Pressure-induced denaturation of monomer beta-lactoglobulin is partially irreversible: comparison of monomer form (highly acidic pH) with dimer form (neutral pH)

This study was conducted to assess the effect of high hydrostatic pressure on monomer beta-lactoglobulin (BLg) at acid pH by fluorescence spectroscopy under pressure and by circular dichroism (CD) and (1)H NMR spectroscopies after release of pressure. The intrinsic (tryptophan) fluorescence measurement and the study of 8-anilinonaphthalene-1-sulfonate (ANS) binding to BLg indicated that at pH 2.0 the recovery of center of spectral mass or ANS fluorescence was almost complete upon pressure release. No difference in (1)H NMR spectra was observed between pressurized and unpressurized BLg. In addition, NMR detection of the H/D exchange of aromatic protein indicated that the conformation of the vicinity of tryptophan residues could be refolded almost completely after release of pressure. These results seemingly confirm that the pressure-induced denaturation of BLg at pH 2.0 is reversible. However, cis-parinaric acid binding ability of pressurized BLg was largely lost, although its retinol binding ability was the same as its unpressurized one. Furthermore, CD spectra of the far-UV region and 2D NMR spectra evidently revealed the difference in the conformation of the molecule between unpressurized and pressurized BLg. These results are interpreted as an existence of partially fragile structure in the BLg molecule by high pressure.     

Leptospiral attachment to extracellular matrix of mouse fibroblast (L929) cells

The attachment of leptospires to the extracellular matrix (ECM) remaining after mouse fibroblast (L929) cells on coverslips had been solubilized with Triton X-100 was examined. Each highly virulent line of Leptospira interrogans serovar copenhageni, canicola and pomona attached to ECM more effectively than intermediately virulent and avirulent lines of the same strains, suggesting a correlation between virulence and attachment to ECM. Inhibition of the attachment of highly virulent copenhageni to ECM was found in the presence of the homologous immunoglobulin G Fab fragment.     

Determination of blood volume in dogs using an enriched stable isotope 50Cr

For the measurement of canine blood volume, various experimental conditions and techniques have been investigated using a non radioactive stable isotope 50Cr. On the basis of the results in this preliminary work, erythrocytes were labeled using 50Cr. Five micrograms of 50Cr per 1 ml of blood was added and incubated for 60 min. The canine erythrocytes were tagged using 50Cr and injected into vein of the same dogs. The blood samples collected at 60 min after the injection were irradiated by thermal neutron for 20 min at the reactor of the JAERI. Activities of 51Cr (the 50Cr concentration method) and 51Cr/59Fe radioactivity ratios (the 51Cr/59Fe ratio method) in the samples were measured. There was a very high correlation (r = 0.97, P less than 0.001) between the blood volumes calculated by the 50Cr concentration method and the 51Cr/59Fe ratio method. The latter method is less complicated than the former, because measurement of the sample weight and correction of thermal neutron flux are unnecessary. The mean blood volumes calculated by the ratio method and the Evans blue method were 89.8 +/- 6.8 ml/kg B.W. (mean +/- SD) and 98.9 +/- 10.6 ml/kg, respectively, showing a significant difference between them (P less than 0.05). However, these values are almost in accord by correction of venous blood PCV values with factor 0.97. As a detection limit of 50Cr was approximately 0.1 ng per 1 ml of blood in this system, this method has been concluded to be applicable to the measurement of the blood volume of cattle.