Molecular cloning of the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene that contributes to the construction of a new transformation system in <Emphasis Type="Italic">Coriolus versicolor</Emphasis>

The white-rot basidiomycete Coriolus versicolor secretes several enzymes that participate in the degradation of lignin and various persistent organic pollutants. In this study, we attempted to establish a genetic transformation system with a homogenous promoter sequence for driving the gene for antibiotic resistance. We succeeded in cloning the promoter sequence of the gene for glyceraldehyde-3-phosphate dehydrogenase (gpd), which is expressed at high levels in C. versicolor. The expression vector pT7GPTHPT was constructed, which included a gene for resistance to hygromycin B under control of the gpd promoter. The successful selection of transformants on medium that contained hygromycin B indicated that the system should be useful not only for the genetic transformation of C. versicolor, but also for the overproduction of useful fungal enzymes such as laccase and peroxidase.     

Vagosympathetic interactions in ischemia-induced myocardial norepinephrine and acetylcholine release

To elucidate the pathophysiological roles of vagosympathetic interactions in ischemia-induced myocardial norepinephrine (NE) and acetylcholine (ACh) release, we measured myocardial interstitial NE and ACh levels in response to a left anterior descending coronary occlusion in the following groups of anesthetized cats: intact autonomic innervation (INT, n = 7); vagotomy (VX, n = 6); local administration of atropine (Atro, n = 6); transection of the stellate ganglia (TSG, n = 5); local administration of phentolamine (Phen, n = 6); and combined vagotomy and transection of the stellate ganglia (VX+TSG, n = 5). The maximum NE release was enhanced in the VX group (141 +/- 30 nmol/l, means +/- SE, P < 0.05) compared with the INT group (61 +/- 12 nmol/l). Neither the Atro (50 +/- 24 nmol/l) nor VX+TSG groups (84 +/- 25 nmol/l) showed enhanced NE release. The maximum ACh release was unaltered in the TSG and Phen groups compared with the INT group (19 +/- 4, 18 +/- 4, and 13 +/- 3 nmol/l, respectively). These findings indicate that the cardiac vagal afferent but not efferent activity reduced the ischemia-induced myocardial NE release. In contrast, the cardiac sympathetic afferent and efferent activities played little role in the ischemia-induced myocardial ACh release.     

Differential dynamic baroreflex regulation of cardiac and renal sympathetic nerve activities

Although regional difference in sympathetic efferent nerve activity has been well investigated, whether this regional difference exists in the dynamic baroreflex regulation of sympathetic nerve activity remains uncertain. In anesthetized, vagotomized, and aortic-denervated rabbits, we isolated carotid sinuses and randomly perturbed intracarotid sinus pressure (CSP) while simultaneously recording cardiac (CSNA) and renal sympathetic nerve activities (RSNA). The neural arc transfer function from CSP to CSNA and that from CSP to RSNA revealed high-pass characteristics. The increasing slope of the transfer gain in the frequencies between 0.03 and 0.3 Hz was significantly greater for CSNA than for RSNA (2.96 +/- 0.72 vs. 1.64 +/- 0.73 dB/octave, P < 0.01, n = 9). The difference was hardly explained by the difference in static nonlinear characteristics of CSP-CSNA and CSP-RSNA relationships or by the difference in conduction velocities in the multifiber recording. These results indicate that the central processing in the brain stem differs between CSNA and RSNA. The neural arc of the baroreflex may exert differential effects on the heart and kidney in response to dynamic baroreflex activation.     

The adapter type protein CMS/CD2AP binds to the proto-oncogenic protein c-Cbl through a tyrosine phosphorylation-regulated Src homology 3 domain interaction

CMS/CD2AP is a cytoplasmic protein critical for the integrity of the kidney glomerular filtration and the T cell function. CMS contains domains and motifs characteristic for protein-protein interactions, and it is involved in the regulation of the actin cytoskeleton. We report here that the individual SH3 domains of CMS bind to phosphotyrosine proteins of approximately 80, 90, and 180 kDa in cell lysates stimulated with epidermal growth factor. The second SH3 domain of CMS bound specifically to a tyrosine-phosphorylated protein of 120 kDa, which we identified as the proto-oncoprotein c-Cbl. The c-Cbl-binding site for CMS mapped to the carboxyl terminus of c-Cbl and is different from the proline-rich region known to bind SH3-containing proteins. CMS binding to c-Cbl was markedly attenuated in a tyrosine phosphorylation-defective c-Cbl mutant indicating that this interaction is dependent on the tyrosine phosphorylation of CMS. It also implies that CMS interacts with c-Cbl in an inducible fashion upon stimulation of a variety of cell-surface receptors. Immunofluorescence analysis revealed that both proteins colocalize at lamellipodia and leading edges of cells, and we propose that the interaction of CMS with c-Cbl offers a mechanism by which c-Cbl associates and regulates the actin cytoskeleton.     

