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.

Effect of angiotensin II on diabetic glomerular hyperpermeability: an in vivo permeability study in rats.

Angiotensin II drives the pathogenesis of diabetic kidney disease, and its systemic administration induces glomerular hyperpermeability in normal rats. However, the response of diabetic glomerular permeability to angiotensin II is largely unknown. In the present study, we investigated the impact of extended systemic administration of angiotensin II on the glomerular permeability of streptozotocin (STZ)-induced late diabetes in rats. We examined the changes in the glomerular permeability after subcutaneous infusion of angiotensin II at 200 ng·kg-1·min-1 for 7 days in male Wistar diabetic rats with 3 mo of STZ-induced diabetes (i.e., blood glucose of ∼20 mmol/L). We also compared these changes with the effects on nondiabetic rats. The sieving coefficients (θ) for inert polydisperse Ficoll molecules, which had a radius of 10-90 Å (Ficoll70-90 Å), were measured in vivo. The θ for large Ficoll molecules was selectively enhanced after infusion of extended angiotensin II in both diabetic (θ for Ficoll70-90 Å = 0.00244 vs. 0.00079, P < 0.001) and nondiabetic animals (θ for Ficoll70-90 Å = 0.00029 vs. 0.00006, P < 0.001). These changes were compatible with the more than twofold increase in the macromolecular glomerular transport through the large-pore pathways after infusion of angiotensin II in both diabetic and nondiabetic animals. Angiotensin II infusion enhanced the large shunt-like glomerular transport pathway of STZ-induced late diabetes. Such defects can account for the large-molecular-weight IgM-uria that is observed in severe diabetic kidney disease.

Effect of Macromolecular Crowding on the FMN-Heme Intraprotein Electron Transfer in Inducible NO Synthase.

Previous biochemical studies of nitric oxide synthase enzymes (NOSs) were conducted in diluted solutions. However, the intracellular milieu where the proteins perform their biological functions is crowded with macromolecules. The effect of crowding on the electron transfer kinetics of multidomain proteins is much less understood. Herein, we investigated the effect of macromolecular crowding on the FMN-heme intraprotein interdomain electron transfer (IET), an obligatory step in NOS catalysis. A noticeable increase in the IET rate in the bidomain oxygenase/FMN (oxyFMN) and the holoprotein of human inducible NOS (iNOS) was observed upon addition of Ficoll 70 in a nonsaturable manner. Additionally, the magnitude of IET enhancement for the holoenzyme is much higher than that that of the oxyFMN construct. The crowding effect is also evident at different ionic strengths. Importantly, the enhancing extent is similar for the iNOS oxyFMN protein with added Ficoll 70 and Dextran 70 that give the same solution viscosity, showing that specific interactions do not exist between the NOS protein and the crowder. Moreover, the population of the docked FMN-heme state is significantly increased upon addition of Ficoll 70 and the fluorescence lifetime values do not correspond to those in the absence of Ficoll 70. The steady-state cytochrome c reduction by the holoenzyme is noticeably enhanced by the crowder, while the ferricyanide reduction is unchanged. The NO production activity of the iNOS holoenzyme is stimulated by Ficoll 70. The effect of macromolecular crowding on the kinetics can be rationalized on the basis of the excluded volume effect, with an entropic origin. The intraprotein electron transfer kinetics, fluorescence lifetime, and steady-state enzymatic activity results indicate that macromolecular crowding modulates the NOS electron transfer through multiple pathways. Such a mechanism should be applicable to electron transfer in other multidomain redox proteins.

Quantitative characterization of temperature-independent polymer-polymer interaction and temperature-dependent protein-protein and protein-polymer interactions in concentrated polymer solutions.

