Monitoring plasmon coupling and SERS enhancement through in situ nanoparticle spacing modulation.

Self-assembled nanoparticle (NP) arrays at liquid interfaces provide a unique optical response which has opened the door to new tuneable metamaterials for sensing and optical applications. NPs can spontaneously assemble at a liquid-liquid interface, forming an ordered, self-healing, low-defect 2D film. The close proximity of the NPs at the interface results in collective plasmonic modes with a spectral response dependent on the distance between the NPs and induces large field enhancements within the gaps. In this study, we assembled spherical and rod-shaped gold NPs with the aim of improving our understanding of NP assembly processes at liquid interfaces, working towards finely controlling their structure and producing tailored optical and enhanced Raman signals. We systematically tuned the assembly and spacing between NPs through increasing or decreasing the degree of electrostatic screening with the addition of electrolyte or pH adjustment. The in situ modulation of the nanoparticle position on the same sample allowed us to monitor plasmon coupling and the resulting SERS enhancement processes in real time, with sub-nm precision.

FOLFOX6 and bevacizumab in non-optimally resectable liver metastases from colorectal cancer.

BACKGROUND: In patients with colorectal liver metastases (CLM) R0 resection significantly improves overall survival (OS). METHODS: In this report, we present the results of a phase II trial of FOLFOX6+bevacizumab in patients with non-optimally resectable CLM. Patients received six cycles of FOLFOX6+ five of bevacizumab. Patients not achieving resectability received six additional cycles of each. A PET-CT was performed at baseline and again within 1 month after initiating treatment. RESULTS: From September 2005 to July 2009, 21 patients were enrolled (Male/Female: 15/6; median age: 65 years). An objective response (OR) was documented in 12 cases (57.1%; complete responses (CRs): 3, partial response (PR): 9); one patient died from toxicity before surgery. Thirteen patients underwent radical surgery (61.9%). Three (23%) had a pathological CR (pCR). Six patients (46.1%) experienced minor postsurgical complications. After a median 38.8-month follow-up, the median OS was 22.5 months. Patients achieving at least 1 unit reduction in Standard uptake value (SUV)max on PET-CT had longer progression-free survival (PFS) (median PFS: 22 vs 14 months, P=0.001). CONCLUSIONS: FOLFOX6+bevacizumab does not increase postsurgical complications, yields high rates of resectability and pCR. Early changes in PET-CT seem to be predictive of longer PFS.

Aromatase mRNA expression in the brain of adult Xenopus laevis exposed to Lambro river water and endocrine disrupting compounds.

Aromatase P450 (P450 arom; Cyp19) is a key enzyme for vertebrate reproduction and brain development that catalyzes the conversion of androgens to estrogens. The aim of this study was to improve the knowledge on EDC effects by analysing their potential impact on brain P450 arom in adult Xenopus laevis exposed for 4 weeks to an environmental sample, the water of the river Lambro (LAM), the most polluted tributary of the Po river in North Italy. Other groups were exposed to individual compounds 10(-8) M tamoxifen (TAM), ethinylestradiol (EE2), flutamide (FLU) and methyldihydrotestosterone (MDHT) known for their (anti)estrogenic and (anti)androgenic modes of action. Expression of CYP19 was evaluated in brain extracts by quantitative RT-PCR, using a pair of primers located in the open reading frame (ORF) that allowed the simultaneous amplification of all transcripts (Aro-ORF) and a pair of primers specific for brain aromatase (Aro-B). Significant increase in Aro-ORF and Aro-B mRNA levels were observed in both females and males exposed to LAM. Different changes were observed for the model compounds using two pairs of primers. Aro-ORF mRNA expression was significantly increased in EE2 and MDHT exposed males and in FLU-exposed females, while it was significantly decreased in TAM exposed females. Aro-B mRNA was significantly increased in both sexes exposed to FLU and decreased in TAM exposed females. In conclusion, aromatase mRNA in the brain of X. laevis was regulated differentially in a gender specific manner by certain (anti)estrogenic and (anti)androgenic EDCs, supporting previous hypotheses that diverse compounds present in the river Lambro may induce feminization and demasculinization effects.

Effects of carnitines on rat sertoli cell protein metabolism.

