bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Wolbachia protein synthesis in Drosophila in vivo
Authors: Sasaki T. , Braig H. R. , O'Neill S. L.
Published: March 2003

Intracellular Wolbachia infections are extremely common in arthropods and exert profound control over the reproductive biology of the host. However, very little is known about the underlying molecular mechanisms which mediate these interactions with the host. We examined protein synthesis by Wolbachia in a Drosophila host in vivo by selective metabolic labelling of prokaryotic proteins and subsequent analysis by 1D and 2D gel electrophoresis. Using this method we could identify the major proteins synthesized by Wolbachia in ovaries and testes of flies. Of these proteins the most abundant was of low molecular weight and showed size variation between Wolbachia strains which correlated with the reproductive phenotype they generated in flies. Using the gel systems we employed it was not possible to identify any proteins of Wolbachia origin in the mature sperm cells of infected flies.
Ribosomal protein L5 of Bombyx mori: cDNA sequence and tissue differences in the L5 mRNA content
Authors: Yang C. , Sehnal F.
Published: March 2003

A gene encoding the ribosomal protein L5, which assembles with the 5S rRNA into one of the components of the 60S ribosomal subunit, was identified in insects for the first time. We report on the isolation of Bombyx mori cDNA, whose putative translation product is 60–76% identical with the L5 sequences known from other animals and about 50% identical with the L5 of rice and yeasts. Bombyx contains a single copy of the L5 gene, which is constitutively expressed as a 1.1 kb mRNA in all tissues. The ratio of L5 mRNA to total RNA appears to reflect the proteosynthetic tissue capacity. A very high level of L5 mRNA is maintained in functional silk glands. A rapid decline of the ratio of L5 mRNA to the rRNA, which occurs in the silk glands after cocoon spinning, indicates differences in the stability of these two RNA classes.
Amplification of a serine esterase gene is involved in insecticide resistance in Sri Lankan Culex tritaeniorhynchus
Authors: Karunaratne S. H. P. P. , Vaughan A. , Paton M. G. , Hemingway J.
Published: March 2003

Culex tritaeniorhynchus, the major vector of Japanese encephalitis in Sri Lanka, is resistant to organophosphorus insecticides, with a 10‐fold resistance ratio at the LC50 for chlorpyrifos, and a high heterogeneity factor in the insect field population. The major mechanism of resistance in this species, as in the mosquito C. quinquefasciatus, is elevation of esterase activity. Basic biochemical, immunological and molecular analysis suggests that the C. tritaeniorhynchus CtrEstβ1 gene is orthologous to the C. quinquefasciatus amplified Est7beta;s. The Estβ21 antiserum cross‐reacts strongly with CtrEstβ11. Its corresponding cDNA, over the 545 base pairs sequenced, has ∼84% identity with the various C. quinquefasciatus Estβs. The gene is amplified in C. tritaeniorhynchus. Amplification of the same esterase in two independent species, along with multiple amplification events involving this esterase gene in C. quinquefasciatus suggests that the location of this gene within the genome predisposes it to amplification.
Phylogenetic analysis of parthenogenesis‐inducing Wolbachia in the genus Aphytis (Hymenoptera: Aphelinidae)
Authors: Gottlieb Y. , Zchori‐Fein E. , Faktor O. , Rosen D.
Published: March 2003

Parthenogenesis‐inducing intracellular bacteria of the genus Wolbachia are found in a variety of parasitoid wasp genera. The presence of Wolbachia in the uniparental Aphytis species A. fingnanensis Compere, A. diaspidis (Howard), A. chilensis Howard, and A. chrysomphali (Mercet) was tested using primers specific for the ftsZ gene. The symbiont was detected in all of these species. Wolbachia ftsZ genes that were sequenced from the four hosts show a high degree of similarity. Both the PCR with specific primers for group ‘A’ and phylogenetic analysis place these Wolbachia in group ‘A’. The fact that the tested Aphytis species belong to different phylogenetic groups and harbour what seem to be almost identical Wolbachia, lends credence to the horizontal transmission hypothesis.
Phylogenetic relationships of seven palearctic members of the maculipennis complex inferred from ITS2 sequence analysis
Authors: Marinucci M. , Romi R. , Mancini P. , Luca M. Di , Severini C.
Published: March 2003

The sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) were determined from seven palearctic mosquitoes species belonging to the Anopheles maculipennis species complex, namely An. atroparvus, An. labranchiae, An. maculipennis, An. messeae, An. melanoon, An. sacharovi and An. martinius. The length of the ITS2 ranged from 280 to 300 bp, with a GC content of 49.4–54.1%. With the exception of An. messeae, negligible levels of intraspecific polymorphism and no intrapopulation variation were observed. The phylogenetic relationships among the members of the maculipennis complex were inferred by maximum‐parsimony analysis of the
paup program and the neighbour‐joining and maximun‐likelihood analysis of the
phylip program. All of the trees obtained were almost identical in topology, although the relationships among three species, i.e. An. maculipennis, An. messeae and An. melanoon, remained unresolved. The phylogenies were in good agreement with the previous gene–enzyme and polytene chromosome banding pattern studies.
Comparative analysis of promoters for transient gene expression in cultured mosquito cells
Authors: Zhao Y‐G. , Eggleston P.
Published: March 2003

