bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired germinal centre formation and humoral immune response in the absence of CD28 and interleukin‐4
Authors: Reiter Rudolph , Pfeffer Klaus
Published: March 2003

The generation of an optimal humoral immune response requires fully activated T‐cells. For complete activation at least two signals are needed. The first one is an antigen dependent one via the T cell receptor, the second one is a costimulatory signal which can be delivered by the CD28 molecule after binding to CD80 (B7.1) or CD86 (B7.2). Fully activated T helper cells are competent to deliver help to B‐cells by secreting cytokines (e.g. interleukin (IL)‐4) or up‐regulating CD40 ligand for proliferation and differentiation of B cells. These interactions mainly take place in germinal centres (GC) that arise after antigen stimulation in B cell‐follicles of peripheral lymphatic tissues and are the sites of massive B‐cell proliferation, affinity maturation and class switch. The roles of CD28 and IL‐4 were investigated in GC formation and antibody production. A markedly diminished humoral immune response was observed in IL‐4−/−×CD28−/− mice whereas in CD28−/− and IL‐4−/− mice the defect was less severe. Especially the formation of germinal centres was significantly reduced in CD28−/− or IL‐4−/− mice and almost undetectable in IL‐4−/−×CD28−/− mice. Taken together these data indicate that CD28 and IL‐4 are synergistically involved in GC formation and immunoglobulin production.
Limitations of HLA‐transgenic mice in presentation of HLA‐restricted cytotoxic T‐cell epitopes from endogenously processed human papillomavirus type 16 E7 protein
Authors: Street Michael D. , Doan Tracy , Herd Karen A. , Tindle Robert W.
Published: March 2003

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen‐binding domains to test vaccines composed of defined HLA A*0201‐restricted cytotoxic T‐lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour‐associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7‐directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization‐introduced E7, by the mouse antigen‐processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide‐pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201‐restricted E7 CTL epitopes; (3) HLA A*0201‐restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7‐specific T cells either in ‘conventional’ HLA A*0201 transgenic (A2.1Kb) mice, or in HHD transgenic mice in which expression of endogenous H‐2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201‐ and E7‐expressing tumour. There are generic implications for the universal applicability of HLA‐class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7‐based therapeutic vaccines for cervical cancer.
Interleukin‐6 promotes the production of interleukin‐4 and interleukin‐5 by interleukin‐2‐dependent and ‐independent mechanisms in freshly isolated human T cells
Authors: Heijink Irene H. , Vellenga Edo , Borger Peter , Postma Dirkje S. , De Monchy Jan G. R. , Kauffman Henk F.
Published: March 2003

T helper 2 (Th2) cytokines [interleukin (IL)‐4 and IL‐5] play a central role in the development of allergic immune responses. After allergen provocation, the expression of Th2 cytokines is rapidly up‐regulated in atopy and asthma. IL‐6 is a multifunctional cytokine that is able to direct Th2 immune responses and is secreted by multiple tissue cell types. This study shows that IL‐6 induces up‐regulation of IL‐4 and IL‐5 after short (5 min) preincubation periods in freshly isolated, α‐CD3/α‐CD28‐stimulated T cells. After longer preincubation periods with IL‐6 (12 and 24 hr), the priming effect on IL‐4 production gradually disappears, whereas the effect on IL‐5 becomes more pronounced. In contrast, a small but significant inhibitory effect is found on the production of the Th1 cytokine interferon‐γ. Additional experiments indicate that the long‐term priming effect of IL‐6 on IL‐5 production is dependent on IL‐2 signalling. This is not the case for the short‐term IL‐6 effect on IL‐5 secretion, where the p38 mitogen‐activated protein kinase‐dependent induction of activator protein‐1 DNA‐binding activity is involved, independent of signal transducer and activator of transcription 3 phosphorylation. In summary, these data demonstrate that the short‐term and long‐term priming effects of IL‐6 on Th2 cytokine production are regulated by different mechanisms.
Tumour necrosis factor‐α but not lipopolysaccharide enhances preference of murine dendritic cells for Th2 differentiation
Authors: Kikuchi Kazuhiro , Yanagawa Yoshiki , Aranami Toshimasa , Iwabuchi Chikako , Iwabuchi Kazuya , Onoé Kazunori
Published: March 2003

Using murine spleen‐derived dendritic cells (DC) and DO11.10 T cells specific for ovalbumin (OVA), the influences of maturational condition and antigen dose on the capability of DC to induce helper T‐cell (Th) differentiation were analysed. Immature DC (iDC) with high‐ or low‐dose OVA323–339 predominantly induced Th1 or Th2 responses in DO11.10 T cells, respectively. DC matured by tumour necrosis factor‐α (TNF/DC) induced a significantly higher Th2 response in the presence of low‐dose OVA323–339 than iDC and DC matured by lipopolysaccharide (LPS) (LPS/DC). In the presence of high‐dose OVA323–339, LPS/DC induced significantly lower levels of Th1 response than iDC. Under these conditions no difference in the Th1 response was noted between TNF/DC and iDC. The enhanced capability of TNF/DC with a low‐dose antigen for Th2 polarization and the decreased preference of LPS/DC with a high‐dose antigen to Th1 polarization were not related to the amount of IL‐12 produced in these cultures. These results demonstrate for the first time that TNF/DC with a low‐dose antigen are potent inducers of Th2 differentiation.
A parallel signal‐transduction pathway for eotaxin‐ and interleukin‐5‐induced eosinophil shape change
Authors: Choi Eun Nam , Choi Moon Kyung , Park Choon‐Sik , Chung Il Yup
Published: March 2003

