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We investigated genes involved in the interleukin-2 activation of cultured lymphocytes using a differential display reverse transcription PCR technique. Three cDNA fragments corresponding to mRNAs differentially amplified in the activated lymphocytes were sequenced and identified. These fragments were identical to the 3' region of the mRNAs encoding for the tumor rejection antigen TRA 1 that is the human homologue of the murine heat shock protein gp96, the DAP12 protein that possesses an immunoreceptor tyrosine-based activation motif, and the human motor protein p87/89 expressed in the heart. These proteins are involved, respectively, in cellular communication, in signal transduction, and in cellular movements. Our findings suggest that the activation of cellular immune response by interleukin-2 is a process analogous to other known phenomena of activation of catabolic reactions of energy transduction for activities which allow adaptation of cells to stress conditions.

Exon trapping was employed to identify coding sequences from a collection of 46 bovine cosmids, previously characterized for the presence of microsatellite markers and physically mapped to chromosomes by FISH. The sequence analysis of 104 clones revealed 18 putative exons, 10 of which showed near identity to known sequences. Among these were the human (cytosine-5)-methyltransferase (DNMT), ATP-citrate lyase (ACLY), the mouse Lbcl1 oncogene, the bovine mitochondrial aconitase (ACO2) and beta-arrestin 1 (ARR1). The chromosomal localization of the cloned exons was inferred from the localization of the parent cosmids. DNMT and ACLY were not previously known in cattle, but the physical localization of the cloned bovine exons is in agreement with the published comparative human and bovine maps. The trapping of exons for bovine ACO2 and ARR1 confirms the available mapping information based on synteny and provides a physical assignment for the genes.

ATG7 is a key autophagy-promoting gene that plays a critical role in the regulation of cell death and survival of various cell types. We report here that microRNAs (miRNAs), a class of endogenous 22–24 nucleotide noncoding RNA molecules able to affect stability and translation of mRNA, may represent a novel mechanism for regulating ATG7 expression and therefore autophagy. We demonstrated that ATG7 is a potential target for miR-17, and this miRNA could negatively regulate ATG7 expression, resulting in a modulation of the autophagic status in T98G glioblastoma cells. Treatment of these tumor cells with the miR-17 mimic decreased, and with the antagomir increased, the expression of ATG7 protein. Dual luciferase reporter assay confirmed that a specific miR-17 binding sequence in the 3′-UTR of ATG7 contributed to the modulation of the expression of the gene by miR-17. Interestingly, our results showed that anti-miR-17 administration activated autophagy through autophagosome formation, as resulted by LC3B and ATG7 protein expression increase, and by the analysis of GFP-LC3 positive autophagosome vesicles in living cells. Furthermore, the autophagy activation by anti-miR-17 resulted in a decrease of the threshold resistance at temozolomide doses in T98G cells, while miR-17 modulation in U373-MG glioblastoma cells resulted in a sensitization to low ionizing radiation doses. Our study of the role of miR-17 in regulating ATG7 expression and autophagy reveals a novel function for this miRNA sequence in a critical cellular event with significant impacts in cancer development, progression and treatment.

Different epidemiological markers were used to characterize 2 Staphylococcus epidermidis and 8 Staphylococcus aureus strains isolated from patients with severe infections. We compared random amplified polymorphic DNA (RAPD) fingerprints, biotypes, antibiotic assays, plasmid profiles and chromosomal DNA restriction endonuclease analysis (REA). Data analysis based on numerical taxonomy methods indicates that RAPD and REA give similar results allowing a good discrimination of the two species and of each isolate. The RAPD method is easier and faster than REA, but the reproducibility of RAPD fingerprints obtained in independent experiments can be problematic. We have found simple technical devices to improve the reproducibility of the RAPD procedure which is therefore a very useful tool in epidemiology for identification and characterization of Staphylococcus spp.

