bluuebunny/crossref_metadata_2025 huggingface dataset:
Heat transfer modelling for both workpiece and tool in the friction stir welding process: A coupled model
Authors: Song M , Kovacevic R
Published: February 2004

In this paper, a three-dimensional transient-heat transfer model for both the tool and the workpiece in friction stir welding (FSW) has been presented. A moving coordinate is introduced to reduce the difficulty of modelling the heat generation due to the movement of the tool pin. The heat transfer process of the tool and workpiece are coupled at the workpiece/tool interface. The finite difference method was used in solving the energy equations. In order to improve the temperature resolution near the tool in the calculation, a non-uniform grid mesh is generated for the calculation. FSW experiments have been done to validate the modelling results. The calculated results are in good agreement with the experimental ones.
Optimization of a gas metal arc welding process using the desirability function and the genetic algorithm
Authors: Kim D , Rhee S
Published: February 2004

The dual response approach is a method of determining the optimal welding conditions in consideration of both the mean value and variance of a characteristic. The definition of the objective function is important, as the results of the optimization problem are influenced by the objective function, which is defined using the mean and variance. This study focuses on the definition and optimization of the objective function in the dual response approach applied to a gas metal arc (GMA) welding process. The objective function is defined using the desirability function. The genetic algorithm, a global optimization algorithm, is used as an optimization algorithm in this study, whereas local optimization algorithms have commonly been used in preceding studies.
On the role of laser-induced microstructures in influencing the surface energy of magnesia partially stabilized zirconia bioceramic
Authors: Hao L , Lawrence J
Published: February 2004

Surface energy must in some way be connected to surface chemistry and is therefore an important determinant of a biomaterial's functions. This work elucidates the basic phenomena and wetting mechanisms associated for a widely used bioinert ceramic, magnesia partially stabilized zirconia (MgO-PSZ) following CO2 laser treatment. Contact angles for a set of test liquids were used to measure the wettability characteristics and to reduce the surface energy of the MgO-PSZ before and after CO2 laser treatment. CO2 laser treatment of the MgO-PSZ surface was seen to effect an improvement in the material's wettability characteristics. Furthermore, it was found that the extent of wettability modification was closely related to the microstructure induced by CO2 laser radiation on the surface of the MgO-PSZ.
A study of porosity formation in pressure die casting using the Taguchi approach
Authors: Tsoukalas V D , Mavrommatis St A , Orfanoudakis N G , Baldoukas A K
Published: February 2004

Porosity is one of the defects most frequently encountered in aluminium die castings. Porosity formation causes costly scrap loss and limits the use of die-cast parts in critical high-strength applications. The amount of porosity is closely related to die casting process parameters. A considerable reduction in porosity formation can be obtained using the Taguchi approach in the die casting process. An example of the off-line technique used in the Greek die casting industry is given, with the aim of illustrating practicality and ease of use by industry engineers and managers. In the thick-walled sample casting studied, the final intensification pressure contributed 69 per cent towards the reduction in porosity. Among the other parameters considered, melt and die temperatures have a smaller effect (about 10 per cent), followed by the plunger velocities in the second and first stages which have only a slight effect on porosity formation. In addition, the expected quality improvement and the cost savings under optimum parameter settings were evaluated. The expected improvement in reduction in porosity formation was found to be 48.6 per cent, while the expected cost savings for multiple casting parts under optimum conditions was calculated to be 71.4 per cent/$1 loss before the experiment.
Linear programming and optimizing lumber quality composition in secondary hardwood dimension mills
Authors: Buehlmann U , Zuo X , Thomas R E
Published: February 2004

Linear programming has been widely applied in the secondary hardwood dimension manufacturing industries to solve the least-cost lumber grade mix problem, which refers to the search for minimal raw material costs. Most existing models are based on the assumption of a simple linear relationship between lumber grade mix and yield. However, this crucial assumption has never been verified.

In this study, the results from a five-factor mixture design statistically proved that none of the cutting bills tested has a simple linear relationship between yield and different lumber grade mixes. It was observed that cutting bill characteristics and lumber quality affect the relationship between yield and lumber grades. Cutting bills that require wider and/or longer parts tend to behave non-linearly. In addition, the more dissimilar lumber grade qualities that are processed, the more likely is the occurrence of a non-linear response.

The inability to predict the relationship between yield and lumber grades, coupled with the high percentage of non-simple linear relationships observed in this study, brings into question the validity of the linearity assumption applied in previous linear programming models. Further efforts are needed to construct a new least-cost lumber grade mix model that does not rely on the assumption of a simple linear relationship between lumber grade mix and yield.
RULES-5: A rule induction algorithm for classification problems involving continuous attributes
Authors: Pham D T , Bigot S , Dimov S S
Published: February 2004

This paper presents RULES-5, a new induction algorithm for effectively handling problems involving continuous attributes. RULES-5 is a ‘covering’ algorithm that extracts IF-THEN rules from examples presented to it. The paper first reviews existing methods of rule extraction and dealing with continuous attributes. It then describes the techniques adopted for RULES-5 and gives a step-by-step example to illustrate their operation. The paper finally gives the results of applying RULES-5 and other algorithms to benchmark problems. These clearly show that RULES-5 generates rule sets that are more accurate than those produced by its immediate predecessor RULES-3 Plus and by a well-known commercially available divide-and-conquer machine learning algorithm.
Meshing analysis of a gear with a ring-involute gear
Authors: Yang S-C
Published: February 2004

