Tyrosine kinase nerve growth factor receptor switches from prosurvival to proapoptotic activity via Abeta-mediated phosphorylation.

The present study shows that increased Abeta production in hippocampal neurons, due to a failure of NGF signal, induces an unexpected phosphorylation of tyrosine kinase receptor A (TrkA), followed by activation of the phospholipase C gamma (PLCgamma) pathway and neuronal death. Such phosphorylation seems causally connected with 2 kinases known be involved in amyloidogenesis, Src and CDK5, and associated with alpha and gamma secretase-mediated p75 processing. Pharmacologic inhibition of TrkA phosphorylation and partial silencing of TrkA and/or p75 receptors prevent PLCgamma activation and protect neurons from death. Concomitantly with these events, TrkA, p75, Abeta peptides, and PS1 protein coimmunoprecipitate, suggesting their direct interplay in the subsequent onset of apoptotic death. Together, these findings depict a cellular mechanism whereby the same cellular transducing system may invert its intracellular message from trophic and antiapoptotic to a death signaling, which could also have relevance in the onset of Alzheimer's disease.

Expression of miR-487b and miR-410 encoded by 14q32.31 locus is a prognostic marker in neuroblastoma.

BACKGROUND: Combination of age at diagnosis, stage and MYCN amplification stratifies neuroblastoma into low-risk and high-risk. We aimed to establish whether a microRNA (miRNA) signature could be associated with prognosis in both groups. METHODS: Microarray expression profiling of human miRNAs and quantitative reverse-transcriptase PCR of selected miRNAs were performed on a preliminary cohort of 13 patients. Results were validated on an independent cohort of 214 patients. The relationship between miRNA expression and the overall or disease-free survival was analysed on the total cohort of 227 patients using the log-rank test and the multivariable Cox proportional hazard model. RESULTS: A total of 15 of 17 miRNAs that discriminated high-risk from low-risk neuroblastoma belonged to the imprinted human 14q32.31 miRNA cluster and two, miR-487b and miR-410, were significantly downregulated in the high-risk group. Multivariable analyses showed miR-487b expression as associated with overall survival and disease-free survival in the whole cohort, independently of clinical covariates. Moreover, miR-487b and miR-410 expression was significantly associated with disease-free survival of the non-MYCN-amplified favourable neuroblastoma: localised (stage 1, 2 and 3) and stage 4 of infant <18 months. CONCLUSION: Expression of miR-487b and miR-410 shows predictive value beyond the classical high-/low-risk stratification and is a biomarker of relapse in favourable neuroblastoma.

Intratumoral vs systemic administration of meta-tetrahydroxyphenylchlorin for photodynamic therapy of malignant gliomas: assessment of uptake and spatial distribution in C6 rat glioma model.

Malignant gliomas, with an incidence of 5 cases per 100,000 population per year, represent the most common primary brain tumour. They have an overall survival length of less than 2 years. Many different adjuvant therapies have been developed. Among them, Photodynamic Therapy (PDT), that is based on photochemical reactions between light and tumoral tissue selectively labelled with exogenous photosensitizing agents. Among photosensitizers, m-THPC (Temoporfin), seems to be the most promising one for the treatment of brain tumors, but, unfortunately, it causes problems of high skin photosensitivity. To by-pass this problem, we devised an intratumoral route of administration of this photosensitizer. The aim of this study is to investigate and compare the uptake of m-THPC in brain tumor and normal tissue after systemic and intratumoral administration of the drug. 30 female Wistar rats received m-THPC 12 days after C6 tumor implantation. Temoporfin was administered intratumorally in 24 rats at two different concentrations. 6 rats constituted the control group and received m-THPC by means of an intraperitoneal injection. The brains were extracted at 4 h, 24 h and 96 h after Temoporfin injection. The samples were examined with a confocal laser scanning microscope. All samples showed high fluorescence emission exclusively in the tumour area, without appreciable differences between the samples taken at the different times of sacrifice and the two routes of administration. No fluorescence whatsoever was detected among normal brain tissue surrounding the tumour. The intratumoral route appears to give comparable results to the systemic one, regarding intracellular uptake efficiency and tumour--normal tissue ratio, with the advantage of a much shorter time needed to reach optimal intratumoural concentration--that is just four hours from m-THPC injection.

Gene dosage alteration of L2 ribosomal protein genes in Saccharomyces cerevisiae: effects on ribosome synthesis.

