Identification of the 190 kD microtubule-associated protein in cultured fibroblasts and its association with interphase and mitotic microtubules

We previously investigated the biochemical characteristics of microtubule-associated proteins (MAPs) of the adrenal medulla and adrenal cortex and found that they contain a new kind of MAP with a molecular weight of 190,000 (190 kD MAP) as a major species (Kotani, S., H. Murofushi, S. Maekawa, C. Sato, and H. Sakai. Eur. J. Biochem. 156, 23-29, 1986). We now have used an affinity purified anti-(190 kD MAP) antibody and show by indirect immunofluorescent microscopy the association of this MAP with microtubules in situ in TIG-3 cells (human embryonic lung fibroblasts). The 190 kD MAP was present along the interphase and mitotic microtubules, and there was no marked difference between the staining pattern with anti-tubulin and that with anti-(190 kD MAP) antibodies, evidence that the localization of 190 kD MAP is not restricted to the subset of microtubules. We also isolated MAPs from TIG-3 cells and identified their 190 kD MAP as a major heat-stable component. Several other unidentified polypeptides were recovered in the MAP fraction specifically.     

Morphological pathway of flagellar assembly in Salmonella typhimurium.

The process of flagellar assembly was investigated in Salmonella typhimurium. Seven types of flagellar precursors produced by various flagellar mutants were purified by CsCl density gradient protocol. They were characterized morphologically by electron microscopy, and biochemically by two-dimensional gel electrophoresis. The MS ring is formed in the absence of any other flagellar components, including the switch complex and the putative export apparatus. Four proteins previously identified as rod components, FlgB, FlgC, FlgF, FlgG, and another protein, FliE, assemble co-operatively into a stable structure. The hook is formed in two distinct steps; formation of its proximal part and elongation. Proximal part formation occurs, but elongation does not occur, in the absence of the LP ring. FlgD is necessary for hook formation, but not for LP-ring formation. A revised pathway of flagellar assembly is proposed based on these and other results.     

Extracellular production of a heat-stable somatic antigen by Bacillus thuringiensis

Extracellular production of a heat-stable somatic antigen (HSSA) by Bacillus thuringiensis subsp. dendrolimus strain T84A1-A [flagellar (H) serotype 4a: 4b] was serologically detected. In Ouchterlony tests, the HSSA antiserum gave single precipitin lines against both untreated and heat-treated culture supernatants. These two precipitin lines fused completely. When colonies of strain T84A1-A were grown on nutrient agar plates containing the homologous HSSA antiserum, precipitin halos were formed around the colonies. Of 27 type strains of B. thuringiensis subspecies tested, only the type strains of B. thuringiensis subsp. sotto (H serotype 4a: 4b) and B. thuringiensis subsp. israelensis (H serotype 14) formed [precipitin halos on nutrient agar plates containing antiserum against the HSSA of strain T84A1-A.     

HLA-DQ system and insulin-dependent diabetes mellitus in Japanese: does it contribute to the development of IDDM as it does in Caucasians?

Fifty-six unrelated Japanese patients with insulin-dependent diabetes mellitus (IDDM) were HLA-typed, and restriction fragment length polymorphism (RFLP) analysis was performed after enzyme digestion with Bam HI and Taq I by using both DR and DQ probes. As previously reported, increased frequencies of Bw54, Cw1, DR4, and DRw53, which are in strong linkage disequilibrium in the Japanese population and make the characteristic Japanese haplotype, were confirmed. DQw4, a new allele of the DQ system recognized by the monoclonal antibody HU-46 and in linkage disequilibrium with this haplotype, presented the highest IDDM association. The RFLP analysis also showed the strongest correlation to IDDM when the DQ probe was applied. These results indicate that HLA-DQ might play the most important role in the development of IDDM in Japanese as well as in Caucasians. The correlation of DQ amino acid sequences strongly associated with IDDM in Japanese are discussed in this study, and contrasting results were found when such sequences were compared with those of Caucasians.Abbreviations used in this paper IDDM insulin-dependent diabetes mellitus - RFLP restriction fragment length polymorphism - Asp aspartic acid - Asp-57 aspartic acid at the 57th residue of the DQ chain - non-Asp-57 nonaspartic acid at the 57th residue of the DQ chain - R.R. relative risk of Woolf and Haldane     

Purification and characterization of kinesin from bovine adrenal medulla

Kinesin was purified from bovine adrenal medulla. The sedimentation coefficient was 8.8 S. Sedimentation equilibrium ultracentrifugation studies showed the molecular weight of kinesin to be 300,000. The calculated axial ratio was 1:16. The Stokes radius was estimated to be 8.9 nm by gel filtration. Circular dichroism showed the alpha-helix content to be about 50%. Purified kinesin preparation contained a major polypeptide with a molecular weight of 120,000 and minor ones with molecular weights of 71,000, 68,000, and 65,000. Bovine adrenal kinesin had an ATPase activity which was stimulated severalfold by microtubules to a specific activity of about 0.1 mumol/min.mg. Kinesin molecules adsorbed to a glass slide promoted the movement of microtubules on the glass surface at a rate of about 0.5 micron/s. Immunostaining of EBTr (bovine embryonic trachea fibroblast) cells and bovine adrenal chromaffin cells in interphase with an affinity-purified antibody against the major polypeptide of kinesin showed that some kinesin was located on microtubules and the rest distributed throughout the cytoplasm in a diffuse manner. EBTr cells in mitotic phase gave a staining pattern showing that kinesin was present throughout the cytoplasm with higher concentration in the region of mitotic apparatus.     

