Evidence for the presence of major peripheral myelin glycoprotein P0 in mammalian spinal cord and a change of its glycosylation state during aging.

Glycoproteins, which react with Lens culinaris agglutinin, in the membrane preparation of various portions of brains and spinal cords, obtained from 9-week-old rats and 29-month-old rats, were comparatively analyzed by SDS-polyacrylamide gel electrophoresis. In contrast to the samples from brain, which showed similar staining patterns in the two different age groups, the glycoprotein patterns of spinal cords showed marked differences by the age of donors. The most prominent evidence is that a glycoprotein with an apparent molecular weight of 30 kDa (gp30) was detected in the aged rats, but not in the young adult rats. Based on the amino acid sequence data around the glycosylation site, the gp30 was identified as P0, which is a member of immunoglobulin superfamily and a major structural component of mammalian peripheral nerve myelin. This is the first report indicating that P0, which has been considered as a peripheral nerve-specific glycoprotein, occurs also in the spinal cord of mammals. In addition, nonglycosylated P0 molecule could be detected in the spinal cord of young adult rats by anti-P0 polyclonal antibody. These results indicate that the glycosylation state of the P0 molecule in the spinal cord changes during aging.     

Protein associated with Myc (PAM) is involved in spinal nociceptive processing

PAM (protein associated with Myc) is a potent inhibitor of adenylyl cyclases (ACs) which is primarily expressed in neurones. Here we describe that PAM is highly expressed in dorsal horn neurones and motoneuron of the spinal cord, as well as in neurones of dorsal root ganglia in adult rats. PAM mRNA expression is differentially regulated during development in both spinal cord and dorsal root ganglia of rats, being strongest during the major respective synaptogenic periods. In adult rats, PAM expression was up-regulated in the spinal cord after peripheral nociceptive stimulation using zymosan and formalin injection, suggesting a role for PAM in spinal nociceptive processing. Since PAM inhibited Galphas-stimulated AC activity in dorsal root ganglia as well as spinal cord lysates, we hypothesized that PAM may reduce spinal nociceptive processing by inhibition of cAMP-dependent signalling. Accordingly, intrathecal treatment with antisense but not sense oligonucleotides against PAM increased basal and Galphas-stimulated AC activity in the spinal cord and enhanced formalin-induced nociceptive behaviour in adult rats. Taken together our findings demonstrate that PAM is involved in spinal nociceptive processing.     

Comparison of endogenous phosphorylation of hen and rat spinal cord proteins and partial characterization of optimal phosphorylation conditions for hen spinal cord

The optimal conditions for the endogenous phosphorylation of hen spinal cord cytosolic and membrane proteins with 5 μM [γ-³²P]ATP, 10 mM MgCl₂, were determined by 10% SDS-polyacrylamide gel electrophoresis, autoradiography, and microdensitometry. Phosphate incorporation increased linearly with concentrations ranging from 35–75 μg/100 μl for cytosolic proteins and 21–125 μg/200 μl for membrane proteins. Optimal incubation times, temperatures, and pH values were 60 s, 30°C, and 6.0, respectively, for spinal cord cytosolic proteins and 15 s, 45°C, and 8.0, respectively, for spinal cord membranes. Prominent species differences in protein phosphorylation between these fractions in hens and similarly prepared fractions in rats, co-electrophoresed, include 80K and 30K protein phosphate acceptors unique to rat spinal cord cytosol, 60K and 16K protein phosphate acceptors characteristic of rat spinal cord membranes, a 50K protein phosphate acceptor present only in hen spinal cord membranes, and greater phosphorylation of a more abundant 20K protein in both hen spinal cord fractions. The functional significance of these differences is presently unclear. However, their characterization provides a basis from which to launch future investigations of the biochemistry, pharmacology, and toxicology of spinal cord protein phosphorylation and indicates that caution should be exercised in the choice of an animal model with characteristics appropriate to those of the system it is representing.     

