Human 14-3-3 Protein: Radioimmunoassay, Tissue Distribution, and Cerebrospinal Fluid Levels in Patients with Neurological Disorders

Abstract: An antiserum to human 14-3-3 protein has been produced in rabbits. The protein was a poor antigen and attempts to improve immunogenicity were unsuccessful. A radioimmunoassay was developed using the antiserum, ¹²⁵I- 14-3-3-2, and unlabelled 14-3-3-2 as standards. The assay had a sensitivity limit of 2.5 ng.m1⁻¹. The minor component of human 14-3-3 protein (14-3-3-1 protein) cross-reacted to approximately 10% in the assay. Human tissues were surveyed for 14-3-3 protein by two-dimensional electrophoresis and by radioimmunoassay. Two-dimensional electrophoresis showed a 14-3-3 protein complex in brain, intestine, and testis, but not in other tissues. Radioimmunoassay showed that although brain had the highest concentration of 14-3-3 (13.3 μg. mg⁻¹ soluble protein), immunoreactivity was present in all tissues, with the concentration in intestine and testis approaching 50% of the brain level. Lower levels (less than 1.0 μg. mg⁻¹ soluble protein) were seen in liver, kidney, skeletal muscle, and erythrocytes. The immunoreactivity present in tissues other than brain showed the same molecular weight and charge characteristics as authentic 14-3-3 protein. The radioimmunoassay also detected 14-3-3 protein in serum (50 ng.m1⁻¹) and in CSF (5-130 ng.ml⁻¹). The immunoreactivity present in CSF appeared to be intact 14-3-3 protein. CSF 14-3-3 levels were measured in 82 patients with various neurological disorders. Measurements of this protein did not appear sufficiently discriminating to be o f diagnostic value.     

Substance P and secretoneurin in vitreous aspirates of patients with various vitreoretinal diseases

By means of highly sensitive radioimmunoassays, the levels of substance P (SP) and secretoneurin (SN) were detected in vitreous aspirates of patients with macular holes which served as controls, in patients with nonproliferative diabetic retinopathy (DR), active proliferative diabetic retinopathy (active PDR), inactive PDR, rhegmatogenous retinal detachment and proliferative vitreoretinopathy (PVR). Furthermore, SN-like immunoreactivities were characterized by reversed phase-HPLC. The concentration of SN was more than 20-fold higher in macular holes when compared with SP and reversed phase HPLC revealed evidence that the vitreous levels of SN represent authentic SN. SN was significantly decreased in patients with nonproliferative DR, active PDR and inactive PDR by more than 70% which seems to result from a reduced expression and/or secretion from the cilary epithelium and a reduced release from the retina both due to diabetes mellitus. By contrast SP was increased in rhegmatogenous retinal detachment most obviously due to an enhanced outflow of the peptide through retinal breaks. Despite their proangiogenic activities, SP and SN are unlikely to be involved in the pathogenesis of neovascularizations in DR because of their unchanged and reduced levels, respectively, but the low levels of both peptides may facilitate the regression of vasoproliferations following laser photocoagulation.     

Free and Conjugated Amines in Human Lumbar Cerebrospinal Fluid

Significant amounts of acid-hydrolyzable conjugates of 3,4-dihydroxyphenylethylamine, norepinephrine, and 5-hydroxytryptamine were detected in lumbar CSF from 22 awake unpremedicated healthy individuals. In the CSF samples, the amounts of conjugated amines almost always exceeded the amounts of free amines, but were less than the amounts of the acid metabolites 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid.     

Development of an Antiserum to the Midportion of Substance P: Applications for Biochemical and Anatomical Studies of Substance P-Related Peptide Species in CNS Tissues

