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Caloric restriction mimetics (CRMs) are promising molecules to prevent age-related diseases as they activate pathways driven by a true caloric restriction. Hydroxycitric acid (HCA) is considered a bona fide CRM since it depletes acetyl-CoA pools by acting as a competitive inhibitor of ATP citrate lyase (ACLY), ultimately repressing protein acetylation and promoting autophagy. Importantly, it can reduce inflammation and tumour development. In order to identify phenotypically relevant new HCA targets we have investigated HCA effects in Saccharomyces cerevisiae, where ACLY is lacking. Strikingly, the drug revealed a powerful anti-aging effect, another property proposed to mark bona fide CRMs. Chronological life span (CLS) extension but also resistance to acetic acid of HCA treated cells were associated to repression of cell apoptosis and necrosis. HCA also largely prevented cell deaths caused by a severe oxidative stress. The molecule could act widely by negatively modulating cell metabolism, similarly to citrate. Indeed, it inhibited both growth reactivation and the oxygen consumption rate of yeast cells in stationary phase. Genetic analyses on yeast CLS mutants indicated that part of the HCA effects can be sensed by Sch9 and Ras2, two conserved key regulators of nutritional and stress signal pathways of primary importance. Our data together with published biochemical analyses indicate that HCA may act with multiple mechanisms together with ACLY repression and allowed us to propose an integrated mechanistic model as a basis for future investigations. Electronic supplementary material The online version of this article (10.1007/s10495-020-01625-1) contains supplementary material, which is available to authorized users.

Aspirin and its main metabolite salicylate are promising molecules in preventing cancer and metabolic diseases. S. cerevisiae cells have been used to study some of their effects: (i) salicylate induces the reversible inhibition of both glucose transport and the biosyntheses of glucose-derived sugar phosphates, (ii) Aspirin/salicylate causes apoptosis associated with superoxide radical accumulation or early cell necrosis in MnSOD-deficient cells growing in ethanol or in glucose, respectively. So, treatment with (acetyl)-salicylic acid can alter the yeast metabolism and is associated with cell death. We describe here the dramatic effects of salicylate on cellular control of the exit from a quiescence state. The growth recovery of long-term stationary phase cells was strongly inhibited in the presence of salicylate, to a degree proportional to the drug concentration. At high salicylate concentration, growth reactivation was completely repressed and associated with a dramatic loss of cell viability. Strikingly, both of these phenotypes were fully suppressed by increasing the cAMP signal without any variation of the exponential growth rate. Upon nutrient exhaustion, salicylate induced a premature lethal cell cycle arrest in the budded-G2/M phase that cannot be suppressed by PKA activation. We discuss how the dramatic antagonism between cAMP and salicylate could be conserved and impinge common targets in yeast and humans. Targeting quiescence of cancer cells with stem-like properties and their growth recovery from dormancy are major challenges in cancer therapy. If mechanisms underlying cAMP-salicylate antagonism will be defined in our model, this might have significant therapeutic implications.

In Saccharomyces cerevisiae the subcellular distribution of Bcy1 is carbon source dependent. In glucose-grown cells, Bcy1 is almost exclusively nuclear, while it appears more evenly distributed between nucleus and cytoplasm in carbon source-derepressed cells. Here we show that phosphorylation of its N-terminal domain directs Bcy1 to the cytoplasm. Biochemical fractionation revealed that the cytoplasmic fraction contains mostly phosphorylated Bcy1, whereas unmodified Bcy1 is predominantly present in the nuclear fraction. Site-directed mutagenesis of two clusters (I and II) of serines near the N terminus to alanine resulted in an enhanced nuclear accumulation of Bcy1 in ethanol-grown cells. In contrast, substitutions to Asp led to a dramatic increase of cytoplasmic localization in glucose-grown cells. Bcy1 modification was found to be dependent on Yak1 kinase and, consequently, in ethanol-grown yak1 cells the Bcy1 remained nuclear. A two-hybrid screen aimed to isolate genes encoding proteins that interact with the Bcy1 N-terminal domain identified Zds1. In ethanol-grown zds1 cells, cytoplasmic localization of Bcy1 was largely absent, while overexpression of ZDS1 led to increased cytoplasmic Bcy1 localization. Zds1 does not regulate Bcy1 modification since this was found to be unaffected in zds1 cells. However, in zds1 cells cluster II-mediated, but not cluster I-mediated, cytoplasmic localization of Bcy1 was found to be absent. Altogether, these results suggest that Zds1-mediated cytoplasmic localization of Bcy1 is regulated by carbon source-dependent phosphorylation of cluster II serines, while cluster I acts in a Zds1-independent manner.

