The Role of Calcium in Differentiation of Human Adipose-Derived Stem Cells to Adipocytes

Differentiation process of mesenchymal stem cells (MSCs) into adipocyte is involved in obesity. Multiple factors such as Ca²⁺ play important roles in different stages of this process. Because of the complicated roles of Ca²⁺ in adipogenesis, the aim of present investigation was to study the influx and efflux of Ca²⁺ into and out of the cells during adipogenesis. Adipose-derived MSCs were used to differentiate into adipocytes. MSCs were exposed to 2.5 mM Ca²⁺ or 1.8 mM Ca²⁺ plus calcium ionophore, A23187, for 3 days. Lipid staining, triglycerides (TG) content, and glyceraldehyde phosphate dehydrogenase (GAPDH) activity were evaluated to confirm the efficiency of the differentiation. Gene expression of GLUT4, PPARγ2, RAR-α, and calreticulin, as well as the protein levels of GLUT4 and PPARγ2 were determined. Ca²⁺ and in particular Ca²⁺ plus A23187 significantly lowered the efficiency of differentiation accompanied by decrease in intracellular TG deposits, GAPDH activity and alleviation of gene, and protein levels of GLUT4 and PPARγ2. While calreticulin and RAR-α were remarkably upregulated in A23187 group. This study showed the inhibitory effects of calcium in adipogenesis. Additionally, it indicated the greater inhibitory effect of calreticulin and RAR-α in controlling adipogenesis by higher levels of calcium.     

Oritavancin exhibits dual mode of action to inhibit cell-wall biosynthesis in Staphylococcus aureus

Solid-state NMR measurements performed on intact whole cells of Staphylococcus aureus labeled selectively in vivo have established that des-N-methylleucyl oritavancin (which has antimicrobial activity) binds to the cell-wall peptidoglycan, even though removal of the terminal N-methylleucyl residue destroys the d-Ala-d-Ala binding pocket. By contrast, the des-N-methylleucyl form of vancomycin (which has no antimicrobial activity) does not bind to the cell wall. Solid-state NMR has also determined that oritavancin and vancomycin are comparable inhibitors of transglycosylation, but that oritavancin is a more potent inhibitor of transpeptidation. This combination of effects on cell-wall binding and biosynthesis is interpreted in terms of a recent proposal that oritavancin-like glycopeptides have two cell-wall binding sites: the well-known peptidoglycan d-Ala-d-Ala pentapeptide stem terminus and the pentaglycyl bridging segment. The resulting dual mode of action provides a structural framework for coordinated cell-wall assembly that accounts for the enhanced potency of oritavancin and oritavancin-like analogues against vancomycin-resistant organisms.     

Expression, purification, and characterization of His-tagged Staphylococcus xylosus lipase wild-type and its mutant Asp 290 Ala

The gene encoding the extracellular lipase of Staphylococcus xylosus (SXL) was cloned using PCR technique. The sequence corresponding to the mature lipase was subcloned in the pET-14b expression vector, with a strong T7 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. High level expression of the lipase by Escherichia coli BL21 (DE3) cells harbouring the lipase gene containing expression vector was observed upon induction with 0.4 mM IPTG at 37 degrees C. One-step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified His-tagged SXL was 1500 or 850 U/mg using tributyrin or olive oil emulsion as substrate, respectively. It has been proposed that the region near the residue Asp290 could be involved in the selection of the substrate. Therefore, we also mutated the residue Asp 290 by Ala using site-directed mutagenesis. The mutant SXL-D290A was overexpressed in E. coli BL21 (DE3) and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged SXL-D290A mutant was 1000 U/mg using either tributyrin or olive oil emulsion as substrate. A comparative study of the wild type (His(6)-SXL) and the mutant (His(6)-SXL-D290A) proteins was carried out. Our results confirmed that Asp290 is important for the chain length specificity and catalytic efficiency of the enzyme.     

Bisphosphonated fluoroquinolone esters as osteotropic prodrugs for the prevention of osteomyelitis

Osteomyelitis is a difficult to treat bacterial infection of the bone. Delivering antibacterial agents to the bone may overcome the difficulties in treating this illness by effectively concentrating the antibiotic at the site of infection and by limiting the toxicity that may result from systemic exposure to the large doses conventionally used. Using bisphosphonates as osteophilic functional groups, different forms of fluoroquinolone esters were synthesized and evaluated for their ability to bind bone and to release the parent antibacterial agent. Bisphosphonated glycolamide fluoroquinolone esters were found to present a profile consistent with effective and rapid bone binding and efficient release of the active drug moiety. They were assessed for their ability to prevent bone infection in vivo and were found to be effective when the free fluoroquinolones were not.     