To study the effect of non-specific interactions arising from proteins being in a crowded environment on physiological processes, the self-interaction of concentrated Dextran T70 and Ficoll 70 and the interactions between a dilute protein and these polymeric macromolecules were quantified using non-ideal tracer sedimentation equilibrium. Sedimentation equilibria of each polymer were measured between 5 and 37 °C, and sedimentation equilibria of 2 mg cm-3 superoxide dismutase (SOD) in 0-0.1 g cm-3 of each polymer was also measured. Results were analyzed using a model-free thermodynamic virial expression of activity coefficients in terms of the concentration of polymer and a structural model using a statistical thermodynamics approximation. The equilibrium gradients of each of the polymers suggest repulsive interaction, which is independent of temperature. However, the net repulsive interaction between superoxide dismutase (SOD) species and the polymers is dependent on temperature. The ratio of the solvation energy of SOD in Dextran T70 to that in Ficoll 70, lnγSOD(Dex)/lnγSOD(Fic) at the same w/v concentration was about 1.8 at 37 °C, 1.6 at the intermediate temperature, and ranges from 1.2 to 1.6 at 5 °C over the entire concentration range. The enthalpy and entropy of interaction of SOD with dilute Dextran T70 are - 14 kJ mol-1 and - 5.6 J K-1 mol-1, respectively. For SOD in dilute Ficoll 70, the enthalpy and entropy are - 8.1 kJ mol-1 and 12.9 J K-1 mol-1, respectively. Thus, Dextran T70 contributes more to the attractive protein-polymer interaction and to self-association of protein than Ficoll 70 and reasons for this are discussed.

A distributed solute model: an extended two-pore model with application to the glomerular sieving of Ficoll.

One of the many unresolved questions regarding the permeability of the glomerular filtration barrier is the reason behind the marked difference in permeability between albumin and polysaccharide probe molecules such as Ficoll and dextran of the same molecular size. Although the differences in permeability have been mainly attributed to charge effects, we have previously shown that this would require a highly charged filtration barrier, having a charge density that is ~10 times more than that on the albumin molecule. In this article, the classic two-pore model was extended by introducing size distributions on the solute molecules, making them conformationally flexible. Experimental sieving data for Ficoll from the rat glomerulus and from precision-made silicon nanopore membranes were analyzed using the model. For the rat glomerulus a small-pore radius of 36.2 Å and a geometric standard deviation (gSD) for the Ficoll size-distribution of 1.16 were obtained. For the nanopore membranes, a gSD of 1.24 and a small-pore radius of 43 Å were found. Interestingly, a variation of only ~16% in the size of the polysaccharide molecule is sufficient to explain the difference in permeability between albumin and Ficoll. Also, in line with previous data, the effects of applying a size distribution on the solute molecule are only evident when the molecular size is close to the pore size. Surely there is at least some variation in the pore radii, and, likely, the gSD obtained in the current study is an overestimation of the "true" variation in the size of the Ficoll molecule.

Calcineurin in a Crowded World.

Calcineurin is a Ser/Thr phosphatase that is important for key biological processes, including immune system activation. We previously identified a region in the intrinsically disordered regulatory domain of calcineurin that forms a critical amphipathic α-helix (the "distal helix") that is required for complete activation of calcineurin. This distal helix was shown to have a Tm close to that of human body temperature. Because the Tm was determined in dilute buffer, we hypothesized that other factors inherent to a cellular environment might modulate the stability of the distal helix. One such factor that contributes to stability in other proteins is macromolecular crowding. The cell cytoplasm is comprised of up to 400 g/L protein, lipids, nucleic acids, and other compounds. We hypothesize that the presence of such crowders could increase the thermal stability of the distal helix and thus lead to a more robust activation of calcineurin in vivo. Using biophysical and biochemical approaches, we show that the distal helix of calcineurin is indeed stabilized when crowded by the synthetic polymers dextran 70 and ficoll 70, and that this stabilization of the distal helix increases the activity of calcineurin.

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.

A distributed two-pore model: theoretical implications and practical application to the glomerular sieving of Ficoll.

