Porcine bone grafts defatted by lipase: efficacy of defatting and assessment of cytocompatibility

Defatting is an important procedure for the preparation of bone grafts because lipids in bone grafts strongly influence the osteointegration. Lipases have been widely used in different fields. However, study on the application to defatting process for bone grafts preparation has never been found so far. In this study, bone samples were treated respectively by lipase, NaHCO₃/Na₂CO₃, acetone and deionized water. The lipids content of processed bone grafts was calculated in Soxhlet extractor method. Surface morphology of the bone grafts was observed under scanning electron microscope (SEM). DNA content of processed bone grafts was measured. Cytocompatibility was evaluated by co-culturing mouse preosteoblasts (MC3T3-E1) on defatted bone cubes. Proliferation rates of MC3T3-E1 were examined by cell counting kit-8 (CCK-8) assay. No statistically significant difference was found between lipids amount of bone processed by lipase (0.46 ± 0.16 %) and acetone (1.11 ± 0.13 %) (P > 0.05). Both of them were significantly lower than that in groups processed by Na₂CO₃/NaHCO₃ (3.46 ± 0.69 %) and deionized water (8.88 ± 0.18 %) (P = 0.000). Only cell debris were discovered over the surface of bone processed by lipase or acetone, while lipid droplets were observed on bone processed by Na₂CO₃/NaHCO₃ or water by SEM. The difference of DNA concentration between the bone processed by lipase (3.16 ± 0.81 ng/μl) and acetone (4.14 ± 0.40 ng/μl) is not statistically significant (P > 0.05). Both of them are significantly lower than that groups processed by Na₂CO₃/NaHCO₃ (5.22 ± 0.38 ng/μl) and water (7.88 ± 0.55 ng/μl) (P < 0.05). MC3T3-E1 cells maintained their characteristic spreading on the trabecular surfaces of bone processed by lipase. There were no statistically significant differences among absorbance of lipase, acetone groups in CCK-8 assay. The application of lipase to bone tissue defatting appears to be a very promising technique for bone grafts preparation.     

Plasmodium falciparum TryThrA antigen synthetic peptides block in vitro merozoite invasion to erythrocytes

Tryptophan-threonine-rich antigen (TryThrA) is a Plasmodium falciparum homologue of Plasmodium yoelii-infected erythrocyte membrane pypAg-1 antigen. pypAg-1 binds to the surface of uninfected mouse erythrocytes and has been used successfully in vaccine studies. The two antigens are characterized by an unusual tryptophan-rich domain, suggesting similar biological properties. Using synthetic peptides spanning the TryThrA sequence and human erythrocyte we have done binding assays to identify possible TryThrA functional regions. We describe four peptides outside the tryptophan-rich domain having high activity binding to normal human erythrocytes. The peptides termed HABPs (high activity binding peptides) are 30884 ((61)LKEKKKKVLEFFENLVLNKKY(80)) located at the N-terminal and 30901 ((401)RKSLEQQFGDNMDKMNKLKKY(420)), 30902 ((421)KKILKFFPLFNYKSDLESIM(440)) and 30913 ((641)DLESTAEQKAEKKGGKAKAKY(660)) located at the C-terminal. Studies with polyclonal goat antiserum against synthetic peptides chosen to represent the whole length of the protein showed that TryThrA has fluorescence pattern similar to PypAg-1 of P. yoelii. All HABPs inhibited merozoite in vitro invasion, suggesting that TryThrA protein may be participating in merozoite-erythrocyte interaction during invasion.     

