bluuebunny/crossref_metadata_2025 huggingface dataset:
Leishmania major lipophosphoglycan modulates the phenotype and inhibits migration of murine Langerhans cells
Authors: Ponte‐Sucre Alicia , Heise Dirk , Moll Heidrun
Published: March 2003

Langerhans cells (LC), members of the dendritic cell family, play a central role in the initiation and regulation of the immune response against the protozoan parasite Leishmania major. LC take up antigens in the skin and transport them to the regional lymph nodes for presentation to T cells. However, it is not known whether LC functions are modulated by parasite antigens. In the present study, we examined the effect of a major parasite surface molecule, L. major lipophosphoglycan (LPG), on the maturation of LC and their migratory properties. The results show that exposure to LPG did not affect the expression of major histocompatibility complex (MHC) class II and B7, but induced an up‐regulation of CD25, CD31 and vascular endothelial (VE)‐cadherin expression and a down‐regulation of Mac‐1 expression, by LC. Importantly, LPG treatment inhibited the migratory activity of LC, as it reduced their efflux from skin explants and their migration in transwell cultures. These results suggest that Leishmania LPG impairs LC migration out of the skin and thus may modulate their immunostimulatory functions, which require LC translocation from skin to lymph nodes.
The regulation of DNAse activities in subcellular compartments of activated thymocytes
Authors: Nagata Takuya , Kishi Hiroyuki , Liu Qin Li , Matsuda Tadashi , Imanaka Tsuneo , Tsukada Kazuhiro , Kang Dongchon , Muraguchi Atsushi
Published: March 2003

Thymocytes expressing self‐reactive T‐cell receptors (TCR) are eliminated in the thymus through a TCR‐mediated signal. This cell death signal (negative selection) generates nuclear morphological change and DNA fragmentation in thymocytes. However, the pathway leading to DNA fragmentation of thymocytes following TCR engagement remains obscure. In this study, we investigated the localization and function of caspase‐activated DNAse (CAD) and its inhibitor (ICAD) in thymocytes prior to or after in vivo TCR stimulation. We showed that CAD and ICAD are co‐localized in microsome, nuclei and cytosol in unstimulated thymocytes. Following in vivo TCR engagement, ICAD located in cytosol and microsome was degraded and the resulting activated CAD induced chromosomal DNA fragmentation. CAD present in cytosol and microsome of unstimulated thymocytes was activated by recombinant caspase‐3, and microsomal CAD was released to the cytosol. These results demonstrate that TCR engagement of thymocytes induces caspase‐3‐dependent activation of CAD localized in both cytosol and microsome, leading to DNA fragmentation in harmony.
Roles of cytotoxic T‐lymphocyte‐associated antigen‐4 in the inductive phase of oral tolerance
Authors: Chen Yiguang , Ma Yuanfang , Chen Youhai
Published: March 2003

To elucidate the roles of cytotoxic T‐lymphocyte‐associated antigen‐4 (CTLA‐4) in oral tolerance, we studied the consequences of CTLA‐4 blockade during the inductive phase of oral tolerance using a transgenic T‐cell transfer model. We found that CTLA‐4 blockade significantly accelerated cell cycle progression of antigen‐specific T cells and dramatically increased their numbers in lymphoid organs following oral administration of ovalbumin (OVA). In mice fed with OVA, only ≈ 35% of specific T cells underwent more than four cycles of cell division. This was increased to 65% in mice fed with OVA and treated with a blocking anti‐CTLA‐4 monoclonal antibody (mAb). The OVA‐specific T cells in the latter group were localized primarily in the T‐cell zones of the mesenteric lymph nodes and Peyer's patches with a few penetrated into B‐cell follicles. Nevertheless, both faecal anti‐OVA immunoglobulin A (IgA) and seral anti‐OVA immunoglobulin G (IgG) were produced in anti‐CTLA‐4 mAb‐treated mice. These results suggest that CTLA‐4 limits the degree of T‐cell activation by blocking cell cycle progression during the inductive phase of oral tolerance. In the absence of the CTLA‐4 signal, mucosal exposure of antigen induces heightened T‐cell activation and expansion, which in turn promotes the production of antigen‐specific antibodies.
Identification and characterization of L‐selectin ligands in porcine lymphoid tissues
Authors: Khan Adil I. , Haskard Dorian O. , Malhotra Rajneesh , Landis R. Clive
Published: March 2003

A human L‐selectin–ZZ fusion protein was used to screen porcine inguinal lymph nodes for the presence of monoclonal antibody (mAb) MECA 79‐negative ligands. Fractionation of lymph node‐conditioned medium by anion‐exchange chromatography revealed two distinct L‐selectin‐binding fractions, one containing a MECA 79 non‐reactive species and the second containing two MECA 79 reactive species of ≈ 84 000 and 210 000 molecular weight. The MECA 79 non‐reactive species exhibited Ca2+‐ and lectin‐dependent binding to L‐selectin–ZZ in a solid‐phase capture enzyme‐linked immunosorbent assay (ELISA), and this was specifically disrupted by the addition of EDTA, mannose‐6‐phosphate and the blocking anti‐L‐selectin mAb, DREG‐56. Enzymatic characterization of the ligand by trypsin, O‐sialoglycoprotease endopeptidase, heparinases I and III, or chondroitinase ABC lyase digestion indicated that L‐selectin binding was predominantly dependent upon a chondroitin sulphate‐modified glycoprotein determinant. Although Coomassie Blue staining of sodium dodecyl sulphate (SDS) polyacrylamide gels did not reveal a detectable protein species, carbohydrate‐specific staining using GlycoTrack™ revealed a single, heavily glycosylated protein of high molecular weight (> 220 000). These studies have revealed the existence of a MECA 79 non‐reactive, chondroitin sulphate glycosaminoglycan‐modified ligand, termed csgp>220, which is secreted by peripheral lymph nodes and may play a role in leucocyte trafficking to the lymph node.
Cross‐reactivity of the anti‐La monoclonal antibody SW5 with early endosome antigen 2
Authors: A. Fouraux Michael , Heijden Annemarie van der , Van Venrooij Walther J. , Pruijn Ger J. M.
Published: March 2003

