Intracrine endorphinergic systems in modulation of myocardial differentiation

A wide variety of peptides not only interact with the cell surface, but govern complex signaling from inside the cell. This has been referred to as an "intracrine" action, and the orchestrating molecules as "intracrines". Here, we review the intracrine action of dynorphin B, a bioactive end-product of the prodynorphin gene, on nuclear opioid receptors and nuclear protein kinase C signaling to stimulate the transcription of a gene program of cardiogenesis. The ability of intracrine dynorphin B to prime the transcription of its own coding gene in isolated nuclei is discussed as a feed-forward loop of gene expression amplification and synchronization. We describe the role of hyaluronan mixed esters of butyric and retinoic acids as synthetic intracrines, controlling prodynorphin gene expression, cardiogenesis, and cardiac repair. We also discuss the increase in prodynorphin gene transcription and intracellular dynorphin B afforded by electromagnetic fields in stem cells, as a mechanism of cardiogenic signaling and enhancement in the yield of stem cell-derived cardiomyocytes. We underline the possibility of using the diffusive features of physical energies to modulate intracrinergic systems without the needs of viral vector-mediated gene transfer technologies, and prompt the exploration of this hypothesis in the near future

Cytochalasin B Influences Cytoskeletal Organization and Osteogenic Potential of Human Wharton’s Jelly Mesenchymal Stem Cells

Among perinatal stem cells of the umbilical cord, human Wharton’s jelly mesenchymal stem cells (hWJ-MSCs) are of great interest for cell-based therapy approaches in regenerative medicine, showing some advantages over other MSCs. In fact, hWJ-MSCs, placed between embryonic and adult MSCs, are not tumorigenic and are harvested with few ethical concerns. Furthermore, these cells can be easily cultured in vitro, maintaining both stem properties and a high proliferative rate for several passages, as well as trilineage capacity of differentiation. Recently, it has been demonstrated that cytoskeletal organization influences stem cell biology. Among molecules able to modulate its dynamics, Cytochalasin B (CB), a cyto-permeable mycotoxin, influences actin microfilament polymerization, thus affecting several cell properties, such as the ability of MSCs to differentiate towards a specific commitment. Here, we investigated for the first time the effects of a 24 h-treatment with CB at different concentrations (0.1–3 μM) on hWJ-MSCs. CB influenced the cytoskeletal organization in a dose-dependent manner, inducing changes in cell number, proliferation, shape, and nanomechanical properties, thus promoting the osteogenic commitment of hWJ-MSCs, as confirmed by the expression analysis of osteogenic/autophagy markers

Cytochalasin B Influences Cytoskeletal Organization and Osteogenic Potential of Human Wharton's Jelly Mesenchymal Stem Cells

Among perinatal stem cells of the umbilical cord, human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) are of great interest for cell-based therapy approaches in regenerative medicine, showing some advantages over other MSCs. In fact, hWJ-MSCs, placed between embryonic and adult MSCs, are not tumorigenic and are harvested with few ethical concerns. Furthermore, these cells can be easily cultured in vitro, maintaining both stem properties and a high proliferative rate for several passages, as well as trilineage capacity of differentiation. Recently, it has been demonstrated that cytoskeletal organization influences stem cell biology. Among molecules able to modulate its dynamics, Cytochalasin B (CB), a cyto-permeable mycotoxin, influences actin microfilament polymerization, thus affecting several cell properties, such as the ability of MSCs to differentiate towards a specific commitment. Here, we investigated for the first time the effects of a 24 h-treatment with CB at different concentrations (0.1-3 mu M) on hWJ-MSCs. CB influenced the cytoskeletal organization in a dose-dependent manner, inducing changes in cell number, proliferation, shape, and nanomechanical properties, thus promoting the osteogenic commitment of hWJ-MSCs, as confirmed by the expression analysis of osteogenic/autophagy markers

Complexity of Bidirectional Transcription and Alternative Splicing at Human RCAN3 Locus

Human RCAN3 (regulator of calcineurin 3) belongs to the human RCAN gene family

Albumin as marker for susceptibility to metal ions in metal-on-metal hip prosthesis patients