Evaluating the fitness of PA/I38T-substituted influenza A viruses with reduced baloxavir susceptibility in a competitive mixtures ferret model

Baloxavir is approved in several countries for the treatment of uncomplicated influenza in otherwise-healthy and high-risk patients. Treatment-emergent viruses with reduced susceptibility to baloxavir have been detected in clinical trials, but the likelihood of widespread occurrence depends on replication capacity and onward transmission. We evaluated the fitness of A/H3N2 and A/H1N1pdm09 viruses with the polymerase acidic (PA) I38T-variant conferring reduced susceptibility to baloxavir relative to wild-type (WT) viruses, using a competitive mixture ferret model, recombinant viruses and patient-derived virus isolates. The A/H3N2 PA/I38T virus showed a reduction in within-host fitness but comparable between-host fitness to the WT virus, while the A/H1N1pdm09 PA/I38T virus had broadly similar within-host fitness but substantially lower between-host fitness. Although PA/I38T viruses replicate and transmit between ferrets, our data suggest that viruses with this amino acid substitution have lower fitness relative to WT and this relative fitness cost was greater in A/H1N1pdm09 viruses than in A/H3N2 viruses.     

Manipulation of thrombus formation in the hamster cheek pouch with drugs that interact with PGI2

A single platelet thrombus was formed in an arteriole of the hamster cheek pouch by electrical stimulation followed by topical application of ADP. The sizes of the thrombi were continuously recorded with a photocell placed on a TV monitor screen and quantified by areas on the record. Repeated application of small doses of ADP (5–15 nmol/10 μl) resulted in very reproducible formation of the thrombi, and the size of the thrombi was reduced dose-dependently by topical application of PGI₂. Three drugs were tested in this model. Cycloxygenase inhibitor (indomethacin 10 mg/kg, i.p.) increased the formatiion of thrombi, while a smaller dose (3 mg/kg) did not have any significant effect. This could be explained by inhibition of the generation of endogemous PGI₂, since aggregation of hamster platelets by ADP was not inhibited by indomethacin . EG-626 (phthalazinol, a phosphodiesterase inhibitor) (300 mg/kg, i.p.) decreased the size of thrombus. AI-122 (1.0 mg/kg, i.p.) which has been proven to enhance PGI₂ biosynthesis from isolated rat aortae, also decreased the formation. Thus, drugs such as EG-626 or AI-122 are quite promising as anti-thrombic drugs.     

Senescence marker protein 30 inhibits angiotensin II-induced cardiac hypertrophy and diastolic dysfunction

Background and objective

Senescence marker protein 30 (SMP30) is assumed to behave as an anti-aging factor. Recently, we have demonstrated that deficiency of SMP30 exacerbates angiotensin II-induced cardiac hypertrophy, dysfunction and remodeling, suggesting that SMP30 may have a protective role in the heart. Thus, this study aimed to test the hypothesis that up-regulation of SMP30 inhibits cardiac adverse remodeling in response to angiotensin II.

Methods

We generated transgenic mice with cardiac-specific overexpression of SMP30 gene using α-myosin heavy chain promoter. Transgenic mice and wild-type littermate mice were subjected to continuous angiotensin II infusion (800 ng/kg/min).

Results

After 14 days, heart weight and left ventricular weight were lower in transgenic mice than in wild-type mice, although blood pressure was similarly elevated during angiotensin II infusion. Cardiac hypertrophy and diastolic dysfunction in response to angiotensin II were prevented in transgenic mice compared with wild-type mice. The degree of cardiac fibrosis by angiotensin II was lower in transgenic mice than in wild-type mice. Angiotensin II-induced generation of superoxide and subsequent cellular senescence were attenuated in transgenic mouse hearts compared with wild-type mice.

Conclusions

Cardiac-specific overexpression of SMP30 inhibited angiotensin II-induced cardiac adverse remodeling. SMP30 has a cardio-protective role with anti-oxidative and anti-aging effects and could be a novel therapeutic target to prevent cardiac hypertrophy and remodeling due to hypertension.     