Coimmunoprecipitation studies with SW5, a frequently used and specific mouse monoclonal antibody (mAb) directed against the human La autoantigen, led to the identification of a functionally unrelated 80 000 MW protein, designated early endosome antigen 2 (EEA2). EEA2 appeared to be directly targeted by mAb SW5. Because an RNA‐binding domain, a structural element of La containing the SW5‐epitope, was not discernable in the primary structure of EEA2, the SW5‐epitope on EEA2 was determined. Coiled‐coil region 3 of EEA2 appeared to contain the epitope recognized by SW5. The SW5 epitope regions of La and EEA2 share a limited sequence homology and probably share a higher degree of structural similarity at the tertiary level. Most likely, the most critical determinants for recognition by SW5 reside in elements adopting alpha‐helical conformations. These data indicate that the application of specific mAbs to purify and characterize (functionally) interacting proteins can be severely obscured by the cross‐reactivity of mAbs with structurally, but not functionally, similar proteins.
A new Groucho TLE4 protein may regulate the repressive activity of Pax5 in human B lymphocytes
Authors: Milili Michèle , Gauthier Laurent , Veran Julie , Mattei Marie‐Geneviève , Schiff Claudine
Published: March 2003

During mouse B‐cell development, Pax5 is an essential transcription factor that acts as an activator of B‐cell‐specific genes and as a repressor of alternative lineage fates. The repressive function is mediated by the recruitment of members of the Groucho co‐repressor family. Using an RNA display approach, we have isolated a transcript, called QD, specifically expressed in human pro‐B and pre‐B cells, which is derived from the human Groucho TLE4 gene. The QD transcript contains the first four TLE4 exons and an intronic sequence 3′ of exon 4, demonstrating that QD is a splice variant of TLE4. The putative resulting protein of 94 amino acids corresponds to approximately half of an N‐terminal tetramerization domain. We also show specific expression of TLE4 transcripts in human B cells and of TLE4 proteins in B‐cell nuclei. Moreover, we demonstrate that recombinant QD protein binds to the TLE4 Q domain and is able to abolish the TLE4/Pax5 interaction. Thus, QD could act as a negative regulator of TLE4 function, in early B‐cell differentiation.
Significant role of Fas ligand‐binding but defective Fas receptor (CD95) in lymph node hyperplasia composed of abnormal double‐negative T cells
Authors: Matsuzawa Akio , Shimizu Motomu , Takeda Yasutaka , Nagase Hisashi , Sayama Kazutoshi , Kimura Mikio
Published: March 2003

The functional differences between two mutations of the Fas (CD95) locus, Faslpr (lpr) and Faslprcg (lprcg), were investigated using bone marrow (BM) transplantation on the C3H mouse background. Both lpr/lpr and lprcg/lprcg BM transferred caused lymph node (LN) hyperplasia in lpr/+ and lprcg/+ recipients, although it was clearly smaller than that in lpr/lpr and lprcg/lprcg recipients of lpr/lpr and lprcg/lprcg BM. In addition, both BM induced significantly larger LN hyperplasia in lprcg/+ than lpr/+ recipients. Appearance of CD4− CD8−[double negative (DN)] T cells in the periphery is the most consistent phenotype of Fas mutations. Importantly, the proportion of DN T cells was higher in larger LN hyperplasia in the order of lpr/+, lprcg/+ and lpr/lpr or lprcg/lprcg recipients. On the other hand, both lpr/lpr and lprcg/lprcg BM transferred into wild‐type (+/+) mice caused marked LN atrophy. The former, but not the latter, induced wasting syndrome. Faslg1d (gld)‐homozygous lpr/lpr BM transferred into +/+ mice elicited LN hyperplasia of the same extent as that in lpr/lpr mice transferred with lpr/lpr BM, but not wasting syndrome. Taken together with the fact that DN T cells massively express Fas ligand (FasL), this study implied that FasL overexpressed on DN cells may be involved in the accumulation of DN T cells in LN, LN atrophy and wasting syndrome, and that lprcg Fas, which can bind to Fas ligand but not transduce apoptosis signal into cells, may modulate these pathological conditions by interfering with the binding of FasL to Fas.
Interaction between nitric oxide and subsets of human T lymphocytes with differences in glutathione metabolism
Authors: Roozendaal Ramon , Kauffman HENK F. , Dijkhuis Anne‐Jan , Ommen Elisabeth T. V. , Postma Dirkje S. , De Monchy Jan G. R. , Vellenga Edo
Published: March 2003

