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C A Marotta

Publications and source records attributed to C A Marotta.

At least 55 records · Page 3Linked to original sources
PubMed1983

In vitro synthesis of polypeptides of moderately large size by poly(A)-containing messenger RNA from postmortem human brain and mouse brain.

Studies were undertaken to optimize the conditions for isolation and in vitro translation of poly(A)-containing mRNA from human postmortem brain. The comparison of several methods for preparation of mRNA from frozen mouse brain indicated that although the yield of mRNA was increased using polysomes prepared in the presence of ribonucleoside vanadyl complexes and subsequently extracted with guanidinium thiocyanate, the translation products were indistinguishable from those synthesized by total cellular RNA directly extracted from tissue with guanidinium thiocyanate. The oligo d(T)-cellulose-purified poly(A)-containing mRNA preparations were translated in vitro in a rabbit reticulocyte lysate in the presence of L-[35S]methionine. Messenger RNA from frozen mouse brain stimulated protein synthesis from 9- to 20-fold over endogenous mRNA. Over 450 polypeptides were reproducibly synthesized and separated by two-dimensional polyacrylamide gel electrophoresis (PAGE); size classes up to 130,000 daltons were present. Direct extraction of RNA from frozen human cerebral cortex and cerebellum with guanidinium thiocyanate followed by oligo d(T)-cellulose chromatography yielded 1.8 micrograms/g and 2.0 micrograms/g, respectively, of poly(A)-containing mRNA; this represents a two- to fourfold increase over our earlier results. In the rabbit reticulocyte translation system human brain mRNA stimulated protein synthesis nearly threefold over endogenous mRNA. Compared with earlier studies, the number of newly synthesized polypeptides was increased by 30%. Over 300 species were separated by two-dimensional PAGE, and size classes up to 130,000 daltons were present, as compared to 70,000 in an earlier report. The polypeptides synthesized by human cerebral cortex and cerebellum were indistinguishable. However, several appeared to be uniquely human when compared with the products synthesized by mouse brain mRNA. The method described for the preparation of postmortem human brain mRNA eliminates the need to prepare polysomes, which are recovered in variable and low yield from the postmortem human brain. The procedure appears applicable to studies on the synthesis of moderately large human brain polypeptides and for investigations of brain protein polymorphism when relatively large numbers of products are required for analysis.

Animals↗
PubMed1983

Axonal polypeptides cross-reactive with antibodies to neurofilament proteins.

Antibodies were prepared to mammalian CNS neurofilament proteins (NFPs) and the antibody specificities were compared using a sensitive immunoblotting method. This procedure was used to detect and characterize cross-reactive proteins and their degradation products in neurofilament preparations. NFPs were prepared by axon flotation. Rabbits were immunized with 200,000, 140,000, and 70,000 NFPs (200K, 140K, and 70K) that had been electrophoretically purified by polyacrylamide gel electrophoresis (PAGE). By immunohistofluorescence it was shown that all antisera stained similar filamentous structures in rat cerebellar neurons. By use of a horseradish peroxidase-conjugated indirect antibody procedure, however, differences were detected in the cross-reactivities of the antisera to rat NFPs, separated by PAGE and electrophoretically transferred to nitrocellulose membranes. Each antiserum exhibited strong binding to the homologous NFP and, thus, was suitable for the detection of cross-reactive polypeptides and proteolytic degradation products derived exclusively from the individual NFPs. Anti-200K, anti-140K, or anti-70K was applied to overloaded two-dimensional nitrocellulose blots of NFPs prepared by axon flotation. Each of the three sera detected a group of unique nonoverlapping polypeptides, some of which were identified as NFP degradation products. A different group of polypeptides was cross-reactive with antiserum to purified glial fibrillary acidic protein. The immunostaining of polypeptides on nitrocellulose was far more sensitive for detecting NFP degradation products than was staining polyacrylamide gels with Coomassie blue. Titers for the antisera were two to three orders of magnitude higher with the immunoblotting procedure than with immunohistologic methods. The sensitivity and the specificity of the described methods suggest their usefulness for examining proteolytic cleavage products of NFPs under a variety of conditions.

