Study of the interaction between actin and antitumor substance aplyronine A with a novel fluorescent photoaffinity probe

The interaction between actin and aplyronine A, a potent antitumor and actin-depolymerizing substance of marine origin, was investigated by photoaffinity labeling experiments. Photoaffinity probes consisting of a side-chain portion of aplyronine A as a ligand, a diazirine moiety as a photoaffinity group, and a fluorophore as a detecting group were synthesized. Photolabeling experiments between actin and the probe were carried out. Actin was successfully photolabeled by the fluorescent probe and visualized clearly. The present results provide the first chemical evidence for the direct interaction between actin and the side-chain portion of aplyronine A.     

Detection of eight antibodies in cancer patients' sera against proteins derived from the adenocarcinoma A549 cell line using proteomics-based analysis

To screen cancer for specific autoantibodies, we applied the approach established by Brichory et al., who reported annexins I and II as specific antigens. Solubilized proteins from a cancer cell line (A549) were separated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by Western blotting (WB) analysis, in which the sera of individual patients were tested for primary antibodies. We found 11 positive spots on PVDF membrane using a WB/enhanced chemiluminescence detection Kit, and identified eight proteins, such as alpha-enolase, inosine-5'-monophosphate dehydrogenase, aldehyde dehydrogenase, 3-phosphoglycerate dehydrogenase, 3-oxoacid CoA transferase, chaperonin, peroxiredoxin 6 and triosephosphate isomerase, that reacted with these antibodies in patients' sera using MALDI-TOF/TOF. All eight antibodies were not detected in the sera derived from lung tuberculosis and healthy controls.     

EPA effect on NOS gene expression and on NO level in endothelin-1-induced hypertrophied cardiomyocytes

Cardiomyocytes release (or metabolize) several diffusible agents (e.g., nitric oxide [NO], endothelin-1 [ET-1], and angiotensin II) that exert direct effects on myocyte function under various pathologic conditions. Although cardiac hypertrophy is a compensatory mechanism in response to different cardiovascular diseases, there can be a pathologic transition in which the myocardium becomes dysfunctional. Recently, NO has been found to be an important regulator of cardiac remodeling. Specifically, NO has been recognized as a potent antihypertrophic and proapoptotic mediator in cultured cardiomyocytes. We demonstrated that ET-1-induced hypertrophic remodeling in neonatal cardiomyocytes was arrested by pretreatment with eicosapentaenoic acid (EPA), a major component of fish oil. In some recent studies, EPA has demonstrated cardioprotective effects by modulating NO. This study investigated the changes in NO synthase (NOS) in ET-1-induced hypertrophied cardiomyocytes and in total levels of nitrates and nitrites. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured in D-MEM/Ham F12 supplemented with 0.1% fatty acid-free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control group, ET-1 (0.1 nM) group, and ET-1 pretreated with EPA (10 microM) group. NOS gene expression was evaluated 24 hrs after treatment using real-time polymerase chain reaction. Endothelial NOS (eNOS) mRNA expression was decreased in the ET-1 group compared with controls and was unchanged by pretreatment with EPA. mRNA expression of inducible NOS (iNOS) was significantly increased in ET-1-treated cardiomyocytes and was suppressed by EPA pretreatment. Neuronal NOS gene expression and total NO level did not exhibit a statistically significant change in any of the groups. There may be some interaction between ET-1, eNOS, and iNOS in ET-1-induced and EPA-regressed hypertrophied cardiomyocytes that suppress iNOS expression without modulating total NO level or eNOS gene expression.     

Time course alterations of myocardial endothelin-1 production during the formation of exercise training-induced cardiac hypertrophy

Endothelin (ET)-1 is produced by endothelial cells and cardiac myocytes. ET-1 has positive inotropic and chronotropic effects on the heart and causes myocardial cell hypertrophy. Exercise training induces a physiologic cardiac hypertrophy. To study whether myocardial ET-1 is involved in the formation of exercise training-induced cardiac hypertrophy, we investigated time-course alterations of myocardial ET-1 gene expression and ET-1 peptide level in the heart of rats during a formative process of exercise training-induced cardiac hypertrophy. We used the hearts of rats that had been exercise-trained for 4 weeks (4WT) or 8 weeks (8WT) and sedentary control rats for 4 weeks (4WC) or 8 weeks (8WC). Exercise-trained rats performed treadmill running for 5 days/week (60 mins/day). Left ventricular mass index and wall thickness and stroke volume index, measured using echocardiography, in the 8WT group were significantly greater than in the 8WC group, although there were no differences between the 4WC and 4WT groups in these parameters. These results indicated that the 8WT rats developed physiologic cardiac hypertrophy, whereas the 4WT rats did not yet have cardiac hypertrophy. Myocardial ET-1 gene expression and tissue ET-1 concentration in the heart were significantly higher in the 8WT group than in the 8WC group, whereas these values did not differ between the 4WC and 4WT groups. The present study suggests that an alternation of myocardial ET-1 production corresponds with the formation of exercise training-induced cardiac hypertrophy. Therefore, the exercise training-induced change in myocardial ET-1 production may participate in a mechanism of exercise training-induced cardiac adaptation (e.g., cardiac hypertrophy).     