Novel human mPGES-1 inhibitors identified through structure-based virtual screening

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase after exposure to pro-inflammatory stimuli and, therefore, represents a novel target for therapeutic treatment of acute and chronic inflammatory disorders. It is essential to identify mPGES-1 inhibitors with novel scaffolds as new leads or hits for the purpose of drug design and discovery that aim to develop the next-generation anti-inflammatory drugs. Herein we report novel mPGES-1 inhibitors identified through a combination of large-scale structure-based virtual screening, flexible docking, molecular dynamics simulations, binding free energy calculations, and in vitro assays on the actual inhibitory activity of the computationally selected compounds. The computational studies are based on our recently developed three-dimensional (3D) structural model of mPGES-1 in its open state. The combined computational and experimental studies have led to identification of new mPGES-1 inhibitors with new scaffolds. In particular, (Z)-5-benzylidene-2-iminothiazolidin-4-one is a promising novel scaffold for the further rational design and discovery of new mPGES-1 inhibitors. To our best knowledge, this is the first time a 3D structural model of the open state mPGES-1 is used in structure-based virtual screening of a large library of available compounds for the mPGES-1 inhibitor identification. The positive experimental results suggest that our recently modeled trimeric structure of mPGES-1 in its open state is ready for the structure-based drug design and discovery.     

Curvature effect on hemodynamic conditions at the inner bend of the carotid siphon and its relation to aneurysm formation

Although high-impact hemodynamic forces are thought to lead to cerebral aneurysmal change, little is known about the aneurysm formation on the inner aspect of vascular bends such as the internal carotid artery (ICA) siphon where wall shear stress (WSS) is expected to be low. This study evaluates the effect of vessel curvature and hemodynamics on aneurysm formation along the inner carotid siphon. Catheter 3D-rotational angiographic volumes of 35 ICA (10 aneurysms, 25 controls) were evaluated in 3D for radius of curvature and peak curvature of the siphon bend, followed by univariate statistical analysis. Computational fluid dynamic (CFD) simulations were performed on patient-derived models after aneurysm removal and on synthetic variants of increasing curvature. Peak focal siphon curvature was significantly higher in aneurysm bearing ICAs (0.36±0.045 vs. 0.30±0.048 mm⁻¹, p=0.003), with no difference in global radius of curvature (p=0.36). In CFD simulations, increasing parametric curvature tightness (from 5 to 3 mm radius) resulted in dramatic increase of WSS and WSS gradient magnitude (WSSG) on the inner wall of the bend. In patient-derived data, the location of aneurysms coincided with regions of low WSS (<4 Pa) flanked by high WSS and WSSG peaks. WSS peaks correlated with the aneurysm neck. In contrast, control siphon bends displayed low, almost constant, WSS and WSSG profiles with little spatial variation. High bend curvature induces dynamically fluctuating high proximal WSS and WSSG followed by regions of flow stasis and recirculation, leading to local conditions known to induce destructive vessel wall remodeling and aneurysmal initiation.     

Delivery of cytoplasmic proteins to autophagosomes

Autophagy represents a signaling-dependent regulated process that allows the degradation of some cellular proteins in autophagosomes, and plays a critical role in the management of cellular homeostasis under various stress conditions. In recent years, selective degradation of cytoplasmic proteins during stress has attracted considerable scientific interest. Here we examined the ability of resveratrol to induce autophagy in a variety of human cancer cell lines. We found that resveratrol-induced autophagy is accompanied by colocalization of proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) with the green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) in autophagosomes. In addition, we found that hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a previously shown PELP1-interacting protein, is co-recruited to autophagosomes in the presence of resveratrol. Although autophagy has been assumed to be a bulk and non-selective degradation process, in recent years, evidence of selective degradation of cytosolic proteins and organelles by autophagy is mounting. These observations suggest that the interaction of the target protein(s) with the delivery protein or proteins such as HRS facilitates the transport of certain cytoplasmic proteins to autophagosomes for their selective degradation, and thus, could influence the cytoplasmic as well as nuclear functions of nuclear receptor coregulators. Since PELP1 and, perhaps, other nuclear receptor coregulators are widely dysregulated in human cancers, these findings highlight the significance of the autophagic selective degradation of PELP1 following resveratrol (or other phytoestrogens) treatment in developing future strategies to use resveratrol under cancer prevention and therapeutic settings.     

Dual regulation of lysophosphatidic acid (LPA1) receptor signalling by Ral and GRK

Lysophosphatidic acid (LPA) is a major constituent of blood and is involved in a variety of physiological and pathophysiological processes. LPA signals via the ubiquitously expressed G protein-coupled receptors (GPCRs), LPA₁ and LPA₂ that are specific for LPA. However, in large, the molecular mechanisms that regulate the signalling of these receptors are unknown. We show that the small GTPase RalA associates with both LPA₁ and LPA₂ in human embryonic kidney (HEK 293) cells and that stimulation of LPA₁ receptors with LPA triggers the activation of RalA. While RalA was not found to play a role in the endocytosis of LPA receptors, we reveal that LPA₁ receptor stimulation promoted Ral-dependent phospholipase C activity. Furthermore, we found that GRK2 is required for the desensitization of LPA₁ and LPA₂ and have identified a novel interaction between RalA and GRK2, which is promoted by LPA₁ receptor activity. Taken together, these results establish RalA and GRK2 as key regulators of LPA receptor signalling and demonstrate for the first time that LPA₁ activity facilitates the formation of a novel protein complex between these two proteins.