Evaluation of the PMMA microlens efficiency for the realization of a solar micro-concentrator array.

In order to assess the performance of solar micro-concentrators, specific methods and protocols need to be developed, tested, and applied. In detail, as in conventional concentration modules, one of the fundamental parameters to consider is the efficiency of optical concentrators. In fact, optical concentrators give fundamental information on the current potentially generated from solar microcells that receive the concentrated light radiation. To develop a measurement method for micrometer-size optical components, a suitable optical system was implemented and used. Moreover, the potential application of the printed microstructures in an optical system for solar micro-concentrators was demonstrated.

Optimizing PANi doped electroactive substrates as patches for the regeneration of cardiac muscle.

In scaffold aided regeneration of muscular tissue, composite materials are currently utilized as a temporary substrate to stimulate tissue formation by controlled electrochemical signals as well as continuous mechanical stimulation until the regeneration processes are completed. Among them, composites from the blending of conductive (CPs) and biocompatible polymers are powerfully emerging as a successful strategy for the regeneration of myocardium due to their unique conductive and biological recognition properties able to assure a more efficient electroactive stimulation of cells. Here, different composite substrates made of synthesized polyaniline (sPANi) and polycaprolactone (PCL) were investigated as platforms for cardiac tissue regeneration. Preliminary, a comparative analysis of substrates conductivity performed on casted films endowed with synthesized polyaniline (sPANi) short fibres or blended with emeraldine base polyaniline (EBPANi) allows to study the attitude of charge transport, depending on the conducting filler amount, shape and spatial distribution. In particular, conducibility tests indicated that sPANi short fibres provide a more efficient transfer of electric signal due to the spatial organization of electroactive needle-like phases up to form a percolative network. On the basis of this characterization, sPANi/PCL electrospun membranes have been also optimized to mimic either the morphological and functional features of the cardiac muscle ECM. The presence of sPANi does not relevantly affect the fibre architecture as confirmed by SEM/image analysis investigation which shows a broader distribution of fibres with only a slight reduction of the average fibre diameter from 7.1 to 6.4 µm. Meanwhile, biological assays--evaluation of cell survival rate by MTT assay and immunostaining of sarcomeric α-actinin of cardiomyocites-like cells--clearly indicate that conductive signals offered by PANi needles, promote the cardiogenic differentiation of hMSC into cardiomyocite-like cells. These preliminary results concur to promise the development of electroactive biodegradable substrates able to efficiently stimulate the basic cell mechanisms, paving the way towards a new generation of synthetic patches for the support of the regeneration of damaged myocardium.

Dietary polyphenols: focus on resveratrol, a promising agent in the prevention of cardiovascular diseases and control of glucose homeostasis.

Plants continuously produce an extraordinary variety of biologically active low-molecular-mass compounds. Among them, resveratrol (3,5,4'-trihydroxystilbene) is endowed with significant positive activities by protecting against cardiovascular diseases and preventing the development and progression of atherosclerosis. Furthermore, the molecule significantly ameliorates glucose homeostasis in obese mice. These beneficial effects have driven considerable interest towards resveratrol molecular activities, and intensive efforts for the identification of the stilbene targets have been made. The molecule shows a pleiotropic mode of action. Particularly, its cellular targets are crucial for cell proliferation and differentiation, apoptosis, antioxidant defence and mitochondrial energy production. The complexity of resveratrol activities might account for its effectiveness in ameliorating multifactorial processes, including the onset and/or progression of several degenerative diseases such as myocardial infarction, atherosclerosis and type 2 diabetes. This article reports the actions of resveratrol on cardiovascular diseases and the molecular bases of its activity. We also discuss recent data on the effect of resveratrol on glucose homeostasis and obesity. Finally, the relevance of the stilbene use in the development of new pharmacological strategies is evaluated.

Electrical bistability in conductive hybrid composites of doped polyaniline nanofibers-gold nanoparticles capped with dodecane thiol.