Interleukin‐5 (IL‐5) and eotaxin are the most important cytokines/chemokines responsible for regulating eosinophil locomotion and are known to play a co‐operative role in the selective recruitment of eosinophils to inflamed tissues. Following exposure to chemoattractants, eosinophils undergo a series of events, including reorganization of actin filaments and subsequent rapid shape changes, culminating in chemotaxis. In this study we examined the signalling pathways for eosinophil shape change regulated by eotaxin and IL‐5, primarily using a gated autofluorescence/forward‐scatter assay. Eotaxin and IL‐5 were able to elicit shape change with peaks at 10 and 60 min, respectively, and IL‐5 triggered the shape change more efficiently than eotaxin. The pharmacological inhibitors of mitogen‐activated protein kinase (MAP kinase) and p38 blocked both eotaxin‐ and IL‐5‐induced eosinophil shape change in a dose‐dependent manner. In addition, depletion of intracellular Ca2+ and inhibition of protein kinase A (PKA) strongly reduced eosinophil shape change. In contrast, even when used at high concentrations, protein tyrosine kinase (PTK) inhibitors caused only a slight reduction in the ability to change shape. However, treatment with protein kinase C (PKC) inhibitors, such as GF109203X and staurosporine, resulted in a striking inhibition of eosinophil shape change by IL‐5, but not eotaxin. Data from the inhibition of activation and chemotaxis of the extracellular signal‐regulated kinases (ERK1/2) by the PKC inhibitors were also consistent with findings from the experiments on shape change. Collectively, two eosinophil‐selective cytokines/chemokines probably regulate eosinophil shape change via a largely overlapping signalling pathway, with involvement of PKC restricted to the IL‐5 signal alone.
Deep structure or emergence theory: contrasting theoretical foundations for information systems development
Authors: Truex Duane P. , Baskerville Richard
Published: March 2003

The linguistic conceptual distinction between deep and surface structures offers an interesting metaphor for developing new theories of information systems. However, the deep structure notion is both richer and more strongly contested in the linguistic field than can be communicated in published articles about new theories of information systems. This article explores the deep structure notion more fully, how faithful information system (IS)‐related articles adhere to the original concept and the implication of alternative linguistic theories for the IS field.
A knowledge‐focused perspective on the diffusion and adoption of complex information technologies: the BPR example
Authors: Newell S. , Swan J. A. , Galliers R. D.
Published: March 2003

Abstract.

This paper presents a knowledge‐focused perspective for the development of a model to explain the diffusion and adoption of complex integrating technologies. Business process re‐engineering (BPR) is used as the example to illustrate the model. However, while BPR is used to illustrate our argument, the model that is developed is relevant to understanding the innovation processes surrounding any complex IT‐based innovation. It is argued that the strength of this diffusion model is that it focuses not on the spread of particular technological artifacts (whether it is BPR or any other IT‐based innovation), but on the spread of the ideas and knowledge underpinning the technology. In particular, the model draws attention to the ways in which technology suppliers commodify knowledge and present ‘packaged’ solutions. This creates problems for potential users who need to unpack this knowledge and integrate it with existing organizational knowledge. The diffusion and adoption of innovations is thus seen as a process of integrating knowledge across disparate communities. Such knowledge integration, however, is difficult. This can help to explain the apparent contradiction between the limited success rate of BPR and its widespread diffusion among western firms.
The mosquito dihydrofolate reductase amplicon contains a truncated synaptic vesicle protein gene
Authors: Wang Z. H. , Fallon A. M.
Published: March 2003

When maintained under continuous selection with the folate inhibitor, methotrexate, cultured Aedes albopictus mosquito cells amplify an ∼200 kb region of DNA containing the dihydrofolate reductase gene. To determine whether the amplicon contained additional coding regions, Southern blots of cosmid clones containing amplicon DNA were probed separately with reverse‐transcribed mRNA from methotrexate‐sensitive and methotrexate‐resistant cells. Cosmid pWED118 contained five EcoRI fragments (A, B, C, F, G) ranging in size from 2 to 5 kb that hybridized with cDNA from resistant cells. Of these, fragments B and F also hybridized to probe representing mRNA from sensitive cells, and all but fragment G hybridized to repetitive DNA from wild‐type cells. Fragment G, which appeared to encode a low copy number gene in wild‐type cells that subsequently became part of the dihydrofolate reductase amplicon in methotrexate‐resistant cells, hybridized strongly to a 7 kb band and more weakly to bands measuring 9 and 3 kb on Northern blots containing RNA from resistant cells. Fragment G contained a 1203 bp open reading frame, encoding 401 amino acids homologous to synaptic vesicle protein SV2, a member of a transmembrane transporter family expressed in neural and endocrine cells. The region of homology included the six N‐terminal transmembrane domains, an internal cytoplasmic loop, a seventh transmembrane domain, and most of an intravesicular loop. This partial sequence, which appears to correspond to a truncated gene generated during formation of the dihydrofolate reductase amplicon, provides a useful basis for more extensive characterization of an important gene family that may be the target of novel insecticides.
Genetic variation within a parthenogenetic lineage
Authors: Lushai G. , De Barro P. J. , David O. , Sherratt T. N. , Maclean N.
Published: March 2003

