Chronic hydroxytyrosol feeding modulates glutathione-mediated oxido-reduction pathways in adipose tissue: a nutrigenomic study.

BACKGROUND AND AIM: Hydroxytyrosol (HT) is the most prominent phenolic component of olives, olive oil, and their by-products, e.g. olive mill waste water. As the link between HT consumption (via extra virgin olive oil intake) and better cardiovascular prognosis is being scientifically validated, HT is entering the market as a potentially useful supplement for cardiovascular disease prevention. One of the target organs in cardiometabolic prevention is the adipose tissue, where inflammation, oxidative stress, and secretion of adipocytokines contribute to cardiovascular risk. METHODS AND RESULTS: We explored the nutrigenomic effects of long-term supplementation with nutritionally-relevant doses of HT, i.e. 0.03 gm% - with specific reference to the adipose tissue and glutathione metabolism - and we explored underlying mechanisms in vitro. We show that HT modulates the antioxidant network in the adipose tissue, as mediated by glutathione (GSH) and associated enzymes. We also confirmed the GSH-modulating activities of HT in cultured adipocytes, where low, physiological HT concentrations were able to blunt the H2O2-induced GSH/GSSG alteration indicative of oxidative stress. In terms of surrogate markers of cardiovascular disease, we recorded significantly decreased circulating leptin concentrations in mice fed with HT as compared with controls. CONCLUSIONS: HT - in nutritionally relevant amounts - is able to positively modulate the glutathione-driven antioxidant enzymatic machinery in the adipose tissue. Because HT is generally recognized as safe (GRAS) and exhibits an excellent safety profile in vitro and in vivo, its future employment as adjunct treatment of metabolic syndrome can be envisioned, pending specific trials.

Design of a custom-made device for real-time optical measurement of differential mineral concentrations in three-dimensional scaffolds for bone tissue engineering.

Monitoring bone tissue engineered (TEed) constructs during their maturation is important to ensure the quality of applied protocols. Several destructive, mainly histochemical, methods are conventionally used to this aim, requiring the sacrifice of the investigated samples. This implies (i) to plan several scaffold replicates, (ii) expensive and time consuming procedures and (iii) to infer the maturity level of a given tissue construct from a cognate replica. To solve these issues, non-destructive techniques such as light spectroscopy-based methods have been reported to be useful. Here, a miniaturized and inexpensive custom-made spectrometer device is proposed to enable the non-destructive analysis of hydrogel scaffolds. Testing involved samples with a differential amount of calcium salt. When compared to a reference standard device, this custom-made spectrometer demonstrates the ability to perform measurements without requiring elaborate sample preparation and/or a complex instrumentation. This preliminary study shows the feasibility of light spectroscopy-based methods as useful for the non-destructive analysis of TEed constructs. Based on these results, this custom-made spectrometer device appears as a useful option to perform real-time/in-line analysis. Finally, this device can be considered as a component that can be easily integrated on board of recently prototyped bioreactor systems, for the monitoring of TEed constructs during their conditioning.

Myogenic commitment of human stem cells by myoblasts Co-culture: a static vs. a dynamic approach.

An in-vitro model of human bone marrow mesenchymal stem cells (hBM-MSCs) myogenic commitment by synergic effect of a differentiation media coupled with human primary skeletal myoblasts (hSkMs) co-culture was developed adopting both conventional static co-seeding and perfused culture systems. Static co-seeding provided a notable outcome in terms of gene expression with a significant increase of Desmin (141-fold) and Myosin heavy chain II (MYH2, 32-fold) at day 21, clearly detected also by semi-quantitative immunofluorescence. Under perfusion conditions, myogenic induction ability of hSkMs on hBM-MSCs was exerted by paracrine effect with an excellent gene overexpression and immunofluorescence detection of MYH2 protein; furthermore, due to the dynamic cell culture in separate wells, western blot data were acquired confirming a successful cell commitment at day 14. A significant increase of anti-inflammatory cytokine gene expression, including IL-10 and IL-4 (15-fold and 11-fold, respectively) at day 14, with respect to the pro-inflammatory cytokines IL-12A (7-fold at day 21) and IL-1ß (1.4-fold at day 7) was also detected during dynamic culture, confirming the immunomodulatory activity of hBM-MSCs along with commitment events. The present study opens interesting perspectives on the use of dynamic culture based on perfusion as a versatile tool to study myogenic events and paracrine cross-talk compared to the simple co-seeding static culture.