Nitric oxide (NO) modulates human T‐lymphocyte responses through several mechanisms. In the current study we show that interactions between NO and glutathione (GSH) metabolism are related to the selective persistent inhibition of interferon‐γ (IFN‐γ) production by NO, which we previously identified. T cells were exposed to NO using the NO‐donor compound Spermine‐nonoate (Sper) and activated using anti‐CD3 plus anti‐CD28 monoclonal antibodies. Persistent inhibition of IFN‐γ by Sper was prevented by addition of the GSH precursor l‐cysteine, which inhibits Sper induced GSH depletion. Subsets of cells were either susceptible (GSHlow) or resistant (GSHhigh) to NO‐induced GSH depletion. The GSHlow subset was characterized by enhanced numbers of CD4+ cells, reduced numbers of activated cells as characterized by CD25 and CD69, and reduced numbers of memory (CD45RO+) cells relative to the GSHhigh population. Rather than directly affecting susceptibility to NO, these surface markers reflected different expression patterns. Particularly, the GSHlow subset was further characterized by decreased activity of the GSH synthesis related enzymes multi‐drug resistance related protein (MRP)‐1 and γ‐glutamyltranspeptidase (γ‐GT). Blocking γ‐GT, using acivicin was shown to exacerbate NO‐induced GSH depletion and NO‐induced apoptosis. Since NO induced apoptosis selectively affects IFN‐γ production these findings implicate GSH metabolism in the modulation and maintenance of the T helper (Th)1/Th2 balance.
In vivo maintenance of T‐lymphocyte unresponsiveness induced by thymic medullary epithelium requires antigen presentation by radioresistant cells
Authors: Hudrisier Denis , Feau Sonia , Bonnet Véronique , Romagnoli Paola , Van Meerwijk Joost P. M.
Published: March 2003

The T‐cell repertoire developing in the thymus is rid of autospecific cells by the process of thymic negative selection. Recognition of major histocompatibility complex (MHC)/self‐peptide complexes expressed by thymic antigen‐presenting cells (APC) of bone marrow origin leads to induction of apoptotic death of autospecific thymocytes. Induction of tolerance to self‐antigens not presented by thymic APC is mediated by medullary thymic epithelial cells (mTEC) which express a very wide range of proteins, e.g. inducible and tissue‐specific proteins. The main type of tolerance induced by mTEC is non‐deletional and the issue of how it is maintained outside the thymus is therefore of crucial interest. We have previously shown that the non‐T‐cell receptor (TCR) ‐transgenic T‐cell repertoire developing in conditions in which tolerance to self‐MHC/peptide ligands is exclusively induced by mTEC is tolerant to syngeneic targets in vivo but lyses such targets in vitro. Here we report that this non‐deletional in vivo self‐tolerance is not due to active tolerance assured by known naturally occurring regulatory or immune‐modulating T lymphocytes. Importantly, we show that in vivo maintenance of this therefore probably anergic state requires continued interaction of autospecific T cells with self‐MHC/peptide ligands expressed by radioresistant cells while APC are incapable of maintaining the tolerant state. Therefore, maintenance of non‐deletional T‐lymphocyte tolerance to the wide range of self‐antigens expressed by mTEC depends on continued interaction with radioresistant cells that very probably express a much more limited repertoire of antigens. Our data may therefore have important consequences for tolerance to tissue‐specific and inducible self‐antigens.
Form and pattern of MUC1 expression on T cells activated in vivo or in vitro suggests a function in T‐cell migration
Authors: Correa Isabel , Plunkett Tim , Vlad Anda , Mungul Arron , Candelora‐Kettel Jessica , Burchell Joy M. , Taylor–papadimitriou Joyce , Finn Olivera J.
Published: March 2003

MUC1 is a transmembrane mucin that is expressed on ductal epithelial cells and epithelial malignancies and has been proposed as a target antigen for immunotherapy. The expression of MUC1 has recently been reported on T and B cells. In this study we demonstrate that following activation in vivo or activation by different stimuli in vitro, human T cells expressed MUC1 at the cell surface. However, the level of expression in activated human T cells was significantly lower than that seen on normal epithelial cells or on breast cancer cells. In contrast, resting T cells did not bind MUC1‐specific monoclonal antibodies (mAbs), nor was MUC1 mRNA detectable by reverse transcription–polymerase chain reaction (RT–PCR) or Northern blot analysis in these cells. The profile of activated T‐cell reactivity with different MUC1‐specific antibodies suggested that the glycoform of MUC1 expressed by the activated T cells carried core 2‐based O‐glycans, as opposed to the core 1 structures that dominate in the cancer‐associated mucin. Confocal microscopy revealed that MUC1 was uniformly distributed on the surface of activated T cells. However, when the cells were polarized in response to a migratory chemokine, MUC1 was found on the leading edge rather than on the uropod, where other large mucin‐like molecules on T cells are trafficked. The concentration of MUC1 at the leading edge of polarized activated human T cells suggests that MUC1 could be involved in early interactions between T cells and endothelial cells at inflammatory sites.
An abnormal but functionally active complement component C9 protein found in an Irish family with subtotal C9 deficiency
Authors: Orren Ann , O'Hara Ann M. , Morgan B. Paul , Moran Anthony P. , Würzner Reinhard
Published: March 2003