In Saccharomyces cerevisiae, the genes coding for the ribosomal protein L2 are present in two copies per haploid genome. The two copies, which encode proteins differing in only a few amino acids, contribute unequally to the L2 mRNA pool: the L2A copy makes 72% of the mRNA, while the L2B copy makes only 28%. Disruption of the L2B gene (delta B strain) did not lead to any phenotypic alteration, whereas the inactivation of the L2A copy (delta A strain) produced a slow-growth phenotype associated with decreased accumulation of 60S subunits and ribosomes. No intergenic compensation occurred at the transcriptional level in the disrupted strains; in fact, delta A strains contained reduced levels of L2 mRNA, whereas delta B strains had almost normal levels. The wild-type phenotype was restored in the delta A strains by transformation with extra copies of the intact L2A or L2B gene. As already shown for other duplicated genes (Kim and Warner, J. Mol. Biol. 165:79-89, 1983; Leeret al., Curr. Genet. 9:273-277, 1985), the difference in expression of the two gene copies could be accounted for via differential transcription activity. Sequence comparison of the rpL2 promoter regions has shown the presence of canonical HOMOL1 boxes which are slightly different in the two genes.

The ABF1 factor is the transcriptional activator of the L2 ribosomal protein genes in Saccharomyces cerevisiae.

The same factor, ABF1, binds to the promoters of the two gene copies (L2A and L2B) coding for the ribosomal protein L2 in Saccharomyces cerevisiae. In vitro binding experiments and in vivo functional analysis showed that the different affinities of the L2A and L2B promoters for the ABF1 factor are responsible for the differential transcriptional activities of the two gene copies. The presence of ABF1-binding sites in front of many housekeeping genes suggests a general role for ABF1 in the regulation of gene activity.

Antibodies elicited by naked DNA vaccination against the complementary-determining region 3 hypervariable region of immunoglobulin heavy chain idiotypic determinants of B-lymphoproliferative disorders specifically react with patients' tumor cells.

Several reports have suggested that the mechanism of protection induced by antiidiotypic vaccination against low-grade lymphoproliferative disorders is likely to be antibody mediated. Here we test the hypothesis that DNA vaccination with the short peptide encompassing the complementary-determining region 3 hypervariable region of immunoglobulin heavy chain (VH-CDR3) may elicit a specific antibody immune response able to recognize the native antigens in the form required for therapy. As a test system, we used the VH-CDR3 sequences derived from two patients with non-Hodgkin's B lymphomas (PA, AS) and one patient with hairy cell leukemia (BA) to immunize outbred Swiss mice. This experimental model could mimic a clinical setting in which different patients present distinct HLA haplotypes. Individual tumor-specific VH-CDR3 sequences were amplified by a two-step procedure and directly cloned into multigenic plasmid vectors (pRC100 and derived) with and without mouse interleukin 2 (mIL-2). Each tumor-specific sequence was characterized by sequencing. Female Swiss mice were vaccinated i.m. with plasmids expressing the tumor-specific VH-CDR3 sequence alone (pRC101-PA), mIL-2 plus the VH-CDR3 sequence (pRC111-PA), or a different unrelated antigen (NS3 of hepatitis C virus; pRC112), the sole mIL-2 (pRC110), and the empty plasmid (pRC100). Boost injections were performed at 3 and 16 weeks from the first vaccination, and sera were drawn before each vaccination and at 6, 9, and 19 weeks. Induction of anti-VH-CDR3s antibodies in the sera and their ability to recognize native antigens on patients' tumor cells were evaluated by FACS analysis. Up to 56% (n = 25) of mice vaccinated with pRC111-PA plasmid and 20% (n = 15) of mice vaccinated with pRC101-PA developed a specific immune response that was maintained throughout 19 weeks of observation in 40% of pRC111-PA-vaccinated mice. No response was detected in sera obtained from mice vaccinated with the other plasmids (n = 45). pRC111-PA injection s.c. was less effective (13%, n = 15) than i.m. injection (53%, n = 15). Indeed, we demonstrated that antibodies elicited by naked DNA vaccination against three different patient-derived VH-CDR3 peptides (pRC111-PA or BA or AS) readily reacted with binding epitopes on the idiotypic proteins expressed on the surface of tumor cells derived from each patient; 60, 40, and 40% of, respectively, PA-, BA-, and AS-vaccinated mice developed specific antibodies. No cross-reactivity was detected among the three different CDR3s against tumor cells derived from the other two patients. The outbred mouse strategy confirmed the significant matching potential of three different VH-CDR3 peptides to be efficaciously presented through different MHCs. We conclude that individual VH-CDR3 DNA vaccination can result in a potentially effective specific immune response against non-Hodgkin's B lymphoma cells by a rapid and low-cost therapeutic approach.