Atrial natriuretic polypeptide in spinal cord and autonomic ganglia

Using a radioimmunoassay for alpha-rat atrial natriuretic polypeptide (alpha-rANP), tissue levels of alpha-rANP-like immunoreactivity (-LI) in the rat spinal cord and autonomic ganglia were investigated. The alpha-rANP-LI level was higher in the more caudal parts of the spinal cord and the highest in the sacral spinal cord. alpha-rANP-LI was also detected in the superior cervical and coeliac ganglia. Gel permeation chromatographic analysis showed that the major peak of alpha-rANP-LI in the spinal cord was a low molecular weight form co-eluted with synthetic alpha-rANP. Reverse-phase high performance liquid chromatographic analysis revealed that alpha-rANP-LI with a low molecular weight in the spinal cord consisted of several components, two major components of which co-migrated with synthetic alpha-rANP (4-28) and alpha-rANP (5-28), whereas little immunoreactivity was eluted at the position of alpha-rANP. These findings suggest the involvement of ANP in the function of the spinal cord and autonomic nervous system.     

The distribution and origin of VIP in the spinal cord of six mammalian species

The distribution of VIP-immunoreactivity was studied in the spinal cord and dorsal root ganglia of 6 mammalian species. Immunoreactive fibres and cell bodies were most apparent in the dorsal horn, dorsolateral funiculus, intermediolateral cell columns and the area around the central canal. The distribution of VIP immunoreactivity was similar in all species studied, mouse, rat, guinea pig, cat, horse and the marmoset monkey. There were fewer VIP fibres in the dorsal horn of cervical and thoracic segments than in lumbosacral segments. Using radioimmunoassay this gradient increase was quantitatively most marked in the sacral spinal cord of the cat. In dorsal root ganglia few nerve cell bodies but numerous fibres were present. A dual origin for VIP in the spinal cord is suggested: (A) Extrinsic, from dorsal root afferent fibres since immunoreactivity was decreased in dorsally rhizotomized animals (cats and rats) and in capsaicin pretreated rats (microinjection of dorsal root ganglia). (B) From local cell bodies intrinsic to the spinal cord which became visible after colchicine pretreatment of rats.     

Lectin microarrays differentiate carcinoma cells from reactive mesothelial cells in pleural effusions

The aim of this study was to evaluate the diagnostic utility of lectin microarrays in pleural effusions of patients with lung cancer. A lectin microarray, LTL, PSA, LCA, UEA-1, AAL, MAL-I, MAL-II, SNA, WGA, ECL, DSA, STL, SWGA, HPA, ConA, GNA, HHL, BPL, EEL, Jacalin, WFA, ACL, MPL, DBA, SBA, was used to determine the glycoprotein profile of cells in pleural effusions from patients with lung cancer (54 cases), and with benign lung disease (54 cases). The A549 cell line, used as an experimental control, was positive for AAL, MAL-I, WGA, STL, Jacalin and ACL binding. Adenocarcinoma cells in pleural effusions were positive for ECL, DSA, AAL, MAL-I, WGA, STL, Jacalin, and ACL binding. AAL, WGA, and ACL positive binding was the most common, found in 54, 48, and 38 samples, respectively. ECL and DSA binding was positive in only 4 samples. In comparison, reactive mesothelial cells displayed positive binding for all markers in the microarray panel. SNA and AAL positive binding was detected in the majority of samples; 50/54 and 48/54 samples, respectively. Positive binding of DBA, MAL-II and EEL was present in only 2, 4 and 4 samples, respectively. SNA binding had the highest sensitivity (92.6 %), specificity (100 %), and accuracy (96.3 %). SNA may be used as a biomarker to distinguish reactive mesothelial cells from adenocarcinoma cells. The lectin microarrays proved able to distinguish carcinoma cells from reactive mesothelial cells in pleural effusions.     

Proteolytic degradation of neuronal benzodiazepine binding sites

The pathway of breakdown of membrane-bound benzodiazepine binding sites has been examined with proteolytic enzymes. Photoaffinity labeled benzodiazepine receptors were degraded for varying amounts of time and at varying enzyme concentrations. The properties of fractions both remaining in the membrane and released into the supernatant were examined for their apparent molecular weight by SDS gel electrophoresis. Trypsin treatment converted the 46K subunits of the GABA/BDZ complex which bind 3H-flunitrazepam into 40K and 27.5K fragments which remained in the membrane and finally a small fragment which was released into the supernatant. An endogenous trypsin-like activity in the membrane fractions has similar proteolytic effects on the membrane bound receptor.     