In the present study, an extended two-pore theory is presented where the porous pathways are continuously distributed according to small- and large-pore mean radii and SDs. Experimental glomerular sieving data for Ficoll were analyzed using the model. In addition, several theoretical findings are presented along with analytic solutions to many of the equations used in distributed pore modeling. The results of the data analysis revealed a small-pore population in the glomerular capillary wall with a mean radius of 36.6 Å having a wide arithmetic SD of ∼5 Å and a large-pore radius of 98.6 Å with an even wider SD of ∼44 Å. The small-pore radius obtained in the analysis was close to that of human serum albumin (35.5 Å). By reanalyzing the data and setting the distribution spread of the model constant, we discovered that a narrow distribution is compensated by an increased mean pore radius and a decreased pore area-to-diffusion length ratio. The wide distribution of pore sizes obtained in the present analysis, even when considering electrostatic hindrance due to the negatively charged barrier, is inconsistent with the high selectivity to proteins typically characterizing the glomerular filtration barrier. We therefore hypothesize that a large portion of the variance in the distribution of pore sizes obtained is due to the molecular "flexibility" of Ficoll, implying that the true variance of the pore system is lower than that obtained using flexible probes. This would also, in part, explain the commonly noted discrepancy between the pore area-to-diffusion length ratio and the filtration coefficient.

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.

Investigating the folding dynamics of NS2B protein of Zika virus.

NS2B protein of the Zika virus acts as a co-factor for NS3 protease and also involves in remodeling NS3 protease structure. Therefore, we investigated the overall dynamics of NS2B protein. We find surprising similarities between selected flavivirus NS2B model structures predicted from Alphafold2. Further, the simulated ZIKV NS2B protein structure shows a disordered cytosolic domain (residues 45-95) as a part of a full-length protein. Since only the cytosolic domain of NS2B is sufficient for the protease activity, we also investigated the conformational dynamics of only ZIKV NS2B cytosolic domain (residues 49-95) in the presence of TFE, SDS, Ficoll, and PEG using simulation and spectroscopy. The presence of TFE induces α-helix in NS2B cytosolic domain (residues 49-95). On the other hand, the presence of SDS, ficoll, and PEG does not induce secondary structural change. This dynamics study could have implications for some unknown folds of the NS2B protein.

Development of a modified vitrification strategy suitable for subsequent scale-up for hepatocyte preservation.

This work explores the design of a vitrification solution (VS) for scaled-up cryopreservation of hepatocytes, by adapting VS(basic) (40% (v/v) ethylene glycol 0.6M sucrose, i.e. 7.17 M ethylene glycol 0.6M sucrose), previously proven effective in vitrifying bioengineered constructs and stem cells. The initial section of the scale-up study involved the selection of non-penetrating additives to supplement VS(basic) and increase the solution's total solute concentration. This involved a systematic approach with a step-by-step elimination of non-penetrating cryoprotectants, based on their effect on cells after long/short term exposures to high/low concentrations of the additives alone or in combinations, on the attachment ability of hepatocytes after exposure. At a second stage, hepatocyte suspension was vitrified and functions were assessed after continuous culture up to 5 days. Results indicated Ficoll as the least toxic additive. Within 60 min, the exposure of hepatocytes to a solution composed of 9% Ficoll+0.6M sucrose (10⁻³ M Ficoll+0.6 M sucrose) sustained attachment efficiency of 95%, similar to control. Furthermore, this additive did not cause any detriment to the attachment of these cells when supplementing the base vitrification solution VS(basic). The addition of 9% Ficoll, raised the total solute concentration to 74.06% (w/v) with a negligible 10⁻³ M increase in molarity of the solution. This suggests main factor in inducing detriment to cells was the molar contribution of the additive. Vitrification protocol for scale-up condition sustained hepatocyte suspension attachment efficiency and albumin production. We conclude that although established approach will permit scaling-up of vitrification of hepatocyte suspension, vitrification of hepatocytes which are attached prior to vitrification is more effective by comparison.

Attractive protein-polymer interactions markedly alter the effect of macromolecular crowding on protein association equilibria.