The influence of carnitine on male reproductive function is well established. However, the molecular mechanism by which carnitines control male fertility have not yet been clearly identified. In the present study, we studied the role of carnitines at testis level by examining their effects on protein metabolism in cultured rat Sertoli cells. Our results demonstrate that L-carnitine and, in particular, L-acetylcarnitine administration affects overall protein synthesis, inducing a dose-dependent decrease in amino acid incorporation and consequently an increase of the intracellular amino-acid pool useful to speed up Sertoli cell energy metabolism. We also observed significant effect on the expression of specific proteins involved in the regulation of glucose uptake and Insulin-like Growth Factor (IGF) biological activity. Actually, an increased expression of the mRNA coding for the membrane Glucose Transporter-1 (GLUT-1) and a decreased expression of mRNA coding for the negative modulator insulin-like growth factor-binding protein-4 (IGFBP-4) were induced by in vitro treatment with L-carnitine and L- acetylcarnitine separately and in combination; in fact, the effects observed were far more pronounced following concomitant treatment with both compounds. Taken together, these data indicate that the influence of carnitines on male fertility could be partly mediated by an activity at Sertoli cell level.

Prolactin effect on pre-pubertal Sertoli cell proliferation and metabolism.

Direct effects of PRL on Sertoli cell proliferation were investigated by using Sertoli cell primary cultures isolated from both prepubertal rat and porcine testes. PRL metabolic effects were analyzed in rat Sertoli cell primary cultures. Exposure to physiological doses of PRL resulted in a significant increase (+50-60%) of basal DNA synthesis, as reflected by the pattern of [3H] thymidine incorporation during culture; significant increases in lactate secretion (about 50%), androgen binding protein (ABP) production (about 30%) and basal protein synthesis (25-30%), as reflected in the augmented [14C] valine incorporation, were also evident. Taken together, our present findings, indicating significant effects of PRL on Sertoli cell proliferation and metabolism, demonstrate that Sertoli cells are a potential target for PRL action at testicular level during pre-pubertal development.

Effect of simulated microgravity conditions on poly(ADP-ribose) polymerase activity in primary cultures of adult rat hepatocytes.

The possible involvement of poly(ADP-ribose) polymerase [PARP; E.C. 2.4.2.30] in the adaptive response to low-g conditions was studied in cultured adult rat hepatocytes exposed to simulated microgravity produced by the random positioning machine (RPM-3D-clinostat). Four different poly(ADP-ribose) polymerases (PARPs) have been identified recently. The best-studied member of this family is PARP-1, a highly conserved, multimodular 113 kDa protein. In multicellular organisms PARPs catalyze poly(ADP-ribose) synthesis from NAD+ to a number of structural and catalytic proteins. Moreover, PARP-1 can control its protein and DNA interactions by catalyzing its automodification with poly(ADP-ribose) molecules that can include up to 200 ADP-ribose residues and several branching points; by these polymers, PARP-1 may nocovalently interact with other proteins and alter their functions. PARP-1 binds to DNA and is activated by free ends interacting with several other DNA damage checkpoint proteins. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions. Poly(ADP-ribosyl)ation plays a central role in genome stability and is involved in DNA replication and repair, gene expression, cell differentiation and transformation. We have shown that a loss of PARP-1 activity is a critical event in the early molecular steps of the hepatocarcinogenesis process. Moreover, a prompt increase in this enzymatic activity is linked not only to the presence of DNA free ends but is linked also to the start of DNA synthesis. More recently, we have reported that PARP-1 is involved in hormone-mediated gene expression in vitro and in vivo during rat liver regeneration.

Poly(ADP-ribose) polymerase is affected early by thyroid state during liver regeneration in rats.

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA synthesis, DNA repair, and cell replication and transformation, also plays a role in the early steps of liver regeneration induced by partial hepatectomy (PH). PARP and DNA topoisomerase I (Topo I) activities and de novo DNA synthesis were studied during liver regeneration in rats with altered thyroid state. Hepatic PARP activity, evaluated as [(32)P]NAD incorporated into isolated liver nuclei, was inhibited in hyperthyroid rats and increased in hypothyroid animals. In both euthyroid and hyperthyroid rats PARP activity was rapidly stimulated, peaking 6 h after PH. In hypothyroid animals, an early decrease in activity was found, at a minimum of 6 h after PH, followed by an early onset of DNA synthesis. An inverse relationship between PARP and Topo I activities was a shared feature among euthyroid, hypothyroid, and hyperthyroid rats. Together these data show that, in replicating hepatocytes, thyroid hormones exert a regulatory role on PARP activity, which reflects the control of a number of nuclear proteins involved in DNA metabolism.

