bluuebunny/crossref_metadata_2025 huggingface dataset:
Opposing cytokine‐specific effects of all trans‐retinoic acid on the activation and expression of signal transducer and activator of transcription (STAT)‐1 in THP‐1 cells
Authors: Chen Qiuyan , Ma Yifan , Ross A. Catharine
Published: March 2003

The regulation of signal transducer and activator of transcription‐1 (STAT‐1) by cytokines and all‐trans‐retinoic acid (RA) was investigated in THP‐1 monocytic cells cultured with RA and stimulated with lipopolysaccharide (LPS), tumour necrosis factor‐α (TNF‐α), interferon‐β (IFN‐β), and IFN‐γ, individually or in combinations. While RA (10−8 m) alone did not alter STAT‐1 activation or expression in THP‐1 cells, RA enhanced or prolonged STAT‐1 activation (tyrosine 701 phosphorylation) and gene expression (mRNA and protein) induced by either IFN‐β or IFN‐γ. However, in contrast, RA reduced STAT‐1 activation and gene expression induced by LPS and/or TNF‐α by about 50–70%, and lowered in vitro DNA binding activity to both a STAT‐1 consensus element and a nuclear factor kappa B (NFκB) binding element. These results imply that RA can significantly rebalance STAT‐1‐dependent responses, and that one of the mechanisms may be through the inhibition of the NFκB pathway.
Cross‐presentation of cell‐associated antigens by CD8α+ dendritic cells is attributable to their ability to internalize dead cells
Authors: Schulz Oliver , Reis e Sousa Caetano
Published: March 2003

In the mouse, cross‐presentation is an exclusive property of the CD8α+ subset of dendritic cells (DC) but the basis for this selectivity remains unclear. Here we report that splenic CD8α+ DC are much superior to other DC subsets in internalizing dying cells in vitro. In contrast, CD8α+, CD8α– CD4+ and CD8α– CD4– DC subsets phagocytose bacteria or latex beads to a similar extent. Although CD8α+ DC are better than CD4+ DC at presenting ovalbumin (OVA)‐loaded splenocytes to naïve OT‐I T lymphocytes, CD4+ DC are better at presenting OVA‐expressing Escherichia coli to the same T cells. In both cases, presentation is abrogated by lactacystin. These results show that both splenic CD8α+ and CD8α– DC can present exogenous antigens on major histocompatibility complex (MHC) class I via a proteasome‐dependent pathway and suggest that the specialized cross‐presenting function of CD8α+ DC is a result of their ability to endocytose dying cells rather than a unique pathway for handling endosomal contents.
Haptoglobin directly affects T cells and suppresses T helper cell type 2 cytokine release
Authors: Arredouani M. , Matthijs P. , Van Hoeyveld E. , Kasran A. , Baumann H. , Ceuppens J. L. , Stevens E.
Published: March 2003

T helper cell type 1 (Th1) and type 2 (Th2) immune responses are characterized by a different pattern of cytokine expression following T‐cell activation. Alterations of the ratio of Th1 to Th2 cells are important determinants of susceptibility to viral and parasitic infections, allergies, anti‐tumour responses, and autoimmunity. In this work we bring new evidence for an effect of haptoglobin (Hp), a positive acute‐phase protein, on T‐lymphocyte functions. We show that Hp specifically interacts with both resting and activated CD4+ and CD8+ T cells. This specific binding results in a strong suppression of induced T‐cell proliferation. In addition, Hp exhibits a strong in vitro inhibitory effect on Th2 cytokine release, while the production of interferon‐γ (IFN‐γ) and interleukin‐2 (IL‐2) is only slightly inhibited at high Hp doses. As a result, the presence of Hp promotes Th1 activation over Th2 activation in vivo as evidenced in Hp‐deficient mice. Anti‐CD3 monoclonal antibody injection indeed resulted in predominant IL‐4 production in Hp−/− mice, in contrast to predominant IFN‐γ production in Hp+/+ mice. We conclude that Hp plays a modulating role on the Th1/Th2 balance by promoting a dominant Th1 cellular response. This points to a role of acute‐phase proteins in balancing immune responses.
Organizational change and virtual teams: strategic and operational integration
Authors: Jackson Paul J.
Published: March 2003

Abstract. This paper focuses on the challenges involved in integrating virtual teams strategically and operationally into organizational systems. It argues that an understanding of virtual team integration must consider both the context within which virtual teams are being introduced and processes involved in their implementation. Such an approach demands that we consider the strategic rationales involved in teamworking generally, and virtual teams in particular. By looking at change processes, attention is drawn to the social, cultural and political dynamics that affect the implementation and operation of virtual teams. This analysis is used to suggest further avenues for research on virtual teams, as well as pointing to practical considerations
Group support systems in Hong Kong: an action research project
Authors: Davison Robert , Vogel Doug
Published: March 2003

Abstract.