Two independently segregating C9 genetic defects have previously been reported in two siblings in an Irish family with subtotal C9 deficiency. One defect would lead to an abnormal C9 protein, with replacement of a cysteine by a glycine (C98G). The second defect is a premature stop codon at amino acid 406 which would lead to a truncated C9. However, at least one of two abnormal proteins was present in the circulation of the proband at 0·2% of normal C9 concentration. In this study, the abnormal protein was shown to have a molecular weight approximately equal to that of normal C9, and to carry the binding site for monoclonal antibody (mAb) Mc42 which is known to react with an epitope at amino acid positions 412–426, distal to 406. Therefore, the subtotal C9 protein carries the C98G defect. The protein was incorporated into the terminal complement complex, and was active in haemolytic, bactericidal and lipopolysaccharide release assays. A quantitative haemolytic assay indicated even slightly greater haemolytic efficiency than normal C9. Epitope mapping with six antihuman C9 mAbs showed the abnormal protein to react to these antibodies in the same way as normal C9. However, none of these mAbs have epitopes within the lipoprotein receptor A module, where the C98G defect is located. The role of this region in C9 functionality is still unclear. In conclusion, we have shown that the lack of a cysteine led to the production of a protein present in the circulation at very much reduced levels, but which was fully functionally active.
The design of information systems: parti, formats and sketching
Authors: Stolterman Erik
Published: March 2003

In this article it is argued that an information system must be treated as an artefact with a format. We need to see information systems design as based on architectonic and not tectonic thinking. Design based on architectonic thinking emphasizes (1) the task of giving form to a design proposal, and (2) the task of creating an overall structure for the information system. These two activities are closely related because they both deal with the issue of seeing the idea or whole of systems, and the problem of how to give form to that whole. The paper begins with an exploration of the concept formative faculties in relation to ‘given’ and ‘non‐given’ objects. The concepts of parti and format are then presented as a way to grasp the ‘whole’ of an information system. How formats can be used in information systems design is discussed as well as how this leads to the act of sketching as an important but neglected activity in information systems design. Sketching is promoted by introducing diathenic graphologue as the art of ‘giving form to the unknown’. The paper ends by suggesting how formats and diathenic graphologue can be practised and included as important aspects of information systems design education and training.
Transforming information systems development through computer‐aided systems engineering (CASE): lessons from practice
Authors: Butler Tom
Published: March 2003

In dealing with the challenges posed by the ongoing problem of developing and integrating an evermore complex and diverse range of information systems in a timely manner, practitioners continue to grapple with important issues such as increasing developer productivity and bringing quality improvements to the process and product of systems development. Many organizations have adopted computer‐aided systems engineering (CASE) tools with such outcomes in mind. With few exceptions, previous research into the phenomenon of CASE adoption and use has been survey based in the main, and has resulted in some confusion over the benefits to be derived from the use of CASE tools within the systems development process. This paper extends previous work as part of an interpretive, case‐based research strategy to examine the adoption and use of integrated CASE (I‐CASE) in a single organization. Such an approach facilitates a deeper understanding of the impact that CASE exerts on the process and product of systems development. Based on the experience of practitioners in the organization studied, a set of recommendations is presented. The findings of this study also lend support to the view that an I‐CASE development platform does indeed have the potential to exert a positive impact on the development process and its product.
The learning organization information system (LOIS): looking for the next generation
Authors: Williamson Adrian , Iliopoulos Constantinos
Published: March 2003

This paper explores the notion that the next generation of information systems will focus on supporting organizational learning. The paper suggests that the increasingly successful automation of procedural work will lead to pressure on organizations to improve performance through enhanced support for knowledge work. A set of outline requirements for the learning organization information system (LOIS) is then proposed using recent research findings from computer supported co‐operative working and organizational learning. The computerized on‐line journal from this research is described. This journal provides transparent capture of episodes of work and it is argued that the general principles established could support LOIS by helping to provide a richly defined organizational memory. The journal supports collaborative working through the use of groupware, which manages the sharing of, and learning from, journal contents. This can facilitate the retention of not only data and information, but also the inquiry process that produced them. The paper concludes that LOIS will be a self‐organizing system, focussing on knowledge work, learning and using advanced technologies drawn from ubiquitous computing. A view of a system that moves towards this aim is presented. Future topics for research are identified, and a natural language approach to knowledge asset management is discussed briefly. In closing, it is argued that LOIS is an important future vision for organizations operating in the information age.
Analysis of a mosquito acetylcholinesterase gene promoter
Authors: Liu H.‐T. , Stilwell G. , Anthony N. , Rocheleau T. , Ffrench‐Constant R. H.
Published: March 2003

Insect acetylcholinesterase is the target site for organo­phosphorus and carbamate insecticides and point mutations in the Ace gene are associated with resistance in Drosophila melanogaster and Musca domestica. However, little is known of the genetic regulation of insect Ace genes. Here we report the isolation of four different cDNAs from an Aedes Ace locus and identification of the gene promoter. Northern analysis reveals two large (>10 kb) transcripts and one smaller transcript of 4 kb. The region containing the initiation of transcription was localized by sequencing the two 5′ most cDNAs and by 5′ RACE. The transcription start point was subsequently identified by primer extension and is flanked by a perfect arthropod initiator consensus sequence. The promoter lacks a TATA box but contains several matches to other consensus sequences for eukaryotic transcription factors. In common with the Drosophila Ace gene, there are also multiple potential initiators of translation (ATGs) upstream of the main open reading frame. The structure of the 5′ leader and promoter is compared to that found in other insect and vertebrate Ace genes and the possibility that this locus is homologous to one of two Ace loci described in another mosquito, Culex pipiens, is discussed.
Molecular cloning and nucleotide sequenceof a cytochrome P450 cDNA from a pyrethroid‐resistant mosquito, Culex quinquefasciatus Say
Authors: Kasai S. , Shono T. , Yamakawa M.
Published: March 2003