A novel electrical bistable hybrid nanocomposite based on doped Polyaniline nanofibers with 1-Dodecanethiol-protected Gold nanoparticle (PAni.AuDT), 3-4 nm in size, as the conductive component and polystyrene as polymer matrix was prepared. The structural morphology of the composite and the dispersion of nanoparticles inside it were evaluated using Transmission Electron Microscopy (TEM). The thermal stability and the ratio Polyaniline/Gold nanoparticles in the composite were determined by using thermogravimetric analysis. The electrical bistability of the PAni.AuDT-PS composite, the influence of the dispersion of the PAni.AuDT conductive network and the basic operation mechanism, have been assessed by measuring the electrical response of planar device architectures, also as a function of the environmental temperature (in the range 200 K < T < 360 K). The basic operation mechanism of the hybrid compound has been then correlated to the combined action of the thermally-induced scattering of charge carriers and the thermal contraction of the hosting polymeric matrix. Moreover, the right compromise between these two effects in terms of the most efficient bistability has been studied, founding the concentration of the conductive component which optimizes the device on-off ratio (I(on)/ I(off)).

A novel Leu153Ser mutation of the Fanconi anemia FANCD2 gene is associated with severe chemotherapy toxicity in a pediatric T-cell acute lymphoblastic leukemia.

Fanconi anemia (FA) is an autosomal recessive disease characterized by pancitopenia, congenital malformations, predisposition to cancers and chromosomal instability. We report the clinical and molecular features of a patient initially identified as a potential FA case only because of chemotherapy toxicity during the treatment of a T-lineage acute lymphoblastic leukemia (ALL). Cells from this patient showed a moderate chromosomal instability, increasing sensitivity to DNA crosslinking agents but normal response to ionizing radiation. The analysis of FA proteins demonstrated a marked reduction of FANCD2 (>95%), but normal levels of FANCA or FANCG. Interestingly, this defect was associated with a homozygous missense mutation of FANCD2, resulting in a novel amino-acid substitution (Leu153Ser) at residue Leu153, which is highly conserved through evolution. The FANCD2(L153S) protein, whose reduced expression was not due to impaired transcription, was detected also in its monoubiquitinated form in the nucleus, suggesting that the mutation does not affect post-translation modifications or subcellular localization but rather the stability of FANCD2. Therefore, the hypomorphic Leu153Ser mutation represents the first example of a FANCD2 defect that might promote clonal progression of tumors, such as T-ALL, and severe chemotherapy toxicity in patients without any clinical manifestations typical of FA.

New established melanoma cell lines: genetic and biochemical characterization of cell division cycle.

BACKGROUND: Cancer might be envisaged as the result of a genetic process causing the unregulated proliferation of a given cell as well as its inability to undergo differentiation and/or apoptosis. Alterations of genes regulating cell division cycle appear to play a key role in the development of human cancer. OBJECTIVE: On the bases of the above considerations, we decided to establish new cell lines from human melanoma specimens, in order to analyse the molecular alterations in primary preparations of malignant cells. RESULTS: The present paper describes two new established cell lines and their genetic and biochemical features. Both the melanoma cell lines show inactivation of the cyclin-dependent kinase inhibitor gene, CDKN2A/p16INK4A, thus demostrating that this alteration occurs in primary human melanomas. No other alterations were observable when we investigated several different cell cycle genes including those encoding cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors. Analyses at protein level by means of immunoblotting confirmed the results obtained at the genetic level. Moreover, the inducibility of a pivotal cyclin-dependent kinase inhibitor gene, namely p21CIP1 gene, was obtained by treating the cells with histone deacetylase inhibitors, namely butyrate and phenylbutyrate. CONCLUSIONS: Our results suggest a primary role of cyclin-dependent kinase inhibitor genes inactivation in the origin of human melanoma and allow the proposal of new therapeutic strategies based on the transcriptional activation of p21CIP1 gene.

Cell division cycle control in embryonal and alveolar rhabdomyosarcomas.