Animals↗
PubMed1982

Posttranslational modification of a neurofilament protein during axoplasmic transport: implications for regional specialization of CNS axons.

The possibility that proteins are modified during axoplasmic transport in central nervous system axons was examined by analyzing neurofilament proteins (200,000, 140,000, and 70,000 mol wt) along the mouse primary optic pathway (optic nerve and optic tract). The major neurofilament proteins (NFPs) exhibited considerable microheterogeneity. At least three forms of the " 140,000" neurofilament protein differing in molecular weight by SDS PAGE (140,000-145,000 mol wt) were identified. The "140,000" proteins, and their counterparts in purified neurofilament preparations, displayed similar isoelectric points and the same peptide maps. The "140,000" NFPs exhibited regional heterogeneity when consecutive segments of the optic pathway were separately examined on polyacrylamide gels. Two major species (145,000 and 140,000 mol wt) were present along the entire length of the optic pathway. The third protein (143,000 mol wt) was absent proximally but became increasingly prominent in distal segments. After intravitreal injection of [(3)H]proline, newly synthesized radiolabeled proteins in the "140,000" mol wt region entered proximal mouse retinal ganglion cell (RGC) axons as two major species corresponding to the 145,000 and 14,000 mol wt NFPs observed on stained gels. When transported NFPs reached more distal axonal regions (30 d postinjection or longer), a 143,000 mol wt protein appeared that was similar in isoelectric point and peptide map to the 145,000 and 140,000 mol wt species. The results suggest that (a) the composition of CNS neurofilaments, particularly the "140,000" component, is more complex than previously recognized, that (b) retinal ganglion cell axons display regional differentiation with respect to these cytoskeletal proteins, and that (c) structural heterogeneity of "140,000" NFPs arises, at least in part, from posttranslational modification during axoplasmic transport. When excised but intact optic pathways were incubated in vitro at pH 7.4, a 143,000 NFP was rapidly formed by a calcium-dependent enzymatic process active at endogenous calcium levels. Changes in major proteins other than those in the 145,000-140,000 mol wt region were minimal. In optic pathways from mice injected intravitreally with L-[(3)H]proline, tritiated 143,000 mol wt NFP formed rapidly in vitro if radioactively labeled NFPs were present in distal RGC axonal regions (31 d postinjection). By contrast, no 143,000 mol wt NFP was generated if radioactively labeled NFPs were present proximally in RGC axons (6 d postinjection). The enzymatic process that generates 143,000 mol wt NFP in vitro, therefore, appears to have a nonuniform distribution along the RGC axons. The foregoing results and other observations, including the accompanying report (J. Cell Biol., 1982, 94:159-164), imply that CNS axons may be regionally specialized with respect to structure and function.

Animals↗
PubMed1982

Posttranslational processing of alpha-tubulin during axoplasmic transport in CNS axons.

Tubulin proteins in mouse retinal ganglion cell (RGC) neurons were analyzed to determine whether they undergo posttranslational processing during axoplasmic transport. Alpha- and beta-tubulin comprised heterogeneous proteins in the primary optic pathway (optic nerve and optic tract) when examined by two-dimensional (2D) PAGE. In addition, however, alpha-tubulin exhibited regional heterogeneity when consecutive 1.1-mm segments of the optic pathway were analyzed separately. In proximal segments, alpha-tubulin consisted of two predominant proteins separable by isoelectric point and several less abundant species. In more distal segments, these predominant proteins decreased progressively and the alpha-tubulin region of the gel was represented by less abundant multiple forms only; beta-tubulin region of the gel was represented by less abundant multiple forms only; beta-tubulin was the same in all segments. After intravitreal injection of [3H]proline to mice, radiolabeled alpha- and beta-tubulin heteroproteins were conveyed together at a rate of 0.1-0.2 mm/d in the slowest phase of axoplasmic transport. At 45 d postinjection, the distribution of radiolabeled heterogeneous forms a alpha- and beta-tubulin in consecutive segments of optic pathway resembled the distribution of unlabeled proteins by 2D PAGE, indicating that regional heterogeneity of tubulin arises during axonal transport. Peptide mapping studies demonstrated that the progressive alteration of alpha-tubulin revealed by PAGE analysis cannot be explained by contamination of the alpha-tubulin region by other proteins on gels. The results are consistent with the posttranslational processing of alpha-tubulin during axoplasmic transport. These observations, along with the accompanying report (J. Cell Biol., 1982, 94:150-158), provide additional evidence that CNS axons may be regionally specialized.

