bluuebunny/crossref_metadata_2025 huggingface dataset:
Ubiquitin ligase Rad18Sc localizes to the XY body and to other chromosomal regions that are unpaired and transcriptionally silenced during male meiotic prophase
Authors: van der Laan Roald , Uringa Evert-Jan , Wassenaar Evelyne , Hoogerbrugge Jos W. , Sleddens Esther , Odijk Hanny , Roest Henk P. , de Boer Peter , Hoeijmakers Jan H. J. , Grootegoed J. Anton , Baarends Willy M.
Published: September 2004

In replicative damage bypass (RDB) in yeast, the ubiquitinconjugating enzyme RAD6 interacts with the ubiquitin ligase RAD18. In the mouse, these enzymes are represented by two homologs of RAD6, HR6a and HR6b, and one homolog of RAD18, Rad18Sc. Expression of these genes and the encoded proteins is ubiquitous, but there is relatively high expression in the testis. We have studied the subcellular localization by immunostaining Rad18Sc and other RDB proteins in mouse primary spermatocytes passing through meiotic prophase in spermatogenesis. The highest Rad18Sc protein level is found at pachytene and diplotene, and the protein localizes mainly to the XY body, a subnuclear region that contains the transcriptionally inactivated X and Y chromosomes. In spermatocytes that carry translocations for chromosomes 1 and 13, Rad18Sc protein concentrates on translocation bivalents that are not fully synapsed. The partly synapsed bivalents are often localized in the vicinity of the XY body, and show a very low level of RNA polymerase II, indicating that the chromatin is in a silent configuration similar to transcriptional silencing of the XY body. Thus, Rad18Sc localizes to unsynapsed and silenced chromosome segments during the male meiotic prophase. All known functions of RAD18 in yeast are related to RDB. However, in contrast to Rad18Sc, expression of UBC13 and polη, known to be involved in subsequent steps of RDB, appears to be diminished in the XY body and regions containing the unpaired translocation bivalents. Taken together, these observations suggest that the observed subnuclear localization of Rad18Sc may involve a function outside the context of RDB. This function is probably related to a mechanism that signals the presence of unsynapsed chromosomal regions and subsequently leads to transcriptional silencing of these regions during male meiotic prophase.
Mechanism of Dronc activation in Drosophila cells
Authors: Muro Israel , Monser Kristin , Clem Rollie J.
Published: September 2004

Proteolytic processing is required for the activation of most caspases. However, recent reports have suggested that the activation of the mammalian initiator caspase caspase-9 occurs during dimerization rather than after processing. Previously, we reported that, in normal living Drosophila S2 cells, the initiator caspase Dronc is continuously processed to a 40 kDa form we called Pr1 and that, during apoptosis, a second processed form of 37 kDa is also observed, which we called Pr2. In this study, we determined that Dronc Pr1 is the result of Dronc autoprocessing at amino acid E352, whereas Pr2 results from Drice cleaving full-length Dronc at amino acid D135. By using purified recombinant proteins and expressing Dronc cleavage mutants in S2 cells, we determined that autoprocessing at E352 is crucial for Dronc caspase activity, whereas Drice cleavage at D135 has little effect on Dronc activity. Suppression of the oligomerizing factor Dark by RNA interference revealed that Dark is required for Dronc autoprocessing at E352, whereas RNA interference of the effector caspase Drice revealed that Drice is also required for apoptosis in S2 cells. These results provide the first details of the mechanisms regulating initiator caspase activation in an invertebrate organism.
Smooth muscle archvillin: a novel regulator of signaling and contractility in vascular smooth muscle
Authors: Gangopadhyay Samudra S. , Takizawa Norio , Gallant Cynthia , Barber Amy L. , Je Hyun-Dong , Smith Tara C. , Luna Elizabeth J. , Morgan Kathleen G.
Published: September 2004

The mechanisms by which protein kinase C (PKC) and extracellular-signal-regulated kinases (ERK1/2) govern smooth-muscle contractility remain unclear. Calponin (CaP), an actin-binding protein and PKC substrate, mediates signaling through ERK1/2. We report here that CaP sequences containing the CaP homology (CH) domain bind to the C-terminal 251 amino acids of smooth-muscle archvillin (SmAV), a new splice variant of supervillin, which is a known actin- and myosin-II-binding protein. The CaP-SmAV interaction is demonstrated by reciprocal yeast two-hybrid and blot-overlay assays and by colocalization in COS-7 cells. In differentiated smooth muscle, endogenous SmAV and CaP co-fractionate and co-translocate to the cell cortex after stimulation by agonist. Antisense knockdown of SmAV in tissue inhibits both the activation of ERK1/2 and contractions stimulated by either agonist or PKC activation. This ERK1/2 signaling and contractile defect is similar to that observed in CaP knockdown experiments. In A7r5 smooth-muscle cells, PKC activation by phorbol esters induces the reorganization of endogenous, membrane-localized SmAV and microfilament-associated CaP into podosome-like structures that also contain F-actin, nonmuscle myosin IIB and ERK1/2. These results indicate that SmAV contributes to the regulation of contractility through a CaP-mediated signaling pathway, involving PKC activation and phosphorylation of ERK1/2.
Early signaling events involved in the entry of Rickettsia conorii into mammalian cells
Authors: Martinez Juan J. , Cossart Pascale
Published: September 2004

