Bioactive glass serves as a substrate for maintenance of phenotype of nucleus pulposus cells of the intervertebral disc.

The objective of the study was to investigate the capability of bioactive glass 45S5 to serve as a substrate for nucleus pulposus cells in vitro. Nucleus pulposus cells were isolated from adult rabbit discs and seeded onto bioactive glass. At selected time intervals, the cells and glass were evaluated. We found that the cells rapidly attached to the substrate, colonizing it within 12 h. By 21 days, they had formed a lawn of cells over the glass substrate. DNA measurements showed a progressive increase in cell number with time. The phenotype was maintained; the cells expressed aggrecan, and collagen type II and I, but were negative for collagen type X. CD44, a cell-surface glycoprotein that binds hyaluronate, was also expressed by these cells. Electron dispersive X-ray analysis and Fourier transform infrared spectroscopy revealed calcium phosphate-rich layer formation on the substrate surface. The results of this study suggest that nucleus pulposus cell proliferation may be an anchorage dependent event, and that the cells use the calcium phosphate-rich layer to facilitate cell adhesion, and subsequent proliferation. These findings point to the importance of the conditioned bioactive glass as a substrate for nucleus pulposus cells.

Rapid plasma clearance of albumin-acrolein adduct in rats.

Protein adducts are used as markers of chemical exposure. Determination of the clearance rate of these adducts from the blood circulation will provide the time frame for their measurement. Radioactive albumin was prepared biosynthetically by repeated intraperitoneal injections of L-[4,5-3H]lysine to a rat. After an affinity purification, an aliquot of this native [3H-lysine]albumin was adducted with 5 mM acrolein. Both the native albumin (A-treated group) and the albumin-acrolein adduct (AAA-treated group) were intravenously injected to separate groups of rats, and the clearance of radioactivity from the plasma was measured as a function of time. At the end of the experiment (33 h after the injection), radioactivity in the whole plasma, and in homogenates of liver, kidney and spleen and their trichloroacetic acid(TCA)-soluble and -insoluble fractions in both A- and AAA-treated groups, was measured. The results, at the initial 11 h after the injection, showed that the radioactivity was cleared from the circulating plasma more rapidly in the AAA-treated group (32% of the injected radioactivity remained) than the A-treated group (52%). At 33 h after the injection, 22% of the injected radioactivity remained in the plasma in the AAA-treated group as compared to 32% in the A-treated group. The whole homogenates of liver and kidney and their corresponding TCA-soluble fractions showed higher radioactivity in the AAA-treated group as compared to the A-treated group. However, the TCA-insoluble fractions from livers and kidneys of the AAA-treated group showed lower radioactivity as compared to the A-treated group. These results indicated that the albumin-acrolein adduct was removed more rapidly from the circulation than the native albumin, and degraded more rapidly by the liver and kidney. There was no preferential removal or degradation of the adducted albumin by the spleen.

Inactivation of plasma alpha 1-proteinase inhibitor by acrolein: adduct formation with lysine and histidine residues.

Four new peaks were observed upon amino acid analysis of alpha 1-proteinase inhibitor (alpha 1-PI), which was inactivated by acrolein under in vitro conditions. The first peak emerged just before ammonia, the second and third between ammonia and lysine, and the fourth between histidine and arginine. The new fourth peak was also observed when model compounds of lysine (N-acetyllysine or polylysine) were reacted with acrolein and subsequently processed for amino acid analysis. This new compound was purified by high-voltage paper electrophoresis and subjected to fast atom bombardment mass spectrometry, which showed a protonated molecule ion at m/z 203 [M + H]+ followed by m/z 186 [M + H+ - NH3]+. This compound was thus identified as 3-oxopropyllysine, a lysine adduct of acrolein. Similarly, when a model polypeptide of histidine, polyhistidine, was reacted with acrolein under the same conditions as alpha 1-PI, three new peaks (besides histidine) emerged from the column. Their elution times corresponded to the first three new peaks found in the hydrolysates of acrolein treated alpha 1-PI.

