Search

Functional and ultrastructural investigations support the concept that altered brain connectivity, exhausted neural plasticity, and synaptic loss are the strongest correlates of cognitive decline in age-related neurodegenerative dementia of Alzheimer's type. We have previously demonstrated that in transgenic mice, expressing amyloid-β precursor protein-Swedish mutation active caspase-3 accumulates in hippocampal postsynaptic compartments leading to altered postsynaptic density (PSD) composition, increased long-term depression (LTD), and dendritic spine loss. Furthermore, we found strong evidence that dendritic spine alteration is mediated by calcineurin activation, a calcium-dependent phosphatase involved in synapse signaling. In the present work, we analyzed the molecular mechanism linking alteration of synaptic plasticity to the increase of calcineurin activity. We found that acute treatment of young and plaque-free transgenic mice with the calcineurin inhibitor FK506 leads to a complete rescue of LTD and PSD composition. Our findings are in agreement with other results reporting that calcineurin inhibition improves memory function and restores dendritic spine density, confirming that calcineurin inhibition may be explored as a neuroprotective treatment to stop or slowdown synaptic alterations in Alzheimer's disease.

Voltage dependent sodium channels are membrane proteins essential for cell excitability. They are composed by a pore-forming α-subunit, encoded in mammals by up to 9 different genes, and 4 different ancillary β-subunits. The expression pattern of the α subunit isoforms confers the distinctive functional and pharmacological properties to different excitable tissues. β subunits are important modulators of channel function and expression. Mutation C121W of the β1-subunit causes an autosomal dominant epileptic syndrome without cardiac symptoms. The C121W mutation may act by a dominant-competition, modifying the expression of α-subunit proteins. To test this hypothesis, we transfected GH3 cells, from neuro-ectoderm origin, with wild-type or mutant β1 subunits and compared them to native cells. To examine the tissue specificity of the C121W-β1 mutation, we compared the effects of the mutation on neural cells with those of H9C2 cells of cardiac origin. We found that in GH3 cells the over-expression of the β1 subunit augments the α subunit mRNA and protein levels, while in the H9C2 cells the enhanced level of β1 subunit not only increases but also qualitatively modifies the sodium channel α isoform expression pattern. Interestingly, the introduction of the epileptogenic C121W-β1 subunit does not alter the sodium channel isoform composition of GH3 cells, while produces additional changes in the α-subunit expression pattern of H9C2 cells. Electrophysiological measurements confirm these molecular results. The expression differences observed could be correlated to the tissue-specific regulatory action of the β1 subunit and to the nervous system specificity of the C121W mutation. Our findings could be helpful for the comprehension of the molecular mechanism of generalised epileptic with febrile seizures plus in patients with identified β1 subunit mutations.

Breast cancer is often fatal during its metastatic dissemination. To unravel the role of microRNAs (miRs) during malignancy, we analyzed miR expression in 77 primary breast carcinomas and identified 16 relapse-associated miRs that correlate with survival and/or distinguish tumor subtypes in different datasets. Among them, miR-148b, down-regulated in aggressive breast tumors, was found to be a major coordinator of malignancy. In fact, it is able to oppose various steps of tumor progression when overexpressed in cell lines by influencing invasion, survival to anoikis, extravasation, lung metastasis formation, and chemotherapy response. miR-148b controls malignancy by coordinating a novel pathway involving over 130 genes and, in particular, it directly targets players of the integrin signaling, such as ITGA5, ROCK1, PIK3CA/p110α, and NRAS, as well as CSF1, a growth factor for stroma cells. Our findings reveal the importance of the identified 16 miRs for disease outcome predictions and suggest a critical role for miR-148b in the control of breast cancer progression.

Oxidative damage plays a pathogenic role in various chronic degenerative diseases. Oxidative damage targeting trabecular meshwork (TM) cells as a consequence of mitochondrial damage is a pathogenic mechanism for glaucoma, the most common cause of irreversible blindness worldwide. Consequences of oxidative damage are attenuated by endocellular activities involved in scavenging reactive oxidative species and DNA repair. Selected bacterial genes are highly efficient at protecting cells from oxidative DNA damage. This situation occurs for Escherichia coli formamidopyrimidine DNA glycosylase (FPG), a major DNA glycosylase that repairs oxidatively damaged DNA. Accordingly, this study was aimed at transfecting human TM cells (HTMC) with Fpg in order to increase their resistance to oxidative damage. This study demonstrates that it is feasible to increase resistance of HTMC to endogenous oxidative damage by gene transfection. These findings bear relevance for primary and secondary prevention of degenerative glaucomas and other degenerative diseases where oxidative damage plays a pathogenic role.

Infection-driven inflammation has been implicated in the pathogenesis of ~15–20% of human tumors. Expression of microRNA-155 ( miR-155 ) is elevated during innate immune response and autoimmune disorders as well as in various malignancies. However, the molecular mechanisms providing miR-155 with its oncogenic properties remain unclear. We examined the effects of miR-155 overexpression and proinflammatory environment on the frequency of spontaneous hypoxanthine phosphoribosyltransferase ( HPRT ) mutations that can be detected based on the resistance to 6-thioguanine. Both miR-155 overexpression and inflammatory environment increased the frequency of HPRT mutations and down-regulated WEE1 (WEE1 homolog-S. pombe), a kinase that blocks cell-cycle progression. The increased frequency of HPRT mutation was only modestly attributable to defects in mismatch repair machinery. This result suggests that miR-155 enhances the mutation rate by simultaneously targeting different genes that suppress mutations and decreasing the efficiency of DNA safeguard mechanisms by targeting of cell-cycle regulators such as WEE1. By simultaneously targeting tumor suppressor genes and inducing a mutator phenotype, miR-155 may allow the selection of gene alterations required for tumor development and progression. Hence, we anticipate that the development of drugs reducing endogenous miR-155 levels might be key in the treatment of inflammation-related cancers.

Objectives. Features of deregulated Notch1 signaling and NF-κB activation have independently been reported in cervical cancers. Here, we have extended these observations and examined both these pathways simultaneously in human cervical cancer tissue. Further, we have investigated the potential cross-talk between these pathways in a human cervical cancer derived cell line CaSki, which mirrors features of Notch activation as in the majority of human cervical cancers. Methods. Cervical tissue samples were analyzed for the expression of Notch1, Jagged 1, Hes1, pAKT, NF-κB p50, NF-κB p65, IκB-α, Bcl-2, CyclinD1, Cdk9, c-Fos, and p53 by immunohistochemistry. A total of 352 samples were analyzed which included 69 normal cervical tissue, 132 preinvasive lesions and 151 squamous cell carcinomas of the uterine cervix. Dual immunofluorescent analysis was performed to evaluate the coexpression of Notch1 and NF-κB. Transcriptional reporter assays and xenografts were undertaken with CaSki cells. Results. Features of Notch1 activation as measured by intracellular Notch1, high levels of Jagged1, Hes1 and Cdk9 were paralleled by nuclear translocation of both NF-κB p50 and p65 with target gene expression (IκB-α, Bcl-2, and CyclinD1) in human cervical cancer sections. Reporter assays in CaSki cells are consistent with Notch being an upstream regulator of NF-κB. Further, the xenografts recreate key aspects of human cancer tissue. Conclusions. Results from this study suggest that there is a co-activation of Notch1 and NF-κB signaling pathways at the cellular level in the majority of human cervical cancers, with Notch as an upstream regulator.

