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PubMed1997

Identification and phenotypic analysis of two glyoxalase II encoding genes from Saccharomyces cerevisiae, GLO2 and GLO4, and intracellular localization of the corresponding proteins.

We have isolated and characterized two genes coding for the glyoxalase II enzyme from Saccharomyces cerevisiae. The coding region of the GLO2 gene corresponds to a protein with 274 amino acids and a molecular mass of 31,306 daltons. The open reading frame of the GLO4 gene could be translated into a protein with 285 amino acids and a molecular mass of 32,325 daltons. The amino acid sequences of the deduced proteins are 59.1% identical and show high similarities to the sequence of the human glyoxalase II. When grown on either glucose or glycerol as a carbon source, a glo2 glo4 double deletion strain contains no glyoxalase II activity at all and shows no obvious phenotype during vegetative growth. However, this strain showed a similar high sensitivity against exogenous methylglyoxal as compared with a glyoxalase I-deficient strain. Whereas the GLO2 gene is expressed on both glucose and glycerol, the GLO4 gene is only active on glycerol. The active Glo2p protein is localized in the cytoplasm and the active Glo4p in the mitochondrial matrix. Heterologous expression of the full-length GLO2 coding region in Escherichia coli resulted in an active protein. However, to get an active Glo4p protein in E. coli, the putative mitochondrial transit peptide at the N-terminal end had to be removed by shortening the 5' end of the GLO4 open reading frame.

Amino Acid Sequence↗
PubMed1999

An open reading frame element mediates posttranscriptional regulation of tropoelastin and responsiveness to transforming growth factor beta1.

Elastin, an extracellular component of arteries, lung, and skin, is produced during fetal and neonatal growth. We reported previously that the cessation of elastin production is controlled by a posttranscriptional mechanism. Although tropoelastin pre-mRNA is transcribed at the same rate in neonates and adults, marked instability of the fully processed transcript bars protein production in mature tissue. Using RNase protection, we identified a 10-nucleotide sequence in tropoelastin mRNA near the 5' end of the sequences coded by exon 30 that interacts specifically with a developmentally regulated cytosolic 50-kDa protein. Binding activity increased as tropoelastin expression dropped, being low in neonatal fibroblasts and high in adult cells, and treatment with transforming growth factor beta1 (TGF-beta1), which stimulates tropoelastin expression by stabilizing its mRNA, reduced mRNA-binding activity. No other region of tropoelastin mRNA interacted with cellular proteins, and no binding activity was detected in nuclear extracts. The ability of the exon-30 element to control mRNA decay and responsiveness to TGF-beta1 was assessed by three distinct functional assays: (i) insertion of exon 30 into a heterologous gene conferred increased reporter activity after exposure to TGF-beta1; (ii) addition of excess exon 30 RNA slowed tropoelastin mRNA decay in an in vitro polysome degradation assay; and (iii) a mutant tropoelastin cDNA lacking exon 30, compared to wild-type cDNA, produced a stable transcript whose levels were not affected by TGF-beta1. These findings demonstrate that posttranscriptional regulation of elastin production in mature tissue is conferred by a specific element within the open reading frame of tropoelastin mRNA.

Animals↗
PubMed1991

Eukaryotic initiation factor (eIF)-4F. Implications for a role in internal initiation of translation.

In order to study the eukaryotic translation initiation mechanisms of "internal initiation," "re-initiation," and/or "coupled internal initiation," a series of model mRNAs have been constructed which contain two non-overlapping open reading frames (ORFs) that encode different lengths of rabbit alpha globin. These mRNAs, along with the bicistronic constructs TK/CAT and TK/P2CAT developed by Pelletier and Sonenberg (Pelletier, J., and Sonenberg, N. (1988) Nature 334, 320-325, 1988), were used to program an in vitro rabbit reticulocyte lysate translation system. Cap-dependent and cap-independent translation were distinguished by monitoring translation in the presence or absence of exogenously added cap analog (m7GTP). Messenger RNAs which translate both ORF1 and ORF2 by a cap-dependent mechanism, as well as mRNAs that translate ORF2 by a cap-independent mechanism while still translating ORF1 in a cap-dependent fashion have been obtained. These same alpha globin mRNAs differ by no more than 45 nucleotides in intercistronic length. Initiation factor addition studies were performed in this same in vitro translation system. Both eukaryotic initiation factor (eIF)-4F and, to a lesser extent, eIF-4B can stimulate translation of an internally located ORF independent of upstream ORF translation and in a manner not dependent on mRNA cap recognition. This indicates that the cap-recognition initiation factor, eIF-4F, and eIF-4B facilitate cap-independent and internal initiation of an open reading frame.