Development and validation of an in-silico tool for the study of therapeutic agents in 3D cell cultures.

Computational models constitute a fundamental asset for cancer research and drug R&D, as they provide controlled environments for testing of hypotheses and are characterized by the total knowledge of the system. These features are particularly useful for 3D cell culture models where a complex interaction among cells and their environments ensues. In this work, we present a programmable simulator capable of reproducing the behavior of cells cultured in 3D scaffolds and their response to pharmacological treatment. This system will be shown to be able to accurately describe the temporal evolution of the density of a population of MDA-MB-231 cells following their treatment with different concentrations of doxorubicin, together with a newly described drug-resistance mechanism and potential re-sensitization strategy. An extensive technical description of this model will be coupled to its experimental validation and to an analysis aimed at identifying which variables and behaviors account for differences in the response to treatment. Comprehensively, this work contributes to the growing field of integrated in-silico/in-vitro analysis of biological processes which has great potential for both the increase of our scientific knowledge and the development of novel, more effective treatments.

minifly, a Drosophila gene required for ribosome biogenesis.

We report here the genetic, molecular, and functional characterization of the Drosophila melanogaster minifly (mfl) gene. Genetic analysis shows that mfl is essential for Drosophila viability and fertility. While P-element induced total loss-of-function mutations cause lethality, mfl partial loss-of-function mutations cause pleiotropic defects, such as extreme reduction of body size, developmental delay, hatched abdominal cuticle, and reduced female fertility. Morphological abnormalities characteristic of apoptosis are found in the ovaries, and a proportion of eggs laid by mfl mutant females degenerates during embryogenesis. We show that mfl encodes an ubiquitous nucleolar protein that plays a central role in ribosomal RNA processing and pseudouridylation, whose known eukaryotic homologues are yeast Cfb5p, rat NAP57 and human dyskerin, encoded by the gene responsible for the X-linked dyskeratosis congenita disease. mfl genetic analysis represents the first in vivo functional characterization of a member of this highly conserved gene family from higher eukaryotes. In addition, we report that mfl hosts an intron encoded box H/ACA snoRNA gene, the first member of this class of snoRNAs identified so far from Drosophila.

Repeated glucose stimulation reveals distinct and lasting secretion patterns of individual rat pancreatic B cells.

To determine whether pancreatic B cells show a constant secretion pattern during repeated stimulations, we have used a sequential hemolytic plaque assay to monitor their individual insulin release during several successive 30-min incubations in the presence of 16.7 mM glucose. We have found that the total B cell secretion did not vary significantly in these successive glucose stimulations and that, under these conditions, the majority of B cells that were stimulated to release insulin during the first incubation also secreted during the second, third, and, when this was tested, during the fourth incubation. Similarly, most of the B cells that did not release detectable amounts of insulin during the first incubation did not secrete also during the two (or three) subsequent secretion tests. Together, the two groups of B cells that showed a constant secretory pattern, represented approximately 75% of the entire B cell population. The remaining 25% of B cells shifted from a secreting to a non-secreting state, or vice versa, from one incubation to another. These observations were made under three different time frames in which we tested single B cells as well as B cell clusters at rather different intervals. These findings support the existence of distinct B cell subpopulations differing lastingly in their ability to secrete insulin in response to glucose.

Cyclic adenosine monophosphate prevents the glucocorticoid-mediated inhibition of insulin gene expression in rodent islet cells.