Cl- apically enters the epithelium of rabbit gallbladder by a Na+-Cl- symport, sensitive to hydrochlorothiazide (HCTZ). Since HCTZ also activates an apical SITS-sensitive Cl- conductance (G(Cl)), the symport inhibition might be merely due to a short circuit of the symport by G(Cl) rather than to a direct action of HCTZ on the symporter. To examine whether the symport is directly inhibited by HCTZ and whether the symporter belongs to the family of thiazide-sensitive cotransporters (TSC), radiochemical measurements of the apical Cl- uptake, electrophysiological determinations of intracellular Cl- and Na+ activities (a(i,Cl) and a(i,Na)) with selective theta microelectrodes and molecular biology methods were used. The 13Cl- uptake proved to be a measurement of the apical unidirectional Cl- influx (Jmc) and of the symport only (without backflux components), with measuring times of 45 sec under all experiment conditions; its inhibition by HCTZ was unaffected by G(Cl) activation or abolition. After HCTZ treatment the decrease in a(i,Cl) (measured as the initial rate or in 3 min) was larger than the decrease in a(i,Na). The difference was reduced to one third in a group of epithelia in which the elicited G(Cl) was reduced to one third; moreover it was abolished in any case when G(Cl) was abolished with 10(-4) M SITS. The SITS-insensitive rate of a(i,Cl) decrease was equal to that of the a(i,Na) decrease in any case. Thus the a(i,Cl) decrease displays a component dependent on G(Cl) activation and a second component dependent on symport inhibition. Using the RT-PCR technique a cDNA fragment was obtained that was 99% identical to the corresponding region of the rabbit renal TSC isoform. The results indicate that in rabbit gallbladder epithelium HCTZ displays a dual action, namely G(Cl) activation and Na+-Cl- symport inhibition. This Na+-Cl- symporter is the first TSC found to be functionally expressed in a nonrenal or nonrenal-like epithelium.

In budding yeast, cAMP-dependent protein kinase (PKA) plays a central role in the nutritional control of metabolism, cell cycle, and transcription. This study shows that both the regulatory subunit Bcy1p and the catalytic subunit Tpk1p associated with it are predominantly localized in the nucleus of rapidly growing cells. Activation of nuclear PKA by cAMP leads to fast entry of a significant part of Tpk1p into the cytoplasm, while the regulatory subunit remains nuclear. In contrast to rapidly proliferating cells, both Bcy1p and Tpk1p are distributed over nucleus and cytoplasm in cells growing on a nonfermentable carbon source or in stationary phase cells. These results demonstrate that at least two different mechanisms determine the subcellular localization of PKA; cAMP controls the localization of Tpk1p, and the carbon source determines that of Bcy1p. The N-terminal domain of Bcy1p serves to target it properly during logarithmic and stationary phase. Studies with Bcy1p mutant versions unable to concentrate in the nucleus revealed that cells producing them are less viable in stationary phase than wild type cells, display delayed reproliferation following transfer to fresh growth medium, and, as diploids, exhibit reduced efficiency of sporulation.

