Regulation of Nervous System-Specific S-100 Protein and Enolase Levels in Adipose Tissue by Catecholamines

The effect of catecholamines on the levels of S-100 protein and nervous system-specific enolase (NSE) in epididymal adipose tissue of Wistar rats in vivo was examined by sensitive enzyme immunoassay methods. Soluble S-100 protein levels in the adipose tissue of 9-12-week-old rats (1.46 +/- 0.19 microgram/mg protein) were decreased to less than 50% of those of controls by serial injection (for 4-7 days) of epinephrine (0.1 mg/day) or norepinephrine (0.15 mg) with, however, little effect on the levels of membrane-bound (pentanol-extractable) S-100 protein. A significant decrease in the soluble S-100 protein levels was observed at 2 h after a single injection of epinephrine (1.04 +/- 0.13 microgram/mg protein). On the other hand, levels of NSE subunit (gamma subunit or 14-3-2 protein) in adipose tissue (0.51 +/- 0.03 gamma gamma-equivalent pmol/mg protein) were increased to 170% of control by serial injection (for 7 days) of epinephrine or norepinephrine with little change of the level of enolase alpha subunit on a mg protein basis. Isoproterenol had no apparent effect on the levels of soluble S-100 protein and NSE subunit. These results suggest that the levels of S-100 protein and NSE in adipose tissue are regulated by catecholamines.     

Effects of Various Forms of Stimulation on the Content of Enolase Isozymes and S-100 Protein in Superior Cervical Sympathetic Ganglia Excised from Rats

Contents of the three forms (alpha alpha, alpha gamma, and gamma gamma) of enolase isozymes and S-100 protein in superior cervical sympathetic ganglia (SCG) excised from rats were determined by the sensitive method of enzyme immunoassay, after application of various forms of stimulation, during incubation for 3 h at 37 degrees C in vitro. The amounts of the three forms of enolase isozymes and of S-100 protein in the SCG were not altered by preganglionic or postganglionic stimulation (10 Hz) or by the addition of acetylcholine (1 mM) or a high concentration of K+ (70 mM) to the incubation medium. Norepinephrine (NE; 50 microM), as well as isoproterenol (200 microM) or 3,4-dihydroxy phenylethylamine (dopamine; 200 microM), increased the ganglionic alpha alpha and alpha gamma enolase content to 1.5 to 2.0 times the control level, whereas NE tended to slightly decrease the gamma gamma enolase content. The increase in the alpha isozymes did not appear until after 2 to 3 h of incubation with this agent as a result of an increase in protein synthesis. Propranolol, an adrenergic antagonist, partly inhibited the NE-induced increase in both alpha alpha and alpha gamma enolases. NE and its agonists also considerably increased the S-100 protein level in the SCG; however, the effect developed within half an hour of incubation as a result of the conversion of the bound S-100 protein to the water-soluble form, and did not greatly increase thereafter. Cyclic AMP (1 mM) produced the same kind of increase in the ganglionic S-100 protein content as NE did.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue distribution, developmental profiles and effect of denervation of enolase isozymes in rat muscles

The tissue distribution of muscle-type alpha beta and beta beta enolases in rats were determined with the sandwich-type enzyme immunoassay method which utilized the purified antibodies specific to the alpha and to the beta subunit of enolase, and beta-D-galactosidase from Escherichia coli as label. All the tissues examined contained detectable levels of both alpha beta and beta beta enolases. The beta beta enolase was found at high levels in the skeletal muscle tissues (tongue, esophagus, diaphragm and leg muscles) and in the cartilages (xipoid process and auricular cartilage). The alpha beta enolase was distributed at a relatively high concentration in the heart and in the above-mentioned tissues. The beta beta enolase in the leg muscles, diaphragm and tongue was present on the day of birth at a concentration higher than that of the alpha alpha and alpha beta enolases, and its concentration further increased in a manner apparently related to the functional state of each tissue. Denervation of the leg muscles by cutting the sciatic nerve in adult rats resulted in a drastic change in the isozymes profile. The concentration of beta beta enolase in the tibialis anterior gastrocnemius lateralis and extensor digitorum longus (about 800 pmol/mg protein) decreased to about a half in a few weeks after denervation. In contrast, the concentrations of alpha alpha (2 pmol/mg) and alpha beta (80 pmol/mg) usually showed a slight increase by the treatment (alpha alpha, 7 pmol/mg; alpha beta, 100 pmol/mg after 2 weeks). As compared with these three muscles, the soleus had normally a low enolase level and the effect of denervation was less drastic. These results seem to suggest that the concentration of beta beta enolase is closely correlated with the functional state of the muscle tissue.     