A clone of the grain aphid Sitobion avenae F. was maintained parthenogenetically over thirty‐two generations (n= 344) in a constant environment: a new generation being set up by a female selected at random from the preceding generation. Genomic DNA from individual aphids was screened for genetic stability using RAPD‐PCR with a previously tested ten‐mer primer. A putative germ‐line mutation was noted in generation 14 and somatic mutations were noted in generations 12, 25, 27 and 29. There were no differences in the RAPD‐PCR profiles of winged and wingless morphs and samples tested for symbiotic DNA. No endobiotic fungal organism was associated with the clone. Southern blotting and hybridization studies indicated that band additions were of aphid origin. However, the RAPD‐PCR profiles of the germ‐line and somatic mutation samples were unique from other aphid clones cultured during the experimental period. This paper documents discernible genetic changes occurring within an animal clonal lineage over time and impacts on the consequences this may have for clonal systems.
An estimate of phylogenetic relationships among culicine mosquitoes using a restriction map of the rDNA cistron
Authors: Kumar A. , Black W. C. , Rai K. S.
Published: March 2003

Restriction maps of the rDNA cistron of twelve species of mosquitoes in six genera of the subfamily Culicinae were constructed using eight 6 bp recognition restriction enzymes. Anopheles albimanus was used as an outgroup. The size of the rDNA cistron ranged from 8.5 kb in Aedes katherinensis to 12.9 kb in Ae. polynesiensis. A total of twenty‐six sites were scored; eighteen were polymorphic among ingroup taxa. The proportion of polymorphic nucleotide sites (Pnuc) was 0.059 and the heterozygosity per nucleotide site (Hnuc) was 0.028. Wagner and Fitch Parsimony, Dollo Parsimony and Nei‐Li distance/neighbour‐joining methods were used to construct phylogenetic trees. The rDNA RFLP dataset did not provide a well‐supported phylogeny among culicine taxa. The RFLP phylogenies are incongruent with the morphology character based and molecular phylogenies and derived relationships did not correspond with current taxonomic classifications. The lack of resolution was due to homoplasy arising from frequent independent loss or gain of restriction sites among unrelated taxa.
The Queensland fruit fly, Bactrocera tryoni, contains multiple members of the hAT family of transposable elements
Authors: Pinkerton A. C. , Whyard S. , Mende H. A. , Coates C. J. , O’Brochta D. A. , Atkinson P. W.
Published: March 2003

Members of the hAT transposable element family are mobile in non‐host insect species and have been used as transformation vectors in some of these species. We report that the Queensland fruit fly, Bactrocera tryoni, contains at least two types of insect hAT elements called Homer and a Homer‐like element (HLE). The Homer element is 3789 bp in size and contains 12‐bp imperfect inverted terminal repeats. The Homer element contains a long open reading frame (ORF) that encodes a putative transposase. Three different copies of this long ORF were recovered from the B. tryoni genome and, upon transcription and translation in an in vitro system, all produced transposase. The HLE is an incomplete element since no 3′ inverted terminal repeat (ITR) was found. Homer and the HLE are as related to one another as either is to the other insect hAT elements such as Hermes, hobo, hermit and hopper. The structure and distribution of these two Homer elements is described.
A P element‐homologous sequence in the house fly, Musca domestica
Authors: Lee Seung Hoon , Clark Jonathan B. , Kidwell Margaret G.
Published: March 2003

Sequences homologous to the P transposable element have been identified in Musca domestica. Sequence analysis of a genomic clone (Md‐P1) indicates that, although the house fly P element has lost its coding capacity, the basic general structure of drosophilid P elements is present. The house fly P element sequence shares a number of structural features with that from the blow fly, Lucilia cuprina, including a large intron separating exons 1 and 2, two additional introns interrupting exon 2 and the apparent absence of inverted repeat termini. Within a relatively well‐conserved central region, the house fly sequence shows 59% similarity to the D. melanogaster P element, but distal regions are more diverged. Southern blot analysis of several strains indicated the presence of at least four P element copies.
Cloning of a family of serine protease genes from the cat flea Ctenocephalides felis
Authors: Gaines P. J. , Sampson C. M. , Rushlow K. E. , Stiegler G. L.
Published: March 2003

Serine protease gene fragments approximately 480 nucleotides in length were amplified from Ctenocephalides felis larval and adult cDNA libraries using degenerate oligonucleotide PCR primers. Partial clones of thirty‐eight distinct serine protease encoding sequences were isolated, and nineteen different full‐length cDNAs encoding mature serine proteases were subsequently cloned and sequenced. All of the mature proteases contained the histidine, aspartic acid and serine amino acids of the catalytic triad characteristic of serine proteases. The mature C. felis serine proteases had amino acid sequences that were at most 29–53% identical to those known insect and arachnid serine proteases. Two of the C. felis gene sequences had similarity with the Drosophila melanogaster developmental genes snake and stubble. mRNA expression of selected serine protease genes was examined in different life stages, tissues, genders, and in response to bloodfeeding.
Is the insect glutathione S‐transferase I gene family intronless?
Authors: Lougarre A. , Bride J. M. , Fournier D.
Published: March 2003

