Analysis of intact bacteriohopanepolyols from methanotrophic bacteria by reversed-phase high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry.

Direct detection of most intact biohopanoids is not possible using conventional GC-MS techniques due to their highly functionalised and amphiphilic nature. Here we report the application of a new reversed-phase high-performance liquid chromatography method for the direct analysis of acetylated, intact bacteriohopanepolyols in solvent extracts of methanotrophic bacteria. Atmospheric pressure chemical ionisation mass spectrometric detection provides structural information relating to the number and types of functional groups present in the four biohopanoids detected: bacteriohopanetetrol, aminobacteriohopanetriol, -tetrol and -pentol. The method should facilitate the assessment of hopanoid composition of both bacteria and environmental samples.

Altered neurofilament phosphorylation and beta tubulin isotypes in Charcot-Marie-Tooth disease type 1.

Charcot-Marie-Tooth disease type 1 (CMT1) is associated with atrophy and degeneration of peripheral nerve axons in addition to prominent changes in the structure of Schwann cells. We have investigated the composition of the axonal cytoskeleton in sural nerve biopsies from patients with CMT1. Compared to controls, CMT1 nerves exhibited marked hypophosphorylation of neurofilament proteins and an increased relative abundance of beta tubulin isotypes 2 and 3. Biopsies from patients with other causes of neuropathy, matched to the CMT1 group for severity of axonal atrophy, exhibited an intermediate degree of neurofilament hypophosphorylation and no abnormality of tubulin isotypes. The axonal cytoskeleton in CMT1 resembles that of immature nerve fibers. A failure of normal Schwann cell-axon interaction in CMT1 may prevent full differentiation of the axonal cytoskeleton of myelinated nerve fibers.

Phosphorylation-dependent immunoreactivity of neurofilaments and the rate of slow axonal transport in the central and peripheral axons of the rat dorsal root ganglion.

The rate of axonal transport of tubulin, actin, and the neurofilament proteins was measured in the peripheral and central projections of the rat L5 dorsal root ganglion (DRG). [35S]Methionine was injected into the DRG, and the "front" of the radiolabeled protein was located 7, 14, and 20 days postinjection. Transport rates calculated for the neurofilament triplet proteins, tubulin, and actin in the peripheral nerve were approximately 1.5-fold faster than those in the dorsal root. A progressive decrease in the rate of transport was observed from 7 to 20 days after radiolabeling in both the central and peripheral directions (neurofilaments, approximately 1.7-fold; tubulin/actin, 2.1-fold). A surgical preparation, leaving the peripheral sciatic nerve with predominantly sensory fibers, was the basis for ELISAs for phosphorylation-dependent immunoreactivity of the high-molecular-weight neurofilament protein. In both dorsal roots and peripheral sensory axons the degree of phosphorylation was greater in nerve segments further away from the cell bodies. The degree of phosphorylation-related immunoreactivity correlates with the slowing of transport of radiolabeled cytoskeletal protein.

Redistribution of cytoskeletal proteins in mammalian axons disconnected from their cell bodies.

Mice of the strain C57/BL/Ola exhibit a delay of Wallerian degeneration, such that axons survive for several weeks after a nerve transection that separates the axons from the cell bodies. In this Ola strain we have examined the distribution of cytoskeletal proteins in a 5 mm segment of the sciatic nerve for as long as 2 weeks after proximal and distal transections that prevent entry or exit of proteins via axonal transport. By 7 d after transections, there was a marked accumulation of alpha- and beta-tubulin, actin, and nonphosphorylated neurofilament epitopes at the proximal and at the distal ends of the transected axons, and loss of these proteins from the center of the isolated nerve segment. Highly phosphorylated neurofilament epitopes did not redistribute along the nerve, but there was a gradual loss of phosphorylated neurofilament immunoreactivity. These observations indicate the potential for bidirectional transport of a substantial portion of certain cytoskeletal proteins within axons of the PNS.