Animals↗
PubMed2005

LOVD: easy creation of a locus-specific sequence variation database using an "LSDB-in-a-box" approach.

The completion of the human genome project has initiated, as well as provided the basis for, the collection and study of all sequence variation between individuals. Direct access to up-to-date information on sequence variation is currently provided most efficiently through web-based, gene-centered, locus-specific databases (LSDBs). We have developed the Leiden Open (source) Variation Database (LOVD) software approaching the "LSDB-in-a-Box" idea for the easy creation and maintenance of a fully web-based gene sequence variation database. LOVD is platform-independent and uses PHP and MySQL open source software only. The basic gene-centered and modular design of the database follows the recommendations of the Human Genome Variation Society (HGVS) and focuses on the collection and display of DNA sequence variations. With minimal effort, the LOVD platform is extendable with clinical data. The open set-up should both facilitate and promote functional extension with scripts written by the community. The LOVD software is freely available from the Leiden Muscular Dystrophy pages (www.DMD.nl/LOVD/). To promote the use of LOVD, we currently offer curators the possibility to set up an LSDB on our Leiden server.

Computational Biology↗
PubMed1993

CLG1, a new cyclin-like gene of Saccharomyces cerevisiae.

A region of chromosome VII adjacent to SKI8 has a 453 amino acid open reading frame whose sequence has significant similarity to that of HCS26, a G1 cyclin. A disruption mutation of this open reading frame has no apparent phenotype under the conditions tested. ORFD, an open reading frame adjacent to the CDC48 gene, is even more similar to HCS26.

Amino Acid Sequence↗
PubMed1990

The DNA sequence and structural organization of the GC2 plasmid from the red alga Gracilaria chilensis.

The complete DNA sequence of the circular GC2 plasmid from the red alga Gracilaria chilensis was obtained. It contains 3827 bp and has a base composition of 75% A + T nucleotides. The sequence revealed that GC2 has two inverted repeats, each of 290 nucleotides, that border four long direct tandem repeats of 216 nucleotides. five short, direct, tandem repeats of 21-22 nucleotides were also found in the plasmid. The plasmid sequence has one major open reading frame that could encode a 411 amino acid polypeptide. Finally, the GC2 plasmid is transcriptionally active.

Amino Acid Sequence↗
PubMed2000

The nucleotide sequence of Indian peanut clump virus RNA 2: sequence comparisons among pecluviruses.

The RNA-2 molecule of an isolate of the L serotype of Indian peanut clump virus (IPCV) was shown to consist of 4,290 nucleotides with five open reading frames (ORF). The arrangement of the ORFs resembled that in RNA-2 of Peanut clump virus (PCV) from West Africa. The proteins encoded by the ORFs in IPCV-L RNA are between 32% and 93% identical to those encoded by PCV RNA. Partial sequence data for the RNA-2 of isolates of the H and T serotypes of IPCV show that the coat and P40 proteins encoded by the 5'-most ORFs of RNA-2 of IPCV-L, IPCV-H and IPCV-T are as similar to each other as any is to the corresponding proteins of PCV. A conserved motif 'F-E-x6-W' is present near the C-termini of the coat proteins of all three IPCV serotypes and of PCV, as it is in the coat proteins of other viruses that have rod-shaped particles, such as Tobacco mosaic virus and Tobacco rattle virus. The results support the distinction of IPCV and PCV as separate virus species, but also raise the question of how the serotypes of IPCV should be classified.

Amino Acid Sequence↗
PubMed2006

Positive influence of robotically assisted laparoscopic prostatectomy on the collaborative-care pathway for open radical prostatectomy.