This report describes the generation and biochemical characterization of a high-affinity antiserum that recognizes an epitope contained in the midportion sequence of substance P, i.e., substance P4-10. Designated A47, this reagent bound a variety of related peptide species containing the substance P4-10 sequence with apparent equipotency. A double radioimmunoassay procedure was developed that utilized A47, in combination with a traditional high-affinity COOH-terminally directed anti-substance P serum, to provide quantification of mature and immature forms of substance P in CNS tissues. Across most rat CNS areas, levels of substance P-like immunoreactivity were consistently 15% higher when monitored by analyses using A47 versus anti-substance P serum. In the dorsal root ganglia, an apparent enhancement in levels of substance P-like immunoreactivity of approximately 40%, when quantified by analyses using A47 versus anti-substance P serum, was observed; this most likely reflected the presence of an active biosynthetic pool of intermediate processing forms of substance P in this tissue. Coordinated HPLC/radioimmunoassay analyses of extracted dorsal root ganglia tissues demonstrated multiple peaks of immunoreactivity corresponding to mature substance P and to several of its precursor forms found in the normal biosynthetic pathway. Of the total recovered HPLC-fractionated immunoreactivities, that corresponding to the putative immediate precursor to substance P, i.e., substance P-glycine, was the predominant peak. In an additional series of HPLC/radioimmunoassay analyses, selective decreases in immunoreactive peaks corresponding to precursor forms of substance P were observed in dorsal root ganglia tissues from rats treated with the neurotoxic agent capsaicin. These results indicated decreased turnover of substance P as a consequence of drug treatment. Finally, initial immunohistochemical analyses employing affinity-purified A47 produced an unusual pattern of labeling characterized by well defined punctate terminal elements within the superficial aspects of the dorsal horn of the spinal cord.     

Presence and Ontogeny of Enkephalin and Substance P in the Chick Ciliary Ganglion

The avian ciliary ganglion has been reported to contain both enkephalin and substance P in preganglionic terminals. However, extensive biochemical characterization of these antigens has not been completed. Using radioimmunoassays specific for Met5- and for Leu5-enkephalin and for substance P we identified immunoreactive substances in ganglionic extracts that comigrate on HPLC columns with standard Met5- and Leu5-enkephalin and with substance P. The ontogeny of Met5-enkephalin and substance P during embryogenesis was determined in ganglionic extracts and we found that the content of Met5-enkephalin in the ganglion reached a peak at embryonic stage 37 whereas the content of substance P in the ganglion reached its maximum in the adult.     

Evidence for Two Cholesterol Ester Hydrolases in Human Cerebrospinal Fluid

Abstract: In the present study, the properties, such as pH optima, detergent requirement, and effects of various lipids, of cholesterol ester hydrolase in human cerebrospinal fluid (CSF) were examined, and the activity levels of the enzyme in CSF from multiple sclerosis (MS) patients and non-MS individuals were compared. Our data indicate that the enzyme in CSF exhibits two pH optima: pH 6.0 in the presence of Triton X-100 and pH 7.0 in the presence of sodium taurocholate. Both phosphatidylethanolamine (PE) and phosphatidylserine (PS) enhanced the hydrolase activity at pH 6.0. The activity at pH 7.0, on the other hand, was enhanced slightly in the presence of PE but was inhibited in the presence of PS. These data suggest the presence of two cholesterol ester hydrolases in CSF and also indicate that the activity at pH 6.0 may be due to microsomal enzyme in brain and that at pH 7.0 may be due to myelin enzyme. The hydrolase activity at pH 7.0 was significantly lower in CSF from MS patients. The activity at pH 6.0 in CSF from MS and non-MS patients, however, did not differ significantly. This indicates that the reduction in pH 7.0 hydrolase activity in CSF may be related to demyelination.     

Substance P in Human Plasma Is Degraded by Dipeptidyl Peptidase IV, Not by Cholinesterase

Human serum cleaves two dipeptides from the N-terminus of the neurohormone substance P. It has been suggested that this degrading activity is inherent to serum cholinesterase. We oppose this, because it turned out that highly purified serum cholinesterase contains traces of dipeptidyl peptidase IV, an enzyme known to attack the N-terminus of substance P. The peptidase is incompletely separated from cholinesterase during the procainamide-gel affinity chromatography as the last step of the usual purification procedure. Physostigmine completely inhibits the hydrolysis of butyrylthiocholine by such purified cholinesterase preparations, but not their substance P-degrading activity. Vice versa, epsilon-carbobenzoxy-lysylproline, an inhibitor of dipeptidyl peptidase IV, inhibits the peptidase activity of these preparations more than their esterase activity. After rechromatography on procainamide gel the peptidase is completely separated and the remaining cholinesterase has lost its substance P-degrading activity. We conclude that the N-terminal region of substance P is not degraded by cholinesterase but by the contaminating dipeptidyl peptidase IV, a different serine enzyme.     

