Distinct membrane mechanical properties of human mesenchymal stem cells determined using laser optical tweezers

The therapeutic efficacy of mesenchymal stem cells (MSCs) in tissue engineering and regenerative medicine is determined by their unique biological, mechanical, and physicochemical characteristics, which are yet to be fully explored. Cell membrane mechanics, for example, has been shown to critically influence MSC differentiation. In this study, we used laser optical tweezers to measure the membrane mechanics of human MSCs and terminally differentiated fibroblasts by extracting tethers from the outer cell membrane. The average tether lengths were 10.6+/-1.1 microm (hMSC) and 3.0+/-0.5 microm (fibroblasts). The tether extraction force did not increase during tether formation, which suggests existence of a membrane reservoir intended to buffer membrane tension fluctuations. Cytoskeleton disruption resulted in a fourfold tether length increase in fibroblasts but had no effect in hMSCs, indicating weak association between the cell membrane and hMSC actin cytoskeleton. Cholesterol depletion, known to decrease lipid bilayer stiffness, caused an increase in the tether length both in fibroblasts and hMSCs, as does the treatment of cells with DMSO. We postulate that whereas fibroblasts use both the membrane rigidity and membrane-cytoskeleton association to regulate their membrane reservoir, hMSC cytoskeleton has only a minor impact on stem cell membrane mechanics.     

Membrane binding and subcellular targeting of C2 domains

C2 domains are a ubiquitous structural module and many of them function in Ca2+ -dependent membrane binding and thereby serve as Ca2+ effectors for divergent Ca2+ -mediated cellular processes. Extensive structural, biochemical, biophysical, and cellular studies of C2 domains and host proteins in the past decade have shown that due to their structural diversity C2 domains have disparate Ca2+ sensitivity, lipid selectivity and membrane binding mechanisms. This review summarizes the basic structural and functional properties of C2 domains as well as recent findings on Ca2+ and membrane binding, lipid selectivity, and subcellular localization of C2 domains and their host proteins.     

Orientation and penetration depth of monolayer-bound p40phox-PX

X-ray reflectivity was used to study the interaction of the PX domain of p40(phox) protein (p40(phox)-PX) with a Langmuir monolayer of a mixture of SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), SOPS (1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine), and DPPtdIns(3)P (1,2-dipalmitoylphosphatidylinositol 3-phosphate) lipids supported on a buffered aqueous solution. The reflectivity is analyzed in terms of the known crystallographic structure of the p40(phox)-PX domain and a slab model that represents the lipid layer, yielding an electron density profile of the lipid layer and bound PX domains. This analysis determines the angular orientation and penetration depth of the p40(phox)-PX domain bound to the SOPC/SOPS/DPPtdIns(3)P monolayer. The best fit orientation is characterized by the following angles: theta = 30 +/- 10 degrees and phi = 140 +/- 30 degrees. These angles describe rotations, about axes in a coordinate system fixed to the domain, that are required to orient the domain with respect to the lipid layer at the interface. The protein penetrated into the lipid layer by 9 +/- 2 A, indicating that the protein penetrated into the headgroup region, but not deeply into the hydrocarbon region of the monolayer. In this analysis, polar Tyr(94) and hydrophobic Val(95) penetrated deepest into the lipid monolayer. The backbone of these residues was approximately 5 A above the headgroup-buffer interface, i.e., at the level of the SOPC/SOPS lipid phosphates. Positively charged Lys(92) and Lys(98) were also near the SOPC/SOPS lipid phosphates. This position of the protein allows for a favorable electrostatic contribution to binding.     

Granulocyte colony-stimulating factor treatment enhances the efficacy of cellular cardiomyoplasty with transplantation of embryonic stem cell-derived cardiomyocytes in infarcted myocardium

We tested the hypothesis that granulocyte colony-stimulating factor (G-CSF) administration would enhance the efficacy of cellular cardiomyoplasty with embryonic stem (ES) cell-derived cardiomyocytes in infarcted myocardium. Three weeks after myocardial infarction by cryoinjury, Sprague-Dawley rats were randomized to receive either an injection of medium, ES cell-derived cardiomyocyte transplantation, G-CSF administration, or a combination of G-CSF administration and ES cell-derived cardiomyocyte transplantation. Eight weeks after treatment, the cardiac tissue formation, neovascularization, and apoptotic activity in the infarct regions were evaluated by histology and immunohistochemistry. The left ventricular (LV) dimensions and function of the treated heart were evaluated by echocardiography. Transplanted ES cell-derived cardiomyocytes survived and participated in the myocardial regeneration in the infarcted heart. A combination of G-CSF treatment and ES cell-derived cardiomyocyte transplantation significantly promoted angiogenesis and reduced the infarct area and cell apoptosis in the infarcted myocardium compared with ES cell-derived cardiomyocyte transplantation alone. The combination therapy also attenuated LV dilation, as compared with ES cell-derived cardiomyocyte transplantation alone. G-CSF treatment can enhance the efficacy of cellular cardiomyoplasty by ES cell-derived cardiomyocyte transplantation to treat myocardial infarction.     