The surface of a gear with ring-involute teeth generated by a rack cutter with ring-involute teeth is a new type of gear. This paper describes a method developed from gear theory for deriving a pinion and a gear with ring-involute teeth. A gear with ring-involute teeth is regarded as an envelope to the family of rack cutter surfaces when the pinion and gear rotate for a cycle. Using a developed mathematical model, the investigation on the undercutting analysis of the proposed gear is studied. Here the kinematic errors are investigated according to the obtained geometric modelling of the designed gear meshing when assembly errors were present. Stress analysis for the proposed gear was performed. Finally, a numerical example is presented to demonstrate the geometric model of a gear with ring-involute teeth and a gear ratio of 3:2.
Generalized coupled transient thermoelastic problem of multilayered spheres by the hybrid numerical method
Authors: Lee Z Y , Chang C L
Published: February 2004

This paper deals with axisymmetric quasi-static coupled thermoelastic problems for multilayered spheres. Laplace transforms and finite difference methods are used to analyse the problems. Using the Laplace transform with respect to time, the general solutions of the governing equations are obtained in the transform domain. The solution is obtained by using the matrix similarity transformation and inverse Laplace transform. Solutions are obtained for the temperature and thermal deformation distributions for the transient and steady state. It is demonstrated that the computational procedures established in this paper are capable of solving the generalized thermoelasticity problem of multilayered spheres.
Bifurcation analysis of externally pressurized porous gas journal bearings
Authors: Wang C-C , Chen C-K
Published: February 2004

This paper studies the bifurcation of a rigid rotor supported by an externally pressurized porous gas journal bearing. A time-dependent mathematical model for porous gas journal bearings is presented. The modified Reynolds equation is solved using the finite difference method and the SOR (successive over relation) method. The system state trajectory, Poincaré maps, power spectra and bifurcation diagrams are used to analyse the dynamic behaviour of the rotor centre in the horizontal and vertical directions under different operating conditions. The analysis reveals a complex dynamic behaviour comprising periodic and quasi-periodic responses of the rotor centre. This paper shows how the dynamic behaviour of systems of this type varies with changes in rotor mass, squeeze number and bearing number. The results of this study contribute to a further understanding of the non-linear dynamics of gas-lubricated, externally pressurized, porous rotor-bearing systems.
Synthesis of l-Cysteine and l-Cysteic Acid by Paired Electrolysis Method
Authors: Wang Xixin , Zhao Jianling
Published: February 2004

l-Cysteine and l-cysteic acid were synthesized by paired eletrolysis method. A high purity over 98% and high yield over 90% of both products were gained. When current density was 7 A/dm2 and concentration of l-cysteine was 0.6 mol/dm3, the highest current efficiency of anode and cathode was achieved. Total current efficiency was over 180%. The cyclic voltammetry behaviors of hydrobromic acid and cystine showed that a typical EC reaction took place in the anodic cell. The anode reaction and successive chemical reaction accelerated each other to get a high speed and current efficiency.
Growth of Sb2O3 Nanotubes via a Simple Surfactant-assisted Solvothermal Process
Authors: Zhang Yunxia , Li Guanghai , Zhang Lide
Published: February 2004

Sb2O3 nanotubes were successfully prepared via a simple surfactant-assisted solvothermal method. The nanotubes have an orthorhombic structure with outer diameter range from 40 to 150 nm, the wall thickness of 10 to 40 nm and a length of up to several micrometers. The formation of nanotubes followed a rolling-up mechanism.
Synthesis of Bamboo-shaped TiO2 Nanotubes in Nanochannels of Porous Aluminum Oxide Membrane
Authors: Peng Tianyou , Yang Huanping , Chang Gang , Dai Ke , Hirao Kazuyuki
Published: February 2004

The bamboo-shaped TiO2 nanotubes with diameters of ca. 100 nm have been fabricated in nanochannels of PAO membranes by upright dipping manner. The bamboo-shaped nanotubes consist of many hollow compartments that are separated by TiO2 layers.
Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits
Authors: ALKEMAR GUNNAR , NYGÅRD ODD
Published: February 2004

The 18S rRNA of the small eukaryotic ribosomal subunit contains several expansion segments. Electron microscopy data indicate that two of the largest expansion segments are juxtaposed in intact 40S subunits, and data from phylogenetic sequence comparisons indicate that these two expansion segments contain complementary sequences that could form a direct tertiary interaction on the ribosome. We have investigated the secondary structure of the two expansion segments in the region around the putative tertiary interaction. Ribosomes from yeast, wheat, and mouse—three organisms representing separate eukaryotic kingdoms—were isolated, and the structure of ES3 and part of the ES6 region were analyzed using the single-strand-specific chemical reagents CMCT and DMS and the double-strand-specific ribonuclease V1. The modification patterns were analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The investigated sequences were relatively exposed to chemical and enzymatic modification. This is in line with their indicated location on the surface at the solvent side of the subunit. The complementary ES3 and ES6 sequences were clearly inaccessible to single-strand modification, but available for cleavage by double-strand-specific RNase V1. The results are compatible with a direct helical interaction between bases in ES3 and ES6. Almost identical results were obtained with ribosomes from the three organisms investigated.
Functional group recognition at the aminoacylation and editing sites of E. coli valyl-tRNA synthetase
Authors: TARDIF KEITH D. , HOROWITZ JACK
Published: February 2004