A novel cDNA clone encoding a cytochrome P450 was screened from a gut cDNA library of permethrin‐resistant­Culex quinquefasciatus larvae. Nucleotide sequence of the clone designated CYP6E1 was determined. This is the first full‐length sequence of a mosquito P450 cDNA. Comparison of the deduced amino acid sequence of CYP6E1 to those of other known cytochrome P450s indicates that this clone belongs to CYP6 family. In addition, a phylogenetic analysis of CYP6E1 primary structure showed that it is related to CYP6A and CYP6C subfamilies. Polymerase chain reaction (PCR) using primers designed on the basis of the nucleotide sequence of the cDNA showed that mRNA encoding CYP6E1 is present in permethrin‐susceptible larvae as well as ‐resistant larvae, suggesting the ubiquitous distribution of CYP6E1.
Novel mutations in the para‐homologous sodium channel gene associated with phenotypic expression of nerve insensitivity resistance to pyrethroids in Heliothine lepidoptera
Authors: Head D. J. , McCaffery A. R. , Callaghan A.
Published: March 2003

Coding sequences from the intracellular segment linking repeat domains III and IV of the para‐homologous sodium channel gene were amplified and cloned from two species of Heliothine larvae using RT‐PCR techniques. Sequence comparisons between standard laboratory susceptible and nerve insensitive strains of the tobacco budworm (Heliothis virescens) and the cotton bollworm (Helicoverpa armigera) identified two mutations. These were an aspartic acid (GAC) to valine (GTC ) and a glutamic acid (GAA) to glycine (GGA) mutation. PCR and sequencing was undertaken only on individuals that were demonstrated to be phenotypically nerve insensitive or susceptible to pyrethroids using a neurophysiological technique. The finding of identical mutations across two species suggests that they may be implicated in resistance.
Molecular analysis and chromosome mapping of the H2A, H3 and H4 histone genes from the malaria vector Anopheles gambiae
Authors: Reynaud E. , Vázquez M. , Zurita M.
Published: March 2003

In this we report the cloning and analysis of PCR generated fragments that encode H2A, H3 and H4 histone genes from the malaria vector An. gambiae. Sequence analysis indicated that some conservative changes are present in the An. gambiae H2A and H4 genes as compared with histone genes from other organisms. Divisional mapping showed that these genes map in division 20 on the left arm of the second chromosome. Southern blot experiments and the molecular characterization of the genomic fragment containing the H2A, H2B, H3 and H4 genes showed that they are organized in a cluster with an orientation different from the one found in other dipterans.
Transposable element interactions in insects: crossmobilization of hobo and Hermes
Authors: Sundararajan P. , Atkinson P. W. , O'brochta D. A.
Published: March 2003

There are four non‐drosophilid insect gene vector systems available that have been constructed from the short inverted repeat‐type transposable elements Minos, piggyBac, mariner and Hermes. These elements (with the possible exception of piggyBac) are members of transposable element families that appear to be widespread in nature. Because these transposable element families are large it is possible that an insect species targeted for transformation will contain related transposable elements. The data presented here begin to address directly the question of interaction between diverged but related members of transposable element families. We tested the ability of the hAT elements hobo and Hermes to interact and cause crossmobilization. Using plasmid‐based and chromosome‐based element mobility assays we found that the terminal sequences of hobo and Hermes were almost equally good substrates for hobo transposase. However, this ability to crossmobilize was not reciprocal. Hermes transposase was only rarely able to cause the excision of hobo elements from plasmids and was never observed from germline chromosomes. These results have important implications for transgenic insect studies in the future.
Recombinant juvenile hormone esterase, an effective tool for modifying juvenile hormone‐dependent expression of the early trypsin gene in mosquitoes
Authors: Edgar K. A. , Noriega F. G. , Bonning B. C. , Wells M. A.
Published: March 2003

The study of the changes in the steady‐state levels of the early trypsin (ET) messenger RNA (mRNA) was used as a sensitive assay for measuring the effects of recombinant juvenile hormone esterase (rJHE) on juvenile hormone (JH)‐dependent gene expression in mosquitoes. ET is a female‐specific protease present in the midgut of the yellow fever mosquito Aedes aegypti during the first few hours after ingestion of a blood meal. Transcription of the early trypsin gene is part of the normal postemergence maturation of the midgut in the adult female, and it is regulated by JH. JHE was cloned from Heliothis virescens and expressed in a baculovirus vector. Injection of rJHE into mosquitoes resulted in an increase of JHE activity in the haemolymph. Injection of rJHE into newly emerged adult females delayed the normal increase in steady‐state levels of ET mRNA observed in controls. Topically applied methoprene (a JH analogue) reversed the effect of rJHE. Injection of increasing concentrations of rJHE into 3‐day‐old unfed females resulted in a dose‐dependent decrease in the steady‐state levels of ET mRNA after 24 h. The effect of rJHE was transient, once the enzyme was cleared (72 h after injection), the steady‐state levels of ET mRNA were restored. The injection of rJHE is an effective tool for modifying JH‐dependent expression of the early trypsin gene in mosquitoes.
Molecular cloning and characterization of a metal responsive Aedes aegypti intestinal mucin cDNA
Authors: Rayms‐Keller A. , McGaw M. , Oray C. , Carlson J. O. , Beaty B. J.
Published: March 2003

