In vivo metabolism of mammalian neurofilament polypeptides in developing and adult rat brain

Calvert, R.; Anderton, Brian H.

doi:10.1016/0014-5793(82)80160-9

Cited by 34 publications

(11 citation statements)

References 10 publications

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“…The antineurofilament antibody, RT97, specifically and exclusively labels the L-neuron population (Lawson et al 1984), which correlates with the finding that L neurons have many neurofilaments while the SD neurons have very few neurofilaments (Yamadori, 1970;Duce & Keen, 1977;Lawson et al 1984). RT97 is a mouse monoclonal antibody which binds predominantly to the 200 kDa neurofilament protein subunit; it also binds to a lesser extent to the 150 kDa but not to the 70 kDa subunits of bovine neurofilament (Wood & Anderton, 1981;Calvert & Anderton, 1982). It has recently been shown to bind to the phosphorylated and not to the dephosphorylated form (Haugh, Probst, Ulrich, Kahn & Anderton, 1986; Dahl, Labkovsky & Bignami, 1988).…”

supporting

confidence: 64%

Soma neurofilament immunoreactivity is related to cell size and fibre conduction velocity in rat primary sensory neurons.

Lawson

Waddell

1991

The Journal of Physiology

403

280

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SUMMARY1. Intracellular recordings were made in dorsal root ganglia in vitro at 37 'C. The L4, L5 and L6 ganglia from 46-to 51-day-old female Wistar rats were used. In each neuron conduction velocity (CV) was measured and fluorescent dye was injected. Later the intensity of the immunoreactivity to RT97 (a monoclonal antibody to the phosphorylated 200 kDa neurofilament subunit) as well as the cell size (crosssectional area at the nuclear level) were measured in the dye-injected neurons. RT97 was used to distinguish between the L (light, neurofilament-rich) and the SD (small dark, neurofilament-poor) neuronal somata.2. Neurons were classified as C neurons (CV < 1-3 m/s), C/Ad neurons (1-3-2 m/s),A neurons (2-12 m/s) or Aa//? neurons (> 12 m/s).3. All A-fibre somata were RT97 positive (L) and all C-fibre somata were RT97 negative (SD), although in the C/M group both positive and negative neurons were seen. Thus, RT97-negative somata had C (unmyelinated) or C/Ad fibres, while RT97-positive somata had A (myelinated) or C/Ad fibres. 4. The size distributions of A neurons and C neurons were consistent with their classification as L-and SD-cell neurons respectively. The size distribution of Ad cells was skewed with a peak of small cells and a tail of medium-sized cells.5. There was a loose positive correlation between cell size and fibre CV. 6. RT97 intensity was positively correlated with CV if all neurons were considered together, but no correlation was seen within the C, Ad or Aoc/, CV groups.7. RT97 intensity was positively correlated with cell size when all neurons were considered together. Although no correlation was seen within the C or the Ad CV groups, a clear positive correlation was seen for Atz// neurons. 8. The relationship of RT97 intensity to cell size was not demonstrably altered by axotomy, time in vitro or the presence of intracellular dye in control experiments.9. RT97-negative and -positive neurons could be seen in neonatal rat ganglia. Their size distributions resembled, respectively, the SD-and L-neuron populations at this age. RT97 immunoreactivity may therefore be a useful predictor of the cell type and myelinated state which a sensory cell is destined to reach in the adult rat.

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supporting

confidence: 64%

Soma neurofilament immunoreactivity is related to cell size and fibre conduction velocity in rat primary sensory neurons.

Lawson

Waddell

1991

The Journal of Physiology

403

280

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“…Monoclonal antibodies that recognize unshared (21) and shared (21,22) determinants on the three polypeptides have been reported.…”

mentioning

confidence: 99%

Microheterogeneity ("neurotypy") of neurofilament proteins.

Goldstein¹,

Sternberger²,

Sternberger³

1983

Proc. Natl. Acad. Sci. U.S.A.