Three heterologous promoters (hsp 70 and actin 5C from Drosophila melanogaster and IE1 from the immediate early gene of the Bombyx mori baculovirus) were assessed for their ability to drive transient luciferase expression in mosquito cells. Overall, the actin 5C promoter was considerably more effective at driving luciferase expression than either hsp70 or IE1 in cell lines derived from Anopheles, Aedes and Culex species. hsp70 functioned well when induced by heat shock and was also induced to a lesser extent by chemicals such as sodium arsenite. IE1 was also an effective initiator of transcription, particularly in two Anopheles cell lines, but generally it performed less well than the actin 5C promoter and was also outperformed by hsp70 in Anopheles gambiae cells.
Insect immunity: molecular cloning, expression, and characterization of cDNAs and genomic DNA encoding three isoforms of insect defensin in Aedes aegypti
Authors: Lowenberger C. A. , Smartt C. T. , Bulet P. , Ferdig M. T. , Severson D. W. , Hoffmann J. A. , Christensen B. M.
Published: March 2003

Aedesaegypti were immune activated by injection with bacteria, and the expression of insect defensins was measured over time. Northern analyses indicated that defensin transcriptional activity continued for at least 21 days after bacterial injection, and up to 10days after saline inoculation. Mature defensin levels in the haemolymph reached approximately 45 μM at 24 h post inoculation. cDNAs encoding the preprodefensins of three previously described mature Ae. aegypti defensins were amplified by PCR, cloned and sequenced. Genomic clones were amplified using primers designed against the cDNA sequence. Sequence comparison indicates that there is significant inter–and intra–isoform variability in the signal peptide and prodefensin sequences of defensin genes. Preprodefensin sequences of isoforms A and B are very similar, consisting of a signal peptide region of twenty amino acids, a prodefensin region of thirty–eight amino acids and a forty amino acid mature peptide domain. The sequence encoding isoform C is significantly different, comprising a signal peptide region of twenty–six amino acids, and the mature protein domain of forty amino acids, Analysis of the genomic clones of each isoform revealed one intron spatially conserved in the prodefensin region of all sequences. The intron in isoforms A and B is 64 nt long, and except for a 4 nt substitution in one clone, these intron sequences are identical. The intron in isoform C is 76 nt long and does not share significant identity with the intron sequences of isoforms A or B. The defensin gene mapped to chromosome 3, between two known loci, blt and LF 168.
Precise excision and transposition of piggyBac in pink bollworm embryos
Authors: Thibault S. T. , Luu H. T. , Vann N. , Miller T. A.
Published: March 2003

Transposable elements such as P, hobo, Hermes, mariner and Minos have been successfully harnessed as gene vectors to achieve the transformation of several dipteran species including Drosophila melanogaster, Ceratitis capitata and Aedes aegypti. Plasmid‐based excision and transposition assays have been useful indicators of an element's ability to be mobilized in vivo and thus potentially serve as a transforming vector. We report that the transposable element piggyBac is capable of precise excision and transposition in the pink bolloworm (Pectinophora gossypiella), a worldwide pest of cultivated cotton. Combined with a suitble marker gene, the piggyBac element may serve as a vector for germline transformation in this and (potentially) other lepidopteran species.
Molecular cloning and expression in Escherichia coli of an aquaporin‐like gene from adult buffalo fly (Haematobia irritans exigua)
Authors: Elvin C. M. , Bunch R. , Liyou N. E. , Pearson R. D. , Gough J. , Drinkwater R. D.
Published: March 2003

A gene fragment encoding a putative member of the aquaporin gene family was amplified using cDNA prepared from unfed adult buffalo fly poly(A)+ RNA and degenerate PCR primers designed from highly conserved regions of amino acids found in all members of the aquaporin gene family. This PCR product was labelled with digoxigenin‐dUTP and used as a probe to screen a λgt‐11 cDNA library constructed from unfed adult buffalo fly. One positively hybridizing clone (AqpBF1), contained an insert of 1878 bp, and DNA sequence analysis revealed an open reading frame of 753 bp encoding a polypeptide of predicted Mr = 26 163 Da. Comparison of the AqpBF1 deduced protein sequence with the GenBank database revealed significant homology to many aquaporin genes, including 72% identity with a partial DNA sequence encoding a member (drip) of the MIP protein family isolated from Drosophila melanogaster. The most closely related, full‐length, GenBank sequence was an aquaporin gene isolated from the digestive tract of the sap‐sucking insect Cicadella viridis, which was 53% identical to the buffalo fly AqpBF1 protein sequence. The full‐length coding sequence of AqpBF1 was cloned into the (His)6‐fusion vector, pQE10, and the recombinant protein was expressed in Escherichia coli following induction by IPTG. The recombinant (His)6‐fusion protein was localized predominantly in the membrane fraction of E. coli. The protein was solubilized from E. coli membranes with n‐octyl β‐d‐glucopyranoside and purified by affinity chromatography on a Ni++–sepharose column in the presence of detergent.
Phylogenetic relationships of phlebotomine sandflies inferred from small subunit nuclear ribosomal DNA
Authors: Aransay A. M. , Scoulica E. , Tselentis Y. , Ready P. D.
Published: March 2003

Relationships among seventy specimens, fifteen species and three genera of phlebotomines were inferred from the phylogenetic analysis of small subunit nuclear rDNA, obtained by the PCR amplification and cloning of almost full‐length genes. Outgroups included fifteen dipterans, and single representatives of four other insect orders. The more distant the taxa compared, the larger were the regions of ambiguous sequence alignment that needed to be deleted in order to avoid circularity in performing parsimony analyses. Phlebotomine sequences formed a monophyletic clade within the suborder Nematocera, with the progressively more basal sister groups of Diptera being Culicomorpha, Tipulomorpha and the suborder Brachycera. Within Phlebotominae, subgeneric relationships were resolved and the genus Phlebotomus was shown to be monophyletic, but markers for intraspecific geographical populations were not found and intergeneric relationships were not resolved.
A gut‐specific serine protease from the malaria vector Anopheles gambiae is downregulated after blood ingestion
Authors: Shen Z. , Edwards M. J. , Jacobs‐Lorena M.
Published: March 2003

A chymotrypsin‐like serine protease gene (AgChyL) was cloned from the mosquito Anopheles gambiae by a polymerase chain reaction (PCR)‐based subtractive cDNA cloning strategy. AgChyL messenger RNA (mRNA) is abundant in the adult female gut prior to, and for 8 h following, a blood meal. During the peak of digestion, from 12 to 24 h following a blood meal, AgChyL mRNA abundance decreased to barely detectable levels. AgChyL mRNA was abundant again by 48 h following a blood meal. Recombinant pro‐AgChyL was expressed in Escherichia coli. The pro‐enzyme can be activated by trypsin. Activated AgChyL cleaves the synthetic chymotrypsin substrate succinyl‐
l‐Ala‐Ala‐Pro‐Phe‐nitroanilide, but not two other synthetic chymotrypsin substrates or synthetic trypsin and elastase substrates. The potential role of AgChyL in the coordination of An. gambiae digestion is discussed.
Germ‐line transformation of pink bollworm (Lepidoptera: Gelechiidae) mediated by the piggyBac transposable element
Authors: Peloquin J. J. , Thibault S. T. , Staten R. , Miller T. A.
Published: March 2003

The pink bollworm, Pectinophora gossypiella, is a world‐wide pest of cultivated cotton. In certain growing regions populations are suppressed by a sterile release strategy. Efforts to improve the sterile insect technique as well as our understanding of lepidopteran biology could benefit greatly from a germ‐line transformation system. We report transformation of pink bollworm with a piggyBac transposable element carrying the enhanced green flourescent protein (EGFP) marker gene. This vector–marker system resulted in recovery of transgenics at a rate of approximately 3.5%. Integration of the transforming construct that was typical of piggyBac was demonstrated by Southern analysis and sequence determination of transposon flanks. Expression of the EGFP marker was visualized by fluorescent microscopy and Western Blot analysis. Maintenance of transformed strains indicates that the transgene segregates in a Mendelian fashion and has been stable over fourteen generations to date.
Genomic organization and immune regulation of the defensin gene from the mosquito, Anopheles gambiae
Authors: Eggleston P. , Lu W. , Zhao Y.
Published: March 2003

The defensin gene from the mosquito, Anopheles gambiae, is present as a single copy per haploid genome. Two exons, encoding a 102 residue preprodefensin, are separated by a 105 bp intron bounded by consensus splice sites. The upstream regulatory sequence includes a TATA box, arthropod initiator and numerous motifs homologous to insect and mammalian immune response elements. This promoter is capable of upregulation by immune challenge in cultured cells and activity is further stimulated by Gambif1, a mosquito Rel protein known to translocate to the nucleus and bind NF‐κB sites in target promoters. Activity is inhibited by p50, a mammalian Rel protein that competitively binds NF‐κB sites, and virtually abolished by p40, an avian IκB protein that inhibits nuclear translocation.
Immune activation upregulates lysozyme gene expression in Aedesaegypti mosquito cell culture
Authors: Gao Y. , Fallon A. M.
Published: March 2003

After stimulation with heat‐killed bacteria, cultured cells from the mosquito Aedesaegypti (Aag‐2 cells) secreted an induced protein with a mass of ≈ 16 kDa that cross‐reacted with antibody to chicken egg lysozyme. To investigate whether lysozyme messenger RNA is induced in bacteria‐treated cells, we used polymerase chain reaction‐based approaches to obtain the complete lysozyme cDNA from Aag‐2 cells. The deduced protein contained 148 amino acids, including a 23 amino acid signal sequence. The calculated mass of the precursor protein is 16 965 Da, which is processed to yield a mature lysozyme of 14 471 Da with a calculated pI of 10.1. The lysozyme from Ae. aegypti shared 50% amino acid identity with lysozymes from Anophelesgambiae and Anophelesdarlingi, which in turn shared 70% identity between each other. Northern analysis with the lysozyme cDNA probe showed induction of a 1.3 kb messenger RNA during the first 3 h after treatment of Aag‐2 cells with heat‐killed bacteria, followed by maximal expression 12–36 h after treatment. Southern analysis suggested that the gene likely occurs as a single copy in the genome of Aag‐2 cells.
Microsatellite loci are not abundant in all arthropod genomes: analyses in the hard tick, Ixodes scapularis and the yellow fever mosquito, Aedes aegypti
Authors: Fagerberg A. J. , Fulton R. E. , Black Iv W. C.
Published: March 2003

