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Biomedical subjects

U Z Littauer

Publications and source records attributed to U Z Littauer.

At least 37 records · Page 2Linked to original sources
PubMed1983

Regulation of mRNA levels for microtubule proteins during nerve regeneration.

The molecular regulation of tubulin synthesis was investigated in the regenerating goldfish retina. Previous in vivo studies pointed to an increase in tubulin synthesis in the retina during regeneration of the injured goldfish optic nerve. Using labeled cDNA probes, we showed that this increase occurs as a result of enhanced tubulin mRNA levels. Analysis of labeled in vivo products revealed enhanced beta 2-tubulin synthesis accompanied by an increase in the level of the low-Mr microtubule-associated proteins identified as TAU factors. The results are discussed with respect to the possible involvement of these proteins in the process of nerve regeneration.

Animals↗
PubMed1983

Biphasic regulation by dibutyryl cyclic AMP of tubulin and actin mRNA levels in neuroblastoma cells.

Blot hybridization analysis that used labeled tubulin cDNA probes revealed that N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate [dibutyryl cyclic AMP (Bt2cAMP)] initially increases and later decreases the level of tubulin mRNA in a neuroblastoma-glioma hybrid cell line as well as in the parent cells. A significant increase in tubulin mRNA sequences is already evident 1 hr after the addition of Bt2cAMP to the neuroblastoma cells, and a maximal induction of 2-fold is seen after 12 hr. Continued treatment with Bt2cAMP for 4 days results in a down-regulation of the initial tubulin mRNA level independently of cell density. In the glioma cells Bt2cAMP also rapidly increases the level of tubulin mRNA sequences, reaching a maximum within 6 hr. However, in these cells the subsequent decrease in tubulin mRNA content depends on the culture's phase of growth: cells at the logarithmic growth phase do not down-regulate the tubulin mRNA content even after prolonged treatment with Bt2cAMP, whereas confluent cells do. The hybrid cell line manifests intermediate characteristics in Bt2cAMP regulation of tubulin mRNA level. The time course of induction and down-regulation of tubulin mRNA content observed in the hybrid cells is similar to that of the parent neuroblastoma, whereas the sensitivity to induction is glioma-like and is 8-fold over the initial level. Blot hybridization with labeled actin cDNA probes showed a similar but not identical induction of actin mRNA synthesis with the hybrid and glioma cells, whereas no significant change was observed with the neuroblastoma cells. Moreover, prolonged treatment with Bt2cAMP of all these cell lines did not result in down-regulation of actin mRNA sequences below the initial control value. It was also observed that the level of tubulin sequences in mRNA isolated from 12-day-old rat brain was higher than that in the newborn brain. However, at an age of 30 days, the level of tubulin sequences decreases to about 75% of the newborn level.

Actins↗
PubMed1982

Mapping of distinct structural domains on microtubule-associated protein 2 by monoclonal antibodies.

Monoclonal antibodies against microtubule-associated protein 2 (MAP2) were prepared and their specificity was verified by visualization of the antigens using the antibody overlay technique and by radioimmunoassay. MAP2 was cleaved by alpha-chymotrypsin to generate a series of high-molecular-mass fragments ranging between 270 and 140 kDa. The precursor-product relationship of these fragments was suggested from the rate of their appearance and from the analysis of the tryptic peptide map of each fragment. A group of monoclonal antibodies was found to react predominantly with the intact 270-kDa MAP2 molecule and a fragment having a mass of 240 kDa and to some extent with a 215-kDa fragment. Another group of monoclonal antibodies reacted with an antigenic determinant which was located on the 270-kDa molecule as well as on fragments as small as 140 kDa. None of the two groups of monoclonal antibodies reacted with the microtubule-binding domain of MAP2. These results suggest that one group of antibodies reacts with sites located at or dependent upon a terminal 60-kDa domain(s) distal to the microtubule-binding site of MAP2. The second group of antibodies, which can still bind to smaller proteolytic products, appear to be associated with the central region of the MAP2 molecule. Indirect immunofluorescence experiments with the antibody preparations indicated that at least some of the antigenic determinants are exposed when MAP2 is associated with microtubules in the cell body and neurite outgrowths of differentiated rat brain neuroblastoma B104 cells.