Dexamethasone negatively regulates insulin gene expression in HIT-15 cells. In vivo, however, an excess of glucocorticoids results in an increase in insulin biosynthesis and peripheral hyperinsulinemia. To resolve this contradiction, we have studied the effects of dexamethasone in primary rat islet cells. We show here that dexamethasone decreases insulin mRNA levels in single islet cells, as in HIT-15 cells, but does not affect these levels in reaggregated islet cells and increases them in intact islets of Langerhans. Because cAMP is an important regulator of insulin gene expression and intracellular cAMP content may be decreased in single beta cells, we investigated whether cAMP could prevent the inhibitory effect of dexamethasone on insulin mRNA levels. In the presence of cAMP analogues, the inhibitory action of dexamethasone was not only prevented, but insulin mRNA increased to levels comparable to those observed when cAMP analogues were used alone. We conclude that the insulin gene is negatively regulated by dexamethasone in single islet cells, but that other factors such as cAMP prevent this effect when the native environment of islet cells is preserved. Our results indicate that insulin gene regulation is influenced by cell to cell contacts within the islet, and that intracellular cAMP levels might be influential in this regulation.

Rapid and reversible secretion changes during uncoupling of rat insulin-producing cells.

To determine whether insulin secretion is affected by a blockage of gap junctions between B cells, we have studied the secretion of rat pancreatic islets of Langerhans, primary dispersed islet cells, and cells of the RINm5F line, during short-term exposure to heptanol. Within minutes, this alkanol blocked gap junctions between the B cells of intact islets and abolished their normal secretory response to glucose. These two changes were rapidly and fully reversible after return of the islets to control medium. We further found that heptanol had no significant effect on the glucose-stimulated secretion of single B cells but inhibited that of B cell pairs. In the clone of RINm5F cells, whose junctional coupling and D-glyceraldehyde-induced stimulation of insulin release by aggregated cells were also inhibited by heptanol, this alkanol did not perturb intracellular pH and Ca2+ and the most distal steps of the secretion pathway. In summary, a gap junction blocker affected the secretion of insulin-producing cells by a mechanism which is dependent on cell contact and is not associated with detectable pleiotropic perturbations of the cell secretory machinery. The data provide evidence for the involvement of junctional coupling in the control of insulin secretion.

Ion channels of glucose-responsive and -unresponsive beta-cells.

To assess whether different electrophysiological characteristics could account for the heterogeneous secretion of individual beta-cells in vitro, we used patch-clamp configurations to study currents in plaque-forming (insulin-secreting) and non-plaque-forming rat pancreatic beta-cells that were distinguished in a reverse hemolytic plaque assay (RHPA) after a 30-min stimulation by 16.7 mM glucose. RHPA showed that the population of single beta-cells under study was stimulated (P less than 0.01-0.001) to secrete insulin by 16.7 mM glucose, 100 microM tolbutamide, 20 microM glyburide, or 30 mM KCl but, under these conditions, also comprised beta-cells that did not secrete detectable amounts of insulin. Under current clamp conditions, secreting and nonsecreting beta-cells showed analogous resting membrane potentials (approximately 60 mV) and were similarly depolarized by 30 mm KCl and 100 microM tolbutamide. Under voltage-clamp conditions, total membrane conductance (approximately 6 nS) was also similar in the glucose-responsive and -unresponsive beta-cells, which, when monitored in the whole-cell configuration after RHPA, showed the following currents: a voltage-dependent Na+ current, a voltage-activated Ba2+ current, a voltage-dependent K+ delayed-rectifier current, a voltage-dependent Ca(2+)-activated K+ current, and a voltage-independent and tolbutamide-sensitive K+ current. In the cell-attached configuration and the presence of 2.8 mM glucose, secreting and nonsecreting beta-cells displayed a similar single-channel activity that was abolished when glucose concentration was raised to 16.7 mM. We conclude that beta-cells studied after RHPA have an electrically normal membrane whether they release insulin in response to 16.7 mM glucose or not.