To correct misactivation and misacylation errors, Escherichia coli valyl-tRNA synthetase (ValRS) catalyzes a tRNAVal-dependent editing reaction at a site distinct from its aminoacylation site. Here we examined the effects of replacing the conserved 3′-adenosine of tRNAVal with nucleoside analogs, to identify structural elements of the 3′-terminal nucleoside necessary for tRNA function at the aminoacylation and editing sites of ValRS. The results show that the exocyclic amino group (N6) is not essential: purine riboside-substituted tRNAVal is active in aminoacylation and in stimulating editing. Presence of an O6 substituent (guanosine, inosine, xanthosine) interferes with aminoacylation as well as posttransfer and total editing (pre- plus posttransfer editing). Because ValRS does not recognize substituents at the 6-position, these results suggest that an unprotonated N1, capable of acting as an H-bond acceptor, is an essential determinant for both the aminoacylation and editing reactions. Substituents at the 2-position of the purine ring, either a 2-amino group (2-aminopurine, 2,6-diaminopurine, guanosine, and 7-deazaguanosine) or a 2-keto group (xanthosine, isoguanosine), strongly inhibit both aminoacylation and editing. Although aminoacylation by ValRS is at the 2′-OH, substitution of the 3′-terminal adenosine of tRNAVal with 3′-deoxyadenosine reduces the efficiency of valine acceptance and of posttransfer editing, demonstrating that the 3′-terminal hydroxyl group contributes to tRNA recognition at both the aminoacylation and editing sites. Our results show a strong correlation between the amino acid accepting activity of tRNA and its ability to stimulate editing, suggesting misacylated tRNA is a transient intermediate in the editing reaction, and editing by ValRS requires a posttransfer step.
Nascent-peptide-mediated ribosome stalling at a stop codon induces mRNA cleavage resulting in nonstop mRNA that is recognized by tmRNA
Authors: SUNOHARA TAKAFUMI , JOJIMA KAORU , YAMAMOTO YASUFUMI , INADA TOSHIFUMI , AIBA HIROJI
Published: February 2004

Recent studies have established that tmRNA-mediated protein tagging occurs at stop codons depending on the C-terminal amino acid sequence of the nascent polypeptide immediately adjacent to those codons. We investigate here how the trans-translation at a stop codon occurs by using model crp genes encoding variants of cAMP receptor protein (CRP). We demonstrate that a truncated crp mRNA is efficiently produced along with a normal transcript from the model gene where tmRNA-mediated protein tagging occurs. The truncated crp mRNA was not detected in the presence of tmRNA, indicating that its degradation was facilitated by tmRNA. The major 3′-ends of the truncated crp mRNA in cells unable to express tmRNA were mapped at and near the stop codon. When RNA derived from the model crp–crr fusion gene was analyzed, crr mRNA was detected as a downstream cleavage product along with the upstream crp mRNA. These results are compatible with the hypothesis that ribosome stalling caused by the tagging-provoking sequences leads to endonucleolytic cleavage of mRNA around the stop codon, resulting in nonstop mRNA. In addition, the data are consistent with the view that mRNA cleavage is the cause of trans-translation at stop codons. Neither the bacterial toxin RelE nor the known major endoribonucleases are required for this cleavage, indicating that either other endoribonuclease(s) or the ribosome itself would be responsible for the mRNA cleavage in response to ribosome stalling caused by the particular nascent peptides.
Secondary structure and function of the 5′-proximal region of the equine arteritis virus RNA genome
Authors: VAN DEN BORN ERWIN , GULTYAEV ALEXANDER P. , SNIJDER ERIC J.
Published: February 2004

Nidoviruses produce an extensive 3′-coterminal nested set of subgenomic mRNAs, which are used to express their structural proteins. In addition, arterivirus and coronavirus mRNAs contain a common 5′ leader sequence, derived from the genomic 5′ end. The joining of this leader sequence to different segments (mRNA bodies) from the genomic 3′-proximal region presumably involves a unique mechanism of discontinuous minus-strand RNA synthesis. Key elements in this process are the so-called transcription-regulating sequences (TRSs), which determine a base-pairing interaction between sense and antisense viral RNA that is essential for leader-to-body joining. To identify RNA structures in the 5′-proximal region of the equine arteritis virus genome that may be involved in subgenomic mRNA synthesis, a detailed secondary RNA structure model was established using bioinformatics, phylogenetic analysis, and RNA structure probing. According to this structure model, the leader TRS is located in the loop of a prominent hairpin (leader TRS hairpin; LTH). The importance of the LTH was supported by the results of a mutagenesis study using an EAV molecular clone. Besides evidence for a direct role of the LTH in subgenomic RNA synthesis, indications for a role of the LTH region in genome replication and/or translation were obtained. Similar LTH structures could be predicted for the 5′-proximal region of all arterivirus genomes and, interestingly, also for most coronaviruses. Thus, we postulate that the LTH is a key structural element in the discontinuous subgenomic RNA synthesis and is likely critical for leader TRS function.
Intracellular expression of engineered RNase P ribozymes effectively blocks gene expression and replication of human cytomegalovirus
Authors: KIM KIHOON , UMAMOTO SEAN , TRANG PHONG , HAI RONG , LIU FENYONG
Published: February 2004