Effects of Denervation and Axotomy on Nervous System-Specific Protein, Ornithine Decarboxylase, and Other Enzyme Activities in the Superior Cervical Sympathetic Ganglion of the Rat

The time courses of changes of three enolase isozymes (alpha alpha, alpha gamma, and gamma gamma), S-100 protein, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), ornithine decarboxylase (ODC), beta-galactosidase, and glucose-6-phosphate dehydrogenase (G6PDH) were examined from 1 to 14 days after cutting of the preganglionic nerve (denervation) or the postganglionic nerve (axotomy) of the superior cervical sympathetic ganglion (SCG) of the rat. The wet weight and protein content in the axotomized SCG increased continuously, to nearly twice those of the denervated SCG for 1-2 weeks after the operations. Among enolase isozymes in the SCG, neuron-specific gamma gamma-enolase decreased rapidly after denervation and stayed at a low level for 2 weeks, whereas the isozyme remained almost unchanged after axotomy. On the contrary, ganglionic alpha alpha-enolase and the alpha gamma-hybrid form increased remarkably to reach a maximum at the second day after axotomy, and remained above control for 1 to 2 weeks; these two enolase isozymes showed little change after denervation. Denervation caused a much larger increase than did axotomy in the ganglionic S-100 protein, an astrocyte-specific protein, during the first week after the operation, while the protein content decreased after 2 weeks of either denervation or axotomy. CNPase, a myelin-associated enzyme, rose suddenly 2 days after axotomy, and remained at a rather high level compared with the denervated ganglion, which showed little variation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of S-100b (beta beta) protein in human teratocarcinoma cells

Human teratocarcinoma NT2/D1 cells undergo differentiation into a variety of cell types, including neurons, treated with retinoic acid. In the present study, the concentrations of alpha S-100 and beta S-100 proteins (alpha and beta subunits of S-100 proteins), and three subunits (alpha, beta and gamma) of enolase in NT2/D1 cells were measured using the sensitive enzyme immunoassay method. The concentration of beta S-100 was markedly increased in the cells after treatment with retinoic acid, whereas the concentration of alpha S-100 was undetectably low, indicating that the S-100b (beta beta) protein was induced by retinoic acid. On the other hand, the concentrations of the three forms of enolase isozymes did not change in the same culture. The induction of S-100b protein was not observed in the NT2/D1 cells after treatment with forskolin, dibutyryl cyclic AMP or cholera toxin. The indirect double-labeled immunofluorescence, using antibodies specific to beta S-100 and monoclonal antibodies specific to neurofilaments, revealed that both the S-100b protein and the neurofilaments were induced in the same subpopulation of cells which underwent neuronal differentiation.     

