Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme.
The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme. (+info)
Separation and properties of two acetylacetoin reductases from Bacillus cereus YUF-4.
The separation and purification of two kinds of acetylacetoin reductases (AACRs) from Bacillus cereus YUF-4 were examined. NADPH-linked AACR (AACR I) and NADH-linked AACR (AACR II) were separated from each other by ammonium sulfate fractionation, DEAE-cellulose chromatography, and hydroxyapatite chromatography. The former was purified 3.4-fold with a yield of 10.0%, and the latter was purified 29-fold with a yield of 15.6%. The two enzymes differ from each other in some enzymic properties such as substrate specificity. (+info)
Coating titanium implants with bioglass and with hydroxyapatite. A comparative study in sheep.
This study compares the osteointegration of titanium implants coated with bioglass (Biovetro GSB formula) and with hydroxyapatite (HAP). Twenty-four bioglass-coated and 24 HAP-coated cylinders were implanted in the femoral diaphyses of sheep, and examined after 2, 4, 6, 8, 12, and 16 weeks. The HAP coating gave a stronger and earlier fixation to the bone than did bioglass. Bioglass formed a tissue interface which showed a macrophage reaction with little new bone formation activity. In contrast, HPA, showed intense new bone formation, with highly mineralised osseous trabeculae in the neighbourhood of the interface. (+info)
Development of calcium phosphate cement for rapid crystallization to apatite.
The purpose of this study was to develop an alpha-tricalcium phosphate (alpha-TCP) cement which transforms to hydroxyapatite (HAP) in a relatively short period. We used calcium and phosphate solutions as the liquid phase for the alpha-TCP cement. The alpha-TCP powder was first mixed with CaCl2 solution, and then mixed with NaH2PO4 or Na2HPO3 solution for a total powder/liquid ratio of 1.8. The setting time became shorter with the increase in the concentration of calcium and phosphate solutions, reaching 5 min, whereas the setting time was longer than 30 min when distilled water was used as the liquid phase. X-ray diffraction analysis revealed that the cement was mostly transformed to HAP within 24 h when kept in an incubator. We concluded that alpha-TCP should be mixed with calcium and phosphate solutions since this results in a moderate setting time and fast transformation to HAP even if the method of mixing becomes a little complex. (+info)
Myelopathy due to calcification of the cervical ligamenta flava: a report of two cases in West Indian patients.
Two cases of cervical myelopathy due to calcification of the ligamenta flava (CLF) are described for the first time in black patients from the French West Indies. A pre-operative CT scan differentiated the diagnosis from one of ossification of the ligamenta flava. Microanalysis on the operatively excised specimen in one patient revealed a mixture of calcium pyrophosphate dihydrate crystals and hydroxypatite crystals. Poor outcome in one patient contrasting with excellent recovery in the other one, who had undergone posterior decompressive laminectomy, emphasizes the importance of surgery in the management of CLF. (+info)
DNA relatedness of Leptospira strains isolated from beef cattle in Zimbabwe.
The DNA relatedness of 17 Leptospira strains isolated from beef cattle in Zimbabwe was determined using the hydroxyapatite method. Similarly to previously speciated African strains, all Zimbabwe isolates belonged to either Leptospira borgpetersenii or Leptospira kirschneri. All serovars within serogroups Pyrogenes (kwale, mombe and a strain closely related to serovar nigeria), Hebdomadis (marondera and mhou), Tarassovi (ngavi) and Sejroe (balcanica and hardjo) were L. borgpetersenii. L. kirschneri contained all strains in serovars of serogroups Icterohaemorrhagiae (zimbabwe), Australis (fugis), Bataviae (paidjan) and Pomona (a strain closely related to mozdok). The species designations of the Zimbabwe fugis and paidjan strains were different from those of the reference strains of these two serovars, both of which belong to Leptospira interrogans. (+info)
Elastin calcification and its prevention with aluminum chloride pretreatment.
Elastin, an abundant structural protein present in the arterial wall, is prone to calcification in a number of disease processes including porcine bioprosthetic heart valve calcification and atherosclerosis. The mechanisms of elastin calcification are not completely elucidated. In the present work, we demonstrated calcification of purified elastin in rat subdermal implants (Ca(2+) = 89.73 +/- 9.84 microgram/mg after 21 days versus control, unimplanted Ca(2+) = 0.16 +/- 0.04 microgram/mg). X-ray diffraction analysis along with resolution enhanced FTIR spectroscopy demonstrated the mineral phase to be a poorly crystalline hydroxyapatite. We investigated the time course of calcification, the effect of glutaraldehyde crosslinking on calcification, and mechanisms of inhibition of elastin calcification by pretreatment with aluminum chloride (AlCl(3)). Glutaraldehyde pretreatment did not affect calcification (Ca(2+) = 89.06 +/- 17.93 microgram/mg for glutaraldehyde crosslinked elastin versus Ca(2+) = 89.73 +/- 9.84 microgram/mg for uncrosslinked elastin). This may be explained by radioactive ((3)H) glutaraldehyde studies showing very low reactivity between glutaraldehyde and elastin. Our results further demonstrated that AlCl(3) pretreatment of elastin led to complete inhibition of elastin calcification using 21-day rat subdermal implants, irrespective of glutaraldehyde crosslinking (Ca(2+) = 0.73-2.15 microgram/mg for AlCl(3) pretreated elastin versus 89.73 +/- 9.84 for untreated elastin). The AlCl(3) pretreatment caused irreversible binding of aluminum ions to elastin, as assessed by atomic emission spectroscopy. Moreover, aluminum ion binding altered the spatial configuration of elastin as shown by circular dichroism (CD), Fourier transform infrared (FTIR), and (13)C nuclear magnetic resonance (NMR) spectroscopy studies, suggesting a net structural change including a reduction in the extent of beta sheet structures and an increase in coil-turn conformations. Thus, it is concluded that purified elastin calcifies in rat subdermal implants, and that the AlCl(3)-pretreated elastin completely resists calcification due to irreversible aluminum ion binding and subsequent structural alterations caused by AlCl(3). (+info)
Involvement of a cellular glycolytic enzyme, phosphoglycerate kinase, in Sendai virus transcription.
In vitro mRNA synthesis of Sendai virus is almost entirely dependent on the addition of cellular proteins (host factors). Previous studies indicated that the host factor activity from bovine brain was resolved into at least two complementary fractions, one of which may be tubulin. In this study, the host factor activity that stimulates the transcription in the presence of tubulin was further purified from bovine brain. This fraction was found to contain at least two complementary factors, and one of them was purified to a single polypeptide chain with an apparent M(r) of 46,000 (p46). From the amino acid sequence, biochemical, and immunological analyses, p46 was identified as a glycolytic enzyme, phosphoglycerate kinase (PGK). Purified native PGK from rabbit and yeast, and a recombinant human PGK substituted for p46. Although, as previously suggested, tubulin was involved in the transcription initiation complex formation by being integrated into the complex, p46 and its complementary factor had little effect on the complex formation. On the other hand, when p46 and the complementary factor were added to the RNA chain elongation reaction from the isolated initiation complex formed with tubulin, mRNA synthesis was dramatically stimulated. The enzymatic activity per se of PGK did not seem to be required for its activity. West-Western blot analysis showed that PGK could directly interact with tubulin. These data suggest that PGK stimulates Sendai virus mRNA synthesis at the elongation step, probably through its interaction with tubulin in the initiation complex. (+info)