bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and identification of a binding protein for pancreatic secretory trypsin inhibitor: a novel role of the inhibitor as an anti-granzyme A
Authors: TSUZUKI Satoshi , KOKADO Yoshimasa , SATOMI Shigeki , YAMASAKI Yoshie , HIRAYASU Hirofumi , IWANAGA Toshihiko , FUSHIKI Tohru
Published: May 2003

Pancreatic secretory trypsin inhibitor (PSTI) is a potent trypsin inhibitor that is mainly found in pancreatic juice. PSTI has been shown to bind specifically to a protein, distinct from trypsin, on the surface of dispersed cells obtained from tissues such as small intestine. In the present study, we affinity-purified the binding protein from the 2% (w/v) Triton X-100-soluble fraction of dispersed rat small-intestinal cells using recombinant rat PSTI. Partial N-terminal sequencing of the purified protein gave a sequence that was identical with the sequence of mouse granzyme A (GzmA), a tryptase produced in cytotoxic lymphocytes. We confirmed the formation of an affinity-cross-linked complex between 125I-labelled PSTI and recombinant rat GzmA (rGzmA). In situ hybridization and immunostaining revealed the existence of GzmA-expressing intraepithelial lymphocytes in the rat small intestine. We concluded that the PSTI-binding protein isolated from the dispersed cells is GzmA that is produced in the lymphocytes of the tissue. The rGzmA hydrolysed the N-α-benzyloxycarbonyl-l-lysine thiobenzyl ester (BLT), and the BLT hydrolysis was inhibited by PSTI. Sulphated glycosaminoglycans, such as fucoidan or heparin, showed almost no effect on the inhibition of rGzmA by PSTI, whereas they decreased the inhibition by antithrombin III. In the present paper, we propose a novel role of PSTI as a GzmA inhibitor.
Hormone-induced modifications of the chromatin structure surrounding upstream regulatory regions conserved between the mouse and rabbit whey acidic protein genes
Authors: MILLOT Benjamin , MONTOLIU Lluís , FONTAINE Marie-Louise , MATA Teresa , DEVINOY Eve
Published: May 2003

The upstream regulatory regions of the mouse and rabbit whey acidic protein (WAP) genes have been used extensively to target the efficient expression of foreign genes into the mammary gland of transgenic animals. Therefore both regions have been studied to elucidate fully the mechanisms controlling WAP gene expression. Three DNase I-hypersensitive sites (HSS0, HSS1 and HSS2) have been described upstream of the rabbit WAP gene in the lactating mammary gland and correspond to important regulatory regions. These sites are surrounded by variable chromatin structures during mammary-gland development. In the present study, we describe the upstream sequence of the mouse WAP gene. Analysis of genomic sequences shows that the mouse WAP gene is situated between two widely expressed genes (Cpr2 and Ramp3). We show that the hypersensitive sites found upstream of the rabbit WAP gene are also detected in the mouse WAP gene. Further, they encompass functional signal transducer and activator of transcription 5-binding sites, as has been observed in the rabbit. A new hypersensitive site (HSS3), not specific to the mammary gland, was mapped 8 kb upstream of the rabbit WAP gene. Unlike the three HSSs described above, HSS3 is also detected in the liver, but similar to HSS1, it does not depend on lactogenic hormone treatments during cell culture. The region surrounding HSS3 encompasses a potential matrix attachment region, which is also conserved upstream of the mouse WAP gene and contains a functional transcription factor Ets-1 (E26 transformation-specific-1)-binding site. Finally, we demonstrate for the first time that variations in the chromatin structure are dependent on prolactin alone.
Effect of caspase cleavage-site phosphorylation on proteolysis
Authors: TÖZSÉR József , BAGOSSI Péter , ZAHUCZKY Gábor , SPECHT Suzanne I. , MAJEROVA Eva , COPELAND Terry D.
Published: May 2003