The dependence of the fluorescence of catalase upon the concentration of added superoxide dismutase (SOD) indicates that SOD binds to saturable sites on catalase. The affinity of SOD for these sites varies with temperature, and with the concentration of each of three nominally inert polymeric additives--dextran 70, Ficoll 70, and polyethylene glycol 2000. At room temperature (25.0 degrees C) and higher, the addition of high concentrations of polymer is found to significantly enhance the affinity of SOD for catalase, but with decreasing temperature the enhancing effect of polymer addition diminishes, and at 8.0 degrees C, addition of polymer has little or no effect on the affinity of SOD for catalase. The results presented here provide the first experimental evidence for the existence of competition between a repulsive excluded volume interaction between protein and polymer, which tends to enhance association of dilute protein, and an attractive interaction between protein and polymer, which tends to inhibit protein association. The net effect of high concentrations of polymer upon protein associations depends upon the relative strength of these two types of interactions at the temperature of measurement, and may vary significantly between different proteins and/or polymers.

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.

Unaltered size selectivity of the glomerular filtration barrier in caveolin-1 knockout mice.

The transfer of albumin from blood to tissue has been found to be increased in caveolin-1 knockout (KO) mice. This has been considered to reflect increased microvascular permeability, conceivably caused by an increased endothelial production of nitric oxide (NO) in these mice. To investigate whether such an increase in NO production would also affect glomerular barrier characteristics, the glomerular sieving coefficients (theta) to neutral FITC-Ficoll 70/400 (molecular radius 13-90 A) were determined in caveolin-1 KO mice vs. their wild-type counterparts. The theta for Ficoll were assessed using high-performance size-exclusion chromatography on blood and urine samples. Furthermore, the transcapillary escape rate (TER) of (125)I-labeled albumin and plasma volume (PV) were determined in both types of mice. The kidney expressed low levels of caveolin-1 compared with the lung and bladder, but immunofluorescence associated with vascular structures was evident. Staining was lost in the caveolin-1 KO kidney, as was caveolin-1 expression in the lung and bladder. Despite an increase in the glomerular filtration rate in caveolin-1 KO mice (0.23 +/- 0.04 vs. 0.10 +/- 0.02 ml/min; both n = 7; P < 0.05), the glomerular Ficoll sieving curves were nearly identical. Furthermore, caveolin-1 KO mice showed an increased PV (6.59 +/- 0.42 vs. 5.18 +/- 0.13 ml/100 g; P < 0.01) but only a tendency toward an increased TER (14.69 +/- 1.59 vs. 11.62 +/- 1.62%/h; not significant). It is concluded that in caveolin-1 KO mice the glomerular permeability was not increased, despite the presence of glomerular hyperfiltration. The present data are in line with the concept that the increased transvascular albumin leakage previously found in mice lacking caveolin-1 may be due to an elevation in systemic microvascular pressure due to precapillary vasodilatation, rather than being a consequence of increased microvascular permeability per se.

Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus.

Fluorescence recovery after photobleaching (FRAP) was used to quantify the translational diffusion of microinjected FITC-dextrans and Ficolls in the cytoplasm and nucleus of MDCK epithelial cells and Swiss 3T3 fibroblasts. Absolute diffusion coefficients (D) were measured using a microsecond-resolution FRAP apparatus and solution standards. In aqueous media (viscosity 1 cP), D for the FITC-dextrans decreased from 75 to 8.4 x 10(-7) cm2/s with increasing dextran size (4-2,000 kD). D in cytoplasm relative to that in water (D/Do) was 0.26 +/- 0.01 (MDCK) and 0.27 +/- 0.01 (fibroblasts), and independent of FITC-dextran and Ficoll size (gyration radii [RG] 40-300 A). The fraction of mobile FITC-dextran molecules (fmob), determined by the extent of fluorescence recovery after spot photobleaching, was >>0.75 for RG << 200 A, but decreased to <<0.5 for RG >> 300 A. The independence of D/Do on FITC-dextran and Ficoll size does not support the concept of solute "sieving" (size-dependent diffusion) in cytoplasm. Photobleaching measurements using different spot diameters (1.5-4 micron) gave similar D/Do, indicating that microcompartments, if present, are of submicron size. Measurements of D/Do and fmob in concentrated dextran solutions, as well as in swollen and shrunken cells, suggested that the low fmob for very large macromolecules might be related to restrictions imposed by immobile obstacles (such as microcompartments) or to anomalous diffusion (such as percolation). In nucleus, D/Do was 0.25 +/- 0.02 (MDCK) and 0.27 +/- 0.03 (fibroblasts), and independent of solute size (RG 40-300 A). Our results indicate relatively free and rapid diffusion of macromolecule-sized solutes up to approximately 500 kD in cytoplasm and nucleus.