OBJECTIVE: To define changes in the clinical pathway for open radical retropubic prostatectomy (RRP) and to determine their impact on patient care under the indirect influence of minimally invasive prostatectomy after implementing robotically assisted laparoscopic prostatectomy (RALP) at our institution and modifying the collaborative-care pathway. PATIENTS AND METHODS: We retrospectively reviewed the Vanderbilt Prostate Database since the initiation of RALP in May 2003, evaluating those patients who had open RRP. Data were analysed in three consecutive 5-month blocks to determine incremental changes in protocol and outcomes, including complications and re-admissions. RESULTS: Between May 2003 and August 2004, 196 patients had RALP and 183 had RRP. After May 2003, there was a progressive reduction for open RRP in the length of stay from 1.6 to 1.3 days. Analysis also showed an elimination of patient-controlled analgesia, a rapid diet advancement, and drain removal at the surgeon's discretion. There were no major complications after surgery, and three minor complications (1.6%). The rate of re-admission after open RRP was low throughout the study period (seven patients, 3.8%), and did not increase progressively. CONCLUSIONS: The management after open RRP was modified substantially after implementing RALP, with the two clinical pathways now similar. Despite these modifications in the treatment of patients having open RRP there was no increase in morbidity or compromise in patient outcome. Indeed, this study shows the feasibility and safety of a 1-day length of stay for open RRP.

Feasibility Studies↗
PubMed1996

The pharmacotherapy of hypochondriasis.

This article addresses the diagnosis and pharmacologic treatment of hypochondriasis. Diagnostic issues are reviewed briefly, focusing on the need for a thorough medical re-consideration of the patient's presenting symptoms. Because the diagnosis rests on the absence of a medical cause to account for the presence or intensity of the physical symptoms, neither self-report forms nor non-medically trained interviewers should be used to definitively make the diagnosis of hypochondriasis. We review the case reports and small uncontrolled series on the pharmacologic treatment of hypochondriasis, emphasizing the growing body of evidence suggesting particular efficacy for the serotonin reuptake inhibitors. Preliminary results from an ongoing placebo-controlled trial of hypochondriasis using fluoxetine are presented. While the controlled trial supports the open treatment data in revealing a high rate of improvement among patients completing treatment with fluoxetine, it also demonstrates that many patients respond to placebo as well. In conclusion, although the traditional nihilistic attitude regarding the possibility of successful treatment of hypochondriacs appears no longer warranted, the question remains open as to whether SSRIs have particular efficacy in patients with hypochondriasis or whether nonspecific treatment effects are the primary cause of improvement.

Humans↗
PubMed2007

Automated mass correction and data interpretation for protein open-access liquid chromatography-mass spectrometry.

Characterization of recombinant protein purification fractions and final products by liquid chromatography-mass spectrometry (LC/MS) are requested more frequently each year. A protein open-access (OA) LC/MS system was developed in our laboratory to meet this demand. This paper compares the system that we originally implemented in our facilities in 2003 to the one now in use, and discusses, in more detail, recent enhancements that have improved its robustness, reliability, and data reporting capabilities. The system utilizes instruments equipped with reversed-phase chromatography and an orthogonal accelerated time-of-flight mass spectrometer fitted with an electrospray source. Sample analysis requests are accomplished using a simple form on a web-enabled laboratory information management system (LIMS). This distributed form is accessible from any intranet-connected company desktop computer. Automated data acquisition and processing are performed using a combination of in-house (OA-Self Service, OA-Monitor, and OA-Analysis Engine) and vendor-supplied programs (AutoLynx, and OpenLynx) located on acquisition computers and off-line processing workstations. Analysis results are then reported via the same web-based LIMS. Also presented are solutions to problems not addressed on commercially available, small-molecule OA-LC/MS systems. These include automated transforming of mass-to-charge (m/z) spectra to mass spectra and automated data interpretation that considers minor variants to the protein sequence-such as common post-translational modifications (PTMs). Currently, our protein OA-LC/MS platform runs on five LC/MS instruments located in three separate GlaxoSmithKline R&D sites in the US and UK. To date, more than 8000 protein OA-LC/MS samples have been analyzed. With these user friendly and highly automated OA systems in place, mass spectrometry plays a key role in assessing the quality of recombinant proteins, either produced at our facilities or bought from external sources, without dedicating extensive amounts of analyst resource.