In this study, we investigated the mRNA level of several genes involved in cell cycle regulation in alveolar (ARMS) and embryonal rhabdomyosarcomas (ERMS). p21(Cip1), Cyclin D1, Cyclin D2, Cyclin D3, CDK2, and CDK4 were evaluated by RT-PCR. All (13 out of 13) ERMS expressed the p21(Cip1) gene compared with only 40% (4 out of 10) of the ARMS. Moreover, the amount of p21(Cip1) mRNA was noticeably higher in the ERMS samples than in the positive ARMS specimens. p27(Kip1) protein were analysed by immunohistochemical and immunoblotting. A noticeable difference was observed, in that ERMS had higher amounts of the cell cycle inhibitor compared with the ARMS. Finally, treatment of two rhabdomyosarcoma cell lines, RH-30 and RD, with butyrate, resulted in complete growth inhibition and in the upregulation of the p21(Cip1) and p27(Kip1) levels. Our results demonstrate that ERMS have a much higher level of p27(Kip1) and p21(Cip1) than the alveolar types, explaining, at least in part, the distinct features and outcomes (i.e. a poor prognosis of the alveolar type) of the two forms of this childhood solid cancer. Moreover, the data on butyrate-treated cell lines suggest that the two genes are potential novel therapeutic targets for the treatment of rhabdomyosarcomas.

Genes modulated by histone acetylation as new effectors of butyrate activity.

A wealth of evidence correlates the chemopreventive activity of a fiber-rich diet with the production of butyrate. In order to identify the genes transcriptionally modulated by the molecule, we analyzed the expression profile of butyrate-treated colon cancer cells by means of cDNA expression arrays. Moreover, the effect of trichostatin A, a specific histone deacetylase inhibitor, was studied. A superimposable group of 23 genes out of 588 investigated is modulated by both butyrate and trichostatin A. Among them, a major target was tob-1, a gene involved in the control of cell cycle. tob-1 is also up-regulated by butyrate in a neuroblastoma-derived cell line, and its overexpression in the colon cells caused growth arrest. Our findings represent an extensive analysis of genes modulated by butyrate and identify completely new effectors of its biological activities.

Hydroxytyrosol, a natural molecule occurring in olive oil, induces cytochrome c-dependent apoptosis.

2-(3,4-Dihydroxyphenyl)ethanol (DPE), a naturally occurring phenolic antioxidant molecule found in olive oil, has been reported to exert several biological and pharmacological activities. We studied the effect of this compound on the proliferation and survival of HL60 cell line. Concentrations from 50 to 100 microM DPE, comparable to its olive oil content, caused a complete arrest of HL60 cell proliferation and the induction of apoptosis. This was demonstrated by flow cytometric analyses, poly(ADP-ribose) polymerase cleavage, and caspase 3 activation. The apoptotic effect requires the presence of two ortho-hydroxyl groups on the phenyl ring, since tyrosol, 2-(4-hydroxyphenyl)ethanol, did not induce either cell growth arrest or apoptosis. DPE-dependent apoptosis is associated with an early release of cytochrome c from mitochondria which precedes caspase 8 activation, thus ruling out the engagement of cell death receptors in the apoptotic process. 2-(3,4-Dihydroxyphenyl)ethanol induced cell death in quiescent and differentiated HL60 cells, as well as in resting and activated peripheral blood lymphocytes, while did not cause cell death in two colorectal cell lines (HT-29 and CaCo2). These results suggest that DPE down-regulates the immunological response, thus explaining the well-known antinflammatory and chemopreventive effects of olive oil at the intestinal level.

Reduced expression of transforming growth factor-beta receptor type III in high stage neuroblastomas.

Transforming growth factor beta (TGF-beta) is a powerful inhibitor of cell proliferation and a potent inducer of differentiation. Resistance to TGF-beta action is a characteristic of many malignancies and has been attributed to alterations of TGF-beta receptors as well as disturbance of downstream transduction pathways. To analyse the TGF-beta response in neuroblastoma, the expression of TGF-beta1 and TGF-beta type I, II and III receptor genes was investigated in 61 cancer samples by means of reverse transcription polymerase chain reaction. The specimens analysed belong to different stages, namely nine samples of stage 1, ten of stage 2, nine of stage 3 and 28 of stage 4. Moreover, five samples were of stage 4S, which represents a tumour form undergoing spontaneous regression. The results obtained show that TGF-beta1 and TGF-beta type I and II receptor genes appear to be almost equally expressed in neuroblastomas of all stages. Conversely, TGF-beta type III receptor gene expression, which is required for an efficacious TGF-beta binding and function, is strongly reduced exclusively in neuroblastomas of stages 3 and 4. These findings were directly confirmed by immunohistochemical analyses of ten neuroblastoma specimens. Our results suggest the occurrence of an altered TGF-beta response in advanced neuroblastomas which might be an important mechanism for escaping growth control and for developing invasiveness. Moreover, our findings allow the proposal of a novel mechanism, namely down-regulation of TGF-beta type III receptor gene expression, to avoid TGF-beta inhibitory activity.