Calcitonin Gene-Related Peptide-Binding Sites of Porcine Cardiac Muscles and Coronary Arteries: Solubilization and Characterization

Calcitonin gene-related peptide (CGRP)-binding sites were solubilized, using digitonin, from the porcine spinal cord, atria, and coronary arteries. The specific binding of 125I-human alpha-CGRP to the solubilized binding sites was inhibited by human alpha- and beta-CGRP and by rat alpha-CGRP, but not by angiotensin II or human calcitonin. Scatchard plot analysis of saturation gave the same KD value for CGRP in the crude membrane fractions of the tissues examined. The affinity of CGRP to the binding sites was decreased by solubilization in the atria and coronary arteries, but not in the spinal cord. Affinity labeling followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed distinct molecular sizes of the specific binding sites among the tissues; 70K for the spinal cord, 70K and 90K for the coronary arteries, and 70K and 120K for the atria. These results indicate that the molecular characteristics of the specific binding sites of CGRP in the cardiovascular system are distinct from those in the central nervous system.     

Expression of phosphorylated high molecular weight neurofilament protein (NF-H) and vimentin in human developing dorsal root ganglia and spinal cord

The expression of vimentin and the phosphorylated variant of high molecular weight neurofilament protein (NF-H) was studied in developing human fetal dorsal root ganglia and spinal cord. The technique used for examination of cryosections was double-label fluorescence with monoclonal antibodies. Both proteins were present in the nerve fibres inside the ganglia of 6- and 8-week-old embryos. During further development the expression of vimentin continued to increase in the satellite cells, but was found to be decreasing in the ganglion cells. Phosphorylated NF-H was found in the processes of ganglion cells, as well as in the perikarya at all developmental stages. In the spinal cord of 6- and 8-week-old embryos, phosphorylated NF-H protein was found in the longitudinal fibres of the marginal layer and in processes of the mantle zone; some of the fibres also contained vimentin. Later the co-expression of the two proteins ceased and vimentin was found only in glial and mesenchymal derivatives. Phosphorylated NF-H was located, at all developmental stages, in the axons of both white and grey matter, but not in the neuronal perikarya. The results indicate that phosphorylation of the NF-H in human dorsal root ganglia starts in the perikarya of the ganglion cells while in the ganglion cells of the spinal cord it takes place in the axons.     

Membrane lipids of human peripheral nerve and spinal cord.

Major membrane lipids were determined in specimens of human peripheral nerve (cauda equina) and spinal cord of 10 subjects aged 20-70 years. The same lipids were also assayed in myelin from the same tissues isolated with two different procedures and in myelin of cauda equina from 3 subjects aged 17-91 years isolated with a third method. The concentrations (mean and standard deviation) of phospholipids were 90 +/- 11 and 96 +/- 9 nmol/g fresh weight; of cholesterol 70 +/- 15 and 101 +/- 16; of cerebroside 19 +/- 3 and 41 +/- 7; of sulfatide 10 +/- 1 and 11 +/- l; and of gangliosides 0.80 +/- 0.08 and 0.40 +/- 0.05 N in cauda equina and spinal cord, respectively. The proportion of ethanolamine phosphoglyceride was lower and that of sphingomyelin higher in cauda equina than in spinal cord. The myelin of peripheral nerve and spinal cord contained almost the same proportions of lipids as the whole tissue. The protein-bound sialic acid content was 3-fold higher than the lipid-bound sialic acid content in cauda myelin. The fatty acid patterns of choline, ethanolamine, inositol and serine phosphoglycerides of spinal cord and its myelin, were very similar to those of cerebral white matter, while the phosphoglycerides of cauda equina had higher proportions of monoenoic acids and lower proportions of polyunsaturated fatty acids. The fatty acid patterns of sphingomyelin, cerebroside and sulfatide of spinal cord were similar to those of cerebral white matter, while those of cauda equina contained significantly more saturated fatty acids. This suggests that the lipid and fatty acid compositions of peripheral nerve are particularly suitable for the formation of a tightly packed myelin membrane which can be a powerful shield against infections and other injuries.     

Lectin binding pattern in normal human labial mucosa

Summary The pattern of lectin binding in normal human labial mucosa was examined by light and electron microscopy using eight different lectins (ConA, LCA, WGA, UEA-1, RCA-1, SBA, DBA and PNA) and compared with the patterns in normal human skin and oesophageal mucosa. As seen by light microscopy, ConA, LCA, and WGA stained cell membranes in all layers of the mucosae. RCA-1 stained the plasma membrane of cells in the basal and middle layers, whereas cells in the superficial layers showed little positive staining. UEA-1, SBA, and PNA stained the cells in the middle layers weakly in some cases. No positive staining for DBA was seen. By electron microscopy, reaction product indicating ConA-binding sites was observed in the plasma membrane, cisternae of the endoplasmic reticulum, nuclear envelope and the Golgi apparatus. Binding of LCA, WGA, and RCA-1 was observed in the plasma membrane. These results show that the binding pattern of PNA, SBA, and RCA-1 in labial mucosa is different from that in the normal skin or oesophageal mucosa, although the labial mucosal epithelium, epidermis, and oesophageal epithelium are all stratified squamous epithelia. These differences in the cell-surface sugar residues are likely to be related to the possible functional differences in these tissues.     