Expression of the enkephalin precursor gene in rat Sertoli cells. Regulation by follicle-stimulating hormone

Searching for somatic cells expressing the preproenkephalin (A) gene in the testis, we have isolated Sertoli cells from the testes of 20-day-old rats. Cultured Sertoli cells contained a single species (about 1.5 kb) of preproenkephalin mRNA, and follicle-stimulating hormone (FSH) transiently increased the mRNA abundance to a maximum (about 30 molecules per cell) at 12 h. Various compounds that activate the cyclic AMP system in Sertoli cells similarly increased the abundance of preproenkephalin mRNA. Moreover, FSH increased intracellular Met-enkephalin immunoreactive peptides in Sertoli cells. Thus, the preproenkephalin gene expression in Sertoli cells is positively regulated by FSH through the cyclic AMP system.     

Study on the possible factors influencing the expression of H-2 restriction specificity and Ir phenotype of antigen-specific proliferative T cells with various types of radiation chimeras

To better understand the factors described previously as influencing the manifestation of H-2 restriction specificity and Ir phenotype of T cells from radiation bone marrow chimeras, we also examined H-2 restriction specificity (Ir phenotype) of antigen (DNP-OVA, (T, G)-A-L, (H, G)-A-L)-specific proliferative T cells generated in various types of H-2 incompatible radiation chimeras prepared under our specific-pathogen-free (SPF) condition. The results indicated the following: (a) T cells generated in F1----parent bone marrow chimeras preferentially manifested host-type H-2 restriction specificity and Ir phenotype, regardless of the radiation dose (8.70 vs 11.59 Gy); (b) T cells recovered from twice-reconstituted F1----(PA----PB) chimeras manifested primary host (PB)-type Ir phenotype; (c) T cells which were recovered from (B10.Thy-1.1 X B10.BR.Thy-1.1)F1----parent (Thy-1.2) bone marrow chimeras and treated with anti-Thy-1.2 plus complement to deplete host-derived T cells still manifested preferentially the restriction specificity for host-type H-2; (d) PA-derived T cells which had differentiated in a fully allogeneic host (PB) environment of (PA + PB)----PB chimeras manifested fully allogeneic host-type Ir phenotype; (e) T cells from F1----parent chimeras that were prepared with 13-day fetal liver cells also manifested host H-2-restricted Ir phenotype; and (f) host preference for Ir phenotype of antigen-specific proliferative T cells was observed even in the case of F1----parent bone marrow chimeras reconstituted with "intact" bone marrow cells. The data suggest that thymic APCs, surviving host T cells or the source of stem cells (adult bone marrow vs 13-day fetal liver), do not necessarily play a significant role in the manifestation of H-2 restriction specificity and Ir phenotype of T cells generated in H-2 incompatible radiation chimeras.     

Microtubule-binding domain of tau proteins

Limited chymotryptic digestion of whole tau proteins produced a fragment of Mr 14,000 (CT14), which was able to bind to microtubules reconstituted from tubulin alone in the presence of taxol. This fragment was also found to persist in microtubules when microtubules consisting of tau proteins and tubulin were digested by chymotrypsin. Analysis of amino acid composition revealed that CT14 was rich in lysine and proline residues, suggesting unique structure of microtubule-binding domain of tau proteins. Amino-terminal sequence of CT14 was determined to be Ser-Ser-Pro-Gly-Ser-Pro-Gly-Thr-Pro-Gly-Ser-Arg-Ser-Arg-X-Pro-Ser-Leu-Pr o. No heterogeneity was detected in this amino-terminal sequence of 19 residues. Five species of polypeptides consisting of tau proteins were separated from each other by gel electrophoresis and subjected to chymotryptic digestion. CT14 was produced from each of the tau polypeptides by chymotryptic digestion, indicating that all tau polypeptides have a common microtubule-binding domain.     

Pairwise perturbation of flagellin subunits. The structural basis for the differences between plain and complex bacterial flagellar filaments

Although plain and complex bacterial flagellar filaments differ in their physical properties and helical symmetry, they both appear to derive from a common underlying structure. Analysis of electron micrographs of complex filaments of Rhizobium lupini revealed that the unit cell has twice the length of that of plain filaments, with a corresponding reduction in helical symmetry whereby the six-start helical family present in plain filaments collapses into a three-start family. Mass per unit length measurements were made by scanning transmission electron microscopy. These, together with the unit cell dimensions and the molecular weight of the flagellin monomer, enabled the number of monomers per unit cell to be estimated. Whereas plain filaments have a single monomer per unit cell, complex filaments have two. These results suggest that complex filament structure differs from plain filament structure by a pairwise perturbation, or interaction, of the flagellin monomers. The additional bonding interactions involved in the perturbation in the complex filament may make it more rigid than the plain filament, which has no such perturbation.