Evidence for oxidative activation of c-Myc-dependent nuclear signaling in human coronary smooth muscle cells and in early lesions of Watanabe heritable hyperlipidemic rabbits: protective effects of vitamin E.

BACKGROUND: Oxidized LDL (oxLDL) promotes atherogenesis, and antioxidants reduce lesions in experimental models. OxLDL-mediated effects on c-Myc are poorly characterized, and those on c-Myc nuclear pathways are completely unknown. c-Myc stimulates smooth muscle cell (SMC) proliferation and could be involved in atherosclerosis. We investigated the early effects of oxLDL and alpha-tocopherol on c-Myc, its binding partner Max, and the carboxy-terminal domain-binding factors activator protein-2 and elongation 2 factor in human coronary SMCs. We also investigated whether 9-week treatment of Watanabe heritable hyperlipidemic (WHHL) rabbits with diet-enriched alpha-tocopherol reduces c-Myc expression and oxLDL in the left coronary artery. METHODS AND RESULTS: OxLDL enhanced c-Myc/Max expression and transcription by cotransfection assay and the nuclear activities of E2F and activator protein-2 by binding shift and supershift in coronary SMCs. alpha-Tocopherol significantly reduced these molecular events. Furthermore, alpha-tocopherol reduced early lesions, SMC density, and the immunohistochemical presence of c-Myc, which colocalized with oxLDL/foam cells in the coronaries of WHHL rabbits. CONCLUSIONS: We provide the first evidence that oxLDL and alpha-tocopherol may influence c-Myc activation and several c-Myc-dependent signaling pathways in human coronary SMCs. The observation that in vivo, an antioxidant reduces both c-Myc and oxLDL in early coronary lesions of rabbits is consistent with, but does not prove, the hypothesis that c-Myc-dependent factors activated by oxidative processes contribute to atherogenesis and coronary heart disease.

Control of neoplastic cell proliferation and differentiation by restoration of 4-hydroxynonenal physiological concentrations.

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. In anaplastic cell lines products of membrane lipid peroxidation are very low or undetectable. Furthermore numerous results demonstrate effect of lipid peroxidation products on central biochemical pathways and intracellular signalling at physiological concentrations. 4-hydroxynonenal (HNE) is one of the most active products of lipid peroxidation. The restoration of HNE physiological concentrations in neoplastic cells may inhibit cell proliferation and modulate cell re-differentiation. This review try to summarize and critically discuss the effects of physiological concentrations of HNE on normal and neoplastic cell line.

Growth inhibition and differentiation induction in murine erythroleukemia cells by 4-hydroxynonenal.

4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation. Here we show that the exposure of murine erythroleukemia (MEL) cells to 1 microM HNE, for 10.5 h over 2 days, induces a differentiation comparable with that observed in cells exposed to DMSO for the whole experiment (7 days). The exposure of MEL cells for the same length of time demonstrates a higher degree of differentiation in HNE-treated than in DMSO-treated MEL cells. The protooncogene c-myc is down-modulated early, in HNE-induced MEL cells as well as in DMSO-treated cells. However, ornithine decarboxylase gene expression first increases and then decreases, during the lowering of the proliferation rate. These findings indicate that HNE, at a concentration physiologically found in many normal tissues and in the plasma, induces MEL cell differentiation by modulation of specific gene expression.

[The relations between atrial natriuretic factor release and adrenergic activation in hypertrophic cardiomyopathy]. / Relazioni tra rilascio di fattore natriuretico atriale ed attivazione adrenergica nella cardiomiopatia ipertrofica.

Atrial natriuretic factor (ANF) is a potent natriuretic and vasoactive (vasorelaxant) peptide localized in the secretory-like atrial specific granules. The main peptide in this storage granules is the 126 amino acid proatrial natriuretic peptide, but the principal circulating form in human plasma is the 28 amino acid, alpha-human natriuretic peptide. Animal and in vitro studies have suggested that ANF modulates autonomic circulatory control, probably with a dose-dependent mechanism. Moreover, recent human studies have resulted contradictory. In particular, it is still unclear if high circulating levels of ANF, which are present in congestive heart diseases constantly, may be correlated with sympathetic nervous system activity in man. Previously we have shown that in congestive diseases there is a relation between ANF and catecholamine secretion. From these basis, the aim of this study was to investigate on the pathophysiological relations between atrial natriuretic factor (ANF) release and adrenergic activation in patients with obstructive hypertrophic cardiomyopathy (n = 6) and non obstructive hypertrophic cardiomyopathy (n = 4). Sympathetic activation in physiologic way was induced by cycloergometer sub-maximal exercise. Then specimens of venous blood were achieved for plasma determination of ANF and catecholamines pre- and post-exercise. Results have shown that in obstructive hypertrophic cardiomyopathy patients basal levels of ANF and catecholamines were higher than levels of these parameters in non obstructive hypertrophic cardiomyopathy patients.(ABSTRACT TRUNCATED AT 250 WORDS)

