Hearing loss in an institutionalized mentally retarded population. Identification by auditory brainstem response.

We report the results of auditory brainstem response testing of 122 profoundly retarded institutionalized children, a segment of the retarded population heretofore generally regarded as untestable by behavioral audiometry. Major findings include: 32% of the study population showed, by auditory brainstem response, hearing loss exceeding 20 decibels of hearing level in one or both ears (12% showed conductive loss and 20% sensorineural loss); of the 15.6% with evidence of bilateral sensorineural loss, 7.37% had losses in the 30- to 50-dB range, and 8.19% had losses of 60 dB or greater; and evidence of abnormal brain-stem function was found in 11%. Results of otologic examinations and audiologic habilitative follow-up in selected children are also reported.

Characterization of tubulin in mouse brain myelin.

Analysis of mouse brain myelin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that in the high-molecular-weight range it contained, besides the Wolfgram protein doublet, proteins comigrating with actin and with both subunits of tubulin. The occurrence of these alpha and beta subunits was confirmed by peptide mapping in myelin analyzed by two-dimensional electrophoresis. This tubulin did not arise from an artifactual binding of soluble brain tubulin to the myelin fraction: addition of exogenously labeled tubulin to brain homogenates proved that during myelin isolation by the procedure of Norton and Poduslo (1973) the contaminating tubulin was washed out. On the other hand, the distribution of tubulin isoforms in myelin was investigated by isoelectric focusing and compared with the distribution of the 21 isoforms listed for the whole brain soluble tubulin. It was shown that many isoforms were found in myelin (three isoforms for the alpha subunit and nine for the beta subunit), and that some isoforms were represented both in myelin and in soluble tubulin, but in different relative proportions.

Microheterogeneity of tubulin proteins in neuronal and glial cells from the mouse brain in culture.

The microheterogeneity of the alpha and beta isoforms of tubulin in brain cells in culture was studied. The cells were prepared from two precise regions of the embryonic mouse brain (ED15), the striatum and the mesencephalon. It was possible to maintain virtually pure cultures of neuronal or glial cells up to 1 and 4 weeks in vitro, respectively. The tubulin heterogeneity of striatal and mesencephalic neurons was found to be very similar after a few days in culture. More precise examination of pure neurons from the striatum revealed that their tubulin content after 7 days in vitro exhibited the same degree of complexity as a control extract from a 4 day-old mouse brain. In fact, we could detect the presence of at least six alpha and nine beta tubulin isoforms. Among these isoforms a specific family of beta proteins (beta' tubulin) and the more acidic alpha proteins were present. Since these isoforms have, up to now, been found only in tubulin extracts prepared from the nervous system, our experiments suggest that they belong to the neuronal subpopulation of this tissue. This point is reinforced by their complete absence from the tubulin proteins extracted from pure glial cells even after several weeks in vitro. These results lead us to propose that brain tubulin microheterogeneity is associated with the presence of neurons and not of glia and may, therefore, play a specific role in maintaining neuronal shape and function.

[Fractionation of a sciatic nerve homogenate from normal, trembler and quaking mice on a continuous sucrose gradient]. / Fractionnement d'un homogénat de nerf sciatique de souris normale, Trembler et quaking sur gradient continu de saccharose.

Two fractions, characterized by biochemical criteria [quantity and density of particulate material, localization of myelin specific protein--Po, P1 and Pr--and of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and of 5'-nucleotidase], were isolated from sciatic nerve homogenate of normal, Trembler and quaking, young and adult Mice, on a continuous sucrose gradient. The fraction, at 0.58-0.6 M, the richest in membrane particles contains the myelin. The fraction at 0.7-0.75 M should contain the plasma membrane of the Schwann cell which synthesizes myelin.

High level of tubulin microheterogeneity in the mouse brain.

Using a high resolution isoelectric focusing method, we have demonstrated a high level of tubulin heterogeneity in mouse brain: 21 isotubulins were identified. Determination of the apparent molecular weight and analysis of the peptide map of each isotubulin species allowed us to identify 7 alpha and 14 beta tubulin subunits. Furthermore, variations in this tubulin population during brain development were confirmed; in particular, an increase in the number of beta isotubulins was observed at the beginning of the postnatal period.

Evolution of tubulin heterogeneity during mouse brain development.

In this report, we have characterized tubulin subunit heterogeneity and its evolution during mouse brain development, from embryonic to adult stages. A modification of the two-dimensional protein analysis was used to specify these events. The number of isotubulins increases from 6 (4 alpha and 2 beta), in the embryonic brain, to 11 (6 alpha and 5 beta), in the adult. The changes occurring in tubulin heterogeneity are developmentally controlled but it seems that alpha and beta isotubulins are independently regulated: changes in alpha tubulin occur only just before birth whereas the major evolution is concerned with the appearance and accumulation of acidic beta isotubulins throughout development.

Changes in some cytoskeletal proteins during neuroblastoma cell differentiation.

