3-Bromopyruvate antagonizes effects of lactate and pyruvate, synergizes with citrate and exerts novel anti-glioma effects

Oxidative stress-energy depletion therapy using oxidative stress induced by D-amino acid oxidase (DAO) and energy depletion induced by 3-bromopyruvate (3BP) was reported recently (El Sayed et al., Cancer Gene Ther., 19, 1–18, 2012). Even in the presence of oxygen, cancer cells oxidize glucose preferentially to produce lactate (Warburg effect) which seems vital for cancer microenvironment and progression. 3BP is a closely related structure to lactate and pyruvate and may antagonize their effects as a novel mechanism of its action. Pyruvate exerted a potent H₂O₂ scavenging effect to exogenous H₂O₂, while lactate had no scavenging effect. 3BP induced H₂O₂ production. Pyruvate protected against H₂O₂-induced C6 glioma cell death, 3BP-induced C6 glioma cell death but not against DAO/D-serine-induced cell death, while lactate had no protecting effect. Lactate and pyruvate protected against 3BP-induced C6 glioma cell death and energy depletion which were overcome with higher doses of 3BP. Lactate and pyruvate enhanced migratory power of C6 glioma which was blocked by 3BP. Pyruvate and lactate did not protect against C6 glioma cell death induced by other glycolytic inhibitors e.g. citrate (inhibitor of phosphofructokinase) and sodium fluoride (inhibitor of enolase). Serial doses of 3BP were synergistic with citrate in decreasing viability of C6 glioma cells and spheroids. Glycolysis subjected to double inhibition using 3BP with citrate depleted ATP, clonogenic power and migratory power of C6 glioma cells. 3BP induced a caspase-dependent cell death in C6 glioma. 3BP was powerful in decreasing viability of human glioblastoma multiforme cells (U373MG) and C6 glioma in a dose- and time-dependent manner.     

Engineering of a novel Ca²⁺-regulated kinesin molecular motor using a calmodulin dimer linker

The kinesin-microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have "on-off" control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesin monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355-M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355-M13 dimerization with CaM dimers, we measured K355-M13 motility and found that it can be reversibly regulated in a Ca(2+)-dependent manner. We also found that velocities of K355-M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca(2+)-dependent dimerization of K355-M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.     

Overexpression of the Escherichia coli catalase gene, katE, enhances tolerance to salinity stress in the transgenic indica rice cultivar, BR5

Salinity stress is a major limiting factor in cereal productivity. Many studies report improvements in salt tolerance using model plants, such as Arabidopsis thaliana or standard varieties of rice, e.g., the japonica rice cultivar Nipponbare. However, there are few reports on the enhancement of salt tolerance in local rice cultivars. In this work, we used the indica rice (Oryza sativa) cultivar BR5, which is a local cultivar in Bangladesh. To improve salt tolerance in BR5, we introduced the Escherichia coli catalase gene, katE. We integrated the katE gene into BR5 plants using an Agrobacterium tumefaciens-mediated method. The introduced katE gene was actively expressed in the transgenic BR5 rice plants, and catalase activity in T₁ and T₂ transgenic rice was approximately 150% higher than in nontransgenic plants. Under NaCl stress conditions, the transgenic rice plants exhibited high tolerance compared with nontransgenic rice plants. T₂ transgenic plants survived in a 200 mM NaCl solution for 2 weeks, whereas nontransgenic plants were scorched after 4 days soaking in the same NaCl solution. Our results indicate that the katE gene can confer salt tolerance to BR5 rice plants. Enhancement of salt tolerance in a local rice cultivar, such as BR5, will provide a powerful and useful tool for overcoming food shortage problems.     