DNA topoisomerase I activity in regenerating liver of hypothyroid rats.

DNA topoisomerase I activity is known to be inhibited by poly(ADP-ribosyl)ation. Both poly(ADP-ribose)polymerase and DNA topoisomerase I participate to major biological events, such as DNA transcription, repair and synthesis. Previously, a 2-fold increase in PARP activity has been shown in hypothyroid animals. Using the regenerating rat liver model, we have studied the behaviour of DNA topoisomerase I activity in hypothyroid rats. PARP activity, was also studied in another set of experiments. DNA topoisomerase I relaxing activity was determined on supercoiled plasmid DNA, and topoisomers separated by agarose gel electrophoresis. An increase in the relaxing activity of Topo I was observed early after hepatectomy. This enhancement well correlates with the reported inhibition of PARP activity at the scheduled times. The data from hypothyroid animals support an inverse relationship between PARP and Topo I. These results are completely reversed with respect to those obtained during liver regeneration in euthyroids.

Poly(ADP-ribose) polymerase activity in regenerating liver of hypothyroid rats.

The role of PARP, a nuclear enzyme involved in DNA synthesis, repair and cell transformation, was studies during liver regeneration in hypothyroid animals. Hypothyroidism was induced by in vivo administration of propylthiouracil. In regenerating euthyroid animals PARP activity is stimulated showing an early and significant increase at 1.5 h with a maximum at 6 h after partial hepatectomy. Such an increase returns to control values within 18 h preceding the onset of DNA synthesis. A markedly different behavior, with respect to euthyroids, has been evidenced in hypothyroid rats. At first, liver PARP level was about 2-fold higher in non regenerating hypothyroid rats with respect to control euthyroids. During regeneration, PTU-treated animals show a net decrease in PARP activity, with a minimum at 6-9 h after partial hepatectomy. The activity returns to control levels within 24 days. The minimum in PARP activity anticipates, also in this case, the onset of DNA synthesis, which exhibits a maximum at 15-18 h. During liver regeneration PARP activity shows modifications related to the beginning of de novo DNA synthesis. Furthermore, these variations in turn undergo the effects of hypothyroidism.

In vitro effect of 3,5,3'-triiodothyronine on poly(ADP-ribosyl)ation of DNA topoisomerase I.

DNA topoisomerase I activity (topo I) is known to be inhibited by poly(ADP-ribosyl)ation. Both poly(ADP-ribose)polymerase (pADPRP) and DNA topoisomerase I participate to major biological events, such as DNA transcription, repair and synthesis. It has been shown that thyroid hormones, such as 3,5,3'-triiodothyronine (T3), stimulate DNA transcription and down-regulate pADPRP activity. Using an in vitro model, we have studied the poly(ADP-ribosyl)ation of topo I, in vitro, in the presence of T3. T3 treatment of pADPRP inhibits the enzyme up to 75-80% of control activity. DNA topoisomerase I relaxing activity was determined on supercoiled plasmid DNA, and topoisomers were separated by agarose gel electrophoresis. Poly(ADP-ribosyl)ation completely inhibits the relaxing activity of topo I, with respect to non-ribosylated controls, but the activity remains unaffected when pADPRP is inactivated by heat or treated with specific inhibitors, such as 3-aminobenzamide (3ABA). In this study we show that treatment of pADPRP with T3 reduces the inhibition on topo I. In this system 10(-8) M T3 was effective in maintaining almost all topo I activity, even though modifications in processivity and distributivity of the reaction were noted. These data support a close relationship between pADPRP and topo I in hormone-stimulated DNA transcription.

Hepatic poly(ADP-ribose) polymerase activity in rat is controlled by thyroid hormones.

