Quantitation and characterization of tau factor in porcine tissues

Using a monospecific antibody against brain tau factor purified by affinity chromatography, we have studied the distribution of tau factor or related polypeptides in different cells. The presence of tau in all cell types tested was demonstrated by a radioimmunoassay. Tau factor-related proteins were found in liver, spleen, pancreas, kidney and lung, although at much lower levels than that found in neural cells. In all cases, they copolymerized with tubulin and were heat-resistant. When the distribution of tau factor-related proteins was studied by Western blotting, tau factor antiserum reacted against peptides with an electrophoretic mobility that was similar to those of brain tau factor peptides. Immunofluorescence studies have also been performed with the same antibody to determine the distribution of tau factor-related peptides in PK15 cells. Our results indicated that these peptides were associated to the microtubule network.     

The removal of the carboxy-terminal region of tubulin favors its vinblastine-induced aggregation into spiral-like structures

Vinblastine induces brain tubulin to assemble into spirals. This process is stimulated by microtubule-associated proteins (MAPs) which copolymerize with brain microtubules assembled in vitro. When the carboxy terminal of tubulin is removed by subtilisin digestion, vinblastine readily induces the aggregation of tubulin into spiral-like or circular structures, even in the absence of MAPs. These results suggest that in the absence of MAPs, the carboxy-terminal domain of tubulin may inhibit vinblastine-induced polymerization of tubulin into spiral-like structures.     

Localization of the high affinity calcium-binding site on tubulin molecule

Tubulin is a calcium-binding protein. Two different modes of interaction of calcium with tubulin have been described: a high affinity interaction to one or two binding sites and lower affinity interactions to several other binding sites. In the present study, we have used limited proteolysis of tubulin with trypsin, chymotrypsin, and subtilisin to localize the high affinity calcium-binding sites. Our results indicate that two sites are located in the carboxyl-terminal region of both tubulin subunits, and that tubulin deprived of its carboxyl-terminal region is able to polymerize in the presence of 0.5 mM calcium.     

The interaction between subunits in the tubulin dimer.

Limited proteolysis and chemical cross-linking techniques have been used to study the interaction between alpha- and beta-tubulin subunits. Trypsin digestion of tubulin dimer resulted in the cleavage of the alpha-subunit into two fragments, whereas chymotrypsin cleaved the beta-subunit into two distinct fragments. All of these fragments have been mapped on the tubulin subunits by further proteolysis with formic acid. Cross-linking of trypsin- and chymotrypsin-cleaved subunits has been performed with two different cross-linker agents of different cross-linking distance. The addition of formaldehyde resulted in the cross-linking of the alpha-tubulin N-terminal fragment with beta-tubulin C-terminal domain. The same result was obtained when methyl 4-mercaptobutyrimidate was used.     

Characterization of a membrane-specific tubulin isoform by peptide mapping

A membrane-specific tubulin-like protein, found in preparations of synaptic plasma membranes and brain mitochondria, was analyzed by chemical and proteolytic peptide mapping to determine which part of the molecule was different from cytoplasmic tubulin. The membrane polypeptide was identical to alpha tubulin in the first two-thirds of the molecule containing the amino terminal, as found by peptide mapping. However, some differences were observed in the peptide maps of the carboxy terminal one third of the molecule which includes a domain that is important in the regulation of tubulin self-assembly.     

Ultrastructural differentiation of glandular stomach (proventriculus) in chick embryo

Cytochemical characterization of mucosubstances of chick glanular stomach (proventriculus) changes from 15 days of development to postnatal and adult stages was studied. To corroborate these data cytochemical, ultrastructural and ultracytochemical study of chick embryo proventriculus from 7 to 20 days of development was performed. At the 7th day several layers of undifferentiated cells formed an epithelium which covered the walls of the glandular stomach. Mocosubstances were not found. Between the 9th and 5th day a single layer of cylindrical cells was encountered forming invaginations which originated deep glands. Three types of cells were separated from the above mentioned layer, dark, clear and undifferentiated. The dark cells had organelles which are involved in protein synthesis and the clear ones were rich in mitochondria. Argentaffine cells appeared at 15th day instead mucosubstances formed a thin coat on the epithelium at 9th day which increased at the end of development in the apical cytoplasm and gland cells. These observations demonstrate that proventriculus of chick embryo has ultrastructurally differentiated cells involved with enzymatic and hydrochloric acid secretion after the 9th day. These progressive events are correlated with the digestion process of yolk during embryogenesis. At the end of development the proventriculus has completely organized the glandular layer.     

Trypanosoma cruzi: 4-aminopyrazolopyrimidine in the treatment of experimental Chagas' disease

An allopurinol metabolite, 4-aminopyrazolopyrimidine, was tested on two different strains of mice (NMRI-IVIC and C57Bl/6J) that had been infected 4 days earlier with the virulent Ya strain of Trypanosoma cruzi. Low doses of 4-aminopyrazolopyrimidine (0.125-0.500 mg/kg body wt/day) for 10 days induced a significant reduction in parasitemia (direct counts and subinoculation experiments) and increased survival time (without any evidence of toxicity) compared with untreated animals. When tested in vitro, 4-aminopyrazolopyrimidine was sixfold more active than allopurinol as a trypanostatic drug. The low therapeutic doses of 4-aminopyrazolopyrimidine suggest that this drug may be useful in the treatment of acute Chagas' disease.     

Microtubule-associated protein MAP2 preferentially binds to a dA/dT sequence present in mouse satellite DNA

Microtubule-associated protein MAP2 binds to the Sau96.1 restriction monomer fragment of mouse satellite DNA. This fragment is also present in a lower proportion in bulk DNA. The digestion of MAP2-Sau96.1 fragment complex by DNase results in the protection of certain nucleotide sequences. The sequence poly(dA)4/poly(dT)4 is mainly protected against DNase digestion.     

Unusual properties of a cold-labile fraction of Atlantic cod (Gadus morhua) brain microtubules

A cold-labile fraction of microtubules with unusual properties was isolated from the brain of the Atlantic cod (Gadus morhua). The yield was low, approximately six times lower than that for bovine brain microtubules. This was mainly caused by the presence of a large amount of cold-stable microtubules, which were not broken down during the disassembly step in the temperature-dependent assembly-disassembly isolation procedure and were therefore lost. The isolated cold-labile cod microtubules contained usually only a low amount of microtubule-associated proteins (MAPs). Three high molecular mass proteins were found, of which one was recognized as MAP2. Cod MAP2 differed from mammalian brain MAP2; it was not heat stable and had a slightly higher molecular mass. In contrast to mammalian MAPs, MAP1 was not found in the cold-labile fraction of microtubules. A new heat-labile MAP of higher molecular mass (400 kilodaltons) was however present, as well as a heat-stable protein of slightly lower molecular mass than MAP2. These MAPs showed similar tubulin-binding characteristics as bovine brain MAPs, since they coassembled with taxol-assembled bovine brain microtubules consisting of pure bovine tubulin. In spite of the fact that Ca2+ bound equally to cod and porcine tubulins, it did not inhibit cod microtubule assembly even at high concentrations (greater than 1 mM). In contrast, rings, spirals, and macrotubules were formed. The results show that there are major differences between this fraction of cod microtubules and microtubules from mammalian brain.