Effect of Oral Administration of Tri-o-cresyl Phosphate on In Vitro Phosphorylation of Membrane and Cytosolic Proteins from Chicken Brain

Abstract: The effects of a single oral dose of 750 mg/kg tri- o -cresyl phosphate (TOCP) on the endogenous phosphorylation of specific brain proteins were assessed in male adult chickens following the development of delayed neurotoxicity. Phosphorylation of crude synaptosomal (P₂) membrane and synaptosomal cytosolic proteins was assayed in vitro by using [γ-³²P]ATP as phosphate donor. Following resolution of brain proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis, specific protein phosphorylation was detected by autoradiography and quantified by microdensitometry. TOCP administration enhanced the phosphorylation of both cytosolic (M_r 65,000 and 55,000) and membrane (20,000) proteins by as much as 146% and 200%, respectively.     

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.     

Biochemical changes in sciatic nerve of hens treated with tri-o-cresyl phosphate: increased phosphorylation of cytoskeletal proteins.

Calcium- and calmodulin-regulated protein phosphorylation has been suggested to play a role in the pathogenesis of organophosphorus compound-induced delayed neurotoxicity (OPIDN). This condition is characterized by ataxia that progresses to paralysis concurrent with a central-peripheral distal axonopathy after a delay period of 1-2 weeks following exposure to an organophosphorus compound causing delayed neurotoxicity, such as tri-o-cresyl phosphate (TOCP). Calcium/calmodulin (CaM) kinase II is involved in the increased phosphorylation of brain microtubule and spinal cord neurofilament triplet proteins following treatment of animals with organophosphorus compounds that are capable of producing OPIDN. In this study, chickens were given a single oral neurotoxic dose of 750 mg TOCP/kg body weight and killed after 1, 6, 14 or 21 days following treatment. Protein kinase-mediated phosphorylation of cytoskeletal proteins was studied in proximal and distal parts of sciatic nerves of control and treated hens. Peripheral nerve proteins were phosphorylated in vitro using [gamma-32P]ATP as a phosphoryl group donor. Phosphorylated proteins were separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein phosphorylation was detected by autoradiography and quantified by laser microdensitometry. The extent of Ca2+-calmodulin dependent phosphorylation of five cytoskeletal proteins was significantly increased in TOCP treated animals, particularly at 1 and 6 days after treatment, in both the proximal and distal portion of the nerve. The identity of these proteins was confirmed by 2-D PAGE as tubulin, the neurofilament triplet proteins and microtubule associated protein-2 (MAP-2). These results confirm earlier observation of the close temporal relationship between increased cytoskeletal protein phosphorylation and the development and OPIDN.     

Reduction of retrograde axonal transport associated-proteins motor proteins, dynein and dynactin in the spinal cord and cerebral cortex of hens by tri-ortho-cresyl phosphate (TOCP)

Tri-ortho-cresyl phosphate (TOCP) can cause a type of neurotoxicity known as organophosphate-induced delayed neuropathy (OPIDN). The characteristic axonal swelling containing aggregations of neurofilaments, microtubules, and multivesicular vesicles is consistent with a disturbance of axonal transport. We hypothesized that there existed a disturbance of molecular motor in the pathogenesis of OPIDN. In the present study, adult hens were treated with a dosage of 750 mg/kg TOCP by gavage, or pretreated 24h earlier with phenylmethanesulfonyl fluoride (PMSF) and subsequently with TOCP, then sacrificed on the time-points of 0, 1, 5, 10, and 21 days after dosing of TOCP, respectively. The level of kinesin-1, dynein, and dynactin in spinal cords and cerebral cortexes of hens was determined. Immunoblotting analysis showed a progressive decline of dynein and dynactin in spinal cords after dosing TOCP. Furthermore, a significant reduction in dynactin and dynein was observed in cerebral cortexes at several time-points post dosing TOCP. In contrast, no significant changes of kinesin-1 were observed throughout the period of experiment. When given before TOCP administration, PMSF could inhibit TOCP-induced motor protein disruption, while it protected hens against the delayed neuropathy. In conclusion, the reduction of the motor proteins, dynein and dynactin, might be associated with the disruption of retrograde neuronal axonal transport in OPIDN.     