The in vivo effect of the thyroidal state on poly(ADP-ribose) polymerase activity was investigated in eu- and hypothyroid rats after treatment with L-triiodothyronine. Untreated hypothyroids showed an increased basal rate of the enzyme. The treatment of both eu- and hypothyroid rats with L-triiodothyronine induced a prompt drop of the endogenous activity not due to a reduction of the catalytic protein. This decrease well evident 1 h after treatment was transient, returning to controls values within 8 h. In isolated liver nuclei from euthyroids the in vitro exposure to increasing L-triiodothyronine concentrations from 10(-18) to 10(-6) M resulted in a progressive inhibition of the enzyme. This loss in activity was not derived from a reduction of the total level of the catalytic protein. The pretreatment with the antagonist amiodarone suppressed the hormone effect, suggesting that nuclear receptors could mediate poly(ADP-ribose) polymerase activity.

Relationship between poly(ADP-ribose) polymerase activity and DNA damage induced by zinc dithiocarbamates in mouse and rat liver.

The genotoxic effects due to in vivo treatment with zinc dithiocarbamates were evaluated in rat and mouse liver. The two pesticides Zineb and Ziram, belonging to this chemical class, induced an increase in single-strand DNA breaks, as measured by the alkaline elution technique. The nuclear enzyme poly(ADP-ribose) polymerase (pADPRP), a chromatin-bound catalytic protein, utilizing NAD+ as a substrate, was tested by a radiometric procedure. A close relationship between the increased extent of DNA damage and the enhanced level of endogenous pADPRP activity was obtained in rat liver, whereas both parameters remained unchanged in mouse liver.

Effect of N-2-acetylaminofluorene on poly(ADP-ribose) polymerase activity and DNA synthesis in cultured rat hepatocytes exposed to epidermal growth factor.

The nuclear enzyme poly(ADP-ribose) polymerase is involved in basic cellular processes such as DNA replication and repair, cell differentiation and transformation, gene expression. We have studied the effect of 2AAF, a genotoxic aromatic amine, on pADPRP activity during DNA synthesis stimulated by EGF, using the cultured rat hepatocytes model. DNA synthesis was measured as [3H]thymidine incorporated/microgram DNA while pADPRP activity was expressed in pmol[32P]NAD incorporated/min/microgram DNA. Our results show that 2AAF treatment of EGF-stimulated rat hepatocytes induces a full block of DNA replication which is preceded and accompanied by a net inhibition of endogenous and total pADPRP activity, respectively. A block in pADPRP activity in normal hepatocytes, exposed to 2AAF in vitro or in vivo, could play a key role in cell transformation. Our data add further information on the possible involvement of this nuclear catalytic activity during DNA replication.

Influence of poly(ADP ribose) polymerase depletion on promotion of liver carcinogenesis.

In previous studies we demonstrated that liver poly(ADP ribose) polymerase (pADPRP) activity was lost in animals exposed to N-2-acetylaminofluorene (2AAF) according to the Teebor and Becker experimental model (Cancer Res 31:1-3, 1971). In addition, we used the resistant hepatocyte model of Solt and Farber (Nature 263:702-703, 1976) to further investigate pADPRP activity during the multistep process of liver carcinogenesis. A marked depletion of the catalytic protein was evidenced after 2AAF exposure, confirming previous results and indicating a specific effect of 2AAF on this nuclear enzyme that controls conformational changes of chromatin and regulates several catalytic activities in the nucleus. The levels of pADPRP mRNA, measured by northern blot analysis using both experimental models, indicate that the enzyme depletion is not due to a loss of transcript. Moreover, these data indicate that pADPRP depletion, caused by 2AAF, was also maintained during liver compensatory growth, which is known to induce a rapid and marked increase in pADPRP activity and protein level. Treatment of 2AAF-exposed animals with N-acetyl-L-cysteine not only efficiently protected against DNA damage, but also prevented a rapid depletion of the catalytic protein. Interestingly, these data indicate that the marked loss of liver pADPRP occurred during the promotion step induced by 2AAF feeding and that this loss was observed using different models for experimental hepatocarcinogenesis. This phenomenon can be ascribed to a highly defective transcript that cannot be correctly translated into the specific protein or to a rapid degradation of the translated protein.

Relationship between DNA topoisomerase I activity and DNA synthesis in cultured hepatocytes: effects of orotic acid.

