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

terapia con micofenolato dell'interstiziopatia polmonare associata a sclerosi sistemica - esperienza monocentrica

Background Interstitial lung disease (ILD) is the most frequent type of organ involvement and the main cause of death in systemic sclerosis (SSc). The trigger of fibrosis is an immune mediated alveolitis, thus in the last years, several immunosuppressant drugs have been put to the test, mostly cyclophosphamide (CYC), azathioprine (AZA) and mycophenolate mofetil (MMF). Therefore, also in the Rheumatology Unit of Padua University, in recent years have been treated in with immunosuppressive therapy. Aim The aim of our study was to evaluate the efficacy of MMF as a first-line drug on pulmonary function in SSc-related ILD patients and compare them to a historical group of SSc patients treated with CYC followed by AZA. Moreover, it has been assessed the safety of each immunosuppressant drugs. Methods Eighteen patients with SSc-related ILD have been investigated and treated with MMF as first-line therapy for two years. Fifteen patients, instead have been treated one year with CYC and for and with AZA for the second years. The two groups of patients have been evaluated at baseline, and then after 12 and 24 months of therapy. The evaluation parameters were: pulmonary function tests (FVC and DLCO), HRCT score and NYHA class. The comparison between the two groups was assessed using Pearson’s chi-square test, student t test and Wilcoxon signed rank test as statistical approaches. Results At baseline, patients characteristics appeared homogeneous between the two groups and non-statistically significant increase of FVC was observed in both groups at month 12 and month 24. In the group treated with MMF no patient has deteriorated in NYHA functional class, respiratory tests showed an average stability pulmonary function and no patient has progressed in the TC score. In the control group, treated sequentially with CYC and AZA, has no been shown significant difference in NYHA class, respiratory tests, or in score TC. Comparing the two groups, no resounding significant difference has been highlighted. for the evaluated clinical parameters, showing similar efficacy of MMF compared to a pattern of traditional immunosuppressive therapy. In the group treated with MMF no patients reported adverse events than can cause discontinuation of treatment, while in the control group, 2 patients had to interrupt the CYC for leukopenia, and 5 had suspended AZA, 2 for hepatotoxicity and 3 for leukopenia. The difference between the two groups respect to adverse events was statistically significant in favor of MMF (p = 0.0046). Conclusions The data of our study suggest that the immunosuppressive therapy with MMF administered for a period of two years has led to a stabilization of ILD in a cohort of SSc patients, as shown by respiratory test, the HRCT score, and functional class NYHA. Similar results have been observed in another group cohort of patients treated sequentially for two years with CYC and AZA. Besides, MMF treated group presented a significant decrease of side effects, compared to the group treated with CYC and AZA

Characterization of the transcription factor encoding gene, KlADR1: metabolic role in Kluyveromyces lactis and expression in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Adr1 is a zinc-finger transcription factor involved in the transcriptional activation of ADH2. Deletion of KlADR1, its putative ortholog in Kluyveromyces lactis, led to reduced growth in glycerol, oleate and yeast extract-peptone medium suggesting, as in S. cerevisiae, its requirement for glycerol, fatty acid and nitrogen utilization. Moreover, growth comparison on yeast extract and peptone plates showed in K. lactis a KlAdr1-dependent growth trait not present in S. cerevisiae, indicating different metabolic roles of the two factors in their environmental niches. KlADR1 is required for growth under respiratory and fermentative conditions like KlADH, alcohol dehydrogenase genes necessary for metabolic adaptation during the growth transition. Using in-gel native alcohol dehydrogenase assay, we showed that this factor affected the Adh pattern by altering the balance between these activities. Since the activity most affected by KlAdr1 is KlAdh3, a deletion analysis of the KlADH3 promoter allowed the isolation of a DNA fragment through which KlAdr1 modulated its expression. The expression of the KlADR1-GFP gene allowed the intracellular localization of the factor in K. lactis and S. cerevisiae, suggesting in the two yeasts a common mechanism of KlAdr1 translocation under fermentative and respiratory conditions. Finally, the chimeric Kl/ScADR1 gene encoding the zinc- finger domains of KlAdr1 fused to the transactivating domains of the S. cerevisiae factor activated in Scadr1D the transcription of ADH2 in a ScAdr1-dependent fashion

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

Fluorometric detection of protein-ligand engagement: The case of phosphodiesterase5