Human erythrocyte glutathione S-transferase: a possible marker of chemical exposure.

We have explored the possibility of using glutathione S-transferase (GST) as a biological marker of chemical exposure. All the model compounds tested in the present study (acrolein, propylene oxide, styrene oxide, ethylene dibromide and ethylene dichloride) showed a dose-dependent inactivation of erythrocyte GST in situ as well as the inhibition of purified erythrocyte GST.

Plausible mechanism of inactivation of plasma alpha 1-proteinase inhibitor by acrolein.

Evidence is presented to show that inactivation of alpha 1-proteinase inhibitor caused by acrolein depends upon the modification of lysine and histidine residues. Approximately 90% activity was lost when twenty three lysine and eight histidine residues were modified.

The inactivation of plasma alpha 1-proteinase inhibitor by nitrous acid.

Exposure of alpha 1-PI to nitrous acid resulted in a complete inactivation of either of its elastase or trypsin inhibitors activities. Amino acid analyses of the nitrous acid treated inhibitor revealed only losses of one tryphanyl and three lysyl residues. Reductive methylation of alpha 1-PI offered no protection against loss of activity by nitrous acid. Since no further loss of lysyl residues was observed upon exposure of fully active reductively methylated alpha 1-PI to nitrous acid, modification of one tryptophanyl residue appears to be responsible for the inhibitor's sensitivity to nitrous acid. Absorption spectral studies of the nitrous acid treated alpha 1-PI indicated that the tryptophanyl residue was modified to its N-nitroso derivative.

Proteinase inhibitory assays of serum using high-performance size exclusion chromatography.

A size exclusion column (Spherogel TSK-2000 SW) was utilized in a high-performance size exclusion chromatographic assay to determine the proteinase inhibitory capacity of human sera. Values from assays using this technique agreed well with the standard spectrophotometric inhibitory assays. Nanogram to milligram amounts of protein, namely, alpha 1-proteinase inhibitor, elastase, trypsin, chymotrypsin and their corresponding complexes with the inhibitor, were fractionated in less than 15 min. The nitrated or oxidized alpha 1-proteinase inhibitor was shown to retain its ability to form stable complexes with trypsin or chymotrypsin; however, they lost the inhibitory activity against elastase and instead they behaved as common protein substrates for this enzyme. The present chromatographic procedure was unable to detect any peptide released when the native inhibitor and any of the proteinases reacted to form a complex. Moreover, dissociation of the alpha 1-proteinase inhibitor--elastase complex in an alkaline pH did not result in the formation or release of any peptide.

Selective loss of elastase inhibitory activity of alpha 1-proteinase inhibitor upon chemical modification of its tyrosyl residues.

Nitration or acetylation of tyrosine residues of human plasma alpha 1-proteinase inhibitor (alpha 1-PI) with tetranitromethane or N-acetylimidazole resulted in a selective loss of its inhibitory activity. While the chymotrypsin- and trypsin-inhibitory activities were unaffected, all of the elastase inhibiting activity was lost. Comparative amino acid analyses of the native inhibitor and the chemically modified derivatives revealed no differences in the amino acid compositions with the exception that in the case of the tetranitromethane-treated material there were losses in the tyrosine content which could be accounted for by the formation of 3-nitrotyrosine. Nitration of alpha 1-PI.trypsin, alpha 1-PI.chymotrypsin, and alpha 1-PI.elastase complexes with excess tetranitromethane indicated that complex formation protected the binding site of the inhibitor for elastase, but not for trypsin or chymotrypsin. Quantitative estimation of the number of nitrotyrosine residues in the alpha 1-PI isolated from the complexes has shown that one tyrosine residue was protected from nitration. Competition studies have also indicated that the elastase inhibitory site is different from that of trypsin or chymotrypsin, although the possibility of overlapping sites could not be disregarded.