Plasmid libraries enriched for microsatellites were generated in the tick, Ixodes scapularis and in the mosquito Aedes aegypti. Libraries were enriched for genomic DNA containing (AC)n, (AG)n, (ATG)n, (CAG)n, (TAG)n, (AAT)n, (CTGY)n or (GATA)n motifs. Clones containing each motif were sequenced in both species for PCR primer design. In I. scapularis, most primers amplified a single locus and alleles varied in the number of microsatellite repeats and segregated as codominant markers. In contrast (AC)n, (TAG)n and (GATA)n microsatellite loci extracted from Ae. aegypti appeared to be members of multigene families. A primer pair designed to amplify a particular TAG locus instead amplified many independently segregating loci, some of which did not contain TAG microsatellites. Alleles at the TAG loci segregated as dominant markers and there was limited evidence for length variation among alleles. These results suggest that microsatellite loci are not universally abundant in arthropod genomes nor do alleles always segregate as codominant markers.
Isolation of cDNA clones encoding putative odourant binding proteins from the antennae of the malaria‐transmitting mosquito, Anopheles gambiae
Authors: Biessmann Harald , Walter Marika F. , Dimitratos Spiros , Woods Daniel
Published: March 2003

One way of controlling disease transmission by blood‐feeding mosquitoes is to reduce the frequency of insect–host interaction, thus reducing the probability of parasite transmission and re‐infection. A better understanding of the olfactory processes responsible for allowing mosquitoes to identify human hosts is required in order to develop methods that will interfere with host seeking. We have therefore initiated a molecular approach to isolate and characterize the genes and their products that are involved in the olfactory recognition pathway of the mosquito Anopheles gambiae, which is the main malaria vector in sub‐Saharan Africa. We report here the isolation and preliminary characterization of several cDNAs from male and female A. gambiae antennal libraries that encode putative odourant binding proteins. Their conceptual translation products show extensive sequence similarity to known insect odourant binding proteins (OBPs)/pheromone binding proteins (PBPs), especially to those of D. melanogaster. The A. gambiae OBPs described here are expressed in the antennae of both genders, and some of the A. gambiae OBP genes are well conserved in other disease‐transmitting mosquito species, such as Aedes aegypti and Culex quinquefasciatus.
Germ‐line transformation of the South American malaria vector, Anopheles albimanus, with a piggyBac/EGFP transposon vector is routine and highly efficient
Authors: Perera O. P. , Harrell II R. A. , Handler A. M.
Published: March 2003

Stable and efficient germ‐line transformation was achieved in the South American malaria vector, Anopheles albimanus, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Transgenic mosquitoes were identified from four independent experiments at frequencies ranging from 20 to 43% per fertile G0. Fluorescence was observable throughout the body of larvae and pupae, and abdominal segments of adults. Transgenic lines analysed by Southern hybridization had one to six germ‐line integrations, with most lines having three or more integrations. Hybridized transposon vector fragments and insertion site sequences were consistent with precise piggyBac‐mediated integrations, although this was not verified for all lines. The piggyBac/PUbnlsEGFP vector appears to be a robust transformation system for this anopheline species, in contrast to the use of a piggyBac vector in An. gambiae. Further tests are needed to determine if differences in anopheline transformation efficiency are due to the marker systems or to organismal or cellular factors specific to the species.
cDNA cloning, functional expression and characterization of kynurenine 3‐hydroxylase of Anopheles stephensi (Diptera: Culicidae)
Authors: Hirai M. , Kiuchi M. , Wang J. , Ishii A. , Matsuoka H.
Published: March 2003

Kynurenine 3‐hydroxylase (K3H) is a NADPH‐dependent flavin monooxygenase involved in the tryptophan pathway. Xanthurenic acid (XA) is a metabolite of this pathway and has recently been identified as a gamete activating factor (GAF) of the malarial parasite. We cloned K3H cDNA from Anopheles stephensi (AsK3H), because anopheline mosquitoes are a vector of the human malaria parasite, Plasmodium falciparum and the catalytic function of AsK3H in XA production. Recombinant AsK3H protein was expressed in Sf‐9 cells using the baculovirus system and its enzymatic properties were characterized. The specific activities of crude cell lysate and affinity purified protein were 94.9 ± 6.2 and 865.6 ± 10.5 nmol/min/mg protein, respectively. The optimum pH of AsK3H was 7.0. Analysis of AsK3H gene expression using RT‐PCR revealed that AsK3H was constitutively expressed in egg, larva, pupa and adult.
A novel family of anticoagulants from the saliva of Ixodes scapularis
Authors: Narasimhan S. , Koski R. A. , Beaulieu B. , Anderson J. F. , Ramamoorthi N. , Kantor F. , Cappello M. , Fikrig E.
Published: March 2003