SUMMARYEight taxa have recently been proposed as being encompassed by the genus Trichinella on the basis of allozyme and biological data. In this paper we show that an analogous 8 taxon structure for this genus results from the random amplified polymorphic DNAs (RAPDs). Five 10-mer or 20-mer primers were used under different polymerase chain reaction (PCR) conditions to produce multiband RAPD fingerprints from muscle larvae of 40 isolates of Trichinella spp. The resulting RAPD data were analysed following the numerical taxonomic approach, and the resulting classification was compared to that derived from allozyme data. The agreement found between allozymes and RAPDs, while supporting the polyspecific structure of the genus Trichinella, confirms the potential of RAPDs as a tool for the detection of cryptic species. The selected primers were tested on individual muscle larvae in an attempt to standardize a RAPD assay for the routine identification of the 8 taxa of Trichinella. Only 1 of the 5 primers yielded reproducible fingerprints from the single larvae. Using this primer, the 5 species and the 3 other taxa of the genus Trichinella can be identified in a single assay without the need for massive in vivo parasite production.

Autophagy is a so-called "self-eating" system responsible for degrading long-lived proteins and cytoplasmic organelles, whose products are recycled to maintain cellular homeostasis. This ability makes autophagy a good candidate for a survival mechanism in response to several stresses, including the tumor cell transformation. In particular, recent studies suggested that autophagy functions as a pro-death mechanism within different tumor contexts. It is, however, widely reported that autophagy represents both a survival mechanism or contributes directly to cell death fate. This interplay of the autophagy functions has been observed in many types of cancers and, in some cases, autophagy has been demonstrated to both promote and inhibit antitumor drug resistance. From a therapeutical point of view, the effects of the modulation of the tumor cell autophagic status, in response to ionizing radiations, are presently of particular relevance in oncology. Accordingly, this review also provides a perspective view on future works for exploring the modulation of autophagic indices in tumor cells as a novel molecular-based adjuvant strategy, in order to improve radiotherapy and chemotherapy effects in cancer patients.

An ImageJ JavaScript, AUTOCOUNTER, was specifically developed to monitor and measure LC3B-GFP expression in living human astrocytoma cells, namely T98G and U373-MG. Discrete intracellular GFP fluorescent spots derived from transduction of a Baculovirus replication-defective vector (BacMam LC3B-GFP), followed by microscope examinations at different times. After viral transgene expression, autophagy was induced by Rapamycin administration and assayed in ph-p70S6K/p70S6K and LC3B immunoblotting expression as well as by electron microscopy examinations. A mutated transgene, defective in LC3B lipidation, was employed as a negative control to further exclude fluorescent dots derived from protein intracellular aggregation. The ImageJ JavaScript was then employed to evaluate and score the dynamics changes of the number and area of LC3B-GFP puncta per cell in time course assays and in complex microscope examinations. In conclusion, AUTOCOUNTER enabled to quantify LC3B-GFP expression and to monitor dynamics changes in number and shapes of autophagosomal-like vesicles: it might therefore represent a suitable algorithmic tool for in vitro autophagy modulation studies.

Doppel is a newly recognized prion-like protein encoded by a novel gene locus, PRND, located on the same chromosomal region of the prion coding gene (PRNP); doppel is considered a prion paralogue and together they constitute the prion-gene family, originated through an ancestral gene duplication event. Prion and doppel have different expression patterns, suggesting that the gene products exhibit different biological functions. Actually, doppel is not involved in the etiology of the transmissible spongiform encephalopathies (TSEs) or “prion diseases” and it is highly expressed only within the testicular tissue, where its important physiological role in the process of spermatogenesis and fertilization in human and mouse has been described. Importantly, doppel is toxic when ectopically overexpressed in the central nervous system (CNS), with concomitant prion protein absence: this evidence suggests deeper investigations within particular pathological contexts, such as in Parkinson and Alzheimer’s diseases as well as in cancer. In the latter scenario several studies are showing that doppel represents a novel and attractive diagnostic molecular marker, and that it might provide insights into the regulatory pathways of tumor-cell transformation. In particular, doppel protein has been recently associated with the ability of tumor cells to migrate, as one of the most important hallmarks of cancer. In conclusion, since its discovery, the intriguing spectrum of biological and pathological functions of this new prion-like protein is constantly considered for novel investigations.