Over the past 50 years, many advances in our understanding of the general principles controlling gene expression during hematopoiesis have come from studying the synthesis of hemoglobin. Discovering how the alpha- and beta-globin genes are normally regulated and documenting the effects of inherited mutations that cause thalassemia have played a major role in establishing our current understanding of how genes are switched on or off in hematopoietic cells. Previously, nearly all mutations causing thalassemia have been found in or around the globin loci, but rare inherited and acquired trans-acting mutations are being found more often. Such mutations have demonstrated new mechanisms underlying human genetic disease. Furthermore, they are revealing new pathways in the regulation of globin gene expression that, in turn, may open up new avenues for improving the management of patients with common types of thalassemia.

The recruitment of lymphocytes across the blood brain barrier (BBB) is mediated by adhesion molecules and chemokines. The expression of activation markers and of chemokine receptors on T cells homing to the nervous system (NS) may help define their functional state. In the cerebrospinal fluid (CSF) of subjects with inflammatory neurological diseases (IND), including multiple sclerosis, we observed an increased number of T cells coexpressing CXCR3 and CCR5 as well as T cells with a CD45RO+ CCR7+ CD27+ memory phenotype. A subset of CCR7+ T cells coexpressed CXCR3 and CCR5. We also detected an increased number of interferon-γ-producing T cells in the CSF compared with peripheral blood, mostly but not exclusively in the CD45RO+ CCR7− CD27− compartment. T helper 1 (Th1) clones, established from the CSF of individuals with IND and from a healthy subject, similarly migrated to CXCL10, CXCL12, and CCL5. CXCL10, CXCL12, and CCL19 were increased in the CSF of individuals with neuroinflammation. These findings suggest that CSF is enriched in Th1-polarized memory T cells capable of differentiating into effector cells upon antigen encounter. These cells are recruited into the CSF by inducible chemokines. Thus, CSF represents a transitional station for T cells trafficking to and from the NS.

Intracellular Ca 2+ levels control both contraction and relaxation in vascular smooth muscle cells (VSMCs). Ca 2+ -dependent relaxation is mediated by discretely localized Ca 2+ release events through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). These local increases in Ca 2+ concentration, termed sparks, stimulate nearby Ca 2+ -activated K + (BK) channels causing BK currents (spontaneous transient outward currents or STOCs). STOCs are hyperpolarizing currents that oppose vasoconstriction. Several RyR isoforms are coexpressed in VSMCs; however, their role in Ca 2+ spark generation is unknown. To provide molecular information on RyR cluster function and assembly, we examined Ca 2+ sparks and STOCs in RyR3-deficient freshly isolated myocytes of resistance-sized cerebral arteries from knockout mice and compared them to Ca 2+ sparks in cells from wild-type mice. We used RT-PCR to identify RyR1, RyR2, and RyR3 mRNA in cerebral arteries. Ca 2+ sparks in RyR3-deficient cells were similar in peak amplitude (measured as F/F 0 ), width at half-maximal amplitude, and duration compared with wild-type cell Ca 2+ sparks. However, the frequency of STOCs (between −60 mV and −20 mV) was significantly higher in RyR3-deficient cells than in wild-type cells. Ca 2+ sparks and STOCs in both RyR3-deficient and wild-type cells were inhibited by ryanodine (10 μmol/L), external Ca 2+ removal, and depletion of SR Ca 2+ stores by caffeine (1 mmol/L). Isolated, pressurized cerebral arteries of RyR3-deficient mice developed reduced myogenic tone. Our results suggest that RyR3 is part of the SR Ca 2+ spark release unit and plays a specific molecular role in the regulation of STOCs frequency in mouse cerebral artery VSMCs after decreased arterial tone.

Chemical lesion of olfactory neuroepitheium induced an up-regulation of the mGlu1a metabotropic glutamate receptor protein in the olfactory bulb, as shown by Western blot analysis. At 2 days after the lesion, the increase in the receptor protein was associated with an increase in mGlu1a mRNA levels; in contrast, at longer times after the lesion (16 days), mRNA levels were reduced in spite of the high expression of the receptor protein, perhaps as a result of product-inhibition of mGlu1 gene expression. Immunohistochemical analysis showed that the increase in mGlu1a induced by olfactory denervation was confined to the glomeruli, which occupy the external portion of the olfactory bulb. Within these structures, mGlu1a receptors are mainly localized on the distal dendrites of mitral cells, which are innervated by the glutamatergic axons of the olfactory nerve. These results demonstrate that the expression of mGlu1a receptors is up-regulated in response to glutamatergic deafferentation, supporting a role for this particular receptor subtype in the physiology of synaptic transmission.

Background The budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses) and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus). We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast. Results To do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5% glucose and 5% galactose. Following such induction, AAV virus like particles (VLPs) were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells. Conclusions Taken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology.

Gene therapy vectors based on the adeno-associated virus (AAV) are extremely efficient for gene transfer into post-mitotic cells of heart, muscle, brain, and retina. The reason for their exquisite tropism for these cells has long remained elusive. Here, we show that upon terminal differentiation, cardiac and skeletal myocytes downregulate proteins of the DNA damage response (DDR) and that this markedly induces permissivity to AAV transduction. We observed that expression of members of the MRN complex (Mre11, Rad50, Nbs1), which bind the incoming AAV genomes, faded in cardiomyocytes at ~2 weeks after birth, as well as upon myoblast differentiation in vitro ; in both cases, withdrawal of the cells from the cell cycle coincided with increased AAV permissivity. Treatment of proliferating cells with short-interfering RNAs (siRNAs) against the MRN proteins, or with microRNA-24, which is normally upregulated upon terminal differentiation and negatively controls the Nbs1 levels, significantly increased permissivity to AAV transduction. Consistently, delivery of these small RNAs to the juvenile liver concomitant with AAV markedly improved in vivo hepatocyte transduction. Collectively, these findings support the conclusion that cellular DDR proteins inhibit AAV transduction and that terminal cell differentiation relieves this restriction.