Muscle mechanoreflex augments arterial baroreflex-mediated dynamic sympathetic response to carotid sinus pressure

Although the muscle mechanoreflex is one of the pressor reflexes during exercise, its interaction with dynamic characteristics of the arterial baroreflex remains to be quantitatively analyzed. In anesthetized, vagotomized, and aortic-denervated rabbits (n = 7), we randomly perturbed isolated carotid sinus pressure (CSP) using binary white noise while recording renal sympathetic nerve activity (SNA) and arterial pressure (AP). We estimated the transfer functions of the baroreflex neural arc (CSP to SNA) and peripheral arc (SNA to AP) under conditions of control and muscle stretch of the hindlimb (5 kg of tension). The muscle stretch increased the dynamic gain of the neural arc while maintaining the derivative characteristics [gain at 0.01 Hz: 1.0 +/- 0.2 vs. 1.4 +/- 0.6 arbitrary units (au)/mmHg, gain at 1 Hz: 1.7 +/- 0.6 vs. 2.7 +/- 1.4 au/mmHg; P < 0.05, control vs. stretch]. In contrast, muscle stretch did not affect the peripheral arc. In the time domain, muscle stretch augmented the steady-state response at 50 s (-1.1 +/- 0.3 vs. -1.7 +/- 0.7 au; P < 0.05, control vs. stretch) and negative peak response (-2.1 +/- 0.5 vs. -3.1 +/- 1.5 au; P < 0.05, control vs. stretch) in the SNA step response. A simulation experiment using the results indicated that the muscle mechanoreflex would accelerate the closed-loop AP regulation via the arterial baroreflex.     

Characterization of the hypotensive effect of S-nitroso-N-acetylcysteine in normotensive and hypertensive conscious rats.

S-Nitrosothiols (RSNOs) are potent vasodilators found naturally in vivo. A variety of synthetic RSNOs have been considered as potential nitric oxide (NO) donors for biomedical applications. We have characterized the hypotensive effect of the RSNO S-nitroso-N-acetylcysteine (SNAC) in normotensive and hypertensive conscious rats. SNAC reduced the medium arterial pressure in a dose-response manner in both normotensive and hypertensive animals. At the same doses (EC(50) of SNAC), SNAC showed a vasodilator effect in normotensive rats more potent and more prolonged than that of sodium nitroprusside (SNP). The hypotensive effect of SNAC was also more potent in methylene blue-treated rats, where the cGMP-dependent pathway had been blockaded. These data indicate that SNAC acts by both cGMP-dependent and cGMP-independent pathways. It was also shown that the thiol N-acetylcysteine (NAC) potentiates the action of SNP in hypertensive rats, pointing to the mediation of thiols in the vasodilator action of SNP in this condition. Such mediation may involve the formation of a more potent thiol complex with the nitroprusside anion or the transfer of NO to NAC, generating SNAC as a primary vasoactive species. The kinetic monitoring of the decomposition reactions of SNAC and SNP showed that both compounds are quite stable under the infusion conditions used. Therefore, their vasodilator action cannot be assigned to their breakdown with release of free NO in solution. As the two compounds are unlikely to cross the plasmalemma of smooth muscle cells, their actions are probably associated with the mediation of endogenous thiols in transnitrosation reactions.     

Cleavage of ultraviolet light-irradiated DNA by single strand-specific S1 endonuclease

It was evidenced that the single strand-specific S1 endonuclease could cleave the ultraviolet light-irradiated T7 DNA. The cleavage of ultraviolet light-irradiated T7 DNA by S1 endonuclease was studied by sucrose density gradient centrifugation. The extent of cleavage was proportional to the dose of ultraviolet light given, the concentration of endonuclease and the ionic strength in the reaction. The cleavage consisted of both single-strand and double-strand breaks. The double-strand breaks were observed even at relatively lower dose of ultraviolet light. It seems likely that S1 endonuclease can recognize the alteration in the double-helical structure produced by ultraviolet light-irradiation rather than specifically attack ultraviolet light-induced photoproducts.     

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

Binding of interleukin-2 (IL-2) to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2 receptor (IL-2R)-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling.