Molecular mechanisms behind the dose-dependent differential activation of MAPK pathways induced by transforming growth factor-beta1 in hematopoietic cells.

Transforming growth factor-beta (TGF-beta) controls a wide range of cellular responses, including cell proliferation, lineage determination, differentiation, and apoptosis, and figures prominently in animal development. It is considered as a pleiotropic factor because it can exert a positive or negative effect on various cellular processes depending on developmental stage of the target cell, its microenvironment, and also its biochemical make up. It has been shown to have a strong inhibitory effect on hematopoietic stem cell proliferation and differentiation. We have earlier shown that TGF-beta1 exerts a bidirectional effect on hematopoietic cell proliferation as a function of its concentration. Although it acted as an inhibitor at high concentrations, at low concentrations it stimulated the stem/progenitor cells. We also provided evidence that the differential activation of mitogen-activated protein kinase pathways was responsible for the observed bidirectional effect. In the present study, we examined the molecular mechanism behind this phenomenon. We observed that the high inhibitory concentrations of TGF-beta1 induced a strong phosphorylation of SMAD 3 and also activated stress kinase-related transcription factors, namely c-Jun and ATF-2. On the other hand, low stimulatory concentrations acted in a SMAD 3-independent pathway and activated STAT proteins. Our results clearly show that differential activation of signal transduction pathways by TGF-beta1 as a function of its concentration underlies its bidirectional effect on hematopoietic cells.

Thermoprecipitation of lysozyme from egg white using copolymers of N-isopropylacrylamide and acidic monomers.

Thermoprecipitation of lysozyme from egg white was demonstrated using copolymers of N-isopropylacrylamide with acrylic acid, methacrylic acid, 2-acryloylamido-2-methylpropane-sulfonic acid and itaconic acid, respectively. Polymers synthesized using molar feed ratio of N-isopropylacrylamide:acidic monomers of 98:2 exhibited lower critical solution temperatures in the range of 33--35 degrees C. These polymers exhibited electrostatic interactions with lysozyme and inhibited its bacteriolytic activity. The concentration of acidic groups required to attain 50% relative inhibition of lysozyme by the polymers, was 10(4)--10(5) times lower than that required for the corresponding monomers. This was attributed to the multimeric nature of polymer-lysozyme binding. More than 90% lysozyme activity was recovered from egg white. Polymers exhibited reusability up to at least 16 cycles with retention of >85% recovery of specific activity from aqueous solution. In contrast, copolymer comprising natural inhibitor of lysozyme i.e. poly (N-isopropylacrylamide-co-O-acryloyl N-acetylglucosamine) lost 50% recovery of specific activity. Thermoprecipitation using these copolymers, which enables very high recovery of lysozyme from egg white, would be advantageous over pH sensitive polymers, which generally exhibit lower recovery.