Alterations of gene expressions of preproET-1 and ET receptors in brains of endotoxemic Sprague-Dawley rats

During severe sepsis, several immunological defense mechanisms initiate a cascade of inflammatory events leading to multiorgan failure, including septic encephalopathy and ultimately death. Endothelin-1 (ET-1) has recently been investigated in different cerebral pathologies. Some reports suggest the involvement of ET-1 in sepsis. However, no study to date has reported the alterations in expression of the genes encoding preproET-1 and ET receptors in the frontal cortex of the septic brain. Male Sprague-Dawley (SD) rats 8 weeks of age were administered either saline or 15 mg/kg lipopolysaccharide (LPS) at different time points (1, 3, 6, and 10 hrs). Rats that did not receive LPS were considered to be controls. The rats were sacrificed with ether, and the brain tissues were harvested. Systolic and diastolic blood pressure decreased 1 hr after LPS administration and then gradually returned to normal, without any change in the heart rate. We confirmed the induction of endotoxemia in the brains of SD rats by measuring the expression of nitric oxide synthase (NOS) mRNA induced in the cerebrum. The expression of inducible NOS (iNOS) mRNA in the brains of SD rat after LPS administration was 30-fold higher than that in the brains of control rats. mRNA expression of preproET-1 in the frontal cortex of SD rats after LPS administration was 2-fold higher than that in control rats. A time-dependent increase in the expression of the gene encoding the ET(A) receptor (vasoconstrictive property) after LPS administration was observed in SD rat brain, whereas expression of the gene encoding the ET(B) receptor (vasodilatatory property) showed an initial upregulation and then gradually decreased as sepsis progressed. In conclusion, we report for the first time that expressions of the genes encoding ET-1 and ET receptors are altered in the endotoxemic brain and that these alterations are time-dependent in SD rats. The alterations in the ET system in brain tissue observed in the present study may contribute to the understanding of the pathophysiological changes in the endotoxemic brain.     

Changes in important apoptosis-related molecules in the endothelin-1-induced hypertrophied cardiomyocytes: effect of the pretreatment with eicosapentaenoic Acid

Human heart failure is preceded by a process called cardiac remodeling, in which heart chambers progressively enlarge and contractile function deteriorates. Programmed cell death (apoptosis) of cardiac muscle cells has been identified as an essential process in the progression to heart failure. The execution of the apoptotic program entails complex interactions between and execution of multiple molecular subprograms. Endothelin (ET)-1, a potent vasoconstrictor peptide, is synthesized and secreted by cardiomyocytes and induces hypertrophy of cardiomyocytes. The cardiovascular benefit of fish oil containing eicosapentaenoic acid (EPA) in humans and experimental animals was reported. Recently, we found that ET-1-induced cardiomyocytic remodeling could be prevented by pretreatment with EPA. The aim of the present study is to investigate whether there would be any alteration in the expression of important apoptosis-related molecules in ET-1-administered hypertrophied cardiomyocytes. We also sought to determine, if there are alterations in apoptotic molecules, what type of role for EPA would then exist. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control, the ET-1 (0.1 nM)-treated group, and the ET-1 group pretreated with EPA (10 microM). Twenty-four hours after the treatment, the gene expressions of three important molecules related to apoptosis (caspase-3, Bax, and Bcl-2) in three experimental groups were evaluated by real-time polymerase chain reaction. The present study could not demonstrate any significant or representative alteration in any of the above three apoptosis-related important markers in either ET-1-induced hypertrophied cardiomyocytes with or without EPA pretreatment. The present study would at least be able to exclude the involvement of some representative molecules related to apoptosis in ET-1-induced hypertrophied cardiomyocytes. In addition, the present study demonstrates that the antihypertrophic effect of EPA to ET-1-administered cardiomyocytes appears not to modulate the apoptosis signaling cascade.     

Chemokine CXCL14/BRAK transgenic mice suppress growth of carcinoma cell xenografts

We reported previously that the forced expression of the chemokine BRAK, also called CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells decreased the rate of tumor formation and size of tumor xenografts compared with mock-vector treated cells in athymic nude mice or in severe combined immunodeficiency mice. This suppression occurred even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that a high expression level of the gene in tumor cells is important for the suppression of tumor establishment in vivo. The aim of this study was to determine whether CXCL14/BRAK transgenic mice show resistance to tumor cell xenografts or not. CXCL14/BRAK cDNA was introduced into male C57BL/6 J pronuclei, and 10 founder transgenic mice (Tg) were obtained. Two lines of mice expressed over 10 times higher CXCL14/BRAK protein levels (14 and 11 ng/ml plasma, respectively) than normal blood level (0.9 ng/ml plasma), without apparent abnormality. The sizes of Lewis lung carcinoma and B16 melanoma cell xenografts in Tg mice were significantly smaller than those in control wild-type mice, indicating that CXCL14/BRAK, first found as a suppressor of tumor progression of HNSCC, also suppresses the progression of a carcinoma of other tissue origin. Immunohistochemical studies showed that invasion of blood vessels into tumors was suppressed in tumor xenografts of CXCL14/BRAK Tg mice. These results indicate that CXCL14/BRAK suppressed tumor cell xenografts by functioning paracrine or endocrine fashion and that CXCL14/BRAK is a very promising molecular target for tumor suppression without side effects.     

Monitoring of duplex and triplex formation by 19F NMR using oligodeoxynucleotides possessing 5-fluorodeoxyuridine unit as 19F signal transmitter

We prepared oligodeoxynucleotides (ODNs) possessing a 5-fluorodeoxyuridine (5-FU) unit as a ¹⁹F-signal transmitter, and characterized their structures including single strand, duplex, and triplex using ¹⁹F NMR. The change in chemical shift induced by incorporation of 5-FU into the ODNs and the formation of higher order structures allowed monitoring of structural changes. Data from UV melting experiments and CD spectra were consistent with the spectral changes in the NMR studies. These ¹⁹F-labeled ODNs may be promising molecular probes for the identification of DNA structures in complicated biological conditions.