Brain Cancer Cell-Derived Matrices and Effects on Astrocyte Migration.

Cell-derived matrices are useful tools for studying the extracellular matrix (ECM) of different cell types and testing the effects on cell migration or wound repair. These matrices typically are generated using extended culture with ascorbic acid to boost ECM production. Applying this technique to cancer cell cultures could advance the study of cancer ECM and its effects on recruitment and training of the tumor microenvironment, but ascorbic acid is potently cytotoxic to cancer cells. Macromolecular crowding (MMC) agents can also be added to increase matrix deposition based on the excluded volume principle. We report the use of MMC alone as an effective strategy to generate brain cancer cell-derived matrices for downstream analyses and cell migration studies. We cultured the mouse glioblastoma cell line GL261 for 1 week in the presence of three previously reported MMC agents (carrageenan, Ficoll 70/400, and hyaluronic acid). We measured the resulting deposition of collagens and sulfated glycosaminoglycans using quantitative assays, as well as other matrix components by immunostaining. Both carrageenan and Ficoll promoted significantly more accumulation of total collagen content, sulfated glycosaminoglycan content, and fibronectin staining. Only Ficoll, however, also demonstrated a significant increase in collagen I staining. The results were more variable in 3D spheroid culture. We focused on Ficoll MMC matrices, which were isolated using the small molecule Raptinal to induce cancer cell apoptosis and matrix decellularization. The cancer cell-derived matrix promoted significantly faster migration of human astrocytes in a scratch wound assay, which may be explained by focal adhesion morphology and an increase in cellular metabolic activity. Ultimately, these data show MMC culture is a useful technique to generate cancer cell-derived matrices and study the effects on stromal cell migration related to wound repair.

In vivo comparison of hard tissue regeneration with ovine mesenchymal stem cells processed with either the FICOLL method or the BMAC method.

INTRODUCTION: The aim of this work was to analyse the suitability of mesenchymal stem cell isolation by FICOLL density centrifugation and the closed bone marrow aspirate concentrate (BMAC) system for sinus augmentation with bovine bone mineral (BBM) in the sheep model. METHODS: 16 sheep underwent sinus augmentation with BBM and MSCs; they were divided between two groups with survival points of 8 and 16 weeks. For the FICOLL control arm three, and for the BMAC test arm, five augmentations were performed for each time point. The derived cell numbers were counted; a colony forming unit (CFU) assay was performed; the pluripotency of the MSCs was proved; histological and histomorphometrical analysis were performed. RESULTS: The approach of using BBM and MSCs in combination with fibrin adhesive was sufficient for new bone formation as the FICOLL experiment indicated. However, due to significantly lower cell numbers isolated using the BMAC in sheep, less new bone was formed in the test arm. CONCLUSIONS: The BMAC system is well suited for human MSC isolation but it needs to be optimized to fit sheep cell characteristics if it is to be used in this animal model.

Modulation of human IAPP fibrillation: cosolutes, crowders and chaperones.

The cellular environment determines the structure and function of proteins. Marginal changes of the environment can severely affect the energy landscape of protein folding. However, despite the important role of chaperones on protein folding, less is known about chaperonal modulation of protein aggregation and fibrillation considering different classes of chaperones. We find that the pharmacological chaperone O4, the chemical chaperone proline as well as the protein chaperone serum amyloid P component (SAP) are inhibitors of the type 2 diabetes mellitus-related aggregation process of islet amyloid polypeptide (IAPP). By applying biophysical methods such as thioflavin T fluorescence spectroscopy, fluorescence anisotropy, total reflection Fourier-transform infrared spectroscopy, circular dichroism spectroscopy and atomic force microscopy we analyse and compare their inhibition mechanism. We demonstrate that the fibrillation reaction of human IAPP is strongly inhibited by formation of globular, amorphous assemblies by both, the pharmacological and the protein chaperones. We studied the inhibition mechanism under cell-like conditions by using the artificial crowding agents Ficoll 70 and sucrose. Under such conditions the suppressive effect of proline was decreased, whereas the pharmacological chaperone remains active.

