Rat mesangial cells are normally resistant to tumor necrosis factor-␣ (TNF-␣)-induced apoptosis. In this report we show that the cells can be made susceptible to the apoptotic effect of TNF-␣ when pretreated with actinomycin D, cycloheximide, or vanadate. c-Jun N-terminal protein kinase (JNK) has been thought to mediate apoptotic processes elicited by some stimuli, but its involvement in TNF-␣-induced apoptosis has been controversial. JNK activation was investigated under conditions where the mesangial cells were either resistant or susceptible to TNF-␣-induced apoptosis. TNF-␣ alone stimulated a single transient JNK activity peak. However, when the cells were pretreated with actinomycin D or cycloheximide, TNF-␣ stimulated a second sustained JNK activity peak. When the cells were pretreated with the phosphatase inhibitor vanadate, TNF-␣-induced JNK activation was greatly prolonged. In all three cases, a sustained JNK activation was associated with the initiation of apoptosis. Our data suggest that a sustained activation of JNK induced by these reagents may be associated with blocking the expression of a phosphatase that inactivates JNK. Further studies reveal that the expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) was induced by TNF-␣, indicating that MKP-1 may be involved in protecting the cells from apoptosis by preventing a prolonged activation of JNK under normal conditions. Additional studies showed that extracellular signal-regulated protein kinase activation stimulated by TNF-␣ was unlikely to contribute to the resistance of mesangial cells to TNF-␣ cytotoxicity.

Narrowly dispersed, temperature-responsive BAB block copolymers capable of forming physical gels under physiological conditions were synthesized via aqueous reversible addition fragmentation chain transfer (RAFT) polymerization. The use of a difunctional trithiocarbonate facilitates the two-step synthesis of BAB copolymers with symmetrical outer blocks. The outer B blocks of the triblock copolymers consist of poly(N-isopropylacrylamide) (PNIPAM) and the inner A block consists of poly(N,N-dimethylacrylamide). The copolymers form reversible physical gels above the phase transition temperature of PNIPAM at concentrations as low as 7.5 wt % copolymer. Mechanical properties similar to collagen, a naturally occurring polypeptide used as a three-dimensional in vitro cell growth scaffold, have been achieved. Herein, we report the mechanical properties of the gels as a function of solvent, polymer concentration, and inner block length. Structural information about the gels was obtained through pulsed field gradient NMR experiments and confocal microscopy.

Cell specific delivery of small interfering ribonucleic acid (siRNA) using well-defined multivalent folate-conjugated block copolymers is reported. Primary amine functional, biocompatible, hydrophilic-block-cationic copolymers were synthesized via aqueous reversible additionfragmentation chain transfer (RAFT) polymerization. N-(2-hydroxypropyl)methacrylamide) (HPMA), a permanently hydrophilic monomer, was copolymerized with a primary amine containing monomer, N-(3-aminopropyl)methacrylamide (APMA). Poly(HPMA) confers biocompatibility while APMA provides amine functionality allowing conjugation of folate derivatives. (HPMA-stat-APMA) was chain extended with a cationic block, poly(N-[3-(dimethylamino)propyl] methacrylamide) in order to promote electrostatic complexation between the copolymer and the negatively charged phosphate backbone of siRNA. Notably, poly(HPMA) stabilizes the neutral complexes in aqueous solution while APMA allows the conjugation of a targeting moiety, thus, dually circumventing problems associated with the delivery of genes via cationically charged complexes (universal transfection). Fluorescence microscopy and gene down-regulation studies indicate that these neutral complexes can be specifically delivered to cancer cells that over-express folate receptors.

Background:The antiviral mechanisms are not known in mESCs. Results: mESCs are susceptible to viral infections and dsRNA-inhibited cell proliferation but do not express type I interferons. Conclusion: mESCs have underdeveloped mechanisms for type I interferon expression. Significance: The findings are important for understanding the development of antiviral mechanisms in ESCs and stem cell physiology.

Oxidative stress, associated with either normal metabolism or disease conditions, affects many cellular activities. Most of our knowledge in this fi eld is derived from fully differentiated cells. Embryonic stem cells (ESCs) have attracted enormous attention for their potential applications in cell therapy, but little is known about how the unique properties of ESCs are affected by oxidative stress. We have investigated the effects of oxidative stress induced by H 2 O 2 on several cellular activities of mouse ESCs. Like differentiated cells, ESCs are sensitive to H 2 O 2 -induced apoptosis when continuously exposed to H 2 O 2 at the concentrations above 150 μM. However, unlike differentiated cells, ESCs are resistant to oxidative stress induced senescence. This is demonstrated by the results that when subjected to a short-term sublethal concentration and duration of H 2 O 2 treatment, fi broblasts enter the senescent state with enlarged fl attened cell morphology concurrent with increased expression of senescence marker p21. On the contrary, ESCs neither show any sign of senescence nor express p21. Instead, ESCs enter a transient cell cycle arrest state, but they have remarkable recovery capacity to resume the normal cell proliferation rate without losing the ability of self-renewal and pluripotency. Our results further revealed that H 2 O 2 inhibits cell adhesion and the expression of cyclin D1, which are early events proceeding apoptosis and cell cycle arrest. In conclusion, our data suggest that ESCs are sensitive to H 2 O 2 toxicity, but may have unique mechanisms that prevent H 2 O 2 -induced senescence and protect self-renewal capacity.