Using biochemical and molecular approaches, we have identified a 9.8 kDa protein in the saliva of Ixodes scapularis that inhibits the intrinsic pathway of coagulation. The 9.8 kDa anticoagulant protein was purified by reverse‐phase HPLC and its N‐terminal amino acid sequence determined. The N‐terminal sequence showed homology with Salp14, an immuno‐dominant antigen present in the saliva of engorging I. scapularis nymphs. Recombinant Salp14 expressed in Escherichia coli prolonged the activated partial thromboplastin time (APTT) of human plasma in a dose‐dependent manner and was a specific inhibitor of factor Xa. A cDNA encoding a 9.3 kDa protein, Salp9Pac, was subsequently isolated from an I. scapularis salivary gland cDNA library. Salp9Pac showed 93% identity to the N‐terminal sequence of the anticoagulant purified by HPLC. These data indicate that the anticoagulant protein purified by HPLC, Salp9Pac and Salp14 are members of a family of novel coagulation protease inhibitors present in tick saliva. While recombinant Salp9Pac did not show biological activity in the assays tested currently, it is likely to be mechanistically different from its paralogues. This raises the possibility that ticks may enhance their adaptive ability to cope with a wide spectrum of proteases, by transcribing such structurally related anticoagulant proteins with different functions.
Cytochrome P450 CYP6X1 cDNAs and mRNA expression levels in three strains of the tarnished plant bug Lygus lineolaris (Heteroptera: Miridae) having different susceptibilities to pyrethroid insecticide
Authors: Zhu Y. C. , Snodgrass G. L.
Published: March 2003

Three cDNAs, cloned from both pyrethroid‐susceptible and ‐resistant strains of Lygus lineolaris, contained a 1548 nucleotide open reading frame encoding a 516 amino acid residue protein. Predicted cytochrome P450s from cDNAs were classified as the first three new members of subfamily CYP6X, CYP6X1v1 for a susceptible strain and CYP6X1v2 and CYP6X1v3 for two resistant strains. Putative cytochrome P450 CYP6X1s from L. lineolaris were highly similar (up to 42% amino acid sequence identity) to several insect CYP6s that are responsible for reduced sensitivity to pyrethroid insecticides. A total of twenty‐six nucleotide substitutions were revealed between cDNAs of susceptible and resistant strains. Two nucleotide substitutions resulted in amino acid changes, Asp373 to Ala373 and Ser487 to Ala487, between susceptible and resistant strains. The resistant laboratory strain contained 2.1‐fold higher cytochrome P450 mRNA per microgram total RNA than the susceptible laboratory strain. Topical treatment with 10 ng permethrin elevated cytochrome P450 mRNA levels by approximately 2‐fold. The results of this study indicated that cytochrome P450 gene mutation, coupled with up‐regulation, was present only in the pyrethroid resistant strains, and was possibly related to resistance development in the tarnished plant bug.
Reparative hard tissue formation following calcium hydroxide application after partial pulpotomy in cariously exposed pulps of permanent teeth
Authors: Nosrat  , Nosrat
Published: March 2003

In a prospective study, partial pulpotomy was performed on six permanent molars with deep carious lesions and pulpal involvement. The bleeding pulp was irrigated with normal tap water until bleeding had stopped and the exposed pulp was covered with calcium hydroxide followed by zinc oxide eugenol, and finally covered with a semipermanent restoration. All teeth showed hard tissue barrier formation, both clinically and radiographically, within three months and were free from subjective and objective symptoms through the observation period (average observation period was 26 months). The patients also experienced the therapy positively. These findings and those of others have helped gain more recognition for partial pulpotomy as a strong possible alternative therapy when pulps are exposed by deep carious lesions and a bleeding pulp is exposed during the excavation process. The rationale for this therapy is to remove the infected and/or inflamed pulpal areas beneath the carious lesion and disintegrated tissue. A rapid and simplified procedure would allow the general practitioner to perform this procedure when necessary at dental clinics, without specialist facilities under conditions that avoid unnecessary contamination of the pulp.
Microbiological, pathological, inflammatory, immunological and molecular biological aspects of periradicular disease
Authors: Takahashi
Published: March 2003

Multimicrobial infection of the dental pulp triggers inflammatory responses and ultimately causes bone destruction in the periradicular tissues. Besides bacteria, noxious substances such as degraded protein components and cholesterol could also act as antigens and elicit a host response, which can be harmful to periradicular tissues. Histologically, a dense infiltration of immunocompetent cells is seen in periradicular lesions and their host reactions may induce bone resorption. Polymorphonuclear leucocytes (PMN) and macrophages migrate to the periapical lesions and phagocytose pathogens as a first line of defence. Dead PMN are quickly phagocytosed by macrophages and this disposal system plays a role in maintaining chronicity of the lesions. The pathological roles of periradicular T cells have been assessed through analysing phenotypic markers of cell types, especially CD antigens, but the results are still controversial. Recently, technical developments in immunology and molecular biology have made it possible to investigate the pathogenesis of many diseases at molecular level. The investigation of functional analysis of the immune cells and their regulatory molecules such as apoptosis‐associated molecules and adhesion molecules, will lead to a better understanding of the pathogenesis of periradicular lesions. The role of inflammatory mediators including antibodies, cytokines, matrix metalloproteinases, growth factors and arachidonic metabolism is becoming known for these lesions. Knowledge from these investigations improve the understanding of the pathological mechanisms of periradicular infections.
A scanning electron microscopic evaluation of root surfaces and the gutta‐percha interface following root‐end resection in vitro
Authors: Weston G. D. , Moule A. J. , Bartold P. M.
Published: March 2003

Weston GD, Moule AJ, Bartold PM. A scanning electron microscopic evaluation of root surfaces and the gutta‐percha interface following root‐end resection in vitro. International Endodontic Journal, 32, 450–458 1999.

Aim The aim of this study was to evaluate the effects that various commonly used instruments have on root surface morphology, the obturating material, and the interface between the obturation and the root canal walls following root‐end resection in vitro.