Isolation of healthy individuals' and rheumatoid arthritis patients' peripheral blood neutrophils by the gelatin and Ficoll-Hypaque methods: comparative efficiency and impact on the neutrophil oxidative metabolism and Fcγ receptor expression.

In vitro assessment of the functional responses of leukocytes sometimes requires their isolation from blood, joint and tissues. In this study, we compared the efficiency of two procedures - the gelatin method and Ficoll-Hypaque density centrifugation gradient - to isolate peripheral blood neutrophils of healthy individuals and patients with active rheumatoid arthritis (RA). We also assessed whether these procedures affect the neutrophil activation status. Both purification procedures were concluded in 90min, and yielded cell populations with similar degrees of purity (80-90%), number of neutrophils (1-2×10(6) cells per mL of blood), and viability (97-100%). In vitro neutrophil priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly increased the reactive oxygen species producing ability of the cells stimulated with n-formyl-methionyl-leucyl-phenylalanine (n-fMLP), soluble immune complexes (s-ICs), and insoluble immune complexes (i-ICs). Isolated neutrophils not treated with GM-CSF responded to n-fMLP and i-IC, but not to s-IC. Almost all of the neutrophils (98-100%) purified by both methods expressed FcγRII/CD32 and FcγRIII/CD16, but they did not express significant levels of FcγRI/CD64. Similar results were obtained for healthy individuals' and RA patients' neutrophils. In summary, the gelatin method was comparable to Ficoll-Hypaque gradient in terms of purity, yield, and viability of the neutrophil preparations. Both methods neither primed or activated the neutrophils, nor affected their functional responsiveness. Therefore, both methods are suitable to isolate peripheral blood neutrophils of healthy individuals and RA patients.

Molecular crowding stabilizes both the intrinsically disordered calcium-free state and the folded calcium-bound state of a repeat in toxin (RTX) protein.

Macromolecular crowding affects most chemical equilibria in living cells, as the presence of high concentrations of macromolecules sterically restricts the available space. Here, we characterized the influence of crowding on a prototypical RTX protein, RC(L). RTX (Repeat in ToXin) motifs are calcium-binding nonapeptide sequences that are found in many virulence factors produced by Gram-negative bacteria and secreted by dedicated type 1 secretion systems. RC(L) is an attractive model to investigate the effect of molecular crowding on ligand-induced protein folding, as it shifts from intrinsically disordered conformations (apo-form) to a stable structure upon calcium binding (holo-form). It thus offers the rare opportunity to characterize the crowding effects on the same polypeptide chain under two drastically distinct folding states. We showed that the crowding agent Ficoll70 did not affect the structural content of the apo-state and holo-state of RC(L) but increased the protein affinity for calcium. Moreover, Ficoll70 strongly stabilized both states of RC(L), increasing their half-melting temperature, without affecting enthalpy changes. The power law dependence of the melting temperature increase (ΔT(m)) on the volume fraction (φ) followed theoretical excluded volume predictions and allowed the estimation of the Flory exponent (ν) of the thermally unfolded polypeptide chain in both states. Altogether, our data suggest that, in the apo-state as found in the crowded bacterial cytosol, RTX proteins adopt extended unfolded conformations that may facilitate protein export by the type I secretion machinery. Subsequently, crowding also enhances the calcium-dependent folding and stability of RTX proteins once secreted in the extracellular milieu.

Existence of G-quadruplex structures in promoter region of oncogenes confirmed by G-quadruplex DNA cross-linking strategy.

Existence of G-quadruplex DNA in vivo always attract widespread interest in the field of biology and biological chemistry. We reported our findings for the existence of G-quadruplex structures in promoter region of oncogenes confirmed by G-quadruplex DNA cross-linking strategy. Probes for selective G-quadruplex cross-linking was designed and synthesized that show high selectivity for G-quadruplex cross-linking. Further biological studies demonstrated its good inhibition activity against murine melanoma cells. To further investigate if G-quadruplex DNA was formed in vivo and as the target, a derivative was synthesized and pull-down process toward chromosome DNAs combined with circular dichroism and high throughput deep sequencing were performed. Several simulated intracellular conditions, including X. laevis oocytes, Ficoll 70 and PEG, was used to investigate the compound's pure cross-linking ability upon preformed G-quadruplex. Thus, as a potent G-quadruplex cross-linking agent, our strategy provided both valuable evidence of G-quadruplex structures in vivo and intense potential in anti-cancer therapy.