Cancer development, progression, and metastasis are highly dependent on angiogenesis. The use of antiangiogenic drugs has been proposed as a novel strategy to interfere with tumor growth, but cancer cells respond by developing strategies to escape these treatments. In particular, animal models show that antiangiogenic drugs currently used in clinical settings reduce tumor tissue oxygenation and trigger molecular events that foster cancer resistance to therapy. Here, we show that semaphorin 3A (Sema3A) expression overcomes the proinvasive and prometastatic resistance observed upon angiogenesis reduction by the small-molecule tyrosine inhibitor sunitinib in both pancreatic neuroendocrine tumors (PNETs) in RIP-Tag2 mice and cervical carcinomas in HPV16/E2 mice. By improving cancer tissue oxygenation and extending the normalization window, Sema3A counteracted sunitinib-induced activation of HIF-1α, Met tyrosine kinase receptor, epithelial-mesenchymal transition (EMT), and other hypoxia-dependent signaling pathways. Sema3A also reduced tumor hypoxia and halted cancer dissemination induced by DC101, a specific inhibitor of the VEGF pathway. As a result, reexpressing Sema3A in cancer cells converts metastatic PNETs and cervical carcinomas into benign lesions. We therefore suggest that this strategy could be developed to safely harnesses the therapeutic potential of the antiangiogenic treatment.

By transactivating expression of miRNAs that repress expression of the ZEB1 and ZEB2 transcription factors, p53 inhibits the epithelial–mesenchymal transition., p53 suppresses tumor progression and metastasis. Epithelial–mesenchymal transition (EMT) is a key process in tumor progression and metastasis. The transcription factors ZEB1 and ZEB2 promote EMT. Here, we show that p53 suppresses EMT by repressing expression of ZEB1 and ZEB2. By profiling 92 primary hepatocellular carcinomas (HCCs) and 9 HCC cell lines, we found that p53 up-regulates microRNAs (miRNAs), including miR-200 and miR-192 family members. The miR-200 family members transactivated by p53 then repress ZEB1/2 expression. p53-regulated miR-192 family members also repress ZEB2 expression. Inhibition or overexpression of the miRNAs affects p53-regulated EMT by altering ZEB1 and ZEB2 expression. Our findings indicate that p53 can regulate EMT, and that p53-regulated miRNAs are critical mediators of p53-regulated EMT.

Learning Objectives: After completing this course, the reader will be able to: Describe the relationship between HER-3 status and response to treatment with cetuximab and irinotecan in patients with advanced colorectal cancer and EGFR+ wild-type K-RAS mutation in this study.Evaluate data that support using HER-3 status as a predictive factor for anti-EGFR treatment response in patients with colorectal cancer. CME This article is available for continuing medical education credit at CME.TheOncologist.com Preclinical data suggested that, in the presence of human epidermal growth factor receptor (HER)-3–altered activation, colorectal cancer cells may escape anti–epidermal growth factor receptor (EGFR)-mediated cell death. HER-3 overexpression may then represent a key factor for resistance to anti-EGFR antibodies in colorectal cancer. The aim of our analysis was to investigate a possible correlation between HER-3 expression and clinical outcome in wild-type K-RAS advanced colorectal cancer patients receiving cetuximab and irinotecan. We retrospectively analyzed immunoreactivity for HER-3 in wild-type K-RAS advanced colorectal cancer patients receiving irinotecan and cetuximab. Eighty-four advanced wild-type K-RAS colorectal cancer patients were available for HER-3 analysis. Forty patients (48%) had a HER-3− colorectal tumor, whereas the remaining 44 cases (52%) were deemed HER-3+. In patients with HER-3− and HER-3+ tumors, we observed a partial response in 17 (42%) and eight (18%) patients respectively; progressive disease occurred in 11 (35%) and 26 (53%) patients with HER-3− and HER-3+ tumors, respectively (p = .003). The median progression-free survival time was 6.3 months in patients with HER-3− tumors and 2.8 months for those who had HER-3–overexpressing tumors (p < .0001). The median overall survival time was 13.6 months in patients showing HER-3− tumors and 10.5 months for those who had HER-3–expressing tumors (p = .01). HER-3 proved to be a predictive factor for clinical outcome in wild-type K-RAS colorectal cancer patients treated with cetuximab. Combined HER-3 and K-RAS analysis may represent an effective strategy for better selection of responding colorectal cancer patients.

The attempts at identifying precise replication origins (ori) in mammalian DNA have been pursued mainly through physico-chemical and biochemical approaches, in view of the essential failure of the search for autonomously replicating sequences in cultured cells. These approaches involve the mapping of short stretches of nascent DNA, the identification of the regions where either leading or lagging strands switch polarity, or the localization of replication intermediates by two-dimensional gel electrophoresis. Due to the complexity of animal cell genomes, most of these studies have been performed on amplified domains and with the use of synchronization procedures. The results obtained have been controversial. In order to avoid the use of experimental procedures potentially affecting the physiological mechanism of DNA replication, we have developed a method for the localization of ori in single-copy loci in exponentially growing cells. This method entails the absolute quantification of the abundance of selected DNA fragments along a genomic region within samples of newly synthesized DNA by competitive polymerase chain reaction (PCR); the latter is immune to all the uncontrollable variables which severely affect the reproducibility of conventional PCR. The application of this method to SV40 ori-driven plasmid replication precisely identifies the known ori localization. Using the same approach, we have mapped an ori for bi-directional DNA replication in a 13.7-kb locus of human chromosome 19 encoding lamin B2.

Amplification of fibroblast growth factor receptor 1 (FGFR1) occurs in ∼10% of breast cancers and is associated with poor prognosis. However, it is uncertain whether overexpression of FGFR1 is causally linked to the poor prognosis of amplified cancers. Here, we show that FGFR1 overexpression is robustly associated with FGFR1 amplification in two independent series of breast cancers. Breast cancer cell lines with FGFR1 overexpression and amplification show enhanced ligand-dependent signaling, with increased activation of the mitogen-activated protein kinase and phosphoinositide 3-kinase–AKT signaling pathways in response to FGF2, but also show basal ligand-independent signaling, and are dependent on FGFR signaling for anchorage-independent growth. FGFR1-amplified cell lines show resistance to 4-hydroxytamoxifen, which is reversed by small interfering RNA silencing of FGFR1, suggesting that FGFR1 overexpression also promotes endocrine therapy resistance. FGFR1 signaling suppresses progesterone receptor (PR) expression in vitro, and likewise, amplified cancers are frequently PR negative, identifying a potential biomarker for FGFR1 activity. Furthermore, we show that amplified cancers have a high proliferative rate assessed by Ki67 staining and that FGFR1 amplification is found in 16% to 27% of luminal B–type breast cancers. Our data suggest that amplification and overexpression of FGFR1 may be a major contributor to poor prognosis in luminal-type breast cancers, driving anchorage-independent proliferation and endocrine therapy resistance. Cancer Res; 70(5); 2085–94