Pyrrolidine dithiocarbamate induces apoptosis by a cytochrome c-dependent mechanism.

Pyrrolidine dithiocarbamate (PDTC) is a synthetic antioxidant molecule, which has been recently proposed as an antitumoral agent on the basis of its capability of inducing apoptosis. We investigated the effect of PDTC on the proliferation and survival of the promyelocitic cell line HL-60. Concentration as low as 10 microM of PDTC induces a significant reduction of the growth rate and the contemporaneous activation of the apoptotic process. Programmed cell death was demonstrated by biochemical analyses, including the activation of procaspase 3 and the cleavage of poly(ADP-ribose) polymerase (PARP). PDTC-dependent apoptosis was associated with an early release of cytochrome c from mitochondria, while the involvement of pathways due to cell death receptors engagement was ruled out by detailed time-course analyses of caspases 3 and 8 activation. Moreover, no up-regulation of p21(CIP1) level, a pivotal cyclin-dependent kinase inhibitor, occurred at PDTC concentration able to induce apoptosis. Finally, in vitro incubation of purified mitochondria with PDTC demonstrated that the molecule is directly able to induce cytochrome c release from the intermembrane space, thus confirming that mitochondria are a primary cellular target of the molecule.

p27Kip1 accumulation is associated with retinoic-induced neuroblastoma differentiation: evidence of a decreased proteasome-dependent degradation.

Development of human neuroblastoma is due to an arrest in the differentiation program of neural crest sympathoadrenal progenitor cells. However, neuroblastomas, as well as their derived cell lines, maintain the potentiality of terminal differentiation. We investigated the molecular mechanisms by which retinoic acid, a molecule introduced in clinical trials for chemotherapy, induces differentiation in neuroblastoma cell lines. Our findings demonstrate that the retinoic acid-dependent growth arrest of LAN-5 neuroblastoma cell line is associated to a very large accumulation (>tenfold) of p27Kip1 protein, a cyclin-dependent kinase inhibitor; the protein binds and inhibits cyclin-dependent kinase 2, 4 and 6 activities, thus hampering pRb and p107 phosphorylation. p27Kip1 build-up was observable as an early phenomenon (12 - 24 h) after retinoic exposure and resulted in a time-dependent accumulation of high quantities of a free p27Kip1 form. Furthermore, retinoic treatment causes an increase of cyclin-dependent kinase 5 level and activity; however, immunoprecipitation studies proved the absence of interaction with p27kip1. No noticeable variation of other components of G1 phase cell cycle engine was observed. Pulse-chase experiments showed a remarkable elongation of p27Kip1 half-life in retinoic-treated LAN-5, while no enhancement of p27Kip1 gene expression and of the translational efficiency of its messenger RNA were demonstrated. In vivo degradation of p27Kip1 was sensitive to two highly specific proteasome inhibitors, LLnL and lactacystin, while the calpain inhibitor II ALLM and the cysteine protease inhibitor E64 did not modify the level of the protein. LLnL treatment caused a very rapid (2 h) build-up of the Cdk inhibitor content and the accumulation of higher molecular weight anti-p27Kip1 immunoreactive bands, which probably represent ubiquitinated forms of the protein. Finally, in vitro experiments demonstrated that extracts prepared from retinoic-treated LAN-5 cells degraded recombinant p27Kip1 at a rate remarkably slower than the untreated cells. Our results indicate that retinoic acid strongly increases p27Kip1 levels by down-regulating the ubiquitin-proteasome p27Kip1 degrading pathway.

Effects of human immunodeficiency virus type 1 on CD4 lymphocyte subset activation.