A ribozyme (M1GS RNA) constructed from the catalytic RNA subunit of RNase P fromEscherichia coliwas used to target the overlapping region of two human cytomegalovirus (HCMV) mRNAs, which encode for the viral essential protease (PR) and capsid assembly proteins (AP), respectively. The results show a reduction of >80% in the expression levels of PR and AP and an inhibition of ~2000-fold of viral growth in cells that stably expressed the ribozyme. In comparison, <10% reduction in the expression of the targets and viral growth was found in cells that either did not express the ribozyme or produced a “disabled” ribozyme carrying mutations that abolished its catalytic activity. Examination of replication of the virus in the ribozyme-expressing cells indicates that packaging of the viral genomic DNA into capsids is blocked, and suggests that the antiviral effects are because the ribozyme specifically inhibits the AP and PR expression and, consequently, abolishes viral capsid formation and growth. Our results show that RNase P ribozymes are highly effective in blocking HCMV growth by targeting the PR and AP mRNAs and demonstrate the feasibility to use these ribozymes in gene therapy for antiviral applications.
miRNP:mRNA association in polyribosomes in a human neuronal cell line
Authors: NELSON PETER T. , HATZIGEORGIOU ARTEMIS G. , MOURELATOS ZISSIMOS
Published: February 2004

MicroRNAs (miRNAs) are small regulatory RNAs that control gene expression by base-pairing with their mRNA targets. miRNAs assemble into ribonucleoprotein complexes termed miRNPs. Animal miRNAs recognize their mRNA targets via partial antisense complementarity and repress mRNA translation at a step after translation initiation. How animal miRNAs recognize their mRNA targets and how they control their translation is unknown. Here we describe that in a human neuronal cell line, the miRNP proteins eIF2C2 (a member of the Argonaute family of proteins), Gemin3, and Gemin4 along with miRNAs cosediment with polyribosomes. Furthermore, we describe a physical association between a let-7b (miRNA)-containing miRNP and its putative human mRNA target in polyribosome-containing fractions. These findings suggest that miRNP proteins may play important roles in target mRNA recognition and translational repression.
Inhibition of 5′ to 3′ mRNA degradation under stress conditions in Saccharomyces cerevisiae: from GCN4 to MET16
Authors: BENARD LIONEL
Published: February 2004

After deadenylation, most cytoplasmic mRNAs are decapped and digested by 5′ to 3′ exonucleases in Saccharomyces cerevisiae. Capped and deadenylated mRNAs are degraded to a lesser extent by 3′ to 5′ exonucleases. We have used a method, based on the electroporation of in vitro synthetised mRNAs, to study the relative importance of these two exonucleolytic pathways under stress conditions. We show that derepression of GCN4 upon amino acid starvation specifically limits the 5′-to-3′-degradation pathway. Because adenosine 3′-5′ biphosphate (pAp), which is produced by Met16p, inhibits this degradation pathway to a comparable extent, we were prompted to analyse the role of Met16p in this phenomenon. We show that the inhibitory effects of amino acid limitation on 5′ to 3′ mRNA degradation are absent in a met16 mutant. We therefore conclude that the GCN4 dependence of MET16 expression is responsible for the decrease in 5′ to 3′ digestion under stress conditions and that cells use pAp as a signal to limit 5′ to 3′ RNA degradation under stress conditions. Because 3′ to 5′ mRNA degradation is unaffected, the relative importance of this pathway in the decay of certain RNAs may be increased under stress conditions.
The Noc proteins involved in ribosome synthesis and export contain divergent HEAT repeats
Authors: DLAKIĆ MENSUR , TOLLERVEY DAVID
Published: February 2004

The Noc1–4p proteins were previously reported to be involved in intranuclear and nucleocytoplasmic transport of pre-ribosomes. Using fold recognition and structural modeling, we show that Noc1–4p are largely comprised of α-helical repeats similar to HEAT repeats. Because other HEAT-repeat proteins play key roles in transport processes, this finding provides a plausible mechanistic explanation for the function of the Noc proteins.
Characterization of two distinct RNA domains that regulate translation of the Drosophila gypsy retroelement
Authors: RONFORT CORINNE , DE BREYNE SYLVAIN , SANDRIN VIRGINIE , DARLIX JEAN-LUC , OHLMANN THÉOPHILE
Published: February 2004