Spinal cord injury (SCI) results in immediate disruption of the spinal vascular network, triggering an ischemic environment and initiating secondary degeneration. Promoting angiogenesis and vascular stability through the induction of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), respectively, provides a possible therapeutic approach in treating SCI. We examined whether supplementing the injured environment with these two factors, which are significantly reduced following injury, has an effect on lesion size and functional outcome. Sustained delivery of both VEGF(165) and Ang-1 was realized using viral vectors based on the adeno-associated virus (AAV), which were injected directly into the lesion epicenter immediately after injury. Our results indicate that the combined treatment with VEGF and Ang-1 resulted in both reduced hyperintense lesion volume and vascular stabilization, as determined by magnetic resonance imaging (MRI). Western blot analysis indicated that the viral vector expression was maintained into the chronic phase of injury, and that the use of the AAV vectors did not exacerbate infiltration of microglia into the lesion epicenter. The combined treatment with AAV-VEGF and AAV-Ang-1 improved locomotor recovery in the chronic phase of injury. These results indicate that combining angiogenesis with vascular stabilization may have potential therapeutic applications following SCI.

Objective Macrophage activation syndrome (MAS) in systemic onset juvenile idiopathic arthritis (SoJIA) is considered to be an acquired form of familial haemophagocytic lymphohistiocytosis (fHLH). FHLH is an autosomal recessive disorder, characterized by diminished NK cell function and caused by mutations in the perforin gene (PRF1) in 20-50% of patients. Interestingly, SoJIA patients display decreased levels of perforin in NK cells and diminished NK cell function as well. Here, we analysed PRF1 and its putative promoter in SoJIA patients with or without a history of MAS. Methods DNA of 56 SoJIA patients (41 Italian and 15 Dutch) was isolated. Of these, 15 (27%) had a confirmed history of MAS. We sequenced PRF1 and 1.5 kb of the 5'-upstream region. DNA sequence variations in the promoter region were functionally tested in transfection experiments using a human NK cell line. Results We detected a previously undescribed sequence variation (-499 C > T) in the promoter of PRF1 in 18% of the SoJIA patients. However, transfection experiments did not show functional implications of this variation. Secondly, we found that 11 of 56 (20%) SoJIA patients were heterozygous for missense mutations in PRF1. In particular, we found a high prevalence of the Ala91Val mutation, a variant known to result in defective function of perforin. Interestingly, the prevalence of Ala91Val in SoJIA patients with a history of MAS (20%) was increased compared with SoJIA patients without MAS (9.8%). One SoJIA patient, heterozygous for Ala91Val, showed profound decreased perforin levels at the time of MAS. Conclusions These findings suggest that PRF1 mutations play a role in the development of MAS in SoJIA patients.

Hairy cell leukaemia (HCL) is a rare B-cell neoplasm for which the molecular mechanisms are largely unknown. High-density genome-wide DNA profiling was performed with Affymetrix 250K arrays to analyse copy number (CN) changes and loss of heterozygosity (LOH) in 16 cases of HCL. Four of 16 cases (25%) demonstrated gross non-recurrent CN deletions. Within the affected regions, we identified genes involved in bone marrow fibrosis (FGF12) and response to treatment (TP53) in individual cases. Large regions (> 5 Mb) of LOH without any concomitant DNA CN changes were identified in 5/16 (31%) HCL and were indicative of uniparental disomy UD. The germline origin of UD was demonstrated in one case for which a matched normal sample was available. Overall analysis of LOH showed that identical loci were recurrently targeted in chromosomes 1, 2 and 6. As a whole, however, HCL showed a remarkably stable genome. This finding adds to several other features that are unique to HCL among mature B-cell tumours.

The reductase activity of 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) plays an important role in the growth and differentiation of adipose tissue via the prereceptorial activation of glucocorticoids. This enzyme colocalizes with hexose-6-phosphate dehydrogenase (H6PD) at the luminal surface of the endoplasmic reticulum membrane, and the latter enzyme provides NADPH to the former, which can thus act as an 11β-reductase. It was suggested that, during adipogenesis, the increased expression of H6PD causes a dehydrogenase-to-reductase switch in the activity of HSD11B1. However, only the expression of the HSD11B1 has been extensively studied, and little is known about the expression of H6PD. Here, we investigated the expression and the activity of H6PD in the course of the differentiation of human adipose-derived mesenchymal stem cells (ADMSCs) and murine 3T3-L1 cells. It was found that H6PD is already present in adipose-derived stem cells and in 3T3-L1 fibroblasts even before the induction of adipogenesis. Moreover, mRNA and protein levels, as well as the microsomal H6PD activities remained unchanged during the differentiation. At the same time a great induction of HSD11B1 was observed in both cell types. The observed constant expression of H6PD suggests that HSD11B1 acts as a reductase throughout the adipogenesis process in human ADMSCs and murine 3T3-L1 cells.

Background and Objectives The prognostic value of myeloid antigen (MyAg) expression in adult acute lymphoblastic leukemia (ALL) is still controversial. The aim of this study was to correlate the expression of MyAg with clinical, hematologic and biological parameters, and to analyze the impact on response to treatment and prognosis in a large series of adult ALL uniformly characterized and treated.Design and Methods We analyzed the expression of the MyAg CD13 and/or CD33 in a cohort of 377 adult patients with de novo ALL enrolled and treated in the GIMEMA ALL 0496 protocol.Results MyAg expression was documented in 35% of the 377 adult ALL cases analyzed. MyAg were significantly more frequently associated with B-lineage ALL (38%) than with T-ALL (24%) (p=0.02). No difference was found with regard to clinical features at presentation; a difference was found only for white cell count (p=0.03), percentage of peripheral blasts (p=0.004) and platelet count (p=0.004). No difference was observed in the expression of MyAg between patients with normal or abnormal cytogenetics or between those with high-risk (BCR-ABL+, ALL1-AF4+, E2A-PBX1+) or low-risk B-lineage ALL. We failed to observe any difference between MyAg-positive and MyAg-negative cases in terms of achievement of complete remission, disease-free survival and overall survival at 5 years.Interpretation and Conclusions Our data indicate that ALL MyAg expression in adults with ALL is not associated with adverse presenting clinical and biological features, and that response to treatment and prognosis is comparable in MyAg-positive and MyAg-negative ALL patients with regards to both complete remission rate and overall survival. We suppose that these result are due to more intensive treatment modalities adopted in the GIMEMA ALL 0496 protocol.

