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

Role of Transcription Factor Modifications in the Pathogenesis of Insulin Resistance

Non-alcoholic fatty liver disease (NAFLD) is characterized by fat accumulation in the liver not due to alcohol abuse. NAFLD is accompanied by variety of symptoms related to metabolic syndrome. Although the metabolic link between NAFLD and insulin resistance is not fully understood, it is clear that NAFLD is one of the main cause of insulin resistance. NAFLD is shown to affect the functions of other organs, including pancreas, adipose tissue, muscle and inflammatory systems. Currently efforts are being made to understand molecular mechanism of interrelationship between NAFLD and insulin resistance at the transcriptional level with specific focus on post-translational modification (PTM) of transcription factors. PTM of transcription factors plays a key role in controlling numerous biological events, including cellular energy metabolism, cell-cycle progression, and organ development. Cell type- and tissue-specific reversible modifications include lysine acetylation, methylation, ubiquitination, and SUMOylation. Moreover, phosphorylation and O-GlcNAcylation on serine and threonine residues have been shown to affect protein stability, subcellular distribution, DNA-binding affinity, and transcriptional activity. PTMs of transcription factors involved in insulin-sensitive tissues confer specific adaptive mechanisms in response to internal or external stimuli. Our understanding of the interplay between these modifications and their effects on transcriptional regulation is growing. Here, we summarize the diverse roles of PTMs in insulin-sensitive tissues and their involvement in the pathogenesis of insulin resistance

Associations between Organochlorine Pesticides and Vitamin D Deficiency in the U.S. Population

Background: Recently low dose organochlorine (OC) pesticides have been strongly linked to various chronic diseases including diabetes and cardiovascular diseases. Both field and animal studies have suggested a possibility that persistent lipophilic chemicals like OC pesticides can cause vitamin D deficiency, but there have been no human studies of exposure to any chemical as a possible cause of vitamin D deficiency. This study was performed to examine if serum concentrations of OC pesticides were associated with serum concentrations of 25-hydroxyvitamin D (25(OH)D) in the U.S. general population. Methodology/Principal Findings: Cross-sectional associations of serum OC pesticides with serum 25(OH)D were investigated in 1,275 subjects aged $20 in the National Health and Nutrition Examination Survey(NHANES), 2003–2004. We selected 7 OC pesticides detectable in$80 % of participants. Among the 7 OC pesticides, p,p9-DDT (b = 20.022, P,0.01), p,p9-DDE (b = 20.018, P = 0.04), and b-hexachlorocyclohexane (b = 20.022, P = 0.02) showed significant inverse associations with serum 25(OH)D levels. When study subjects were stratified by age, race, and the presence of various chronic diseases, p,p9-DDT showed consistent inverse associations in all subgroups, although stronger associations tended to be observed among subjects with old age, white race, or chronic diseases. Conclusion/Significance: The current study suggests that the background exposure to some OC pesticides leads to vitamin D deficiency in human. Considering the importance of vitamin D deficiency in the development of chronic diseases

Interactions between Food Additive Silica Nanoparticles and Food Matrices

Nanoparticles (NPs) have been widely utilized in the food industry as additives with their beneficial characteristics, such as improving sensory property and processing suitability, enhancing functional and nutritional values, and extending shelf-life of foods. Silica is used as an anti-caking agent to improve flow property of powered ingredients and as a carrier for flavors or active compounds in food. Along with the rapid development of nanotechnology, the sizes of silica fall into nanoscale, thereby raising concerns about the potential toxicity of nano-sized silica materials. There have been a number of studies carried out to investigate possible adverse effects of NPs on the gastrointestinal tract. The interactions between NPs and surrounding food matrices should be also taken into account since the interactions can affect their bioavailability, efficacy, and toxicity. In the present study, we investigated the interactions between food additive silica NPs and food matrices, such as saccharides, proteins, lipids, and minerals. Quantitative analysis was performed to determine food component-NP corona using HPLC, fluorescence quenching, GC-MS, and ICP-AES. The results demonstrate that zeta potential and hydrodynamic radius of silica NPs changed in the presence of all food matrices, but their solubility was not affected. However, quantitative analysis on the interactions revealed that a small portion of food matrices interacted with silica NPs and the interactions were highly dependent on the type of food component. Moreover, minor nutrients could also affect the interactions, as evidenced by higher NP interaction with honey rather than with a simple sugar mixture containing an equivalent amount of fructose, glucose, sucrose, and maltose. These findings provide fundamental information to extend our understanding about the interactions between silica NPs and food components and to predict the interaction effect on the safety aspects of food-grade NPs