Immunocytochemical localization of S-100b protein in degenerating and regenerating rat sciatic nerves

We studied the cellular and subcellular distribution of S-100b protein in normal, crushed, and transected rat sciatic nerves by an immunocytochemical procedure. In uninjured nerves, S-100b protein was restricted to the cytoplasm and membranes of Schwann cells, with no reaction product present in the nucleus or in axons. Similar images were seen from the first to the thirtieth day after the crush in activated Schwann cells during the degeneration period, i.e., up to the seventh post-lesion day, and in normal Schwann cells reappearing during the regeneration period, i.e., after the seventh post-lesion day, in the zone of the crush and proximal and distal to it. By the technique employed, there seemed to be no differences in the intensity of the immune reaction product in normal and activated Schwann cells. Also, similar images were seen in the proximal stump of transected nerves. Only a slight S-100b protein immune reaction product could be observed in the rare activated Schwann cells present in the distal stump around the seventh post-lesion day, the majority of cell types being represented by fibroblasts and elongated cells at this stage and thereafter. By immunochemical assays, similar results as those presented here have been reported and interpreted as indicative of the presence of S-100 protein in axons or, alternatively, of axonal control over expression of S-100 protein in Schwann cells. Our immunocytochemical data clearly show that the strong reduction in the S-100 protein content of the distal stump of transected nerves is owing to the paucity of Schwann cells and to the decrease in the S-100 protein content of these cells, rather than to degeneration of axons.     

Glial and Neuronal Marker Proteins in the Silicone Chamber Model for Nerve Regeneration

Abstract: In the present study, neuronal and Schwann cell marker proteins were used to biochemically characterize the spatiotemporal progress of degeneration/regeneration in the silicone chamber model for nerve regeneration. Rat sciatic nerves were transected and the proximal and distal stumps were inserted into a bridging silicone chamber with a 10-mm interstump gap. Using dot immunobinding assays, S-100 protein and neuronal intermediate filament polypeptides were measured in different parts of the nerve 0–30 days after transaction. In the most proximal nerve segment, all the measured proteins were transiently increased. In the proximal and distal stumps adjacent to the transection, the studied proteins were decreased indicating degeneration of the nerve. Within the silicone chamber, the regenerating nerve expressed the Schwann cell S-100 protein already at 7 days, whereas the neurofilament polypeptides appeared later. These observations are corroborated by previous morphological studies. The biochemical method described provides a new and fast approach to the study of nerve regeneration.     

Tropic 1808基因在大鼠损伤神经组织中的表达

目的观察Tropic 1808基因在大鼠正常和损伤坐骨神经组织中的表达,探讨Tropic 1808基因在周围神经损伤与再生过程中的作用.方法采用地高辛标记的Tropic 1808 cDNA探针、抗大鼠S-100蛋白抗体,以原位杂交和免疫组织化学双重染色法,观察Tropic 1808基因在正常和损伤大鼠坐骨神经组织中的表达.结果免疫组化结果显示,大鼠正常坐骨神经可表达S-100蛋白,但表达量较低;神经损伤后,其远侧端S-100蛋白的表达量明显增加.原位杂交结果显示,大鼠正常坐骨神经组织未见Tropic 1808 mRNA杂交信号;损伤神经的远侧端呈现较强的阳性信号,而且在部分S-100强阳性反应区可见Tropic 1808 mRNA杂交信号.结论 Tropic 1808基因在正常坐骨神经组织中未见表达;坐骨神经损伤后,其远侧端增殖的雪旺氏细胞可表达Tropic 1808 mRNA.提示,Tropic 1808是一种周围神经损伤后特异表达的基因.     