Angiogenesis plays a crucial role in tumor growth and progression. Low expression of mineralocorticoid receptor (MR) in several malignant tumors correlates with disease recurrence and overall survival. Previous studies have shown that MR expression is decreased in colorectal cancer (CRC). Here we hypothesize that decreased MR expression can contribute to angiogenesis and poor patient survival in colorectal malignancies. In a cohort of CRC patients, we analyzed tumor MR expression, its correlation with tumor microvascular density and its impact on survival. Subsequently, we interrogated the role of MR in angiogenesis in an in vitro model, based on the colon cancer cell line HCT116, ingenierized to re-express a physiologically controlled MR. In CRC, decreased MR expression was associated with increased microvascular density and poor patient survival. In pchMR transfected HCT116, aldosterone or natural serum steroids largely inhibited mRNA expression levels of both VEGFA and its receptor 2/KDR. In CRC, MR activation may significantly decrease angiogenesis by directly inhibiting dysregulated VEGFA and hypoxia-induced VEGFA mRNA expression. In addition, MR activation attenuates the expression of the VEGF receptor 2/KDR, possibly dampening the activation of a VEGFA/KDR dependent signaling pathway important for the survival of tumor cells under hypoxic conditions.

A YEAST cell becomes committed to the cell division cycle only if it grows to a critical size1–4 and reaches a critical rate of protein synthesis5,6. The coordination between growth and division takes place at a control step during the Gl phase of the cell cycle called Start4. It relies on the Gl-specific cyclins encoded by CLN1, 2 and 3, which trigger Start through the activation of the Cdc28 protein kinase. In fact, the Cln cyclins are rate-limiting for Start execution and depend on growth7–10. Here we report that the cyclic AMP signal pathway11 modulates the dependency of Cln cyclins on growth. In particular, more growth is required to trigger Start because CLN1 and CLN2 are repressed by the cAMP signal, thus explaining the previously observed cAMP-dependent increase of the critical size and critical rate of protein synthesis12. Cln3 is not inhibited by the cAMP pathway and counteracts this mechanism by partially mediating the growth-dependent expression of other Gl cyclins.

Barrett's esophagus metaplasia is a pre-cancerous condition caused by chronic esophagitis. Chromosomal instability (CIN) is common in Barrett's cells: therefore, we investigated the possible presence of centrosomal aberrations (a main cause of CIN) by centrosomal protein immunostaining in paraffined esophageal samples of patients who developed a Barrett's adenocarcinoma. In most (55%) patients, alterations of the pericentriolar material (PCM) signals were evident and consistently marked the transition between normal epithelium to metaplasia. The alterations could even be found in adjacent native squamous epithelium, Barrett's mucosa and submucosal gland cells, as well as in the basal/epibasal layers of the mucosa and submucosal gland duct, which are the regions hosting esophageal stem and progenitor cells. These findings strongly support the hypothesis that the three esophageal histotypes (one being pathological) can have a common progenitor. Surprisingly, PCM defective signal eventually decreased with neoplastic progression, possibly to enhance the genome stability of advanced cancer cells. Importantly, PCM altered signals in Barrett's mucosa and their apparent evolution in successive histopathological steps were correlated to adenocarcinoma aggressiveness, suggesting PCM as a possible prognostic marker for tumor relapse. Extending our observations in a prospective study might help in the development of new prevention protocols for adenocarcinoma patients.

Skeletal muscle regeneration relies on satellite cells, a population of myogenic precursors. Inflammation also plays a determinant role in the process, as upon injury, macrophages are attracted by the damaged myofibers and the activated satellite cells and act as key elements of dynamic muscle supportive stroma. Yet, it is not known how macrophages interact with the more profound stem cells of the satellite cell niche. Here we show that in the presence of a murine macrophage conditioned medium (mMCM) a subpopulation of multipotent cells could be selected and expanded from adult rat muscle. These cells were small, round, poorly adhesive, slow-growing and showed mesenchymal differentiation plasticity. At the same time, mMCM showed clear myogenic capabilities, as experiments with satellite cells mechanically isolated from suspensions of single myofibers showed that the macrophagic factors inhibited their tendency to shift towards adipogenesis. In vivo, intramuscular administrations of concentrated mMCM in a rat model of extensive surgical ablation dramatically improved muscle regeneration. Altogether, these findings suggest that macrophagic factors could be of great help in developing therapeutic protocols with myogenic stem cells.