The last dozen years have seen a considerable investment of resources into the research and development of group support systems (GSS) technology. This paper describes how GSS was used to support a process improvement project in a Hong Kong accounting firm. Although the project encountered many difficulties, the application of action research facilitated the adaptation of the GSS to the shifting circumstances, and the project was successfully completed. A variety of lessons concerning the use of GSS are presented, while increased use of action research in complex organizational contexts is recommended.
Asynchronous and distributed process improvement: the role of collaborative technologies
Authors: Kock Ned
Published: March 2003

Abstract. The recent proliferation of low‐cost computer networks has driven the development of a new type of organization, in which geographical and time constraints to collaboration among process teams have been gradually removed. As these organizations have to cope with a fast pace of change, they rely increasingly on distributed and asynchronous process improvement (PI) groups to redesign their processes. Yet little is known about the effects of asynchronous group support systems (GSSs) on PI groups. We investigate the effects of asynchronous GSSs on PI groups through a two‐stage action research study of 38 such groups in three organizations, one in Brazil and two in New Zealand, over 4 years and 4 months. Different PI groups voluntarily conducted all, part or none of their communication through an e‐mail conferencing (EC) tool. The research suggests that EC support causes a decrease in the organizational costs associated with PI groups, which, combined with an increase in the number of possible simultaneous PI groups, generates an increase in overall organizational PI efficiency. The research also suggests a neutral overall effect of EC support on PI group outcome quality. Two explanatory causal models summarizing these effects are developed, and implications for research and industry practice are discussed.
The user–developer communication process: a critical case study
Authors: Gallivan Michael J. , Keil Mark
Published: March 2003

Abstract. Although user participation in systems development is widely believed to have positive impacts on user acceptance, it does not guarantee success and there is still much that we do not know about how and why user participation sometimes delivers positive benefits, but not always. Much of the prior research on user participation assumes that user–developer communication will ensure that the resulting system will be designed to meet users’ needs and will be accepted by them. The nature and quality of the communication between users and developers, however, remains an understudied aspect of user participation. In this paper, we focus on the user–developer communication process. We propose a process model that delineates four stages of communication between users and software developers, and we argue that these stages must occur for user participation to lead to effective outcomes. To illustrate our model, we apply it to analyse a ‘critical case study’ of a software project that failed despite high levels of user involvement. We show that when ‘communication lapses’ occurred in several of the user–developer communication stages, developers failed to be informed regarding the underlying reasons that users avoided the system. Based on the insights from this case study, we advise researchers and practitioners how to leverage the potential benefits of user participation, rather than take them for granted.
In vivo regulation of tissue‐specific and LPS‐inducible expression of the Drosophila Cecropin genes
Authors: Roos E. , Björklund G. , Engström Y.
Published: March 2003

The inducible production of antibacterial cecropins in Drosophila fat body and haemocytes is controlled at the level of transcriptional induction. We demonstrate using germ‐line transformation that a short, highly conserved, DNA region, including the insect κB motif, is necessary for tissue‐specific expression in larvae and adults. Quantitative measurements of reporter gene activity in extracts from transgenic larvae confirmed the requirement of this proximal region for LPS‐inducible expression in vivo. Transient expression in a blood cell line indicates the existence of positively acting elements further upstream of the conserved region. Furthermore, our in vivo data suggests that the distal upstream region contains negatively acting element(s).
Evidence for two juvenile hormone esterase‐related genes in the Colorado potato beetle
Authors: Vermunt A. M. W. , Koopmanschap A. B. , Vlak J. M. , de Kort C. A. D.
Published: March 2003

Juvenile hormone esterase (JHE) activity in the haemolymph of the Colorado potato beetle is necessary to initiate pupation in larvae as well as diapause in adults. The enzyme appears in the haemolymph as a dimer consisting of two 57 kDa subunits. The sequence of an encoding cDNA, JHE.A, is distinct from lepidopteran JHEs. In this study, RT‐PCR using primers designed on the basis of the 5′‐ and 3′‐ends of the coding region revealed the existence of a related gene, JHE.B. The presence of two JHE‐related genes was also shown by PCR amplification on genomic DNA from different individual beetles followed by restriction enzyme analysis. Both forms, probably paralogues, were transcribed since they could be amplified on messenger RNA from fat bodies. The size of the PCR products generated with mRNA and genomic DNA were both 1.6 kb, suggesting the absence of introns in the genomic JHE coding sequence. The sequence of a genomic clone, which encoded JHE.B, was 77% identical and 82% similar in amino acids compared to JHE.A. No introns were found in the coding sequence of these coleopteran JHE‐related genes, in contrast to lepidopteran JHE genes. Southern blot analysis of digested genomic DNA confirmed the presence of two JHE‐related genes.
Tissue tropism, transmission and expression of foreign genes in vivo in midgut symbionts of tsetse flies
Authors: Cheng Q. , Aksoy S.
Published: March 2003

Tsetse flies (Diptera: Glossinidae) harbour three different symbiotic organisms in addition to the pathogenic African trypanosomes they transmit. The two gut‐associated symbionts (primary, P; secondary, S) are enteric and are nutritionally required, whereas the third microorganism Wolbachia (family Rickettsiaceae) affects the reproductive biology of the insects it infects. The bacteriome‐associated P‐symbiont (Wigglesworthia glossinidia) displays a concordant phylogeny with its host tsetse species, whereas midgut S‐symbionts characterized from distant tsetse have identical 16S rDNA sequences and therefore may either represent recent independent acquisitions or horizontal transfer between species. The S‐symbionts have been cultured in vitro and a genetic transformation system has been developed. Here we report on their density and tissue tropism in different species (G. m. morsitans, G. p. palpalis, G. austeni and G. brevipalpis) and on their maternal route of transmission to tsetse progeny. Using a bacterium‐specific PCR‐assay, the S‐symbionts were found primarily in the midgut, haemolymph, milk gland and in G. palpalis also in salivary glands of teneral flies. In older flies these infections were found to spread to other tissues including muscle, testes and fat body. The S‐symbionts were transformed to express the marker gene product, Green Fluorescent Protein (GFP) in vitro. When the recombinant symbionts were introduced into the haemoceal of fertile female flies via intrathoracic microinjection, they were detected in the intrauterine progeny, indicating that haemolymph may provide a possible route for their transmission. The implications of these results for symbiont‐host interactions and for transgenic strategies in tsetse are discussed.
Characterization of a carboxypeptidase A gene from the mosquito, Aedes aegypti
Authors: Edwards M. J. , Moskalyk L. A. , Donelly‐Doman M. , Vlaskova M. , Noriega F. G. , Walker V. K. , Jacobs‐Lorena M.
Published: March 2003