Animals↗
PubMed1992

LEU2 gene homolog in Kluyveromyces lactis.

A DNA fragment that can complement the leu2 mutation of Saccharomyces cerevisiae was cloned from the genomic library of Kluyveromyces lactis. The nucleotide sequence revealed an open reading frame of 362 codons, 75% homologous to S. cerevisiae LEU2 gene. The upstream region contained a CCGGAACCGG sequence identical to the site of leucine-specific control of LEU2. Further upstream, there is a partial open reading frame homologous to rat ribosomal protein L7.

Amino Acid Sequence↗
PubMed1998

Full-length GBV-C/HGV genomes from nine Japanese isolates: characterization by comparative analyses.

The genomes of nine GBV-C/HGV isolates from Japanese chronic hepatitis patients were fully sequenced and characterized. They shared 85% nucleotide sequence homology with previously characterized isolates from the US and West Africa. Homology studies and phylogenetic analyses showed that the Japanese isolates formed a third group distinct from the established groups 1 and 2. The genetic distances between the three groups of GBV-C/HGV were very similar to the distances between the two classical swine fever virus (CSFV) serotypes, which suggested that they might belong to a separate GBV-C/HGV serotype. Plot similarity analysis comparing the three groups exposed relatively conserved terminal non-coding regions. Hairpin structures predicted in the Japanese isolates are probably involved in viral replication. The region coding E1-E2-NS-2 showed the least similarity (80%); in HCV the similarity here is only 50% due to its hypervariability. NS-3 and NS-5b that respectively encode the helicase/protease and RNA-dependent RNA polymerase, had a high degree of amino acid homology, suggesting a high degree of functional constraint in this region. The NS-5b nucleotide sequence was highly conserved perhaps because of constraints from RNA secondary structure and/or an open reading frame in the negative strand.

Base Sequence↗
PubMed1995

Evolution in action.

The alpha/beta barrel enzyme phosphotriesterase from soil bacteria appears to have evolved the ability to hydrolyze the insecticide paraoxon at the diffusion limit in only a few decades. A newly-identified open reading frame from Escherichia coli may offer a clue to its origins.

Amino Acid Sequence↗
PubMed1999

Nucleotide and peptide sequences of the open reading frame encoding a truncated toxin A gene of Clostridium difficile strain CCUG 20309.

The open reading frame encoding a putative truncated toxin A gene of Clostridium difficile in strain CCUG 20309 (ATCC 8864), a strain that produces toxin B but not toxin A, was sequenced by cycle sequencing method. The coding region contains 2097 base pairs and has a GC content of 26.4%. The deduced polypeptide is 50 kDa and is generally hydrophilic. Although strain CCUG 20309 of C. difficile was reported not to produce toxin A, it is enterotoxic, an inherent property of toxin A in pathogenic strains of C. difficile. It therefore remains to be determined whether a truncated form of toxin A could actually be expressed from the open reading frame by this strain of C. difficile.

Amino Acid Sequence↗
PubMed2004

Minimally invasive radical prostatectomy.

The development of advanced laparoscopic techniques and robot-assisted technology has resulted in several new surgical approaches for treating organ-confined prostate cancer. Outcomes with these new or minimally Invasive techniques should be assessed carefully to ensure that they are similar to or surpass patients' oncologic and functional outcomes after open radical prostatectomy. This article reviews the current published experience with minimally Invasive approaches to increase awareness about viability. Several of the larger series of patients who have undergone laparoscopic (transperitoneal and extraperitoneal) or robot-assisted laparoscopic radical prostatectomies are discussed and evaluated critically. Comparisons to published data on open radical prostatectomy are included for completeness. The different minimally invasive techniques are described and contrasted in regard to prostate-specific antigen progression-free survival, surgical margin status, blood loss, transfusion rates, postoperative pain, length of hospitalization, duration of urinary catheterization, potency, continence, and complications. The relative costs of each method are provided. The coexistence of multiple surgical approaches should and can challenge surgeons who perform open and minimally invasive procedures to strive for a new standard of care above and beyond what is accepted today to minimize patient morbidity while maximizing functional and oncologic outcomes.

Adult↗