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.

An optimized strategy to measure protein stability highlights differences between cold and hot unfolded states.

Macromolecular crowding ought to stabilize folded forms of proteins, through an excluded volume effect. This explanation has been questioned and observed effects attributed to weak interactions with other cell components. Here we show conclusively that protein stability is affected by volume exclusion and that the effect is more pronounced when the crowder's size is closer to that of the protein under study. Accurate evaluation of the volume exclusion effect is made possible by the choice of yeast frataxin, a protein that undergoes cold denaturation above zero degrees, because the unfolded form at low temperature is more expanded than the corresponding one at high temperature. To achieve optimum sensitivity to changes in stability we introduce an empirical parameter derived from the stability curve. The large effect of PEG 20 on cold denaturation can be explained by a change in water activity, according to Privalov's interpretation of cold denaturation.

Influence of molecular configuration on the passage of macromolecules across the glomerular capillary wall.

The influence of molecular configuration on the filtration of macromolecules across glomerular capillary walls was examined by comparing fractional clearances of two uncharged polysaccharides of distinctly different molecular configuration in the Munich-Wistar rat. The macromolecules employed were dextran, a slightly branched polymer of glucopyranose, and ficoll, a highly cross-linked copolymer of sucrose and epichlorohydrin. Differences in effective shape between these two polymers were determined from measurements of several physical properties of aqueous solutions containing either dextran or ficoll. It was found that dextran is best represented as a prolate ellipsoid with axial ratios of 4, 9, and 16 for molecules with Stokes-Einstein radii of 22, 32, and 40 A, respectively. On the other hand, ficoll is more closely approximated as spherical since the axial ratio was found to be between 1 and 2 for all molecular sizes. Fractional clearances of dextran and ficoll ranging in effective radius from 18 to 44 A were determined in each of seven Munich-Wistar rats. Fractional clearances of dextran were found to be greater than those of ficoll, the difference being significant for molecular radii ranging from 24 to 44 A. In addition, as shown previously for dextran, ficoll was found to be neither secreted nor reabsorbed by the renal tubules. These results, therefore, suggest that in addition to molecular size and charge, molecular configuration is also a determinant of the filtration of macromolecules across the glomerular capillary wall.

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.

Role of viscogens on the macromolecular assemblies of fibrinogen at liquid/air and solid/air interfaces.

In this study, an attempt has been made to understand the organization and association of fibrinogen (Fg) in solvent environment induced by viscogens such as 1-ethyl 3-methyl imidazolium ethyl sulfate (IL-emes), Ficoll, and Trehalose. The author observed that Fg in IL-emes adsorbed on solid surface shows higher ß-sheet conformation. Shear viscosity measured using quartz crystal microbalance, for Fg in IL-emes was highest with a corresponding higher adsorbed mass 3.26 µg/cm(2). Associated assemblies of the protein at the liquid/air interface were monitored with changes in surface tension and were used to calculate work of adhesion. Changes in work of adhesion were used as a tool to measure the adsorption of Fg to solid surfaces in presence of viscogens and highest adsorption was observed for hydrophilic surfaces. Scanning electron microscopy images show Fg in trehalose forms elongated bead like structures implying organization of the protein at the interface. Crowding in the solvent environment induced by viscogens can slow down organization of Fg, leading to macromolecular assemblies near the interface.