Gene expression analysis may help the management of cancer patients, allowing the selection of subjects responding to treatment. The aim of this study was the characterization of expression pattern of genes involved in gemcitabine activity in pancreas tumor specimens and its correlation with treatment outcome. The role of drug transport and metabolism on gemcitabine cytotoxicity was examined with specific inhibitors, whereas transcription analysis of human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (dCK), 5′-nucleotidase (5′-NT), cytidine deaminase (CDA), and ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2) was done by quantitative reverse transcription-PCR in tumor tissue isolated by laser microdissection from surgical or biopsy samples of 102 patients. Association between clinical outcome and gene expression levels was estimated using Kaplan-Meier method and Cox's proportional hazards model. Transport and metabolism had a key role on gemcitabine sensitivity in vitro; moreover, hENT1, dCK, 5′-NT, CDA, RRM1, and RRM2 were detectable in most tumor specimens. hENT1 expression was significantly correlated with clinical outcome. Patients with high levels of hENT1 had a significantly longer overall survival [median, 25.7; 95% confidence interval (95% CI), 17.6-33.7 months in the higher expression tertile versus median, 8.5; 95% CI, 7.0-9.9 months in the lower expression tertile]. Similar results were obtained with disease-free survival and time to disease progression, and the multivariate analysis confirmed the prognostic significance of hENT1. This study suggests that the expression levels of hENT1 may allow the stratification of patients based on their likelihood of survival, thus offering a potential new tool for treatment optimization. (Cancer Res 2006; 66(7): 3928-35)

Background/Aims In this study, we determined the genotypic and allelic frequencies of the Interleukin (IL)-10 −1082G/A IL-10 −592A/C , and IL-10 −819C/T polymorphisms, and their association with the risk to develop B cell Non Hodgkin Lymphoma (NHL) in hepatitis virus C (HCV) carriers. Results Genetic polymorphisms in the IL-10 gene promoter were studied in 250 consecutive patients with B-cell NHL with no clinical and/or laboratory findings of cryoglobulinemia, 142 NHL/HCV− and 108 NHL/HCV+ with chronic hepatitis (CH), 120 consecutive subjects with HCV-related CH, and 110 age, sex-matched healthy blood donors. The frequency of the IL-10 −1082GG genotype vs remaining genotypes (IL-10 −1082GA/AA ) was higher in NHL/HCV+ patients than HCV-related CH patients ( P =0.0002, OR=2.89, CI: 1.62–5.15) and in NHL/HCV+ than NHL/HCV− patients ( P =0.0001, OR=2.99, CI: 1.72–5.19). Moreover, the IL-10 −1082GG genotype was more prevalent in indolent NHL/HCV+ cases than aggressive NHL/HCV+ ( P =0.0004, OR=4.97, CI: 2.10–11.79). Finally, we confirmed that IL-10 −1082GG genotype is associated with higher IL-10 production compared to AA homozygous ( P =0.037). Conclusions The high IL-10 production, due to IL-10 −1082GG genotype, influences the clinical expression of the HCV infection by increasing susceptibility to develop NHL and might contribute to the indolent form of the disease.

p53 and p185 expression in primary breast cancer with microsatellite instability (MSI) is still largely unexplored. To investigate the relationship between these oncoproteins and the pathways of genomic instability, we examined 52 primary invasive breast cancers stratified by the presence and absence of MSI. We determined the status of eight microsatellite loci using radioactive and silver staining methods, and evaluated the immunohistochemical expression of p53 and p185 in a consecutive series of Italian cancer patients characterized by clinical-pathological and biological parameters. Nineteen cases (36.5%) were MSI-positive in at least two loci. p53 was expressed in 15 cases (28.8%) and p185 in eight (15.4%). MSI-positive tumors were inversely correlated with p53 expression ( p = 0.0007); in addition, the percent of p53-expressing cells decreased as the number of MSI-positive loci increased. MSI-positive tumors were correlated with a larger tumor size ( p = 0.04), lymph-node metastasis ( p = 0.001), and advanced clinical stage ( p = 0.0006). These data demonstrate the existence of two subsets of primary breast cancers: one characterized by MSI, the other by p53 expression. MSI-positive patients had a more advanced and/or aggressive disease.

Systemic administration of antiinflammatory molecules to patients affected by immune-mediated inflammatory demyelinating diseases of the central nervous system (CNS) has limited therapeutic efficacy due to the presence of the blood-brain barrier (BBB). We found that three of five rhesus monkeys injected intrathecally with a replication-defective herpes simplex virus (HSV) type 1-derived vector engineered with the human interleukin 4 (IL-4) gene were protected from an hyperacute and lethal form of experimental autoimmune encephalomyelitis induced by whole myelin. The intrathecally injected vector consistently diffused within the CNS via the cerebrospinal fluid and infected ependymal cells, which in turn sustained in situ production of IL-4 without overt immunological or toxic side effects. In EAE-protected monkeys, IL-4-gene therapy significantly decreased the number of brain as well as spinal cord inflammatory perivenular infiltrates and the extent of demyelination, necrosis, and axonal loss. The protective effect was associated with in situ downregulation of inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein 1 (MCP-1), upregulation of transforming growth factor beta (TGF-beta), and preservation of BBB integrity. Our results indicate that intrathecal delivery of HSV-1-derived vectors containing antiinflammatory cytokine genes may play a major role in the future therapeutic armamentarium of inflammatory CNS-confined demyelinating diseases and, in particular, in the most fulminant forms where conventional therapeutic approaches have, so far, failed to achieve a satisfactory control of the disease evolution.

IL-12 activates murine and human B cells, but little information is available as to the expression and function of IL-12R on human B lymphocytes. Here we show that the latter cells, freshly isolated from human tonsils, expressed the transcripts of both β1 and β2 chains of IL-12R and that β2 chain mRNA was selectively increased (4- to 5-fold) by incubation with Staphylococcus aureus Cowan I bacteria or IL-12. B cell stimulation with IL-12 induced de novo expression of the transcripts of the two chains of IL-18R, i.e., IL-1 receptor-related protein and accessory protein-like. Functional studies showed that both IL-12 and IL-18 signaled to B cells through the NF-κB pathway. In the case of IL-12, no involvement of STAT transcription factors, and in particular of STAT-4, was detected. c-rel and p50 were identified as the members of NF-κB family involved in IL-12-mediated signal transduction to B cells. IL-12 and IL-18 synergized in the induction of IFN-γ production by tonsillar B cells, but not in the stimulation of B cell differentiation, although either cytokine promoted IgM secretion in culture supernatants. Finally, naive but not germinal center or memory, tonsillar B cells were identified as the exclusive IL-12 targets in terms of induction of NF-κB activation and of IFN-γ production.

p21WAF1/CIP1 protein is a cyclin-dependent kinase inhibitor, able to prevent the CDK2/cyclin E induced retinoblastoma protein (pRB) phosphorylation, thus inhibiting cell cycle progression at G1 phase. p21WAF1/CIP1 protein levels were examined in a series of 102 ovarian tissue samples including normal ovary, primary ovarian tumors, omental metastasis, recurrent disease and residual tumor after chemotherapy exposure, by Western blot analysis. The association of p21WAF1/CIP1 status with clinicopathological parameters and clinical outcome was also investigated. p21WAF1/CIP1 protein was detectable in 76 out of 102 (74%) ovarian tissue samples. We observed a significant trend of p21 levels to gradually increase from normal ovarian tissues (median 0 a.u.) through primary ovarian cancers (median 0.19 a.u.), omental metastases (median 0.33 a.u.) and recurrence of disease (median 0.44 a.u.) (p=0.015). In the group of stage III-IV ovarian cancer patients, p21-positive cases showed a more favourable prognosis with respect to p21-negative cases: the 3-year time to progression (TTP) rate was 58% for p21-positive compared with 33% of p21-negative cases (p=0.036). In conclusion, p21WAF1/CIP1 expression levels seem to be correlated with tumor status at the time of diagnosis and can predict TTP in a selected group of patients.