cDNA cloning and sequencing of Ca2+ calmodulin-dependent protein kinase IIα subunit and its mRNA expression in diisopropyl phosphorofluoridate (DFP)-treated hen central nervous system

Diisopropyl phosphorofluoridate (DFP) produces delayed neurotoxicity, known as organophosphorus ester-induced delayed neurotoxicity (OPIDN), in hen, human, and other sensitive species. A single dose of DFP (1.7 mg/kg, se.) produces first mild ataxia followed by paralysis in 7-14 days in hens. DFP treatment also increases in vitro autophosphorylation of Ca²⁺ calmodulin-dependent protein kinase II (CaM kinase II) and the phosphorylation of several cytoslceletal proteins in the hen brain. To investigate whether increase in CaM kinase II activity is associated with increased expression of its mRNA, we cloned and sequenced CaM kinase II a subunit cDNA, and used it to study CaM kinase II expression in brain regions and spinal cord. Hen CaM kinase II subunit differs in 7 amino acids from that of rat CaM kinase II. Its mRNA occurs predominantly as a 6.7 kb message, which is very close to that of human CaM kinase II a subunit. Northern blot analysis showed a transient increase in CaM kinase II subunit mRNA in the cerebellum and spinal cord of DFP-treated chickens. The increase in CaM kinase II mRNA expression is consistent with the previously reported increase in its activity in brain and spinal cord, and its increased expression only in cerebellum and spinal cord, which are sensitive to the Wallerian-type degeneration characteristic of OPIDN, suggests the probable role of this enzyme in delayed neurotoxicity.     

三甲基苯基磷酸酯对神经毒性酯酶的抑制及其酶促动力学分析

以可使人和敏感动物产生迟发性神经毒性的有机磷化合物三甲基苯基磷酸酯（TOCP）为测试药物,研究其在体外对成年产卵来航母鸡不同神经组织神经毒性酯酶（NTE）活性抑制的敏感性及其抑制的动力学．结果表明,外周神经NTE对于TOCP的抑制比中枢神经NTE敏感得多．TOCP对鸡脑、脊髓和坐骨神经中NTE抑制的I50值．分别为：1．9323、2．3950和0．0035mmol／L．NTE酶促动力学研究显示,鸡脑NTE催化分解底物戊酸苯酯（PV）的Vmax为62．10nmol·min-1·mg-1,Km为0．92mmol／L．TOCP对鸡脑NTE的抑制属竞争性抑制类型,并有"底物抑制"现象．     

Reduced Protein Kinase C Activity in Ischemic Spinal Cord

Protein phosphorylation was evaluated in a rabbit spinal cord ischemia model under conditions where cyclic AMP-dependent protein kinase (PK-A) and calcium/phospholipid-dependent protein kinase (PK-C) were activated. One hour of ischemia did not affect PK-A activity significantly; however, PK-C activity was reduced by more than 60%. In vitro phosphorylation of endogenous proteins by endogenous PK-C revealed that eight particulate and five cytosolic proteins showed stimulated phosphorylation by PK-C activators in control tissue, although this stimulation was virtually absent in ischemic samples. When control and ischemic particulate fractions were combined, the endogenous protein phosphorylation pattern under PK-C-activating conditions was similar to the ischemic sample, which suggests that inhibitory molecules may be present in the ischemic particulate fraction. In vitro phosphorylation of endogenous proteins under PK-A-activating conditions in ischemic tissue was similar to that in control tissue. The results suggest that the PK-C phosphorylation system is selectively impaired in ischemic spinal cord. In addition to reduced PK-C-dependent phosphorylation, an Mr 64,000 protein was phosphorylated in ischemic cytosolic samples, but not in control samples. The phosphorylation of the Mr 64,000 protein was neither PK-C-dependent nor PK-A-dependent. These altered phosphorylation reactions may play critical roles in neuronal death during the course of ischemia.     