Rickettsia conorii, the causative agent of Mediterranean spotted fever, is able to attach to and invade a variety of cell types both in vitro and in vivo. Although previous studies show that entry of R. conorii into non-phagocytic cells relies on actin polymerization, little else is known about the molecular details governing Rickettsia-host cell interactions and actin rearrangements. We determined that R. conorii recruits the Arp2/3 complex to the site of entry foci and that expression of an Arp 2/3 binding derivative of the WASP-family member, Scar, inhibited bacterial entry into Vero cells, establishing that Arp2/3 is an active component of this process. Using transient transfection with plasmids expressing dominant negative versions of small GTPases, we showed that Cdc42, but not Rac1 is involved in R. conorii invasion into Vero cells. Using pharmacological approaches, we show that this invasion is dependent on phosphoinositide (PI) 3-kinase and on protein tyrosine kinase (PTK) activities, in particular Src-family kinases. C-Src and its downstream target, p80/85 cortactin, colocalize at entry sites early in the infection process. R. conorii internalization correlated with the tyrosine phosphorylation of several other host proteins, including focal adhesion kinase (FAK), within minutes of R. conorii infection. Our results reveal that R. conorii entry into nonphagocytic cells is dependent on the Arp2/3 complex and that the interplay of pathways involving Cdc42, PI 3-kinase, c-Src, cortactin and tyrosine-phosphorylated proteins regulates Arp2/3 activation leading to the localized actin rearrangements observed during bacterial entry. This is the first report that documents the mechanism of entry of a rickettsial species into mammalian cells.
Cortactin associates with N-cadherin adhesions and mediates intercellular adhesion strengthening in fibroblasts
Authors: El Sayegh Tarek Y. , Arora Pamela D. , Laschinger Carol A. , Lee Wilson , Morrison Charlotte , Overall Christopher M. , Kapus Andras , McCulloch Christopher A. G.
Published: September 2004

The regulation of N-cadherin-mediated intercellular adhesion strength in fibroblasts is poorly characterized; this is due, in part, to a lack of available quantitative models. We used a recombinant N-cadherin chimeric protein and a Rat 2 fibroblast, donor-acceptor cell model, to study the importance of cortical actin filaments and cortactin in the strengthening of N-cadherin adhesions. In wash-off assays, cytochalasin D (1 μM) reduced intercellular adhesion by threefold, confirming the importance of cortical actin filaments in strengthening of N-cadherin-mediated adhesions. Cortactin, an actin filament binding protein, spatially colocalized to, and directly associated with, nascent N-cadherin adhesion complexes. Transfection of Rat-2 cells with cortactin-specific, RNAi oligonucleotides reduced cortactin protein by 85% and intercellular adhesion by twofold compared with controls (P<0.005) using the donor-acceptor model. Cells with reduced cortactin exhibited threefold less N-cadherin-mediated intercellular adhesion strength compared with controls in wash-off assays using N-cadherin-coated beads. Immunoprecipitation and immunoblotting showed that N-cadherin-associated cortactin was phosphorylated on tyrosine residue 421 after intercellular adhesion. While tyrosine phosphorylation of cortactin was not required for recruitment to N-cadherin adhesions it was necessary for cadherin-mediated intercellular adhesion strength. Thus cortactin, and phosphorylation of its tyrosine residues, are important for N-cadherin-mediated intercellular adhesion strength.
New clinical tests of unilateral vestibular dysfunction
Authors: Halmagyi G. Michael
Published: September 2004

Vertigo is caused by unilateral vestibular dysfunction. Unilateral vestibular dysfunction represents either vestibular overactivity as benign positional vertigo or underactivity as in labyrinthitis or both, at different times, as in a Ménière's attack. Unilateral dysfunction could also be central rather than peripheral, such as in a lateral medullary syndrome. Unilateral vestibular dysfunction could affect any of the five different sensory areas in the labyrinth, the three semicircular canals and the two otoliths, or their brainstem connections. For rigorous diagnosis of the cause of vertigo, ideally one would have robust, reproducible, quantitative vestibular function tests sensitive to dysfunction of each of the five sensory regions in each ear. In working towards this ideal we have, over the last 15 years, developed three new vestibular function tests: (1) impulsive tests of individual semicircular canal function, (2) evoked potential tests of saccular function and (3) subjective visual horizontal tests of utricular function. The physiological rationale of these three tests is reviewed as well as the interpretation of their results in various diseases.
Topical vancomycin for chronic suppurative otitis media with methicillin-resistant Staphylococcus aureus otorrhoea
Authors: Jang Chul Ho , Song Chang-Hun , Wang Pa-Chun
Published: September 2004

There has been a steady increase in the number of cases of methicillin-resistant Staphylococcus aureus (MRSA) otorrhoea; this is a growing concern. The purpose of this study was to evaluate the efficacy of topical vancomycin treatment in patients with MRSA otorrhoea. Fifty-five patients with MRSA otorrhoea were prospectively enrolled into the study. Thirty-five patients were treated with vancomycin eardrops as outpatients. The concentration of the locally prepared vancomycin solution was 25 mg/ml. The dose of vancomycin was two drops three times daily for 10 days. As a control group, 20 patients were treated with gentamicin 0.3% solution. Data were analysed by the Mann-Whitney U test to compare the efficacy of vancomycin eardrops and gentamicin eardrops. In the vancomycin group, the otorrhoea was significantly reduced in 33 ears (94%); in the gentamicin group, in four ears (20 per cent); this reduction was statistically significant (P < 0.03). The use of topical vancomycin treatment was effective for patients with MRSA otorrhoea refractory to conventional antibiotic treatment.
Detection of laryngeal carcinoma and epithelial hyperplastic laryngeal lesions via a rapid-access dysphonia clinic
Authors: Moore P.L.A. , Kim D. , Selby G. , Proops D.W.
Published: September 2004