Previous studies have demonstrated that DNA topoisomerase I activity can be closely related to DNA replication and active transcription in different experimental models. This relationship was further investigated by studying the time course of DNA topoisomerase I activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. DNA topoisomerase I activity was assayed quantifying ATP-independent relaxation of a negatively supercoiled plasmid, substrate for the enzyme. Incubation of cellular extracts was carried out at 30 degrees C for 15'. Products were electrophoresed and analyzed by densitometry. DNA synthesis was measured as [3H]thymidine incorporation in the hepatocytes, after a 2 hrs pulse labelling. An increase in DNA topoisomerase I activity was observed early after epidermal growth factor addition, before the onset of DNA synthesis. Concomitant administration of orotic acid, which has recently shown mitoinhibitory effect, abolished the EGF-induced activity as well as DNA synthesis. Taken together these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by DNA topoisomerase I with a precise time schedule. Orotic acid administration abolishes this response and causes an overall loss in DNA topoisomerase I activity.

Differential assay and biological significance of poly(ADP-ribose) polymerase activity in isolated liver nuclei.

Poly(ADP-ribosyl)ation of nuclear proteins is catalyzed by poly(ADP-ribose) polymerase. This enzyme is involved in the regulation of basic cellular functions of DNA metabolism. DNA breaks induced by DNA-damaging agents trigger the activation of poly(ADP-ribose) polymerase increasing its endogenous level. This increase modifies the pattern of poly(ADP-ribosyl)ated chromatin proteins. In this paper we describe a procedure for the isolation of intact nuclei from rat liver to be used for the endogenous activity assay. Artifactual activation of the enzyme was avoided since a very low level of DNA-strand breaks occurs during the isolation of nuclei. We present a series of experiments which prove the ability of this procedure to detect increases in endogenous liver activity without modification of the total level. The application of this technique can be useful for a better understanding of the role of early changes in poly(ADP-ribose) polymerase level in physiological conditions and during exposure to DNA-damaging agents.

Changes in activity and mRNA levels of poly(ADP-ribose) polymerase during rat liver regeneration.

ADP-ribosylation of nuclear proteins, catalysed by the enzyme poly(ADP-ribose) polymerase, is involved in the regulation of different cellular processes of DNA metabolism. To further clarify the role of the enzyme during proliferating activity of mammalian cells, we have studied the control of gene expression in regenerating rat liver. The changes in activity and mRNA levels were analysed during the early and late phases of the compensatory model. When enzyme activity was measured in isolated liver nuclei obtained at different times after hepatectomy, two different phases were observed: an early wave occurring before the onset of DNA synthesis, and a second one, starting several hours after the onset of DNA synthesis and returning to control values at later times. The evaluation of the enzymatic level in nuclear extracts and by activity gel analysis showed a more gradual increase starting 1 day after hepatectomy, in concomitance with the peak of DNA synthesis. By using a specific murine cDNA probe, a significant enhancement of mRNA levels for poly(ADP-ribose) polymerase was observed during liver regeneration, slightly preceding the onset of DNA synthesis. The results obtained show that changes in poly(ADP-ribose) polymerase activity, during liver regeneration, are associated both to early events preceding the increase in DNA synthesis and to later phases of the cell proliferation process.

Relationship between poly(ADP-ribose) polymerase activity and DNA synthesis in cultured hepatocytes.

Previous studies have demonstrated that an increase in poly(ADP-ribose) polymerase activity could be closely related to DNA replication during liver regeneration and to DNA repair synthesis in different experimental systems. This relationship was further investigated by studying the time course of endogenous and total poly(ADP-ribose) polymerase activity in cultured rat hepatocytes stimulated by epidermal growth factor. This mitogen has been shown to stimulate DNA synthesis in liver cells both in vivo and in vitro. A 6-fold increase in endogenous activity was observed early after epidermal growth factor addition, just before DNA synthesis. A subsequent 4-fold increment in total enzyme activity, concomitant with DNA synthesis, was detected. Orotic acid, which has recently shown mitoinhibitory effect, abolished the epidermal-growth-factor-induced increase in endogenous and total poly(ADP-ribose) polymerase activity, as well as DNA synthesis. On the contrary, 3-aminobenzamide inhibitor of poly(ADP-ribose) polymerase completely suppressed the endogenous activity but only partially modified the increase in total catalytic level and the overall pattern of thymidine incorporation. Taken together, these data indicate that, in cultured hepatocytes, the induction of DNA synthesis is supported by an increased poly(ADP-ribose) polymerase activity.