A new method for high yield purification of type beta transforming growth factor from human platelets

A new method was developed for the purification of type beta transforming growth factor from human platelets. This method is a three-step procedure including gel filtration, weak cation exchange HPLC and reverse phase HPLC. All steps are carried out at low pH using exclusively volatile acidic buffer solutions. The sterile conditions and easy removal of salt by lyophilization facilitate the quantification of the growth factor in biological assays. Based on immunological characterization the purified acid-stable, highly basic transforming growth factor beta is the beta 1 form. Using the present method pure platelet TGF beta 1 is obtained in very high yield. 40 units of outdated human platelets yield 800 micrograms pure TGF beta 1, which is about a 10-20 fold higher yield than reported for other purification procedures.     

Simple computer method for evaluation of lateral diffusion coefficients from fluorescence photobleaching recovery kinetics.

A method is presented for the analysis of fluorescence photobleaching recovery curves. Based on the simplified kinetic expression of Yguerabide, J., J.A. Schmidt, and E.E. Yguerabide (1982, Biophys. J., 40:69-75), a linearization procedure is described that permits unequivocal determination of all diffusion parameters. The presence of additional membrane flow or multiple diffusion coefficients can easily be detected by this method, and simple corrections for the presence of these alternative recovery processes can be made by the use of a regular mini-computer. The validity of the method is tested on simulated recovery curves, varying the contribution of flow, multiple diffusion coefficients, and statistical noise due to counting error.     

Purification process monitoring in monoclonal antibody preparation: contamination with viruses, DNA and peptide growth factors.

Administration in vivo of monoclonal antibodies to humans is challenged by considerations regarding their safety. Contamination with viruses, potentially oncogenic nucleic acids and biologically active components like growth factors and hormones forms a serious point of concern in this respect. We have investigated the potential risk of viral contamination by measuring the reduction of 12 different viruses (after spiking) in the standard downstream purification process of ascitic fluid. Depending on the type of virus added and the purification step employed, the reduction of infectious virus particles varies considerably. The overall reduction ranges from about 10(3), observed for a member of the family of Papovaviridae, to more than 10(12) for members of the families of Herpesviridae and Orthomyxoviridae. Using hybridization analysis with a mouse (genomic) DNA probe, we show that the amount of residual DNA in ascitic fluids may also vary considerably, ranging from 75 ng/ml to 1 microgram/ml. In crude preparations produced in cell culture, much lower DNA concentrations are found (0.3 ng/ml). When standard downstream purification procedures are applied to ascitic fluid, a significant reduction of residual DNA levels is observed in the purified monoclonal antibody preparations and in intermediate fractions. The overall reduction factors vary from about 10(3) to 10(4), which is also confirmed by spiking experiments with either purified DNA or crude chromatin-like DNA. Using in-vitro cellular assays, we further show that peptide growth factors like PDGF and TGF beta are present in considerable amounts in ascitic fluids. The observed biological activities, however, are completely eliminated during the purification steps applied.     

Effects of cell heterogeneity on production of polypeptide growth factors and mesoderm-inducing activity by Xenopus laevis XTC cells

The Xenopus laevis XTC cell line has been analyzed for the production of polypeptide growth factors and mesoderm-inducing activity. By the use of specific biological assays, it is shown that XTC cells produce a growth factor functionally related to the platelet-derived growth factor (PDGF) and two transforming growth factor (TGF)β-like activities. Mesoderm-inducing activity, as measured on X. laevis ectodermal explants from stage 10 embryos, was found to coelute on a Bio-Gel P-100 column with one of the TGFβ-like activities at an apparent molecular weight of 6–10 kDa. Analysis of the DNA content from XTC cells by flow cytometry demonstrated that the cell line is heterogeneous and consists of both tetraploid and diploid cells. Cloning of the XTC cells and selecting single-cell colonies on the basis of their ability to grow in soft agar resulted in the isolation of several homogeneous, morphologically different clonal derivatives. Analysis of conditioned medium from these clonal derivatives showed that only one of them, the only diploid line among six investigated, produced a strong heat- and acid-stable mesoderm-inducing activity that induced notochord and muscle formation in stage 10 X. laevis ectodermal expiants. The relation between this activity and a recently described TGFβ-like mesoderm-inducing factor obtained from XTC-conditioned medium will be discussed. In conclusion, a clonal cell line derived from X. laevis XTC cells which provides a good source for further characterization of mesoderm-inducing factors has been established.     

Effect of external ATP on the plasma membrane permeability and (Na++K+)-ATPase activity of mouse neuroblastoma cells

1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na⁺ relative to K⁺, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K⁺ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na⁺. Under these conditions the pumping activity of the Neuro-2A (Na⁺+K⁺)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na⁺+K⁺)-ATPase for ouabain, as measured by direct [³H]ouabain-binding studies and by inhibition studies of active K⁺ uptake. In the presence of 3.5 mM ATP and the absence of external K⁺ both techniques indicate an apparent dissociation constant for ouabain of 2·10^?6 M. Neuro-2A cells contain (3.5±0.7)·10⁵ ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7±0.4)·10^?20 mol K⁺/min per copy of (Na⁺+K⁺)-ATPase at room temperature.     