Recent government initiatives in the UK have focused on streamlining oncology services by reducing waiting times between urgent referral, assessment and treatment of patients with possible cancer. The performance of the Quick Early Diagnosis Dysphonia Clinic of the Queen Elizabeth Hospital, Birmingham, between May 1997 and April 2001 was reviewed. Of 721 patients reviewed, 123 (17 per cent) had clinically suspicious laryngeal lesions. Thirteen cases of epithelial hyperplastic laryngeal lesions and 27 laryngeal malignancies were diagnosed. There was no statistical link between early cancer detection and assessment within two weeks of referral. However, rapid-access clinics for dysphonia serve an important role in the reassurance and multidisciplinary management of patients with persistent hoarseness. Greater financial commitments are necessary to achieve compliance with objectives for a maximum two-week wait for patients with suspected laryngeal malignancy.
Laryngeal cleft type I: a novel method of repair using Bioplastique™
Authors: Ahluwalia S. , Pothier D. , Philpott J. , Sengupta P. , Frosh A.
Published: September 2004

The authors report a novel treatment of a case of type I laryngeal cleft diagnosed in an adult. They describe a technique of endoscopic obliteration of the posterior commissure defect. Initially, a test implant of starch and adrenaline was used, followed by permanent staged injections of Bioplastique™ to the posterior commissure. This rare congenital anomaly usually presents in childhood but late presentation should be considered when adults present with lifelong dysphonia especially when associated with gastro-oesophageal reflux disease.
Exceptionally elevated creatine kinase levels in a laryngectomized patient: hypothyroid myopathy
Authors: Cinamon Udi
Published: September 2004

We present a laryngectomized patient with unspecific complaints of fatigue whose laboratory findings were out of proportion with the clinical presentation. The enormously high blood levels of creatine kinase (CPK) (8000 IU/l, normal range 30–190 IU/l) and thyroid-stimulating hormone (100 mU/l, normal range 0.5–4.5 mU/l) led to diagnosis and treatment of and recovery from hypothyroid myopathy. Hypothyroidism reduces the ability of the muscle to maintain its adequate energetic economy, via several suggested mechanisms. This may lead to injury (myopathy) that allows enzymes such as CPK to leak out of cells and causes elevation of their serum levels. To our knowledge, this is the first reported case of a patient previously treated for head and neck cancer who developed hypothyroid myopathy, presenting with exceptionally elevated CPK levels. This is noteworthy, since hypothyroidism may be easily avoided by a comprehensive follow-up of patients treated for head and neck cancer.
WKY/Ztm-ter: a new rat inbred strain on the WKY/Ztm genetic background with congenital teratomas
Authors: Länger B. , Dorsch M. , Gärtner K. , Wedekind D. , Kamino K. , Hedrich H. J.
Published: September 2004

We describe a new rat model for teratomas (WKY/Ztm- ter) which arose through a spontaneous mutation in the inbred WKY/Ztm rat strain. When the tumours of the gonads became clinically apparent, affected males were 14 to 224 days of age, whereas the females only developed tumours between days 21 and 63. Tumour incidence is not gender-dependent. However, almost all females develop bilateral tumours, while 50% of the males show unilateral tumours. Histologically, all examined tumours ( n = 65) represent partially undifferentiated teratocarcinomas.
Polymorphic microsatellite markers in the outbred CFW and ICR stocks for the generation of speed congenic mice on C57BL/6 background
Authors: Teppner I. , Aigner B. , Schreiner E. , Müller M. , Windisch M.
Published: September 2004

Reliable definition of the phenotype of particular alleles is carried out in the genetic background of inbred strains. Appearance of mutations in outbred mice therefore requires the generation of congenic mice. The aim of this study was the establishment of a list of polymorphic microsatellite markers which can be used in a polymerase chain reaction (PCR)-based marker-assisted selection protocol (MASP) to allow the use of the two common outbred stocks, CFW and ICR, as donor animals for the fast generation of congenic C57BL/6 mice. The selection of informative microsatellite markers was carried out to provide a simple evaluation of the PCR products by conventional agarose gel electrophoresis. Outbred mice from three suppliers were examined. In total, 153 microsatellite loci were analysed. Here we present 76 and 70 microsatellite markers polymorphic for the outbred ICR and CFW stocks compared to C57BL/6. At least three microsatellite loci per chromosome were chosen as informative markers for the autosomal genome, giving rise to a maximum marker distance of 58 cM. Thus, additional individual markers have to be selected for the respective outbred mouse which is chosen as a donor animal.
A natural asymptomatic herpes B virus infection in a colony of laboratory brown capuchin monkeys (Cebus apella)
Authors: Coulibaly C. , Hack R. , Seidl J. , Chudy M. , Itter G. , Plesker R.
Published: September 2004

Herpes B virus (BV) infection of macaques persists in the natural host, but is mainly asymptomatic. However, BV can cause fatal disease in humans and in several non-macaque species such as capuchin monkeys ( Cebus apella). The BV infection described here in a colony of capuchin monkeys was persistent but asymptomatic. Initially the infection was detected serologically in five out of seven animals. However, using polymerase chain reaction (PCR) developed specifically for BV, we found the virus in all seven clinically healthy animals. It is probable that the infection was transferred from BV-infected macaques housed in different cages but in the same room for several years. We have no evidence to indicate that similar asymptomatic infections may occur in other New World species but the possibility should not be discounted. We recommend that the housing of capuchin monkeys in close proximity to macaques should be avoided and that greater caution should be used when handling capuchin monkeys and possibly other New World species that have been in contact with macaques. All may act as a source of BV infection in humans, hence routine, repeated testing of all primates is essential.
A model for acute iron overload in sea bass (Dicentrarchus labrax L.)
Authors: Rodrigues P. N. S. , Pereira F. A.
Published: September 2004

Evaluation of several parameters involved in iron metabolism was carried out after intraperitoneal (i.p.) injection with iron dextran (IDx) in sea bass ( Dicentrarchus labrax L.). After treatment, a rapid mobilization of IDx from the peritoneal cavity to other organs was observed. This was followed by a modification of normal peripheral blood iron parameters. Total iron (TI) and transferrin saturation (TS) rose rapidly, to 4.14 μg/ml and 83.7%, respectively, on day 3. In contrast, unsaturated iron binding capacity (UIBC) dropped from 3.19 μg/ml (at day 0) to 0.90 μg/ml on day 3. Tissue iron content was determined by atomic absorption spectometry (AAS). Three days post-IDx injection, values of iron concentration in liver, spleen and head kidney were significantly higher than control values (15, 6 and 9-fold increase, respectively). Samples of liver, spleen and head kidney were processed for routine histology, and the Perl's method was used for iron staining. Histological sections of the IDx-treated animals showed iron deposition in all tissues studied. In the liver, the iron was evenly distributed over the whole organ, being present in the hepatocytes. In the head kidney and spleen, the iron deposition was mainly observed in the melanomacrophage centres (MMCs). The present study characterizes several parameters involved in iron metabolism, and develops a fish model, of iron overload, which can be used in further studies of iron toxicity and iron-induced susceptibility to bacterial infections.
Novel restraint system for neuroendocrine studies of socially living common marmoset monkeys
Authors: Schultz-Darken N. J. , Pape R. M. , Tannenbaum P. L. , Saltzman W. , Abbott D. H.
Published: September 2004

We describe a novel soft jacket and sling-harness restraint that permits species-typical postures for small-bodied primates, such as the common marmoset ( Callithrix jacchus), during long-term (>6 h), continuous restraint. The restraint system is straightforward to use and manipulate, it is easily repaired, and the materials used are readily available. The soft jacket allows for increased versatility and longevity, and the sling-harness provides for greater movement and much longer duration of continuous restraint (up to 3 days) compared to a previously described, more conventional chair restraint for small-bodied primates. The new restraint system prevents the normal diurnal decrease in plasma cortisol levels across the daylight hours; however, it does not disrupt ovulatory cycles. Unlike the previously available techniques, therefore, this new restraint system is applicable to many neurobiological and neuroendocrine studies involving small-bodied, non-human primates and is especially suited to investigations requiring the maintenance of relationships within social groups.
The use of sodium lamps to brightly illuminate mouse houses during their dark phases
Authors: McLennan I. S. , Taylor-Jeffs J.
Published: September 2004

The human and murine diurnal rhythms are out of phase. Consequently in conventionally-lit mouse houses the mice's deep sleep is often disrupted, the daily welfare monitoring of the mice is limited by their inactivity, and scientific data obtained from the mice model the sleeping rather than awake human. Sodium light is bichromatic, with both wavelengths being in the human visual field but at the margin of murine vision. We report here that sodium lamps can be used to light mouse houses to a level that is comfortable for humans, but still sufficiently dull to permit nocturnal behaviour in mice. The response of mice to sodium light was initially monitored by recording the locomotory activity of BALB/c mice. The movement of mice in their cages greatly increased at the start of the nocturnal phase. Alterations in the white light cycle caused an acute change in the onset of nocturnal behaviour. In contrast, sodium light did not suppress the onset of nocturnal locomotory behaviour, even though the lighting was sufficiently bright for humans to read without light adaptation. The sodium lighting was then used to observe the nocturnal behaviour of over 150 mice of various strains, for over 1.5 years. Mice were invariably awake and alert during the nocturnal/sodium light phase. All exhibited high locomotory activity, except for nursing mothers. Some tasks, such as cage cleaning and minor surgery, were more easily done under white than sodium lighting. We therefore adjusted the timing of the light cycles to provide white light in the morning and sodium light (nocturnal phase) in the afternoon. This provided for easy operation of the mouse house, while yielding both animal welfare and scientific advantages.
Thymectomy should be the first choice in the protection of diabetes-prone BB rats for breeding purposes
Authors: Visser Jeroen , Klatter Flip , Hillebrands Jan-Luuk , Jansen Ar , Vijfschaft Lucas , Rozing Jan
Published: September 2004