Animals↗
PubMed1982

ADP-ribosylation of microtubule proteins as catalyzed by cholera toxin.

Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8-3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5'-AMP, respectively labeled with the corresponding isotope. Two-dimensional separation by isoelectric focusing and SDS-PAGE of labeled and native tubulin revealed that labeling occurs at least in four different isotubulins. The isoelectric point of the labeled isotubulins was slightly lower than that of native purified tubulin. This shift in mobility is probably due to additional negative charges involved with the incorporation of ADP-ribosyl residues into the tubulin subunits. SDS-PAGE of peptides derived from [32P]ADP-ribosylated alpha and beta tubulin subunits by Staphylococcus aureus protease cleavage showed a peptide pattern identical with that of native tubulin. Microtubule-associated proteins (MAP1 and MAP2) of high molecular weight were also shown to undergo ADP-ribosylation. Incubation of permeated rat neuroblastoma cells in the presence of [32P]NAD and cholera toxin results in the labeling of only a few cell proteins of which tubulin is one of the major substrates.

Adenosine Diphosphate Ribose↗
PubMed1982

Modulation of tubulin mRNA levels by interferon in human lymphoblastoid cells.

Blot hybridization with labeled tubulin cDNA showed that treatment of Ramos cells, a human cell line of lymphoblastoid origin, with either alpha or beta interferon (IFN) induced a marked increase in the amount of tubulin mRNA sequences. The level of tubulin mRNA sequences increased rapidly after exposure of cells to IFN-alpha and reached a maximum after 1 h of treatment, which was four times the control level. Treatment with IFN-beta induced a maximal increase after 4 h; the amount of tubulin mRNA sequences was seven times higher than the control level. The mRNA extracted from IFN-treated and nontreated cells was translated in vitro in a reticulocyte lysate cell-free system containing [35S]methionine. Electrophoretic analysis of the labeled cell-free products showed an increase in the amount of translatable tubulin mRNA that parallels the time course of induction of tubulin mRNA sequences. Two-dimensional gel electrophoresis of the labeled protein products directed by mRNA indicates that IFN caused a more pronounced increase in the level of alpha-tubulin than beta-tubulin mRNA. Treatment with colchicine, which disrupts the cell microtubules, caused a marked decrease in the tubulin mRNA content. Concomitant treatment of the cells with colchicine and IFN abolished the interferon-dependent induction of tubulin mRNA.

Actins↗
PubMed1982

Modulation of mRNA for microtubule-associated proteins during brain development.

The heterogeneity of tau microtubule-associated proteins from rat brain is developmentally determined. Newborn rat brain contains two tau polypeptides (tau 0) with somewhat different molecular weights than the five tau components associated with microtubules from 12-day-old brain (tau 12). tau 0 and tau 12 are immunologically related and crossreact with antibodies against tau 12 proteins. Enrichment of the tau mRNA was achieved by prior hybridization of unfractionated poly(A)-containing mRNA to cDNA preparations containing tubulin and actin sequences. The remaining unhybridized mRNA was further fractionated by electrophoresis on methylmercury hydroxide agarose gels. Experiments involving cell-free translation of mRNA indicated that the major differences in the composition of tau proteins from newborn and developing brain are controlled at the mRNA level. The mRNA from newborn rat brain directed the synthesis of five tau proteins, two of which are specific for newborn brain, whereas the other three forms are characteristic of the developing brain. Thus, the appearance in newborn brain of mRNA species specific for three tau 12 forms precedes the phase of the synthesis of these proteins in the cell. By contrast, mRNA from 12-day brain directed the synthesis of four tau proteins specific for the developing brain, one of which is not synthesized by mRNA from newborn brain. None of the newborn tau 0 forms were synthesized with mRNA isolated from 12-day brain.

Animals↗
PubMed1981

The nucleotide sequence of rat alpha-tubulin: 3'-end characteristics, and evolutionary conservation.