Effects of dodecacalcium heptaaluminate content on the setting time, compressive strength, alkalinity, and cytocompatibility of tricalcium silicate cement

Objective: This study aimed to investigate the effects of dodecacalcium hepta-aluminate (C12A7) content on some physicochemical properties and cytocompatibility of tricalcium silicate (C3S) cement using human dental pulp cells (hDPCs). Material and Methods: High purity C3S cement was manufactured by a solid phase method. C12A7 was mixed with the cement in proportions of 0, 5, 8, and 10 wt% (C12A7-0, -5, -8, and -10, respectively). Physicochemical properties including initial setting time, compressive strength, and alkalinity were evaluated. Cytocompatibility was assessed with cell viability tests and cell number counts. Statistical analysis was performed by using one-way analysis of variance (ANOVA) and Tukey’s test (p&lt;0.05). Results: The initial setting time of C3S-based cement was shorter in the presence of C12A7 (p&lt;0.05). After 1 day, C12A7-5 showed significantly higher compressive strength than the other groups (p&lt;0.05). After 7 days, the compressive strength of C12A7-5 was similar to that of C12A7-0, whereas other groups showed strength lower than C12A7-0. The pH values of all tested groups showed no significant differences after 1 day (p&gt;0.05). The C12A7-5 group showed similar cell viability to the C12A7-0 group (p&gt;0.05), while the other experimental groups showed lower values compared to C12A7-0 group (p&lt;0.05). The number of cells grown on the C12A7-5 specimen was higher than that on C12A7-8 and -10 (p&lt;0.05). Conclusions: The addition of C12A7 to C3S cement at a proportion of 5% resulted in rapid initial setting time and higher compressive strength with no adverse effects on cytocompatibility

Anti-inflammatory effect of essential oil extracted from Pinus densiflora (Sieb. et Zucc.) wood on RBL-2H3 cells

The aim of this study is to identify the active compounds of the essential oil extracted from the Pinus densiflora (Sieb. et Zucc.) wood using the hydrodistillation method and evaluate their anti-inflammatory activity. The chemical composition of the oil was identified by GC–MS analysis, and its anti-inflammatory activity was assessed by investigating its effect on the expression of interleukin-4 (IL-4), interleukin-13 (IL-13), and β-hexosaminidase in lipopolysaccharide (LPS)-stimulated RBL-2H3 cells. Treatment of the LPS-stimulated RBL-2H3 cells with the oil and its fractions downregulated the production of pro-inflammatory cytokines such as IL-4 and IL-13 and further attenuated the secretion of β-hexosaminidase out of the cells to a significant level. Among the five obtained fractions, fraction E exhibited the best anti-inflammatory activity, and its main constituent, longifolene, was considered as the active compound. Moreover, the inhibitory effect of longifolene on the expression levels of IL-4 and IL-13 and the β-hexosaminidase secretion was similar to that of the P. densiflora wood oil, indicating longifolene as the active constituent of the P. densiflora wood oil with immunosuppressive effects on inflammation

Omega-3 index and smoking in patients with acute ST-elevation myocardial infarction taking statins: a case-control study in Korea