The genomic RNA of the gypsy retroelement from Drosophila melanogaster exhibits features similar to other retroviral RNAs because its 5′ untranslated (5′ UTR) region is unusually long (846 nucleotides) and potentially highly structured. Our initial aim was to search for an internal ribosome entry site (IRES) element in the 5′ UTR of the gypsy genomic RNA by using various monocistronic and bicistronic RNAs in the rabbit reticulocyte lysate (RRL) system and in cultured cells. Results reported here show that two functionally distinct and independent RNA domains control the production of gypsy encoded proteins. The first domain corresponds to the 5′ UTR of the env subgenomic RNA and exhibits features of an efficient IRES (IRESE) both in the reticulocyte lysate and in cells. The second RNA domain that encompasses the gypsy insulator can function as an IRES in the rabbit reticulocyte lysate but strongly represses translation in cultured cells. Taken together, these results suggest that expression of the gypsy encoded proteins from the genomic and subgenomic RNAs can be regulated at the level of translation.
Biostable aptamers with antagonistic properties to the neuropeptide nociceptin/orphanin FQ
Authors: FAULHAMMER DIRK , ESCHGFÄLLER BERND , STARK SANDRA , BURGSTALLER PETRA , ENGLBERGER WERNER , ERFURTH JEANNETTE , KLEINJUNG FRANK , RUPP JOHANNA , VULCU SEBASTIAN DAN , SCHRÖDER WERNER , VONHOFF STEFAN , NAWRATH HERMANN , GILLEN CLEMENS , KLUSSMANN SVEN
Published: February 2004

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, has been shown to play a prominent role in the regulation of several biological functions such as pain and stress. Here we describe the isolation and characterization of N/OFQ binding biostable RNA aptamers (Spiegelmers) using a mirror-image in vitro selection approach. Spiegelmers arel-enantiomeric oligonucleotide ligands that display high affinity and specificity to their targets and high resistance to enzymatic degradation compared tod-oligonucleotides. A representative Spiegelmer from the selections performed was size-minimized to two distinct sequences capable of high affinity binding to N/OFQ. The Spiegelmers were shown to antagonize binding of N/OFQ to the ORL1 receptor in a binding-competition assay. The calculated IC50values for the Spiegelmers NOX 2149 and NOX 2137a/b were 110 nM and 330 nM, respectively. The competitive antagonistic properties of these Spiegelmers were further demonstrated by their effective and specific inhibition of G-protein activation in two additional models. The Spiegelmers antagonized the N/OFQ-induced GTPγS incorporation into cell membranes of a CHO-K1 cell line expressing the human ORL1 receptor. In oocytes fromXenopus laevis,NOX 2149 showed an antagonistic effect to the N/OFQ-ORL 1 receptor system that was functionally coupled with G-protein-regulated inwardly rectifying K+channels.
Eukaryotic protein synthesis inhibitors identified by comparison of cytotoxicity profiles
Authors: CHAN JENNY , KHAN SHAKILA N. , HARVEY ISABELLE , MERRICK WILLIAM , PELLETIER JERRY
Published: February 2004

The National Cancer Institute (NCI) Human Tumor Cell Line Anti-Cancer Drug Screen has evaluated the cytotoxicity profiles of a large number of synthetic compounds, natural products, and plant extracts on 60 different cell lines. The data for each compound/extract can be assessed for similarity of cytotoxicity pattern, relative to a given test compound, using an algorithm called COMPARE. In applying a chemical biology approach to better understand the mechanism of eukaryotic protein synthesis, we used these resources to search for novel inhibitors of translation. The cytotoxicity profiles of 31 known protein synthesis inhibitors were used to identify compounds from the NCI database with similar activity profiles. Using this approach, two natural products, phyllanthoside and nagilactone C, were identified and characterized as novel protein synthesis inhibitors. Both compounds are specific for the eukaryotic translation apparatus, function in vivo and in vitro, and interfere with translation elongation. Our results demonstrate the feasibility of utilizing cytotoxicity profiles to identify new inhibitors of translation.
A guide to ions and RNA structure
Authors: DRAPER DAVID E.
Published: February 2004

RNA folding into stable tertiary structures is remarkably sensitive to the concentrations and types of cations present; an understanding of the physical basis of ion-RNA interactions is therefore a prerequisite for a quantitative accounting of RNA stability. This article summarizes the energetic factors that must be considered when ions interact with two different RNA environments. “Diffuse ions” accumulate near the RNA because of the RNA electrostatic field and remain largely hydrated. A “chelated” ion directly contacts a specific location on the RNA surface and is held in place by electrostatic forces. Energetic costs of ion chelation include displacement of some of the waters of hydration by the RNA surface and repulsion of diffuse ions. Methods are discussed for computing both the free energy of the set of diffuse ions associated with an RNA and the binding free energies of individual chelated ions. Such calculations quantitatively account for the effects of Mg2+ on RNA stability where experimental data are available. An important conclusion is that diffuse ions are a major factor in the stabilization of RNA tertiary structures.
Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing
Authors: MEISTER GUNTER , LANDTHALER MARKUS , DORSETT YAIR , TUSCHL THOMAS
Published: February 2004

A large number of miRNAs have recently been discovered in plants and animals. Development of reverse genetic approaches that act to inhibit microRNA function would facilitate the study of this new class of noncoding RNA. Here we show that 2′-O-methyl oligoribonucleotides, but not 2′-deoxyoligonucleotides specifically inactivate the RNAi activity associated with miRNA–protein complexes in human cell extracts as well as in cultured human cells.
Modifications and deletions of helices within the hairpin ribozyme–substrate complex: An active ribozyme lacking helix 1
Authors: PINARD ROBERT , LAMBERT DOMINIC , POTHIAWALA GULNAR , MAJOR FRANÇOIS , BURKE JOHN M.
Published: February 2004