A gut‐specific carboxypeptidase A gene (AeCPA) from the mosquito, Aedes aegypti, was cloned and characterized. The gene has an open reading frame that predicts a protein of 427 amino acids, 61% of which are identical to an Anopheles gambiae carboxypeptidase A sequence. AeCPA messenger RNA (mRNA) was not detected during larval and pupal development. In situ hybridization experiments indicated that AeCPA mRNA is expressed by posterior midgut epithelial cells. In sharp contrast to An. gambiae carboxypeptidase A gene expression, AeCPA mRNA accumulates to high levels only late (≈ 16–24 h) after ingestion of a blood meal. The temporal profile of AeCPA gene induction is similar to that of Ae. aegypti late trypsin, suggesting the existence of common regulatory elements.
Expression patterns of the larval and adult hexamerin genes of Musca domestica
Authors: Capurro M. de L. , Moreira‐Ferro C. K. , Marinotti O. , James A. A. , De Bianchi A. G.
Published: March 2003

Hexamerins are proteins found in high abundance in the haemolymph of larval and adult insects. The expression patterns of the genes encoding the house fly, Musca domestica, hexamerins were determined by Northern analyses using cDNAs as probes. A cDNA, A1, hybridized to a fat body‐specific messenger RNA (mRNA) which is detectable in larvae until pupation. Antibodies raised to the larval‐specific hexamerin, Hex‐L, bind recombinant protein encoded by a 5′ rapid amplification of cDNA ends (
race) product of A1, A2, indicating that the A cDNAs likely represent the genes encoding Hex‐L. The F1, F2 and F3 cDNAs, corresponding to genes encoding an adult, female‐enriched hexamerin, Hex‐F, hybridized with an mRNA isolated from protein‐fed females which has a temporal expression profile similar to that observed for the accumulation of Hex‐F. Furthermore, expression of the mRNAs hybridizing to the F cDNAs is correlated with the abundance of Hex‐F protein during the gonotrophic cycles. The mRNA transcription profiles indicate that the Hex‐L and Hex‐F genes are regulated in a sex‐, tissue‐ and developmental phase‐dependent manner. This stage‐specific expression of hexamerins contrasts with the expression patterns of hexamerins seen in other insects. The conceptual translation products of larval hexamerin cDNAs showed identity with larval serum protein 1 (LSP1)‐type hexamerins while the deduced products of the female hexamerin cDNAs showed the highest identity with LSP2‐type hexamerins. Genomic analyses showed that the larval hexamerin and female hexamerin genes from M. domestica belong to two distinct multigenic families.
Ribosomal DNA internal transcribed spacer (ITS2) sequences differentiate Anopheles funestus and An. rivulorum, and uncover a cryptic taxon
Authors: Hackett B. J. , Gimnig J. , Guelbeogo W. , Costantini C. , Koekemoer L. L. , Coetzee M. , Collins F. H. , Besansky N. J.
Published: March 2003

Differentiation among the closely related Afrotropical species comprising the Funestus Group is difficult by traditional taxonomic measures. Anopheles rivulorum is the second most abundant and widespread species in the Funestus Group, and is occasionally collected indoors along with the dominant member and major malaria vector, An. funestus. The prospect of misidentification of An. rivulorum as An. funestus prompted the development of a rapid, polymerase chain reaction (PCR)‐based method for identifying these two species. The ribosomal internal transcribed spacer 2 (ITS2) was amplified from thirty‐five specimens of An. rivulorum collected from the extremes of its range: Eastern Africa (Kenya), Southern Africa (South Africa) and Western Africa (Burkina Faso). The ITS2 region of An. rivulorum (≈ 380 bp) is sufficiently different in size from the ITS2 of An. funestus (≈ 700 bp) that these species can be distinguished by agarose gel electrophoresis of PCR products without further manipulation. Comparison of the An. rivulorum and An. funestus ITS2 nucleotide sequences revealed such extensive divergence that meaningful alignment was impossible, except for a 25 bp island near the 5′ end. Intraspecific sequence comparisons revealed no variation among An. rivulorum individuals collected from the same country. However, sequence divergence was 2% between specimens from South Africa and Kenya, and nearly tenfold higher (≈ 19%) between specimens from Burkina Faso and either South Africa or Kenya, an unprecedented level of intraspecific ITS2 divergence in Anopheles. Taken together, these data suggest that the Burkina Faso sample is not An. rivulorum, but rather a cryptic taxon within the Funestus Group.
Microsatellite DNA markers reveal genetic differentiation among populations of Glossina palpalis gambiensis collected in the agro‐pastoral zone of Sideradougou, Burkina Faso
Authors: Solano P. , De La Rocque S. , De Meeus T. , Cuny G. , Duvallet G. , Cuisance D.
Published: March 2003