Diabetes-prone (DP)-BB rats spontaneously develop diabetes and are widely used as an animal model for the study of type 1 diabetes. Since DP-BB rats develop diabetes before or at the time of breeding, such rats used for breeding need to be protected against diabetes development by the transfer of regulatory T cells obtained from diabetes-resistant (DR)-BB rats, by insulin treatment or by thymectomy. Thymectomy of juveniles is not commonly used to protect DP-BB rats, and we investigated whether breeding with thymectomized DP-BB rats was a realistic alternative to the two other methods. No differences in pregnancy rates, numbers of pups per litter or growth rates of pups were found. Moreover, no differences were found in diabetes development in the offspring. Protection of juvenile DP-BB rats by thymectomy is comparable to the other established procedures, is simple and safe, and the rats recover well from the procedure. Breeding with thymectomized animals will reduce the number of animals needed, and it improves the well-being of the animals because it reduces the negative side effects associated with the other procedures such as episodes of hypo and hyperglycaemia. Therefore, although thymectomy is an invasive procedure, we would like to recommend weanling thymectomy as the first choice for the protection of DP-BB rats for breeding purposes.
Chronic implantation of transit-time flow probes on the ascending aorta of rodents
Authors: Smith Lane , Tiba M. Hakam , Goldberg Mary Ellen , Barbee R. Wayne
Published: September 2004

This study describes the implantation of transit-time flow probes on the ascending aorta of rats while minimizing the risk of postoperative complications. Special emphasis is placed on our new method of rat intubation as well as the production of materials necessary for the implantation procedure such as endotracheal tubes and heparin bonded vessel catheters. The effects of these devices on the response to acute hypoxia were studied in rats following a 5–7 day recovery from the implantation procedure. Systemic and microvascular measurements were made on instrumented rats ( n = 5) and non-instrumented controls ( n = 3) that were ventilated with 21%, 15%, 10%, 8% and 5% oxygen. Arterial pressure, PO2, lactate, and base deficit were not different between the implanted and control animals at any inspired oxygen concentration. Microvascular flow in the primary arterioles of the spinotrapezius muscle was also similar between the two groups at all inspired oxygen concentrations. We conclude that this novel methodology facilitates the measurement of whole body oxygen delivery in resting and haemodynamically-stressed rats.
The organization and evolution of the Dipteran and Hymenopteran Down syndrome cell adhesion molecule (Dscam) genes
Authors: GRAVELEY BRENTON R. , KAUR AMARDEEP , GUNNING DORIAN , ZIPURSKY S. LAWRENCE , ROWEN LEE , CLEMENS JAMES C.
Published: September 2004

The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor and can generate 38,016 different isoforms via the alternative splicing of 95 variable exons. Dscam contains 10 immunoglobulin (Ig), six Fibronectin type III, a transmembrane (TM), and cytoplasmic domains. The different Dscam isoforms vary in the amino acid sequence of three of the Ig domains and the TM domain. Here, we have compared the organization of the Dscam gene from three members of the Drosophila subgenus (D. melanogaster, D. pseudoobscura, and D. virilis), the mosquito Anopheles gambiae, and the honeybee Apis mellifera. Each of these organisms contains numerous alternative exons and can potentially synthesize tens of thousands of isoforms. Interestingly, most of the alternative exons in one species are more similar to one another than to the corresponding alternative exons in the other species. These observations provide strong evidence that many of the alternative exons have arisen by reiterative exon duplication and deletion events. In addition, these findings suggest that the expression of a large Dscam repertoire is more important for the development and function of the insect nervous system than the actual sequence of each isoform.
Complex signals in the genomic 3′ nontranslated region of bovine viral diarrhea virus coordinate translation and replication of the viral RNA
Authors: ISKEN OLAF , GRASSMANN CLAUS W. , YU HAIYING , BEHRENS SVEN-ERIK
Published: September 2004

The genomes of positive-strand RNA viruses strongly resemble cellular mRNAs. However, besides operating as a messenger to generate the virus-encoded proteins, the viral RNA serves also as a template during replication. A central issue of the viral life cycle, the coordination of protein and RNA synthesis, is yet poorly understood. Examining bovine viral diarrhea virus (BVDV), we report here on the role of the variable 3′V portion of the viral 3′ nontranslated region (3′NTR). Genetic studies and structure probing revealed that 3′V represents a complex RNA motif that is composed of synergistically acting sequence and structure elements. Correct formation of the 3′V motif was shown to be an important determinant of the viral RNA replication process. Most interestingly, we found that a proper conformation of 3′V is required for accurate termination of translation at the stop-codon of the viral open reading frame and that efficient termination of translation is essential for efficient replication of the viral RNA. Within the viral 3′NTR, the complex 3′V motif constitutes also the binding site of recently characterized cellular host factors, the so-called NFAR proteins. Considering that the NFAR proteins associate also with the 5′NTR of the BVDV genome, we propose a model where the viral 3′NTR has a bipartite functional organization: The conserved 3′ portion (3′C) is part of the nascent replication complex; the variable 5′ portion (3′V) is involved in the coordination of the viral translation and replication. Our data suggest the accuracy of translation termination as a sophisticated device determining viral adaptation to the host.
Nematode m7GpppG and m32,2,7GpppG decapping: Activities in Ascaris embryos and characterization of C. elegans scavenger DcpS
Authors: COHEN LEAH S. , MIKHLI CLAUDETTE , FRIEDMAN CASSANDRA , JANKOWSKA-ANYSZKA MARZENA , STEPINSKI JANUSZ , DARZYNKIEWICZ EDWARD , DAVIS RICHARD E.
Published: September 2004