Antisense-mediated exon skipping holds great potential for the treatment of DMD. In mdx mice, functional recovery of skeletal muscle has been obtained upon systemic delivery of ‘‘naked” oligonucleotides or viral vectors encoding for antisense snRNAs. However, amongst the studies reported so far, which used either neonatal or young adult animals – only one achieved dystrophin restoration in cardiac muscle, using an adeno-associated vector. Here we report the in vivo delivery of morpholino oligos in aged mdx mice, both in skeletal muscle, via intra-arterial injection, and in cardiac muscle, via intra-muscular injection. Localized intra-arterial delivery yielded high levels of dystrophin restoration and just two doses of 100 lg each resulted into detectable force recovery in the EDL muscles of treated limbs. On the other hand, upon intra-cardiac injections in the left ventricle wall the skipping effect was much lower than what obtained in tibialis anterior muscles injected with comparable amounts of oligos. This latter finding suggests that even upon direct delivery antisensemediated dystrophin restoration in cardiac muscle might suffer from limitations that do not exist in skeletal muscle. 2008 Elsevier B.V. All rights reserved.

Myoblast transplantation is a potentially useful therapeutic tool in muscle diseases, but the lack of an efficient delivery system has hampered its application. Here we have combined cell biology and polymer processing to create an appropriate microenvironment for in vivo transplantation of murine satellite cells (mSCs). Cells were prepared from single muscle fibers derived from C57BL/6-Tgn enhanced green fluorescent protein (GFP) transgenic mice. mSCs were expanded and seeded within micro-patterned polyglycolic acid 3-dimensional scaffolds fabricated using soft lithography and thermal membrane lamination. Myogenicity was then evaluated in vitro using immunostaining, flow cytometry, and reverse transcription polymerase chain reaction analyses. Scaffolds containing mSCs were implanted in pre-damaged tibialis anterior muscles of GFP-negative syngenic mice. Cells detached from culture dishes were directly injected into contra-lateral limbs as controls. In both cases, delivered cells participated in muscle regeneration, although scaffold-implanted muscles showed a much higher number of GFP-positive fibers in CD57 mice. These findings suggest that implantation of cellularized scaffolds is better than direct injection for delivering myogenic cells into regenerating skeletal muscle.

In Saccharomyces cerevisiae the CDC25 protein is a positive regulator of RAS/cAMP pathway [1-4], enhancing the GDP-releasing rate of RAS2 protein [5]. In this work we have tried to detect a direct interaction between CDC25 and RAS2 gene products. The results indicate that both the whole RAS2 protein and a truncated version that lacks approximately 25 C-terminal residues interact specifically with the CDC25 protein. On the contrary, a derivative of RAS2 that lacks the 112 C-terminal residues as well as the p21TI-ras is not able to bind the CDC25 protein in our assay conditions. The 310 C-terminal aminoacids of CDC25 bind RAS2 while a C-terminus deletion within this aminoacid stretch abolishes the binding. The possible physiological significance of these findings is discussed.

In Saccharomyces cerevisiae, cyclic AMP is required for cellular growth. In this study we show that cAMP also specifically inhibits the G1-S transition of the S. cerevisiae cell cycle by increasing the critical cell size required at start, the major yeast cell cycle control step. In fact: (a) addition of cAMP delays the time of entering into the S budded phase of small G1 cells, while it is ineffective on large fast-growing cells. (b) If cell growth is strongly depressed, cAMP permanently inhibits cell cycle commitment of cells arrested at the α-factor-sensitive step. The cell fraction inhibited by cAMP is inversely correlated with the average cell size of treated populations. (c) The critical protein content (Ps) and the critical cell volume (VB) required for budding in unperturbed exponentially growing yeast populations are largely increased by cAMP. On these bases, we propose a new cAMP role at start.