Aberrant activation of CDK5 is involved in the pathogenesis of OPIDN

Exposure to triorthocresyl phosphate (TOCP) may result in a late neurological complication, i.e. organophosphate-induced delayed neuropathy (OPIDN). The aim of this study was to examine changes in levels of cyclin-dependent kinase 5 (CDK5) and of its activator, p35/p25, in the spinal cord of hens treated by TOCP. After exposure to a single dose of TOCP, groups of adult hens were examined in 3, 5, 7, 9, 14, and 18 days after exposure. CDK5, p35/p25 expression and distribution in the lumbar spinal cord were evaluated by immunohistochemistry and Western blotting. The hens showed signs of OPIDN around day 9 after exposure. The number of p (phosphorylated) -CDK5 and p35 positive cells increased significantly. Co-localization and mislocalization of p-CDK5 and p35/p25 was identified and became evident in neurons around the 9th day. Meanwhile, CDK5, p-CDK5, p35, p25 protein levels and p25/p35 ratio were increased, and peaked around the 9th day, then decreased. Some hens' unilateral common peroneal was treated by roscovitine 3 days after TOCP exposure. Axonal transport of these nerves was faster than of their opposite side and of those simply treated by TOCP. These findings indicate aberrant activation of CDK5 may be involved in the pathogenesis of OPIDN.     

Activation of mitochondria-mediated apoptotic pathway in tri-ortho-cresyl phosphate-induced delayed neuropathy

Previous studies suggest that abnormal neurons death has been implicated in organophosphate-induced delayed neuropathy (OPIDN). However, the precise mechanism of neuronal death in OPIDN remains largely unknown. In this study, adult hens were treated with a dosage of 750 mg/kg tri-ortho-cresyl phosphate (TOCP) by gavage, and then sacrificed on the time-points of 1, 5, 10, and 21 days after dosing TOCP, respectively. The apoptotic change of spinal cord neurons induced by TOCP was examined, and the role of mitochondria-mediated apoptosis of neurons during OPIDN was investigated. TUNEL assays showed that apoptotic neurons in hen spinal cords began to appear on day 5 following TOCP exposure. Immunohistochemistry and western blot analysis revealed a translocation of cytochrome C from mitochondria to cytoplasm after dosing TOCP. Moreover, the level of Bcl-2, Bcl-xl, Pro-caspase3 and Pro-caspase9 in hen spinal cord was significantly decreased, whereas that of Bax and cleaved-PARP was significantly elevated. Taken together, these findings indicate that the administration of TOCP can induce neuron apoptosis in hen spinal cords, which might be mediated by the activation of mitochondrial apoptotic pathway.     

An acute and subacute neurotoxicity assessment of trichlorfon

The toxicity of trichlorfon (O,O-dimethyl-2,2,2,-trichloro-1-hydroxyethylphosphonate, Dipterex, Dylox), reported to elicit delayed neurotoxicity in man and chickens, was studied by administering single subcutaneous doses of 100 or 300 mg/kg to adult White Leghorn hens. At 24 h posttreatment, the birds were observed for visible signs of neurotoxicity, were euthanized, and samples of blood plasma, brain, and spinal cord (cervical and thoracic regions) were obtained for quantification of cholinesterase and neurotoxic esterase (NTE) activities. In subacute studies, hens were dosed with trichlorfon (100 mg/kg) every 72 h for a total of six doses. Seventy-two hours after the final dose the hens were euthanized, the brains, spinal cords, and distal sciatic nerves were removed for enzymatic and (or) histological examination. Parallel acute and subacute studies were conducted using diisopropyl phosphorofluoridate (DFP), a known neurotoxic agent, at subcutaneous dosages of 1.0 mg/kg. In the acute studies, both DFP and trichlorfon markedly inhibited tissue cholinesterase activities but only DFP elicited a significant inhibition of NTE. In the subacute studies, DFP produced a characteristic central-peripheral distal axonopathy in the 18-day period of study which was confirmed by clinical and morphological evidence and by marked inhibition of neuronal NTE. Trichlorfon caused little or no obvious neurotoxicity, an observation that was supported by minimal morphological changes and impairment of walking ability and no inhibition of brain or spinal cord NTE.     