Methodology Sixty human single‐rooted teeth with fully formed apices were collected and decoronated. The root canals were instrumented, and then obturated with thermoplasticized gutta‐percha using AH‐26 as the sealer. The roots were randomly divided into 12 different groups, and apical root‐end resections were performed using eight different instrument configurations, and two different directions in which the bur moved across the root face in relation to its direction of rotation. Epoxy resin replicas of the resected root ends were examined using scanning electron microscopy.

Results Each instrument produced a characteristic surface finish on the resected root end that mirrored its cutting profile. Irrespective of the design of the bur used, smearing and shredding of the gutta‐percha across the root face occurred only when the handpiece was moved across the root face in reverse direction in relation to the direction of rotation of the bur.

Conclusions To ensure minimal disruption and distortion of the root filling and to prevent shredding of the gutta‐percha interface, care should be taken to ensure that the final pass of the bur across the root canal is in the correct direction in relation to its direction of rotation.
Preclinical endodontology: an international comparison
Authors: Qualtrough A. J. E. , Whitworth J. M. , Dummer P. M. H.
Published: March 2003

Aim The aim of this study was to compare preclinical endodontic teaching in Europe, Scandinavia and North America, and to place recent UK data in an international context.

Methodology A postal questionnaire was sent to all undergraduate dental schools in North America, Scandinavia, and Europe. Data were requested on a range of issues relating to endodontic teaching.

Results Forty‐three percent of the 170 schools surveyed returned completed questionnaires. There was considerable international consensus on the content of preclinical courses, with most schools advocating preflaring canal preparation techniques, sodium hypochlorite for irrigation, and cold lateral condensation as the standard obturation method. There was little consensus on the standard use of intracanal medicaments, though calcium hydroxide was universally popular. The practice of single visittreatment was advocated by at least 70% of schools in all geographical areas.

A number of innovations appear to be gaining acceptance in preclinical teaching, with more than 20% of schools teaching the use of electronic apex locators, and a quarter of Western European, Scandinavian and North American schools embracing nickel‐titanium instrumentation. Regional differences in the priority and resource given to endodontic teaching were striking. On average, UK schools had the worst staff:student ratios for preclinical endodontic teaching, and allocated substantially less time allocation for this teaching compared with Western European, Scandinavian and North American schools.

Conclusions It was concluded that although teachers in the UK were broadly advocating techniques recommended elsewhere, the academic infrastructure and priority given for endodontic teaching in the UK was limited in the international context. This may have some impact on the quality of endodontic provision within the UK General Dental Services.
The preparation of periapical lesions for flow cytometry
Authors: Fernando K. , Gulabivala K. , Barrett A. W.
Published: March 2003

Aim  To devise an optimal protocol and to analyse the leucocyte composition of periapical (PA) lesions by flow cytometry.

Methodology PA lesions were mechanically agitated, with and without proteolysis. This was with either 0.2% collagenase alone, or in combination with 0.02% DNA‐ase in serial incubations until all tissue was digested. The efficacy of each method was assessed by counting total cell yield and cell viability. Phenotype stability was gauged by the percentage of peripheral blood leucocytes (PBL) which expressed CD45RB, CD3, CD20, CD4 and CD8 before and after mechanical and collagenase treatment.

Results  Disaggregation of PA lesions was superior if collagenase was present, but cell clumping was problematic unless the DNA‐ase was also added, and serial digestion with this combination produced optimal cell yield and viability. Nevertheless, the total number of cells released rarely exceeded 105, though viability was in excess of 80%. Mechanical agitation and proteolysis adversely affected PBL phenotypes, but collagenase digestion limited to 10 min caused least damage. Flow cytometric analysis of disaggregated PA lesions failed to identify more than 7.9% (mean, range 6–10%) CD45RB + cells.

Conclusions  Because of the necessity for single cell suspensions, flow cytometry is not easily applied to the analysis of leucocytes in PA lesions, and further refinements in tissue disaggregation and cell preparation are required.
A survey of interfacial forces used during endosonic instrumentation of root canals
Authors: Regan J. D. , Sherriff M. , Meredith N. , Gulabivala K.
Published: March 2003

Aim

The aim of thisin‐vitrostudy was to measure interfacial forces acting between root canal dentine and ultrasonic files during endosonic instrumentation.

Methodology

Single‐rooted teeth were mounted on a cantilevered aluminium beam to which two pairs of single element strain gauges were joined in a half‐bridge configuration mounted at right angles to each other. The strain gauges were connected to an analogue‐to‐digital converter fitted in a microcomputer via a conditioning amplifier. This enabled strains to be recorded as a function of interfacial forces over a period of time. Twenty operators instrumented root canals using sizes 15, 20 and 25 ultrasonically energized K‐type files. The lateral forces generated were calculated.

Results

The mean interfacial forces used varied widely between operators and files, ranging from 18 g to 149 g, but there was a consistency in the relative magnitude for each operator. The average force used by the operators increased with file size; the differences between file sizes were significant (P < 0.05).

Conclusions

The range of forces measured is broader than previously reported and may have a bearing on possible uncontrolled dentine removal, even during ultrasonically activated irrigation.
Inactivation of root canal medicaments by dentine, hydroxylapatite and bovine serum albumin
Authors: Portenier I. , Haapasalo H. , Rye A. , Waltimo T. , Ørstavik D. , Haapasalo M.
Published: March 2003

Aim This study examined and compared the inhibition of the antibacterial effect of saturated calcium hydroxide solution, chlorhexidine acetate and iodine potassium iodide by dentine, hydroxylapatite and bovine serum albumin.

