The purification of alpha 1-antichymotrypsin from human serum using DNA-cellulose chromatography

By exploiting its capacity for binding to DNA, the protease inhibitor alpha 1-antichymotrypsin has been isolated from human serum by ammonium sulfate fractionation and successive chromatography on QAE-Sephadex, DNA-cellulose, and Sephacryl S-300. This experimental procedure compares favorably with existing methods for preparing alpha 1-antichymotrypsin in terms of overall yield and practical convenience. The purified alpha 1-antichymotrypsin was homogeneous as judged by electrophoretic and immunoelectrophoretic criteria. From its inhibition of the fluorimetric titration of chymotrypsin with 4-methylumbelliferyl-p-trimethylammonium cinnamate it was shown to combine with chymotrypsin in a 1:1 molar ratio and thus to retain its biological activity.     

Wnt signalling mediates the cross-talk between bone marrow derived pre-adipocytic and pre-osteoblastic cell populations

The mechanisms underlying the inverse relationship between osteogenic and adipogenic differentiation of bone marrow stromal cells (MSC) are not known in detail. We have previously established two cell lines from mouse bone marrow that are committed to either osteogenic (osteoblasts and chondrocytes) (mMSC^Bone) or adipogenic (mMSC^Adipo) lineage. To identify the molecular mechanism determining the lineage commitment, we compared the basal gene expression profile of mMSC^Bone versus mMSC^Adipo using Affymetrix GeneChip® MG430A 2.0 Array. Gene annotation analysis based on biological function revealed an over-representation of skeletal development genes in mMSC^Bone while genes related to lipid metabolism and immune response were highly expressed in mMSC^Adipo. In addition, there was a significant up-regulation of canonical Wnt signalling genes in mMSC^Bone compared to mMSC^Adipo (p < 0.006). Dual-luciferase assay and expression analysis of genes related to Wnt signalling demonstrated significant activation of Wnt signalling pathway in mMSC^Bone compared to mMSC^Adipo. Reduced Wnt activity in mMSC^Adipo was associated with increased expression of the Wnt inhibitor, secreted frizzled-related protein 1 (sFRP-1) at both mRNA and protein levels in mMSC^Adipo. Interestingly, conditioned medium (CM) collected from mMSC^Adipo (mMSC-CM^Adipo) inhibited osteoblast differentiation of mMSC, while depletion of sFRP-1 protein from mMSC-CM^Adipo abolished its inhibitory effect on osteoblast differentiation. Furthermore, treatment of mMSC with recombinant sFRP-1 resulted in a dose-dependent inhibition of osteoblast and stimulation of adipocyte differentiation. In conclusion, cross-talk exists between different populations of MSC in the bone marrow, and Wnt signalling functions as a molecular switch that determines the balance between osteoblastogenesis and adipogenesis.     

Coenzyme Q10, Copper,Zinc, and Lipid Peroxidation Levels in Serum of Patients with Chronic Obstructive Pulmonary Disease

Severity of chronic obstructive pulmonary disease (COPD) exacerbation is associated with increased level of copper (Cu), zinc (Zn), and lipid peroxidation (malodialdehyde, MDA). The aim of this study was to investigate the levels of lipid peroxidation, Coenzyme Q10 (CoQ10), Zn, and Cu in the COPD exacerbations. Forty-five patients with COPD acute exacerbation and 45 healthy smokers as control group were used in the study. Forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) were lower in exacerbation group than in control. C- reactive protein levels, white blood cell count, and sedimentation rate were significantly (p < 0.001) higher in patients than in control. CoQ10 level and Cu/Zn ratio was significantly (p < 0.05) lower in patients than in control, although MDA, Cu, and Zn levels were significantly (p < 0.05) higher in patients than in control. Negative correlations were found among MDA, Cu, Zn, FEV1, and FVC values in exacerbation and control subjects (p < 0.05). In conclusion, we observed that oxidative stress in the exacerbation period of COPD patients was increased. The decrease in CoQ10 level and Cu/Zn ratio and elevation in Cu and Zn levels observed in the patients probably result from the defense response of organism and are mediated by inflammatory-like substances.     

Crosstalk of EGF-directed MAPK signalling pathways and its potential role on EGF-induced cell proliferation and COX-2 expression in human mesenchymal stem cells

Epidermal growth factor (EGF) promotes proliferation in human mesenchymal stem cells (hMSCs) during in vitro propagation. In this study, we investigated the effects of PI3K/AKT, ERK1/2, P38 and JNK on EGF signalling in hMSCs. The effects of EGF on MAPKs and PI3K/AKT crosstalk were investigated by immunoblotting; cyclooxygenase-2 (COX-2) expression was studied by real-time RT-PCR; and cell proliferation was evaluated by methylthiazolyl tetrazolium bromide assay. Our results showed that EGF immediately activated all four pathways, induced proliferation and increased COX-2 expression. Interestingly, inhibition of PI3K/AKT-enhanced EGF-stimulated ERK1/2 activity, and inhibition of ERK1/2 and JNK reduced AKT phosphorylation. Furthermore, EGF-induced proliferation as well as EGF-augmented COX2 expression was hindered by ERK1/2 and p38 inhibitors. The results of this study provide evidences to be used in extended proliferation of hMSCs for cell therapy.     