The genes coding for class I glutathione S‐transferases in insects were believed to be intronless because the coding sequence was not interrupted by an intron. But sequences of the untranslated 5′ end of transcripts revealed the presence of an intron in housefly and Drosophila genes suggesting that most insect GSTI genes are in fact interrupted.
Sequence analysis and expression of a mRNA for a larval‐specific cuticular protein, LCP1, from Helicoverpa armigera
Authors: Henry S. , Hobbs A.
Published: March 2003

Several cDNA clones for a larval cuticular protein from Helicoverpa armigera were isolated and sequenced. The cDNA clones contain an open reading frame encoding a 109 residue protein which is homologous to other known cuticular proteins. The predicted protein appears to have a signal peptide which would be removed to give a mature protein of 91 amino acid residues with an M r of 10 127. The mature protein, LCP1 (Larval Cuticular Protein), would be highly acidic, as is found for other cuticular proteins from flexible insect cuticles. The mRNA appears to be expressed throughout larval development although it is more highly expressed in the integument of the late final instar.
Cloning and characterization of the ribosomal protein CcP0 of the medfly Ceratitis capitata
Authors: Gagou M.‐E. , Ballesta J. P. G. , Kouyanou S.
Published: March 2003

The gene of the ribosomal protein CcP0, the third member of the ribosomal P‐protein family of the medfly Ceratitis capitata, was identified by genomic and cDNA sequence analysis. It codes for a polypeptide of 317 amino acids and its predicted amino acid sequence shows great similarity to the P0 proteins of other eukaryotic organisms. The CcP0 gene was expressed in Escherichia coli and the 34‐kDa recombinant protein was identical to the P0 protein of purified medfly ribosomes. Both proteins reacted positively with a specific monoclonal antibody against the highly conserved C terminus of eukaryotic ribosomal P proteins. Interestingly, the medfly CcP0 seems to be the only P0 protein of higher eukaryotic organisms with basic character (pI 8.5), as shown by electrofocusing of purified ribosomes.
A member of the Hsp60 gene family from the peach potato aphid, Myzus persicae (Sulzer.)
Authors: Stanley K. , Fenton B.
Published: March 2003

A novel cDNA clone encoding a mitochondrial Hsp60 was isolated from a Myzus persicae cDNA library. The nucleotide sequence consisted of 2348 bp and contained an open reading frame (ORF) of 1722 bases. The putative protein encoded by this ORF consisted of 574 amino acids and was designated MPHSP60. Comparison of MPHSP60 with other Hsp60s found that it was most similar to Hsp60 from Culicoides variipennis (85.6% similarity). Phylogenetic analysis revealed that MPHSP60 is clustered together with other insect Hsp60s. Comparisons were also made between MPHSP60 and SymL, the GroEL homologue of Myzus persicae endosymbionts.
Molecular systematics of the Phlebotomine sandflies of the subgenus Paraphlebotomus (Diptera, Psychodidae, Phlebotomus) based on ITS2 rDNA sequences. Hypotheses of dispersion and speciation
Authors: Depaquit J. , Ferté H. , Léger N. , Killick‐Kendrick R. , Rioux J.‐A. , Killick‐Kendrick M. , Hanafi H. A. , Gobert S.
Published: March 2003

Phylogenetic Paraphlebotomus relationships are inferred by a study based on the sequences of ITS2, which has been sequenced in nine Paraphlebotomus species: P. alexandri, P. andrejevi, P. jacusieli, P. kazeruni, P. mireillae, P. mongolensis, P. saevus, P. sergenti and P. similis and in two out‐groups species of the subgenus Phlebotomus: P. papatasi and P. duboscqi. Paraphlebotomus alexandri appears as the sister group of all other Paraphlebotomus sandflies. Among the other species, three groupings are clearly highlighted: andrejevi and mongolensis;mireillae and saevus;jacusieli, kazeruni, sergenti and similis. These groupings are related to speculations about the migration of Paraphlebotomus from a centre of dispersion located in the Middle East sometime from the early Eocene to the late Miocene.
Molecular cloning and expression of Tenebrio molitor ultraspiracle during metamorphosis and in vivo induction of its phosphorylation by 20‐hydroxyecdysone
Authors: Nicolaï M. , Bouhin H. , Quennedey B. , Delachambre J.
Published: March 2003

Using a RT‐PCR approach, the Tenebrio molitor homologue of Drosophila Ultraspiracle (TmUSP) was characterized. Its DNA binding domain shows a degree of identity with those of the other insect USPs. However, the ligand binding domain is closer to those of retinoid X receptors. Using an antibody raised against DmUSP, Western blot analysis of proteins from epidermis and other tissues revealed five immunoreactive bands, corresponding to different phosphorylated forms of a unique polypeptide, as shown by λ‐phosphatase treatment. The nuclear form of TmUSP seems unphosphorylated. An in vivo 20‐hydroxyecdysone treatment increases considerably and rapidly the phosphorylated forms of TmUSP. This post‐translational modification may play a role in the 20‐hydroxyecdysone response.
Extrachromosomal transposition of the transposable element Minos occurs in embryos of the silkworm Bombyx mori
Authors: Shimizu K. , Kamba M. , Sonobe H. , Kanda T. , Klinakis A. G. , Savakis C. , Tamura T.
Published: March 2003