Animals↗
PubMed1981

The subunit composition of cerebellar tubulin: evidence for multiple beta tubulin messenger RNAs.

Cytoplasmic proteins were isolated from adult rat forebrain and cerebellum and analyzed by two-dimensional gel electrophoresis under conditions which the major subunits of tubulin were separated. Forebrain cytoplasmic tubulin consisted of two groups of alpha subunits (alpha 1 and alpha 2) and a minimum of two beta subunits (beta 1 and beta 2). However, the rat cerebellar cytoplasmic proteins contained greatly decreased amounts of the beta 1 tubulin subunit relative to the analysis of forebrain proteins. Messenger RNA (mRNA) was purified from cerebellum and forebrain and translated in wheat germ homogenate. Analysis of the translation products of cerebellar mRNA indicated only a trace amount of the beta 1 subunit, whereas the expected amount of beta 1 was found among the translation products of forebrain mRNA. This data is consistent with the conclusion that the beta 1 and beta 2 subunits of tubulin are synthesized from different mRNAs. A decrease in beta 1 mRNA relative to other tubulin mRNAs may be one of the factors responsible for the low steady state amounts of beta 1 tubulin in the cerebellum.

Animals↗
PubMed1981

Myelin basic protein in Alzheimer disease neuronal fractions and mammalian neurofilament preparations.

We previously reported a marked increase of a 20,000 molecular weight (MW) protein, P20, in some neuronal fractions and whole cortical homogenates isolated from affected cortex in Alzheimer disease; P20 comigrated electrophoretically with an unidentified, major 20,000 MW protein present in human neurofilament (NF) fractions. We now report that the 20,000 MW protein is a major constituent of rodent as well as human NF fractions and that it comigrates by one- and two-dimensional gel electrophoresis with purified myelin basic protein (MBP). Peptide mapping and staining with amido black confirmed the identity of the 20,000 MW protein of mammalian NF fractions as MBP. One- and two-dimensional gel electrophoresis of neuronal perikaryal fractions from human cortex indicated that the increased P20 protein in Alzheimer neuronal fractions comigrates with human MBP. Deliberate contamination of cortical samples with adjacent subcortical white matter (i.e., myelin) prior to neuronal separation did not result in an increase of P20 in the neuronal fraction. On the basis of these and additional experiments, we conclude that the increase of a 20,000 MW protein in neuronal fractions and whole homogenates from affected cortex in Alzheimer disease represents MBP of intracortical origin.

Alzheimer Disease↗
PubMed1981

Tubulin synthesis in rat forebrain: studies with free and membrane-bound polysomes.