Within the hairpin ribozyme, structural elements required for formation of the active tertiary structure are localized in two independently folding domains, each consisting of an internal loop flanked by helical elements. Here, we present results of a systematic examination of the relationship between the structure of the helical elements and the ability of the RNA to form the catalytically active tertiary structure. Deletions and mutational analyses indicate that helix 1 (H1) in domain A can be entirely eliminated, while segments of helices 2, 3, and 4 can also be deleted. From these results, we derive a new active minimal ribozyme that contains three helical elements, an internal loop, and a terminal loop. A three-dimensional model of this truncated ribozyme was generated using MC-SYM, and confirms that the catalytic core of the minimized construct can adopt a tertiary structure that is very similar to that of the nontruncated version. A new strategy is described to study the functional importance of various residues and chemical groups and to identify specific interdomain interactions. This approach uses two physically separated and truncated domains derived from the minimal motif.
Oral Antivirals for the Acute Treatment of Recurrent Herpes Labialis
Authors: Jensen Lori A , Hoehns James D , Squires Cindy L
Published: February 2004

OBJECTIVE

To evaluate the use and benefit of oral antivirals in the acute treatment of episodic, recurrent herpes labialis.

DATA SOURCES

A literature search was performed in MEDLINE (1966—August 2003) using acyclovir, famciclovir, valacyclovir, cold sores, herpes labialis, and HSV-1 as search terms.

DATA SYNTHESIS

We reviewed 5 placebo-controlled and 2 comparative studies evaluating oral antivirals for acute treatment of recurrent herpes labialis. No studies directly compared different antivirals. Studies discussing the efficacy of antivirals for chronic suppression of herpes simplex virus-1 infection were not included.

CONCLUSIONS

Treatment with oral antivirals decreases the duration of lesion episodes and pain by approximately one day; however, the antivirals do not abort lesions from developing. Clinical implications of these results appear relatively modest.
Pulmonary and Hepatic Toxicity Due to Nitrofurantoin and Fluconazole Treatment
Authors: Linnebur Sunny A , Parnes Bennett L
Published: February 2004

OBJECTIVE

To reemphasize potential risks associated with chronic nitrofurantoin use and to report a case of combined pulmonary and hepatic toxicity precipitated from acute use of fluconazole concomitantly with chronic nitrofurantoin.

CASE SUMMARY

A 73-year-old white man taking nitrofurantoin 50 mg/day for 5 years developed combined hepatic and pulmonary toxicity after taking fluconazole acutely for onychomycosis. Two months after starting fluconazole, the patient's hepatic enzymes showed elevation 5 times the upper limits of normal. In addition, the patient reported fatigue, dyspnea on exertion, pleuritic pain, burning trachea pain, and a cough. Chest X-rays showed bilateral pulmonary disease consistent with nitrofurantoin toxicity. Both drugs were determined to be the cause of the patient's pulmonary and hepatic toxicity, so they were discontinued. Pulmonary function tests measured after discontinuation were abnormal and also consistent with nitrofurantoin toxicity. The patient's hepatic and pulmonary toxicity resolved upon discontinuation of both drugs and use of inhaled corticosteroids.

DISCUSSION

Changes in hepatic enzyme measurement, pulmonary function measurements, and chest X-rays indicate that our patient developed hepatic and pulmonary toxicity due to his drug therapy. An objective causality assessment revealed that these adverse events were probably due to fluconazole given with nitrofurantoin. Either drug may have caused the hepatic toxicity. However, it is possible that pharmacokinetic changes induced by an interaction with fluconazole precipitated the nitrofurantoin-induced pulmonary toxicity.

CONCLUSIONS

Our patient developed pulmonary and hepatic toxicity after starting fluconazole in combination with chronic nitrofurantoin. A potential drug interaction of unknown mechanism may have been the cause of the toxicities.
Detoxification from High-Dose Zolpidem Using Diazepam
Authors: Rappa Leonard R , Larose-Pierre Margareth , Payne Deidre R , Eraikhuemen Nathaniel E , Lanes Douglas M , Kearson Margaretta L
Published: February 2004

OBJECTIVE

To report a rare case of physical and psychological addiction to an excessive dose of zolpidem and subsequent completed detoxification using diazepam.

CASE SUMMARY

A 46-year-old white man with a history of polysubstance abuse received a prescription for zolpidem 2 years prior to his hospital detoxification. During that time, he gradually escalated the total dosage to an amount of 400 mg/day in divided doses. Upon hospitalization, he was detoxified using a standard benzodiazepine 7-day diazepam tapering regimen.

DISCUSSION

Zolpidem is a nonbenzodiazepine medication approved for the short-term treatment of insomnia. Its mechanism is a selective benzodiazepine type 1 receptor agonist. The selectivity of the drug for the type 1 receptor may not be absolute and is inversely dose dependent. Compared with the benzodiazepines, zolpidem addiction is rare. However, at higher than recommended doses for extended periods of time, its addictive potential may be similar to that of the benzodiazepines.