High-Resolution Proton Magnetic Resonance Analysis of Human Cerebrospinal Fluid

High-resolution proton magnetic resonance spectroscopy was used to analyze human cerebrospinal fluid obtained from patients with several neurological problems. The major metabolites measured included glucose, lactate, glutamine, citrate, inositol, acetate, creatine, creatinine, beta-hydroxybutyrate, alanine, and pyruvate. A drug vehicle, propylene glycol, was also measured. Alterations in the cerebrospinal fluid of these metabolites provided information concerning metabolism of the brain. Magnetic resonance spectroscopy offered a simple and rapid means of assessing these and other exogenous and endogenous compounds in diseases affecting the nervous system.     

Molecular Forms of Acetylcholinesterase and Butyrylcholinesterase in Human Plasma and Cerebrospinal Fluid

The measurement of cholinesterase activities in either plasma or cerebrospinal fluid (CSF) may ultimately prove to be relevant in the diagnosis of neurological and neuropsychiatric disorders. However, studies to date have examined only total enzyme activities. Therefore in the present study we have examined the distribution of the individual molecular forms of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in plasma and CSF using sucrose density gradient centrifugation. Although the total activities of AChE were of the same order of magnitude in plasma and CSF, there was a considerable difference (120-500-fold) between total BChE activity in the CSF and the BChE-rich plasma. The analysis of the individual molecular forms revealed that the predominant molecular species of AChE and BChE in the CSF--both lumbar and ventricular--was the G4 form. The G4 form also constituted the majority of the plasma BChE activity and, on average, over half (56%) of the plasma AChE activity. The significance of the AChE and BChE molecular form compositions of both plasma and CSF and their possible relationship to pathological states are discussed.     

Gas Chromatographic-Mass Spectrometric Determination of myo-Inositol in Human Cerebrospinal Fluid

The concentration of free myo-inositol in CSF was determined with a gas chromatographic-mass spectrometric method using deuterated myo-inositol as an internal standard after conversion to the hexa-O-acetyl derivative with acetic anhydride and pyridine. Twenty microliters of CSF is sufficient for the analysis which has a coefficient of variation of 9%. Identical analytical results were obtained on two different mass numbers. Schizophrenic patients were compared with healthy control persons. In addition, patients with rheumatoid arthritis or with neurological illnesses were studied. No consistent differences related to the illness could be found. The mean concentration of myo-inositol was about 25 micrograms/ml. Treatment of schizophrenic patients with chlorpromazine or sulpiride had no significant effect on the concentration of myo-inositol in CSF.     

Diazepam Increases γ-Aminobutyric Acid in Human Cerebrospinal Fluid

In 11 neurological patients, levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) were determined in cerebrospinal fluid (CSF) before and 1, 3, 5, and 8 min after intravenous injection of diazepam (2 or 5 mg). GABA levels increased progressively after intravenous injection of 5 but not 2 mg of the benzodiazepine, the differences from preinjection values being significant at 3, 5, and 8 min. Furthermore, when relative CSF GABA alterations determined after injection of diazepam were compared to those determined in sequential CSF aliquots of 10 patients without diazepam injection, mean GABA increases after diazepam were significantly different from controls in all CSF fractions. The data suggest that, in addition to its well-known effects on postsynaptic GABA function, diazepam may exert effects on endogenous GABA concentrations and/or on GABA release in the human CNS as reflected by elevation of GABA levels in human CSF.     

[3H] Diazepam Displacing Activity in Human Cerebrospinal Fluid

Pooled human cerebrospinal fluid was separated by Sephadex G-50 chromatography. The presence of three peaks, A, B and C, was demonstrated by monitoring absorbance at 254 and 280 nm. All peaks showed [3H]diazepam displacing activity in the membrane receptor test. Peak B was further separated on Bio-Gel P-4. At least two major fractions free of salt and GABA in the molecular weight range of approximately 700--3600 were shown to displace [3H]diazepam in the receptor test. This activity was enhanced by a factor of 3 in the presence of 10 microM-GABA.     