Transport of cytoskeletal proteins in axons of hippocampal pyramidal cells.

Axonal transport of cytoskeletal proteins has not yet been extensively studied in the brain proper, in contrast to the peripheral nerves and optic nerves. The authors have developed a means for the study of transport of cytoskeletal proteins in axons of hippocampal pyramidal cells. Proteins of intrinsic neurons of the dorsal hippocampus were labeled by microinjection of 35S methionine, and the subsequent transport of labeled proteins was characterized in the axons projecting into the fimbria-fornix. A peak of labeled proteins was present in the fimbria-fornix at 4-12 days after labeling, corresponding to transport rates 0.2-0.7 mm/day. The most abundant proteins at each time studied exhibited one-dimensional electrophoretic mobilities of actin and tubulin; neurofilaments were less intensely labeled. The observed specializations of cytoskeletal transport, especially the paucity of tubulin transport at rates of 2-4 mm/day, may predispose hippocampal pyramidal cells to accumulate tubulin and microtubule-associated proteins in their cell bodies in various disease states.

Prenatal diagnosis of Charcot-Marie-Tooth disease type 1A by multicolor in situ hybridization.

Genetic heterogeneity within the most common genetic neuropathy, Charcot-Marie-Tooth disease (CMT) results in about 70% slow nerve conduction CMT1 and 30% normal nerve conduction CMT2. Autosomal dominant CMT1A on chromosome 17p11.2 represents about 70% of CMT1 cases and about 50% of all CMT cases. Three different size CMT1A duplications with variable flanking breakpoints were characterized by multicolor in situ hybridization and confirmed by pulsed field gel electrophoresis and quantitative polymerase chain reaction (PCR) amplification. These different size duplications result in the same CMT1A phenotype confirming that trisomy of a normal gene region results in CMT1A. The smallest duplication does not include the 409 locus used previously to screen for CMT1A duplications. Direct analysis of interphase nuclei from fetuses and at-risk patients by multicolor in situ hybridization to a commonly duplicated CMT1A probe is informative more often than polymorphic PCR analysis, faster than pulsed field gel electrophoresis (PFGE), and faster, more informative, and more reliable than restriction enzyme analysis. CMT1B restriction enzyme analysis of CMT pedigrees without CMT1A is expected to diagnose another 8% of at-risk CMT1 patients (total: 78%).

Axonal transport of class II and III beta-tubulin: evidence that the slow component wave represents the movement of only a small fraction of the tubulin in mature motor axons.

Pulse-labeling studies demonstrate that tubulin synthesized in the neuron cell body (soma) moves somatofugally within the axon (at a rate of several millimeters per day) as a well-defined wave corresponding to the slow component of axonal transport. A major goal of the present study was to determine what proportion of the tubulin in mature motor axons is transported in this wave. Lumbar motor neurons in 9-wk-old rats were labeled by injecting [35S]methionine into the spinal cord 2 wk after motor axons were injured (axotomized) by crushing the sciatic nerve. Immunoprecipitation with mAbs which recognize either class II or III beta-tubulin were used to analyze the distributions of radioactivity in these isotypes in intact and axotomized motor fibers 5 d after labeling. We found that both isotypes were associated with the slow component wave, and that the leading edge of this wave was enriched in the class III isotype. Axotomy resulted in significant increases in the labeling and transport rates of both isotypes. Immunohistochemical examination of peripheral nerve fibers demonstrated that nearly all of the class II and III beta-tubulin in nerve fibers is located within axons. Although the amounts of radioactivity per millimeter of nerve in class II and III beta-tubulin were significantly greater in axotomized than in control nerves (with increases of +160% and +58%, respectively), immunoassay revealed no differences in the amounts of these isotypes in axotomized and control motor fibers. We consider several explanations for this paradox; these include the possibility that the total tubulin content is relatively insensitive to changes in the amount of tubulin transported in the slow component wave because this wave represents the movement of only a small fraction of the tubulin in these motor fibers.