Phosphodiesterases (PDEs) regulate the intracellular levels of cAMP and cGMP. The great clinical success of the PDE5 inhibitors, Sildenafil (Viagra), Vardenafil (Levitra) and Tadalafil (Cialis) has led to an increasing interest for this class of enzymes. Recent studies have shown a correlation between tumor growth and PDE5 overexpression, making PDE5-selective inhibitors promising candidates for cancer treatment. The search for such inhibitors rests today on radioactive assays. In this work, we exploit the conserved catalytic domain of the enzyme and propose a faster and safer method for detecting the binding of ligands and evaluate their affinities. The new approach takes advantage of Förster Resonance Energy Transfer (FRET) between, as the donor, a fluorescein-like diarsenical probe able to covalently bind a tetracysteine motif fused to the recombinant PDE5 catalytic domain and, as the acceptor, a rhodamine probe covalently bound to the pseudosubstrate cGMPS. The FRET efficiency decreases when a competitive ligand binds the PDE5 catalytic site and displaces the cGMPS-rhodamine conjugate. We have structurally investigated the PDE5/cGMPS-rhodamine complex by molecular modelling and have used the FRET signal to quantitatively characterize its binding equilibrium. Competitive displacement experiments were carried out with tadalafil and cGMPS. An adaptation of the competitive-displacement equilibrium model yielded the affinities for PDE5 of the incoming ligands, nano- and micromolar, respectively

Single-cell imaging of α and β cell metabolic response to glucose in living human Langerhans islets

Here we use a combination of two-photon Fluorescence Lifetime Imaging Microscopy (FLIM) of NAD(P)H free/bound ratio in living HIs with post-fixation, immunofluorescence-based, cell-type identification. FLIM allowed to measure variations in the NAD(P)H free/bound ratio induced by glucose; immunofluorescence data allowed to identify single α and β cells; finally, matching of the two datasets allowed to assign metabolic shifts to cell identity. 312 α and 654 β cells from a cohort of 4 healthy donors, 15 total islets, were measured. Both α and β cells display a wide spectrum of responses, towards either an increase or a decrease in NAD(P)H free/bound ratio. Yet, if single-cell data are averaged according to the respective donor and correlated to donor insulin secretion power, a non-random distribution of metabolic shifts emerges: robust average responses of both α and β cells towards an increase of enzyme-bound NAD(P)H belong to the donor with the lowest insulin-secretion power; by contrast, discordant responses, with α cells shifting towards an increase of free NAD(P)H and β cells towards an increase of enzyme-bound NAD(P)H, correspond to the donor with the highest insulin-secretion power. Overall, data reveal neat anti-correlation of tissue metabolic responses with respect to tissue insulin secretion power

MANP Activation Of The cGMP Inhibits Aldosterone Via PDE2 And CYP11B2 In H295R Cells And In Mice

Background: Aldosterone is a critical pathological driver for cardiac and renal diseases. We recently discovered that mutant atrial natriuretic peptide (MANP), a novel atrial natriuretic peptide (ANP) analog, possessed more potent aldosterone inhibitory action than ANP in vivo. MANP and natriuretic peptide (NP)-augmenting therapy sacubitril/valsartan are under investigations for human hypertension treatment. Understanding the elusive mechanism of aldosterone inhibition by NPs remains to be a priority. Conflicting results were reported on the roles of the pGC-A (particulate guanylyl cyclase A receptor) and NP clearance receptor in aldosterone inhibition. Furthermore, the function of PKG (protein kinase G) and PDEs (phosphodiesterases) on aldosterone regulation are not clear. Methods: In the present study, we investigated the molecular mechanism of aldosterone regulation in a human adrenocortical cell line H295R and in mice. Results: We first provided evidence to show that pGC-A, not NP clearance receptor, mediates aldosterone inhibition. Next, we confirmed that MANP inhibits aldosterone via PDE2 (phosphodiesterase 2) not PKG, with specific agonists, antagonists, siRNA silencing, and fluorescence resonance energy transfer experiments. Further, the inhibitory effect is mediated by a reduction of intracellular Ca2+ levels. We then illustrated that MANP directly reduces aldosterone synthase CYP11B2 (cytochrome p450 family 11 subfamily b member 2) expression via PDE2. Last, in PDE2 knockout mice, consistent with in vitro findings, embryonic adrenal CYP11B2 is markedly increased. Conclusions: Our results innovatively explore and expand the NP/pGC-A/3',5', cyclic guanosine monophosphate (cGMP)/PDE2 pathway for aldosterone inhibition by MANP in vitro and in vivo. In addition, our data also support the development of MANP as a novel ANP analog drug for aldosterone excess treatment