Measurement of Substance P Metabolites in Rat CNS

A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.     

Determination of N-Acetylaspartic Acid in Human Cerebrospinal Fluid by Gas Chromatography–Mass Spectrometry

N-Acetyl-L-aspartic acid was identified and determined in human cerebrospinal fluid. The concentration in lumbar fluid was about 2 nmol/ml and about 20 nmol/ml in ventricular fluid. There was no difference between healthy subjects and schizophrenic patients.     

Oxidative Stress in Cerebrospinal Fluid of Patients with Aseptic and Bacterial Meningitis

This study aimed to determine whether patients with aseptic and bacterial meningitis presented alterations in oxidative stress parameters of cerebrospinal fluid (CSF). A total of 30 patients were used in the research. The CSF oxidative stress status has been evaluated through many parameters, such as lipid peroxidation through thiobarbituric acid reactive substances (TBARS) and antioxidant defense systems such as superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and ascorbic acid. TBARS levels, SOD and GST activity increase in aseptic meningitis and in bacterial meningitis. The ascorbic acid concentration increased significantly in patients with both meningitis types. The reduced glutathione levels were reduced in CSF of patients with aseptic and bacterial meningitis. In present study we may conclude that oxidative stress contributes at least in part to the severe neurological dysfunction found in meningitis.     

Ventricular Cerebrospinal Fluid Monoamine Transmitter and Metabolite Concentrations Reflect Human Brain Neurochemistry in Autopsy Cases

Concentrations of dopamine (DA), its metabolites 3-methoxytyramine and homovanillic acid (HVA), noradrenaline (NA), its metabolites normetanephrine (NM) and 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxytryptamine (5-HT, serotonin), and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured in 14 brain regions and in CSF from the third ventricle of 27 human autopsy cases. In addition, in six cases, lumbar CSF was obtained. Monoamine concentrations were determined by reversed-phase liquid chromatography with electrochemical detection. Ventricular/lumbar CSF ratios indicated persistence of rostrocaudal gradients for HVA and 5-HIAA post mortem. Ventricular CSF concentrations of DA and HVA correlated positively with striatal DA and HVA. CSF NA correlated positively with NA in hypothalamus, and CSF MHPG with levels of MHPG in hypothalamus, temporal cortex, and pons, whereas CSF NM concentration showed positive correlations with NM in striatum, pons, cingulate cortex, and olfactory tubercle. CSF 5-HT concentrations correlated positively with 5-HT in caudate nucleus, whereas the concentration of CSF 5-HIAA correlated to 5-HIAA levels in thalamus, hypothalamus, and the cortical areas. These data suggest a specific topographic origin for monoamine neurotransmitters and their metabolites in human ventricular CSF and support the contention that CSF measurements are useful indices of central monoaminergic activity in man.     

Preincubation with Substance P Induces Substance P-Stimulated Phosphatidylinositol Turnover in Cultured Cerebellar Astrocytes

In this study we have investigated the effects of preincubation of cultured astrocytes with substance P (SP) on subsequent 125I-Bolton-Hunter conjugated SP (125I-BHSP) receptor binding, and SP-stimulated phosphatidyl-inositol (PI) accumulation. Spinal cord astrocytes preincubated for up to 96 h with SP (0.001-1,000 nM) suffered a dose-dependent decrease in both subsequent 125I-BHSP and SP-stimulated PI turnover. In contrast, preincubation of cerebellar astrocytes with SP resulted in an increase in SP-stimulated PI turnover, with no change in 125I-BHSP receptor binding. SP-induced PI turnover in cerebellar astrocytes was maximal after 72 h of preincubation with 0.1 nM SP. These data suggest that increased coupling between receptor and second messenger occurs in response to chronic exposure to SP.     