Caspases are important mediators of apoptotic cell death. Several cellular protein substrates of caspases contain potential phosphorylation site(s) at the cleavage-site region, and some of these sites have been verified to be phosphorylated. Since phosphorylation may affect substantially the substrate susceptibility towards proteolysis, phosphorylated, non-phosphorylated and substituted oligopeptides representing such cleavage sites were studied as substrates of apoptotic caspases 3, 7 and 8. Peptides containing phosphorylated serine residues at P4 and P1′ positions were found to be substantially less susceptible towards proteolysis as compared with the serine-containing analogues, while phosphoserine at P3 did not have a substantial effect. P1 serine as well as P1-phosphorylated, serine-containing analogues of an oligopeptide representing the poly(ADP-ribose) polymerase cleavage site of caspase-3 were not hydrolysed by any of these enzymes, whereas the P1 aspartate-containing peptides were efficiently hydrolysed. These findings were interpreted with the aid of molecular modelling. Our results suggest that cleavage-site phosphorylation in certain positions could be disadvantageous or detrimental with respect to cleavability by caspases. Cleavage-site phosphorylation may therefore provide a regulatory mechanism to protect substrates from caspase-mediated degradation.
Synthesis of paucimannose N-glycans by Caenorhabditis elegans requires prior actions of UDP-N-acetyl-d-glucosamine:alpha-3-d-mannoside beta1,2-N-acetylglucosaminyltransferase I, alpha3,6-mannosidase II and a specific membrane-bound beta-N-acetylglucosaminidase
Authors: ZHANG Wenli , CAO Pinjiang , CHEN Shihao , SPENCE Andrew M. , ZHU Shaoxian , STAUDACHER Erika , SCHACHTER Harry
Published: May 2003

We have previously reported three Caenorhabditis elegans genes (gly-12, gly-13 and gly-14) encoding UDP-N-acetyl-d-glucosamine:α-3-d-mannoside β1,2-N-acetylglucosaminyltransferase I (GnT I), an enzyme essential for hybrid and complex N-glycan synthesis. GLY-13 was shown to be the major GnT I in worms and to be the only GnT I cloned to date which can act on [Manα1,6(Manα1,3)Manα1,6](Manα1,3)Manβ1, 4GlcNAcβ1,4GlcNAc-R, but not on Manα1,6(Manα1,3)Manβ1-O-R substrates. We now report the kinetic constants, bivalent-metal-ion requirements, and optimal pH, temperature and Mn2+ concentration for this unusual enzyme. C. elegans glycoproteins are rich in oligomannose (Man6–9GlcNAc2) and ‘paucimannose’ Man3–5GlcNAc2(±Fuc) N-glycans, but contain only small amounts of complex and hybrid N-glycans. We show that the synthesis of paucimannose Man3GlcNAc2 requires the prior actions of GnT I, α3,6-mannosidase II and a membrane-bound β-N-acetylglucosaminidase similar to an enzyme previously reported in insects. The β-N-acetylglucosaminidase removes terminal N-acetyl-d-glucosamine from the GlcNAcβ1, 2Manα1,3Manβ- arm of Manα1,6(GlcNAcβ1,2Manα1,3) Manβ1,4GlcNAcβ1,4GlcNAc-R to produce paucimannose Man3GlcNAc2 N-glycan. N-acetyl-d-glucosamine removal was inhibited by two N-acetylglucosaminidase inhibitors. Terminal GlcNAc was not released from [Manα1,6(Manα1,3)Manα1,6] (GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-R nor from the GlcNAcβ1,2Manα1,6Manβ- arm. These findings indicate that GLY-13 plays an important role in the synthesis of N-glycans by C. elegans and that therefore the worm should prove to be a suitable model for the study of the role of GnT I in nematode development.
Differential interaction of the tyrosine phosphatases PTP-SL, STEP and HePTP with the mitogen-activated protein kinases ERK1/2 and p38alpha is determined by a kinase specificity sequence and influenced by reducing agents
Authors: MUÑOZ Juan José , TÁRREGA Céline , BLANCO-APARICIO Carmen , PULIDO Rafael
Published: May 2003

The protein tyrosine phosphatases (PTPs) PTP-SL, STEP and HePTP are mitogen-activated protein kinase (MAPK) substrates and regulators that bind to MAPKs through a kinase-interaction motif (KIM) located in their non-catalytic regulatory domains. We have found that the binding of these PTPs to the MAPKs extracellular-signal-regulated kinase 1 and 2 (ERK1/2), and p38α is differentially determined by the KIM-adjacent C-terminal regions of the PTPs, which have been termed kinase-specificity sequences, and is influenced by reducing agents. Under control conditions, PTP-SL bound preferentially to ERK1/2, whereas STEP and HePTP bound preferentially to p38α. Under reducing conditions, the association of p38α with STEP or HePTP was impaired, whereas the association with PTP-SL was unaffected. On the other hand, the association of ERK1/2 with HePTP was increased under reducing conditions, whereas the association with STEP or PTP-SL was unaffected. In intact cells, PTP-SL and STEP distinctively regulated the kinase activity and the nuclear translocation of ERK1/2 and p38α. Our results suggest that intracellular redox conditions could modulate the activity and subcellular location of ERK1/2 and p38α by controlling their association with their regulatory PTPs.
Poly(2-alkylacrylic acid) polymers deliver molecules to the cytosol by pH-sensitive disruption of endosomal vesicles
Authors: JONES Rachel A. , CHEUNG Charles Y. , BLACK Fiona E. , ZIA Jasmine K. , STAYTON Patrick S. , HOFFMAN Allan S. , WILSON Mark R.
Published: May 2003