Protein interactions are crucial in most biological processes. Several in silico methods have been recently developed to predict them. This paper describes a bioinformatics method that combines sequence similarity and structural information to support experimental studies on protein interactions. Given a target protein, the approach selects the most likely interactors among the candidates revealed by experimental techniques, but not yet in vivo validated. The sequence and the structural information of the in vivo confirmed proteins and complexes are exploited to evaluate the candidate interactors. Finally, a score is calculated to suggest the most likely interactors of the target protein. As an example, we searched for GRB2 interactors. We ranked a set of 46 candidate interactors by the presented method. These candidates were then reduced to 21, through a score threshold chosen by means of a cross-validation strategy. Among them, the isoform 1 of MAPK14 was in silico confirmed as a GRB2 interactor. Finally, given a set of already confirmed interactors of GRB2, the accuracy and the precision of the approach were 75% and 86%, respectively. In conclusion, the proposed method can be conveniently exploited to select the proteins to be experimentally investigated within a set of potential interactors.

In this study the mRNA levels of fiveEGFRindirectly related genes,EGFR,HB-EGF,ADAM17,PTEN, andMMP9, have been assessed by Real-time PCR in a panel of 37 glioblastoma multiforme specimens and in 5 normal brain samples; as a result, in glioblastoma,ADAM17andPTENexpression was significantly lower than in normal brain samples, and, in particular, a statistically significant inverse correlation was found betweenPTENandMMP9mRNA levels. To verify if this correlation was conserved in gliomas,PTENandMMP9expression was further investigated in an additional panel of 16 anaplastic astrocytoma specimens and, in parallel, in different human normal and astrocytic tumor cell lines. In anaplastic astrocytomasPTENexpression was significantly higher than in glioblastoma multiforme, but no significant correlation was found betweenPTENandMMP9expression.PTENandMMP9mRNA levels were also employed to identify subgroups of specimens within the different glioma malignancy grades and to define a gene expression-based diagnostic classification scheme. In conclusion, this gene expression survey highlighted that the combined measurement ofPTENandMMP9transcripts might represent a novel reliable tool for the differential diagnosis of high-grade gliomas, and it also suggested a functional link involving these genes in glial tumors.

Doppel (Dpl) is a homologue of the prion protein (PrPC). In contrast to PrP(C), Dpl is dispensable for prion disease, but appears to have an essential function in male spermatogenesis. Recently, Dpl has been found to be aberrantly expressed in astrocytic and leukaemic tumor specimens, showing a peculiar cytosolic cellular localization. The aim of this study was to clarify some of the putative Dpl interacting proteins.A yeast two hybrid system was employed and the results were verified by co-immunoprecipitation using transfected cells.Several potential Dpl-binding candidates were identified and, among them, the receptor for activated C-kinase (RACK1) protein was further investigated. RACK1 deletion mutants showed that some of its WD containing domains were directly involved in the binding with Dpl. Our data showed that Dpl interacts with RACK1 by means of its structured globular carboxyl-terminal region.This new Dpl interacting partner might suggest functional hypotheses about the role of this protein in an astrocytoma context where Dpl was found ectopically expressed.

The doppel protein (Dpl) is a newly recognized cellular prion protein (PrP(C))-like molecule encoded by a novel gene locus, PRND, located on the same chromosomal region of the PrP(C) coding gene. Recently, Dpl was shown to be aberrantly expressed in astrocytic tumor specimens and in astrocytoma-derived cell lines, showing a peculiar cytoplasmic localization. Here, Dpl interactions with some of the prion-interacting proteins were studied. In particular, whether the tumor astrocytic environment is suitable for doppel interaction with GFAP and Grb2 proteins, as well as with the PrPC protein itself was investigated.In order to verify our hypothesis, an innovative mammalian two-hybrid system and co-immunoprecipitation assays were employed.The results reported the absence of protein interactions. Our findings provided evidence that, in our astrocytoma cell-based model, Dpl does not share with PrP(C) the ability to interact with GFAP and Grb2.Identifying Dpl ligands may provide new insights into the involvement of Dpl in astrocytoma tumor progression.