Free and membrane-bound polysomes were prepared from rat forebrain and added to a cell-free system containing rabbit reticulocyte factors and L-[35S]methionine. The translation products were analyzed by two-dimensional gel electrophoresis followed by autoradiography. The free polysomes synthesized actin and at least four major tubulin subunits (alpha 1, alpha 2, beta 1, and beta 2) that are found in rat forebrain cytoplasm. The membrane-bound polysomes synthesized predominantly one protein (MB) in the tubulin region of the two-dimensional gel. MB has a molecular weight and isoelectric point similar to alpha-tubulin. Only trace amounts of alpha- and beta-tubulin and action were synthesized by the membrane-bound polysomes. MB co-purified with cytoplasmic tubulin after two cycles of aggregation and disaggregation. MB synthesized in vitro (from membrane-bound polysomes) and alpha- and beta-tubulin and actin subunits (synthesized from free polysomes) were digested with Staphylococcus aureus V8 protease, and the resulting peptides were separated by slab gel electrophoresis followed by autoradiography. The peptide pattern of MB was similar but not identical to the peptide patterns of alpha- and beta-tubulin; MB yielded peptides not found in tubulin. We conclude that membrane-bound polysomes from rat forebrain do not synthesize significant amounts of the predominant tubulin subunits synthesized by free polysomes. A major protein (MB) is synthesized by membrane-bound polysomes and is similar, but not identical, to alpha-tubulin synthesized by free polysomes on the basis of molecular weight, isoelectric point, and peptide analysis.

Animals↗
PubMed1981

In vitro synthesis of human brain proteins including tubulin and actin by purified postmortem polysomes.

Polysomes were prepared from human brain tissue 2-6 h postmortem; the polysomes were active in a cell-free protein synthesis system containing rabbit reticulocyte factors. Protein synthesis was totally dependent upon added MgCl2, ATP, the reticulocyte factor fraction, and the human polysome fraction. Human brain proteins synthesized in the presence of L-[35S]methionine were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Over 250 proteins were synthesized and they extended in size up to 250,000 d; many of the most abundant native human brain proteins were synthesized, including tubulin and actin. It was shown that human brain alpha and beta tubulin and actin isomers synthesized in vitro from human postmortem polysomes have the same apparent molecular weights and isoelectric points as the corresponding proteins synthesized by rat polysomes from fresh cortices. The corresponding tubulin and actin synthesized by human and rat brain polysomes also yield the same radioactive methionine-containing peptides after digestion with Staphylococcus aureus V8 protease. These analyses indicate that postmortem polysomes contain active messenger RNA which can direct the partial and/or complete synthesis of actin and tubulin subunits and other human brain proteins.

Actins↗
PubMed1981

The preparation of biologically active messenger RNA from human postmortem brain tissue.

Messenger RNA (mRNA) was extracted from human postmortem brain tissue by alkaline phenol extraction of polysomes followed by oligo (dT)-cellulose chromatography. The mRNA preparations stimulated protein synthesis in a cell-free system containing wheat germ homogenate. The products of protein synthesis were analyzed by one- and two-dimensional gel electrophoresis. These analyses indicated that numerous polypeptides, including tubulin subunits and actin isomers, were synthesized by the human mRNA. The molecular weight range of polypeptides synthesized by human mRNA fractions from two brain specimens were identical, and analysis by two-dimensional gel electrophoresis indicated qualitatively similar products. The yield of mRNA extracted per gram of human tissue was less than the yield obtained with rat forebrains from animals sacrificed immediately before brain removal and mRNA purification. A decrease in the amount of polysomes isolated from human tissue relative to rat brain tissue was a major factor contributing to the low yield. The molecular weight distribution of polypeptides synthesized by human and rat brain mRNA fractions in wheat germ homogenate was similar; thus, there was no indication for selective breakdown or inactivation of high molecular weight mRNA species in the human tissue. Our studies indicate that it is possible to utilize postmortem tissue for molecular biological investigations of human brain mRNA.

Animals↗
PubMed1981

Characterization and comparison of neurofilament proteins from rat and mouse CNS.