The molecular basis for heart failure is unknown, but oxidative stress is associated with the pathogenesis of the disease. We tested the hypothesis that the activity of xanthine oxidoreductase (XOR), a free-radical generating enzyme, increases in hypertrophied and failing heart. We studied XOR in two rat models: (1) The monocrotaline-induced right ventricular hypertrophy and failure model; (2) coronary artery ligation induced heart failure, with left ventricular failure and compensatory right ventricular hypertrophy at different stages at 3 and 8 weeks post-infarction, respectively. XOR activity was measured at 30 degrees C and the reaction products were analysed by HPLC. In both models XOR activity in hypertrophic and control ventricles was similar. In the monocrotaline model, the hearts showed enhanced XOR activity in the failing right ventricle (65+/-5 mU/g w/w), as compared to that in the unaffected left ventricle (47+/-3 mU/g P

Interleukin-12 (IL-12) is a pivotal cytokine representing the link between the cellular and humoral branches of an effective host immune defense apparatus. IL-12 is a heterodimer produced by phagocytic, B, dendritic, and possibly other accessory cells in both innate and adaptive immune responses. It is a key factor in the induction of T cell-dependent and independent activation of macrophages, generation of T helper type 1 (Th1) and cytotoxic T cells, suppression of IgG1 and IgE production, induction of organ-specific autoimmunity, and resistance to bacterial and parasitic infections [1]. IL-12 has a powerful anti-tumor and anti-metastatic activity against many murine tumors [2-5] as well as human tumors [6-17]. The genes encoding the two heterologous chains of IL-12, p40 and p35 are located on different human chromosomes. Together, p40 and p35 form the biologically active IL-12. Their expressions are highly coordinated during an effective immune response. However, under some pathological conditions, IL-12 is under- or overexpressed, resulting either in a lack of resistance to microbial infection and to uncontrolled tumor growth, or in destructive inflammation, respectively. A transient or irreversible dysregulation of IL-12 production may reflect a pathogen/tumor cell-induced disruption in the highly coordinated expression of p40 and p35. The understanding of the molecular mechanisms governing the expression of IL-12 p40 and p35 genes in the context of interactions between pathogens and the immune system is essential in efforts aimed at designing therapeutic strategies to treat infectious and malignant diseases.

The poly(A) signal of the human Lamin B2 gene was previously shown to lie 600 bp upstream of the cap site of a gene of unknown function (ppv 1). However, using RNase protection analysis, we show that ppv 1 has two clusters of multiple initiation sites, so that the 5"cap site lies only approximately 280 nt downstream of the Lamin B2 poly(A) signal. We analysed nascent transcription across this unusually short intergenic region using nuclear run-on analysis of both the endogenous locus and of transiently transfected hybrid constructs. Surprisingly, transcription of the Lamin B2 gene does not appear to terminate prior to any of the mapped ppv 1 start sites, although pausing of the elongating polymerase complexes is observed downstream of the Lamin B2 poly(A) signal. We suggest that this pausing may be sufficient to protect the downstream gene from transcriptional interference. Finally, we have also investigated the sequences required for efficient recognition of the Lamin B2 poly(A) signal. We show that sequences upstream of the AAUAAA element are required for full activity, which is an unusual feature of mammalian poly(A) signals.

Interleukin-12 (IL-12) is a proinflammatory cytokine produced by antigen-presenting cells in response to many microbial infections. IL-12 plays an important role in the generation of T helper type-1 cells, which favor cell-mediated immune response. IL-12 is composed of two different subunits, p40 and p35, whose expression can be regulated concomitantly or differentially. Monocytic cells, the major producers of IL-12, can be primed by interferon-gamma (IFN-gamma) to produce optimal amounts of IL-12 in response to LPS stimulation as a consequence of bacterial infection. The priming effect is exerted primarily at the transcriptional level on the p40 promoter in conjunction with the effects of LPS, possibly by inducing specific transcription factors, which individually have no direct effect but which cooperatively can activate the promoter. We examined in detail one of these DNA-protein interactions observed around an Ets-2 element situated at -211/-207 of the p40 promoter, which is known to be a functionally critical site. This region interacts with a nuclear complex termed F1 that appears to be highly inducible by either IFN-gamma treatment for 16 h or lipopolysaccharide stimulation for 8 h. F1 binding to the Ets-2 site requires a considerable amount of spacing around the Ets-2 site, as revealed by gel mobility shift and in vitro methylation assays. Supershift experiments and DNA affinity purification indicated that both Ets-2 and a novel, antigenically related protein with an approximate molecular mass of 109 kDa are part of the F1 complex, together with additional components including IRF-1 and c-Rel. This novel protein is designated GLp109 for its inducibility by IFN-gamma or lipopolysaccharide. Its possible role in the activation of the IL-12 p40 promoter is discussed.

