Neonatal FcR expression in bone marrow-derived cells functions to protect serum IgG from catabolism

The neonatal FcR (FcRn) is a receptor that protects IgG from catabolism and is important in maintaining high serum Ab levels. A major site of expression of FcRn is vascular endothelial cells where FcRn functions to extend the serum persistence of IgG by recycling internalized IgG back to the surface. Because FcRn is expressed in other tissues, it is unclear whether endothelial cells are the only site of IgG protection. In this study, we used FcRn-deficient mice and specific antiserum to determine the tissue distribution of FcRn in the adult mouse. In addition to its expression in the vascular endothelium of several organs, we found FcRn to be highly expressed in bone marrow-derived cells and professional APCs in different tissues. Experiments using bone marrow chimeras showed that FcRn expression in these cells acted to significantly extend the half-life of serum IgG indicating that in addition to the vascular endothelium, bone marrow-derived phagocytic cells are a major site of IgG homeostasis.     

Expression of conserved signalling pathway genes during spontaneous vascular differentiation of R1 embryonic stem cells and in Py-4-1 endothelial cells

Embryonic stem (ES) cells are an invaluable model for identifying subtle phenotypes as well as severe outcomes of perturbing gene function that may otherwise result in lethality. However,though ES cells of different origins are regarded as equally pluripotent,their in vitro differentiation potential varies, suggesting that their response to developmental signals is different. The R1 cell line is widely used for gene manipulation due to its good growth characteristics and highly efficient germline transmission. Hence, we analysed the expression of Notch, Wnt and Sonic Hedgehog (Shh) pathway genes during differentiation of R1 cells into early vascular lineages. Notch-, Wnt- and Shh-mediated signalling is important during embryonic development. Regulation of gene expression through these signalling molecules is a frequently used theme, resulting in context-dependent outcomes during development. Perturbing these pathways can result in severe and possibly lethal developmental phenotypes often due to primary cardiovascular defects. We report that during early spontaneous differentiation of R1 cells, Notch-1 and the Wnt target Brachyury are active whereas the Shh receptor is not detected. This expression pattern is similar to that seen in a mouse endothelial cell line. This temporal study of expression of genes representative of all three pathways in ES cell differentiation will aid in further analysis of cell signalling during vascular development.     

FcRn: the neonatal Fc receptor comes of age

The neonatal Fc receptor for IgG (FcRn) has been well characterized in the transfer of passive humoral immunity from a mother to her fetus. In addition, throughout life, FcRn protects IgG from degradation, thereby explaining the long half-life of this class of antibody in the serum. In recent years, it has become clear that FcRn is expressed in various sites in adults, where its potential function is now beginning to emerge. In addition, recent studies have examined the interaction between FcRn and the Fc portion of IgG with the aim of either improving the serum half-life of therapeutic monoclonal antibodies or reducing the half-life of pathogenic antibodies. This Review summarizes these two areas of FcRn biology.     

Forces during bacteriophage DNA packaging and ejection

The conjunction of insights from structural biology, solution biochemistry, genetics, and single-molecule biophysics has provided a renewed impetus for the construction of quantitative models of biological processes. One area that has been a beneficiary of these experimental techniques is the study of viruses. In this article we describe how the insights obtained from such experiments can be utilized to construct physical models of processes in the viral life cycle. We focus on dsDNA bacteriophages and show that the bending elasticity of DNA and its electrostatics in solution can be combined to determine the forces experienced during packaging and ejection of the viral genome. Furthermore, we quantitatively analyze the effect of fluid viscosity and capsid expansion on the forces experienced during packaging. Finally, we present a model for DNA ejection from bacteriophages based on the hypothesis that the energy stored in the tightly packed genome within the capsid leads to its forceful ejection. The predictions of our model can be tested through experiments in vitro where DNA ejection is inhibited by the application of external osmotic pressure.     

Rudhira is a cytoplasmic WD40 protein expressed in mouse embryonic stem cells and during embryonic erythropoiesis

We describe a novel murine gene rudhira that is expressed at high levels in embryonic stem cells and is restricted to blood islands and the erythroid lineage during embryonic development. Rudhira is expressed in angiogenic precursors but is excluded from the differentiated endothelium. Rudhira-expressing cells are seen in close proximity to endothelial cells in angiogenic blood vessels. Rudhira encodes a predicted cytoplasmic WD40 protein that is 98% identical to human BCAS3. The gene encoding BCAS3 maps to a breakpoint of hematological neoplasms on human chromosome 17q23, but its expression and function remain to be determined. We demonstrate that mouse Rudhira is a novel marker for analysis of the erythroid lineage.     

A Model to Evaluate the Cytotoxicity of the Fungal Volatile Organic Compound 1-octen-3-ol in Human Embryonic Stem Cells

Microbial growth in damp indoor environments has been correlated with risks to human health. This study was aimed to determine the cytotoxicity of 1-octen-3-ol (“mushroom alcohol”), a major fungal volatile organic compound (VOC) associated with mushroom and mold odors. Using an airborne exposure technique, human embryonic stem cells were exposed for 1 h to different concentrations (0–1,000 ppm) of racemic 1-octen-3-ol and its enantiomers, (R)-(−)-1-octen-3-ol and (S)-(+)-1-octen-3-ol. Cytotoxicity was assayed using both the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and the fluorescently tagged Calcein AM-mediated “live and dead” assay. Racemic 1-octen-3-ol and (S)-(+)-1-octen-3-ol exhibited greater cytotoxicity to the undifferentiated human cell line H1 than did (R)-(−)-1-octen-3-ol. The inhibition concentration 50 (IC₅₀) values assessed by the MTS assay for racemic 1-octen-3-ol, (S)-(+)-1-octen-3-ol and (R)-(−)-1-octen-3-ol were, respectively, 109, 98, and 258 ppm. These IC₅₀ values were 40–80-fold lower than that of vapor phase toluene, an industrial chemical used as a positive control in this study. Our report pioneers the modeling of human embryonic stem cells as an in vitro approach to screen the potential toxicity of fungal VOCs. Human embryonic stem cells exposed to 1-octen-3-ol, and its enantiomers in the vapor phase showed more cytotoxicity than those exposed to toluene.     