Insights on the association between thyroid diseases and colorectal cancer

Benign and malignant thyroid diseases (TDs) have been associated with the occurrence of extrathyroidal malignancies (EMs), including colorectal cancers (CRCs). Such associations have generated a major interest, as their characterization may provide useful clues regarding diseases’ etiology and/or progression, with the possible identification of shared congenital and environmental elements. On the other hand, elucidation of the underlying molecular mechanism(s) could lead to an improved and tailored clinical management of these patients and stimulate an increased surveillance of TD patients at higher threat of developing EMs. Here, we will examine the epidemiological, clinical, and molecular findings connecting TD and CRC, with the aim to identify possible molecular mechanism(s) responsible for such diseases’ relationship

Study of the diffuse gamma-ray emission from the Galactic plane with ARGO-YBJ

The events recorded by ARGO-YBJ in more than five years of data collection have been analyzed to determine the diffuse gamma-ray emission in the Galactic plane at Galactic longitudes 25{\deg} < l < 100{\deg} and Galactic latitudes . The energy range covered by this analysis, from ~350 GeV to ~2 TeV, allows the connection of the region explored by Fermi with the multi-TeV measurements carried out by Milagro. Our analysis has been focused on two selected regions of the Galactic plane, i.e., 40{\deg} < l < 100{\deg} and 65{\deg} < l < 85{\deg} (the Cygnus region), where Milagro observed an excess with respect to the predictions of current models. Great care has been taken in order to mask the most intense gamma-ray sources, including the TeV counterpart of the Cygnus cocoon recently identified by ARGO-YBJ, and to remove residual contributions. The ARGO-YBJ results do not show any excess at sub-TeV energies corresponding to the excess found by Milagro, and are consistent with the predictions of the Fermi model for the diffuse Galactic emission. From the measured energy distribution we derive spectral indices and the differential flux at 1 TeV of the diffuse gamma-ray emission in the sky regions investigated.Comment: 11 pages, 6 figures, published in AP

Desenvolvimento de itens de ensaio de proficiência para pesquisa de Salmonella spp. em matriz chocolate

The aim of this study was to develop lyophilized test items (TI) containing Salmonella spp., in chocolate matrix to be used in proficiency testing programs (PTP). Microbial analysis was conducted on samples of granulated chocolate to verify that the sample was free of the target microorganisms. Homogeneity and stability studies in long and short term were carried out to monitor TI quality; the presence of vacuum in the samples was also verified, to ensure the efficiency of the lyophilization process. The results of the microbial testing indicated no contamination by Salmonella spp.; thus, the sample was appropriate to be used as matrix. The lyophilization technique, using trehalose as cryoprotectant, has proven to be effective for desiccation of TI produced. The Salmonella batch proved to be sufficiently homogeneous, because the microorganism was present in all analyzed flasks. The batch was held stable at -20°C (five weeks) and -70°C (26 weeks). As for the transportation stability, the batch was considered stable at 4°C (in four days). The TI produced batch in this study showed a quality level that makes it suitable to be used in PTP, to contribute to the increasing reliability of the test results from laboratories and to provide subsidies for identification of problems and troubleshooting.O objetivo desse estudo foi desenvolver itens de ensaio (IE) liofilizados contendo Salmonella spp., em matriz chocolate, para utilização em ensaio de proficiência (EP). Foi realizada a análise microbiológica de uma amostra de chocolate granulado para verificar se estava livre do micro-organismo alvo. Para monitoramento da qualidade dos IE, realizou-se estudos de homogeneidade e estabilidade em longo e curto prazo, bem como verificou-se a presença de vácuo nas amostras garantindo a eficiência do processo de liofilização. A análise microbiológica do chocolate indicou ausência de contaminação por Salmonella spp., estando apto para ser utilizado como matriz. A técnica de liofilização, com uso de trealose como crioprotetor, se mostrou eficaz para dessecação dos IE produzidos. O lote produzido se apresentou suficientemente homogêneo, pois o micro-organismo estava presente em todos os frascos analisados. O lote se apresentou estável à temperatura de -20ºC (em cinco semanas) e -70ºC (em 26 semanas); na estabilidade de transporte, foi considerado estável a 4ºC (em quatro dias). O lote de IE produzido nesse estudo apresentou qualidade que o torna apto para uso em EP, o que visou contribuir para o aumento da confiabilidade dos resultados das análises dos laboratórios e propiciar subsídios para a identificação e solução de problemas