Intraspecific genetic variability of Glossina palpalis gambiensis in the area of Sideradougou, Burkina Faso, was studied using polymorphic microsatellite DNA markers. This genetic study was combined with other epidemiological information on the same tsetse: bloodmeal identification, dissection of tsetse and molecular characterization of the trypanosomes detected. There was significant genetic differentiation among flies caught only a few kilometers apart, within the same riverine habitat. These distinct subpopulations were also differentially infected by trypanosomes. In part of the study area, a Factorial Correspondence Analysis undertaken on the genotypes allowed us to detect a Wahlund effect, suggesting the presence of tsetse originating from different source populations coming from two distinct drainage systems. The apparent structuring of populations of G. palpalis gambiensis is discussed relative to appropriate strategies to control African Trypanosomosis.
The piggyBac transposon mediates germ‐line transformation in the Oriental fruit fly and closely related elements exist in its genome
Authors: Handler A. M. , McCombs S. D.
Published: March 2003

Germ‐line transformation of a white eye strain of the Oriental fruit fly, Bactrocera dorsalis, was achieved with the piggyBac vector, derived from a transposon originally isolated from the cabbage looper moth, Trichoplusia ni. The vector was marked with the medfly white+ gene cDNA, and three transgenic lines were identified at a frequency of approximately 2% per fertile G0. Vector integrations were verified by Southern DNA hybridization, which also revealed the presence of endogenous genomic elements closely related to piggyBac. Approximately 10–20 elements per genome were evident in several B. dorsalis strains, and sequence analysis of 1.5 kb gene amplification products from two wild strains and the white eye host strain indicated 95% nucleotide and 92% amino acid sequence identity among resident elements and the T. ni element. PiggyBac was not evident by hybridization in other tephritid species, or insects previously transformed with the transposon. This is the first discovery of piggyBac beyond T. ni, and its existence in a distantly related species has important implications for the practical use of the vector and insects transformed with it.
Wolbachiainfections in native and introduced populations of fire ants (Solenopsisspp.)
Authors: Shoemaker D. DeWayne , Ross Kenneth G. , Keller L. , Vargo E. L. , Werren John H.
Published: March 2003

Wolbachiaare cytoplasmically inherited bacteria that induce a variety of effects with fitness consequences on host arthropods, including cytoplasmic incompatibility, parthenogenesis, male‐killing and feminization. We report here the presence ofWolbachiain native South American populations of the fire antSolenopsisinvicta, but the apparent absence of the bacteria in introduced populations of this pest species in the USA. TheWolbachiastrains in nativeS. invictaare of two divergent types (A and B), and the frequency of infection varies dramatically between geographical regions and social forms of this host. Survey data reveal thatWolbachiaalso are found in other native fire ant species within theSolenopsis saevissimaspecies complex from South America, includingS. richteri. This latter species also has been introduced in the USA, where it lacksWolbachia. Sequence data reveal complete phylogenetic concordance between mtDNA haplotype inS.invictaandWolbachiainfection type (A or B). In addition, the mtDNA and associated group AWolbachiastrain inS.invictaare more closely related to the mtDNA andWolbachiastrain found inS. richterithan they are to the mtDNA and associated group BWolbachiainS. invicta. These data are consistent with historical introgression ofS. richtericytoplasmic elements intoS.invictapopulations, resulting in enhanced infection and mtDNA polymorphisms inS. invicta.Wolbachiamay have significant fitness effects on these hosts (either directly or by cytoplasmic incompatibility) and therefore these microbes potentially could be used in biological control programmes to suppress introduced fire ant populations.
Molecular cloning of two prophenoloxidase genes from the mosquito Aedes aegypti
Authors: Taft A. S. , Chen C. C. , Li J. , Christensen B. M.
Published: March 2003

The biosynthesis of melanotic materials is an important process in the life of a mosquito. Melanin production is critical for many diverse processes such as egg chorion tanning, cuticular sclerotization, and melanotic encapsulation of metazoan parasites. Prophenoloxidase plays a critical role in this biochemical cascade. Two cDNAs, one full length and one partial clone, and two genomic clones encoding prophenoloxidase (pro‐PO) were isolated from the yellow fever mosquito, Aedes aegypti. The full‐length cDNA, pAaProPO1, is 2286 bp long with a 2055 bp open reading frame encoding a 685 amino acid protein that shares 89% identity with Armigeres subalbatus pro‐PO. It contains two putative copper binding domains (amino acids 197–243 and 346–423) that are homologous to other insect pro‐POs. AaProPO1 messenger RNA (mRNA) was detected by reverse transcription polymerase chain reaction (RT‐PCR) only from third‐stage larvae and not in adult mosquitoes after blood feeding, during the melanotic encapsulation of Dirofilaria immitis microfilariae or following exposure to bacteria. A 750 bp fragment of the second cDNA (pAaProPO2) was cloned using RT‐PCR from mRNA obtained from 14‐day postovipostional eggs. AaProPO2 mRNA was not found in any other life stages, and may be in low abundance or transiently expressed. AaProPO2 and AaProPO1 each contain three introns that are 60, 68 and 58 bp and 61, 69 and 59 bp long, respectively, and the intron sequences of these two genes are not similar.
Hermes‐mediated germ‐line transformation of the Mediterranean fruit fly Ceratitis capitata
Authors: Michel K. , Stamenova A. , Pinkerton A. C. , Franz G. , Robinson A. S. , Gariou‐Papalexiou A. , Zacharopoulou A. , O'Brochta D. A. , Atkinson P. W.
Published: March 2003