To assess the ability of the transposable element Minos to act as a vector for genetic manipulation of the silkworm Bombyx mori, an extrachromosomal transposition assay based on three plasmids was performed. The three plasmids – helper, donor and target – were co‐injected into preblastoderm embryos. Low molecular weight DNA was extracted from the embryos at the stage of blastokinesis and used to transform Escherichia coli. High frequency of transposition was observed in the presence of a helper plasmid possessing an intronless Minos transposase gene, whereas transposition did not occur in the presence of a helper plasmid with the intron‐bearing transposase gene. Sequence analysis of the insertion sites showed that Minos always inserts into a TA dinucleotide. Although the insertions are distributed throughout the target gene, there was a preference for certain insertion sites. However, no consensus could be identified in the sequence flanking the target site. The results strongly suggest that the transposable element Minos has the potential to be used as a vector in the silkworm and probably in other lepidopteran insects.
Molecular cloning and characterization of a putative cyclic nucleotide‐gated channel from Drosophila melanogaster
Authors: Miyazu M. , Tanimura T. , Sokabe M.
Published: March 2003

We have cloned a cDNA encoding a putative cyclic nucleotide‐gated (CNG) channel from Drosophila melanogaster. The N‐terminal half of the predicted protein, designated as CNGL, shows a high degree of sequence similarity with the known CNG channel proteins. CNGL has a long hydrophilic C‐terminal stretch that is absent in other CNG channels. Northern blot analysis revealed that the messenger RNA (mRNA) corresponding to the size of the cloned cDNA is expressed in Drosophila heads. Immunolocalization studies showed that CNGL is expressed in the brain, including the medulla, lobulla and lobulla plate, the antennal lobe glomeruli, and mushroom bodies. These results suggest a possible role of the putative CNGL channel in the processing of visual and olfactory information in the nervous system of Drosophila.
Sibling species in the Lutzomyia longipalpis complex differ in levels of mRNA expression for the salivary peptide, maxadilan
Authors: Yin H. , Norris D. E. , Lanzaro G. C.
Published: March 2003

Maxadilan is a small (≈ 7 kDa) protein found in the saliva of sand fly species in the Lutzomyia longipalpis complex, vectors of the parasite causing visceral leishmaniasis, Leishmania chagasi. It is a potent vasodilator and also has immunomodulatory affects. Maxadilan recovered from different sibling species of the Lu. longipalpis complex differ in amino acid content by as much as 23%, however all variants possess equivalent vasodilatory activity. Therefore, the dramatic differences in vasodilatory activity of the saliva from different sibling species is probably due to differences in the amounts of maxadilan in their saliva. This is significant because it has been suggested that maxadilan may influence the pathogenesis of leishmanial infections. In this study we measured the amount of maxadilan messenger RNA (mRNA) per pair of salivary glands from individual sand flies by quantitative reverse transcription polymerase chain reaction (RT‐PCR) using a competitive method. We report a method using the gene of interest, in this case maxadilan, amplified by the PCR from genomic DNA, as a competitor in the quantitative RT‐PCR, taking advantage of differences in the size of these products due to the presence of an intron. Significant differences in amounts of maxadilan mRNA among colonies from Central and South America are described. We found a strong correlation between the amount of maxadilan mRNA detected in salivary glands of different Lu. longipalpis sibling species and previously described differences in the size of erythemas produced by the bite of these species. Therefore, variation in the amount of mRNA suggests that differences in the vasodilatory properties of saliva among the different sibling species are the result of differences in the amount of maxadilan present in the saliva and not differences in the potency of maxadilan peptide variants. The geographical distribution of species with high or low levels of maxadilan gene expression are concordant with the distribution of atypical cutaneous leishmaniasis resulting from infection with Le. chagasi, lending credence to earlier suggestions that maxadilan may be involved with visceralization of this parasite.
Molecular identification of Anopheles minimus s.l. beyond distinguishing the members of the species complex
Authors: Van Bortel W. , Trung H. D. , Roelants P. , Harbach R. E. , Backeljau T. , Coosemans M.
Published: March 2003

Correct species identification is the starting point for studying the epidemiological role of vectors. Identification is mostly achieved using morphological criteria, but this fails when sibling species and species with overlapping morphological characters are involved. The problem with the identification of Anopheles minimus s.l., one of the most widespread malaria vectors in South‐East Asia, is twofold: it is a complex of at least two isomorphic species, and based on morphology, members of the complex are difficult to distinguish from closely related species. An identification method was developed for An. minimus species A and C, and four related species, An. aconitus, An. pampanai, An. varuna and An. jeyporiensis. PCR‐amplified internal transcribed spacer 2 (ITS2) ribonuclear DNA (rDNA) fragments were digested with restriction endonuclease BsiZI. Clear diagnostic banding patterns for the six species were obtained on agarose gels. Testing field‐collected specimens from different regions in South‐East Asia indicated that the technique will be applicable over a wide geographical area. From this it is clear that molecular identification has to focus not only on the species of complexes, but also on related species if they hamper the morphological identification of the ‘sensu lato species’.
Identification and analysis of Lydia, a LTR retrotransposon from Lymantria dispar
Authors: Pfeifer T. A. , Ring M. , Grigliatti T. A.
Published: March 2003