Cell Mechanics,Structure, and Function Are Regulated by the Stiffness of the Three-Dimensional Microenvironment

This study adopts a combined computational and experimental approach to determine the mechanical, structural, and metabolic properties of isolated chondrocytes cultured within three-dimensional hydrogels. A series of linear elastic and hyperelastic finite-element models demonstrated that chondrocytes cultured for 24 h in gels for which the relaxation modulus is <5 kPa exhibit a cellular Young’s modulus of ～5 kPa. This is notably greater than that reported for isolated chondrocytes in suspension. The increase in cell modulus occurs over a 24-h period and is associated with an increase in the organization of the cortical actin cytoskeleton, which is known to regulate cell mechanics. However, there was a reduction in chromatin condensation, suggesting that changes in the nucleus mechanics may not be involved. Comparison of cells in 1% and 3% agarose showed that cells in the stiffer gels rapidly develop a higher Young’s modulus of ～20 kPa, sixfold greater than that observed in the softer gels. This was associated with higher levels of actin organization and chromatin condensation, but only after 24 h in culture. Further studies revealed that cells in stiffer gels synthesize less extracellular matrix over a 28-day culture period. Hence, this study demonstrates that the properties of the three-dimensional microenvironment regulate the mechanical, structural, and metabolic properties of living cells.     

Acceleration of neutrophil precursors' maturation and immunostimulation of CD3+, CD4+ lymphocytes by stanozolol in mice

The abuse of anabolic-androgenic steroids (AAS) for improved physical performance is associated with many deleterious effects. The present study aims to evaluate the short-term effect of an AAS compound stanozolol, on lipoprotein profile, granulopoiesis and immune response in adult female mice. The mice were assigned to five experimental groups and different doses of stanozolol (low - 0.05 mg, medium - 0.5 mg, high - 5 mg and highest dose - 7.5 mg/kg bwt or only vehicle respectively) were administered s.c. for 15 days. A decrease in high density lipoprotein cholesterol (HDL-c) as well as total cholesterol (TC) in all the stanozolol treated groups and an increase in low density lipoprotein (LDL-c) in high and the highest dose treated groups indicate that stanozolol alters serum lipoprotein profile. A significant increase in the percentage of myelocytes, metamyelocytes and neutrophils in all the treated mice unveils the stimulation of granulopoiesis through the acceleration of neutrophil precursors' maturation in the bone marrow of mice. The stimulation of erythropoiesis was also noted in all the treated groups. The flow cytometry analysis of lymphocyte subpopulations (CD3(+) and CD4(+)) revealed immunoenhancing response of stanozolol at optimum physiological dose, however, it is immunosuppressive at supraphysiologic level. We conclude that stanozolol accelerates granulopoiesis and stimulates immune response (at physiologic level only), though it alters the lipoprotein profile in mice.     

Monoclonal antibodies directed against human FcRn and their applications

The MHC class I-like Fc receptor (FcRn) is an intracellular trafficking Fc receptor that is uniquely responsible for the extended serum half-life of antibodies of the IgG subclass and their ability to transport across cellular barriers. By performing these functions, FcRn affects numerous facets of antibody biology and pathobiology. Its critical role in controlling IgG pharmacokinetics has been leveraged for the design of therapeutic antibodies and related biologics. FcRn also traffics serum albumin and is responsible for the enhanced pharmacokinetic properties of albumin-conjugated therapeutics. The understanding of FcRn and its therapeutic applications has been limited by a paucity of reliable serological reagents against human FcRn. Here, we describe the properties of a new panel of highly specific monoclonal antibodies (mAbs) directed against human FcRn with diverse epitope specificities. We show that this antibody panel can be used to study the tissue expression pattern of human FcRn, to selectively block IgG and serum albumin binding to human FcRn in vitro and to inhibit FcRn function in vivo. This mAb panel provides a powerful resource for probing the biology of human FcRn and for the evaluation of therapeutic FcRn blockade strategies.Key words: FcRn, IgG, monoclonal antibody, albumin, therapy     

Cdc25A serine 123 phosphorylation couples centrosome duplication with DNA replication and regulates tumorigenesis

The cell division cycle 25A (Cdc25A) phosphatase is a critical regulator of cell cycle progression under normal conditions and after stress. Stress-induced degradation of Cdc25A has been proposed as a major way of delaying cell cycle progression. In vitro studies pointed toward serine 123 as a key site in regulation of Cdc25A stability after exposure to ionizing radiation (IR). To address the role of this phosphorylation site in vivo, we generated a knock-in mouse in which alanine was substituted for serine 123. The Cdc25 S123A knock-in mice appeared normal, and, unexpectedly, cells derived from them exhibited unperturbed cell cycle and DNA damage responses. In turn, we found that Cdc25A was present in centrosomes and that Cdc25A levels were not reduced after IR in knock-in cells. This resulted in centrosome amplification due to lack of induction of Cdk2 inhibitory phosphorylation after IR specifically in centrosomes. Further, Cdc25A knock-in animals appeared sensitive to IR-induced carcinogenesis. Our findings indicate that Cdc25A S123 phosphorylation is crucial for coupling centrosome duplication to DNA replication cycles after DNA damage and therefore is likely to play a role in the regulation of tumorigenesis.