Stimulated single-fiber electromyography in Lambert-Eaton myasthenic syndrome.

The Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder of neuromuscular transmission. Electrodiagnosis is confirmed by an increase in compound muscle action potential amplitude during high-frequency repetitive nerve stimulation or following brief exercise. We describe the results of stimulated single-fiber electromyography in 4 patients with disorders of neuromuscular transmission: LEMS (2), LEMS/myasthenia gravis (MG) overlap (1), and MG (1). Stimulated SFEMG was performed in the extensor digitorum communis muscle with axonal intramuscular suprathreshold stimulation at low and high rates. In all 4 patients, a rate dependence of jitter was found. In LEMS and LEMS/MG, jitter and blocking improved with high stimulation rates, as compared with the opposite effect in MG. We conclude that stimulated SFEMG is a valuable technique in the diagnosis of LEMS.

Phosphorylation-related immunoreactivity and the rate of transport of neurofilaments in chronic 2,5-hexanedione intoxication.

Axonal transport of neurofilaments and the phosphorylation of epitopes on neurofilament proteins was studied in rats chronically intoxicated with 2,5-hexanedione. Sensory axons arising from the L5 dorsal root ganglion exhibited accelerated transport and a reduced abundance of neurofilament proteins. The binding of an antibody to phosphorylated neurofilament epitopes was compared to the binding of an antibody to non-phosphorylated epitopes by quantitative ELISA. This immunochemical index of neurofilament phosphorylation was reduced in dorsal roots, proximal peripheral sensory axons and ventral roots, but not in a distal nerve (the nerve to the soleus). Axotomy produced a reduction in neurofilament protein abundance comparable to hexanedione without any change in the immunochemical phosphorylation index. These results are consistent with the hypothesis that the state of phosphorylation of neurofilaments in proximal axons is correlated with the rate of neurofilament transport.

The cold stability of microtubules increases during axonal maturation.

The development of cold-stable microtubules (MTs) was studied in maturing rat sciatic nerves. From 4 to 10 weeks of age, there was a large increase in the proportion of tubulin associated with stable MTs. A greater fraction of axonal tubulin than nonaxonal tubulin was associated with stable MTs. The labeled tubulin remaining behind the peak of slow axonal transport was more highly associated with stable MTs than tubulin in the peak itself. Immunoassay confirmed that the sciatic nerve contains a pool of stable MTs not identical to the peak of tubulin transport. The developmental increase in MT stability is not associated with any increase in the acetylation of tubulin or with alterations in the major MT-associated proteins. One aspect of maturation of the axonal cytoskeleton may involve deposition of tubulin into stable MTs that are either stationary or moving slowly with regard to the peak of transported tubulin.

Neurofilament phosphorylation in peripheral nerve: changes with axonal length and growth state.

We have previously reported that the intrinsic ability of motor axons to sprout can vary considerably from nerve to nerve with an inverse correlation to axonal length. In this study we asked whether this variation might be associated with differences in one axonal component, neurofilaments, near the site of outgrowth. The phosphorylation of epitopes on the heavy subunit of neurofilaments (NF-H) was compared in normal and regenerating axons from long and short nerves in the rat. Quantitative determination of phosphorylation states on NF-H epitopes was made by measuring immunoreactivity to monoclonal antibodies using an enzyme-linked immunosorbent assay system. Our results showed a much higher degree of phosphorylation of epitopes on NF-H in terminal portions of short compared to long axons. There was a significant inverse correlation between phosphorylation of NF-H epitopes and axonal length. In newly formed sprouts NF-H phosphorylation was reduced compared to normal. However, the absolute levels were related to the degree of NF-H phosphorylation in the parent axons. The ability to phosphorylate axonal proteins near the site of outgrowth may correlate with the potential for plastic changes in the axon such as sprouting.

Phosphorylation-dependent immunoreactivity of neurofilaments increases during axonal maturation and beta,beta'-iminodipropionitrile intoxication.

