Observation of Scaling Violations in Scaled Momentum Distributions at HERA

Charged particle production has been measured in deep inelastic scattering (DIS) events over a large range of $x$ and $Q^2$ using the ZEUS detector. The evolution of the scaled momentum, $x_p$, with $Q^2,$ in the range 10 to 1280 $GeV^2$, has been investigated in the current fragmentation region of the Breit frame. The results show clear evidence, in a single experiment, for scaling violations in scaled momenta as a function of $Q^2$.Comment: 21 pages including 4 figures, to be published in Physics Letters B. Two references adde

D* Production in Deep Inelastic Scattering at HERA

This paper presents measurements of D^{*\pm} production in deep inelastic scattering from collisions between 27.5 GeV positrons and 820 GeV protons. The data have been taken with the ZEUS detector at HERA. The decay channel $D^{*+}\to (D^0 \to K^- \pi^+) \pi^+$ (+ c.c.) has been used in the study. The $e^+p$ cross section for inclusive D^{*\pm} production with $5<Q^2<100 GeV^2$ and $y<0.7$ is 5.3 \pms 1.0 \pms 0.8 nb in the kinematic region {$1.3<p_T(D^{*\pm})<9.0$ GeV and $| \eta(D^{*\pm}) |<1.5$}. Differential cross sections as functions of p_T(D^{*\pm}), $\eta(D^{*\pm}), W$ and $Q^2$ are compared with next-to-leading order QCD calculations based on the photon-gluon fusion production mechanism. After an extrapolation of the cross section to the full kinematic region in p_T(D^{*\pm}) and $\eta$(D^{*\pm}), the charm contribution $F_2^{c\bar{c}}(x,Q^2)$ to the proton structure function is determined for Bjorken $x$ between 2 $\cdot$ 10$^{-4}$ and 5 $\cdot$ 10$^{-3}$.Comment: 17 pages including 4 figure

Observation of Events with an Energetic Forward Neutron in Deep Inelastic Scattering at HERA

In deep inelastic neutral current scattering of positrons and protons at the center of mass energy of 300 GeV, we observe, with the ZEUS detector, events with a high energy neutron produced at very small scattering angles with respect to the proton direction. The events constitute a fixed fraction of the deep inelastic, neutral current event sample independent of Bjorken x and Q2 in the range 3 · 10-4 \u3c xBJ \u3c 6 · 10-3 and 10 \u3c Q2 \u3c 100 GeV2

A subtle alternative splicing event gives rise to a widely expressed human RNase κ isoform

Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase k transcript (RNase κ-02) which lacks four consecutive bases compared to the previously isolated RNase k isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase k -02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms. © 2014 Karousis, Sideris

Isolation and sequencing of a cDNA encoding for a ribonuclease from the insect Ceratitis capitata

Two overlapping clones encoding for a ribonuclease from six-day-old larvae of the insect Ceratitis capitata (Cc-RNase) have been isolated by immunoscreening a cDNA library and by 5&apos; RACE. The sequence of the Cc-RNase cDNA contains an open reading frame of 414 nucleotides encoding for a precursor protein of 138 amino acids long with a putative signal peptide consisting of 19 amino acids. The calculated M(r) of the mature protein was found to be 13.7 kDa. Multiple alignments of the deduced amino acid Cc-RNase sequence with other ribonucleases revealed an approximate 25% average identity. Despite the low percentage of identity, histidine and lysine residues which are essential for its catalytic activity, were found to be completely conserved. Furthermore, expression of the clone in E. coli resulted in the production of a recombinant product that showed strong immunoreactivity with anti-RNase specific antibodies. These results support the hypothesis that the identified clone encodes for a protein which is a new member of the RNase superfamily. (C) 2000 Elsevier Science Ltd

Further characterization of a poly(U), poly(C) ribonuclease from the insect Ceratitis capitata

A ribonuclease from 6 day old larvae of Ceratitis capitata has been purified to homogeneity by a four step procedure. The enzyme appears as a single polypeptide chain of approx. 34 kDa. Poly(U) and poly(C) are the only polyribonucleotides degraded under standard assay conditions. The enzyme degrades the mRNA present in polysomes inducing a shift of polysomes to monosomes. However, no major cleavage could be observed in the mRNA and the ribosomal RNA when the incubation with the ribonuclease took place under protein synthesis conditions of polysomes. © 1992

Genomic structure and expression analysis of the RNase κ family ortholog gene in the insect Ceratitis capitata