Loss of size selectivity of the glomerular filtration barrier in rats following laparotomy and muscle trauma.

Posttraumatic microalbuminuria may be caused by either charge- or size-selective alterations in the glomerular filtration barrier, or both, and/or to a reduction in proximal tubular protein reabsorption. This study was performed to elucidate the pathophysiology of the increases in glomerular permeability occurring in rats exposed to a laparotomy or to a laparotomy and muscle trauma. In anesthetized Wistar rats (250-280 g), the left ureter was cannulated for urine collection, while simultaneously blood access was achieved. Rats were exposed to trauma by a laparotomy (L; n = 8), or by a combination of L and muscle trauma (MT; L+MT) induced by topical blunt injury of the abdominal muscles bilaterally. After L, muscles were crushed using hemostatic forceps at either 2 x 2 sites ("small" MT; n = 9), or at 2 x 5 sites ("large" MT; n = 9). Sham groups (n = 16), not exposed to a laparotomy, were used as controls. The glomerular sieving coefficients (theta) to polydisperse FITC-Ficoll-70/400 (molecular radius 13-80 A) were determined at 5 or 60 min after L and L+MT, respectively, from plasma and urine samples, and analyzed by high-performance size-exclusion chromatography. A tissue-uptake technique was used to assess theta for (125)I-labeled serum albumin. L, with or without MT, increased theta for Ficoll(55-80A) and albumin rapidly and markedly. Theta-Ficoll(70A) thus increased approximately threefold, and theta for albumin significantly, for all trauma groups. According to the "two-pore model" of glomerular permeability, these changes mainly reflect an increase in the number of large pores in the glomerular filter without any primary changes in the charge-selective properties of the filter.

Effects of glomerular filtration rate on Ficoll sieving coefficients (theta) in rats.

The purpose of the present study was to assess the role of diffusion and convection during filtration of Ficoll across the glomerular filter by comparing glomerular sieving coefficients (theta) to neutral fluorescein isothiocyanate (FITC)-Ficoll 70/400 obtained at low (hydropenic) vs raised (normal) glomerular filtration rates (GFRs). The theta for FITC-Ficoll was determined in anesthetized Wistar rats (304 +/- 18 g) following laparotomy and cannulation of the ureters, used for urine sampling. After surgery, GFR was 1.2 +/- 0.16 ml/min (+/- s.e.), assessed using the plasma to urine clearance of FITC-inulin and (51)Cr-ethylenediaminetetraacetic acid. FITC-Ficoll 70/400 was infused intravenously (i.v.) following an initial bolus dose. To raise GFR, to an average of approximately 2 ml/min, 5 ml of serum together with glucagon (3 microg/min) was given i.v. FITC-inulin and FITC-Ficoll were determined in plasma and urine using size-exclusion high-performance liquid chromatography. The theta for Ficoll as a function of Stokes-Einstein radius was significantly reduced in the range of 13-43 A when GFR was raised. The maximal theta lowering effect, in relative terms, of raising GFR was obtained for a Ficoll a(e) of approximately 32 A. For Ficoll(36 A) (cf. albumin), theta was reduced from 0.111+/- 0.009 to 0.081+/- 0.012 (P < 0.05; n = 7) for the GFR increment imposed. The reduction in theta for Ficoll after raising GFR indicates the presence of a high diffusive component of glomerular Ficoll filtration in rats in vivo and contradicts the notion of a significant concentration polarization effect in the glomerular filter upon Ficoll molecules < 50 A in radius.

BOB.1/OBF.1 deficiency affects marginal-zone B-cell compartment.