Analysis of oxidized and chlorinated lipids by mass spectrometry and relevance to signalling

Oxidized and chlorinated phospholipids are generated under inflammatory conditions and are increasingly understood to play important roles in diseases involving oxidative stress. MS is a sensitive and informative technique for monitoring phospholipid oxidation that can provide structural information and simultaneously detect a wide variety of oxidation products, including chain-shortened and -chlorinated phospholipids. MSn technologies involve fragmentation of the compounds to yield diagnostic fragment ions and thus assist in identification. Advanced methods such as neutral loss and precursor ion scanning can facilitate the analysis of specific oxidation products in complex biological samples. This is essential for determining the contributions of different phospholipid oxidation products in disease. While many pro-inflammatory signalling effects of oxPLs (oxidized phospholipids) have been reported, it has more recently become clear that they can also have anti-inflammatory effects in conditions such as infection and endotoxaemia. In contrast with free radical-generated oxPLs, the signalling effects of chlorinated lipids are much less well understood, but they appear to demonstrate mainly pro-inflammatory effects. Specific analysis of oxidized and chlorinated lipids and the determination of their molecular effects are crucial to understanding their role in disease pathology.     

In silico analysis of single nucleotide polymorphism (SNPs) in human β-globin gene

Single amino acid substitutions in the globin chain are the most common forms of genetic variations that produce hemoglobinopathies--the most widespread inherited disorders worldwide. Several hemoglobinopathies result from homozygosity or compound heterozygosity to beta-globin (HBB) gene mutations, such as that producing sickle cell hemoglobin (HbS), HbC, HbD and HbE. Several of these mutations are deleterious and result in moderate to severe hemolytic anemia, with associated complications, requiring lifelong care and management. Even though many hemoglobinopathies result from single amino acid changes producing similar structural abnormalities, there are functional differences in the generated variants. Using in silico methods, we examined the genetic variations that can alter the expression and function of the HBB gene. Using a sequence homology-based Sorting Intolerant from Tolerant (SIFT) server we have searched for the SNPs, which showed that 200 (80%) non-synonymous polymorphism were found to be deleterious. The structure-based method via PolyPhen server indicated that 135 (40%) non-synonymous polymorphism may modify protein function and structure. The Pupa Suite software showed that the SNPs will have a phenotypic consequence on the structure and function of the altered protein. Structure analysis was performed on the key mutations that occur in the native protein coded by the HBB gene that causes hemoglobinopathies such as: HbC (E→K), HbD (E→Q), HbE (E→K) and HbS (E→V). Atomic Non-Local Environment Assessment (ANOLEA), Yet Another Scientific Artificial Reality Application (YASARA), CHARMM-GUI webserver for macromolecular dynamics and mechanics, and Normal Mode Analysis, Deformation and Refinement (NOMAD-Ref) of Gromacs server were used to perform molecular dynamics simulations and energy minimization calculations on β-Chain residue of the HBB gene before and after mutation. Furthermore, in the native and altered protein models, amino acid residues were determined and secondary structures were observed for solvent accessibility to confirm the protein stability. The functional study in this investigation may be a good model for additional future studies.     

Association of Obesity and Hypertension With Left Ventricular Geometry and Function in Children and Adolescents

Obesity, especially when complicated with hypertension, is associated with structural and functional cardiac changes. Recent studies have focused on the prognostic impact of the type of left ventricular (LV) geometric remodeling. This study looked at the prevalence and clinical correlates of LV geometric patterns and their relation to cardiac function in a sample of predominantly African‐American (AA) youth. Echocardiographic data was collected on 213 obese (BMI of 36.53 ± 0.53 kg/m²) and 130 normal‐weight subjects (BMI of 19.73 ± 0.21 kg/m²). The obese subjects had significantly higher LV mass index (LVMI; 49.6 ± 0.9 vs. 46.0 ± 1.0 g/m^2.7, P = 0.01), relative wall thickness (RWT; 0.45 ± 0.00 vs. 0.40 ± 0.00, P < 0.001), left atrial (LA) index (33.2 ± 0.7 vs. 23.5 ± 0.6 ml/m, P < 0.001), more abnormal diastolic function by tissue Doppler E/Ea septal (7.5 ± 0.14 vs. 6.5 ± 0.12 ms, P < 0.001), E/Ea lateral (5.7 ± 0.12 vs. 4.8 ± 0.1 ms, P < 0.001), myocardial performance index (MPI; 0.43 ± 0.00 vs. 0.38 ± 0.00, P < 0.001), and Doppler mitral EA ratio (2.0 ± 0.04 vs. 2.4 ± 0.07, P < 0.001) but similar systolic function. Concentric remodeling (CR) was the most prevalent pattern noted in the obese group and concentric hypertrophy (CH) in the obese and hypertensive group. Obesity, hypertension, and CH were independent predictor of diastolic dysfunction. Systolic (SBP) and diastolic blood pressures (DBP) were the prime mediators for CH whereas obesity and diastolic blood pressure were predictors of CR. No significant association was observed between the geometric patterns and systolic function. Tracking LV hypertrophy (LVH) status and geometric adaptations in obesity may be prognostic tools for assessing cardiac risk and therapeutic end points with weight loss.     

Centaurea bruguierana inhibits cell proliferation,causes cell cycle arrest,and induces apoptosis in human MCF-7 breast carcinoma cells

Molecular Biology Reports - Centaurea bruguierana, of the Asteraceae family, has a long history of use in traditional medicines for the treatment of various ailments. However, the anticancer...     

Optimization of caulogenesis in Populus nigra under lead (Pb) stress via response surface methodology (RSM) and desirability function analysis

Plant Cell, Tissue and Organ Culture (PCTOC) - Polyethylene glycol (PEG)-mediated transient expression system in plant protoplasts has been widely used in a variety of plants for gene function...