THE EFFECT OF HYPOCHOLESTEREMIC AGENTS ON MYELINOGENESIS

Abstract— Three drugs known to inhibit biosynthesis of cholesterol, Clofibrate, 20, 25-diazacholesterol and AY-9944 were administered by stomach intubation to suckling rats. At weaning the rats were killed and subcellular fractions, including myelin, were prepared from the brains and spinal cords and analysed for sterol content. Central nervous tissue fractions from Clofibrate-treated rats showed some decrease in total sterols, but the sterol species were qualitatively normal. AY-9944 given to rats caused high amounts of 7-dehydro-cholesterol to accumulate in all brain and spinal cord fractions with the highest amounts (32–38 percent of total sterols) in myelin. In diazasterol-treated rats desmosterol reached 48 per cent of the sterols of myelin. A group of rats was allowed to survive after the final drug intake (21 days) and their brain and spinal cord sterol content followed up to 60 days. At 30 days the proportion of dehydrocholesterol or desmosterol comprised over half the total myelin sterol. By 60 days of age the 7-dehydrocholesterol had almost completely disappeared from all fractions while substantial amounts of desmosterol were retained in myelin. Myelination was retarded by treatment with AY-9944 and 20, 25-diazasterol, possibly by the limited amount of sterols available. The metabolism of the abnormal myelin constituents in drug-treated animals is discussed in relation to the molecular structure of the myelin membrane.     

Changes in In Vitro Brain and Spinal Cord Protein Phosphorylation After a Single Oral Administration of Tri-o-Cresyl Phosphate to Hens

The effect of a single oral 750 mg/kg dose of tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of brain and spinal cord proteins was assessed in hens during the development of and recovery from delayed neurotoxicity. Crude membrane and cytosolic fractions were prepared from the brains and spinal cords of control and TOCP-treated hens at 1, 7, 14, 21, 35, and 55 days after treatment. Brain and spinal cord protein phosphorylation with [gamma-32P]ATP was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), autoradiography, and microdensitometry. TOCP administration conferred calcium and calmodulin dependence on the phosphorylation of a few brain cytosolic proteins and caused an increase in the phosphorylation of a number of other cytosolic and membrane proteins. This effect of TOCP was large in magnitude, and its time course reflected the onset of and recovery from the signs of ataxia and paralysis associated with delayed neurotoxicity in the hen. The molecular weights (Mr) and maximal phosphorylation (percent of control) for the most prominently affected bands were as follows: brain cytosol--50K (183%), 55K (575%), 60K (529%), 65K (273%), and 70K (548%); brain membranes--50K (622%) and 60K (697%); and spinal cord cytosol--20K (182%). The role of endogenous phosphorylation reactions in and their potential usefulness as biochemical indicators of delayed neurotoxicity are being explored further.     

Identification of two binding sites for wheat-germ agglutinin on polylactosamine-type oligosaccharides.

The carbohydrate-binding properties of wheat-germ agglutinin (WGA) have been studied by using glycopeptides isolated from the cell surfaces of a cultured murine myeloid cell line (416B). The glycopeptides were passed through affinity columns of lentil lectin (LCA), concanavalin A (Con A) and WGA arranged in series so that material reaching the WGA column had failed to bind to LCA or Con A. WGA-binding glycopeptides were step-eluted with 0.01 M, 0.1 M and 0.5 M-N-acetylglucosamine (GlcNAc), to yield weak (WGA-W), intermediate (WGA-I) and strong (WGA-S) affinity fractions. WGA-W and WGA-I contained 'N'- and 'O'-linked oligosaccharides bound to separate polypeptides. WGA-S consisted almost entirely of N-linked components. Our analytical work was concentrated mainly on the N-linked fractions. In these carbohydrates WGA affinity was directly proportional to molecular size but inversely related to N-acetylneuraminic acid content. The binding of the weak-affinity fraction was dependent on N-acetylneuraminic acid, but the intermediate- and strong-binding species interacted with the lectin by N-acetylneuraminic acid-independent mechanisms. N-linked glycopeptides in each WGA-binding class were almost totally degraded to monosaccharides by the concerted action of the exoglycosidases neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase. Treatment with endo-beta-galactosidase caused partial depolymerization, yielding some disaccharides but also a heterogeneous population of partially degraded components. These findings suggest that WGA binds with high affinity to internal GlcNAc residues in large oligosaccharides containing repeat sequences of Gal beta(1----4)GlcNAc beta(1----3) (i.e. polylactosamine-type glycans). N-Acetylneuraminic acid is involved only in low-affinity interactions with WGA. WGA therefore displays an intricate pattern of saccharide specificities that can be profitably utilized for structural analysis of complex carbohydrates.     