A spliced leader contributes the mature 5′ends of many mRNAs in trans-splicing organisms. Trans-spliced metazoan mRNAs acquire an m32,2,7GpppN cap from the added spliced leader exon. The presence of these caps, along with the typical m7GpppN cap on non-trans-spliced mRNAs, requires that cellular mRNA cap-binding proteins and mRNA metabolism deal with different cap structures. We have developed and used an in vitro system to examine mRNA degradation and decapping activities in nematode embryo extracts. The predominant pathway of mRNA decay is a 3′ to 5′ pathway with exoribonuclease degradation of the RNA followed by hydrolysis of resulting mRNA cap by a scavenger (DcpS-like) decapping activity. Direct decapping of mRNA by a Dcp1/Dcp2-like activity does occur, but is ~15-fold less active than the 3′ to 5′ pathway. The DcpS-like activity in nematode embryo extracts hydrolyzes both m7GpppG and m32,2,7GpppG dinucleoside triphosphates. The Dcp1/Dcp2-like activity in extracts also hydrolyzes these two cap structures at the 5′ ends of RNAs. Interestingly, recombinant nematode DcpS differs from its human ortholog in its substrate length requirement and in its capacity to hydrolyze m32,2,7GpppG.
Efficient stimulation of site-specific ribosome frameshifting by antisense oligonucleotides
Authors: HOWARD MICHAEL T. , GESTELAND RAYMOND F. , ATKINS JOHN F.
Published: September 2004

Evidence is presented that morpholino, 2′-O-methyl, phosphorothioate, and RNA antisense oligonucleotides can direct site-specific -1 translational frameshifting when annealed to mRNA downstream from sequences where the P- and A-site tRNAs are both capable of repairing with -1 frame codons. The efficiency of ribosomes shifting into the new frame can be as high as 40%, determined by the sequence of the frameshift site, as well as the location, sequence composition, and modification of the antisense oligonucleotide. These results demonstrate that a perfect duplex formed by complementary oligonucleotides is sufficient to induce high level -1 frameshifting. The implications for the mechanism of action of natural programmed translational frameshift stimulators are discussed.
Evidence for auto-inhibition by the N terminus of hADAR2 and activation by dsRNA binding
Authors: MACBETH MARK R. , LINGAM ARUNTH T. , BASS BRENDA L.
Published: September 2004

Adenosine deaminases that act on RNA (ADARs) catalyze adenosine to inosine conversion in RNA that is largely double stranded. Human ADAR2 (hADAR2) contains two double-stranded RNA binding motifs (dsRBMs), separated by a 90-amino acid linker, and these are followed by the C-terminal catalytic domain. We assayed enzymatic activity of N-terminal deletion constructs of hADAR2 to determine the role of the dsRBMs and the intervening linker peptide. We found that a truncated protein consisting of one dsRBM and the deaminase domain was capable of deaminating a short 15-bp substrate. In contrast, full-length hADAR2 was inactive on this short substrate. In addition, we observed that the N terminus, which was deleted from the truncated protein, inhibits editing activity when added in trans. We propose that the N-terminal domain of hADAR2 contains sequences that cause auto-inhibition of the enzyme. Our results suggest activation requires binding to an RNA substrate long enough to accommodate interactions with both dsRBMs.
Evaluation of Bleeding Complications Associated with Glycoprotein IIb/IIIa Inhibitors
Authors: Brouse Sara D , Wiesehan Vanessa G
Published: September 2004

BACKGROUND

Platelet glycoprotein IIb/IIIa inhibitors are used with aspirin and heparin to decrease rates of death, myocardial infarction, and urgent revascularization in patients with acute coronary syndromes.

OBJECTIVE

To determine whether bleeding event rates differed among 3 glycoprotein IIb/IIIa inhibitors prescribed in a high-risk, elderly veteran population and identify risk factors for bleeding.

METHODS

A retrospective chart analysis of patients who received abciximab, eptifibatide, or tirofiban was conducted. χ2 Analysis evaluated the incidence of bleeding complications, and stepwise regression analysis was utilized to identify bleeding risk factors. Parameters evaluated as possible risk factors for bleeding included age, renal function, weight, heparin and glycoprotein IIb/IIIa inhibitor dosing and infusion duration, concomitant antiplatelet and/or anticoagulant medications or disease states, and baseline hemoglobin, hematocrit, or platelet counts.

RESULTS

Of 348 patients whose charts were reviewed, 79 patients were included. There were 37.9% who received abciximab (n = 30), 30.3% who received eptifibatide (n = 24), and 31.6% who received tirofiban (n = 25). Twenty-one bleeding events not related to coronary artery bypass grafting occurred: only 1 major bleed (tirofiban) and 20 minor bleeds. Bleeding rates were not significantly different between groups: abciximab 30% (n = 9), eptifibatide 21% (n = 5), and tirofiban 28% (n = 7). Significant risk factors for bleeding included patient weight, infusion duration, and baseline platelet count (p = 0.024). Patients who bled received significantly more transfusions of packed red blood cells and platelets than patients with no bleeding (p = 0.047).

CONCLUSIONS

Although bleeding complications did not differ significantly between the 3 drugs, several risk factors for bleeding events were identified. A considerable rate of bleeding events occurred in this high-risk patient population.
Hypotension Due to Interaction Between Lisinopril and Tizanidine
Authors: Kao Chuen-Der , Chang Jiun-Bin , Chen Jen-Tse , Wu Zin-An , Shan Din-E , Liao Kwong-Kum
Published: September 2004

OBJECTIVE

To report a case in which significant hypotension occurred after initiation of tizanidine in a patient using the antihypertensive agent lisinopril.