We have isolated a cDNA from Aedes aegypti that is transcribed in the larval midgut in response to metal exposure, and in the adult female midgut in response to iron or cadmium exposure, or a blood meal. The cDNA encodes a protein, designated Aedes aegypti intestinal mucin 1 (AEIMUC1), which has similarities with invertebrate intestinal mucins and peritrophins, and vertebrate mucins. Proline, serine and threonine comprise 30% of the amino acid composition of AEIMUC1, a characteristic of mucins. AEIMUC1 contains three cysteine‐rich domains, two of which flank a proline/serine/threonine‐rich domain, a feature shared by many mucin genes. This is the first report on the isolation of a metal‐responsive gene from an aquatic insect.
Distribution of genetic variation among chromosomal forms of Anopheles gambiae s.s.: introgressive hybridization, adaptive inversions, or recent reproductive isolation?
Authors: Black W. C. , Lanzaro G. C.
Published: March 2003

A series of four papers in this issue explores the reproductive status of the five chromosomal forms of An. gambiae s.s. using molecular techniques to examine the variation among twelve different genes located throughout the An. gambiae s.s. genome. Results of these and previous studies are consistent with a hypothesis of at least partial barriers to gene flow between some chromosomal forms in the Ivory Coast and other West African countries to the north and west, but introgression between S and M types in Benin and countries to the east. Collectively, these studies indicate the need for a broader geographical sampling of An. gambiae s.s., increased research on mechanisms of prezygotic reproductive isolation and field‐based studies of survival and fecundity in hybrids to test for postzygotic reproductive isolation.
Expression and induction of three family 4 cytochrome P450 (CYP4)* genes identified from insecticide‐resistant and susceptible western corn rootworms, Diabrotica virgifera virgifera
Authors: Scharf M. E. , Parimi S. , Meinke L. J. , Chandler L. D. , Siegfried B. D.
Published: March 2003

We have previously determined that cytochrome P450‐based oxidation is involved in resistance to the insecticides methyl parathion and carbaryl in geographically distinct Nebraska western corn rootworm populations. Three new family 4 cytochrome P450 (CYP4) gene fragments (CYP4AJ1, CYP4G18 and CYP4AK1) were cloned and sequenced from insecticide‐resistant and ‐susceptible western corn rootworms. Insecticide bioassays indicated the resistant population employed in this study was significantly resistant to the insecticides methyl parathion and carbaryl. CYP4AJ1 and CYP4G18 were cloned from both genomic PCR and RT‐PCR products, although only CYP4AJ1 contains an intronic region. Alignments of inferred amino acid sequences with other homologous insect CYP4 genes indicates a high degree of similarity. Northern analysis concurrently employing mixed probes representing each of the three rootworm CYP4 fragments identified increased mRNA transcript signals (i) in resistant rootworms and (ii) following induction by the P450 inducer pentamethyl benzene. These results support our previous documentation of P450‐based insecticide resistance and suggest increased CYP4 transcript abundance can serve as a molecular resistance‐associated marker.
Expression and function of the Drosophila melanogaster ADH in male Ceratitis capitata adults: a potential strategy for medfly genetic sexing based on gene‐transfer technology
Authors: Christophides G. K. , Savakis C. , Mintzas A. C. , Komitopoulou K.
Published: March 2003

The aim of development of a Mediterranean fruit fly Ceratitis capitata genetic sexing strain derives from the large scale SIT programmes being carried out to control this pest. Toward this direction, we present here the male‐specific expression of the Drosophila melanogaster alcohol dehydrogenase (ADH) in medfly transgenic adults generated by Minos‐mediated germ line transformation. This expression pattern is obtained by using a promoter fragment of the male‐specific gene MSSP‐α2 of the medfly. We show that the heterologous enzyme is functional in the medfly oxidizing both ethanol and 2‐propanol. Although leading to an approximately twofold increase of total ADH activity in male compared to female transgenic adults, these expression levels are not enough for performing genetic sexing when high doses of environmental alcohol are applied. This could be achieved either by further enhancement of the transgene expression or by generating an Adh− line to host the Minos insertions.
Paraphyly in Hawaiian hybrid blowfly populations and the evolutionary history of anthropophilic species
Authors: Stevens J. R. , Wall R. , Wells J. D.
Published: March 2003

Complementary nuclear (28S rRNA) and mitochondrial (COI + II) gene markers were sequenced from the blowflies,Lucilia cuprinaandLucilia sericata, from Europe, Africa, North America, Australasia and Hawaii. Populations of the two species were phylogenetically distinct at both genes, with one exception. HawaiianL. cuprinapossessed typicalL. cuprina‐type rRNA, but hadL. sericata‐type mitochondrial (COI + II) sequences. An explanation for this pattern is that Hawaiian flies are hybrids and comparison of observed levels of sequence divergence to possible introduction events, e.g. Polynesian colonization, suggests that HawaiianL. cuprinamay be evolving rapidly. Moreover, the monophyly of these flies also suggests that theL. sericatamtDNA haplotype was apparently fixed in HawaiianL. cuprinaby lineage sorting, indicating a population bottleneck in the evolutionary history of these island flies.
Induction of the white egg 3 mutant phenotype by injection of the double‐stranded RNA of the silkworm white gene
Authors: Quan G. X. , Kanda T. , Tamura T.
Published: March 2003