Cc RNase is the founding member of the recently identified RNase κ family, which is represented by a single ortholog in a wide range of animal taxonomic groups. Although the precise biological role of this protein is still unknown, it has been shown that the recombinant proteins isolated so far from the insect Ceratitis capitata and from human exhibit ribonucleolytic activity. In this work, we report the genomic organization and molecular evolution of the RNase κ gene from various animal species, as well as expression analysis of the ortholog gene in C. capitata. The high degree of amino acid sequence similarity, in combination with the fact that exon sizes and intronic positions are extremely conserved among RNase κ orthologs in 15 diverse genomes from sea anemone to human, imply a very significant biological function for this enzyme. In C. capitata, two forms of RNase κ mRNA (0.9 and 1.5 kb) with various lengths of 3′ UTR were identified as alternative products of a single gene, resulting from the use of different polyadenylation signals. Both transcripts are expressed in all insect tissues and developmental stages. Sequence analysis of the extended region of the longer transcript revealed the existence of three mRNA instability motifs (AUUUA) and five poly(U) tracts, whose functional importance in RNase κ mRNA decay remains to be explored. © 2008 The Authors

Essential cysteine residues for human RNase κ catalytic activity

Human RNase κ is an endoribonuclease expressed in almost all tissues and organs and belongs to a highly conserved protein family bearing representatives in all metazoans. To gain insight into the role of cysteine residues in the enzyme activity or structure, a recombinant active form of human RNase κ expressed in Pichia pastoris was treated with alkylating agents and dithiothreitol (DTT). Our results showed that the human enzyme is inactivated by DDT, while it remains fully active in the presence of alkylating agents. The unreduced recombinant protein migrates on SDS/PAGE faster than the reduced form. This observation in combination with the above findings indicated that human RNase κ does not form homodimers through disulfide bridges, and cysteine residues are not implicated in RNA catalysis but participate in the formation of intramolecular disulfide bond(s) essential for its ribonucleolytic activity. The role of the cysteine residues was further investigated by expression and study of Cys variants. Ribonucleolytic activity experiments and SDS/PAGE analysis of the wild-type and mutant proteins under reducing and non-reducing conditions demonstrated that Cys7, Cys14 and Cys85 are not essential for RNase activity. On the other hand, replacement of Cys6 or Cys69 with serine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of human RNase κ by forming a disulfide bond. Due to the absolute conservation of these cysteine residues, the Cys6-Cys69 disulfide bond is likely to exist in all RNase κ family members. © 2012 FEBS

A hybrid-specific, polymerase chain reaction-based amplification approach for chromosomal walking

Here we report the development of an accurate and efficient genome walking approach based on ligation-mediated polymerase chain reaction (PCR) and magnetic capture hybrid selection technique. This approach overcomes the nonspecific amplification products that often occur in similar PCR-based methods. Our strategy was successfully applied for the cloning of the promoter region of the Cc RNase gene. This rapid, cost-effective, and sensitive protocol can easily be extended for use in the isolation and amplification of any target sequences for which only partial information is known such as identification of the position of transposable elements and integrated viral DNA sequences. © 2009 Elsevier Inc. All rights reserved

Effect of cytostatic drugs on the mRNA expression levels of ribonuclease k in breast and ovarian cancer cells

Breast and ovarian cancers remain a major public health issue, constituting a major cause of female morbidity and mortality worldwide. Even though systemic chemotherapy remains the main course of action for both types of cancer, current research studies aim at the discovery of novel therapeutic targets. Ribonucleases, due to their apparent implication in gene expression regulation, may serve as potential anticancer drugs. Human RNase κ is an endoribonuclease belonging in a recently identified family of proteins. Recent data from expression microarray studies reveal that the RNase κ gene is found either up- or down regulated in a number of human cancers, indicating a possible diagnostic and/or prognostic utility. The aim of this study was to investigate modulations in the expression levels of RNase κ gene, in breast and ovarian cancer cells, as a response to treatment with different chemotherapeutic agents. BT-20 breast cancer cells and SKOV-3 ovarian cancer cells were treated with the cytotoxic drugs paclitaxel, docetaxel, cisplatin, carboplatin, epirubicin and vinorelbine. Gene expression analysis was performed by the comparative CT method also known as 2-ΔΔCT method. The results revealed a distinct increase in the expression levels of RNase κ mRNA (up to 9-fold) after treatment with the antineoplastic agent paclitaxel in both cell lines, while treatment with the remaining anticancer drugs did not alter drastically the mRNA levels of RNase κ. Based on the fact that paclitaxel exerts its cytotoxic action by inducing apoptosis, the results could be indicative of a potential implication of RNase κ in apoptosis-related pathways. © 2014 Bentham Science Publishers