CASE SUMMARY

A 48-year-old woman was admitted due to cerebral hemorrhage at the midbrain and pons, with extension to the fourth ventricle. Consciousness disturbance (Glasgow coma scale 4) with a decerebrate posture improved 5 days after stroke onset. As the BP was fairly high, antihypertensive agents, including lisinopril, were initiated. Three weeks later, the decerebrate rigidity and high BP remained, and tizanidine was initiated to see whether the decrease in muscle tone could facilitate hypertension control and motor recovery. However, the BP dropped dramatically within 2 hours after the first dose of tizanidine. The tizanidine and all of the antihypertensive medications were withdrawn. Tizanidine was used again after her BP had stabilized, but did not produce similar problems.

DISCUSSION

A similar event was reported in 2000. The reaction in our patient appeared after tizanidine initiation and improved after both lisinopril and tizanidine were discontinued. According to the Naranjo probability scale, this was classified as a possible drug interaction. This kind of reaction is seldom mentioned as occurring during co-administration with tizanidine. With its characteristics, tizanidine has the potential to compromise hemodynamic stability during concomitant angiotensin-converting enzyme inhibitor use.

CONCLUSIONS

Based upon the literature review, the hypotension in this patient was possibly due to the interaction between tizanidine and lisinopril.
Recurrent Acute Hepatitis Associated with Use of Cetirizine
Authors: Pompili Maurizio , Basso Maria , Grieco Antonio , Vecchio Fabio M , Gasbarrini Giovanni , Gian Ludovico Rapaccini
Published: September 2004

OBJECTIVE

To describe a case of recurrent acute hepatitis related to the use of cetirizine, a selective histamine1-receptor antagonist approved for the treatment of common allergic diseases.

CASE SUMMARY

A 26-year-old man was hospitalized with a week-long history of weakness, nausea, anorexia, and hyperchromic urine, which had developed after 6 days of therapy with oral cetirizine 10 mg/day for allergic rhinitis. Admission laboratory testing revealed evidence of acute hepatitis and seropositivity for liver–kidney microsome antibodies. Liver biopsy findings of diffuse portal tract and lobular inflammation with a prominent eosinophilic infiltrate were consistent with drug-related hepatitis. The patient was discharged after one week of treatment with tocopherol and glutathione. Three months after discharge, transaminase levels were normal. At 6 months, seropositivity for liver–kidney microsome antibodies was still present, but considerably less intense. The patient had suffered 2 previous episodes of “acute hepatitis of unknown origin,” and both had occurred after cetirizine use.

DISCUSSION

Use of the Naranjo probability scale indicated cetirizine as the probable cause of acute hepatitis, and the positivity for liver–kidney microsome antibodies is suggestive of an autoimmune mechanism for liver damage. As of September 13, 2004, ours is the fourth reported case of acute hepatitis associated with cetirizine and the second in which liver–kidney microsome antibodies have been documented.

CONCLUSIONS

Although cetirizine is considered to have low potential for severe hepatic toxicity, the possibility that it can provoke autoimmune-mediated hepatotoxicity should be considered.
Combating the Obesity Epidemic: Community Pharmacists Counseling on Obesity Management
Authors: Dastani Homa B , Brown Carolyn M , O'Donnell Dannielle C
Published: September 2004

BACKGROUND

Obesity is reaching an epidemic proportion in the US. Nearly two-thirds (64.7%) of US adults are overweight or obese. Given the myriad of issues related to the management of obesity, community pharmacists can facilitate weight loss among their patients.

OBJECTIVE

To identify factors that influence the frequency of counseling obese patients by community pharmacists.

METHODS

A mail survey was sent to a random sample of 400 Texas community pharmacists. The questionnaire collected information on frequency of counseling obese patients, perceived comfort level with counseling obese patients, perceived effectiveness of various obesity treatments, and perceived confidence in achieving positive outcomes as a result of counseling. Demographic information was also collected. Descriptive statistics, correlational analyses, and t-tests were used to examine the data.

RESULTS

A usable response rate of 35.2% was obtained (139 completed surveys out of 395 assumed delivered). Overall, pharmacists counseled patients rarely to sometimes about various aspects of obesity management. Correlational analyses revealed that pharmacists’ frequency of counseling about obesity management was significantly and positively associated with their comfort level with counseling obese patients (r = 0.47; p < 0.001). Higher levels of confidence in achieving positive outcomes as a result of counseling (r = 0.39; p < 0.001) and higher levels of perceived effectiveness of obesity management options (r = 0.18; p = 0.037) were also significantly associated with higher levels of counseling about obesity management.

CONCLUSIONS

Obesity counseling by pharmacists was positively correlated with their perceived comfort with counseling obese patients, confidence in achieving positive outcomes, and effectiveness of obesity management options.
Heterogeneous mixed-layer clays from the Cretaceous Greensand, Isle of Wight, southern England
Authors: McCarty Douglas K. , Drits Victor A. , Sakharov Boris , Zviagina Bella B. , Ruffell Alastair , Wach Grant
Published: September 2004

The sea-cliffs of the Isle of Wight were deposited during a period of overall sea-level rise starting in the Barremian (Lower Cretaceous) and continuing into the Aptian and Albian. They consist of fluvial, coastal and lagoonal sediments including greensands and clays. Numerous episodes of erosion, deposition and faunal colonization reflect condensation and abandonment of surfaces with firmgrounds and hardgrounds. This study focused mainly on shallow marine cycles where variations in clay mineralogy would not be expected, because overall system composition, sediment source, and thermal history are similar for all the samples in the studied section. Instead we found a wide variety of clay assemblages even in single samples within a 200 m interval.

