Computational study on the polymerization reaction of d-aminopeptidase for the synthesis of d-peptides

Almost all natural proteins are composed exclusively of l-amino acids, and this chirality influences their properties, functions, and selectivity. Proteases can recognize proteins composed of l-amino acids but display lower selectivity for their stereoisomers, d-amino acids. Taking this as an advantage, d-amino acids can be used to develop polypeptides or biobased materials with higher biostability. Chemoenzymatic peptide synthesis is a technique that uses proteases as biocatalysts to synthesize polypeptides, and d-stereospecific proteases can be used to synthesize polypeptides incorporating d-amino acids. However, engineered proteases with modified catalytic activities are required to allow the incorporation of d-amino acids with increased efficiency. To understand the stereospecificity presented by proteases and their involvement in polymerization reactions, we studied d-aminopeptidase. This enzyme displays the ability to efficiently synthesize poly d-alanine-based peptides under mild conditions. To elucidate the mechanisms involved in the unique specificity of d-aminopeptidase, we performed quantum mechanics/molecular mechanics simulations of its polymerization reaction and determined the energy barriers presented by the chiral substrates. The enzyme faces higher activation barriers for the acylation and aminolysis reactions with the l-stereoisomer than with the d-substrate (10.7 and 17.7 kcal mol⁻¹ higher, respectively). The simulation results suggest that changes in the interaction of the substrate with Asn155 influence the stereospecificity of the polymerization reaction.

We studied the molecular mechanism of d-aminopeptidase for the synthesis of polypeptides incorporating d-amino acids.     

Efficacy and safety of polaprezinc as a preventive drug for radiation-induced stomatitis

BACKGROUND: Radiation-induced stomatitis is one of the adverse effects of total body irradiation(TBI). We examined the usefulness of oral polaprezinc as a preventive drug for stomatitis. PATIENTS AND METHODS: The present study was conducted with 19 patients who were diagnosed with hematologic malignancy and who underwent TBI as pretreatment for bone marrow transplantation, peripheral blood stem transplantation, or cord blood stem cell transplantation. Eleven patients ingested the suspension of polaprezinc and 2% carmellose sodium (carboxymethylcellulose sodium: CMC) beginning the day before TBI(P-CMC group), while the other eight patients did not ingest P-CMC(control group). The severity of stomatitis was assessed in each group during a four-weeks period. RESULTS: Stomatitis (Grade: > or = 3) developed in one of 11 patients in the polaprezinc group and in 4 of 8 patients in the control group(P = 0.046). The times at which stomatitis development ranged between weeks 1 and 2 after the onset of TBI in the two groups. No adverse reaction owing to the ingestion of P-CMC was observed. CONCLUSIONS: These results suggested the efficacy and safety of polaprezinc as a preventive drug for radiation-induced stomatitis.     

Progenitor analysis of primitive erythropoiesis generated from in vitro culture of embryonic stem cells

OBJECTIVE: A variety of hematopoietic lineage cells have been produced from embryonic stem (ES) cells, but their differentiation processes have not been elucidated well, especially from the point of view of progenitor analysis. In this study, we utilized our coculture system, in which ES-derived Flk-1+ cells differentiated into TER-119+ primitive erythroid (EryP) cells on OP9 cells, and looked for progenitors in primitive erythropoiesis. MATERIALS AND METHODS: We studied the kinetics of TER-119+ erythroblast generation from Flk-1+ cells by monitoring the expression of TER-119, CD41, VE-cadherin, CD34, and c-kit antigens. Multicolor analysis was performed to detect CD41+TER-119+ cells and the stained cells were sorted to examine their morphology and EryP-producing potential in colony formation. RESULTS: Kinetic studies showed that the CD41+ population appeared early in the coculture and its expression pattern implied a role as an immediate progenitor of TER-119+ EryP cells. Multicolor analysis and colony-formation study supported this notion. Other progenitor markers such as VE-cadherin, CD34, and c-kit did not seem to define an immediate progenitor of EryP cells. One interesting observation is the detection of unique populations, CD41dim and CD41bright, detectable after 48 hours of the coculture. Majority of the CD41dim population progressed to the EryP lineage, whereas the CD41bright population seemingly advanced on a pathway distinct from the CD41dim population. CONCLUSIONS: CD41 expression was a useful marker to trace hematopoietic progenitors in ES-derived differentiation system. In particular, the CD41dim but not CD41bright population could serve as immediate precursors of EryP cells.     