Rat and mouse CNS neurofilament proteins (NFPs) were characterized and compared, in terms of electrophoretic properties on polyacrylamide gels and by peptide mapping, with one another and with other co-purifying lower-molecular-weight CNS proteins, including alpha and beta tubulin. NFPs were partially purified by modification of the axon flotation procedure of Norton and co-workers and were demyelinated with Triton X-100. On one-dimensional SDS polyacrylamide gels the molecular weights of the triad of NFPs from both rat and mouse were approximately 200,000, 140,000, and 70,000. Prominent lower-molecular-weight proteins (63,000-16,000) as well as minor amounts of tubulin and actin were observed after gel electrophoresis. On two-dimensional gels (isoelectric focusing followed by SDS gel electrophoresis) each of the NFPs appeared to be composed of more than one component and the corresponding NFPs from rat and mouse had similar isoelectric points. Gel electrophoresis peptide mapping using Staphylococcus aureus V8 protease indicated the following: (1) the triad of NFPs of different sizes have different peptide maps; (2) alpha and beta tubulin have nonidentical digestion products, which are dissimilar to those of the NFPs; (3) other proteins that co-purify by the axon flotation procedure also have nonidentical peptide maps; and(4) the corresponding NFPs from rat and mouse have similar peptide maps. The co-purifying proteins examined in detail (63,000-49,000) do not appear to be derived by proteolytic cleavage of NFPs and may represent other cytoskeletal constituents.

Animals↗
PubMed1981

Variations in brain cortical polysome translation products among rats of the same strain.

Studies were undertaken to determine whether there exist variations among the translation products of polysomes from different brains of animals of the same strain. Polysomes were prepared from individual rat cortices and translated in a reticulocyte protein-synthesizing system containing rabbit reticulocyte factors and L-[35S]methionine; he resulting radioactive proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis autoradiography. Comparison of the autoradiographs revealed that two acidic proteins, A and B, of apparent 54,000 M. W. occur as three phenotype: A only, B only, or A plus B. These proteins were not detectable by Coomassie brilliant blue staining of two-dimensional electrophoretograms of brain protein preparations. Messenger RNA was extracted from pooled cortices and translated in a wheat germ extract, and both A and B proteins were detected among the products of translation. Cyclic AMP affinity chromatography of the translation products of cortical polysomes showed that both A and B proteins bind to cyclic AMP. Our data are consistent with the conclusion that there are qualitative differences in the polysome translation products that bind to cyclic AMP among individual cortices of rats of the same strain.

Animals↗
PubMed1979

Sununit structure of synaptosomal tubulin.

The subunit structure of rat brain synaptosomal tubulin was examined by high resolution two-dimensional gel fractionation. Whole brain cytoplasmic tubulin consists of two groups of alpha subunits (alpha1 and alpha2), and a minimum of two beta subunits (beta1 and beta2). Both alpha subunits consist of a major relatively acidic form and minor relatively basic forms. In contrast, tubulin purified from synaptoplasm contains an additional subunit, alpha3, which has the same isoelectric point but slightly faster electrophoretic mobility than alpha1 and alpha2. All synaptosomal alpha subunits are the relatively acidic forms and the minor basic forms are absent. The synaptosomal beta subunits have electrophoretic properties similar to the corresponding cytoplasmic forms. The alpha3 synaptosomal tubulin subunit has affinity for colchicine, has a tryptic peptide map similar to whole brain cytoplasmic alpha tubulin, and can be purified by a standard tubulin purification method.

Animals↗
PubMed1977

Human beta-globin messenger RNA. III. Nucleotide sequences derived from complementary DNA.

Sequences of human beta-globin mRNA were determined by analysis of complementary DNA. beta-mRNA was transcribed into double-stranded cDNA by RNA-dependent DNA polymerase. cDNA was cut by restriction endonucleases and the fragments were terminally labeled by means of polynucleotide kinase and [gamma-32P]ATP. After purification, fragments were degraded by snake venom phosphodiesterase. Alternatively single-stranded [32P]cDNA was prepared by transcription in the presence of [alpha-32P]dCTP and actinomycin D; the product was digested by endonuclease IV and degraded by snake venom phosphodiesterase. cDNA tracts obtained by both labeling methods enabled us to construct a sequence for the translated and 3'-terminal untranslated regions of human beta-mRNA.

Anemia, Sickle Cell↗