Methodology Enterococcus faecalis strain A197A prepared to a suspension of 3 × 108 cells per ml in 0.5% peptone water was used. Fifty µL of saturated calcium hydroxide solution, 0.05% chlorhexidine acetate or 0.2/0.4% iodine potassium iodide were incubated at 37 °C with 28 mg dentine powder (DP), hydroxylapatite (HA) or bovine serum albumin (BSA) in 50 µL water for 1 h before adding 50 µL of the bacterial suspension. Samples for bacterial culturing were taken from the suspension 1 and 24 h after adding the bacteria. In further experiments, the amount of dentine was stepwise reduced from 28 mg 150 µL−1 to 2.8 mg 150 µL−1.

Results Calcium hydroxide was totally inactivated by the presence of 28 mg of DP, HA or BSA. Chlorhexidine (0.05%) was strongly inhibited by BSA and slowed down by dentine. However, HA had little or no inhibitory effect on chlorhexidine. The antibacterial effect of 0.2/0.4% iodine potassium iodide on E. faecalis was totally inhibited by dentine (28 mg), but was practically unaffected by HA or BSA. A stepwise reduction of dentine from 28 mg 150 µL−1 to 2.8 mg 150 µL−1 was followed by a similar reduction of the inhibition of the antibacterial activity of chlorhexidine. Iodine potassium iodide was not inhibited at all with dentine amounts less than 28 mg. However, the effect of saturated calcium hydroxide solution was totally eliminated by dentine, in all four concentrations.

Conclusion Inhibition by dentine of the antibacterial activity of calcium hydroxide, chlorhexidine and iodine potassium iodide occurs by different mechanisms. Different components of dentine may be responsible for the inhibition of these three medicaments. Calcium hydroxide was particularly sensitive to inhibition by both inorganic and organic compounds.
The outcome of endodontic resurgery: a systematic review
Authors: Peterson J. , Gutmann J. L.
Published: March 2003

Aim The aim of this paper is to establish an outcome standard for the assessment of healing radiographically after resurgery of persistent periradicular lesions by systematically reviewing the results from published studies.

Methodology The systematic review process requires the definition of predetermined criteria delineating the inclusion parameters of studies reviewed. Of 42 papers that were reviewed, eight qualified for inclusion. A weighted‐average was calculated from the results taken from the eight eligible, peer‐reviewed studies, published between 1970 and 1997.

Results Three hundred and thirty patients out of 2375 (14%) from the included studies underwent resurgery for failure of healing as determined radiographically. Of this population, 35.7% healed successfully after resurgery, 26.3% healed with uncertain results and 38% did not heal at the one‐year follow‐up.

Conclusions Although there is nearly equal distribution of results between all categories, a 35.7% rate of healing as assessed radiographically is essentially equivalent to the 38% failure rate. This paper will allow an evaluation of current research results to establish an outcome standard and enable techniques and filling materials to be evaluated and compared. Furthermore, the outcome standard can assist in defining demographic and aetiological factors that contribute to the potential outcome of resurgery cases.
An in vitro evaluation of the antimicrobial efficacy of irrigants on biofilms of root canal isolates
Authors: Spratt D. A. , Pratten J. , Wilson M. , Gulabivala K.
Published: March 2003

Aim

The bactericidal effect of four antimicrobial agents was investigated against single‐species biofilms derived from a range of root canal isolates.

Methodology

Single‐species biofilms of Prevotella intermedia, Peptostreptococcus micros, Streptococcus intermedius, Fusobacterium nucleatum and Enterococcus faecalis were generated on membrane filter discs and subjected to 15 min or 1 h incubation with 5 p.p.m. colloidal silver, 2.25% sodium hypochlorite (NaOCl), 0.2% chlorhexidine, 10% iodine or phosphate buffered saline (PBS) as a control. The antimicrobial activity of the agents was neutralized and the bacterial cells were harvested from the discs by vortexing, serially diluted in reduced transport fluid, plated on fastidious anaerobe agar containing 5% horse blood, incubated anaerobically and colony‐forming units calculated.

Results

Iodine and NaOCl were more effective than chlorhexidine except against P. micros and P. intermedia where they were all 100% effective. Iodine and NaOCl elicited a 100% kill after 1 h incubation for all strains used. However, after 15 min, they showed differing bactericidal effects depending on the strain. None of the agents were effective against F. nucleatum after 15 min but NaOCl, iodine and chlorhexidine were all effective after 1 h. Colloidal silver was generally ineffective.

Conclusions

The effectiveness of a particular agent was dependent on the nature of the organism in the biofilm and on the contact time. NaOCl was generally the most effective agent tested, followed by iodine. However the clinical efficacy of these agents must be considered in light of the complex root canal anatomy and polymicrobial nature of root canal infections.
The influence of preparation technique and sodium hypochlorite on removal of pulp and predentine from root canals of posterior teeth
Authors: Evans G. E. , Speight P. M. , Gulabivala K.
Published: March 2003

Aim

To test the null hypothesis that removal of pulp tissue and predentine from root canals of multirooted teeth is not significantly different when performed by one of two mechanical preparation techniques in conjunction with either water or 3% sodium hypochlorite (NaOCl) irrigation.