After in vitro microtubule assembly of mouse neuroblastoma crude extracts, six protein species migrate in the tubulin region of two-dimensional electrophoregrams. The evolution of these forms after morphological cell differentiation of the clone NIE115 shows two major modifications. Form 5 decreased drastically while form 6 increases during neurite formation. Peptide mapping analysis reveals that forms 5 and 6 are vimentin, a component of intermediate filaments, and beta-tubulin subunit, respectively. Sodium butyrate treatment of NIE115 cells or serum starvation of NIA103 cells, conditions blocking cell division and failing to induce morphological differentiation, prevent any modifications in the relative proportion of these proteins. It is concluded that the changes in the distribution of the tubulin isoforms and vimentin are directly related to neurite formation.

Partial purification of the alpha-tubulin and beta-tubulin messenger RNAs from rat brain.

Poly(A)-containing RNA from frozen adult rat brain were fractionated by centrifugation in a formamide/sucrose gradient. Individual fractions were used to program protein synthesis in vitro in a reticulocyte lysate. The cell-free translation products were analyzed by two-dimensional electrophoresis in polyacrylamide slab gels. We observed a heterodispersion of the mRNA translation activity coding for the beta-tubulin subunit which contrasts with a relatively homogeneous distribution of the alpha-tubulin subunit mRNA. These last mRNA species are present in a peak which sediments near the 18-S region of the gradient whereas the beta-tubulin mRNA activity is predominant in the fractions corresponding to the heaviest mRNA species. When these heaviest RNAs were separated again by centrifugation in a second formamide/sucrose gradient, a poly(A)-rich RNA population was obtained that was enriched in RNA for programming the beta-tubulin subunit. Analysis of the products whose synthesis in vitro was directed by this mRNA population revealed that beta tubulin was the main protein formed, the ratio beta/alpha being more than tenfold greater than in the products translated in vitro using total poly(A)-rich RNA.

Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and alpha and beta tubulin subunits.

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a run-off reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.

Function of individual E. coli 30 S ribosomal proteins as determined by in situ immunospecific neutralization: a tentative classification.

The accessibility of each 30S subunit protein to their cognate antibodies (IgG or Fabs) having been previously well established, the effect of their in situ specific neutralization by monovalent IgG fragments (FabI) are reported for five reactions: 1) T4 and R17 RNA directed protein synthesis: 2) polyphenylalanine synthesis: 3) enzymatic Phe-tRNA binding in the presence of 30S + 50W subunits: 4) fMet-tRNAf binding to the 30S subunit in the presence of initiation factors IF1, IF2, IF3; 5) coupling with lambda plac DNA transcription of the initial translation step (i.e., interaction of IF3 activated 30S subunits with nascent mRNA, in the absence of tRNA). According to evident similarities in their inhibition pattern concerning the five reactions tested, 30S subunit proteins can be classified in five categories which are discussed in terms of functional topography.

Phenotypic modulation in mouse neuroblastoma cells: the case of acetylcholinesterase.

Mouse neuroblastoma clone NIE-115 contains two species of acetylcholinesterase (AChE) which sediment at 4S and 11S. The former is predominant in growing cells (70%), whereas the 11S is more abundant in cells with neurites (55--65%). When cell replication is arrested by sodium butyrate, there is an increase in AChE activity but no modification in the proportion of 4S:11S species. Inhibition of protein synthesis by cycloheximide provokes a slow increase in the 11S form and a parallel decrease in 4S. Neurite retraction in presence of colchicine or excess serum does not lead to a concomitant reversion in the high 11S proportion. We postulate that the 11S AChE is formed by a conversion of the 4S and that the shift in the S-value ratio of the enzyme reflects changes in membrane remodeling during neurite extension.

Changes in the sedimentation properties of acetycholinesterase during neuroblastoma differentiation.

The adrenergic mouse neuroblastoma clone NIE 115 contains two species of AChE which sediment at 4S and 11S. These two species are found both in logarithmic growing phase cells (round neuroblast morphology) and in cells which have undergone morphological differentiation due to the elimination of serum. The 4S form is predominant in the growing cells (70 per cent) whereas the 11S form is more abundant (55 per cent to 65 per cent) in the cells with neurites. When protein synthesis is inhibited by cycloheximide, there is an increase states : we postulate that the 11S species is formed by a conversion of the 4S species. When cell division is blocked by sodium butyrate, there is an increase in AChE activity but no modification in the proportion of 4S/11S species as compared to the cells in growing phase. We were unable to associate temporally the increase in the 11S species with neurite outgrowth ; when cells were allowed to retract their neurites and were subsequently maintained in a GROWING state for 10 to 15 days, the 11s species still predominates. Although the presence of the 11S molecule cannot always be correlated with the state of morphological differentiation, the shift in sedimentation coefficient of AChE form 4S to 11S might eventually constitute a reference in the study of biochemical events leading to terminal differentiation in this system.