SummaryWe investigated the expression patterns and distribution of Doppel (Dpl), theproduct of the prion-like gene PRND, in the leukaemic cell lines HL-60 andK562 and in bone marrow cells from 44 patients with acute myeloidleukaemia (AML) and 63 patients with myelodysplastic syndrome (MDS).Whereas normal samples were negative or showed very weak expression thatwas restricted to CD34 + cells, Dpl was detected in both cell lines and in mostAML and MDS cases by immunocytochemistry and Western blotting.Quantitative reverse transcription polymerase chain reaction revealedvariable mRNA levels in almost all AML and MDS cases, but barelydetectable levels in normal bone marrow. These differences were confirmedby in situ hybridization. PRND expression was higher in advanced comparedwith early MDS (P ¼ 0AE01), but Dpl levels did not predict diseaseprogression. In AML there was no correlation between Dpl levels andclinical or laboratory findings. In conclusion, this is the first time that theexpression of PRND has been demonstrated in human bone marrow. Themolecular mechanism of the observed overexpression is unknown; however,the differential Dpl distribution in AML and MDS versus healthy subjectsmakes it a possible leukaemia-associated antigen that could be useful fordiagnostic and therapeutic purposes.Keywords: Doppel, PRND gene expression, myelodysplastic syndrome, acutemyeloid leukaemia, real-time PCR.

Restriction site analysis of Polymerase Chain Reaction (PCR) products of cytochrome b mitochondrial DNA was applied to identify species in meat meal and animal feedstuffs. PCR was used to amplify a variable region of cytochrome b mitochondrial DNA gene. Species differentiation was determined by digestion of the obtained 359 bp amplicon with restriction enzymes, which generated species-specific electrophoresis patterns; the sequencing of PCR products was used as confirming analysis. PCR-RFLP analysis revealed the presence of meat meal in animal feedstuffs and distinguished species of interest. The results supported the application of the method in control measures which should be adopted for meat-meal-based animal feed, as suggested by EU law. As a technical improvement, to simplify the analysis, the number of enzymes presented in this study for the detection of different species was smaller than others described in the literature; discrimination between ruminant and nonruminant species and between mammalian and poultry species was possible with few digestions.

We investigated the possible application of RAPD (Random Amplified Polymorphic DNA) analysis to the study of the systematic relationships of five cervid taxa. Amplifications with eight different primers gave reproducible electrophoretic patterns which could be regarded as a data-set consisting of monomorphic and polymorphic characters. Some of these characters are species- and subspecies-specific. Band-sharing analysis and numerical taxonomy methods allowed us to generate a phenetic tree. Our results point out new possible systematic considerations within the examined taxa.

A procedure is described to isolate DNA probes from the bovine genome that are enriched in sites for the so-called rare cutter restriction endonucleases. A collection of SacII (CvCGCGG)-Hin-dIII fragments from bovine sperm was established in the plasmid Bluescript. 180 clones were picked at random and analysed for the presence of inserts with sites for the following rare cutters: EagI, BsshII, NarI, MluI, NruI, NaeI: 70% of the clones contained at least 1 site and 5% contained four different such sites. 22.8% had multiple sites for one or more of the rare cutters tested. Sequence analysis for 16 clones confirmed the cloning of DNA with a G+C content and a proportion of CpG vs GpCs indicative of CpG islands.

Doppel (PRND) is a paralogue of the mammalian prion (PRNP) gene. It is abundant in testis and, unlike PRNP, it is expressed at low levels in the adult central nervous system (CNS). Besides, doppel overexpression correlates with some prion-disease pathological features, such as ataxia and death of cerebellar neurons. Recently, ectopic expression of doppel was found in two different tumor types, specifically in glial and haematological cancers. In order to address clinical important issues, PRND mRNA expression was investigated in a panel of 111 astrocytoma tissue samples, histologically classified according to the World Health Organization (WHO) criteria (6 grade I pilocytic astrocytomas, 15 grade II low-grade astrocytomas, 26 grade III anaplastic astrocytomas and 64 grade IV glioblastoma multiforme). Real-time PRND gene expression profiling, after normalisation with GAPDH, revealed large differences between low (WHO I and II) and high grade (III and IV) of malignancy (P