Methodology

Forty‐six freshly extracted premolar or molar teeth, with pulps vital at the time of extraction, were assigned to four groups balanced by anatomy (group 1, step‐back filing/NaOCl; group 2, step‐back filing/water; group 3, automated rotary/NaOCl; group 4, automated rotary/water). Preparation was undertaken by a single operator and the volumes of irrigant recorded for each tooth. One root from each tooth was prepared for transverse histological assessment at eight levels, including one at and two apical to the working length. A validated index was used to measure the presence and location of residual pulp tissue and predentine.

Results

No statistically significant differences were found in the frequency with which residual pulp tissue or predentine were observed amongst the groups. Coronal parts were more frequently devoid of pulp tissue and predentine than the apical. Canals were frequently occluded with residual pulp tissue apical to the working length. There was a trend for better pulp and predentine removal in accessible parts of the canal as a function of rotary preparation and NaOCl irrigation. The isthmus had residual pulp tissue less frequently in the NaOCl groups. Accessory anatomy was unaffected regardless of technique or irrigant.

Conclusions

Pulp tissue and predentine removal were not significantly different between a step‐back filing and an automated rotary preparation technique in conjunction with water or NaOCl.
Root and canal morphology of Burmese maxillary molars
Authors: Ng Y‐L. , Aung T. H. , Alavi A. , Gulabivala K.
Published: March 2003

Aim

To investigate the root and canal morphology of Burmese maxillary molars using a canal staining and tooth clearing technique.

Methodology

Maxillary molars (239) were collected from indigenous Burmese patients and designated; first (90), second (77), third (72) molars. Following pulp tissue removal and canal system staining with Indian ink, the teeth were decalcified with 10% nitric acid, dehydrated and cleared with methyl salicylate. The following features were evaluated: (i) number of roots and morphology; (ii) number of canals per root; (iii) root canal configuration (Vertucci's classification); (iv) number of apical foramina per root; (v) number and location of lateral canals; and (vi) the presence of intercanal communications.

Results

All first and second molars had three separate roots, whilst third molars had one of five different root forms, of which three separate (25%) or fused (31%) roots were most prevalent. The majority of palatal (100%) and disto‐buccal (96%) roots possessed one canal (type I). The prevalence of mesio‐buccal roots with two canals decreased from first to third molars (68%, 49%, 39%, respectively); type II (two orifices, one apical foramen) and IV (two orifices, two apical foramina) configurations were the most common. Single/fused rooted third molars had a range of number and type of canals. Most roots in all molars had one apical foramen, those with four apical foramina were confined to third molars. Intercanal communications were most prevalent in mesio‐buccal roots and two/three fused rooted third molars. Lateral canals were most prevalent in the apical third of the roots of all molars.

Conclusions

The mesio‐buccal roots of Burmese maxillary molars possessed a variety of canal system types. Over 50% of the first and second molars had a second mesio‐buccal canal, of which over 20% had intercanal communications. The palatal and disto‐buccal canals mainly had type I canals. Lateral canals were equally prevalent in all tooth types but were most common in the apical third.
Accuracy of a new apex locator: an in vitro study
Authors: Kaufman A. Y. , Keila S. , Yoshpe M.
Published: March 2003

Aim The purpose of this study was to test in an in vitro model the accuracy of a Bingo 1020 electronic apex locator, to compare the results to those of a well known apex locator, Root ZX, as well as to those of the radiographic method of tooth length determination.

Methodology A total of 120 extracted teeth, preserved in Thymol solution and kept refrigerated, was used for the study. The experiment was performed on single‐rooted teeth and on one‐root canal, chosen randomly, in multirooted teeth. The teeth were randomly divided into 12 groups of 10 teeth each. After access preparation, the actual length (AL) was measured. The teeth were embedded in an alginate model specially developed for testing apex locators. Electronic tooth length measurements (EL) were carried out prior to root canal preparation using the two electronic apex locators (EAL) – Root ZX and Bingo 1020; three measurements were taken and an average computed. After the third measurement, the file was left in the root canal and a periapical radiograph was taken. The radiographic length (RL) was recorded by measuring the file length from the coronal reference point to the tip of the file. Each root canal was then prepared to a no. 40 K‐file diameter using a standardized technique; saline was used for irrigation. Upon completion of the root canal preparation, EL measurements were taken by each EAL in dry conditions and with different irrigation solutions. Each measurement was repeated three times. The RL was recorded according to the last EL measurement. Results were subjected to statistical analysis.

Results In all parameters tested, a significant statistical difference was found between Bingo 1020 and the Root ZX. Measurements obtained using the Bingo 1020 were consistently closer to the AL (0.08 mm) than those obtained using the Root ZX. Both EALs measured the tooth length with great accuracy and a positive correlation of 0.76 (P = 0.00) existed between the two devices. No significant difference was found between the two apex locators when measurements were taken with the different irrigants (P = 0.34) and the content of the root canal did not affect the accuracy of the measurements. Lengths obtained by calculations from the radiographs were longer than the AL as well as the length obtained by both EALs (P = 0.00).

Conclusions The Bingo 1020 proved to be as reliable as Root ZX and was user friendly. Under the experimental conditions, electronic measurements were more reliable than radiographs in the process of root length determination.