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Neurofilaments purified from adult rat brainstem by two methods were electrophoresed on NaDodSO4/polyacrylamide gels to separate the triplet proteins (approximate Mrs of 200,000, 155,000, and 68,000) which, in turn, were electroblotted onto nitrocellulose paper. On Coomassie blue-stained gels that were not electroblotted, the same banding pattern was seen with both methods of preparation. Immunocytochemical staining of the electroblots with each of five monoclonal antibodies revealed that three of the monoclonal antibodies were specific for the Mr 200,000 neurofilament protein and two, for both the Mrs 200,000 and 155,000 neurofilament proteins. None of the antibodies reacted with the Mr 68,000 band. The Mr 200,000 band could be resolved into doublet bands. Individual monoclonal antibodies reacted with either one or both of the Mr 200,000 doublets. The immunocytochemical staining of the neurofilament triplets on electroblots was compared to that of adult rat cerebellar paraffin sections. Each monoclonal antibody had a unique pattern of staining, reacting only with certain subpopulations of neurons or their processes. Correlation of the staining patterns in cerebellar tissue sections with those of neurofilament polypeptides on electroblots suggested that different neurofilament polypeptides can be localized to different structures and subpopulations of neurons and that molecular heterogeneity ("neurotypy") may be revealed within the MrS 200,000 and 155,000 neurofilament polypeptides.Neurofilaments are neuron-specific intermediate filaments with a diameter of 10 nm and consist of the neurofilament triplet, three major polypeptides with Mrs of 200,000, 150,000, and 68,000 (1-5). The origin of the triplet is uncertain. Immunological crossreactivity between the neurofilament polypeptides with polyclonal antibodies suggests common antigenic sites (1,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and implies that the neurofilament proteins are derived from a common precursor (15). On the other hand, peptide maps reveal little homology among the neurofilament polypeptides (14-20) and point to an independent origin for each.Monoclonal antibodies to a single neurofilament polypeptide would be useful to explore the origin, structure, and organization of neurofilaments and their polypeptides. Monoclonal antibodies that recognize unshared (21) and shared (21, 22) determinants on the three polypeptides have been reported.In the present study, we have produced five monoclonal antibodies, each of which recognizes one or more of the neurofilament polypeptides. The staining, by each monoclonal antibody, of neurofilament proteins separated by gel electrophoresis and electroblot transfer onto nitrocellulose paper has provided additional evidence for the existence of one or more common antigenic sites. Comparison of these electroblots to the staining of rat cerebellar tissue sections has allowed for closer examination of the structure and distribution of neurofilaments and their polypeptides in the brain. The different staining patter...

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“…Although NF is known to localize in paraneurons, (such as gut endocrine cells, pancreatic islet cells, thyroid parafollicular cells) they did not influence the results of the present study. This was probably due to the small quantity of NF in paraneurons [22][23][24].…”

Section: Discussionmentioning

confidence: 99%

Identification of human brain from a tissue fragment by detection of neurofilament proteins

Takata

Miyaishi

Kitao

et al. 2004

Forensic Science International

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We developed a method for identifying human brain from a tissue-like fragment by detection of neurofilament protein (NF) using enzyme-linked immunosorbent assay (ELISA). NF was extracted from 0.1 g of organ/tissue homogenized with Tris-HCl buffer (pH 7.2) containing urea, phenylmethylsulfonyl fluoride (PMSF), EDTA and, EGTA. It was necessary to dilute the extract at more than 2(3)-fold to avoid immunosuppression by urea. Positive reaction was always obtained for NF-H in 2(3)-fold diluted extract of brain tissue, however, NF-L and NF-M were not always detected when a brain fragment contained gray matter. Human cerebral white matter could be easily distinguished from other organs/tissues by detecting any of the NF-subunits. Brains of human and some animals could be discriminated by detecting NF-L or NF-M, although the species specificity of NF-H was not good. Our findings suggested that detection of NF-H was more useful than NF-L and NF-M for identifying a brain from a tissue-like fragment. The present ELISA method for NF-H could identify human brain specimens under the following conditions: putrefied at 4 degrees C for up to 3 weeks, dried at 37 degrees C for at least 4 months, heated at 50 degrees C for at least 4 weeks. Our results showed that our method is useful for identification of brain tissue in forensic stain analysis. Two practical cases are described.

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In vivo metabolism of mammalian neurofilament polypeptides in developing and adult rat brain

Cited by 34 publications

References 10 publications

Soma neurofilament immunoreactivity is related to cell size and fibre conduction velocity in rat primary sensory neurons.

Soma neurofilament immunoreactivity is related to cell size and fibre conduction velocity in rat primary sensory neurons.

Microheterogeneity ("neurotypy") of neurofilament proteins.

Identification of human brain from a tissue fragment by detection of neurofilament proteins

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