The Schizosaccharomyces pombe replication inhibitor Spd1 regulates ribonucleotide reductase activity and dNTPs by binding to the large Cdc22 subunit

Ribonucleotide reductase (RNR) is an essential enzyme that provides the cell with a balanced supply of deoxyribonucleoside triphosphates for DNA replication and repair. Mutations that affect the regulation of RNR in yeast and mammalian cells can lead to genetic abnormalities and cell death. We have expressed and purified the components of the RNR system in fission yeast, the large subunit Cdc22p, the small subunit Suc22p, and the replication inhibitor Spd1p. It was proposed (Liu, C., Powell, K. A., Mundt, K., Wu, L., Carr, A. M., and Caspari, T. (2003) Genes Dev. 17, 1130-1140) that Spd1 is an RNR inhibitor, acting by anchoring the Suc22p inside the nucleus during G1 phase. Using in vitro assays with highly purified proteins we have demonstrated that Spd1 indeed is a very efficient inhibitor of fission yeast RNR, but acting on Cdc22p. Furthermore, biosensor technique showed that Spd1p binds to the Cdc22p with a KD of 2.4 microM, whereas the affinity to Suc22p is negligible. Therefore, Spd1p inhibits fission yeast RNR activity by interacting with the Cdc22p. Similar to the situation in budding yeast, logarithmically growing fission yeast increases the dNTP pools 2-fold after 3 h of incubation in the UV mimetic 4-nitroquinoline-N-oxide. This increase is smaller than the increase observed in budding yeast but of the same order as the dNTP pool increase when synchronous Schizosaccharomyces pombe cdc10 cells are going from G1 to S-phase.     

Urea, fluorofamide, and omeprazole treatments alter helicobacter colonization in the mouse gastric mucosa

BACKGROUND: Helicobacter pylori is a causative agent of gastric and duodenal ulcers and gastric cancer. Its urease enzyme allows survival in acid conditions and drives bacterial intracellular metabolism. We aimed to investigate the role of urease in determining the intragastric distribution of Helicobacter species in vivo. MATERIALS AND METHODS: The C57BL/6 mouse model of gastritis was used for infection with Helicobacter felis (CS1) or H. pylori (SS1). Urease-modulating compounds urea and/or fluorofamide (urease inhibitor) were administered to mice over 7 days. Concurrent gastric acid inhibition by omeprazole was also examined. Bacterial distribution in the antrum, body, antrum/body, and body/cardia transitional zones was graded "blindly" by histologic evaluation. Bacterial colony counts on corresponding tissue were also conducted. RESULTS: Urease inhibition by fluorofamide decreased H. pylori survival in most gastric regions (p < .05); however, there were no marked changes to H. felis colonization after this treatment. There was a consistent trend for decreased antral colonization, and an increase in antrum/body transitional zone and body colonization with excess 5% or 6% (w/v) urea treatment. Significant reductions of both Helicobacter species were observed with the co-treatment of urea and fluorofamide (p < .05). Collateral treatment with omeprazole did not alter H. pylori colonization patterns caused by urea/fluorofamide. CONCLUSIONS: Urease perturbations affect colonization patterns of Helicobacter species. Combined urea and fluorofamide treatment reduced the density of both Helicobacter species in our infection model.     

Quantification of isosorbide 5-mononitrate in human plasma by liquid chromatography-tandem mass spectrometry using atmospheric pressure photoionization

Isosorbide 5-mononitrate (5-ISMN) is an organic nitrate widely used for its vasodilating properties in the treatment of angina pectoris. In the present study, an efficient, sensitive, robust method was developed for the determination and quantification of isosorbide 5-mononitrate, in human plasma, by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), using photospray ionization. Isosorbide 5-mononitrate was extracted from 0.5 mL human plasma by liquid-liquid extraction (LLE). The method had a chromatographic run of 2.0 min using a C(8) analytical column (100 mm x 2.1 mm i.d.) and the linear calibration curve over the range was linear from 20 to 2000 ng mL(-1) (r(2)>0.995). The between-run precision, based on the relative standard deviation replicate quality controls, was 7.9% (60 ng mL(-1)), 5.2% (300 ng mL(-1)) and 7.0% (1800 ng mL(-1)). The between-run accuracy was 94.9%, 94.1% and 88.8% for the above-mentioned concentrations, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of isosorbide 5-mononitrate 40 mg.