Phosphorylation of membrane proteins by cytosolic casein kinases in human erythrocytes. Effect of monovalent ions, 2,3-bisphosphoglycerate and spermine

Summary Membrane proteins of human erythrocytes can be phosphorylated not only by membrane casein kinase (MS) but also by cytosolic casein kinases CS and CTS, resembling casein kinase I and II, respectively.Casein kinase CS, like membrane casein kinase MS, preferentially phosphorylates membrane proteins such as band 2 (spectrin, -subunit) and band 3, which are the major phosphate-acceptor proteins in the endogenous phosphorylation of isolated ghosts in the presence of [-³²P]ATP.By contrast, cytosolic casein kinase CTS phosphorylates, in addition to band 2, some membrane proteins, whose endogenous phosphorylation in isolated ghosts under the same conditions is negligible, if any.The CS- and CTS-catalyzed phosphorylations exhibit different response to increasing NaCl (or KCI) concentrations up to physiological levels (140 mM KCI, 20 mM NaCI); i.e. CS-and MS-catalyzed phosphorylations are strongly inhibited by 75–150 mM KCI (or NaCl), while CTS-catalyzed phosphorylation is practically unaffected.In the absence of added NaCl, CS- and MS-catalyzed phosphorylations are markedly inhibited by 1.5-3 mM 2,3-bisphosphoglycerate, whereas CTS-catalyzed phosphorylation appears to be practically unaffected.Finally, CS- and MS-catalyzed phosphorylations are slightly inhibited also by 1 mM spermine, while CTS-catalyzed phosphorylation is enhanced by this polycation concentration.     

Novel Endogenous Protein Kinase C Substrate Proteins, Neutrinins, in Brain Synaptic Membranes of Maturing Rat

Abstract: A new family of membrane phosphoproteins designated as P9, P12, P15, P16, and P20 with corresponding apparent molecular weights of 9K, 12K, 15K, 16K, and 20K was characterized from rat brain by using in vitro exogenous or endogenous phosphorylation and autoradiography. As the phosphorylation was selectively inhibited by the protein kinase C (PKC) inhibitor PKC_19–31 or Ca²⁺-chelating reagents and again stimulated by the PKC activator phorbol 12,13-dibutyrate, these proteins are thought to be the natural PKC substrates. Because P12, P15, P16, and P20 were neutral proteins (pl 7.0) and specifically distributed in neuronal membranes, the new family of membrane-associated PKC substrate proteins was referred to as neutrinins. Neutrinins were widely distributed in rat brain, being especially plentiful in the spinal cord, medulla oblongata, cerebellum, and midbrain, relatively scanty in the cerebral cortex, but lacking in cytosol of brain areas and cell membrane preparations of peripheral tissues. The expression of the developmental changes of neutrinins has been monitored by the in vitro exogenous phosphorylation approach, i.e., adding purified PKC to a deactivated synaptosomal plasma membrane system. Levels of all the neutrinin proteins in rat cerebral cortex, as represented by P12, P15, and P16, showed an ontogenetic increase from the early postnatal days to the adult. This appears to be correlated with the commencement of synaptogenesis.     

Characterization of Infant Rat Cerebral Cortical Membrane Proteins Phosphorylated In Vivo: Identification of the ACTH-Sensitive Phosphoprotein B-50