SUMMARYThe random amplified polymorphic DNA (RAPD) technique was successfully used to produce genetic fingerprints distinguishing between Trichinella spiralis and Trichinella britovi. The same patterns were obtained from purified and crude DNA preparations of pooled and single muscle larvae. RAPD fingerprinting was applied to muscle larvae preserved under different conditions and recovered from different hosts. Larvae recovered from fresh and frozen meat and stored at – 20 °C for a long time or under 70% ethyl alcohol at room temperature for 30 d gave good and reproducible results. Single larvae recovered from a naturally infected wild boar and from a human biopsy gave fingerprints congruent to those obtained from T. britovi reference strains. The results prove that RAPD analysis is a quick method to distinguish between the autochthonous Trichinella species of Central-Southern Europe in less than 1 d after the detection of the infection. If necessary, the biological material can be frozen or stored under 70% ethyl alcohol at room temperature and sent to laboratories able to perform the RAPD analysis. The RAPD technique requires no prior knowledge of the molecular biology of the organism to be investigated and therefore appears to be a promising tool in parasitology for the identification of sibling species.

Doppel (Dpl) is a paralogue of the mammalian Prion (PrP) protein. It is abundant in testis and, unlike PrP, it is expressed at low levels in the adult central nervous system (CNS). Besides, Dpl overexpression correlates with some prion-disease pathological features, such as ataxia and death of cerebellar neurons. Recently, ectopic expression of doppel was found in two different tumor types, specifically in glial and haematological cancers. In this study the doppel gene (PRND) mRNA and protein expression in PRT-HU2 and IPDDC-A2 astrocytoma-derived cell lines was investigated. Northern blot analysis revealed two equally abundant PRND mRNA isoforms, while real-time PCR, on nuclear and cytoplasmic RNA fractions, and cRNA in situ hybridization, on astrocytoma cells and bioptical specimens, showed a nuclear retention of PRND transcripts. Western blot analysis showed that the amount of protein expressed is low compared to the level of mRNA. Moreover deglycosylation studies indicated that Dpl undergoes unusual glycosylation processes. Immunohistochemistry experiments demonstrated that Dpl was mainly localised in the cytoplasm of the astrocytic tumor cells, and that it failed to be GPI-anchored to the cell membrane. This unusual cellular localization was also confirmed through EGFP-Dpl expression in astrocytomas; on the contrary, HeLa cells exhibited the expected Dpl membrane localization. Our findings suggest an aberrant doppel gene expression pattern, characterized by a substantial nuclear retention of the transcript, an altered post-translational modification of the protein and an unusual cytoplasmic localization.

Summary Three microsatellites—IDVGA-2, IDVGA-3 and IDVGA-46, composed of a (AC) motif, were associated to beef performance traits of 54 female crossbred cattle from two specialized Italian beef breeds, Piemontese for muscling and meat quality and Chianina for high daily gain and size. Association was tested by a general linear mixed model in which the genotypes for each microsatellite were fixed factors and which considered the additive genetic random effect. Microsatellite IDVGA-46 is associated to most conformation parameters while IDVGA-2 and IDVGA-3 do not seem to affect much performance traits. In fact homozygous individuals carriers of allele 205 (IDVGA-46) show a global better structure because they have bigger body size which is one of the goals of improving beef. Zusammenfassung Verwendung von Mikrosatelliten als genetische Marker der Fleischleistungsmerkmale in Kreuzungstieren der Rassen Piemontese × Chianina Drei aus einem (AC) Motiv gebildete Mikrosatelliten—IDVGA-2, IDVGA-3 und IDVGA-46—wurden Fleischleistungsmerkmalen von 54 Kreuzungskuhen aus zwei spezialisierten italienischen Fleischrassen zugeordnet, Piemontese zur Verbesserung der Muskelentwicklung und Fleischqualitat und Chianina zur Verbesserung des Tageszuwachses. Die Assoziation wurde mit einem linearen Modell getestet, mit fixen Faktoren fur die Genotypen eines jeden Mikrosatelliten. Bei Mikrosatelliten IDVGA-2 und 3 wurden keine Beziehungen zu Fleischleistungsmerkmalen festgestellt, wohingegen Mikrosatellit IDVGA-46 einen signifikanten Einflus auf Korperbeschaffenheit zeigt. Der 205 homozygote Genotyp (IDVGA-46) hat eine bessere Muskel- und Skelettentwicklung aufgewiesen, was eines der Zuchtziele bei der Produktion von Fleischrindern darstellt.