The pathogenesis of the decline of CD4 lymphocyte counts accompanying the typical course of HIV-1 infection is not completely defined and might be related to a differential susceptibility of naive and memory cells to HIV-1 exposure. Here, we examined the effects induced by heat-inactivated HIV-1 virions on these lymphocyte populations. Exposure of CD45RA naive T cells to inactivated viral particles induced a marked decrease of both mitogenic responses and activation-induced apoptosis. Conversely, the growth of CD45RO cells was less severely restrained. Analysis of intracellular levels of cell cycle regulatory proteins revealed an arrest at the G1/S restriction point of the naive but not memory subset. This effect was associated with alterations in phosphotyrosine profile and with a marked decrease of ERK and NJK kinase activation. Finally, up-regulation of the cAMP-dependent protein kinase A (PKA) activity induced by mitogens was not affected by virus. Altogether, these findings show that interaction of HIV-1 with the T cell surface is sufficient to inhibit the proliferative response of the CD4CD45RA subset by disturbing proximal TCR signaling. This mechanism would affect renewal of naive lymphocytes, contributing in such a way to the impairment of T cell turnover during the course of HIV-1 infection.

Resveratrol arrests the cell division cycle at S/G2 phase transition.

Resveratrol (3,5,4'-trihydroxystilbene) is a naturally occurring phytoalexin, found in grapes and wine, which has been reported to exert a variety of important pharmacological effects. We have investigated the activity of resveratrol on proliferation and differentiation of the promyelocitic cell line HL-60. A concentration as low as 30 microM causes a complete arrest of proliferation and a rapid induction of differentiation towards a myelo-monocytic phenotype. Analyses by flow cytometry showed the absence of the G2/M peak and the accumulation of cells in G1 and S phases. Moreover, at the concentrations employed, a very low amount of apoptotic cells was evidenced. A detailed biochemical analysis demonstrated that the G1 phase of the cell division cycle engine was completely unmodified by resveratrol addition, thus indicating that the G1 --> S transition occurs normally. Conversely, after only 24 h treatment, a significant increase of cyclins A and E could be observed along with the accumulation of cdc2 in the inactive phosphorylated form. These data demonstrate that resveratrol causes a complete and reversible cell cycle arrest at the S phase checkpoint.

Analysis of CDKN2A, CDKN2B, CDKN2C, and cyclin Ds gene status in hepatoblastoma.

The status and the expression of cyclin-dependent kinase inhibitor A (CDKN2A) family genes, named CDKN2A, CDKN2B, and CDKN2C and of cyclin Ds (D1, D2, and D3) genes were investigated in 14 cases of human hepatoblastomas. These genes were selected because: 1) CDKN2A and CDKN2B are very frequently inactivated in human cancers; 2) cyclin Ds are overexpressed in several tumors and 3) CDKN2A is posttranscriptionally silenced in hepatocellular carcinomas. Structural analysis of the CDKN2A, CDKN2B, and CDKN2C genes in hepatoblastoma cases showed the absence of deletions and/or point mutations. Moreover, a detailed investigation of loss of heterozygosity at 9p21 and 1p32 (the chromosomal regions where CDKN2A genes are located) rules out the possible loss of one allele. Messenger RNA (mRNA) analysis showed that CDKN2C is expressed in all hepatoblastoma samples studied, while both CDKN2A and CDKN2B genes are not transcribed in the cancer specimens as well as in the matched normal liver tissues. Interestingly, an alternative mRNA expressed by the CDKN2A gene (beta-transcript) is detectable in 100% of the samples investigated. The analysis of cyclin D genes expression revealed that cyclin D1 is highly transcribed in normal hepatic tissue while cyclin D2 or D3 genes were extensively expressed in the matched transformed samples. Investigation at protein level confirmed the data obtained on RNA analysis. Indeed, p16INK4A and p15INK4B (products of expression of CDKN2A and CDKN2B respectively) were not observable while pl8INK4C (which is codified by CDKN2C) was clearly detectable in the samples analyzed. Moreover, a noticeable decrease of cyclin D1 content and increase of cyclin D3 level were observable in tumor tissues versus normal counterparts. Our findings demonstrated the following: 1) CDKN2A, CDKN2B, and CDKN2C genes are structurally unmodified in human hepatoblastoma, and 2) CDKN2A (alpha-transcript) and CDKN2B are transcriptionally silenced in normal liver whereas CDKN2A (beta-transcript) and CDKN2C were clearly expressed. Finally, a clear shift in cyclin D type expression was observable during malignant transformation. These results show that CDKN2A gene family alterations are not involved in hepatoblastoma development, whereas changes in cyclin D types might play a role in this type of tumor. Furthermore, a highly regulated expression of CDKN2A seems to occur in normal hepatic tissue.