The permeability barrier posed by cell membranes represents a challenge for the delivery of hydrophilic molecules into cells. We previously proposed that poly(2-alkylacrylic acid)s are endocytosed by cells into acidified vesicles and are there triggered by low pH to disrupt membranes and release the contents of endosomes/lysosomes to the cytosol. If this hypothesis is correct, these polymers could be valuable in drug-delivery applications. The present paper reports functional comparisons of a family of three poly(2-alkylacrylic acid)s. Poly(2-propylacrylic acid) (PPAA), poly(2-ethylacrylic acid) (PEAA) and poly(2-methylacrylic acid) (PMAA) were compared in red-blood-cell haemolysis assays and in a lipoplex (liposome–DNA complex) assay. We also directly examined the ability of these polymers to disrupt endosomes and lysosomes in cultured human cells. Our results show that: (i) unlike membrane-disruptive peptides, the endosomal-disruptive ability of poly(2-alkylacrylic acid)s cannot necessarily be predicted from their haemolytic activity at low pH, (ii) PPAA (but not PEAA or PMAA) potently facilitates gene transfection by cationic lipoplexes and (iii) endocytosed poly(2-alkylacrylic acid)s are triggered by luminal acidification to selectively disrupt endosomes (not lysosomes) and release their contents to the cytosol. These results will facilitate the rational design of future endosomal-disrupting polymers for drug delivery.
Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus
Authors: KANAI Akio , OIDA Hanako , MATSUURA Nana , DOI Hirofumi
Published: May 2003

We systematically screened a genomic DNA library to identify proteins of the hyperthermophilic archaeon Pyrococcus furiosus using an expression cloning method. One gene product, which we named FAU-1 (P. furiosus AU-binding), demonstrated the strongest binding activity of all the genomic library-derived proteins tested against an AU-rich RNA sequence. The protein was purified to near homogeneity as a 54 kDa single polypeptide, and the gene locus corresponding to this FAU-1 activity was also sequenced. The FAU-1 gene encoded a 472-amino-acid protein that was characterized by highly charged domains consisting of both acidic and basic amino acids. The N-terminal half of the gene had a degree of similarity (25%) with RNase E from Escherichia coli. Five rounds of RNA-binding-site selection and footprinting analysis showed that the FAU-1 protein binds specifically to the AU-rich sequence in a loop region of a possible RNA ligand. Moreover, we demonstrated that the FAU-1 protein acts as an oligomer, and mainly as a trimer. These results showed that the FAU-1 protein is a novel heat-stable protein with an RNA loop-binding characteristic.
Intracellular metabolism and bioactivity of quercetin and its in vivo metabolites
Authors: SPENCER Jeremy P. E. , KUHNLE Gunter G. C. , WILLIAMS Robert J. , RICE-EVANS Catherine
Published: May 2003

Understanding the cellular effects of flavonoid metabolites is important for predicting which dietary flavonoids might be most beneficial in vivo. Here we investigate the bioactivity in dermal fibroblasts of the major reported in vivo metabolites of quercetin, i.e. 3′-O-methyl quercetin, 4′-O-methyl quercetin and quercetin 7-O-β-d-glucuronide, relative to that of quercetin, in terms of their further metabolism and their resulting cytotoxic and/or cytoprotective effects in the absence and presence of oxidative stress. Uptake experiments indicate that exposure to quercetin led to the generation of two novel cellular metabolites, one characterized as a 2′-glutathionyl quercetin conjugate and another product with similar spectral characteristics but 1 mass unit lower, putatively a quinone/quinone methide. A similar product was identified in cells exposed to 3′-O-methyl quercetin, but not in the lysates of those exposed to its 4′-O-methyl counterpart, suggesting that its formation is related to oxidative metabolism. There was no uptake or metabolism of quercetin 7-O-β-d-glucuronide by fibroblasts. Formation of oxidative metabolites may explain the observed concentration-dependent toxicity of quercetin and 3′-O-methyl quercetin, whereas the formation of a 2′-glutathionyl quercetin conjugate is interpreted as a detoxification step. Both O-methylated metabolites conferred less protection than quercetin against peroxide-induced damage, and quercetin glucuronide was ineffective. The ability to modulate cellular toxicity paralleled the ability of the compounds to decrease the level of peroxide-induced caspase-3 activation. Our data suggest that the actions of quercetin and its metabolites in vivo are mediated by intracellular metabolites.
Activation of C-terminal Src kinase (Csk) by phosphorylation at serine-364 depends on the Csk-Src homology 3 domain
Authors: YAQUB Sheraz , ABRAHAMSEN Hilde , ZIMMERMAN Bastian , KHOLOD Natalya , TORGERSEN Knut Martin , MUSTELIN Tomas , HERBERG Friedrich W. , TASKÉN Kjetil , VANG Torkel
Published: May 2003