We report the use of the Hermes transposable element for germ‐line transformation of the Mediterranean fruit fly, Ceratitis capitata. Hermes was able to genetically transform this insect at an estimated frequency between 0.6 and 1.1%, which is comparable to the transformation frequencies obtained for this species when using other transposable elements. Hermes integrates into the medfly genome by a cut‐and‐paste mechanism and the sequences integrated into the genome are delimited by the terminal nucleotides of the Hermes inverted terminal repeats. Integration resulted in the generation of 8 bp target site duplications, the sequences of which conformed to the target site duplications generated by hAT element transposition in insects. The Hermes element is one additional genetic tool that can be deployed in manipulating and characterizing the medfly genome.
Expression and immunolocalization of a Boophilus microplus cathepsin L‐like enzyme
Authors: Renard Gaby , Lara Flávio Alves , De Cardoso Felipe Cardoso , Miguens Flávio Costa , Dansa‐Petretski Marílvia , Termignoni Carlos , Masuda Aoi
Published: March 2003

Efforts are being undertaken to control tick infestations that cause important economic losses. A cathepsin L‐like endopeptidase of Boophilus microplus was expressed in Escherichia coli; the recombinant enzyme was capable of hydrolysing gelatin, tick vitellin and bovine haemoglobin. In this paper we focus on the expression and local of synthesis of this enzyme in the tick. RT‐PCR experiments showed that this endopeptidase is transcribed in the gut of partially engorged tick females. In immunoblotting, polyclonal antibodies against the recombinant enzyme reacted with proteins of larvae older than 5 days, of fully and partially engorged female gut. In immunolocalization experiments the enzyme was localized in probable secretory cells of the gut. Based on our findings we postulate that BmCL1 may be involved in haemoglobin degradation in the B. microplus gut. This enzyme may be used as target for the control of this parasite.
Linkage analysis of maternal EST cDNA clones covering all twenty‐eight chromosomes in the silkworm, Bombyx mori
Authors: Kadono‐Okuda K. , Kosegawa E. , Mase K. , Hara W.
Published: March 2003

A cDNA library of maternal messages was constructed from non‐fertilized Bombyx mori eggs collected just after oviposition without mating. cDNA clones were identified by their nucleotide sequences and evaluated as probes for RFLP linkage analysis. Back crossed F1 segregants – by crossing an F1 female (RF02 × RF50) and a male (RF02) – were used, which takes advantage of the phenomenon that no crossing over occurs in Bombyx females. Fifteen ordered BF1 segregants gave either homozygous (homo) or heterozygous (hetero) RFLP patterns with each cDNA probe. cDNA probes on the same linkage group gave the same homo/hetero order. One hundred and fifty one out of 248 cDNA clones showed polymorphisms between RF02 and RF50, and were therefore suitable as probes for RFLP linkage analysis in the present BF1 cross. They were sorted into twenty‐seven linkage groups and one independent group, by the homo/hetero pattern matrix, covering all twenty‐eight chromosomes in B. mori.
Expression of proteasome subunit isoforms during spermatogenesis in Drosophila melanogaster
Authors: Ma J. , Katz E. , Belote J. M.
Published: March 2003

In this study, we sought to identify and characterize all the proteasome genes of Drosophila melanogaster. Earlier work led to the identification of two genes encoding α4‐type 20S proteasome subunit isoforms that are expressed exclusively in the male germline. Here we extend these results and show that six of the 20S proteasome subunits, and four of the 19S regulatory cap subunits, have gene duplications encoding male‐specific isoforms. More detailed analyses of two of these male‐specific subunits (Prosα3T and Prosα6T), using GFP‐tagged reporter transgenes, revealed that they are predominantly localized to the nucleus at later stages of spermatogenesis and are present there in mature, motile sperm. These results suggest a possible role of a ‘spermatogenesis‐specific’ proteasome in sperm differentiation and/or function.
Cloning and characterization of a 70 kDa heat shock cognate gene (HSC70) from two species of Chironomus
Authors: Karouna‐Renier N. K. , Yang W.‐J. , Rao K. Ranga
Published: March 2003

In the present study we carried out the isolation and characterization of an HSC70 gene from two midges, Chironomus tentans and C. yoshimatsui. The HSC70 cDNAs are approximately 2424 (C. tentans) and 2464 bp (C. yoshimatsui) long, and contain 1950 and 1956 bp open reading frames, respectively. Analysis of genomic DNA revealed the presence of two introns in these genes. The 5′ untranslated regions of the HSC70 genes are adenosine‐rich, a feature found in inducible HSP70 genes. The nucleotide and amino acid sequences exhibit high identity with cytosolic HSC70s from other Dipterans. Northern hybridization indicated that HSC70 is expressed at all developmental stages, from embryo to adult, and Southern hybridization confirmed the presence of multiple HSP70 genes in Chironomus.
Isolation and expression of a sterol carrier protein‐2 gene from the yellow fever mosquito, Aedes aegypti
Authors: Krebs K. C. , Lan Q.
Published: March 2003

Trafficking of cholesterol in insects is a very important process due to the fact that insects depend on dietary cholesterol to fulfil their physiological needs. We identified a putative mosquito sterol carrier protein‐2 (SCP‐2) cDNA from fourth instar subtracted cDNA library. The AeSCP‐2 protein has high degree homology in the sterol transfer domain to both rat and human SCP‐2. Transcripts of AeSCP‐2 in fourth instars were detected strongly in the midgut, and weakly in the head and hindgut. In the early pupae, AeSCP‐2 transcription was observed in the thorax, head and body wall of abdomen, but not in the gut.