The τ Protein in Human Cerebrospinal Fluid in Alzheimer's Disease Consists of Proteolytically Derived Fragments

Abstract: Previous studies have shown that the levels of the microtubule-associated protein τ in the CSF of patients with Alzheimer's disease (AD) are elevated compared with age-matched controls. In spite of these findings, the nature of τ in CSF has not been well documented. In the present study, τ was immunoprecipitated from CSF of patients with AD or acute stroke, as well as normal elderly controls, followed by immunoblot analysis. In all cases, CSF τ consisted primarily of a band migrating at 26–28 kDa. In AD and stroke patients, several smaller τ fragments were also detected. No intact τ was detected in any of the CSF samples examined. Further immunoprecipitation studies showed that the majority of the τ fragments contained the amino terminus of the molecule. Treatment of CSF τ with alkaline phosphatase did not alter the electrophoretic properties of the fragments. These studies clearly demonstrate that CSF τ is truncated rather than intact.     

Erythropoietin in Cerebrospinal Fluid: Age-related Reference Values and Relevance in Neurological Disease

We aimed to establish age-related reference values for Erythropoietin (EPO) in cerebrospinal fluid (CSF) and to evaluate concentrations in neurological diseases. CSF and serum EPO was measured in controls with tension-type headache (CTTH), in patients with ALS, dementia and depression using ELISA technique. Stability experiments showed CSF EPO to be stable for two and a half months and over two thaw/freeze cycles. A positive correlation of CSF EPO with age was found (P < 0.01). We found a CSF/serum EPO concentration ratio of 0.126, pointing towards an intrathecal synthesis of EPO. The ALS group showed significantly lowered CSF EPO compared to age-matched CTTH (P < 0.012), whereas the dementia and depression group showed no significant differences compared to CTTH. The establishment of age-related reference values in a large cohort of controls will improve the interpretation of future CSF EPO evaluations in neurological diseases. The authors have not reported any conflicts of interest.     

Functional Studies with Substance P Analogues: Effects of N-Terminal, C-Terminal, and C-Terminus-Extended Analogues of Substance P on Nicotine-Induced Secretion and Desensitization in Cultured Bovine Adrenal Chromaffin Cells

Abstract: Substance P (SP) and SP analogues, including C-terminal, N-terminal, and C-terminus-extended analogues, have been investigated for their ability to modulate nicotine-induced secretion from bovine adrenal chromaffin cells in culture. Secretion was monitored by measuring the release of endogenous catecholamines by electrochemical detection following separation on HPLC and the release of endogenous ATP with an on-line luciferin-luciferase bioluminescence technique. SP is known to have the following two effects on nicotine-induced secretion of catecholamines (see Livett and Zhou, 1991): inhibition of the nicotinic response and protection against nicotinic desensitization. Secretion induced by 10^-5M nicotine was inhibited 70-80% by SP, SP-methyl ester, and the C-terminus-extended analogue SP-Tyr¹²-NH₂, 65% by (Ala³)SP-NH₂, 45% by the C-terminal analogue SP(4-11), and 20 and 5% by the N-terminal analogues SP(1-7) and SP(1-5), respectively, when these peptides were present at 3 ×; 10^-5M concentrations. The order of potency was SP = SP-methyl ester = SP-Tyr¹²-NH₂ > (Ala³)SP-NH₂ > SP(4-11) > SP(1-7) > SP(1-5). SP, SP-methyl ester, and (Ala³)SP-NH₂ protected against nicotinic desensitization by 40-55%, and SP(4-11) protected by 20% (all at 3 ×; 10^-5M). In contrast, the N-terminal analogues SP(1-7) and SP(1-5) and the C-terminus-extended analogue SP-Tyr¹²-NH₂ at 3 × 10^-5M did not protect against nicotinic desensitization. Cyclo-SP(3-9), Ac-SP(3-9)-NH₂, SP(3-9), and SP(3-6) had neither inhibitory nor facilitatory effects on secretion. Of the 20 SP analogues extended at the C terminus by one amino acid, there were only three that protected against nicotinic desensitization, whereas the majority inhibited nicotine-evoked catecholamine secretion. The present work indicates that for inhibition of nicotine-evoked secretion, both the C terminus and N terminus of SP are necessary. For the protection against nicotine-induced desensitization, the C terminus of SP is important. This suggests that the two mechanisms, inhibition of nicotine-evoked secretion and protection against nicotinic desensitization, are regulated independently.