Kinetic studies of the interaction of synthetic RNAs with quinacrine and its analogs.

Temperature-jump relaxation method has been used to study the interaction of synthetic RNAs with quinacrine (QAC) and its analog. Two relaxation times were observed. The dependence of relaxation times on the RNA concentration and optical properties of the RNA-dye complexes suggests that (1) QAC binds to poly-(rA).poly(rU) through two bimolecular reactions including isomerization from one complex form to another and (2) the 2-methoxy group of the acridine ring plays a significant role in the isomerization.     

Stem cell properties and the side population cells as a target for interferon-alpha in adult T-cell leukemia/lymphoma

The cancer stem cell theory suggests that chemoresistance and recurrence of tumors are often due to the similarity of stem cell properties between normal and cancer cells. Adult T-cell leukemia/lymphoma (ATLL) has poor prognosis, suggesting that ATLL cells possess common stem cell properties. We analyzed side population (SP), a characteristic stem cell phenotype, and CD markers in ATLL cell lines. We found that several lines contained SP with expressions of some hematopoietic stem cell markers. On the other hand, treatment with interferon (IFN)-α is sometimes effective in ATLL, particularly combined with other drugs. We examined its effect on ATLL cells and found that IFN-α significantly reduced the SP proportion. Moreover, CD25-positive cells and phosphorylation of STAT1/5 and ERK were upregulated during this process. These data suggest that their stem cell properties render ATLL cells therapy-resistant, and IFN-α exerts its clinical effect through a reduction of the SP cell population.     

Construction of a new cloning vector utilizing a cryptic plasmid and the highly expressed melanin-synthesizing gene operon from Streptomyces castaneoglobisporus

Streptomyces castaneoglobisporus HUT6202 overproduces a diffusible melanin pigment and harbors a cryptic 7.4-kb plasmid, pHY6202. We constructed a Streptomyces cloning vector, pSY10CMM, consisting of the HUT6202 rep gene, the thiostrepton resistance gene and an operon encoding the synthesis of melanin pigment, abbreviated mel, from S. castaneoglobisporus. The vector, which has SphI and BamHI sites as cloning sites with insertional inactivation of the mel, is a more convenient cloning vector than commonly used pIJ702, because of its broad host range for antibiotic-producing Streptomyces strains and its much greater production of diffusible melanin pigment.     

Resveratrol,a red wine constituent polyphenol,prevents superoxide-dependent inflammatory responses induced by ischemia/reperfusion,platelet-activating factor,or oxidants

Moderate consumption of red wine has been shown to exert cardioprotection against ischemia/reperfusion. Because oxidant-dependent leukocyte infiltration plays a critical role in ischemia/reperfusion-induced tissue injury, we hypothesized that resveratrol, a red wine constituent polyphenol would attenuate postischemic leukocyte recruitment and subsequent endothelial dysfunction. Intravital microscopic approaches were used to quantify leukocyte/endothelial cell interactions and venular protein leakage in rat mesenteries exposed to either 20 min ischemia and 60 min reperfusion (I/R), oxidants generated by the reaction of hypoxanthine and xanthine oxidase (HX/XO), platelet-activating factor (PAF), or leukotriene B4 (LTB4). I/R or HX/HX produced marked increases in the number of adherent (LA) and emigrated (LE) leukocytes, which were associated with significant increases in venular albumin leakage (VAL). Intravenous administration of resveratrol or superoxide dismutase (SOD) attenuated these increases in LA, LE, and VAL. Superfusion of the mesentery with PAF or LTB4 also markedly increased LA, LE, and VAL. While resveratrol attenuated the proinflammatory effects of PAF, LTB4-induced changes were not affected by resveratrol. Resveratrol prevents leukocyte recruitment and endothelial barrier disruption induced by a number of superoxide-dependent proinflammatory stimuli, including I/R, HX/XO, or PAF. These salutary effects appear to be related to the antioxidant properties of resveratrol and contribute to the cardioprotective actions associated with consumption of red wine.     

A Non-Neuronal Cardiac Cholinergic System Plays a Protective Role in Myocardium Salvage during Ischemic Insults

Background

In our previous study, we established the novel concept of a non-neuronal cardiac cholinergic system–cardiomyocytes produce ACh in an autocrine and/or paracrine manner. Subsequently, we determined the biological significance of this system–it played a critical role in modulating mitochondrial oxygen consumption. However, its detailed mechanisms and clinical implications have not been fully investigated.

Aim

We investigated if this non-neuronal cardiac cholinergic system was upregulated by a modality other than drugs and if the activation of the system contributes to favorable outcomes.

Results

Choline acetyltransferase knockout (ChAT KO) cells with the lowest cellular ACh levels consumed more oxygen and had increased MTT activity and lower cellular ATP levels compared with the control cells. Cardiac ChAT KO cells with diminished connexin 43 expression formed poor cell–cell communication, evidenced by the blunted dye transfer. Similarly, the ChAT inhibitor hemicholinium-3 decreased ATP levels and increased MTT activity in cardiomyocytes. In the presence of a hypoxia mimetic, ChAT KO viability was reduced. Norepinephrine dose-dependently caused cardiac ChAT KO cell death associated with increased ROS production. In in vivo studies, protein expression of ChAT and the choline transporter CHT1 in the hindlimb were enhanced after ischemia-reperfusion compared with the contralateral non-treated limb. This local effect also remotely influenced the heart to upregulate ChAT and CHT1 expression as well as ACh and ATP levels in the heart compared with the baseline levels, and more intact cardiomyocytes were spared by this remote effect as evidenced by reduced infarction size. In contrast, the upregulated parameters were abrogated by hemicholinium-3.

Conclusion

The non-neuronal cholinergic system plays a protective role in both myocardial cells and the entire heart by conserving ATP levels and inhibiting oxygen consumption. Activation of this non-neuronal cardiac cholinergic system by a physiotherapeutic modality may underlie cardioprotection through the remote effect of hindlimb ischemia-reperfusion.     

Endogenous AP-1 levels necessary for oncogenic activity are higher than those sufficient to support normal growth

We investigated the role of endogenous AP-1 in human tumor cell lines by introducing SupJunD-1, a dominant-negative mutant of AP-1, using vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors. Single inoculation of six human tumor cell lines, originating from osteosarcomas, non-small cell lung carcinomas or cervical carcinomas, with recombinant SupJunD-1 virus at a high multiplicity of infection readily inhibited colony formation in soft agar. We detected no significant changes in expression levels of AP-1 components c-Jun or Fra-1, adhesion molecules CD44 or E-cadherin, or cell cycle regulator p53, which are encoded by genes previously reported to be under the control of AP-1 in some mouse or human cell lines. By varying the dosage of VSV-G-pseudotyped retrovirus, we were able to change the proviral copy number of supjunD-1 from 1 to approximately 10 and monitor suppression of endogenous AP-1 function as assessed by growth characteristics of the tumor cell lines, we found a SupJunD-1 dosage which significantly suppressed anchorage-independent growth without affecting the cellular growth in monolayer cultures at all. We conclude that endogenous AP-1 levels necessary for oncogenic activity are much higher than those sufficient to support normal growth.     

Identification of Mutations Involved in the Requirement of Potassium for Growth of Typical Melissococcus plutonius Strains

Melissococcus plutonius is a fastidious honeybee pathogen, and the addition of KH₂PO₄ to culture medium is required for its growth. Using genome sequences and a newly developed vector, we showed that mutations in genes encoding Na⁺/H⁺ antiporter and cation-transporting ATPase are involved in the potassium requirement for growth.