Sensory and autonomic innervation of non-hairy and hairy human skin

Summary Non-hairy and hairy human skin were investigated with the use of the indirect immunohistochemical technique employing antisera to different neuronal and non-neuronal structural proteins and neurotransmitter candidates. Fibers immunoreactive to antisera against neurofilaments, neuron-specific enolase, myelin basic protein, protein S-100, substance P, neurokinin A, neuropeptide Y, tyrosine hydroxylase and vasoactive intestinal polypeptide (VIP) were detected in the skin with specific distributional patterns. Neurofilament-, neuron-specific enolase-, myelin basic protein-, protein S-100-, substance P-, neurokinin A-and vasoactive intestinal polypeptide (VIP)-like immunoreactivities were found in or in association with sensory nerves; moreover, neuron-specific enolase-, myelin basic protein-, protein S-100, neuropeptide Y-, tyrosine hydroxylase- and vasoactive intestinal polypeptide (VIP)-like immunoreactivities occurred in or in association with autonomic nerves. It was concluded that antiserum against neurofilaments labels sensory nerve fibers exclusively, whereas neuron-specific enolase-, myelin basic protein- and protein S-100-like immunoreactivities are found in or in association with both sensory and autonomic nerves. Substance P- and neurokinin A-like immunoreactivities were observed only in sensory nerve fibers, and neuropeptide Y- and tyrosine hydroxylase-like immunoreactivities occurred only in autonomic nerve fibers, whereas vasoactive intestinal polypeptide (VIP)-like immunoreactivity was seen predominantly in autonomic nerves, but also in some sensory nerve fibers.     

CYCLOHEXIMIDE ALTERS AXONAL TRANSPORT AND SUBCELLULAR DISTRIBUTION OF DOPAMINE-β-HYDROXYLASE ACTIVITY

—Administration of cycloheximide, 10 mg/kg s.c. led within 4 h to an approx 30% reduction of dopamine-β-hydroxylase (DBH) activity in the abdominal portion of rat sciatic nerves. At least two more hours elapsed before DBH activity in the distal part of these nerves began to fall. This pattern suggests reduced synthesis or delivery of DBH into axons but continued transport of previously delivered enzyme. Coinciding with the time at which DBH activity began to fall in distal segments of sciatic nerve, there was a marked reduction in the accumulation of DBH activity above a ligature in this region. Between 4 and 8 h after administration of cylcoheximide, 10 mg/kg, accumulation above a ligature was 70% less than in untreated nerves (P < 0.001), a reduction significantly greater (P < 0.05) than the accompanying 28% loss of baseline DBH activity. At the same time, the clearance of DBH activity from nerve regions distal to a ligature was greatly reduced. This pattern is consistent with the depletion of a minor but rapidly transported compartment of DBH. Six hours after administration of cylcoheximide, 10 mg/kg, the apparent subcellular distribution of DBH in distal regions of sciatic nerve was altered by a significant 36% loss in sedimentable DBH activity, with non-significant changes in othcr fractions. This suggests that rapidly transported DBH, depleted from the nerve by cycloheximide-induced inhibition of protein synthesis, is more highly associated with intraneuronal particles than is slowly transported or stationary DBH.     

Inhibition by Forskolin of Excitatory Amino Acid-Induced Accumulation of Cyclic AMP in Guinea Pig Hippocampal Slices

Left sciatic nerves of adult male Sprague-Dawley rats were crushed and allowed to recover for 0, 1, 2, 4, 7, or 14 days. At each of these times both L-5 dorsal root ganglia were injected with 100 microCi of [3H]glucosamine. Two days later, dorsal root ganglia, lumbosacral trunks, and sciatic nerves were removed bilaterally. The amounts of radiolabelled ganglioside in crushed lumbosacral trunks were consistently higher than in the controls, with the largest difference occurring within 2 days from simultaneous crush and injection to killing (specimens labelled day 0). The largest difference in the amount of radiolabelled ganglioside between crushed and control sciatic nerve (4-9 days from crush to killing) occurred later than that of lumbosacral trunk, but no significant difference occurred within the first 3 days following crush. There was only a slightly higher radioactivity in gangliosides totalled from all three anatomical specimens of crushed than in control nerves. The neutral nonganglioside lipid and acid-precipitable fraction followed patterns of synthesis and accumulation similar to those of the gangliosides. These findings indicate that after nerve crush gangliosides, glucosamine-labelled neutral nonganglioside lipids, and glycoproteins accumulate close to the proximal end of the regenerating axon. This accumulation could serve as a reservoir to increase the ganglioside concentration in the growth cone membrane.     