[Angiotensin II stimulates endothelin-1 release from human endothelial cells]. / L'angiotensina II stimola il rilascio di endotelina-1 da cellule endoteliali umane.

Endothelin-1 (ET-1) is an endothelium-derived vasoconstrictor peptide isolated from the culture supernatant of porcine aortic endothelial cells. This 21 amino-acid residue peptide has potent vasoconstrictive properties in vitro and in vivo. ET-1 action involves phosphatidylinositol turnover, calcium mobilization and protein kinase C activation. Endothelial cells have distinct receptors for different operating through hydrosoluble hormones. The aim of this study was to investigate on a possible role of angiotensin II (ANG II) to modulate the release ET-1 from human endothelial cells in vitro. These data revealed a time- and a dose-dependent increase of ET-1 production in response to ANG II. This mechanism may have important pathophysiological implications in vivo. In fact, a double-mechanism of secretion of ET-1 from endothelial cells could exist: one active in a physiological condition and an other in response to a vasoconstrictor stimuli (as well as ANG II). Furthermore, these results may suggest an additional favourable effect of ACE-inhibition in human hypertension therapy.

Does the term 'trophic' actually mean anti-amyloidogenic? The case of NGF.

The term trophic is widely used to indicate a general pro-survival action exerted on target cells by different classes of extracellular messengers, including neurotrophins (NTs), a family of low-molecular-weight proteins whose archetypal member is the nerve growth factor (NGF). The pro-survival action exerted by NTs results from a coordinated activation of multiple metabolic pathways, some of which have only recently come to light. NGF has been shown to exert a number of different, experimentally distinguishable effects on neurons, such as survival, differentiation of target neurons, growth of nerve fibers and their guidance (tropism) toward the source of its production. We have proposed a more complete definition of the NGF trophic action that should also include its newly discovered property of inhibiting the amyloidogenic processing of amyloid precursor protein (APP), which is among the first hypothesized primary trigger of Alzheimer's disease (AD) pathogenesis. This inhibitory action appears to be mediated by a complex series of molecular events and by interactions among NGF receptors (TrkA and p75), APP processing and tau metabolic fate and function.

Extensive modulation of a set of microRNAs in primary glioblastoma.

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles in animals and plants by repressing translation or cleaving RNA transcripts. The specific modulation of several microRNAs has been recently associated to some forms of human cancer, suggesting that these short molecules may represent a new class of genes involved in oncogenesis. In our study, we examined by microarray the global expression levels of 245 microRNAs in glioblastoma multiforme, the most frequent and malignant of primary brain tumors. The analysis of both glioblastoma tissues and glioblastoma cell lines allowed us to identify a group of microRNAs whose expression is significantly altered in this tumor. The most interesting results came from miR-221, strongly up-regulated in glioblastoma and from a set of brain-enriched miRNAs, miR-128, miR-181a, miR-181b, and miR-181c, which are down-regulated in glioblastoma.

The ribosomal protein L2 in S. cerevisiae controls the level of accumulation of its own mRNA.

The expression of the yeast L2 r-protein gene is controlled at the level of mRNA accumulation. The product of the gene appears to participate in this regulation by an autogenous feedback mechanism. This control does not operate at the level of transcription but instead affects L2 mRNA accumulation. This autogenous regulation of mRNA accumulation provides an interesting analogy to the autogenous translational regulation of r-proteins in Escherichia coli.

Selection of novel Mg(2+)-dependent self-cleaving ribozymes.

Four RNA motifs are known that catalyse site-specific cleavage in the presence of Mg2+ ions, all discovered in natural RNAs. In a single in vitro selection experiment we have isolated representatives of five novel classes of Mg(2+)-dependent ribozymes. Small versions of three of these showed that a very simple internal loop type of secondary structure is responsible for the activity. One of these was synthesized in a bimolecular form, and compared directly with the hammerhead ribozyme; for the new ribozyme, the cleavage step of the reaction is much faster than the spontaneous rate of phosphodiester bond cleavage, yet substantially slower than that for the hammerhead. The results suggest that many more Mg(2+)-dependent self-cleaving RNA sequences can be found.