Injection of double‐stranded RNA (dsRNA) corresponding to the silkworm white gene (Bmwh3) into preblastoderm eggs of the wild‐type silkworm induced phenotypes similar to those observed with mutants of the white egg 3 locus (10–19.6). The induced phenotypes were characterized by the presence of white eggs and translucent larval skin. Northern analysis showed that the expression of the endogenous Bmwh3 gene in the injected embryos was distinctly depressed. Furthermore, the injection of the GFP dsRNA inhibited the expression of the GFP gene from a plasmid co‐injected with the dsRNA but did not depress the expression of the Bmwh3 gene. These findings demonstrate that sequence‐specific RNA interference occurred in the silkworm. We conclude from the results that the RNA interference can be applied as a tool for the analysis of the gene function in the lepidopteran insects.
cDNA cloning of biotransformation enzymes belonging to the cytochrome P450 family in the antennae of the noctuid moth Mamestra brassicae
Authors: Maïbèche‐Coisne M. , Jacquin‐Joly E. , François M. C. , Nagnan‐Le Meillour P.
Published: March 2003

The involvement of cytochrome P450 enzymes in olfaction was demonstrated in vertebrates some time ago. In insects these enzymes are well known for their role in insecticide resistance, but the involvement of P450 in pheromone degradation was only recently demonstrated. Using a PCR strategy, we have isolated two cDNAs from the antennae of the cabbage armyworm Mamestra brassicae–CYP4L4 and CYP4S4– which encode microsomal P450s. CYP4S4 expression is restricted to the antennae, whereas CYP4L4 is also found in the proboscis and legs. Moreover, the two genes are strongly expressed in one type of sensory unit of the antennae – the sensilla trichodea – which are tuned to the detection of odourants. The putative function of the corresponding enzymes is discussed with regard to their respective expression patterns.
Acute apical periodontitis: incidence of post‐operative pain using two different root canal dressings
Authors: Fava
Published: March 2003

A clinical study using non‐vital maxillary central incisors exhibiting acute apical periodontitis was carried out to evaluate the incidence of post‐operative pain after biomechanical preparation and dressing with a calcium‐hydroxide paste or a corticosteroid‐antibiotic solution. Sixty teeth from 48 patients were prepared and dressed on the first visit and re‐evaluated clinically 7 days later. No difference was observed in the incidence of post‐operative pain between the two groups.
Incidence of four canals in root‐canal‐treated mandibular first molars in a Saudi Arabian sub‐population
Authors: Al‐Nazhan S.
Published: March 2003

Aim The aim of this in‐vivo study was to assess the incidence of three roots and four root canals in clinical cases of root‐treated mandibular first molars in a Saudi Arabian population.

Methodology A clinical study of 251 root‐canal treated permanent mandibular first molars was conducted The teeth were examined clinically and radiographically.

Results The results showed that 5.97% of the examined teeth had three roots, 57.76% had four root canals (two mesial and two distal) and 42.3% had three root canals (two mesial and one distal).

Conclusions The occurrence of four root canals in the mandibular first molar of a Saudi Arabian subpopulation was high.
The effects of Ledermix paste on discolouration of mature teeth
Authors: Kim S. T. , Abbott P. V. , McGinley P.
Published: March 2003

Aim

The aims of this study were to: (i) investigate the effects of Ledermix paste as an intracanal medicament on discolouration of mature teeth, (ii) examine whether the discolouring effects were related to the method of application, and (iii) examine the effects of sunlight upon discolouration of mature teeth.

Methodology

The root canals of 45 mature extracted human teeth were prepared and filled with either Ledermix paste, calcium hydroxide [Ca(OH)2], or saline. In group 1, Ledermix paste was placed apical to the cemento‐enamel junction (CEJ), whilst in groups 2 and 3 the paste filled the entire pulp chamber and root canals. In group 4, a Ca(OH)2 and methyl cellulose paste and, in group 5, saline (control) were allowed to fill the pulp chamber and the root canals. Group 3 teeth were kept in the dark and the other groups were exposed to indirect sunlight for 12 weeks.

Results

After 12 weeks, sunlight exposure had caused dark grey‐brown staining of the teeth in the Ledermix groups, but this did not occur when the teeth were kept in the dark. More severe staining was noted when Ledermix paste filled the pulp chamber than when the paste was restricted to below the CEJ.

Conclusions

It was concluded that Ledermix paste may cause discolouration of teeth. Such effects can be minimized if placement of the paste is restricted to below the gingival margin. Clinicians should ensure that Ledermix paste is not left on the walls of access cavities.
In vitro evidence that lipopolysaccharide of an oral pathogen leaks from root‐end filled teeth
Authors: Laghios C. D. , Cutler C. W. , Gutmann J. L.
Published: March 2003

Aim

The ability to achieve a complete apical seal of the root canal system is thought to be important in the success of non‐surgical and surgical endodontics. The aim of this study was to establish whether or not root‐end filled teeth allow leakage of lipopolysaccharide (LPS) from a known oral pathogen in vitro.