Cross-talking among Drosophila nuclear receptors at the promiscuous response element of the ng-1 and ng-2 intermolt genes.

In Drosophila, peaks of the titer of the steroid hormone ecdysone act as molecular signals that trigger all the major developmental transitions occurring along the life cycle. The EcR/USP heterodimer, known to constitute the functional ecdysone receptor, binds with high affinity to specific target sequences, the ecdysone response elements (EcREs), whose repertoire still remains to be fully characterized at both the molecular and functional levels. In order to investigate the properties of EcREs composed of directly repeated half-sites (DRs), we have analysed the binding properties of the ng-EcRE, a DR element located within the coding region of ng-1 and ng-2, two highly homologous genes mapping at the ecdysone-regulated 3C intermolt puff. We report here that the ng-EcRE contacts the ecdysone receptor through its directly repeated half-sites spaced by 12 bp, and that this element may interact efficiently with at least three Drosophila orphan receptors, namely DHR38, DHR39 and beta FTZ-F1. Interestingly, DHR38 is bound alone or in combination with USP, providing the first evidence that the EcR-USP and DHR38-USP may directly compete for binding to a common response element. These results suggest that EcREs composed of widely spaced DRs may contribute to the establishment of extensive nuclear receptors cross-talking along the development, a mechanism that might play a relevant role in determining the temporal and spatial specificity of the ecdysone response. Finally, we show that the ng-EcRE can promote functional interactions in vitro as well as in vivo, acting as a transcriptional enhancer able to confer a specific developmental expression profile to a minimal promoter in transgenic flies.

Functional analysis of regulatory elements controlling the expression of the ecdysone-regulated Drosophila ng-1 gene.

The steroid hormone ecdysone controls multiple aspects of insect development, including larval moults and metamorphosis, and can induce specific genetic responses in different tissues. The definition of the molecular mechanisms able to mediate this tissue-specific responsiveness may greatly contribute to understanding how such an accurate genetic response is achieved. In this work we have identified, by transgenic analysis, the regulatory elements directing the expression of ng-1, an ecdysone-regulated Drosophila gene showing a highly specific developmental expression profile. Our results show that an ecdysone-responsive element located within the ng-1 coding region is necessary for high-level gene expression, whereas the gene's spatial and temporal expression profile is fully controlled by a distinct upstream regulatory region. This region binds a set of transcriptional factors, including the FKH regulatory protein, which can potentially modulate the ecdysone genetic regulated response.

Polyamines, NO and cGMP mediate stimulation of DNA synthesis by tumor necrosis factor and lipopolysaccharide in chick embryo cardiomyocytes.

OBJECTIVE: We have recently shown that tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS) stimulate DNA synthesis in chick embryo cardiomyocytes (CMs). The aim of the present research was to investigate the pathways involved in this mitogenic response. METHODS: CMs were isolated from 10-day-old chick embryos and grown to confluence. After 20 h of serum starvation the cells were treated with TNFalpha and LPS, and/or specific agonists and antagonists to manipulate the levels of polyamines, NO, cGMP and their biosynthetic enzymes ornithine decarboxylase (ODC), nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC). ODC, NOS, sGC activities and cGMP contents were determined by radiochemical procedures. DNA synthesis was determined by incorporation of [3H]-thymidine. RESULTS: Treatment of CMs with TNFalpha and LPS increased cell number and [3H]-thymidine incorporation. Addition of TNFalpha and LPS provoked an induction of ODC, with consequent polyamine accumulation, and a more delayed enhancement of NOS activity, which appeared to be independent of the activation of the ODC-polyamine system. TNFalpha and LPS treatment also enhanced cGMP level in CMs and both polyamine and NO biosyntheses appeared to be required. Experiments with specific inhibitors of ODC and NOS, as well as with inhibitors of sGC and cGMP-dependent protein kinase (PKG), showed that polyamine-, NO- and cGMP-dependent pathways are required for the mitogenic action of TNFalpha and LPS. Moreover, addition of exogenous polyamines to untreated cells raised the cGMP level in a NO-dependent fashion, and enhanced [3H]-thymidine incorporation. The latter effect was inhibited by sGC or PKG inhibitors. Treatment of quiescent cells with NO donors, 8-bromo-cGMP or YC-1, an sGC activator, also promoted DNA synthesis. Furthermore, putrescine and NO donor can additively activate sGC in cell-free extracts. CONCLUSION: TNFalpha and LPS stimulate DNA synthesis in chick embryo CMs and this effect is mediated by polyamines, NO and intracellular cGMP.