Characterization of pancreatic islet Ca2+-ATPase

Distribution of three isoenzymes of brain enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11) (alpha alpha, alpha gamma and gamma gamma forms) in clonal cell lines of neuroblastoma (NS20Y and N18TG-2), glioma (C6BU-1), and hybrid cells NG108-15, NCB20, Nbr10A, Nbr20A, N4G-B-a and N4G-C-a) was examined with a sensitive enzyme immunoassay system, that uses a rabbit antibody to rat brain enolase alpha alpha or gamma gamma. All cell lines tested were found to possess the enolase which contains gamma subunit (a neuron-specific protein), although the alpha alpha enolase (non-neuronal enolase) was the dominant from in these cells. A clonal rat glioma (C6BU-1) cell contained about 40, 1 and 0.07 microgram/mg protein of alpha alpha, alpha gamma and gamma gamma enolases, respectively, at the confluent stage. Inclusion of 1 mM dibutyryl cyclic AMP or 10 micrometers prostaglandin E1 plus 1 mM theophylline in the culture medium of a hybrid cell (NG108-15, mouse neuroblastoma x rat glioma) resulted in a more than 2-fold increase in the concentrations of alpha gamma and gamma gamma in the cell within a few days, with little change in the alpha alpha enolase concentration. A similar increase in the concentration of gamma subunit by the nucleotide (but not by prostaglandin E1 plus theophylline) was also observed in the glioma cell (C6BU-1) line. The results suggest that the gamma subunit or the neuron-specific protein can be regulated in NG108-15 and C6BU-1 cells in a cyclic AMP-dependent fashion.     

Selective Increase in S-100β Protein by Aging in Rat Cerebral Cortex

Changes in the concentrations of nervous tissue-related proteins and their isoproteins, such as S-100 proteins (S-100 alpha and S-100 beta), enolase isozymes (alpha-enolase and gamma-enolase), and GTP-binding proteins (Go alpha, Gi2 alpha, and beta-subunits), were determined in the CNS of male rats of various ages (from 2 to 30 months old) by means of enzyme immunoassay. The weights of brains and the concentrations of soluble proteins in the cerebral cortex, cerebellum, and brainstem were constant during the observation period. The concentration of S-100 beta protein, which is predominantly localized in glial cells, increased gradually in the cerebral cortex with age; levels in the 25-month-old rats increased to approximately 150% of the levels in the young (2-month-old) rats. However, the S-100 beta concentrations in the cerebellum and brainstem were relatively constant, showing similar values in rats 2-30 months old. Levels of other proteins, including both neuronal (gamma-enolase and Go alpha) and glial (alpha-enolase and S-100 alpha) marker proteins, did not change significantly with age in the cerebral cortex, cerebellum, and brainstem. These results suggest that there is a close relation between the age-dependent changes of the CNS function and S-100 beta protein levels in the cerebral cortex.     

Posttranslational Protein Modification by Amino Acid Addition in Intact and Regenerating Axons of the Rat Sciatic Nerve

Abstract: Experiments were performed to determine whether ppsttranslational addition of amino acids to axonal proteins occurs in axons of the rat sciatic nerve. Two ligatures were placed 1 cm apart on sciatic nerves. Six days later, segments proximal to each ligature were removed, homogenized, centrifuged at 150,000 · g , and analyzed for the ability to incorporate ³H-amino acids into proteins. No incorporation of amino acids into proteins was found in the high-speed supernatant, but when the supernatant was passed through a Sephacryl S-200 chromatography column (removing molecules less than 20 kD), [³H]arginine, lysine, leucine and aspartic acid were incorporated into proteins in both proximal and distal nerve segments. Small but consistently greater amounts of radioactivity were incorporated into proteins in proximal segments compared with distal segments, indicating that the components necessary for the reaction are transported axonally. This reaction represents the posttranslational incorporation of a variety of amino acids into proteins of rat sciatic nerve axons. Other experiments showed that the incorporation of amino acids into proteins is by covalent bonding, that the amino acid donor is likely to be tRNA, and that the reaction is inhibited in vivo by a substance whose molecular mass is less than 20 kD. This inhibition is not affected by incubation with physiological concentrations of unlabeled amino acids, by boiling, or by treatment with Proteinase K. When the axonally transported component of the reaction was determined in regenerating nerves, the amount of incorporation of amino acids into protein was 15–150 times that in intact nerves. The results indicate that the components of this reaction are transported axonally in rat sciatic nerves and that the reaction is increased dramatically in growing axons during nerve regeneration.     

