A biomechanical and spectroscopic study of bone from rats with selenium deficiency and toxicity

Selenium, being an essential mineral in the mammalian diet, is important in providing protection against oxidative damage. Numerous in vitro studies of selenium compounds reveal a very high correlation between catalytic activity of selenium compounds and toxicity. The present study was designed to investigate the effects of dietary selenium on the biomechanical properties of bone. New born rats of both sexes were fed with either a control, or a selenium- and vitamin E-deficient, or a selenium-excess and vitamin E-adequate diet. We obtained the stiffness (modulus of elasticity) of bones (femur and tibia) by tensile test for all groups considered. Both the deficient and the excess groups have decreased biomechanical strength with respect to the control group. To support our biomechanical results for both experimental groups, X-ray diffraction analysis and FTIR spectroscopic study were performed on the femurs and tibiae. The X-ray diffraction analysis showed that the intensities of the peak observed at around 2°=31.820, in the control femur and tibia are stronger than the intensities of the corresponding peak of two experimental groups. In FTIR spectroscopy, the disappearance and/or reduction of the intensities of some carbonate bands in the two experimental groups indicate that there is a decrease in crystallinity and mineral contents which, together with X-ray diffraction analysis, correlate very well with the biomechanical data.     

Differentiation of myoblasts is accelerated in culture in a magnetic field

Summary We developed a new cell stimulation method in which magnetic microparticles (MPs) were introduced into the cytoplasm of cultured myoblasts and the cells were cultured in a magnetic field. The differentiation of myoblasts was examined from the viewpoint of their morphology and myogenin production. After exposure to the magnetic field, the cells containing MPs became larger and were elongated along the axis of the magnetic poles. Myogenin, a muscle-specific regulatory factor involved in controlling myogenesis, was formed earlier, and myotubes were seen earlier and more frequently in this group of myoblasts than in the other groups (cells alone without magnetic field, cells containing MPs but without magnetic field, and cells alone with magnetic field). Moreover, we succeeded in differentiation of early muscle cells with striated myofibrils in culture at 0.05 T. The precisely quantitative and stable stimulus induced by a magnetic field developed in the present study offers a new approach to elucidate the entire process of myoblast differentiation into myotubes.     

Differences in Elasticity of Vinculin-Deficient F9 Cells Measured by Magnetometry and Atomic Force Microscopy

We have investigated a mouse F9 embryonic carcinoma cell line, in which both vinculin genes were inactivated by homologous recombination, that exhibits defective adhesion and spreading [Collet al.(1995)Proc. Natl. Acad. Sci. USA92, 9161–9165]. Using a magnetometer and RGD-coated magnetic microbeads, we measured the local effect of loss and replacement of vinculin on mechanical force transfer across integrins. Vinculin-deficient F9Vin(−/−) cells showed a 21% difference in relative stiffness compared to wild-type cells. This was restored to near wild-type levels after transfection and constitutive expression of increasing amounts of vinculin into F9Vin(−/−) cells. In contrast, the transfection of vinculin constructs deficient in amino acids 1–288 (containing the talin- and α-actinin-binding site) or substituting tyrosine for phenylalanine (phosphorylation site, amino acid 822) in F9Vin(−/−) cells resulted in partial restoration of stiffness. Using atomic force microscopy to map the relative elasticity of entire F9 cells by 128 × 128 (n= 16,384) force scans, we observed a correlation with magnetometer measurements. These findings suggest that vinculin may promote cell adhesion and spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, thereby affecting the elastic properties of the cell.     

The T-cell receptor Vβ repertoire in naive and human immunodeficiency virus-1 (HIV-1) infected chimpanzees

Using a panel of human T-cell receptor (TCR) variable region β chain (Vβ) polymerase chain reaction (PCR) primers, we performed cross-sectional and longitudinal analyses of the TCR Vβ repertoire in naive and HIV-1 infected chimpanzees. We demonstrate that our TCR PCR primer panel will support amplification of chimpanzee cDNA from most of the TCR Vβ families. However, no differences in TCR Vβ expression were found between the naive and HIV-1 infected chimpanzees, unlike the TCR Vβ repertoire perturbation found in HIV-1 infected human subjects. This finding suggests that a complete TCR repertoire in HIV-1 infected chimpanzees is associated with the maintenance CD4 + T-cell numbers and lack of progression to AIDS.     

Elucidating the exact role of engineered CRABPII residues for the formation of a retinal protonated Schiff base

Cellular Retinoic Acid Binding Protein II (CRABPII) has been reengineered to specifically bind and react with all‐trans‐retinal to form a protonated Schiff base. Each step of this process has been dissected and four residues (Lys132, Tyr134, Arg111, and Glu121) within the CRABPII binding site have been identified as crucial for imine formation and/or protonation. The precise role of each residue has been examined through site directed mutagenesis and crystallographic studies. The crystal structure of the R132K:L121E‐CRABPII (PDB‐3I17) double mutant suggests a direct interaction between engineered Glu121 and the native Arg111, which is critical for both Schiff base formation and protonation. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Inflating bacterial cells by increased protein synthesis

Understanding how the homeostasis of cellular size and composition is accomplished by different organisms is an outstanding challenge in biology. For exponentially growing Escherichia coli cells, it is long known that the size of cells exhibits a strong positive relation with their growth rates in different nutrient conditions. Here, we characterized cell sizes in a set of orthogonal growth limitations. We report that cell size and mass exhibit positive or negative dependences with growth rate depending on the growth limitation applied. In particular, synthesizing large amounts of “useless” proteins led to an inversion of the canonical, positive relation, with slow growing cells enlarged 7‐ to 8‐fold compared to cells growing at similar rates under nutrient limitation. Strikingly, this increase in cell size was accompanied by a 3‐ to 4‐fold increase in cellular DNA content at slow growth, reaching up to an amount equivalent to ~8 chromosomes per cell. Despite drastic changes in cell mass and macromolecular composition, cellular dry mass density remained constant. Our findings reveal an important role of protein synthesis in cell division control.     

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.     

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.