In this interval, distinct clay mineral assemblages were found and can be described as consisting of Al-rich, Fe-richand intermediate Fe and Al compositions withrespect to 2:1 and 1:1 layers in mixed-layer arrangements. Nearly pure glauconite-nontronite clays exist in the <2 µm fraction only when the bulk rock is free of K- and plagioclase feldspar. Conditions favorable to glauconite-nontronite formation are interpreted to result from a hiatus in volcanoclastic sedimentation, thus providing a stable substrate for glauconitization.

The Fe-bearing mixed-layer clay assemblages consist of glauconite, nontronite and berthierine-like layers in various proportions with several mixed-layer clays often coexisting in the same sample. In different samples, Al-richand Fe-Mg-rich mixed-layer clays are similar in their content and distribution of 1:1 and 2:1 layers. This suggests that the original clay assemblages were similar and later diagenesis affected certain horizons resulting in substitution of Al by Fe + Mg while preserving the original layer structure and arrangement.

Structural formulae for the berthierine-like phase and berthierine-like layers in these mixed-layer clays show their layer cation composition is intermediate between odinite and standard berthierine. The total sum of octahedral cations varies from 5.26 to 5.55 whereas the amount of Fe2+ cations varies from 2.12 to 2.22 per O10(OH)8. A feature of the berthierine-like phase as well as of berthierine-like layers is that they are di-trioctahedral and Fe2+ and Fe3+ are the prevalent cations. Moreover, in these berthierine-like components, the amount of Fe2+ is greater than that of Mg (in contrast to odinite) and Fe3+ cations prevail over Al (in contrast to berthierine). The presence of authigenic ferrous Fe clays and the relationship between glauconite-nontronite and bulk mineralogy has implications for sedimentological processes and geochemical conditions during and shortly after deposition.
Measurement of clay surface areas by polyvinylpyrrolidone (PVP) sorption and its use for quantifying illite and smectite abundance
Authors: Blum A. E. , Eberl D. D.
Published: September 2004

A new method has been developed for quantifying smectite abundance by sorbing polyvinylpyrrolidone (PVP) on smectite particles dispersed in aqueous solution. The sorption density of PVP-55K on a wide range of smectites, illites and kaolinites is ∼0.99 mg/m2, which corresponds to ∼0.72 g of PVP-55K per gram of montmorillonite. Polyvinylpyrrolidone sorption on smectites is independent of layer charge and solution pH. PVP sorption on SiO2, Fe2O3and ZnO normalized to the BET surface area is similar to the sorption densities on smectites. γ-Al2O3, amorphous Al(OH)3and gibbsite have no PVP sorption over a wide range of pH, and sorption of PVP by organics is minimal. The insensitivity of PVP sorption densities to mineral layer charge, solution pH and mineral surface charge indicates that PVP sorption is not localized at charged sites, but is controlled by more broadly distributed sorption mechanisms such as Van der Waals’ interactions and/or hydrogen bonding. Smectites have very large surface areas when dispersed as single unit-cell-thick particles (∼725 m2/g) and usually dominate the total surface areas of natural samples in which smectites are present. In this case, smectite abundance is directly proportional to PVP sorption. In some cases, however, the accurate quantification of smectite abundance by PVP sorption may require minor corrections for PVP uptake by other phases, principally illite and kaolinite. Quantitative XRD can be combined with PVP uptake measurements to uniquely determine the smectite concentration in such samples.
Na biotite and intermediate Na-K biotite in schists from the Betic Cordilleras (Spain)
Authors: Ruiz Cruz María Dolores
Published: September 2004

Submicroscopic intergrowths of K biotite, Na biotite and intermediate Na-K biotite from a schist near Málaga (Betic Cordilleras, Spain) were discovered using high-resolution transmission electron microscopy and analytical electron microscopy. The sample was also studied with X-ray diffraction, electron microprobe analysis, and scanning electron microscopy. Scanning electron microscopy revealed that the Na-enriched biotite is concentrated in albite-rich microdomains, albite being partially replaced by biotite. These images also revealed that both K and Na-K biotite grains appear locally retrograded to kaolinite. Transmission electron microscopic data indicated that K biotite, Na biotite and Na-K biotite form parallel or subparallel packets with interfaces parallel to the basal planes of biotite. Potassium biotite forms thick packets, chemically homogeneous, with a basal spacing of 10.1 Å. Sodium biotite also occurs as chemically homogeneous stacks of layers with a 9.78 Å periodicity. Sodium-K biotite shows, on the contrary, variable composition and basal spacings intermediate between K and Na biotites. Analytical electron microscopic data revealed important chemical differences between Na and K biotites, which affect both the tetrahedral and the octahedral sheets. Both electron microprobe analysis and analytical electron microscopy indicated that the trioctahedral micas show relatively low interlayer occupancy, suggesting the presence of H3O+ replacing the interlayer cations. Partial hydration of biotite explains the presence of a weak 14 Å reflection in the X-ray patterns. Both chemical and textural data suggested that these trioctahedral micas grew during a common prograde metamorphic episode, the phases with intermediate composition probably being metastable.