Metal-on-metal (MoM) hip prostheses are known to release chromium and cobalt (Co), which negatively affect the health status, leading to prosthesis explant. Albumin (ALB) is the main serum protein-binding divalent transition metals. Its binding capacity can be affected by gene mutations or modification of the protein N-terminal region, giving the ischaemia-modified albumin (IMA). This study evaluated ALB, at gene and protein level, as marker of individual susceptibility to Co in MoM patients, to understand whether it could be responsible for the different management of this ion. Co was measured in whole blood, serum and urine of 40 MoM patients. A mutational screening of ALB was performed to detect links between mutations and metal binding. Finally, serum concentration of total ALB and IMA were measured. Serum total ALB concentration was in the normal range for all patients. None of the subjects presented mutations in the investigated gene. Whole blood, serum and urine Co did not correlate with serum total ALB or IMA, although IMA was above the normal limit in most subjects. The individual susceptibility is very important for patients' health status. Despite the limited results of this study, we provide indications on possible future investigations on the toxicological response to Co

Identification and analysis of human RCAN3 (DSCR1L2) mRNA and protein isoforms

Human RCAN3 (Regulator of calcineurin 3; previously known as DSCR1L2, Down syndrome critical region gene 1-like 2) is a five-exon gene mapped on chromosome 1 and belongs to the human RCAN gene family which also includes RCAN1 and RCAN2. The novel denomination RCAN for genes and proteins, instead of DSCR1L (Down syndrome critical region gene 1-like) has recently been widely discussed. The aim of the present work was to perform a multiple approach analysis of five RCAN3 mRNA and encoded protein isoforms, two of which have been identified for the first time in this research. The two new RCAN3 mRNA isoforms, RCAN3-2,4,5, which lacks exon 3, and RCAN3-2,3,5, which lacks exon 4, were identified during RCAN3 RT-PCR (reverse transcription-polymerase chain reaction) cloning, the product of which unexpectedly revealed the presence of five isoforms as opposed to the three previously known. In order to analyze the expression pattern of the five RCAN3 mRNA isoforms in seven different human tissues, a quantitative relative RT-PCR was performed: interestingly, all isoforms are present in all tissues investigated, with a statistically significant constant prevalence of RCAN3 isoform (the most complete, \u201creference\u201d isoform). The RCAN3 locus expression level was comparable in all seven tissues analyzed, considering all isoforms, which indicates a ubiquitous expression of this human RCAN family member. To date two possible interactors have been described for this protein: human cardiac troponin I (TNNI3) and calcineurin. Here we report the interaction between the new RCAN3 variants and TNNI3, demonstrated by both yeast cotransformation and by the GST (glutathione-sepharose transferase) fusion protein assay, as was to be expected from the presence of exon 2 whose product has been seen to be sufficient for binding to TNNI3

Comparison of oxidative stress effects on senescence patterning of human adult and perinatal tissue-derived stem cells in short and long-term cultures

Human Mesenchymal Stem Cells (hMSCs) undergo senescence in lifespan. In most clinical trials, hMSCs experience long-term expansion ex vivo to increase cell number prior to transplantation, which unfortunately leads to cell senescence, hampering post-transplant outcomes. Hydrogen peroxide (H 2 O 2 ) in vitro represents a rapid, time and cost-effective tool, commonly used as oxidative stress tantalizing the stem cell ability to cope with a hostile environment, recapitulating the onset and progression of cellular senescence. Here, H 2 O 2 at different concentrations (ranging from 50 to 400 μM) and time exposures (1 or 2 hours-h), was used for the first time to compare the behavior of human Adipose tissue-derived Stem Cells (hASCs) and human Wharton’s Jelly-derived MSCs (hWJ-MSCs), as representative of adult and perinatal tissue-derived stem cells, respectively. We showed timely different responses of hASCs and hWJ-MSCs at low and high subculture passages, concerning the cell proliferation, the cell senescence-associated β-Galactosidase activity, the capability of these cells to undergo passages, the morphological changes and the gene expression of tumor protein p53 (TP53, alias p53) and cyclin dependent kinase inhibitor 1A (CDKN1A, alias p21) post H 2 O 2 treatments. The comparison between the hASC and hWJ-MSC response to oxidative stress induced by H 2 O 2 is a useful tool to assess the biological mechanisms at the basis of hMSC senescence, but it could also provide two models amenable to test in vitro putative anti-senescence modulators and develop anti-senescence strategies

Identification of housekeeping genes suitable for gene expression analysis in the zebrafish

Housekeeping (HK) genes are constitutively expressed in order to maintain cellular function. They produce the minimal essential transcripts necessary for normal cellular physiology. Wide range expression, stable expression level and high expression level are independent features of a single gene expression and are all desirable for the definition of an "ideal" HK. Recent studies have questioned the possible existence of "ideal" HK mRNAs, mainly because of the wide expression conditions variability. This would imply that for each investigated organism the suitability of a putative HK should be verified. We perform a systematic analysis to identify "optimal" HK genes in Danio rerio (zebrafish), to be used in expression analyses conducted on embryos/larvae at different developmental stages, as well as on differentiated adult tissues from single donors. The expression pattern of candidate genes, selected on the basis of the literature available and of ad hoc bioinformatics analysis, was assessed by quantitative relative RT-PCR in an RNA panel, including six different embryo/larvae developmental stages and six adult tissues. Statistical analysis was performed to identify genes with the lowest expression standard deviation in the studied panel. Our results showed that beta-actin 2 (bactin2) is the mRNA with the lowest variability of expression