Binding and cleavage of pre-tRNA by the Xenopus splicing endonuclease: two separable steps of the intron excision reaction.

We have constructed three base-substitution mutants of the yeast tRNALeu3 gene. In two of them the ability to form an extended anticodon stem is lost. In the first mutant the bases encoding the anticodon change from TTG to GAC (positions 37, 36, 35); in the second, the nucleotides encoding the region of the intron that base-pair with the anticodon change from CAA to GTC (positions 48, 47, 46). The third is a double mutant characterized by both substitutions described above so that its ability to form an extended anticodon stem is restored. The precursors derived from the two single mutants are accurately spliced in the X. laevis germinal vesicles (GV) extract: pairing of the anticodon with the intron, therefore, is not required for the splicing reaction. The precursor derived from the double mutant is not spliced, indicating that the new extended anticodon stem exerts an inhibitory action. Since the double mutant precursor binds to the purified splicing endonuclease, binding and cleavage occur as two separable steps in the intron excision reaction.

Site selection by the tRNA splicing endonuclease of Xenopus laevis.

To investigate the mechanism by which the purified Xenopus tRNA splicing endonuclease recognizes its splice sites, we utilized yeast pre-tRNA(3Leu) and pre-tRNA(Phe) variants constructed by in vitro mutagenesis. We found that the endonuclease interacts with conserved features of the mature tRNA domain. In particular, U8 and C56 may be examples of contact points between protein and RNA. Given that there are no conserved sequences at the splice junctions, the specificity of cutting at both splice sites is determined by the length of the anticodon stem. Although in general, the sequence of the intron is unimportant for splicing, there are some structural requirements.

A tricistronic retroviral vector expressing natural antiangiogenic factors inhibits angiogenesis in vitro, but is not able to block tumor progression in vivo.

Angiogenesis, the formation of new blood vessels out of pre-existing capillaries, is essential for tumor progression. Many factors have been identified that are able to inhibit angiogenesis. Here, we report the construction of a tricistronic retroviral vector encoding two inhibitors of angiogenesis expressed in mammals: the N-terminal fragment of rat prolactin (16KrPRL) and a secreted form of human platelet factor 4 (sPF4). When transduced by this retroviral vector, a rat glioblastoma cell line loses its ability of promoting endothelial cell locomotion, the initial step of angiogenesis, and the formation of an endothelial cell tube network. In spite of this encouraging in vitro result, however, the anti-angiogenic vector cannot block glioblastoma progression in animal models. These results suggest that therapeutic strategies aiming to block tumor progression through the inhibition of tumor-associated angiogenesis, should not only provide large numbers of angiogenesis inhibitors, but also target the angiogenic factors produced by tumor cells. Moreover, the data described herein may confirm recent findings from other groups which indicate that in order to successfully counteract tumor progression, drugs inhibiting new blood vessel formation should be employed in combination with traditional anti-tumor strategies, such as chemotherapy or radiotherapy.

Histological findings and evidence of lipid conjugated dienes and malonyldialdehyde in human fetal aortas.

Recent evidence strongly suggests that peroxidative modification of lipids may play a significant role in atherogenesis. In our present research, we investigated if the oxidative stress mediated by oxygen free radicals was a pathophysiologic condition that occurred in the early stages of human development. Thus the aim of this research was to examine lipid peroxidation in human fetal aortas. Human fetal aortas and proximal iliac arteries (n = 8) were obtained from fetuses aged 7 +/- 2 months, immediately after autopsy. Lipids from the initial fatty streak lesions (LFS) and the vessels uninvolved (LUV) were extracted by the chloroform/methanol method. Lipid peroxidation levels were measured by two different methods: determination of lipid conjugate dienes (the spectrum trend was recorded from 320 to 200 nm with a spectrophotometer) and malonyldialdehyde (MDA) content (TBA method). We observed that lipid conjugated dienes were present in LFS, but not in LUV, with a characteristic absorption peak at 233 nm. In addition, MDA levels were significantly higher when the LFS = 3.85 +/- 0.91 nmol than when the LUV = 0.41 +/- 0.12 nmol (p < 0.001 versus LUV). The presence of lipid peroxidation in our samples could be mediated by free radical production in the first stages of human development. Thus these data suggest that LFS peroxidation mediated by free radicals occurs in the vascular circulation in the early stages of human development. This could influence the progression of vascular damage and atherosclerotic disease.