Methodology

Lipopolysaccharide (LPS) from a virulent strain of Porphyromonas gingivalis (P. gingivalis) (A7436 from patient with refractory periodontitis), was isolated by the Westphall and Jann technique, dialysed extensively, lyophilyzed, resuspended in distilled water and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Root canals from 10 teeth were instrumented endodontically and the apical 3 mm of resected roots were filled with gutta‐percha. The teeth were mounted in 12 mL polypropylene vials by using sticky wax and root surfaces were covered with two layers of nail varnish. Teeth were filled with 3.3 mg mL–1 LPS and the vials filled with 11 mL of Tris Buffered Saline (TBS) containing 0.05% sodium azide. Both positive and negative controls were run in parallel with the experimental specimens. Aliquots were removed each day and subjected to slot blot analysis to quantitate the amount of LPS that had leaked into the bottom of the vials. The density of slots was analyzed using a laser densitometer and regression analysis was used to generate a standard curve, confidence intervals and experimental values.

Results

The data indicated that teeth obturated apically with gutta‐percha leaked, whilst no LPS leakage was detected in teeth covered completely with nail varnish (P < 0.05).

Conclusions

In vitro teeth with gutta‐percha root‐end fills can permit leakage of LPS from an identified oral pathogen.
Efficacy of Quantec rotary instruments for gutta‐percha removal
Authors: Bramante C. M. , Betti L. V.
Published: March 2003

Aim

The purpose of this study was to evaluate Quantec SC rotary instruments for removal of gutta‐percha during retreatment of straight root canals.

Methodology

The root canals of 30 central incisors were instrumented and obturated before the teeth were randomly divided into three groups of 10 specimens each. Quantec SC rotary instruments and a 16 : 1 reduction handpiece powered by an electric motor were then used to remove the gutta‐percha and sealer from canals. A different speed was used in each group (group 1, 350 r.p.m.; group 2, 700 r.p.m.; and group 3, 1500 r.p.m.). The followings factors were evaluated: time taken to reach working length, time for gutta‐percha removal, total time, apically extruded material during filling removal and number of fractured instruments. Radiographs were taken after the filling removal and after the canal wall cleanliness was evaluated. The teeth were grooved longitudinally, divided, and the walls of each half were evaluated visually for cleanliness. They were then digitized using a scanner and the residual debris measured.

Results

The group in which a speed of 1500 r.p.m. was used had significantly faster treatment than the other groups. The amount of apically extruded material was not significantly different between groups. The only significant difference between groups for canal cleanliness was the middle third by radiographic evaluation: the group of 350 r.p.m. had larger amount of debris than the others. In group 1, six instruments fractured; in group 2, four instruments fractured; and in group 3, one instrument fractured.

Conclusions

Cleanliness and residual debris were equivalent for each group, but the use of 1500 r.p.m. speed was more rapid and fewer instruments fractured.
The standardized‐taper root canal preparation – Part 1. Concepts for variably tapered shaping instruments
Authors: Buchanan L. S.
Published: March 2003

Buchanan LS. The standardized‐taper root canal preparation – Part 1. Concepts for variably tapered shaping instruments. International Endodontic Journal, 33, 516–529, 2000.

Aim

To introduce the concept of variable taper instruments for predictable and ergonomic root canal preparation, and demonstrate the design features of Greater Taper files.

Optimal root canal shaping is difficult to practice and teach with traditional instruments. Instrument sequences are complex, with up to 18 instruments and 63 procedural stages, providing almost limitless scope for poor results and iatrogenic error.

In the first of six articles, Dr Buchanan describes the Variable Taper concept, which grew from such frustrations, and represents a new concept in file design. Milled from NiTi alloys in tapers of 0.06, 0.08, 0.10 and 0.12 mm mm−1, with accessory files for wide canals, their design embodies the key shaping features of adequate coronal enlargement, full deep shape, and predictable apical resistance form case after case. The ease and simplicity of their use is described, and enhanced cleaning and obturation outcomes discussed in relation to their unique design features.

Key learning points

Canal preparation is difficult to practice and teach with traditional K‐files and Gates Glidden drills.

Variable Taper files are designed to offer the optimal preparation features of adequate (not excessive) coronal enlargement, full deep shape, and apical resistance form in a simple instrument sequence.

Variable Taper technique is simple to master, and offers predictable cleaning and obturation outcomes, even in inexperienced hands.
Root canal systems of the mandibular and maxillary first permanent molar teeth of South Asian Pakistanis
Authors: Wasti F. , Shearer A. C. , Wilson N. H. F.
Published: March 2003

Aim

The purpose of the present ex vivo study was to investigate variations in the root canal systems of mandibular and maxillary first permanent molar teeth of South Asian Pakistanis.

Methodology

The root canal systems of a sample of 30 mandibular and 30 maxillary first permanent molar teeth extracted from South Asian Pakistanis were studied using a clearing technique.

Results

The mesial roots of the mandibular molar teeth typically presented with two canals (97%) of type II, IV or VI configuration. The distal roots of these teeth presented with a single canal (50%) of type I or V configuration or with two canals (50%) of type II, IV or VI. The prevalence of four root canals in two‐rooted mandibular first permanent molar teeth was 47%. In maxillary molar teeth the mesial roots with a single canal (47%) were type I or type V; those with two canals (53%) were type II, IV or type VI. The distal and palatal roots that presented as a single canal (100%) were type I or type V configuration. The prevalence of four root canals in three‐rooted maxillary first permanent molar teeth was 53%.

Conclusion

It is concluded that four root canals in mandibular and maxillary first permanent molar teeth of South Asian Pakistanis is a common occurrence. The distribution of the different configurations of root canal systems in this population differed from that in Caucasian groups, suggesting that variations in root canal systems may be attributed to racial divergence.