a prolonged period of prolactin release which is dependent on the influx of external calcium. A causal relationship between diglyceride release and increased action potential frequency was demonstrated since local application (via a U-tube apparatus) of either 2 microM phorbol ester (phorbol 12,13-dibutyrate or phorbol 12-myristate 13-acetate) or 60 microM 1-oleoyl-2-acetyl-glycerol to patch-clamped cells could mimic this aspect of the TRH effect. In contrast, the inactive phorbol ester, 4 alpha-phorbol, was unable to elicit this response.(ABSTRACT TRUNCATED AT 250 WORDS)

Myelin synthesis during postnatal nutritional deprivation and subsequent rehabilitation.

Newborn Long-Evans rats were undernourished by maternal deprivation so that by 20 days of age their body and brain weights were about 45 and 80%, respectively, of the values obtained for control (well-nourished) values. Proteins from myelin of undernourished and control rats were separated by polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate. At 15 and 20 days of age the proportion of basic and proteolipid protein was reduced in the starved animals relative to controls, indicative of a delay in maturation. However, by 30 days of age the composition of myelin from starved and control animals appeared similar. At all ages the yield of myelin from brains of starved rats was less than 25% of that obtained from control animals. A series of isotope labeling experiments, using a double label design, was carried out to compare relative rates of incorporation of radioactive amino acids into individual proteins of various brain subcellular fractions. In 20-day-old rats the incorporation of [3H] OR [14C] leucine or glycine into myelin proteins, relative to incorporation into proteins of other subcellular fractions, is preferentially depressed (about 60%) in starved animals. Synthesis of all the myelin proteins was depressed, supporting the hypothesis that the high molecular weight proteins isolated with myelin are true myelin constituents. Similar experiments were conducted using [3H]-and [14C] acetate, choline, or glycerol as precursors of lipids. Incorporation of isotope into lipids of myelin, relative to lipids of other subcellular fractions, was also depressed by about 60% in starved animals. In several experiments we studied synthesis during rehabilitation (ad libitum feeding) following 20 days of postnatal starvation. After 6 days of rehabilitation, incorporation of radioactive precursors into myelin, relative to other subcellular fractions, was still depressed. This result was true for both proteins and lipids, and was interpreted as evicence against the initiation of a process leading to a net recovery of myelin (i.e., an irreversible deficit of myelin synthesis is induced by this regime of nutritional deprivation).

The postnatal development of glial fibrillary acidic protein and neurofilament triplet proteins in rat brain stem.

Cytoskeletal preparations containing both the glial fibrillary acidic protein and the neurofilament triplet proteins were prepared from brain stems of rats at different ages and the individual peptides separated in polyacrylamide gels. Stained peptide bands were quantitated as the area under peaks generated by densitometric scanning. Peak areas were converted to grams of protein based on total gel dye binding and total protein applied to the gels. Between 5 and 30 days, the concentration of the peptide (g of peptide/mg of tissue protein) of apparent molecular weight 51,000 (corresponding to the glial fibrillary acidic protein), increased 3 fold. The corresponding increase in total concentration of the three peptides corresponding to the neurofilament proteins was 4.5 fold. However, the increase in concentration of the individual neurofilament peptides was each different. Very little of the apparent molecular weight 210,000 neurofilament peptide was present at 5 days and its concentration increased 11 fold by 30 days compared to about 3.5 fold for the other two neurofilament peptides. These results are in general agreement with studies using immunological techniques and the methods have the advantages of using readily available techniques and allowing the simultaneous comparison of both neuronal and glial specific filaments during development.

Schwann cells as targets for neurotoxicants.

Schwann cells subserve a variety of roles in the peripheral nervous system (PNS), including ionic homeostasis, and protection and possible metabolic support of axons. It is, however, the myelinating subtype of these glia which appear most sensitive to toxic insults. Myelinating Schwann cells must synthesize large amounts of myelin proteins (P0 is the major myelin protein) and lipids (cholesterol is most prominent) within a short, tightly-programmed developmental window. Schwann cells are preferentially vulnerable to neurotoxic insults during this period of maximal metabolic stress. The hydrophobicity of myelin (reservoir for lipid-soluble toxicants) and possible specialized energy-requiring mechanisms for maintenance of myelin structure are points of vulnerability for the mature myelin sheath. Fortunately, Schwann cells are highly plastic; they dedifferentiate to more primitive precursor cells following a demyelinating insult, but are able to redifferentiate and remyelinate axons during subsequent nerve regeneration. For study of such processes, a useful model system is exposure of developing rats to the element tellurium; this produces a highly synchronous primary demyelination of PNS which is followed closely by rapid remyelination. Interpretation of the metabolic events involved in simplified by the nearly complete lack of axonal degeneration. We have uncovered the primary lesion (block in cholesterol biosynthesis) and elucidated some of the steps involved in the demyelination-remyelination response. Particularly useful have been studies of gene expression of certain proteins (nerve growth factor receptor, myelin proteins, macrophage-specific lysozyme) which have enabled us to define some of the cellular responses to this toxicant-induced injury. A generally applicable result that has emerged from these and other similar studies is that upregulation of NGF-R mRNA is a sensitive marker of nerve damage; it may be useful as a screen for potentially neurotoxic compounds.

Gene expression during tellurium-induced primary demyelination.

A compound may be "developmentally neurotoxic" because it interferes with a metabolic step exclusively or preferentially expressed during development in a particular class of neural cells. The initial metabolic specificity is often complicated by: (1) secondary responses in the affected cells, (2) involvement of other functionally-related cell types, and (3) the presence of compensatory and/or regenerative responses. In this context we study tellurium, which systemically blocks cholesterol biosynthesis at the squalene epoxidase step. Because of the high demand in developing peripheral nerves for newly synthesized cholesterol required for myelin assembly, this metabolic block leads to demyelination of the sciatic nerve. This insult is confounded by the fact that the myelin-forming Schwann cells do not upregulate their cholesterol biosynthetic pathway. This is contrary to expectations; liver (the main source of cholesterol for many tissues outside the nervous system) upregulates synthesis of cholesterol and overcomes the metabolic block. The shortage of cholesterol in Schwann cells results in an immediate secondary response down-regulation of steady-state mRNA levels for specific myelin proteins. Remyelination occurs after cessation of tellurium exposure. This model of primary demyelination allows study of Schwann-cell specific responses during the processes of myelin breakdown and subsequent steps leading to remyelination, without the complications of axonal degeneration and regeneration. Because tellurium specifically blocks the synthesis of a major required membrane component, it is also well suited for examining the coordinate control of membrane synthesis and assembly at the genomic level.