Interleukin (IL) 12 is a proinflammatory cytokine produced by phagocytic cells, B cells, and other antigen-presenting cells that modulates adaptive immune responses by favoring the generation of T helper type 1 cells. IL-12 mediates some of its physiological activities by acting as a potent inducer of interferon (IFN) gamma production by T and natural killer cells. IFN-gamma enhances the ability of the phagocytic cells to produce IL-12 and other proinflammatory cytokines. Thus, IL-12-induced IFN-gamma acts in a positive feedback loop that represents an important amplifying mechanism in the inflammatory response to infections. We show here that IFN-gamma enhances IL-12 production mostly by priming phagocytic cells for lipopolysaccharide (LPS)-induced transcription of the IL-12 p40 gene, which encodes the heavy chain of the IL-12 heterodimer; furthermore, IFN-gamma directly induces transcription of the IL-12 p35 gene, which encodes the light chain of IL-12, and has at least an additive effect with LPS stimulation in inducing its transcription. The priming effect of IFN-gamma on the LPS-induced p40 gene transcription requires preincubation of the cells with IFN-gamma for at least 8 h to obtain a maximal effect. The priming effect of IFN-gamma for IL-12 production is predominantly at the transcriptional level for both the p40 and the p35 gene, and no evidence for a major role of posttranscriptional or translational mechanisms was found. A 3.3-kb human IL-12 p40 promoter construct transfected into cell lines recapitulated the tissue specificity of the endogenous gene, being silent in two human T cell lines, constitutively active in two human Epstein-Barr virus-positive B lymphoblastoid cell lines, and LPS inducible in the human THP-1 and mouse RAW264.7 monocytic cell lines. Because the RAW264.7 cell line is easily transfectable and regulates the endogenous IL-12 p40 gene in response to IFN-gamma or LPS similarly to human monocytes, it was used for analysis of the regulation of the cloned human IL-12 p40 promoter. A requirement for the region between -222 and -204 in both LPS responsiveness and IFN-gamma priming was established. This region contains an ets consensus sequence that was shown to mediate activation of the promoter by IFN-gamma and LPS, as well as by a cotransfected ets-2. The -222 construct was also regulated in a tissue-specific manner. Two other elements, IRF-1 located at -730 to -719, and NF-IL6 at -520 to -512, were also studied by deletion analysis, which did not result in decreased response to IFN-gamma and LPS stimulation.

The nonstructural protein NS-1 of minute virus of mice (MVMp), an autonomous parvovirus, trans-inhibits the replication of a chimeric plasmid containing the SV40 origin of replication (ori) embedded in the MVMp genome. It appears that a 157-bp 5' proximal sequence of MVMp DNA is sufficient, in the presence of NS-1, to cause the inhibition of DNA replication driven by the SV40 ori placed on the same molecule. This effect is not dependent on the orientation of the MVMp target sequence and results from both a reduced level of utilization of SV40 ori and the blockage of progressing replication forks at the level of the target. Furthermore, replication driven by Epstein-Barr virus origin (oriP) is trans-inhibited by MVMp but this inhibition does not require the presence of parvoviral sequences in cis. On the basis of sequence homologies between EBV oriP and MVMp 5' terminal sequence, it is proposed that the direct or indirect interaction of NS-1 with parvovirus-like sequences present in heterologous viral and possibly also cellular genomes may result in an inhibition of DNA replication.

The effect of transforming growth factor beta (TGF beta) on the expression of a group of liver genes has been investigated in the hepatoma cell line Hep 3B. TGF beta induces a decrease of the basal level of apolipoprotein A-II (ApoA-II), retinol binding protein (RBP) and alpha-fetoprotein (alpha Fp). Furthermore, TGF beta efficiently antagonizes the IL-6-induction of hemopexin (Hpx) and haptoglobin (Hp) and alpha 1-acid glycoprotein (AGP). These effects of TGF beta are apparently mediated by post-transcriptional mechanism(s). These findings, together with previously reported data on the inhibitory effect of TGF beta on acute phase genes (e.g. ApoA-I and albumin), suggest a role for TGF beta in the regulation of expression of liver genes.

In liver cells recovering from reversible ischaemia, total protein synthesis by postmitochondrial supernatant and membrane-bound and free polyribosomes is not different from that in sham-operated controls. However, the relative proportion of specific proteins is changed, since the incorporation of [3H]leucine in vivo into liver albumin, relative to incorporation into total protein, as determined by precipitation of labelled albumin with the specific antibody, decreases by 40-50% in post-ischaemic livers. Cell-free synthesis by membrane-bound polyribosomes and poly(A)-enriched RNA isolated from unfractionated liver homogenate shows that the decrease in albumin synthesis in liver of rats recovering from ischaemia is due to the relative decrease in translatable albumin mRNA.

The synthesis of heat shock proteins (HSP) was studied in rat liver and in a series of transplantable Morris hepatomas with different growth rates, subjected to heat shock in vivo and in vitro. Different from the liver, hepatomas synthesized HSP constitutively, i.e., also before exposure to heat. This constitutive synthesis was low and limited to one HSP in the slowest-growing tumor, more marked and involving other HSP in the intermediate- and fast-growing hepatomas. In tumor that synthesized HSP constitutively, the induction of HSP in response to heat was proportionately reduced. These patterns of reaction were essentially similar in vivo ad in vitro. The amount of HSP 68 was well correlated to the levels of its mRNA in liver and in all hepatomas, whereas the increase in HSP 89 was accompanied by a corresponding increase in the related mRNA in liver and in slow-growing hepatoma, not in the other tumors, thus suggesting a different mechanism of control of HSP 89 synthesis in the more malignant hepatomas.

We report that a 2.4-kilobase (kb) pim-1 transcript is expressed in the germ cells of mouse testis. Analysis of purified populations of spermatogenic cell types indicates that the 2.4-kb transcript is selectively expressed in haploid postmeiotic early spermatids. The evidence for a developmentally regulated expression of pim-1 in haploid spermatids suggests a possible developmental role for this protooncogene product. The 2.4-kb pim-1 transcript present in postmeiotic cells differs in size from the 2.8-kb transcript usually detected in somatic tissues. Similar testis-specific transcripts have been seen for mos and abl genes. These data suggest specificity in transcription or processing of certain genes in haploid male germ cells. We have also analyzed other representative protooncogenes, including examples of protein kinases, the ras family, and the "nuclear" protooncogenes. The results indicate that additional protooncogenes are preferentially expressed in either meiotic pachytene cells or postmeiotic early spermatids. These findings suggest a differential regulation of gene expression in these two developmental stages of germ cells. In particular, analysis of expression of the three members of the ras gene family indicates a distinct temporal differential regulation in the expression of the Harvey, Kirsten, and N-ras genes in these germ cells.

The different amounts of H-rich and L-rich isoferritins found in malignant and non malignant lymphoid cells are accompanied by proportional variations in the relative quantity of messenger RNAs for the H and L subunits of ferritin. The correlation between levels of messenger RNAs and proteins suggests that the amount of messenger RNA plays an important role in ferritin biosynthesis in these cells. The enhanced expression of ferritin messenger RNAs in some neoplastic cells is not caused by gross alterations in the structure of ferritin genes.

We describe the purification and cloning of human DNA replicated at the onset of S phase in HL60 cells synchronized with aphidicolin. A survey of the overall structural properties of these sequences did not show any distinctive features except for an enrichment in Cot0 DNA. The two longer fragments were completely sequenced and studied in more detail. Both were shown to contain transcriptional signals associated with promoters and/or enhancers, such as the binding sites of Sp1, T antigen and nuclear factor III. In one instance, a binding site for a known cellular transcription factor (USF/MLTF) was located inside the sequence by footprinting. Accordingly, by CAT assay and Northern blot, the same sequence was shown to contain an active promoter. The significance of these findings with respect to the role of transcription in initiation of DNA replication at the origin is discussed. None of the tested fragments exhibited autonomously replicating sequence (ARS) activity in transfected cells. The problems connected with the detection of ARS activity in human cells are critically examined.