Purification and characterization of alpha 1-proteinase inhibitor from microsomal fraction of normal human liver.

A plasma alpha 1-proteinase inhibitor was purified, for the first time, from the microsomal fraction of normal human liver by combined procedures of deoxycholate extraction, (NH4)2-SO4 precipitation, gel filtration and immunoadsorbent column chromatographies. The final yield of the material was approx. 15 mg/300 g liver. The preparation was shown to be homogeneous according to polyacrylamide gel electrophoretic and immunological criteria. The estimated molecular weight of the glycoprotein as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was 53 000, a value comparable to that reported for plasma alpha 1-proteinase inhibitor. The liver material was, however, devoid of trypsin-, chymotrypsin- or elastase-inhibitory activity. The loss of activity could be due to deoxycholate treatment during the extraction procedure. SDS-polyacrylamide gel electrophoresis of reduced (beta-mercaptoethanol) and alkylated (iodoacetamide) preparation revealed no change in mobility suggesting the presence of only polypeptide chain. The amino acid and carbohydrate compositions of the microsomal material were in close agreement with values reported in the literatures for the corresponding glycoprotein purified from the plasma. The liver microsomal alpha 1-proteinase inhibitor has a single amino terminal residue, namely, glutamic acid, as determined by the dansyl procedure, and a single carboxyl terminal residue, lysine as shown by the carboxypeptidase digestion method. These terminal amino acids are the same as those found in alpha 1-proteinase inhibitor purified from normal human plasma.

Circular dichroism of chemically modified human plasma alpha1-antitrypsin. Interaction with porcine elastase.

Chemical modifications of human plasma alpha1-antitrypsin with reagents which modify lysyl residues (citraconic anhydride, acetic anhydride, formaldehyde and 2,4,6-trinitrobenzenesulfonic acid) and arginyl residued (1,2-cyclohexanedione) were examined with regard to their effect upon the elastase inhibitory capacity of the glycoprotein. 2,4,6-Trinitrobenzenesulfonic acid was employed to quantitate the remaining free amino groups (epsilon-NH2 groups of lysine) and the extent of modifications. Amino acid analysis was utilized in the same capacity for the guanidino groups of arginyl residues. The elastase inhibitory capacity of alpha1-antitrypsin was destroyed following trinitrophenylation, citraconylation and acetylation. Circular dichroism of the native and modified derivatives revealed major changes in conformation following trinitrophenylation and citraconylation while CD profiles of acetylated and reductively methylated derivatives differed from that of the native profile considerably less. Reductively methylated alpha1-antitrypsin retained its elastatse inhibitory capacity. The reaction of 1,2-cyclohexanedione with alpha1-antitrypsin did not effect in a loss in inhibitory capacity. Gel filtration studies of native and modified alpha1-antitrypsin on Sephadex G-100 demonstrated an increased molecular weight presumably through molecular aggregation, in the citraconylated and trinitrophenylated derivatives, but not in the cases of the other derivatives. Based upon these studies and previous investigations of our laboratory, it was concluded that (1) alpha1-antitrypsin is a lysyl inhibitor type (i.e., the reactive site is a Lys-X bond), (2) its interaction with elastase follows a pattern similar to trypsin and chymotrypsin, and (3) the positively charged epsilon-NH2 group of lysine is essential for the maintenance of elastase inhibitory capacity.

Metabolism of D-mannosamine in bovine thyroid gland slices.

1. The nature of the acid-soluble mannosamine intermediates was investigated in bovine thyroid gland slices after incubation with a tracer amount of [14C]mannosamine plus 10 mM carrier mannosamine. 2. These radioactive intermediates were separated by ion exchange, columns, characterized by paper chromatography and were identified as N-acetylmannosamine-6-P. The radioactivity associated with these acid-soluble compounds constituted about 17 percent of the added [14C]mannosamine. 3. [14C]Mannosamine was incorporated as sialic acid only into thyroglobulin.