This study on the phosphorylation in vivo of membrane proteins in cerebral cortices of infant rats reports the identification of the adrenocorticotropin (ACTH)-sensitive phosphoprotein B-50 as one of the substrate proteins that are rapidly phosphorylated in vivo following intracisternal administration of 2 mCi [32P]orthophosphate. Rats were sacrificed 30 min after isotope injection. A fraction enriched in membranes, designated neural membranes (NM), was isolated from the cerebral cortices according to the procedure used for preparation of synaptic plasma membranes (SPM) from adult brain. This NM fraction was characterized by electron microscopy. The proteins of NM were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Numerous protein bands of NM in infant rat brain were phosphorylated in vivo. Attention was focussed on the 32P-labeled protein bands in the molecular weight range of 47K-67K. In this region one phosphoprotein band (MW 48K) was more highly labeled than the other bands. The electrophoretic behavior of three of these labeled bands, designated a, c, and e (MW 48K, 55K, and 62K, respectively) was compared with that of protein bands that were phosphorylated in vitro in cerebral membranes isolated from noninjected infant rats. The effects of ACTH1-24 and cyclic AMP in the in vitro system were also studied to probe for the presence of specific membrane proteins known to be sensitive to these modulators. On incubation of NM with [gamma-32P)ATP in the presence and absence of ACTH1-24 in vitro, phosphorylation of a 48K protein band was inhibited in a dose-dependent fashion by the neuropeptide. Two-dimensional electrophoretic separation of NM proteins labeled in vivo indicated that the 48K band had an isoelectric point of 4.5, identical to that of the ACTH-sensitive B-50 protein previously identified. Cyclic AMP stimulated phosphorylation in vitro of two protein bands (MW 55K and 59K) in NM preparations. This result indicates that the in vivo labeled band c may correspond to the cyclic AMP-sensitive 55K protein, whereas phosphoprotein band e, labeled in vivo, appears to be different from the cyclic AMP-sensitive 59K protein band. These observations indicate that neural membranes isolated from infant rat cerebral cortices contain a variety of proteins that can be phosphorylated in vivo. Several of these, for example, the 48K protein band, have the properties of synaptic plasma membrane proteins of adult rat brain that have been characterized by their sensitivity to neuromodulators in endogenous phosphorylating systems in vitro.     

Neuropathy target esterase in hens after sarin and soman

To estimate the potential of small doses of sarin (types I and II) and soman to cause delayed neuropathic effects, 400, 200, 61, and 0 micrograms/kg of sarin-I, 280, 140, 70, and 0 micrograms/kg of sarin-II, and 14.2, 7.1, 3.5, and 0 micrograms/kg of soman by gavage were compared with 510 mg/kg tri-o-cresyl phosphate (TOCP) in 14- to 18-month-old SPF white leghorn hens (4/dose) protected with atropine (100 mg/kg). The neuropathy target esterase (NTE) activity 24 hr after dosing was determined in brain, spinal cord, and lymphocytes and in plasma and brain for cholinesterase and carboxylesterase. None of the compounds showed statistically significant NTE decreases. Sarin-II showed a dose-related trend in the lymphocyte NTE (to 33% of control at 280 micrograms/kg), suggesting that longer exposure to lower doses might cause a cumulative neurotoxic insult. All of the agents decreased the activity of plasma and brain cholinesterase and carboxylesterase. Using more than 70% inhibition of brain NTE as a biochemical predictor of delayed neuropathy, sarin and soman appear unable to cause delayed neuropathy at nonlethal doses within this protocol.     

Myelin-Deficient Rat: Analysis of Myelin Proteins

Myelin basic protein (BP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity were quantitated in the brains and spinal cords of normal and myelin-deficient (md) rats at 8, 12, 18, and 25 days of age. The levels of BP, MAG, and CNP in 25-day-old md brain were 1.1, 1.8, and 11% of those in controls, respectively. In spinal cord, the levels were higher, at 9, 15, and 12% of control values, respectively. Although BP content in the mutant rats was a lower percentage of the control level than MAG and CNPase contents at all ages, the absolute level of BP increased steadily between 8 and 25 days of age in both brain and spinal cord, whereas there was little change in the amounts of MAG and CNPase during this period. Immunoblotting analysis did not reveal an increased apparent Mr for MAG, as has been observed in quaking and trembler mice. There was little difference in the relative distributions of the 14K, 17K, 18.5K, and 21.5K forms of BP between control and md rat spinal cord homogenates at the ages examined. PLP content was reduced more than that of the other proteins in the md mutants, because it could not be detected by a technique capable of detecting 0.2% of the control brain level and 0.1% of control spinal cord level. This suggests that the expression of PLP may be preferentially affected in the md mutation.     

