Crystal structure determinations of oxidized and reduced pseudoazurins from Achromobacter cycloclastes. Concerted movement of copper site in redox forms with the rearrangement of hydrogen bond at a remote histidine.

The crystal structures of oxidized and reduced pseudoazurins from a denitrifying bacterium, Achromobacter cycloclastes IAM1013, have been determined at 1.35- and 1.6-A resolutions, respectively. The copper site in the oxidized state exhibits a distorted tetrahedral structure like those of other pseudoazurins. However, not only a small change of the copper geometry, but concerted peptide bond flips are identified. The imidazole ring of remote His6 has a hydrogen bonding distance of 2.73 A between N-delta1(His6) and O-gamma1(Thr36) in the oxidized protein. When the protein is reduced at pH 6.0, the imidazole ring rotates by 30.3 degrees and moves 1.00 A away from the position of the oxidized state. A new hydrogen bond between N-epsilon2(His6) and O-epsilon1(Glu4) is formed with a distance of 3.03 A, while the hydrogen bond between N-delta1(His6)-O-gamma1(Thr36) is maintained with an interatomic distance of 2.81 A. A concomitant peptide bond flip of main chain between Ile34 and Thr36 occurs.     

Conformational study of 13C-enriched fibroin in the solid state, using the cross polarization nuclear magnetic resonance method

Silk fibroin with the alanyl carboxyl carbon enriched with 13C was obtained by giving a diet containing 13C-enriched alanine to the larvae of Bombyx mori and Antheraea pernyi at the fifth instar. Sericin-free fibroin fibers were prepared from cocoons, and gut was made from the liquid silk in the gland. The final 13C content was about 13%. Cross polarization/magic angle sample spinning spectra at 25 MHz and 75 MHz were measured for each sample at different orientations. Spectra were simulated using the principal values and orientations of the shielding tensor in the alanine crystal. The results indicate that the beta-structure of the fibroin may be a little more flattened than the typical pleated sheet beta-structure.     

Repetitive sequences in class-switch recombination regions of immunoglobulin heavy chain genes

Immunoglobulin class switch involves a unique recombination event that takes place at the region 5′ to each heavy chain constant region gene during B lymphocyte differentiation. Such regions that are responsible for the class-switch recombination are defined as S regions (Kataoka et al., Proc. Natl. Acad. Sci. USA 77, 919, 1980). We have cloned a rearranged γ2b gene from a mouse myeloma (MPC11) and compared its structure with the germ line counterparts. The rearranged γ2b gene contained the 5′ flanking region of the γ3 gene (S_γ3 region) which are linked to the 5′ flanking region of the γ2b gene (S_γ2b region). We have determined nucleotide sequences surrounding the recombination site of the rearranged and germ line γ2b genes, which include the S_γ2b and S_γ3 regions. Both _γ2b and S_γ3 regions comprise tandem repetition of conserved units of 49 bp. Similar 49 bp repeating units are also found in the previously determined sequence of the S_γ1 region in which class-switch recombination took place in MC101 myeloma. The nucleotide sequences of the S_γ1, S_γ2b and S_γ3 repeating units share significant homology with each other. The Sμ region, partial nucleotide sequence of which was previously determined, contains abundant short sequences such as AGCT, TGGG and AGCTGGGG which are shared in common by repeating sequences in S_γ regions. These results suggest that the recombination responsible for class switch from μ to γ or from a γ to another γ, may be facilitated directly or indirectly by homology of repeating sequences in S regions.     

Purification of a fibrolast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4

Abstract A factor showing inhibitory activity against human gingival fibrolasts was extracted from the cytosol fraction of Actinobacillus actinomycetemcomitans Y4. The activity markedly inhibited the proliferation of human gingival fibrolasts, but had no effect on cell viability or gross morphology. No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101. The extract from A. actinomycetemcomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa. The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts.     

Lipids and cell fusion in vitro: effect of amphotericin B.

Fusion rate of Cl-1D cells mediated by Sendai virus was decreased by preincubating the cells or the virus in the presence of lysolecithin. However, a significant increase occurred when the cells were preincubated in the presence of amphotericin B. This increase was suppressed by exogenous cholesterol.