Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis

Background: Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/ cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological efect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specifc hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine PDE5 isoforms. Results: Using the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purifed A1, A2 and A3 isoforms displaying Km, Vmax and Sildenafl inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by K. lactis cells without major growth defciencies and interferences with the endogenous cAMP/cGMP signal transduction pathways. Conclusions: To our knowledge, this is the frst time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specifc PDE inhibitors for therapeutic applications in many pathologies

Transduction of the arginine-vasopressin signal in skeletal myogenic cells

Arginine vasopressin (AVP) induced concentration-dependent (10(-9) to 10(-6) M) stimulation of inositol phosphate production and a biphasic increment of cytosolic free Ca2+ concentration ([Ca2+]i) in skeletal myogenic cells in culture. These effects were almost completely abolished when the cells were pretreated with the AVP antagonist [deamino-Pen1,Val4,D-Arg8]-vasopressin before stimulation with AVP, thus confirming a V1 receptor-mediated effect. Inositol 1,4,5-trisphosphate production was maximally stimulated within 2-3 s of treatment with AVP, immediately followed by release of Ca2+ from intracellular deposits. Both effects were inhibited by treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA). Such effect of TPA was reversed by the protein kinase C inhibitor staurosporine. Vasopressin also regulated the intracellular pH of responsive cells with mechanisms involving both Na+ and anion transport across the plasma membrane. However, unlike in other cell types, AVP stimulated the Na(+)-H+ antiport only simultaneously with a dramatic cell acidification or after treatment with TPA. Response to AVP was observed in L6 and L5 and, to a lesser extent, in chick embryo myogenic cells, regardless of the stage of differentiation (myoblast or myotube). Comparison of different subclones of the L6 cell line demonstrated that the responsiveness to AVP correlated positively with their myogenic potential

Model of murine ventricular cardiac tissue for in vitro kinematic-dynamic studies of electromagnetic and beta2-adrenergic stimulation

In a model of murine ventricular cardiac tissue in vitro, we have studied the inotropic effects of electromagnetic stimulation (frequency, 75 Hz), isoproterenol administration (10 μM), and their combination. In particular, we have performed an image processing analysis to evaluate the kinematics and the dynamics of beating cardiac syncytia starting from the video registration of their contraction movement. We have found that the electromagnetic stimulation is able to counteract the β-adrenergic effect of isoproterenol and to elicit an antihypertrophic response

Atrophy, oxidative switching and ultrastructural defects in skeletal muscle of the ataxia telangiectasia mouse model

Ataxia telangiectasia is a rare, multi system disease caused by ATM kinase deficiency. Atm-knockout mice recapitulate premature aging, immunodeficiency, cancer predisposition, growth retardation and motor defects, but not cerebellar neurodegeneration and ataxia. We explored whether Atm loss is responsible for skeletal muscle defects by investigating myofiber morphology, oxidative/glycolytic activity, myocyte ultrastructural architecture and neuromuscular junctions. Atm-knockout mice showed reduced muscle and fiber size. Atrophy, protein synthesis impairment and a switch from glycolytic to oxidative fibers were detected, along with an increase of in expression of slow and fast myosin types (Myh7, and Myh2 and Myh4, respectively) in tibialis anterior and solei muscles isolated from Atm-knockout mice. Transmission electron microscopy of tibialis anterior revealed misalignments of Z-lines and sarcomeres and mitochondria abnormalities that were associated with an increase in reactive oxygen species. Moreover, neuromuscular junctions appeared larger and more complex than those in Atm wild-type mice, but with preserved presynaptic terminals. In conclusion, we report for the first time that Atm-knockout mice have clear morphological skeletal muscle defects that will be relevant for the investigation of the oxidative stress response, motor alteration and the interplay with peripheral nervous system in ataxia telangiectasia

Sonographic measurement of the umbilical cord and fetal anthropometric parameters

Objective: To determine reference ranges for the diameter and the cross-sectional area of the umbilical cord during pregnancy and to determine if umbilical cord morphometry is related to fetal size. Methods: A prospective cross-sectional study was designed to assess the sonographic cross-sectional diameter and area of the umbilical cord. The sonographic umbilical cord measurements were obtained in a plane adjacent to the insertion of the cord into the fetal abdomen. Nomograms for the umbilical cord diameter and area were computed. Fetal biometry included: biparietal diameter, abdominal circumference, and femur length. Polynomial regression analysis was conducted. Results: Five hundred and fifty seven patients were included into the study. The regression equation for the umbilical cord diameter (y) according to gestational age (x) was y=-10.0563+1.4265x+0.0194x2 and for the umbilical cord area (y') was y'=91.6-3.3x+0.03x2-0.00007x3. A significant relationship was found between umbilical cord measurements and fetal anthropometric parameters. Conclusion: Reference ranges for umbilical cord diameter and area have been generated. The sonographic diameter and cross-sectional area of the umbilical cord increase as a function of gestational age and both diameter and area correlate with fetal size. Copyright (C) 1999 Elsevier Science Ireland Ltd

Anti-inflammatory effect of PDE5 inhibition in diabetic cardiomyopathy

In patients with type 2 diabetes PDE5 inhibition (PDE5i) is associated with cardiac remodeling and reduced inflammatory cytokines1. In this study it was investigated if PDE5 inhibition prevents the development of cardiomyocyte hypertrophy associated with diabetes by modulation of specific subsets of circulating monocytes and tissue macrophages. Leptin receptor–deficient db/db mice develop diabetes mellitus and cardiac hypertrophy. The db/db mice were treated with the PDE5 inhibitor sildenafil (SILD) for 8 weeks. In the hearts of SILD treated mice cardiomyocyte showed a reduced cross-sectional area compared to the cardiomyocyte of untreated mice. Expres- sion of the hypertrophic marker β-myosin was up-regulated in db/db mice hearts and this increase was prevented by SILD treatment. Circulating pro-inflammatory CD11b+Gr-1+ myeloid cells and the CD11b+Gr-1− monocyte cells, were monitored during SILD treatment. CD11b+Gr-1+ cells were reduced after SILD treatment in db/ db mice compared with untreated ones. No significant changes of CD11b+Gr-1− cells were detected in both control and SILD treated animals. After tissue dissociation, cardiac macrophages infiltration was characterized using specific markers such as F4/80 and TIE2. F4/80+ macrophages in diabetic mice were 2-fold increased compared to untreated mice and they were reduced by SILD treatment. Proangiogenic TIE2 expressing monocytes/macrophages (TEMs) percentage was highly increased in SILD treated db/db mice and correlated with an increase of vessel density, measured by the expression of the endothelial marker CD31. These data suggest that PDE5 inhibition attenuates inflammation in diabetic cardiomyopathy reducing pro-inflammatory monocytes and in parallel increasing TEMs percentage and tissue vascularization

Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL-1 cardiac cell line

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2 and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N-terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT-PCR analysis showed that mPde5a1, mPde5a2 and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL-1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL-1 areas and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy. This article is protected by copyright. All rights reserved