Inhibitory effect of pitavastatin (NK-104) on the C-reactive-protein-induced interleukin-8 production in human aortic endothelial cells

Recent data have indicated that CRP (C-reactive protein) plays a role in atherosclerosis, in addition to being a marker for inflammatory diseases. IL-8 (interleukin-8), a CXC chemokine, is present in human coronary atheroma and promotes monocyte-endothelial cell adhesion. In the present study, we examined the effect of pitavastatin (NK-104), a synthetic statin (3-hydroxy-3-methylglutaryl CoA reductase inhibitor), on IL-8 production induced by CRP in human AoEC (aortic endothelial cells). We also investigated whether CRP can induce IL-8 production and if the activation of signalling pathways are functionally related. The concentrations of IL-8 in the media after stimulation with CRP were measured by ELISA, and the expression of IL-8 mRNA was assessed by Northern blot. The phosphorylation of MAPKs (mitogen-activated protein kinases) was determined by Western blot. The production of IL-8 induced by CRP (10 microg/ml) was enhanced significantly and was inhibited by pitavastatin. The expression of IL-8 mRNA was increased in a dose-dependent manner after stimulation with CRP (1-100 microg/ml), whereas expression of IL-8 mRNA induced by CRP (50 microg/ml) was significantly diminished by 5 microM pitavastatin. Furthermore, specific MAPK inhibitors (PD98059, SB203580 and SP600125) inhibited the expression of IL-8 mRNA induced by CRP (50 microg/ml). The phosphorylation of all three MAPKs [ERK (extracellular-signal-regulated kinase), p38 MAPK and JNK (c-Jun N-terminal kinase)] induced by CRP (10 microg/ml) was also significantly inhibited by pitavastatin. Our results suggest that CRP may play a role in atherosclerosis via IL-8 production and pitavastatin may prevent the progression of atherosclerosis not only by lowering plasma low-density lipoprotein cholesterol levels, but also by suppressing IL-8 production in endothelial cells through the inhibition of MAPK (ERK, p38 MAPK and JNK) pathways.     

Successful treatment of refractory cardiac tamponade due to rheumatoid arthritis using pericardial drainage

Rheumatoid pericarditis occurs in patients with rheumatoid arthritis (RA). However, cardiac tamponade due to rheumatoid pericarditis is rare; we describe a case of a 72-year-old man with a 6-year history of rheumatoid arthritis who developed rheumatoid pericarditis with recurrent cardiac tamponade. The patient experienced relapse of the cardiac tamponade despite treatment with pericardiocentesis. Therefore, the patient underwent surgical pericardial drainage. The patient was also subsequently treated with increasing doses of corticosteroid, methotrexate and leukocytapheresis. These treatments resulted in a successful outcome without any complication. This case suggests that in addition to immunosuppressive therapy, pericardial drainage should be considered in the treatment of life-threatening refractory cardiac tamponade caused by rheumatoid arthritis.     

Transforming growth factor-beta(1) augments granulocyte-macrophage colony-stimulating factor-induced proliferation of umbilical cord blood CD34(+) cells with an associated tyrosine phosphorylation of STAT5

OBJECTIVE: Several investigators have reported that transforming growth factor (TGF)-beta(1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) synergistically support cell proliferation. However, the mechanisms involved have not been elucidated. To clarify the mechanisms of the synergistic action of TGF-beta(1) and GM-CSF, we compared the activation states of STAT5 and mitogen-activated protein kinase in CD34(+) cells and in GM-CSF-dependent hematopoietic cell lines. MATERIALS AND METHODS: Human CD34(+) cells and GM-CSF-dependent cell lines (FKH-1, YNH-1, and M-07e) were stimulated with 1.25 ng/mL GM-CSF and/or 0.25 ng/mL TGF-beta(1), and 1.25 ng/mL GM-CSF and/or 0.25 ng/mL, 0.025 ng/mL TGF-beta(1), respectively, and cell proliferation was analyzed by [3H]thymidine uptake. Expression of signal transduction proteins and their phosphorylation states were determined by Western blotting. RESULTS: TGF-beta(1) synergistically enhanced the GM-CSF-augmented growth of CD34(+) cells and FKH-1 cells, but inhibited the growth of YNH-1 and M-07e cells. Tyrosine phosphorylation of STAT5 induced by GM-CSF was enhanced by stimulation with the combination of TGF-beta(1) and GM-CSF (TGF-beta(1)/GM-CSF) compared with that induced by GM-CSF alone in CD34(+) cells and FKH-1 cells. However, combinations of TGF-beta(1)/GM-CSF caused inhibition of GM-CSF-induced tyrosine phosphorylation in M-07e cells. No significant difference was observed in mitogen-activated protein kinase activation between CD34(+) cells and FKH-1 cells stimulated with GM-CSF/TGF-beta(1) or GM-CSF alone. CONCLUSIONS: Results suggest that TGF-beta(1) may augment GM-CSF-induced proliferation of CD34(+) cells in association with enhanced tyrosine phosphorylation of STAT5. Our data suggest a novel mechanism for the synergistic enhancement of cellular growth induced by the combination of TGF-beta(1) and GM-CSF.