Reversal of axonal transport in regenerating nerves.

Abstract— Orthograde and retrograde axonal transport were studied in rat sciatic nerves which had been crushed and either allowed to regenerate, or prevented from doing so by tightly ligaturing the nerve. At various intervals after crushing the nerve. L-[³H]leucine was injected into the lumbosacral spinal cord. and the subsequent transport of labeled protein in motoneuron axons was quantitated by measuring the accumulation of labeled protein at collection crushes made proximal to the original nerve crush. Accumulations proximal to the collection crushes (orthograde transport) 9-11 h after injection (p.i.). decreased within I day of nerve injury, but returned to normal values as regeneration proceeded. In non-regenerating nerves accumulations remained depressed for at least 30 days. Accumulations distal to the collection crushes (retrograde transport) 9-11 h pi. increased over the first 5 days following injury but returned to normal values as regeneration proceeded. In non-regenerating nerves accumulations remained elevated. The time-course of retrograde transport of newly-synthesized protein also returned to normal during nerve regeneration. It is suggested that changes in retrograde transport during regeneration may inform the neuron cell body of the progress of regeneration and elicit appropriate metabolic responses. among which may be the changes in orthograde transport that follow axotomy.     

Ontogenetic development of the guinea pig uterine innervation. An immunohistochemical study of different neuronal markers, neuropeptides and S-100 protein

The ontogenetic development of the guinea pig uterine autonomic innervation was studied immunohistochemically using neurofibrillary protein (NF) and neuron specific enolase (NSE) as general neuronal markers, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) as specific markers for adrenergic innervation and S-100 protein as marker for Schwann cell structure and/or function. In addition, comparisons were made of the development of the different populations of peptide-containing nerves. The structure and time of appearance were similar for nerves with NF-, NSE-, TH- and DBH-immunoreactivities, which were first present in the organ periphery as coarse nerve trunks, then extending centrally and branching into non-varicose nerves. From these, varicose nerves developed first in relation to vessels and then in association with the myometrial smooth musculature. Development was completed earlier in the cervix than in the uterine horns suggesting differences in local environment. In comparison, S-100 nerve-immunoreactivity appeared later but attained complete development more rapidly than axonal structures. Neuropeptide Y-immunoreactive nerves showed a similar developmental pattern to presumed adrenergic nerves, further verifying the assumption of intraneuronal localization of NPY in uterine adrenergic nerves. Other peptide-containing nerves were developed later probably reflecting differences in neuronal growth properties.     

S-100 alpha-like immunoreactivity in tubules of rat kidney. A clue to the function of a "brain-specific" protein

The localization of the alpha and beta subunits of S-100 protein was studied in normal tissue where the identification of three subclasses of S-100 containing cells was derived: i) cells that contain both alpha and beta subunits; ii) cells that contain only the alpha subunit; and iii) cells that contain only the beta subunit. In this study monospecific antibodies against the S-100 alpha and beta subunits were used to characterize the S-100-like immunoreactivity in the rat kidney: Certain cells in the distal nephron, i.e., the connecting piece, collecting ducts, and the thin limb of Henle's loop, contained S-100 alpha immunoreactivity. Proximal tubules, the thick ascending limb of Henle's loop, the distal tubules, and the juxtaglomerular apparatus were negative. No S-100 beta immunoreactivity was found in kidney tubules. However, Schwann cells of renal pelvic nerves contained S-100 beta immunoreactivity. The presence of S-100 alpha antigen in certain cells of the kidney gives further support to the assumption that the alpha subunit of S-100a is related to cells that are highly involved in pH, electrolyte, and water regulation.     