We present an application of image analysis for the direct quantification of PCR products after gel electrophoresis and ethidium bromide staining of DNA. This procedure has been applied to the development of an assay based on competitive PCR for the measurement of the degree of amplification of c-erbB-2 oncogene in DNA from human tumours. In this method two DNA species (genomic and competitor) compete for PCR amplification. Since results are calculated from the final competitor/genomic ratio any variable affecting the rate of PCR amplification has no effect on the accuracy of the ratio measurement. Results are reported which show that even large variations in the experimental conditions (number of PCR cycles, sample volumes and extracted DNA quality) did not interfere with the precision of the measurement of the competitor/genomic ratio.

By the use of a highly sensitive mapping procedure allowing the identification of the start sites of DNA replication in single-copy genomic regions of untreated, exponentially growing cultured cells (M. Giacca, L. Zentilin, P. Norio, S. Diviacco, D. Dimitrova, G. Contreas, G. Biamonti, G. Perini, F. Weighardt, S. Riva, and A. Falaschi, Proc. Natl. Acad. Sci. USA 91:7119-7123, 1994), the pattern of DNA replication of the Chinese hamster dihydrofolate reductase (DHFR) gene domain was investigated. The method entails the purification of short stretches of nascent DNA issuing from DNA replication origin regions and quantification, within this sample, of the abundance of different adjacent segments by competitive PCR. Distribution of marker abundance peaks around the site from which newly synthesized DNA had emanated. The results obtained by analysis of the genomic region downstream of the DHFR single-copy gene in asynchronous cultures of hamster CHO K1 cells are consistent with the presence of a single start site for DNA replication, located approximately 17 kb downstream of the gene. This site is coincident with the one detected by other studies using different techniques in CHO cell lines containing an amplified DHFR gene domain.

BACKGROUND: As for rhabdomyosarcoma (RMS) of other anatomic regions, the evaluation of traditional clinicopathological parameters does not allow the unequivocal outcome prediction of the single cases of orbital RMS. We investigated the role of DNA ploidy and immunohistochemical expression of p53, bcl-2, MDR-1 and Ki67 (MIB1) in the prognostic evaluation of orbital rhabdomyosarcomas. MATERIALS AND METHODS: The study population consisted of 11 selected cases. Serial sections of each tumor, stained with Feulgen's technique, were analyzed for the DNA content, using the QUANTIMET 500c Leica analyzer, QWINVO200A software. The results were compared with the immunohistochemical expression of p53 (wild plus mutated, W&M and mutated), bcl2, MDR-1 and Ki67 (MIB1), and with follow-up data. RESULTS: The statistical analysis of results showed that the cases of tetraploid and/or multiploid RMS, overexpressing p53 (W&M and mutated) and MDR-1, were characterized by an overall worse prognosis. On the contrary, the tumors with a favourable clinical course showed hyperexpression of MIB1 and absence of mutated p53 expression. Significantly higher MIB1 expression was found in the relapse-free group of tumors, with respect to the RMS with relapse (both in primary tumors and relative relapses, p0.05). CONCLUSION: The evaluation of DNA ploidy, p53, MIB1 and MDR-1 expression could be used for subtyping of orbital RMS into two prognostically different subcategories, respectively RMS responder to the therapy, with favourable clinical outcome, and RMS with a worse prognosis, requiring more aggressive therapeutic protocols.

The present study aims to assess the protective role of the antioxidant enzyme catalase (CAT) with relation to hydrogen peroxide (H2O2) degradation in oxygen plus water on electrophysiological and fluorescence changes induced by in vitro ischemia and on brain damage produced by transient in vivo ischemia. Neuroprotective effects of CAT were determined by means of electrophysiological recordings and confocal fluorescence microscopy in the hippocampal slice preparation. Ischemia was simulated in vitro by oxygen/glucose deprivation (OGD). In vivo ischemia was produced by transient middle cerebral artery occlusion (MCAo). A protection of the rat CA1 field excitatory postsynaptic potential (fEPSP) loss caused by a prolonged OGD (40 min) was observed after exogenous CAT (500 U/mL) bath-applied before a combined exposure to OGD and H2O2 (3 mM). Of note, neither H2O2 nor exogenous CAT alone had a protective action when OGD lasted for 40 min. The CAT-induced neuroprotection was confirmed in a transgenic mouse model over-expressing human CAT [Tg(CAT)]. In the presence of H2O2, the hippocampus of Tg(CAT) showed an increased resistance against OGD compared to that of wild-type (WT) animals. Moreover, CAT treatment reduced for about 50 min fEPSP depression evoked by repeated applications of H2O2 in normoxia. A lower sensitivity to H2O2-induced depression of fEPSPs was also indicated by the rightward shift of concentration-response curve in Tg(CAT) compared to WT mice. Noteworthy, Tg(CAT) mice had a reduced infarct size after MCAo. Our data suggest new strategies to reduce neuronal damage produced by transient brain ischemia through the manipulation of CAT enzyme.

To define the relationship between the two ryanodine receptor (RyR) isoforms present in chicken skeletal muscle, we cloned two groups of cDNAs encoding the chicken homologues of mammalian RyR1 and RyR3. Equivalent amounts of the two chicken isoform mRNAs were detected in thigh and pectoral skeletal muscles. RyR1 and RyR3 mRNAs were co-expressed in testis and cerebellum whereas RyR3 mRNA was expressed also in cerebrum and heart. The full-length sequence of the chicken RyR3 cDNA was established. The RyR3 receptor from chicken had the same general structure as mammalian and amphibian RyRs. The 15089 nt cDNA encoded a 4869-amino-acid-long protein with a molecular mass of 552445. The predicted amino acid sequence of the chicken RyR3 showed 86.9% identity to mammalian RyR3 and 85.6% to frog RyR3. Antibodies specific for chicken RyR1 and RyR3 recognized two different proteins with an apparent molecular mass of about 500 kDa. The two proteins differ slightly in their apparent molecular mass on SDS/PAGE: the protein recognized by antibodies against RyR3 had a higher mobility than the protein recognized by the antiserum against RyR1. Antibodies against RyR1 detected a protein already present in chicken skeletal muscle from 12-day-old embryos and older, while antibodies against RyR3 isoform detected a protein in muscle from only 18-day-old embryos and older. The expression patterns of RyR1 and RyR3 superimpose with those previously reported for the α and the β isoforms respectively. We conclude that α and β isoforms present in chicken skeletal muscle are the homologues of mammalian RyR1 and RyR3.