Appearance and phosphorylation of the 210 kDalton neurofilament protein in newborn rat brain,spinal cord,and sciatic nerve

The appearance and in vivo phosphorylation of the 210 kDalton (kD) neurofilament protein (NF210K) in newborn rat brain, spinal cord, and sciatic nerve were invetigated. Electron microscopic examination of neurofilaments isolated from newborn rat brain and spinal cord demonstrated morphologically distinct filaments which contained cross-bridging side arms. Neurofilament proteins, phosphorylated in vivo, were separated by sodium dodecyl sulfate slab gel electrophoresis and were transferred from acrylamide gels to nitrocellulose sheets. The nitrocellulose sheets were treated with antiserum to the 70 kD, 145 kD and 210 kD neurofilament proteins by the immunoblot technique. The three neurofilament proteins were found to be present in newborn brain, spinal cord and sciatic nerve. The presence of NF210K in newborn rat brain was further confirmed by 2-dimensional gel electrophoresis followed by indentification of this protein by the immunoblot technique. Exposure of the immunostained nitrocellulose sheets to x-ray film revealed that the NF210K, NF145K, and NF70K proteins were phosphorylated in filaments prepared from newborn rat central and peripheral nervous systems. These results suggest that the synthesis and posttranslational modification of the neurofilament proteins may be synchronized or developmentally regulated. It is feasible that phosphorylation of the NF210K subunit may be a prerequisite for the formation of neurofilament cross-bridging elements which are necessary for radial growth of axons.     

缺血与正常小鼠脑内蛋白磷酸化脱磷酸化的比较研究

以小鼠断头脑缺血为模型,研究缺血小鼠脑内蛋白磷酸化脱磷酸化的改变。对缺血１ｍｉｎ、５ｍｉｎ、１５ｍｉｎ和３０ｍｉｎ及对照小鼠脑内蛋白磷酸化脱磷酸化的研究表明,有些磷蛋白如１４５ｋＤ、８４ｋＤ、５９ｋＤ和５０ｋＤ的磷酸化随缺血时间延长而减弱,还有些磷蛋白如１１９ｋＤ、１０５ｋＤ、７８ｋＤ和５５ｋＤ的磷酸化随缺血时间延长而增加。对磷酸化程度变化显著的缺血１５ｍｉｎ小鼠脑内胞浆及膜上ＰＫＡ、ＰＫＣ、Ｃａ~（２＋）／ＣａＭＰＫ底物的磷酸化进行了研究,发现胞浆组分中与钙相关的ＰＫＣ、Ｃａ~（２＋）／ＣａＭＰＫ底物磷酸化在缺血鼠脑中明显减弱。同时研究了脑内唯一依赖于Ｃａ~（２＋）／ＣａＭ的钙调神经磷酸酶（Ｃａｌｃｉｎｅｕｒｉｎ,ＣａＮ）底物的变化,发现缺血小鼠脑内ＣａＮ的某些底物磷酸化降低。     

Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse

In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous [³²P]ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using [³H]methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the [³H] methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.     

C-fos mRNA Induction in the Central and Peripheral Nervous Systems of Diisopropyl Phosphorofluoridate (DFP)-Treated Hens

A single dose of diisopropyl phosphorofluoridate (DFP), an organophosphorus ester, produces delayed neurotoxicity (OPIDN) in hen. DFP produces mild ataxia in hens in 7–14 days, which develops into severe ataxia or paralysis as the disease progresses. Since, OPIDN is associated with alteration in the expression of several proteins (e.g., Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) -subunit, tau, tubulin, neurofilament (NF) protein, vimentin, GFAP) as well as their mRNAs (e.g., NF, CaM kinase II -subunit), we determined the effect of a single dose of DFP on the expression of one of the best known immediate-early gene (IEG), c-fos. C-fos expression was measured by Northern hybridization in cerebrum, cerebellum, brainstem, midbrain, spinal cord, and the sciatic nerves of hens at 0.5 hr, 1 hr, 2 hr, 1 day, 5 days, 10 days, and 20 days after a single 1.7 mg/kg, sc. injection of DFP. All the tissues (cerebrum, 52%; cerebellum, 55%; brainstem, 49%; midbrain, 23%; spinal cord, 80%; sciatic nerve, 157%;) showed significant increase in c-fos expression in 30 min and this elevated level persisted at least up to 2 hr. Expressions of -actin mRNA and 18S RNA were used as internal controls. The significant increase in c-fos expression in DFP-treated hens suggests that c-fos may be one of the IEGs involved in the development of OPIDN.Both of them equally contributed towards this work