Degenerative PCR primers to conserved amino acid motifs were used to identify an LTR retrotransposon from Lymantria dispar. The isolated retrotransposon, Lydia, is 6655 base pairs (bp) in length and contains perfect 300 bp terminal repeats. The identified gag and pol related ORFs have a high degree of similarity to the corresponding regions of the retrotransposon Ted from Trichoplusia ni, although several reading frameshifts and missense mutations are evident. The high degree of similarity between Lydia and Ted LTRs lends support for a family of lepidopteran retrotransposons. Southern blot analysis of individuals from two geographically distinct gypsy moth populations demonstrates that Lydia is found in both populations and the position of this element within the genome of these isolated populations is variable.
Cloning and transcriptional expression of a leucokinin‐like peptide receptor from the Southern cattle tick, Boophilus microplus (Acari: Ixodidae)
Authors: Holmes S. P. , He H. , Chen A. C. , Lvie G. W. , Pietrantonio P. V.
Published: March 2003

Leucokinins are invertebrate neuropeptides that exhibit myotropic and diuretic activity. Only one leucokinin‐like peptide receptor is known, the lymnokinin receptor from the mollusc Lymnaeastagnalis. A cDNA encoding a leucokinin‐like peptide receptor was cloned from the Southern cattle tick, Boophilus microplus, a pest of cattle world‐wide. This is the first neuropeptide receptor known from the Acari and the second known in the subfamily of leucokinin‐like peptide G‐protein‐coupled receptors. The deduced amino acid sequence exhibits 40% identity to the lymnokinin receptor. The receptor transcript is present in all tick life stages as determined by semiquantitative reverse transcription polymerase chain reaction. We also propose that the sequence AAF50775.1 from the Drosophila melanogaster genome (CG10626) encodes the first identified insect leucokinin receptor.
Heat‐shock protein 90 is down‐regulated during pupal diapause in the flesh fly, Sarcophaga crassipalpis, but remains responsive to thermal stress
Authors: Rinehart Joseph P. , Denlinger David L.
Published: March 2003

Heat‐shock protein 23 (hsp23) and hsp70 are both known to be strongly up‐regulated during pupal diapause in the flesh fly Sarcophaga crassipalpis. This prompted us to investigate whether hsp90 was also up‐regulated during diapause. To test this possibility, we developed a partial clone of a hsp90 family member for use as a probe in Northern blot hybridization. Both high and low temperature exposure up‐regulated hsp90 transcripts in nondiapausing individuals. In contrast to hsp23 and hsp70, hsp90 was down‐regulated following entry into diapause, and returned to prediapause levels after diapause termination. The response of hsp90 to heat shock and cold shock remained intact during diapause: both shocks evoked elevated expression. The results indicate differential regulation of hsps during diapause and in response to thermal injury inflicted on diapausing pupae.
Molecular cloning of a second prophenoloxidase cDNA from the mosquito Armigeres subalbatus: prophenoloxidase expression in blood‐fed and microfilariae‐inoculated mosquitoes
Authors: Huang L. H. , Christensen B. M. , Chen C. C.
Published: March 2003

Melanization constitutes an important component in various aspects of insect life, including cuticular sclerotization, egg‐shell tanning, melanization of parasites and wound healing. Recently, a cDNA encoding prophenoloxidase (pro‐PO), a key enzyme in the biosynthesis of melanotic material in insects, was cloned from microfilariae (mf)‐inoculated mosquitoes, Armigeres subalbatus. However, results of Northern blot analyses indicated that two pro‐POs might be present in Ar. subalbatus and these pro‐POs might be responsible for two distinct physiological functions, egg‐shell tanning and melanization of parasites. Subsequently, the second pro‐PO cDNA (As‐pro‐PO II) was cloned from blood‐fed Ar. subalbatus by rapid amplification of cDNA ends polymerase chain reaction. The 2210 bp As‐pro‐PO II cDNA contains a 41 bp 5′‐non‐coding region, a 2064 bp open reading frame and a 105 bp 3′‐non‐coding region. A hydrophobic signal peptide for endoplasmic reticulum targeting is not found in the N‐terminal region. The deduced amino acid sequence of As‐pro‐PO II shares a high degree of identity (81.5%) with that of the As‐pro‐PO I obtained from mf‐inoculated Ar. subalbatus. Both Northern blot and reverse transcription polymerase chain reaction analysis demonstrated that these two mosquito pro‐POs are persistently expressed in mosquito haemocytes and not in fat body, midgut, or ovaries. The expression of As‐pro‐PO I and As‐pro‐PO II in mosquitoes is associated with melanization of mf and blood feeding, respectively.
The rate of terminal nucleotide loss from a telomere of the mosquito Anopheles gambiae
Authors: Walter M. F. , Bozorgnia L. , Maheshwari A. , Biessmann H.
Published: March 2003