Identificazione ed analisi delle isoforme geniche e proteiche di RCAN3 (DSCR1L2)

Il gene umano RCAN3 (Regulator of Calcineurin 3, anche conosciuto come DSCR1L2), localizzato sul cromosoma 1 (1p36.11), appartiene alla famiglia genica RCAN, di cui gli altri membri sono i geni RCAN1 ed RCAN2. Recentemente il Comitato internazionale HUGO ha proposto per i geni e le proteine di questa famiglia la nomenclatura presentata per la prima volta in questo lavoro; nomenclatura che \ue8 stata ampiamente discussa anche nell\u2019ambito di un \u201cForum article\u201d in via di pubblicazione. Il gene RCAN3 \ue8 costituito da 5 esoni e ad oggi sono state identificate due isoforme alternative: RCAN3-2,3,4b,5, che manca di 30 nucleotidi all\u2019inizio dell\u2019esone 4 e codifica per una proteina priva di 10 amminoacidi nella sua porzione centrale (RCAN3-2,3,4b,5) ed RCAN3-2,5 che manca degli esoni 3-4 e codifica per una proteina di 115 amminoacidi, con una porzione proteica C-terminale diversa da RCAN3, a causa di uno scivolamento del modulo di lettura a partire dall\u2019esone 5 (RCAN3-2,5). Ad oggi pi\uf9 di un centinaio di lavori sono stati pubblicati sulla famiglia RCAN e nella maggior parte sono presentati studi sulle funzioni di RCAN1 ed RCAN2. Solo due lavori si sono occupati dei possibili ruoli di RCAN3. Recentemente il nostro gruppo di ricerca, mediante un saggio dei due ibridi condotto in una genoteca di cDNA di cuore umano, ha dimostrato che RCAN3 ed RCAN3-2,5 sono in grado di interagire con la troponina inibitoria cardiaca (TNNI3), una delle principali componenti dell\u2019apparato contrattile muscolare; inoltre Mulero e colleghi hanno identificato nella calcineurina, una fosfatasi calcio-calmodulina dipendente, un altro possibile interattore di RCAN3. Lo scopo del presente lavoro \ue8 stato condurre una analisi delle diverse isoforme di RCAN3 e delle rispettive proteine da un punto di vista molecolare, bioinformatico, di espressione genica e funzionale. In primo luogo sono state identificate grazie ad un clonaggio mediante RT-PCR due nuove isoforme di RCAN3, in accordo con l\u2019elevato numero di splicing dimostrato nei geni umani: RCAN3-2,4,5 che manca dell\u2019esone 3 ed RCAN3-2,3,5, che manca dell\u2019esone 4. Sequenze di cDNA per le isoforme alternative di RCAN3 sono state identificate solo in Homo sapiens, utilizzando come strumenti per tale ricerca il programma BLASTN, ECgene Browser e Genome Browser; solo per l\u2019isoforma RCAN3-2,4,5 sono state ritrovate 2 EST. In seguito \ue8 stata condotta una RT-PCR quantitativa relativa per analizzare il pattern di espressione delle 5 isoforme note in 7 tessuti umani normali (cuore, cervello, polmone, prostata, testicolo, leucociti, intestino tenue), basandosi su dati precedentemente raccolti che dimostravano come il gene RCAN3 fosse espresso in diversi tessuti.Tutte le isoforme sono risultate espresse nei singoli tessuti e in tutti i tessuti si \ue8 dimostrato che l\u2019isoforma pi\uf9 espressa \ue8 RCAN3. Inoltre il livello di espressione dell\u2019intero locus RCAN3 \ue8 risultato confrontabile in tutti i tessuti, dimostrandone l\u2019ubiquitariet\ue0. Infine, grazie ad un saggio di cotrasformazione in lievito e ad un saggio in vitro di legame alla GST, \ue8 stata dimostrata l\u2019interazione delle due nuove isoforme identificate RCAN3-2,4,5 e RCAN3-2,3,5 con la TNNI3, cos\uec come si poteva ipotizzare grazie alla presenza dell\u2019esone 2, il cui prodotto \ue8 ritenuto essere sufficiente per il legame a tale proteina cardiaca