Genetic Polymorphism of Interferon-γ, Interferon-γ Receptor, and Interferon Regulatory Factor-1 Genes in Patients with Hepatitis B Virus Infection

The natural history of hepatitis B virus (HBV) infection is probably related to host immune factors. Interferon-γ (IFN-γ) plays significant roles in immune defense. This study was undertaken to investigate the association between HBV infection and single nucleotide polymorphisms (SNPs) of IFN-γ, IFN-γ receptor (IFNGR)-1 and 2, and interferon regulatory factor (IRF)-1 genes. Between March 2002 and December 2002, 614 Korean patients were enrolled in two different groups: an HBV clearance group (n = 201), who were hepatitis B surface antigen (HBsAg) negative with antibodies to HBsAg and hepatitis B core antigen, and an HBV persistence group (n = 413), who were repeatedly HBsAg positive. We assessed polymorphisms in the IFN-γ gene at position +874, in the IFNGR-1 gene at positions −56 and +95, in the IFNGR-2 gene at the second position of codon 64 (Gln64Arg), and in the IRF-1 gene promoter (−410, −388), and the genotype distributions of the HBV clearance and persistence groups were compared. On the basis of unconditional logistic regression analysis with adjustment for age and sex, no statistically significant association with susceptibility to persistent HBV infection was observed with the IFN-γ, IFNGR-1 and 2, and IRF-1 gene polymorphisms under the codominant, dominant, and recessive models.     

Molecular dynamics simulations of trehalose as a 'dynamic reducer' for solvent water molecules in the hydration shell

Systematic computational work for a series of 13 disaccharides was performed to provide an atomic-level insight of unique biochemical role of the alpha,alpha-(1-->1)-linked glucopyranoside dimer over the other glycosidically linked sugars. Superior osmotic and cryoprotective abilities of trehalose were explained on the basis of conformational and hydration characteristics of the trehalose molecule. Analyses of the hydration number and radial distribution function of solvent water molecules showed that there was very little hydration adjacent to the glycosidic oxygen of trehalose and that the dynamic conformation of trehalose was less flexible than any of the other sugars due to this anisotropic hydration. The remarkable conformational rigidity that allowed trehalose to act as a sugar template was required for stable interactions with hydrogen-bonded water molecules. Trehalose made an average of 2.8 long-lived hydrogen bonds per each MD step, which was much larger than the average of 2.1 for the other sugars. The stable hydrogen-bond network is derived from the formation of long-lived water bridges at the expense of decreasing the dynamics of the water molecules. Evidence for this dynamic reduction of water by trehalose was also established based on each of the lowest translational diffusion coefficients and the lowest intermolecular coulombic energy of the water molecules around trehalose. Overall results indicate that trehalose functions as a 'dynamic reducer' for solvent water molecules based on its anisotropic hydration and conformational rigidity, suggesting that macroscopic solvent properties could be modulated by changes in the type of glycosidic linkages in sugar molecules.     

Molecular dynamics simulations of a cyclic-beta-(1-->2) glucan containing an alpha-(1-->6) linkage as a 'molecular alleviator' for the macrocyclic conformational strain

The conformational preferences of a cyclic osmoregulated periplasmic glucan of Ralstonia solanacearum (OPGR), which is composed of 13 glucose units and linked entirely via beta-(1-->2) linkages excluding one alpha-(1-->6) linkage, were characterized by molecular dynamics simulations. Of the three force fields modified for carbohydrates that were applied to select a suitable one for the cyclic glucan, the carbohydrate solution force field (CSFF) was found to most accurately simulate the cyclic molecule. To determine the conformational characteristics of OPGR, we investigated the glycosidic dihedral angle distribution, fluctuation, and the potential energy of the glucan and constructed hypothetical cyclic (CYS13) and linear (LINEAR) glucans. All beta-(1-->2)-glycosidic linkages of OPGR adopted stable conformations, and the dihedral angles fluctuated in this energy region with some flexibility. However, despite the inherent flexibility of the alpha-(1-->6) linkage, the dihedral angles have no transition and are more rigid than that in a linear glucan. CYS13, which consists of only beta-(1-->2) linkages, is somewhat less flexible than other glycans, and one of its linkages adopts a higher energy conformation. In addition, the root-mean-square fluctuation of this linkage is lower than that of other linkages. Furthermore, the potential energy of glucans increases in the order of LINEAR, OPGR, and CYS13. These results provide evidence of the existence of conformational constraints in the cyclic glucan. The alpha-(1-->6)-glycosidic linkage can relieve this constraint more efficiently than the beta-(1-->2) linkage. The conformation of OPGR can reconcile the tendency for individual glycosidic bonds to adopt energetically favorable conformations with the requirement for closure of the macrocyclic ring by losing the inherent flexibility of the alpha-(1-->6)-glycosidic linkage.     

Heat shock protein 60 modified with O-linked N-acetylglucosamine is involved in pancreatic beta-cell death under hyperglycemic conditions

The objective of this study was to identify proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) in pancreatic beta-cells and to understand their roles in cell death under hyperglycemic conditions. Here we report that heat shock protein 60 (HSP60) is modified with O-GlcNAc. Levels of O-GlcNAcylated HSP60 increased twofold in response to hyperglycemic conditions. HSP60 is a chaperonin known to bind to Bax in the cytoplasm under normoglycemic conditions. Under hyperglycemic conditions, Bax detached from O-GlcNAcylated HSP60 and translocated to mitochondria. Hyperglycemic conditions were also associated with cytochrome c release, caspase-3 activation, and cell death, suggesting that elevated O-GlcNAcylation of HSP60 interferes with HSP60-Bax interactions, leading to pancreatic beta-cell death.