Using a single copy pUChsneo transgene insertion at the Anopheles gambiae 2L telomere, this chromosome end was monitored by genomic Southern blots for forty‐four mosquito generations. During this time, the chromosome end lost terminal nucleotides at an apparently constant rate of 55 bp/generation, which can be accounted for by incomplete DNA replication and does not imply exonuclease activity. No telomere elongation events were detected, suggesting that a previously described gene conversion event at this transgene does not occur very frequently. Moreover, no evidence for elongation by transposable elements was found, as described in Drosophila melanogaster. These results are consistent with the proposal that gene conversion between complex terminal satellite repeats that are present at natural telomeres, represents the major telomere elongation mechanism in A. gambiae. Such recombination events between repetitive sequences would occur more frequently than between the single copy pUChsneo transgene on the 2L homologues.
Evolutionary change in the structure of the regulatory region that drives tissue and temporally regulated expression of alcohol dehydrogenase gene in Drosophila funebris
Authors: Amador A. , Papaceit M. , Juan E.
Published: March 2003

The Adh locus of Drosophilidae is organized as a single gene transcribed from two spatially and temporally regulated promoters except in species of the repleta group, which have two single promoter genes. Here we show that in Drosophila funebris the Adh gene is transcribed from a single promoter, in both larva and adult, with qualitative and quantitative species specific‐differences in tissue distribution. The gene is expressed in larval fat body but in other tissues such as gastric caeca, midgut and Malpighian tubules its expression is reduced compared to most Drosophilidae species, and in adults it is almost limited to the fat body. The comparative analysis of gene expression of two strains, which differ by a duplication, indicates that the cis elements necessary for this pattern of expression in larvae are included in the region of 1.55 kb upstream of the transcription initiation site. This new organization reveals the evolution of a different regulatory strategy to express the Adh gene in the subgenus Drosophila.
cDNA cloning of two different serine protease inhibitor precursors in the migratory locust, Locusta migratoria
Authors: Simonet G. , Claeys I. , Vanderperren H. , November T. , De Loof A. , Vanden Broeck J.
Published: March 2003

Recently, a novel serine protease‐inhibiting peptide family, designated as the ‘pacifastin family’, has been described in locusts and crayfish. All members of this family possess a characteristic cysteine‐rich domain. The present study describes the cDNA cloning, sequencing and transcript distribution of two novel pacifastin‐related peptide precursors in the migratory locust, Locusta migratoria. Only one of the encoded peptides (HI) was identified previously, whereas six others represent new members of the pacifastin family. Northern blot analysis showed that both precursor transcripts are present in adult locust fat body. These could not be detected in the midgut. Interestingly, an in silico data mining approach of the expressed sequence tags (EST) database revealed the existence of Manduca sexta and Bombyx mori cDNAs that display pronounced sequence similarities with these locust pacifastin‐related transcripts.
The involvement of ovarian tumour in the intracellular localization of Sex‐lethal protein
Authors: Hinson S. , Nagoshi R. N.
Published: March 2003

The Drosophila ovarian tumour gene is required at multiple times in the germline for oogenesis. A second gene, Sex‐lethal, controls sex determination in the soma and also has a separate germline function affecting similar oogenic stages as ovarian tumour. We demonstrate that ovarian tumour is not required for early Sex‐lethal gene expression in the female germline, as had been previously reported. Instead, we provide evidence that ovarian tumour has a specific role in the developmentally regulated accumulation of SEX‐LETHAL protein within the cytoplasm and nucleus. Furthermore, the examination of nurse cell polytene chromosomes produced by certain ovarian tumour mutations showed that SEX‐LETHAL protein can associate with discrete chromosomal sites in the germline and that this pattern appears to change as the egg chamber matures. This is the first indication that SEX‐LETHAL is capable of direct physical interactions with chromosomes (albeit in a mutant background) and is consistent with the developmentally regulated nuclear localization of SEX‐LETHAL being important for oogenesis.
An intron enhancer activates the immunoglobulin‐related Hemolin gene in Hyalophora cecropia
Authors: Roxström‐Lindquist K. , Lindström‐Dinnetz I. , Olesen J. , Engström Y. , Faye I.
Published: March 2003

Hemolin is the only insect member of the immunoglobulin (Ig) superfamily reported to be up‐regulated during an immune response. In diapausing pupae of Hyalophora cecropia the gene is expressed in fat body cells and in haemocytes. Like the mammalian Ig κ light chain gene, the Hemolin gene harbours an enhancer including a κB motif in one of its introns. This motif binds the H. cecropia Rel factor Cif (Cecropia immunoresponsive factor). The Hemolin third intron also mediates transient reporter gene expression in immunoresponsive Drosophila mbn‐2 cells. Co‐transfections of Drosophila SL2 cells showed that the Drosophila Rel factor Dif (Dorsal‐related immunity factor), transactivates reporter gene constructs through the intron. Moreover, a 4.8‐fold synergistic activation was obtained when Dif is combined with the rat C/EBP (CCAAT/enhancer element‐binding protein) and human HMGI (high mobility group protein I). This is the first report of an insect immune‐related gene that is up‐regulated by an enhancer activity conferred through an intron.
Molecular cloning and sequencing of salivary gland‐specific cDNAs of the blood‐sucking bug Triatoma brasiliensis (Hemiptera: Reduviidae)
Authors: Sant Anna M. R. V. , Araújo J. G. V. C. , Pereira M. H. , Pesquero J. L. , Diotaiuti L. , Lehane S. M. , Lehane M. J.
Published: March 2003

