Amino acids responsible for flagellar shape are distributed in terminal regions of flagellin.

The shape of the flagellar filaments of the bacterium Salmonella typhimurium under ordinary conditions is a left-handed helix. In addition to the normal wild-type filament, non-helical (i.e. straight), right-handed helical (early), or circular (semi-coiled and coiled) filaments and filament with small amplitude (fl-type) have been found in mutants or in filaments reconstituted in vitro. We analysed wild-type flagellin and flagellins from 17 flagellar-shape mutants (6 with straight filaments, 6 with curly filaments, 4 with coiled filaments and 1 with fl-type filament) by amino acid sequencing to identify the mutational sites. All mutant flagellins except that of the fl-type filament had single mutations; the fl-type flagellin had two mutations in the molecule. The sites of these mutations were localized in alpha-helical segments of the terminal regions of flagellin. A possible mechanism of the polymorphism of the flagellar filament is discussed.

Terminal regions of flagellin are disordered in solution.

Limited proteolysis of flagellin from Salmonella typhimurium SJW1103 by subtilisin, trypsin and thermolysin results in homologous degradation patterns. The terminal regions of flagellin are very sensitive to proteolysis. These parts are degraded into small oligopeptides at the very early stage of a mild digestion that yields a relatively stable fragment with a molecular weight of 40,000. Further proteolytic degradation results in a stable 27,000 Mr fragment. The 40,000 Mr tryptic fragment has been identified as residues 67 to 446 of the flagellin sequence, while the 27,000 Mr fragment involves the 179 to 418 segment. The NH2-terminal sequence positions for the corresponding fragments produced by subtilisin are 60 and 174 for the 40,000 Mr and 27,000 Mr fragments, respectively. The fragments lost their polymerizing ability. Structural properties of flagellin and its 40,000 Mr tryptic fragment were compared by circular dichroism spectroscopy and differential scanning calorimetry. Analysis of the calorimetric melting profiles suggests that terminal parts of flagellin have no significant internal stability and they are in extensive contact with water. However, these regions contain some secondary structure, probably alpha-helices, as revealed by comparison of the circular dichroic spectra in the far-ultraviolet region. Our results indicate that, although the terminal regions of flagellin may contain some alpha-helical secondary structure of marginal stability, they have no compact ordered tertiary structure in solution. On the contrary, the central region of the molecule involves at least two compact structural units.

The PEBP2beta/CBF beta-SMMHC chimeric protein is localized both in the cell membrane and nuclear subfractions of leukemic cells carrying chromosomal inversion 16.

The chromosomal inversion (16)(p13q22), which is associated with the M4-eosinophilia subtype of human acute myeloid leukemia, causes the fusion of two distinct genes. The polypeptide encoded by the chimeric gene, PEBP2p/CBFp-SMMHC, retains the ability to interact with, and dominantly interfere with the function of proteins possessing the Runt homology domain. The Runt protein homologs constitute the DNA binding subunit of the PEBP2/CBF transcription factor. We examined the subcellular localization of PEBP2beta/CBFbeta-SMMHC, as well as that of Runt protein homologs in leukemic cells carrying inversion 16 by immunoblot analysis. A significant amount of the PEBPbeta/CBFbeta-SMMHC protein was recovered from the nuclear fraction along with the Runt protein homologs. Furthermore, some of both polypeptides was retained in the DNA pellet that represents the material remaining after extraction of nuclear fraction with high salt. These observations suggest that the so-called dominant interfering effect of PEBPbeta/CBFbeta-SMMHC on PEBP2/CBF occurs inside the nucleus. In addition, we could detect PEBP2beta/CBFbeta-SMMHC in the cytoplasmic membrane fraction as well. The function of this membrane-located PEBP2beta/CBFbeta-SMMHC, if any, appears to be unrelated to that of Runt protein homologs.

[Study on serum TSH and its response to TRH measured by a high sensitive immunoradiometric assay during and after pregnancy].

With a new highly sensitive immunoradiometric assay (IRMA), serum thyrotropin (TSH) concentrations were determined in 213 healthy pregnant women. Serum free thyroxine (FT4) and human chorionic gonadotropin (HCG) levels were also measured in the same individuals. The mean serum TSH value in the 1st trimester was 0.8 microU/ml and significantly lower than those of the other three periods and non-pregnant women. Seventeen of 77 (22.1%) and 2 of 128 (1.6%) subjects in the 1st and 3rd trimester, respectively, had an undetectable serum TSH value (less than 0.1 microU/ml) with normal or slightly elevated serum FT4 levels and these pregnant women had no clinical signs or symptoms of thyrotoxicosis. Significant positive correlations were found between serum FT4 and HCG in the 1st and 3rd trimester. In 13 subjects whose serum basal TSH values had been less than 0.1 microU/ml, the thyrotropin-releasing hormone (TRH) stimulation test was performed. Two of 4 subjects in the 1st trimester and one of 8 subjects in the 2nd trimester did not respond to TRH and their serum TSH values before TRH administration were less than 0.1 microU/ml. Although the exact mechanism of blunted TSH response to TRH is not clear, the feedback suppression of serum TSH by slightly elevated serum FT4 concentrations may occur early in pregnancy. However, in the 3rd trimester serum FT4 values fell below the reference range without an elevation of serum TSH. Other factors regulating the secretion of TSH during pregnancy can be postulated.