Glycophorin facilitates the transbilayer movement of phosphatidylcholine in vesicles

The rate of transbilayer movement of dioleoylphosphatidylcholine in sonicated lipid vesicles is enhanced by at least two orders of magnitude upon incorporation of glycophorin in the bilayer.     

Non-electrolyte permeability as a tool for studying membrane fluidity

1. The reflection coefficient for the permeation of thiourea through bilayers of phosphatidylcholine is a function of the fatty-acid composition of the lipid molecules. By means of these reflection coefficients an index for membrane fluidity has been given to each of those lipids, relative to that of egg phosphatidylcholine. 2. The maximum number of water molecules that can copermeate with each molecule of solute by means of solute-solvent interaction is a function of the packing of the lipid molecules in the bilayer. This parameter has been used in this paper for characterizing the fluidity of cholesterol-containing membranes and for membranes with their lipids in the gel state.     

Multi-generational evaluation of genetic diversity and parentage in captive southern pygmy perch (<Emphasis Type="Italic">Nannoperca australis</Emphasis>)

Maintaining genetic diversity within captive breeding populations is a key challenge for conservation managers. We applied a multi-generational genetic approach to the captive breeding program of an endangered Australian freshwater fish, the southern pygmy perch (Nannoperca australis). During previous work, fish from the lower Murray-Darling Basin were rescued before drought exacerbated by irrigation resulted in local extinction. This endemic lineage of the species was captive-bred in genetically designed groups, and equal numbers of F1 individuals were reintroduced to the wild with the return of favourable habitat. Here, we implemented a contingency plan by continuing the genetic-based captive breeding in the event that a self-sustaining wild population was not established. F1 individuals were available as putative breeders from the subset of groups that produced an excess of fish in the original restoration program. We used microsatellite-based parentage analyses of these F1 fish to form breeding groups that minimized inbreeding. We assessed their subsequent parental contribution to F2 individuals and the maintenance of genetic diversity. We found skewed parental contribution to F2 individuals, yet minimal loss of genetic diversity from their parents. However, the diversity was substantially less than that of the original rescued population. We attribute this to the unavoidable use of F1 individuals from a limited number of the original breeding groups. Alternative genetic sources for supplementation or reintroduction should be assessed to determine their suitability. The genetic fate of the captive-bred population highlights the strong need to integrate DNA-based tools for monitoring and adaptive management of captive breeding programs.     

Phenotypic transformation of normal rat kidney fibroblasts by endothelin-1. Different mode of action from lysophosphatidic acid, bradykinin, and prostaglandin f2alpha

In the present study, we compared the effects of endothelin (ET)-1 on cell proliferation and second messenger induction in normal rat kidney (NRK) fibroblasts, with those of other activators of G-protein-coupled receptors such as prostaglandin (PG)-F2alpha, bradykinin (BK), and lysophosphatidic acid (LPA). LPA is mitogenic by itself, while the other factors require the presence of EGF. In density-arrested NRK cells, ET-1 and LPA induce phenotypic transformation rapidly, with similar kinetics as retinoic acid (RA) and transforming growth factor (TGF)-beta, while BK and PGF2alpha only do so with delayed kinetics. ET-1 and PGF2alpha are strong inducers of anchorage-independent growth, with a similar level of induction as TGFbeta, in contrast to LPA and BK. When investigating the second messenger generation, we found that ET-1 is the strongest activator of arachidonic acid release and phosphatidylinositol diphosphate hydrolysis. Only in the case of ET-1 the cell depolarization is not reversible upon removal of the factor. Similarly, only the ET-1-induced transient enhancement of intracellular calcium concentration is paralleled by both homologous and heterologous desensitization. In conclusion, these data show that ET-1 is a potent inducer of second messengers and phenotypic transformation in NRK cells, with characteristics that clearly differ from those of other activators of G-protein-coupled receptors, most likely as a result of prolonged receptor activation.     

Structural analysis of an epidermal growth factor/transforming growth factor-alpha chimera with unique ErbB binding specificity

Various chimeras of the ErbB1-specific ligands epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) display an enlarged repertoire as activators of ErbB2.ErbB3 heterodimers. Mutational analysis indicated that particularly residues in the N terminus and B-loop region of these ligands are involved in the broadened receptor specificity. In order to understand the receptor specificity of T1E, a chimeric ligand constructed by the introduction of the linear N-terminal region of TGFalpha into EGF, we determined in this study the solution structure and dynamics of T1E by multidimensional NMR analysis. Subsequently, we studied the structural characteristics of T1E binding to both ErbB1 and ErbB3 by superposition modeling of its structure on the known crystal structures of ErbB3 and liganded ErbB1 complexes. The results show that the overall structure of T1E in solution is very similar to that of native EGF and TGFalpha but that its N terminus shows an extended structure that is appropriately positioned to form a triple beta-sheet with the large antiparallel beta-sheet in the B-loop region. This conformational effect of the N terminus together with the large overall flexibility of T1E, as determined by 15N NMR relaxation analysis, may be a facilitative property for its broad receptor specificity. The structural superposition models indicate that hydrophobic and electrostatic interactions of the N terminus and B-loop of T1E are particularly important for its binding to ErbB3.