Signaling pathways leading to the induction of ornithine decarboxylase: opposite effects of p44/42 mitogen-activated protein kinase (MAPK) and p38 MAPK inhibitors.

Treatment of serum-starved, human ECV304 cells with histamine or ATP elicited a transient induction of ornithine decarboxylase (ODC), a key enzyme in polyamine synthesis, to an extent similar to that provoked by phorbol myristate acetate or serum re-addition. All these agents also provoked an increase in active phosphorylated p44/42 mitogen-activated protein kinase (MAPK) and p38 MAPK. The involvement of p44/42 MAPK and p38 MAPK in the induction of ODC was investigated by using selective inhibitors. U0126 and PD98059, two specific p44/42 MAPK kinase inhibitors, prevented the induction of ODC elicited by any stimulus employed, whereas SB203580 and SB202190, which are widely used as p38 MAPK inhibitors, enhanced ODC induction in a way that appeared dependent on p44/42 MAPK activation. By using inhibitors of other key signaling proteins that may lead to activation of p44/42 MAPK, we provide evidence that protein kinase C, but not phosphoinositide 3-kinase, is involved in histamine-stimulated ODC induction. These results show that the p44/42 MAPK pathway, but not p38 MAPK, is essential for ODC induction stimulated either by agonists of G-protein-coupled receptors, phorbol esters, or serum, and suggest that the inhibition of ODC induction may be an important event in the antiproliferative response to p44/42 MAPK pathway inhibitors.

Peptides of the APUD system in amphibian skins.

Methanol extracted skins from 84 species of amphibia were screened, measuring by RIAs: gastrin-CCK, VIP, calcitonin, GIP, PP and motilin. G-CCK-like immunoreactivity was found in 97.6%; VIP-like immunoreactivity in 41%; CT-like immunoreactivity in 34%; GIP-like immunoreactivity in 10%; PP-like immunoreactivity in 40% and MT-like immunoreactivity in 60% of the samples. The use of a sequence-specific radioimmunoassay and of gel-chromatography confirmed the caerulein-CCK-8-like nature of the immunoreactive material. Detected amounts of the other peptides (VIP, CT, GIP, PP, MT) were too low for bioassay or chromatographic studies, thus leaving the question open if they are due to some kind of unspecific interferences or, most likely, to species-specificity differences of the used antisera.

Dose-response effect of somatostatin-14 on human basal pancreatic hormones.

The effect of increased doses of Somatostatin-14 (3, 10, 30, 100, 300 micrograms/h) on basal release of insulin, pancreatic glucagon and pancreatic polypeptide (PP) was investigated on eight normal volunteers. Levels of Somatostatin-like immunoreactivity (SLI) was determined in order to correlate the increased SLI levels with the degree of islet hormone inhibition (r = 0.9947, p less than 0.01). By increasing the basal levels of SLI by one-third, a significant inhibition (p less than 0.01) of insulin, glucagon, and PP was noted (78.5, 78.6, 75.2%, respectively, on basal levels). The maximal effect was obtained with 300 micrograms/h for insulin, with 30 micrograms/h for glucagon and 100 micrograms/h for PP. In evaluating the relative inhibitory potency of somatostatin, expressed as ED50, the theoretic potency of somatostatin on each peptide had similar values, ranging from 30 to 10 micrograms/h. The present data show that a minimal peripheric increase in SLI is able to regulate basal islet pancreatic hormones.