Bioavailability and Efficacy of a Gap Junction Enhancer (PQ7) in a Mouse Mammary Tumor Model

The loss of gap junctional intercellular communication is characteristic of neoplastic cells, suggesting that the restoration with a gap junction enhancer may be a new therapeutic treatment option with less detrimental effects than traditional antineoplastic drugs. A gap junction enhancer, 6-methoxy-8-[(2-furanylmethyl) amino]-4-methyl-5-(3-trifluoromethylphenyloxy) quinoline (PQ7), on the normal tissue was evaluated in healthy C57BL/6J mice in a systemic drug distribution study. Immunoblot analysis of the vital organs indicates a reduction in Cx43 expression in PQ7-treated animals with no observable change in morphology. Next the transgenic strain FVB/N-Tg(MMTV-PyVT) 634Mul/J (also known as PyVT) was used as a spontaneous mammary tumor mouse model to determine the biological and histological effects of PQ7 on tumorigenesis and metastasis at three stages of development: Pre tumor, Early tumor, and Late tumor formation. PQ7 was assessed to have a low toxicity through intraperitoneal administration, with the majority of the compound being detected in the heart, liver, and lungs six hours post injection. The treatment of tumor bearing animals with PQ7 had a 98% reduction in tumor growth, while also decreasing the total tumor burden compared to control mice during the Pre stage of development. PQ7 treatment increased Cx43 expression in the neoplastic tissue during Pre-tumor formation; however, this effect was not observed in Late stage tumor formation. This study shows that the gap junction enhancer, PQ7, has low toxicity to normal tissue in healthy C57BL/6J mice, while having clinical efficacy in the treatment of spontaneous mammary tumors of PyVT mice. Additionally, gap junctional intercellular communication and neoplastic cellular growth are shown to be inversely related, while treatment with PQ7 inhibits tumor growth through targeting gap junction expression.     

Fractionation of the Leuconostoc mesenteroides NRRL B-1299 Dextran and Preliminary Characterization of the Fractions

The extracellular dextran elaborated by Leuconostoc mesenteroides NRRL B-1299, which was shown to be heterogeneous, was separated into five fractions by assorted fractionation methods. Each fraction turned out to be homogeneous by ultracentrifugation and/or electro-phoretic analysis. Gel filtration analysis suggested that these fractions were divided into two groups on the basis of their average molecular weights, one of which had comparatively low molecular weight of 150,000~200,000 (BPS and CPS) and the other had high molecular weight of about 2,000,000 (BPP, CPP and CS). From the results of periodate oxidation, each fraction was shown to contain 41~46% of 1,6-glucosidic linkage (including non-reducing terminal group) as well as 1,4- like (1,4- and/or 1,2-) and 1,3-like linkages. Partial acid hydrolysis of each fraction yielded a series of α-1,6-linked oligosaccharides and acetolysis gave koji-biose and nigerose. Moreover, these fractions gave characteristic precipitation patterns, when incubated with concanavalin A. On the basis of these results, the structural features of the fractions were discussed.     

In vitro evaluation of the safe margin, antithrombotic and antiproliferative actions for the treatment of restenosis: Nitric oxide donor and polymers

Drug‐eluting stents (DES) were developed to combat the problem of in‐stent restenosis, and evaluating the biological activity from DES systems is critical for its safety and efficacy. To test the cytotoxicity of nitric oxide (NO) donor‐containing polymers for their potential use in DES applications, S‐nitrosoglutathione (GSNO) or in combination with poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) in an aqueous polymeric solution (PVA/PVP/GSNO) was investigated using Balb/c 3T3 and Rabbit arterial smooth muscle (RASM) cells. The sensitivity of 3T3 cells to the cytotoxicity effects induced by GSNO was higher than that of RASM cells, while RASM cells were more susceptible to alterations in membrane permeability. Cell growth assays showed that GSNO and PVA/PVP/GSNO induced antiproliferative effects in RASM cells. Moreover, the presence of polymers can reduce the cytotoxicity and enhance the antiproliferative effects of GSNO. Dose‐dependent inhibition of platelet aggregation was similar for both PVA/PVP/GSNO (EC50 of 3.4 ± 2.3 µM) and GSNO (EC50 of 2.8 ± 1.1 µM) solutions. Platelet adhesion assays showed that the inhibition caused by GSNO (EC50 of 5.0 mM) was dependent on the presence of plasma. These results demonstrate that the methodology adopted here is suitable to establish safety margins and evaluate the antithrombotic potential and antiproliferative effects of NO‐eluting biomaterials and polymeric solutions for the new cardiovascular devices, and also to emphasize the importance of using more specific cell lines in these evaluations. Copyright © 2011 John Wiley & Sons, Ltd.