The interaction of mosquito sterol carrier protein‐2 (AeSCP‐2) with cholesterol was examined. The Kd of purified recombinant AeSCP‐2 to cholesterol was 5.6 ± 0.6 × 10−9 m using radiolabelled cholesterol‐binding assay. The results suggest that AeSCP‐2 has high affinity to cholesterol and may function as a carrier protein in mosquitoes.
Characterization of three Toll‐like genes from mosquito Aedes aegypti
Authors: Luna C. , Hoa N. T. , Zhang J. , Kanzok S. M. , Brown S. E. , Imler J.‐L. , Knudson D. L. , Zheng L.
Published: March 2003

Three Toll‐related genes (AeToll1A, AeToll1B and AeToll5) were cloned and characterized from the yellow fever vector mosquito, Aedes aegypti. All three genes exhibited high levels of amino acid sequence similarity with Drosophila melanogaster (Dm)Toll1 and DmTehao (Toll5). AeToll1A and AeToll1B are 1124 and 1076 amino acid residues long, respectively. Both contain a carboxyl extension downstream of the Toll/interleukin‐1 receptor (TIR) domain. AeToll5 is 1007 residues long and, like DmTehao, lacks the carboxyl terminal extension. Expression of these three genes was examined throughout development and after immune challenge. Both AeToll1A and AeToll5, like their Drosophila counterparts, activate transcription of drosomycin promoter in both Aedes and Drosophila cell lines. Deletion of the carboxyl extension of AeToll1A did not result in a further elevated level of the antifungal response. The intracellular signalling process appears to be species specific based on two observations. (1) DmToll is completely inactive in an Aedes cell line, suggesting a higher specificity requirement for DmToll in the intracellular signalling process. (2) Only one of three amino acid residues essential for DmToll function is required for AeToll1A function.
Application of 16S rDNA‐DGGE to examine the microbial ecology associated with a social wasp Vespula germanica
Authors: Reeson A. F. , Jankovic T. , Kasper M. L. , Rogers S. , Austin A. D.
Published: March 2003

Invertebrates host numerous bacteria, with interactions ranging from pathogenesis to symbiosis. While certain symbiotic relationships have been well studied, little is known about the dynamics of these bacterial communities. Denaturing gradient gel electrophoresis (DGGE) was used to examine the bacterial microflora associated with the eusocial wasp Vespula germanica. DGGE profiles of larval guts revealed a variable microflora, suggesting that V. germanica is not dependent on a particular suite of mutualists. The variation in profiles was not related to season, nest size or macrohabitat. Sequences corresponding to Lactococcus, Lactobacillus, a novel Leuconostoc and two Rickettsiella grylli strains were obtained. DGGE proved to be a useful technique for characterizing the wasp microflora. Given the importance of microbial communities to invertebrates, there is much to be gained from the application of such techniques.
Effect of different root canal sealers on periapical repair of teeth with chronic periradicular periodontitis
Authors: Tanomaru Filho  , Leonardo  , Silva  , Utrilla
Published: March 2003

Teeth with induced chronic periradicular periodontitis in dogs were root canal treated. After the biomechanical preparation, using K files and 5.25% sodium hypochlorite as the irrigant solution, all root canals were dressed with an antibacterial dressing based on calcium hydroxide, which was left in place for 7 days. After this time, the root canals were obturated with lateral condensation of cold gutta‐percha with either a calcium hydroxide root canal filling material (Sealapex) or a zinc oxide‐eugenol sealer (Fill Canal). After 270 days, histopathological analysis showed better apical and periapical repair in the teeth obturated with Sealapex (P < 0.05).
The influence of storage conditions on the clonogenic capacity of periodontal ligament cells: implications for tooth replantation
Authors: Lekic  , Kenny  , Barrett
Published: March 2003

Viable periodontal ligament (PL) cells are required for PL healing of avulsed teeth following replantation. If immediate replantation cannot be accomplished, the ability of PL progenitor cells to reproduce (clonogenic capacity) and recolonize the wound may be extended by prevention of desiccation and storage in physiological media. This investigation examined the effects of storage in saliva, milk, Hank's balanced salt solution (HBSS) and Eagle's medium (αMEM) on the clonogenic capacity of human PL progenitor cells at 30 and 60 min extra‐alveolar time. Twenty erupted human premolar teeth extracted as atraumatically as possible for orthodontic purposes were used in the present study. Fifteen premolars were placed immediately in freshly collected autologous saliva at room temperature, (+ 23°C) for 15 min. These 15 premolars were next divided into three groups of five and stored in either saliva, milk or HBSS at + 4°C in plastic cups surrounded by ice. The remaining five teeth served as positive controls and were immediately placed in αMEM at + 4°C. PL tissue was scraped from one‐half of the root surface with a scalpel at 30 and 60 min total extra‐alveolar duration. Cells were released from the tissue sample with a 30 min enzymatic digestion procedure and the cells from the tissue samples analyzed for clonogenic capacity. There was a reduction in clonogenic capacity with time for all protocols. Periodontal ligament cells stored in αMEM showed the least reduction between 30 and 60 min and the greatest reduction was observed for PL cells stored in saliva. The difference in clonogenic capacity following transfer from saliva to milk or HBSS was not significant at 30 min. At 60 min, cells transferred from saliva to HBSS had a statistically higher percentage of clonogenic cells than those transferred to milk (5.9% vs. 3.5%; P < 0.05). We conclude that immediate storage of avulsed teeth in autologous saliva, followed by transfer to chilled milk, preserves the presence of sufficient progenitor cells in the PL to warrant replantation and the possibility of PL healing at 60 min extra‐alveolar duration.
Effects of root canal sealers on immunocompetent cells in vitro and in vivo
Authors: Bratel  , Jontell  , Dahlgren  , Bergenholtz
Published: March 2003