Strain 112 (RAS1 RAS2) contains a naturally occurring mutation which significantly retards processing of the RAS2 gene product. This mutation, resulting in the accumulation of precursor forms of RAS2 protein, has been assigned by genetic analysis to a single chromosomal locus distinct from the RAS2 locus. In addition to the known precursor molecule of 41000 daltons (p41), 112 cells accumulate within the soluble fraction an intermediate form of RAS2 (p40-1), which migrates, in SDS-polyacrylamide gel, between p41 and the fully processed, membrane-bound 40,000 daltons (p40) product. We propose for RAS2 protein processing the following sequence of events: p41 greater than p40-1 greater than p40 where p40-1 represents a RAS2 intermediate required for the targeting of the protein to the plasma membrane.

The MET2 gene of Saccharomyces cerevisiae, which codes for homoserine-O-acetyltransferase, a key enzyme in methionine biosynthesis, was isolated by complementation of a met2 mutant strain of S. cerevisiae with a yeast gene bank. A 3.9-kb genomic fragment contains the entire gene, as demonstrated by genetic and molecular analysis of the integrative transformants. A polyadenylated mRNA of 1700 nt is detected by Northern blot hybridization with a MET2 probe. The level of this mRNA decreases by addition of exogenous methionine or of S-adenosylmethionine, suggesting a transcriptional regulation. The level of specific mRNA and the enzyme activity found in transformants that bear the MET2 gene on a multicopy plasmid suggest that also a post-transcriptional regulatory mechanism may be operative in budding yeast.

A detailed kinetic analysis of the cell cycle of cdc25-1, RAS2Val-19, or cdc25-1/RAS2Val-19 mutants during exponential growth is presented. At the permissive temperature (24 degrees C), cdc25-1 cells show a longer G1/unbudded phase of the cell cycle and have a smaller critical cell size required for budding without changing the growth rate in comparison to an isogenic wild type. The RAS2Val-19 mutation efficiently suppresses the ts growth defect of the cdc25-1 mutant at 36 degrees C and the increase of G1 phase at 24 degrees C. Moreover, it causes a marked increase of the critical cell mass required to enter into a new cell division cycle compared with that of the wild type. Since the critical cell mass is physiologically modulated by nutritional conditions, we have also studied the behavior of these mutants in different media. The increase in cell size caused by the RAS2Val-19 mutation is evident in all tested growth conditions, while the effect of cdc25-1 is apparently more pronounced in rich culture media. CDC25 and RAS2 gene products have been showed to control cell growth by regulating the cyclic AMP metabolic pathway. Experimental evidence reported herein suggests that the modulation of the critical cell size by CDC25 and RAS2 may involve adenylate cyclase.

Despite growing concern about electromagnetic radiation, the interaction between 50- to 60-Hz fields and biological structures remains obscure. Epidemiological studies have failed to prove a significantly correlation between exposure to radiation fields and particular pathologies. We demonstrate that a 50- to 60-Hz magnetic field interacts with cell differentiation through two opposing mechanisms: it antagonizes the shift in cell membrane surface charges that occur during the early phases of differentiation and it modulates hyperpolarizing K channels by increasing intracellular Ca. The simultaneous onset of both mechanisms prevents alterations in cell differentiation. We propose that cells are normally protected against electromagnetic insult. Pathologies may arise, however, if intracellular Ca regulation or K channel activation malfunctions.

Addition of exogenous cAMP to cultures of the start mutant cdc25-1 of Saccharomyces cerevisiae shifted to restrictive temperature causes a partial reversion of the mutated phenotype, with a marked increase of the percentage of budded cells. This effect is coupled to a progression in the cell cycle, as demonstrated by DNA histograms obtained by flow cytometry. Moreover cdc25 cells have a high intracellular cAMP content also at restrictive temperature, and no change in the cAMP content was seen during a transition from restrictive to permissive temperature. These data suggest that CDC25 gene product allows cell proliferation by interacting with a cAMP-mediated mechanism.