CONCLUSIONS

Given the similarities in receptor binding and pharmacologic activities of zolpidem and the benzodiazepines, we chose to use a standard benzodiazepine detoxification protocol to treat zolpidem withdrawal. Confirmation of this has been evidenced by successful zolpidem detoxification using a standard 7-day benzodiazepine/diazepam taper regimen.
Factors that Influence Prescribing Decisions
Authors: Schumock Glen T , Walton Surrey M , Park Hayley Y , Nutescu Edith A , Blackburn Juan C , Finley Jamie M , Lewis Richard K
Published: February 2004

BACKGROUND

Strategies to control the quality and cost of medication use are largely dependent on the ability to alter selection of medications. Previous models of prescribing behavior have focused on physicians. In the hospital setting, clinical pharmacists and formulary committee members are also key players in drug therapy decision-making. Differences between physicians, formulary committee members, and clinical pharmacists have not been compared. Knowledge of these differences could have importance in predicting the effectiveness of strategies designed to influence drug use in this setting.

OBJECTIVE

To describe and compare the opinions of physicians, clinical pharmacists, and formulary committee members with respect to key factors that influence medication prescribing in community hospitals.

METHODS

Physicians, clinical pharmacists, and formulary committee members were solicited to participate. A trained interviewer administered a standardized questionnaire designed to elicit opinions of participants regarding the importance of factors thought to influence drug prescribing. Responses were described using descriptive statistics, and differences between the groups were determined by post hoc analysis.

RESULTS

A total of 150 individuals participated in the study. Safety, effectiveness, formulary status, and restrictions on prescribing were considered highly influential by all participants. Physicians rated the availability of drug samples and personal experience higher (more influential on prescribing) than clinical pharmacists and formulary committee members. Clinical pharmacists and formulary committee members rated the influence of recommendations by clinical pharmacists, prescribing guidelines, and cost or cost comparisons higher than physicians. Factors that were drug-related or that involved policy-related programs tended to be more influential than indirect factors.

CONCLUSIONS

Those who seek to implement programs to alter medication use should recognize and employ factors that are most influential in the decision-making process. Further, it may be important to consider differences that exist between key participants in the medication use process.
Lupus Anticoagulant (LA) Testing: Performance of Clinical Laboratories Assessed by a National Survey Using Lyophilized Affinity-purified Immunoglobulin with LA Activity
Authors: Tripodi Armando , Biasiolo Alessandra , Chantarangkul Veena , Pengo Vittorio
Published: February 2004

Background: Lupus anticoagulant (LA) screens are frequently ordered in the workup of thrombophilic patients or women with fetal loss. The sensitivity and specificity of LA detection vary depending on the choice of tests, cutoff values, and results interpretation. This variation is detrimental to patient management because persistent LA positivity in patients with a history of thrombosis is a requisite for long-term anticoagulation therapy. Numerous surveys have been performed to assess the state of the art for LA diagnosis. The control plasmas used in these surveys were from LA-positive or -negative patients or were normal plasmas with monoclonal antibodies against human β2-glycoprotein I (β2-GPI) added.

Methods: A large number of laboratories were asked to test a common set of lyophilized plasmas for LA, including three normal plasmas, to which increasing amounts of affinity-purified IgG from a patient positive for anti-β2-GPI had been added, and three LA-negative plasmas: one normal, one with a coagulation deficiency, and one with heparin.

Results: Overall, 69, 68, and 59 of 70 participants were able to detect LA in plasmas with high, intermediate, and low potency (sensitivity, 99%, 97%, and 84%). Conversely, 69, 50, and 53 of 70 were able to rule out LA in the normal, heparinized, and deficient plasma (specificity, 99%, 71%, and 76%).

Conclusions: Sensitivity for LA detection is satisfactory, whereas specificity could be improved. Surveys for LA detection should be carried out on a regular basis because they may help improve performance. Plasmas containing graded amounts of affinity-purified human anti-β2-GPI may be used as a convenient source of well-characterized naturally occurring LA material.
Diagnosis of Huntington Disease
Authors: Margolis Russell L , Ross Christopher A
Published: February 2004

Background: Huntington disease (HD) is a rare, progressive, and fatal autosomal dominant neurodegenerative disorder, typically of adult onset.

Methods: We reviewed the literature concerning the molecular diagnosis of HD.

Results: The discovery of the genetic etiology of HD, a trinucleotide expansion mutation on chromosome 4p, has led to the development of increasingly reliable and valid diagnostic tests that can be applied to symptomatic patients, individuals at risk for HD but currently asymptomatic, fetuses, and embryos. However, the unstable nature of the HD mutation, the lack of effective treatments for HD, the mid-adulthood age of disease onset, and the existence of disorders with the same clinical presentation but different etiology all complicates diagnostic testing.

Conclusion: Conscientious laboratory work, knowledgeable interpretation of genetic test results, and the availability of pre- and posttest counseling are essential components of HD diagnosis.
Endothelial Nitric Oxide Synthase Gene Polymorphisms and Risk of Coronary Artery Disease
Authors: Colombo Maria Giovanna , Paradossi Umberto , Andreassi Maria Grazia , Botto Nicoletta , Manfredi Samantha , Masetti Serena , Biagini Andrea , Clerico Aldo
Published: February 2004

Background: Endothelial nitric oxide synthase (eNOS) could be a candidate gene for coronary artery disease (CAD). This study investigated the relationship of the eNOS Glu298→Asp and T786→C polymorphisms with the presence and severity of CAD in the Italian population.