Over the years of testing biocompatibility of endodontic filling materials, little attention has been paid to the potential adverse influences on the function of the immune system. Therefore, the purpose of this study was to investigate the extent to which extractable components of some commonly used root canal sealing materials (ERCS) may interfere with immunocompetent cells in vitro. The potential of these materials to cause delayed‐type hypersensitivity (DTH) was also addressed in a rat model system. Extractable components were drawn in cell culture medium from freshly mixed or set material of AH 26, Grossman's sealer, Endométhasone, and Apexit. In‐vitro assays included either spleen cells or rat pulp tissue cells that were released following enzymatic digestion with collagenase. Purified T cells for the pulpal cell assay were obtained from rat mesenteric lymph nodes. The effect of ERCS on the proliferation of concanavalin A (con A) stimulated spleen cell was measured by 3H‐hymidine incorporation. Pulpal accessory cell function was monitored by the capacity of pulpal cells, pretreated with components of ERCS, to provide signals to con A stimulated T cells. DTH was tested after subcutaneous implantation of root canal sealers (RCS) in rats and challenge by ear injection. Pretreatment of pulpal cells with low dilutions of eluates from extracted AH 26 and Endomethasone resulted in a strong reduction of the T cell proliferation rate. The effect was considerably reduced (P < 0.01) when extracts of the solid material were employed. Extracts of Grossmans' sealer and Apexit affected T cell proliferation only to a limited extent in the pulpal cell assay. In general, assays on spleen cells showed a similar profile, although increased cell division was induced by Grossman's sealer at high eluate dilutions and a concentration‐dependent decrease of cell division at lower concentrations of this material. ERCS evoked both immunosuppression and, in some instances, immunostimulation, but they did not release DTH.
Effects of sodium hypochlorite on nickel‐titanium Lightspeed® instruments
Authors: Busslinger  , Sener  , Barbakow
Published: March 2003

Sodium hypochlorite is a major irrigant in endodontics, and nickel‐titanium instruments are gaining in popularity. This paper investigated the corrosion of nickel‐titanium Lightspeed® instruments in 1% and 5 % NaOCl solutions. The instruments were immersed in ultrasonicated NaOCl solutions for varying times up to 1 h. Corrosion was determined by electrothermal absorption spectrometry in 100 μL aliquots of NaOCl. Background contamination of nickel in the 1% and 5% NaOCl solutions used was low, but high enough to interfere in detecting any increases in nickel after immersing the instruments. The amounts of titanium recorded in the 1% NaOCl solutions were insignificant. However, a statistically significant amount of titanium was detected from the Lightspeed instruments after immersion times of 30 and 60 min in 5% NaOCl. Clinically such instruments do not have an ‘in situ’ time of 30 min, and this corrosion may be considered irrelevant clinically.
Human teeth with periapical pathosis after overinstrumentation and overfilling of the root canals: a scanning electron microscopic study
Authors: Gutiérrez J. H. , Brizuela C. , Villota E.
Published: March 2003

Thirty‐five human teeth with infected root canals were overinstrumented and overfilled approximately 45 min after their extraction. The experimental teeth were enlarged up to size 40, and the overinstrumentation and overfilling were checked with the aid of a magnifying glass. The specimens were fixed in glutaraldehyde plus sodium cacodylate solutions and prepared for scanning electron microscope examination. Bacteria were detected on the flute of the files and mostly at the root apices around the main foramen, remaining firmly attached to resorptive lacunae, in spite of the fact that the apices had undergone great changes, including fracture or zipping. A control group consisting of 10 human teeth root canals containing vital pulps were also overinstrumented and overfilled. No bacteria were detected on the flutes of the files, at the apices, or on the extruded master cone overfilling these samples.
Canal configuration of the mesiobuccal root of the maxillary first molar of a Japanese sub‐population
Authors: Weine F. S. , Hayami S. , Hata G. , Toda T.
Published: March 2003

Aim The aim of this study was to determine the percentage of anatomical canal configurations of the mesiobuccal root of the maxillary first molar in Japanese patients.

Methodology Three hundred teeth were obtained from general dentists who knew absolutely that they were extracted from Japanese patients. The distobuccal and palatal roots were amputated for radiographic convenience. Preoperative radiographs were taken of the remaining crown and mesiobuccal root (MBR) from mesio‐distal and bucco‐palatal directions for each tooth. Routine endodontic access cavities were prepared and size 08 files were placed through the orifice into the MBR until they were seen at the apex. In some cases preparation of the canal orifice with a long shank round burr was necessary to gain access. In seven teeth, no access to the apex was possible and these teeth were eliminated. In the other 293 teeth, the MBR canal(s) was (were) enlarged up to size 15 file. If another canal opening was found at the apex a 08 file was inserted into the second opening and passed coronally. Postoperative radiographs with file(s) in place were taken from the two directions, as before.