A major control point of the cell cycle in Saccharomyces cerevisiae is a G1 event called ‘start’. At start a yeast cell integrates external and internal signals and decides to progress toward mitosis or to choose alternative pathways such as sporulation, conjugation etc. cdc25 is a class II temperature-sensitive start mutant that blocks at restrictive temperature in G1 as round unbudded cells. The arrest of the cell cycle appears to be independent of the carbon and nitrogen sources, and the cell wall of cdc25-arrested cells shows changes similar to those found in cells undergoing entry in to the stationary phase. After a shift to 36 °C the increase in cell number of cdc25 cultures is gradually inhibited. The nuclear division cycle appears to be inhibited immediately after the shift and the percentage of budded cells decreases, while cytoplasmic growth, monitored either as increase of adsorbance at 450 nm or as protein accumulation, continues for many hours leading to a progressive increase of mean cell volume and mean protein content per cell. The stable RNA accumulation instead is immediately inhibited and this is partially due to a 50% inhibition of ribosomal RNA synthesis, while the rate of synthesis of ds-killer RNA is relatively unaffected. These data suggest that the CDC25 gene product could be a part of a mechanism that leads yeast cells to choose between the progression towards DNA replication and cell division or to enter into the stationary phase. This mechanism appears to turn off both rRNA accumulation and cell-cycle progression and to activate differentiative pathways in response to environmental restriction.

To isolate the CDC25 gene of Saccharomyces cerevisiae we have transformed a cdc25-1, trp1 strain with a yeast gene bank constructed in YRp7 vector, selecting trp+ clones able to grow at restrictive temperature. From several independent positive clones we have recovered a plasmid, called pDGEm-1, that bears a 5-kb genomic fragment and is able to give a full complementation of the cdc25-1 mutation. The genomic sequence has been subcloned and a good complementation obtained with a 2-kb fragment. Several stable integrative trp+, cdc+ transformants have been constructed. Their genetic and molecular analysis indicates that we have cloned the true CDC25 gene. Northern blot hybridization has revealed the presence of a 5-kb mRNA transcribed by the CDC25 gene. This mRNA is also present in nitrogen-starved cells and during the re-enter in cell cycle from starvation, suggesting a constitutive transcription. Transformants bearing the cloned sequence on multicopy plasmid and integrative transformants that bear the CDC25 gene, flanked by plasmid sequences, show an altered control of the cell cycle and fail to arrest in G1 unbudded phase in stationary phase conditions.

Background Barrett's mucosa is the precursor of esophageal adenocarcinoma. The molecular mechanisms behind Barrett's carcinogenesis are largely unknown. Experimental models of longstanding esophageal reflux of duodenal-gastric contents may provide important information on the biological sequence of the Barrett's oncogenesis. Methods The expression of CDX2 hox-gene product was assessed in a rat model of Barrett's carcinogenesis. Seventy-four rats underwent esophago-jejunostomy with gastric preservation. Excluding perisurgical deaths, the animals were sacrificed at various times after the surgical treatment (Group A: 30 weeks). Results No Cdx2 expression was detected in either squamous epithelia of the proximal esophagus or squamous cell carcinomas. De novo Cdx2 expression was consistently documented in the proliferative zone of the squamous epithelium close to reflux ulcers (Group A: 68%; Group B: 64%; Group C: 80%), multilayered epithelium and intestinal metaplasia (Group A: 9%; Group B: 41%; Group C: 60%), and esophageal adenocarcinomas (Group B: 36%; Group C: 35%). A trend for increasing overall Cdx2 expression was documented during the course of the experiment (p = 0.001). Conclusion De novo expression of Cdx2 is an early event in the spectrum of the lesions induced by experimental gastro-esophageal reflux and should be considered as a key step in the morphogenesis of esophageal adenocarcinoma.