The immunoreactivity of the high-molecular-weight neurofilament (NF) subunit toward antibodies that react with phosphorylation-related epitopes was determined at different anatomic sites in the PNS of rats during normal maturation and after intoxication with beta,beta'-iminodipropionitrile (IDPN). A maturational increase in the relative binding of phosphorylation-dependent antibodies compared to phosphorylation-inhibited antibodies occurred from age 3 to 12 weeks. An increase in phosphorylation-related immunoreactivity with increasing distance from the cell bodies was present in ventral and dorsal roots at all ages. The degree of phosphorylation-related immunoreactivity was greater for centrally directed axons in the dorsal roots of the L5 ganglion than for peripherally directed axons. IDPN, a toxin that impairs NF transport, caused a marked increase in reactivity toward the phosphorylation-dependent antibody. NFs from IDPN-treated rats also bound less of an antibody that is normally phosphorylation independent and this inhibition of binding was sensitive to phosphatase digestion. In each instance, greater degrees of phosphorylation-dependent immunoreactivity correlate with conditions known to exhibit slower net rates of axonal transport of NF proteins.

Neurofilament and tubulin transport slows along the course of mature motor axons.

The rate of axonal transport of tubulin and of neurofilament proteins was measured in mature, non-growing, axons of rat lumbar ventral roots and sciatic nerves. A progressive decrease in the velocity of transport was observed along the course of the axons. The rate of advance of the 'front' of labeled neurofilaments decreased from 1.40 +/- 0.07 mm/day proximally to 0.64 +/- 0.04 mm/day distally. The rate for tubulin decreased from 3.10 +/- 0.13 proximally to 1.01 +/- 0.04 mm/day distally. These results demonstrate that a gradient of the velocity of cytoskeletal transport along axons is not a specific adaptation for radial growth of axons, as previously suggested. Local degradation of excess cytoskeletal proteins may permit axons to accommodate a net slowing of cytoskeletal transport without accumulation of neurofilaments or microtubules. Alternatively, the transport kinetics of radiolabeled proteins may be influenced by mixing of labeled and unlabeled cytoskeletal proteins within the axoplasm; in this case, a slowing of the movement of the labeled proteins may not reflect net slowing of transport of the total pool of cytoskeletal proteins.

Axonal transport in neurological disease.

The axonal transport systems have a wide variety of primary roles and secondary responses in neurological disease processes. Recent advances in understanding these roles have built on the increasingly detailed insights into the cell biology of the axon and its supporting cells. Fast transport is a microtubule-based system of bidirectional movement of membranous organelles; the mechanism of translocation of these organelles involves novel proteins, including the recently described protein of fast anterograde transport, kinesin. Slow transport conveys the major cytoskeletal elements, microtubules, and neurofilaments. Several types of structural changes in diseased nerve fibers are understood in terms of underlying transport abnormalities. Altered slow transport of neurofilaments produces changes in axonal caliber (swelling or atrophy) and is involved in some types of perikaryal neurofibrillary abnormality. Secondary changes in slow axonal transport--for example, the reordered synthesis and delivery of cytoskeletal proteins after axotomy--also can produce changes in axonal caliber. Secondary demyelination can be a prominent late consequence of a sustained alteration of neurofilament transport. Impaired fast transport is found in experimental models of distal axonal degeneration (dying back). Retrograde axonal transport provides access to the central nervous system for agents such as polio virus and tetanus toxin, as well as access for known and hypothetical trophic factors. Correlative studies of axonal transport, axonal morphometry, cytoskeletal ultrastructure, and molecular biology of cytoskeletal proteins are providing extremely detailed reconstructions of the pathogenesis of experimental models of neurological disorders. A major challenge lies in the extension of these approaches to clinical studies.

Vacor neuropathy: ultrastructural and axonal transport studies.