Results Of the 293 teeth, 123 (42.0%) were Type II, 89 (30.4%) showed Type III systems and 10 (3.4%) were Type IV. Suggestions for identification and treatment of the second canal in the MBR are presented.

Conclusions The proportion of cases with two canals in the mesiobuccal root of maxillary first molars from Japanese patients was high and similar to that described from studies of other ethnic populations.
Inducible nitric oxide synthase activity by interferon‐g‐producing cells in human radicular cysts
Authors: Takeichi O. , Hayashi M. , Tsurumachi T. , Tomita T. , Ogihara H. , Ogiso B. , Saito T.
Published: March 2003

Aim In this study, the interaction of interferon‐γ‐ (IFN‐γ) and inducible nitric oxide synthase (iNOS)‐producing cells in human radicular cysts were investigated.

Methodology Inflamed periapical tissues were obtained from patients at the time of endodontic surgical treatments and were cut into two pieces. After fixing with acetone or 4% paraformaldehyde in phosphate‐buffered saline, 5‐m‐thick paraffin and cryostat sections were prepared. The paraffin sections of the inflamed tissues were evaluated histologically with haematoxylineosin stains. The specimens diagnosed as radicular cysts were then examined by immunostaining. Immunohistochemistry for iNOS and fluoresence microscopy for IFN‐γ using the cryostat sections were performed with a mixture of affinity purified human iNOS antiserum and human IFN‐γ monoclonal antibodies.

Results The results revealed that iNOS‐γ producing cells localized adjacent to IFN‐γ‐producing cells. In addition, some of iNOS‐producing cells exhibited immunoreactive IFN‐γ. On the other hand, epithelial cells showed significant levels of iNOS production, but not IFN‐γ.

Conclusions The data would suggest the possibility that iNOS production could be precisely controlled by autocrine or paracrine effects of IFN‐γ producing cells in radicular cysts and might play a pivotal role in periapical lesions. These findings are consistent with a hypothesis suggesting that NO inhibitors could be used through the root canals as a pharmacological treatment for periapical lesions.
Black‐pigmented anaerobic rods in closed periapical lesions
Authors: Bogen G. , Slots J.
Published: March 2003

Aim This study determined the frequency of Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens in 20 closed periapical lesions associated with symptomatic and asymptomatic refractory endodontic disease.

Methodology To deliniate possible oral sources of P. endodontalis, the presence of the organism was assessed in selected subgingival sites and saliva in the same study patients. Periapical samples were obtained by paper points during surgical endodontic procedures using methods designed to minimize contamination by non‐endodontic microorganisms. Subgingival plaque samples were obtained by paper points from three periodontal pockets and from the pocket of the toothassociated with the closed periapical lesion. Unstimulated saliva was collected from the surface of the soft palate. Bacterial identification was performed using a species‐specific polymerase chain reaction (PCR) detection method.

Results P. endodontalis was not identified in any periapical lesion, even though subgingival samples from eight patients (40%) revealed the P. endodontalis‐specific amplicon. P. gingivalis occurred in one periapical lesion that was associated with moderate pain. P. nigrescens, P. endodontalis and P. intermedia were not detected in any periapical lesion studied.

Conclusions Black‐pigmented anaerobic rods appear to be infrequent inhabitants of the closed periapical lesion.
The shear bond strength of glass ionomer cement sealers to bovine dentine conditioned with common endodontic irrigants
Authors: Lalh M. S. , Titley K. , Torneck C. D. , Friedman S.
Published: March 2003

Lahl MS, Titley K, Torneck CD, Friedman S. The shear bond strength of glass ionomer cement sealers to bovine dentine conditioned with common endodontic irrigants. International Endodontic Journal, 32, 430–435 1999.

Aim The aim of this in vitro study was to evaluate the shear bond strength of commercially available and experimental glass ionomer cement (GIC) sealers to dentine exposed to common endodontic irrigants.

Methodology The enamel of 90 bovine incisor crowns, randomly divided into nine equal groups, was ground to expose the superficial dentine layer. The exposed surface was conditioned with either: (i) distilled H2O; (ii) 2.6% NaOCl; or (iii) 17% EDTA followed by 2.6% NaOCl. Five cc of each irrigant was applied over a 30‐second period. The following GIC sealers were tested: (i) Ketac‐Endo; (ii) KT‐308, an experimental sealer; and (iii) ZUT, a combination of KT‐308 and an antibacterial agent (0.2% by weight). The test sealers were applied to form cylinders with a standardized contact surface area (17.8mm2) on the conditioned dentine surfaces. Specimens were bench set for 90min and stored in distilled water at 37°°C for 48h, then tested to failure for shear bond strength (MPa) in an Instron machine.

Results KT‐308 and ZUT had significantly higher MPa values than Ketac‐Endo (two‐factor ANOVA, P 0.0001), regardless of the dentine conditioning. All specimens conditioned with 17% EDTA and 2.6% NaOCl combined had significantly lower MPa values than those conditioned with water or 2.6% NaOCl alone (P 0.02).

Conclusions It was concluded that the experimental sealers KT‐308 and ZUT bonded better to bovine dentine than Ketac‐Endo, and that the bond of all three GIC sealers was better with the smear layer present.