<p>Abstract</p> <p>Background</p> <p>n-3 fatty acids and lifestyle also are closely related to risk of CVD. Most Koreans have higher fish consumption than people of Western populations. However, little is known about the recommended value of omega-3 index in Korean patients with acute ST-elevation myocardial infarction (STEMI) taking statins. Here, we tested the hypothesis that lower omega-3 fatty acids and/or smoking are associated with acute STEMI, even though patients with dyslipidemia who were taking statins and who attained their LDL-C goals.</p> <p>Methods</p> <p>We conducted a case-control study in which omega-3 fatty acids and lifestyle factors were determined in 24 consecutive Korean patients taking statins with angiographically confirmed acute STEMI and 68 healthy controls without acute STEMI. The omega-3 index was calculated by the sum of eicosapentaenoic acid and docosahexaenoic acid in erythrocyte membranes. Multivariable adjusted regression analysis was used to assess independent associations between acute STEMI, omega-3 index, and lifestyle factors after adjusting for age, sex, and body mass index (BMI).</p> <p>Results</p> <p>The mean age of total subjects was 59.9 years, and 57.6% of the subjects were male. The omega-3 index was significantly lower in cases (8.83%) than controls (11.13%; P < 0.001); however, total <it>trans</it>-fatty acids were not different between the two groups. The omega-3 index was inversely associated with odds for being a case (OR 0.16 (95% CI 0.03-1.14); P = 0.047), while smoking was positively associated with odds for being a case (OR 6.67 (95% CI 1.77-25.23); P = 0.005) after adjusting for all confounding variables.</p> <p>Conclusion</p> <p>This study shows that relative to controls, acute STEMI cases are more likely to be smokers and to have a lower omega-3 index, even though the cases were taking statins. An omega-3 index of at least 11% and abstinence from smoking are associated with cardioprotection for Koreans.</p

Enhanced cardiac expression of two isoforms of matrix metalloproteinase-2 in experimental diabetes mellitus.

BackgroundDiabetic cardiomyopathy (DM CMP) is defined as cardiomyocyte damage and ventricular dysfunction directly associated with diabetes independent of concomitant coronary artery disease or hypertension. Matrix metalloproteinases (MMPs), especially MMP-2, have been reported to underlie the pathogenesis of DM CMP by increasing extracellular collagen content.PurposeWe hypothesized that two discrete MMP-2 isoforms (full length MMP-2, FL-MMP-2; N-terminal truncated MMP-2, NTT-MMP-2) are induced by high glucose stimulation in vitro and in an experimental diabetic heart model.MethodsRat cardiomyoblasts (H9C2 cells) were examined to determine whether high glucose can induce the expression of the two isoforms of MMP-2. For the in vivo study, we used the streptozotocin-induced DM mouse heart model and age-matched controls. The changes of each MMP-2 isoform expression in the diabetic mice hearts were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical stains were conducted to identify the location and patterns of MMP-2 isoform expression. Echocardiography was performed to compare and analyze the changes in cardiac function induced by diabetes.ResultsQuantitative RT-PCR and immunofluorescence staining showed that the two MMP-2 isoforms were strongly induced by high glucose stimulation in H9C2 cells. Although no definite histologic features of diabetic cardiomyopathy were observed in diabetic mice hearts, left ventricular systolic dysfunction was determined by echocardiography. Quantitative RT-PCR and IHC staining showed this abnormal cardiac function was accompanied with the increases in the mRNA levels of the two isoforms of MMP-2 and related to intracellular localization.ConclusionTwo isoforms of MMP-2 were induced by high glucose stimulation in vitro and in a Type 1 DM mouse heart model. Further study is required to examine the role of these isoforms in DM CMP

Eikenella Corrodens Cervical Spinal Epidural Abscess Induced by a Fish Bone

Cervical spinal epidural abscess, caused by fish bone injury and a secondary infection by Eikenella Corrodens which is part of the normal flora, has not been reported. A 72-yr-old man came to the hospital with pain in his posterior neck and both shoulders for 2 months. He also was experiencing weakness on his right side for 3 days. A fish bone had been stuck in his throat for about 2 months. Neurological examination revealed right hemiparesis, hypesthesia on the left extremities and neck stiffness. Laboratory findings showed an elevated ESR/CRP and leukocytosis, and magnetic resonance imaging revealed a retropharyngeal abscess and cervical myelitis. The patient was treated with emergency surgical decompression and antibiotics. A fish bone was removed from the C3-C4 intervertebral disc space. In the culture of chocolate blood agar and 5% sheep blood agar plate, E. corrodens was detected as a causative organism