Characterization of the enolase isozymes of rabbit brain: kinetic differences between mammalian and yeast enolases

Two isozymes of enolase, alpha alpha and gamma gamma, have been purified from rabbit brain and characterized. The kinetic properties of alpha alpha and gamma gamma (pH optimum, Km for phosphoglycerate and phosphoenolpyruvate, requirement for a divalent cation) are very similar to those of rabbit enolase, form beta beta, and to those of enolase isozymes from other species. However, several novel properties were observed. (i) All the enolases studied were inhibited by Na+ and Li+. (ii) The rabbit enolases, but not yeast enolase, were activated by K+, NH4+, Cs+, and Rb+. (iii) Rabbit enolase is more susceptible to inhibition by excess Mg2+ than is the yeast enolase; the increased inhibition by Mg2+ above pH 7.1 accounts, at least in part, for the observed differences between mammalian and yeast enolases in their pH optima for activity.     

Lysosulfatide (Sulfogalactosylsphingosine) Accumulation in Tissues from Patients with Metachromatic Leukodystrophy

We describe here a sensitive assay method for lysosulfatide (sulfogalactosylsphingosine) in human tissues using HPLC. The method involves extraction of lipids, saponification, isolation using a C18 Sep-Pak column, derivatization with o-phthalaldehyde, and detection of the fluorescent lysosulfatide using HPLC. In control subjects, a small amount of lysosulfatide was detected in the cerebral white matter (9-35 pmol/mg of protein), spinal cord (35 pmol/mg of protein), sciatic nerve (14 pmol/mg of protein), and kidney (approximately 2 pmol/mg of protein) but not in the cerebral gray matter and liver. A marked accumulation of the lipid was noted in tissues from six patients with metachromatic leukodystrophy (MLD). The concentration of lysosulfatide was high in the cerebral white matter, spinal cord, and sciatic nerve (223-1,172 pmol/mg of protein). Even in the cerebral gray matter, kidney, and liver, where lysosulfatide was hardly detected in the control sample, a considerable amount (3-45 pmol/mg of protein) accumulated in MLD patients. The concentration and distribution pattern of lysosulfatide were similar to those of galactosylsphingosine (psychosine) accumulated in patients with Krabbe disease. Therefore, the accumulation of lysosulfatide may explain the demyelination in patients with MLD, as is the case with Krabbe disease.     

Antibodies to neurofilament protein and other brain proteins reveal the innervation of peripheral organs

Monoclonal and polyclonal antibodies to neurofilament proteins, neuron-specific enolase, glial fibrillary acidic protein and S-100 have been used to demonstrate nerves, ganglion cells and the supportive glial system of the innervation of various organs. The female genitalia, the urinary tract, the respiratory system, the pancreas, the heart and the skin of several mammalian species, including rat, mouse, guinea pig, cat, pig, monkey and man were fixed in para-benzoquinone and portions of each organ were snap frozen. Serial or free-floating thick cryostat sections were stained using indirect immunofluorescence and peroxidase anti-peroxidase immunocytochemistry. In addition, the newly described and highly sensitive immunogold-silver staining technique was used on Bouin's-fixed and wax-embedded tissues. Antibodies to neurofilament proteins seemed to react with neuronal structures in all the species studied. Alternately stained serial sections showed a similar distribution of neurofilament proteins and neuron-specific enolase-containing nerves. Neuron-specific enolase staining had a diffuse appearance and was found to be highly variable, indicating that the neuron-specific enolase content might be related to the physiological state of the nerves and ganglion cells, whereas antibodies to neurofilament protein gave a consistently intense and very clear picture of the ganglion cells and nerve fibres. Antibodies to S-100 stained supportive elements of the peripheral nervous system in all tissues examined, whereas antibodies to glial fibrillary acidic protein were more selective.