The structure and sequence of rat alpha-tubulin cDNA clone is being described. The 3'-end of the coding region contains the codon for a C-terminal tyrosine, which was previously considered to be post-translationally added to the completed polypeptide chain. A close homology in the coding sequence is observed when a-tubulin from rat and chick are compared, while the 3-non-translated region had diverged considerably.

Amino Acid Sequence↗
PubMed1980

Brain tubulin and actin cDNA sequences: isolation of recombinant plasmids.

Rat brain mRNA enriched for tubulin and actin sequences was used to prepare double stranded cDNA. A library of recombinant clones was constructed by inserting the dsDNA into the Pst1 site of pBR322 plasmid and transformation of E. coli chi 1776 host. Clones bearing sequences coding for tubulin and actin were identified and characterized.

Actins↗
PubMed1980

A glycoprotein from neurites of differentiated neuroblastoma cells.

Neurites were prepared by a novel method from differentiating mouse neuroblastoma cells. When electrically differentiated cells were labeled metabolically with L-[3H]fucose or D-[3H]glucosamine, both the neurites and the surface membranes showed the presence of a glycoprotein of apparent Mr = 200,000. In contrast, the level of this glycoprotein was reduced in the surface membranes from nondifferentiated cells and a radioactive glycoprotein of similar molecular weight was found in the growth medium. The method for the isolation of neurites is of potential usage in distinguishing specific proteins associated with growing neurites.

Animals↗
PubMed1980

Translation in vitro of rat brain mRNA coding for a variety of tubulin forms.

Prenatal rat brain tubulin was resolved by isoelectric focusing into five to six components (isotubulins 1--6) while in mature brain nine distinct forms were evident (isotubulins 1--9). Tubulin, isolated from various brain regions, displayed different proportions of these nine isotubulins which may result from the heterogeneity in brain cell population. Mature brain mRNA, when translated in vitro, in the reticulocyte lysate cell-free system, directed the synthesis of five tubulin forms, namely isotubulins 1, 3, 4 (or 5), 6 and 7. The mRNA species coding for isotubulins 3 and 7 could be partially separated on formamide/sucrose gradients, while in the absence of formamide the mRNA species directing the synthesis of isotubulins 1, 4 (or 5) and 6 showed differences in mobility. It therefore appears that brain mRNA may consist of five different species coding for distinct tubulin forms. Moreover, a marked age-dependent enhancement in the relative translation of the mRNA coding for isotubulin 7, which is not apparent among the translation products directed by the prenatal mRNA, was detected. Our results indicate that some of the age-dependent variations in tubulin microheterogeneity may be controlled at the mRNA level.

Aging↗
PubMed1979

Expression of external-surface membrane proteins in differentiated and undifferentiated mouse neuroblastoma cells.

Murine neuroblastoma cultures were labeled externally with the cationic reagent N,N,N-[3H]-trimethylamino-beta-alanyl-N-hydroxy-succinimide ester ([3H]Me3N-beta Ala-NSuc) or with 125I/lactoperoxidase. The cells were labeled in the logarithmic and confluent growth phases as well as in a highly differentiated state following treatment with 2% dimethylsulfoxide. The labeled exterior membrane proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Major changes in the exterior membrane proteins were observed during maturation and differentiation of the cells. Most of these changes were clonal-specific, while others were common to several clones. Two proteins of Mr 55,000 and 65,000 were labeled by both 125I/lactoperoxidase and Me3N-[3H]-beta Ala-NSuc. The level of labeling was dependent on the clonal lines used and the state of the cell maturation. A group of proteins displaying a molecular weight between 150,000 and 200,000 was found to be related to the transition of a culture from logarithmic to confluent growth phases. An additional protein, with an apparent molecular weight of 95,000, was common to differentiated cells of the two inducible clones used. In general the maturation of logarithmic phase cells into confluent cells resulted in a less complex electrophoretic distribution of the pattern of labeling. After dimethyl-sulfoxide treatment, further reduction in the complexity of the externally labeled proteins was observed.

Animals↗