Marginal-zone (MZ) B cells represent a first line of defense against particulate blood-borne antigens. Together with the B1 cells, they are responsible for the early response against type II T-independent antigens. The molecular pathways controlling the development of MZ B cells are only poorly understood. We found that these cells are virtually absent in mice deficient in the BOB.1/OBF.1 coactivator. Loss of these B cells was demonstrated by the lack of cells showing the appropriate cell surface phenotype but also by histological analyses and tri-nitro-phenol-Ficoll capturing. The lack of these cells is a B-cell-intrinsic defect, as shown by bone marrow complementation experiments. We also show that the expression of BOB.1/OBF.1 in peripheral B cells is required for the development of MZ B lymphocytes. Our analysis of BOB.1/OBF.1-deficient splenic B cells reveals alterations in cell motility, tumor necrosis factor receptor expression, and B-cell receptor (BCR) signaling. These changes could contribute to the loss of MZ B lymphocytes by altering the maturation of the cells. Interestingly, development of and BCR signaling in B1 B cells are completely normal in BOB.1/OBF.1 mutant mice.

Bullatacin, a potent antitumor annonaceous acetogenin, inhibits proliferation of human hepatocarcinoma cell line 2.2.15 by apoptosis induction.

Bullatacin, isolated from the fruit of Annona atemoya, is one of the most potentially effective antitumor annonaceous acetogenins. Bullatacin was studied here for its ability to inhibit the proliferation of 2.2.15 cells, hepatitis B virus (HBV) DNA transfected human hepatocarcinoma cell line. It was found that bullatacin induced cytotoxicity of 2.2.15 cells in a time- and dose-dependent manner. Fifty percent effective dose (ED50) on day 1 of exposure to bullatacin were 7.8 +/- 2.5 nM for 2.2.15 cells. [3H]-Thymidine incorporation assays showed almost the same results. Bullatacin-treatment also reduced concentrations of hepatitis B surface antigen (HBsAg) in the cultured medium released from 2.2.15 cells, coincident with the decrease in the cell proliferation. Analysis of mophological changes of bullatacin-treated 2.2.15 by inverted phase-contrast microscope and eletron microscopy revealed a possible model of action for bullatacin to inhibit proliferation of 2.2.15 cells by inducing apoptosis. Most of the bullatacin-induced cell death was found to be due to apoptosis, as determined by double staining with fluorescein-isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI).

Antigen-receptor induced clonal expansion and deletion of lymphocytes are impaired in mice lacking HS1 protein, a substrate of the antigen-receptor-coupled tyrosine kinases.

HS1, an intracellular protein expressed specifically in hematopoietic cells, is rapidly tyrosine phosphorylated after cross-linking of antigen receptors on B and T lymphocytes, implicating involvement of this molecule in the signal transduction pathways from the antigen receptors as a substrate of membrane-associated tyrosine kinase(s). The development of lymphoid cells in HS1-deficient mice, generated through gene targeting, appeared normal. However, antibody production to T-independent antigen and proliferative responses of splenic B and T cells after cross-linking of the antigen receptors were impaired in these mutant mice. Furthermore, B cells in the peritoneal cavity of the mutant mice were resistant to multivalent cross-linking of the antigen receptor, which causes apoptosis of such cells in normal mice. Crossing the HS1-deficient mice with the mice harboring transgenes encoding alpha and beta chains of T-cell antigen receptor against a male H-Y antigen resulted in a progeny that demonstrated a significantly impaired ability of thymic negative selection. These results indicate that HS1 is a novel molecule involved in the antigen-receptor-derived signaling pathways and plays important roles not only in clonal expansion, but also in clonal deletion of B and T cells.

Marginal zone macrophages identified by a monoclonal antibody: characterization of immuno- and enzyme-histochemical properties and functional capacities.

In the present study, the non-lymphoid cell of the marginal zone is described: the marginal zone macrophage, identified by a monoclonal antibody, ERTR9. The characteristics of these cells were investigated by use of a two-step immunoperoxidase method (on light and ultrastructural level) in combination with enzyme-cytochemical techniques and functional tests. The marginal zone macrophage appears to be a large cell with close contacts between its cell processes and surrounding marginal zone B cells, and with a well-developed phagocytic capacity. These ERTR9-positive macrophages are capable of selective uptake and retention of i.v. injected FITC-Ficoll and are negative for Ia antigen as well as for the macrophage-related F4/80 antigen. By these characteristics, they constitute a subpopulation of non-lymphoid cells of spleen unique for the marginal zone.