眼镜蛇毒对大鼠脊髓和脊神经节一氧化氮合酶表达的影响

目的 观察眼镜蛇毒对脊髓和脊神经节一氧化氮合酶(NOS)表达的影响。方法 将眼镜蛇毒注入大鼠右侧大腿后部,采用还原型尼克酰胺嘌呤二核苷酸脱氢酶(NADPH．d)法显示NOS的表达。结果 在眼镜蛇毒注射组,脊髓和脊神经节内的NOS阳性神经元和深染NOS阳性神经元明显多于注入生理盐水组和正常对照组。结论 注入眼镜蛇毒能上调大鼠脊髓和脊神经节NOS表达。     

A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack

We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ.     

Immunocytochemical localization of neuromedin K (neurokinin B) in rat spinal ganglia and cord.

Specific antisera directed against substance P and neuromedin K (neurokinin B) have been used in double-label immunofluorescence studies to unambiguously localize these two neuropeptides of the tachykinin family in single tissue sections of rat spinal cord and dorsal root ganglia. Substance P-like immunoreactivity (SPLI) is present but neuromedin K-like immunoreactivity (NMKLI) is undetectable in dorsal root ganglia. Both peptides are present in the spinal cord, but NMKLI is largely restricted to the dorsal gray while SPLI shows a broader distribution. In the spinal gray, NMKLI coexists with SPLI in some, but not all, fibers. While substance P in the dorsal spinal cord is largely of primary afferent origin, neuromedin K appears to originate largely from intrinsic spinal neurons.     

Decrease in serum receptor-reactive somatomedin in diabetes.

Somatomedin in rat serum has been measured by a sensitive radioreceptor assay using 125I-labelled human somatomedin and human placental membrane. In rats made diabetic with strepotzotocin, receptor-reactive somatomedin levels were decrease by up to 75%. The decrease followed the time course of increasing serum glucose and occurred to the same extent in rats aged between 4 and 40 weeks. Endogenous serum receptor-reactive somatomedin appeared exclusively in high molecular weight fractions on gel chromatography. In diabetes the decreased somatomedin was due to a fall in this high molecular weight activity, but was not accompanied by a fall in somatomedin binding protein. These results suggest a role for insulin in maintaining serum somatomedin levels.     

Contribution to histochemical distribution of non-specific cholinesterase activity in sensory ganglia of some mammals

The activity of non-specific cholinesterase was demonstrated histochemically in satellite cells of the spinal ganglia from adult rat, cat, rabbit and baboon. The spinal ganglia of newborn rats displayed distinct intraneuronal reactivity for non-specific cholinesterase while a low reactivity was observed in satellite cells. The spinal and trigeminal ganglia of adult mice contained satellite cells with non-specific cholinesterase reactivity only sporadically. Most of reaction product for non-specific cholinesterase activity (from low to high intensity) was found in perikarya of the neurons. Spinal and trigeminal ganglia of the same mice embryo exhibited diffuse staining for non-specific cholinesterase activity remaining in the spinal ganglia of newborn mice. The trigeminal ganglia of newborn mice exhibited, however, more differentiated pattern of the positive reaction for non-specific cholinesterase like adult animals. The pattern of histochemical distribution of non-specific cholinesterase activity in trigeminal and spinal ganglia from mice of various ages corresponds with morphological differentiation and maturation undergoing in a rostrocaudal wave. Intraneuronal presence of non-specific cholinesterase activity in sensory ganglia during development and in adult animals gives a new possibilities for explanation of the functional involvement of this enzyme in the nervous system.