Structural and functional analysis of cyclin-dependent kinase inhibitor genes (CDKN2A, CDKN2B, and CDKN2C) in neuroblastoma.

The status of the CDKN2A gene family, including CDKN2A, CDKN2B, and CDKN2C, was investigated in 24 cases of neuroblastoma. These genes were selected on the basis of 1) high incidence of their inactivation in several human cancers and 2) their localization on chromosomal regions (9p and 1p) frequently rearranged in neuroblastomas. Detailed molecular analyses indicated the absence of homozygous deletions and point mutations involving these genes in all investigated tumor samples. However, when loss of heterozygostity for chromosome 9p21 (the region where CDKN2A and CDKN2B are localized) was investigated, 16% of cases showed abnormalities in an area telomeric to the CDKN2A locus. To study transcriptional silencing of the CDKN2A gene, the methylation status of exon 1 was examined. In about 35% of cases, a partial methylation was evidenced. Analysis of the CDKN2A mRNA expression, however, did not show any relationship between methylation status and gene transcription. Finally, expression of the CDKN2B gene was demonstrated in all stage IV neuroblastomas, whereas none of stage I tumors expressed this gene. This finding suggests the occurrence of a correlation between CDKN2B transcription and tumor phenotype.

Expression of G1-phase cell cycle genes during hematopoietic lineage.

Characterization of proteins that control the passage through the G1 phase of the cell cycle is of particular interest because virtually all stimuli regulating cell proliferation or differentiation act primarily during this phase. We have analyzed the G1 phase proteic machinery, including cyclin D types, cyclin-dependent kinases (CDKs) and CDK inhibitors, of cell populations obtained at different stages of hematopoietic cell lineage. In particular, five cellular phenotypes, namely CD34+ cells (which contain stem cells), BFU-E, CFU-E, CFU-GM and peripheral lymphocytes were studied as representatives of distinct differentiation pathways. The results obtained indicated that all the cellular preparations express cyclin D2 and D3, while cyclin D1, which is the major cyclin D occurring in mesenchimal tissues, is not expressed. Moreover, CDK6 (but not CDK4) was detectable in all the populations investigated. Among the CDK inhibitors studied, p18INK4C and p19INK4D signals were clearly evidentiable in the various cell types. Interestingly, high levels of p15INK4B, a putative tumor suppressor protein, were detectable especially in granulocyte-monocyte precursors. Our results indicate that a specific hematopoietic G1 phase machinery occurs, which is conserved during the various steps of the different maturation processes.

Purification and characterization of recombinant human 5'-methylthioadenosine phosphorylase: definite identification of coding cDNA.

5'-Methylthioadenosine phosphorylase gene maps on the 9p21 chromosome, strictly linked to the important tumor suppressor gene p16INK4A. Chromosomal deletions encompassing both the phosphorylase and p16INK4A genes cause the complete absence of the enzymatic activity in a large number of tumors, thus resulting in well-defined metabolic differences between malignant and normal cells. Recently, the cloning of the phosphorylase gene has been reported on the basis of indirect evidence. In order to demonstrate definitely the identification of 5'-methylthioadenosine phosphorylase gene, we have cloned the putative enzyme coding sequence in a prokaryotic expression vector and expressed the protein in bacteria. The recombinant phosphorylase has been purified to homogeneity and its physicochemical, immunological and kinetic features have been characterized. The results obtained allowed the conclusive demonstration of 5'-methylthioadenosine phosphorylase gene cloning and the use of recombinant protein for further characterization.