Methods: We enrolled 415 unrelated individuals who underwent coronary angiography. The severity of CAD was expressed by means of the Duke score. The eNOS Glu298→Asp and T786→C variants were analyzed by PCR.

Results: There was significant linkage disequilibrium between the two eNOS polymorphisms (P <0.0001). Both variants were significantly associated with the occurrence and severity of CAD (P = 0.01 and 0.004 for Glu298→Asp and T786→C, respectively). The risk of CAD was increased among individuals homozygous for the C allele of the T786→C polymorphism compared with individuals homozygous for the T allele (odds ratio = 2.5; P <0.01) and was independent of the other common risk factors (P = 0.04). Moreover, individuals with both the Asp/Asp genotype of the Glu298→Asp polymorphism and at least one C allele of the T786→C variant in the promoter region of the eNOS gene had an increased risk of CAD (odds ratio = 4.0; P <0.001) and a significantly higher mean Duke score (26.2 ± 2.9 vs 45.2 ± 3.7; P = 0.002) compared with individuals with the TT genotype and the Glu allele.

Conclusions: The present study provides evidence that the Glu298→Asp and T786→C polymorphisms of the eNOS gene are associated with the presence and severity of angiographically defined CAD in the Italian population and that those individuals carrying both eNOS variants simultaneously might have a higher risk of developing CAD.
Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry-based Detection of Microsatellite Instabilities in Coding DNA Sequences: A Novel Approach to Identify DNA-Mismatch Repair-deficient Cancer Cells
Authors: Bonk Thomas , Humeny Andreas , Gebert Johannes , Sutter Christian , Doeberitz Magnus von Knebel , Becker Cord-Michael
Published: February 2004

Background: Inherited defects in the DNA mismatch repair system lead to increased loss or gain of repeat units in microsatellites, commonly referred to as microsatellite instability (MSI). MSIs in coding regions of critical genes contribute to the pathogenesis of DNA-mismatch repair-deficient cancers, particularly those associated with the hereditary nonpolyposis colorectal cancer syndrome (HNPCC). MSI typing is therefore increasingly used to guide the molecular diagnosis of HNPCC.

Methods: We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to identify MSIs in mononucleotide repeats within the coding sequences of genes relevant to the pathogenesis of MSI+ neoplastic lesions. After a primer extension reaction of PCR products encompassing the microsatellites, the molecular masses of the extension products were determined by MALDI-TOF-MS.

Results: MSIs were detected by MALDI-TOF-MS in the GART, AC1, TGFBR2, MSH3, and MSH6 genes in neoplastic tissues and MSI+ colorectal cancer cell lines but not in MSI− control tissues. The analysis of peak-integral ratios in a single spectrum of the peaks representing insertions or deletions compared with the full-length microsatellites allowed relative quantification of MSIs. MALDI-TOF-MS-based genotyping results were confirmed by conventional DNA sequencing and electrophoresis.

Conclusions: Because of its reliability, short run times, and low costs, this semiquantitative procedure represents an effective alternative, in particular for diagnostic high-throughput typing of MSIs in neoplastic lesions.
Genotype-specific Influence on Nitric Oxide Synthase Gene Expression, Protein Concentrations, and Enzyme Activity in Cultured Human Endothelial Cells
Authors: Song Junghan , Yoon Yeomin , Park Kyoung Un , Park Junwan , Hong Young Joon , Hong Seung Ho , Kim Jin Q
Published: February 2004

Background: The results of studies on the association of ecNOS polymorphisms and vascular diseases are inconsistent. To explore the nature of this interaction in the absence of confounding factors, such as smoking, we measured ecNOS mRNA, protein, and enzyme activity in cultured human umbilical vein endothelial cells (HUVECs) with and without ecNOS polymorphisms.

Methods: We identified a T−786→C polymorphism in the promoter region, the intron 4 variable number of tandem repeats (VNTR), the E298A polymorphism in exon 7, and the G10-T polymorphism in intron 23 of the ecNOS gene in the DNA from 43 human umbilical cords. We measured ecNOS and GAPDH mRNA from the cultured HUVECs by reverse transcription-PCR and ecNOS protein and enzyme activity by Western blotting (as ratio to positive control band) and by determining the conversion of [3H]arginine to [3H]citrulline, respectively.

Results: The T−786→C polymorphism showed the same allelic distribution as the intron 4 VNTR. Mean (SD) ecNOS protein from the cultured HUVECs was significantly lower in the 4a/4b genotype [0.84 (1.23); n = 9] of the intron 4 VNTR than in the 4b/4b genotype [2.14 (2.26); n = 34; P = 0.0300]. The enzyme activity was also significantly lower in the 4a/4b genotype [0.84 (0.21) pmol · min−1 · mg protein−1; n = 9] than in the 4b/4b genotype [1.07 (0.31) pmol · min−1 · mg protein−1; n = 34; P = 0.0197].

Conclusions: ecNOS gene expression, protein concentrations, and enzyme activity are genotype-dependent in HUVECs. The intron 4 VNTR has a consistent influence that may be mediated by the T−786→C polymorphism in the promoter region.