Distal axonal degeneration has been correlated with abnormalities of fast axonal transport in several toxic neuropathies. We have investigated axonal transport in experimental PNU (N-3-pyridylmethyl-N'-p-nitrophenylurea; Vacor) neuropathy, because of the rapid and synchronous degeneration of many terminal axons after a single dose of PNU. Almost all axon terminals at neuromuscular junctions in hindfoot muscles degenerated by 24 hours after the administration of PNU. Fewer affected axons were found in intramuscular nerve twigs, and fewer still in the posterior tibial nerves. No abnormal myelinated axons were found in the sciatic nerve in the thigh. Fast axonal transport in the sciatic nerve remained normal to the mid-thigh, but a reduced amount of labeled transported material reached the posterior tibial nerve at the ankle (27% reduction). Autoradiography showed that nearly no transported material reached the intramuscular nerves and neuromuscular junctions of the hindfeet. These results suggest that toxic impairment of fast anterograde axonal transport may contribute to the axonal degeneration produced by PNU.

Isolated cerebral phycomycosis presenting as focal encephalitis.

A patient with features of a focal encephalitis was found to have isolated cerebral phycomycosis. No risk factors for phycomycosis were present, and the diagnosis was made by brain biopsy.

Control of food intake in the rat by dietary protein concentration.

Male, weanling Sprague-Dawley rats were fed isocaloric diets containing graded levels of protein or amino acid mixtures. Food intakes and weight gains were recorded daily or every other day. Both short-term and long-term (64 days) experiments were carried out. Linear regressions of food intake versus time and weight gain versus time were used to establish daily weight gains and food intakes. The four-parameter mathematical model for physiological responses was used to predict daily food intake, daily weight gain, daily food intake per 100 g weight and efficiency of food conversion (daily weight gain/daily food intake) as functions of dietary protein concentration. The changes in the four parameters generated from the four-parameter model were examined as a function of time. Several aspects of food intake, weight gain and efficiency were shown to be functions of concentration of dietary protein.

Axoplasmic transport with velocities induced by pargyline.

The axoplasmic transport of proteins in spinal motor neurons is altered by pargyline, a drug that causes increased release of monoamines. Two new peaks of transported protein were detected in the sciatic nerves of rats treated with pargyline (75 mg/kg/day ip for three days). These peaks moved with velocities of 595 mm/day (peak I) and 1,230 mm/day (peak II). The bulk of labeled protein was still transported at the control rate of 362 mm/day. Electrophoresis of transported polypeptides labeled with [35S] methionine showed that peak I material was qualitatively similar to material transported at the normal rate in controls, but peak Ii material contained fewer labeled polypeptides. Both peak I and peak II differed from controls in the relative intensity of labeling of various polypeptides. Fast axoplasmic transport in sensory neurons was unaffected by pargyline. Intraspinal injection of pargyline (50 microgram/day for three days) caused changes in axoplasmic transport similar to those induced by intraperitoneal pargyline. These results show that transport of certain proteins along a peripheral nerve can be accelerated by a mechanism initiated in the region of the nerve cell bodies.

Maytansine action on fast axoplasmic transport and the ultrastructure of vagal axons.

Maytansine, an ansa macrolide now in clinical trials as an antineoplastic drug, is a potent inhibitor of microtubule polymerization. Since microtubules are involved in axoplasmic transport, the effect of maytansine on transport was examined. Fast axoplasmic transport of proteins and the axonal ultrastructure was studied in the vagus nerve of cats exposed in vitro to maytansine. Tritiated leucine was microinjected into the nodose ganglion; after 2 hr for incorporation into proteins, nerves were dissected out for transport and ultrastructural studies and incubated for 2.5 hr in Krebs-Ringer solution with 100, 20, 10, 5, or 1 micron maytansine. A reduction in the number of microtubules and a partial blockage of fast axoplasmic transport was observed at 20 and 100 micron maytansine; at 10 micron no detectable changes were observed. These findings show that maytansine in vitro induces alterations of the neurofibrillar elements concomitant with a partial blockage of fast axoplasmic transport.