Forced expression of OCT4 influences the expression of pluripotent genes in human mesenchymal stem cells and fibroblasts.

Genetic reprogramming of adult cells to generate induced pluripotent stem (iPS) cells is a new and important step in sidestepping some of the ethical issues and risks involved in the use of embryonic stem cells. iPS cells can be generated by introduction of transcription factors, such as OCT4, SOX2, KLF4, and CMYC. iPS cells resemble embryonic stem cells in their properties and differentiation potential. The mechanisms that lead to induced pluripotency and the effect of each transcription factor are not completely understood. We performed a critical evaluation of the effect of overexpressing OCT4 in mesenchymal stem cells and fibroblasts and found that OCT4 can activate the expression of other stemness genes, such as SOX2, NANOG, CMYC, FOXD3, KLF4, and ßCATENIN, which are not normally or are very weakly expressed in mesenchymal stem cells. Transient expression of OCT4 was also performed to evaluate whether these genes are affected by its overexpression in the first 48 h. Transfected fibroblast cells expressed around 275-fold more OCT4 than non-transfected cells. In transient expression, in which cells were analyzed after 48 h, we detected only the up-regulation of FOXD3, SOX2, and KLF4 genes, suggesting that these genes are the earlier targets of OCT4 in this cellular type. We conclude that forced expression of OCT4 can alter cell status and activate the pluripotent network. Knowledge gained through study of these systems may help us to understand the kinetics and mechanism of cell reprogramming.

Motor nerve blockade potency and toxicity of non-racemic bupivacaine in rats.

BACKGROUND: Racemic [RS(+/-)] bupivacaine can be associated with severe cardiotoxicity. The S(-) isomer is known to be less neuro- and cardiotoxic, but demonstrates a lower potency to block motor activity than RS(+/-) bupivacaine. Thus, the potency and toxicity of a non-racemic bupivacaine mixture were studied. METHODS: Gastrocnemic muscle twitches induced by electrical stimulation of sciatic nerves in rats were used to compare the impact by bupivacaine solutions on motor activity. Field stimulation at 1 Hz eliciting ventricular muscle twitches was used to investigate the effects on cardiac contractility. The lethal dose of each local anesthetic agent was determined following drug infusions during general anesthesia in mechanically ventilated rats. RESULTS: Non-racemic (75S:25R) bupivacaine was more potent (P<0.05) than S(-) or R(+) enantiomers to block motor nerve activity. The concentrations of RS(+/-), 75S:25R, R(+) and S(-) bupivacaine to inhibit nerve conduction by 50% were 0.84 (0.37- 2.20), 0.84 (0.47-2.48), 2.68 (0.98-3.42) and 2.11 mM (1.5-4.03), respectively. Pronounced reductions in ventricular muscle twitches were observed with RS(+/-) and R(+) bupivacaine at low concentrations (0.5-4 microM). Lethal doses for 75S:25R (39.9 mg kg(-1)), and S(-) (34.7 mg kg(-1)) were higher (P<0.05) than for R(+) (16.2 mg kg(-1)) and RS(+/-) bupivacaine (18.4 mg kg(-1)), respectively. DISCUSSION: The potency of S(-) bupivacaine to block the motor activity in the sciatic nerve was enhanced when 25% of the S(-) isomer was replaced by the antipode R(+) bupivacaine. This effect was not associated with increased toxicity.