Haematophagous insects produce pharmacological substances in their saliva to counteract vertebrate host haemostasis events such as coagulation, vasoconstriction and platelet aggregation. To investigate the bioactive salivary molecules of the triatomine bug Triatoma brasiliensis, we produced subtraction‐enriched cDNAs of salivary‐gland specific genes using suppression subtractive hybridization. Six full‐length differentially expressed cDNAs (Tb113, Tb125, Tb152, Tb169, Tb180 and Tb198) were selected, cloned and sequenced. Sequence similarity searches of the databases using the putative amino acid sequence of our clones gave the following results: Tb152 – Triabin, an antithrombin induced platelet aggregation factor found in salivary gland extracts of T. pallidipennis. Tb169 – Pallidipin, an anticollagen induced platelet aggregation factor also found in T. pallidipennis salivary homogenates. Tb180 – Procalin, the major allergen of T. protracta saliva. The other three salivary‐gland specific cDNAs produced no obvious homologies. Comparison of these salivary gland‐specific cDNAs of with those of other triatomines combined with functional studies using recombinant proteins will allow a better understanding of the co‐evolutionary process occurring between these insects and their vertebrate hosts, and may also lead to the discovery of novel antihaemostatic agents.
Sealing ability of composite resin placed over calcium hydroxide and calcium sulphate plugs in the repair of furcation perforations in mandibular molars: a study in vitro
Authors: Imura  , Mie Otani  , Hata  , Toda  , Zuolo
Published: March 2003

The purpose of this study was to evaluate in vitro the sealing ability of various materials in the repair of furcation perforations in mandibular molars by measuring coronal microleakage with Indian ink. Ninety extracted mandibular molars were embedded individually into a plaster of Paris block, with the roots surrounded by a simulated periodontal ligament of silicone. Subsequently, a standard coronal access opening was prepared, the root canal orifices were located and a perforation was made with a size 012 round bur in a water‐cooled high‐speed handpiece directly into the centre of the floor of the pulp chamber. The perforations were repaired with amalgam, composite resin, calcium sulphate under composite resin and calcium hydroxide under composite resin. The teeth were coated with two layers of nail polish, leaving the access opening area uncovered, and immersed in Indian ink for 4 days at 37°C. The teeth were sectioned longitudinally and dye penetration measured from the coronal level of the repair material to the apical end of the perforation. All experimental groups revealed dye penetration in varying degrees, but there was no significant difference amongst them (Kruskal‐Wallis test P < 0.05). Calcium sulphate and calcium hydroxide prevented overextrusion of composite resin when used under this repair material.
The behaviour and proliferation of human dental pulp cell strains in vitro, and their response to the application of platelet‐derived growth factor‐BB and insulin‐like growth factor‐1
Authors: Denholm  , Moule  , Bartold
Published: March 2003

Human dental pulp fibroblast strains were established from explants of dental pulps using identical culture techniques. To determine proliferative activity, a 3H‐thymidine uptake and a crystal violet dye‐binding assay were performed at passage numbers seven and eight. Assays were performed in the presence of either 0% fetal calf serum (FCS), 0.2% FCS or 10% FCS. Considerable variation in the overall proliferative activity of the different pulp cell strains (when averaged over all other variables) was noted. All dental pulp cell strains demonstrated significantly different proliferative activity from each other. In addition, the level of proliferative response and 3H‐thymidine incorporation decreased as the passage number of the cells increased. This was in accordance with the findings of Tardieu‐Moreau et al. (1992). It is proposed that the differences in proliferative activity are most likely attributable to inherent variability within the established pulp cell strains. Platelet derived growth factor‐BB (PDGF‐BB) and insulin‐like growth factor‐1 (IGF‐1) were added to the human pulp cells both separately and in combination. All of the pulp cells exhibited increased proliferative activity in the presence of the growth factors with the combination of PDGF‐BB/IGF‐1 having the greatest mitogenic effect. There was also significant variability in the level of response of all pulp cell strains to the different growth factors. This study identified significant variability in the responsiveness to the growth factors between the pulp cell strains when the results of the 3H‐thymidine and dye binding assays were compared. These findings reinforce the thesis that different assay procedures may also influence the findings of biological investigations involving the human dental pulp. The results of this study confirm that when comparing the findings of different in vitro studies involving human pulp cells, variations in experimental data can be strongly influenced by the pulp cell strain used and the culture technique employed. Indeed, studies of human pulp cell proliferation using pulp cells which are not of the same transfer number may not be relevant.
Cure behaviour of visible light‐activated pattern materials
Authors: Whitworth J. M. , Makhani S. H. S. , McCabe J. F.
Published: March 2003

Aim This study tested the hypothesis that the cure behaviour (depth of cure and polymerization contraction) of light‐activated pattern materials was no worse than that of light‐activated composite resins, allowing them to be handled in a similar fashion.

Methodology Depth of cure was measured by a penetrometer method.

Results Cure depths were comparable to those of composite resins, ranging from 3.52mm (Lumin‐X paste) to 6.76mm (Visioform) after visible lightactivation for 30s. There were significant differences in the depth of cure of the three materials tested (P0.001). Polymerization contraction was assessed by a minimal load transducer method. Values ranged from 0.45% (Lumin‐X paste) to 1.89% (Visioform), lower than that of composite resins. There were significant differences in the polymerization contraction values for each of the three materials (P0.001).

Conclusions It was concluded that light‐activated pattern materials cure in a manner comparable to composite resins, and may be built up incrementally in a similar fashion.