Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

<p>Abstract</p> <p>Background</p> <p>Caspase-3, a principal apoptotic effector that cleaves the majority of cellular substrates, is an important medicinal target for the treatment of cancers and neurodegenerative diseases. Large amounts of the protein are required for drug discovery research. However, previous efforts to express the full-length caspase-3 gene in <it>E. coli </it>have been unsuccessful.</p> <p>Results</p> <p>Overproducers of thrombin-activatable full-length caspase-3 precursors were prepared by engineering the auto-activation sites of caspase-3 precursor into a sequence susceptible to thrombin hydrolysis. The engineered precursors were highly expressed as soluble proteins in <it>E. coli </it>and easily purified by affinity chromatography, to levels of 10–15 mg from 1 L of <it>E. coli </it>culture, and readily activated by thrombin digestion. Kinetic evaluation disclosed that thrombin digestion enhanced catalytic activity (<it>k</it>_cat/<it>K</it>_<it>M</it>) of the precursor proteins by two orders of magnitude.</p> <p>Conclusion</p> <p>A novel method for a large-scale preparation of active caspase-3 was developed by a strategic engineering to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in <it>E. coli</it>. The precursor protein was easily purified and activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3 in high yields.</p

Culture supernatant of adipose stem cells can ameliorate allergic airway inflammation via recruitment of CD4+CD25+Foxp3 T cells

SDS-PAGE of supernatant after ASC cultivation. Comparison of protein composition of con sup (concentrated medium for ASCs cultivation) and ASC sup (concentrated culture supernatant after ASC cultivation for 3 days) using SDS-PAGE. Thirty micrograms of each sample was loaded into an SDS-PAGE gel. After electrophoresis, the gel was stained by Coomassie Blue (M molecular marker, arrow indicated extra proteins compared to control). (PPT 370 kb

Hu.4-1BB-Fc fusion protein inhibits allergic inflammation and airway hyperresponsiveness in a murine model of asthma

Purpose4-1BB (CD 137) is a costimulatory molecule expressed on activated T-cells. Repression by 4-1BB is thought to attenuate Th2-mediated allergic reactions. The aim of this study was to investigate the effect of 4-1BB on allergic airway inflammation in a murine asthma model.MethodsBALB/c mice were sensitized to and challenged with ovalbumin (OVA). Hu.4-1BB-Fc was administered 1 day before the first OVA sensitization or 1 day after the second OVA sensitization. Following antigen challenge, airway responsiveness to methacholine was assessed and bronchoalveolar lavage (BAL) fluid was analyzed. Total immunoglobulin (Ig) E, OVA-specific IgE, IgG1, and IgG2a levels in sera were measured by enzyme-linked immunosorbent assay. Lung pathology was also evaluated.ResultsIn mice treated with Hu.4-1BB-Fc before the first OVA sensitization, there was a marked decrease in airway hyperresponsiveness, total cell count, and eosinophil count in the BAL fluid. In addition, Hu.4-1BB-Fc treatment decreased serum OVA-specific IgG1 levels and increased serum IgG2a level significantly compared with the corresponding levels in mice sensitized to and challenged with OVA. Hu.4-1BB-Fc-treated mice also showed suppressed peribronchial and perivascular inflammatory cell infiltration. In contrast, treatment with Hu.4-1BB-Fc 1 day after sensitization had no effect on airway hyperresponsiveness and showed less suppression of inflammation in lung tissue.ConclusionAdministration of Hu.4-1BB-Fc can attenuate airway inflammation and hyperreactivity in a mouse model of allergic airway inflammation. In addition, administration before sensitization may be more effective. These findings suggest that 4-1BB may be a useful therapeutic molecule against asthma

Chemokine Lkn-1/CCL15 enhances matrix metalloproteinase-9 release from human macrophages and macrophage-derived foam cells

Atherosclerosis is characterized by a chronic inflammatory disease, and chemokines play an important role in both initiation and progression of atherosclerosis development. Leukotactin-1 (Lkn-1/CCL15), a new member of the human CC chemokine family, is a potent chemoattractant for leukocytes. Our previous study has demonstrated that Lkn-1/CCL15 plays a role in the initiation of atherosclerosis, however, little is currently known whether Lkn-1/CCL15 is associated with the progression of atherosclerosis. Matrix metalloproteinases (MMPs) in human coronary atherosclerotic lesions play a crucial role in the progression of atherosclerosis by altering the vulnerability of plaque rupture. In the present study, we examined whether Lkn-1/CCL15 modulates MMP-9 release, which is a prevalent form expressed by activated macrophages and foam cells. Human THP-1 monocytic cells and/or human peripheral blood monocytes (PBMC) were treated with phorbol myristate acetate to induce their differentiation into macrophages. Foam cells were prepared by the treatment of THP-1 macrophages with human oxidized LDL. The macrophages and foam cells were treated with Lkn-1/CCL15, and the levels of MMP-9 release were measured by Gelatin Zymography. Lkn-1/CCL15 significantly enhanced the levels of MMP-9 protein secretion from THP-1 monocytic cells-derived macrophages, human PBMC-derived macrophages, as well as macrophage-derived foam cell in a dose dependent manner. Our data suggest that the action of Lkn-1/CCL15 on macrophages and foam cells to release MMP-9 may contribute to plaque destabilization in the progression of atherosclerosis

Clinical implications in laboratory parameter values in acute Kawasaki disease for early diagnosis and proper treatment

PurposeThis study aimed to analyse laboratory values according to fever duration, and evaluate the relationship across these values during the acute phase of Kawasaki disease (KD) to aid in the early diagnosis for early-presenting KD and incomplete KD patients.MethodsClinical and laboratory data of patients with KD (n=615) were evaluated according to duration of fever at presentation, and were compared between patients with and without coronary artery lesions (CALs). For evaluation of the relationships across laboratory indices, patients with a fever duration of 5 days or 6 days were used (n=204).ResultsThe mean fever duration was 6.6±2.3 days, and the proportions of patients with CALs was 19.3% (n=114). C-reactive proteins (CRPs) and neutrophil differential values were highest and hemoglobin, albumin, and lymphocyte differential values were lowest in the 6-day group. Patients with CALs had longer total fever duration, higher CRP and neutrophil differential values and lower hemoglobin and albumin values compared to patients without CALs. CRP, albumin, neutrophil differential, and hemoglobin values at the peak inflammation stage of KD showed positive or negative correlations each other.ConclusionThe severity of systemic inflammation in KD was reflected in the laboratory values including CRP, neutrophil differential, albumin, and hemoglobin. Observing changes in these laboratory parameters by repeated examinations prior to the peak of inflammation in acute KD may aid in diagnosis of early-presenting KD patients