In the present study, we investigate the mechanism for the protein kinase A (PKA)-mediated activation of C-terminal Src kinase (Csk). Although isolated Csk kinase domain was phosphorylated at Ser364 by PKA to the same stoichiometry as wild-type Csk, significant activation of the isolated Csk kinase domain by PKA was observed only in the presence of the purified Src homology 3 domain (SH3 domain). Furthermore, the interaction between the SH3 and kinase domains was facilitated by PKA-mediated phosphorylation of the kinase domain, as evaluated by surface plasmon resonance. This suggests that an overall structural domain organization and interaction between the kinase and SH3 domains are important for the activity of Csk and its regulation by PKA.
Transmembrane segments 1, 5, 7 and 8 are required for high-affinity glucose transport by Saccharomyces cerevisiae Hxt2 transporter
Authors: KASAHARA Toshiko , KASAHARA Michihiro
Published: May 2003

Hxt2 is a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae and belongs to the major facilitator superfamily. Hxt1 shares ≈ 70% amino acid identity with Hxt2 in its transmembrane segments (TMs) and inter-TM loops, but transports d-glucose with an affinity about one-tenth of that of Hxt2. To determine which TMs of Hxt2 are important for high-affinity glucose transport, we constructed chimaeras of Hxt2 and Hxt1 by randomly replacing each of the 12 TMs of Hxt2 with the corresponding segment of Hxt1, for a total of 4096 different transporters. Among > 20000 yeast transformants screened, 39 different clones were selected by plate assays of high-affinity glucose-transport activity and sequenced. With only two exceptions, the selected chimaeras contained Hxt2 TMs 1, 5, 7 and 8. We then constructed chimaeras corresponding to all 16 possible combinations of Hxt2 TMs 1, 5, 7 and 8. Only one chimaera, namely that containing all four Hxt2 TMs, exhibited transport activity comparable with that of Hxt2. The Km and Vmax values for d-glucose transport, and the substrate specificity of this chimaera were almost identical with those of Hxt2. These results indicate that TMs 1, 5, 7 and 8 are necessary for exhibiting high-affinity glucose-transport activity of Hxt2.
Mammalian molybdo-flavoenzymes, an expanding family of proteins: structure, genetics, regulation, function and pathophysiology
Authors: GARATTINI Enrico , MENDEL Ralf , ROMÃO Maria João , WRIGHT Richard , TERAO Mineko
Published: May 2003

The molybdo–flavoenzymes are structurally related proteins that require a molybdopterin cofactor and FAD for their catalytic activity. In mammals, four enzymes are known: xanthine oxidoreductase, aldehyde oxidase and two recently described mouse proteins known as aldehyde oxidase homologue 1 and aldehyde oxidase homologue 2. The present review article summarizes current knowledge on the structure, enzymology, genetics, regulation and pathophysiology of mammalian molybdo–flavoenzymes. Molybdo–flavoenzymes are structurally complex oxidoreductases with an equally complex mechanism of catalysis. Our knowledge has greatly increased due to the recent crystallization of two xanthine oxidoreductases and the determination of the amino acid sequences of many members of the family. The evolution of molybdo–flavoenzymes can now be traced, given the availability of the structures of the corresponding genes in many organisms. The genes coding for molybdo–flavoenzymes are expressed in a cell-specific fashion and are controlled by endogenous and exogenous stimuli. The recent cloning of the genes involved in the biosynthesis of the molybdenum cofactor has increased our knowledge on the assembly of the apo-forms of molybdo–flavoproteins into the corresponding holo-forms. Xanthine oxidoreductase is the key enzyme in the catabolism of purines, although recent data suggest that the physiological function of this enzyme is more complex than previously assumed. The enzyme has been implicated in such diverse pathological situations as organ ischaemia, inflammation and infection. At present, very little is known about the pathophysiological relevance of aldehyde oxidase, aldehyde oxidase homologue 1 and aldehyde oxidase homologue 2, which do not as yet have an accepted endogenous substrate.
Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy
Authors: CALLEJA Véronique , AMEER-BEG Simon M. , VOJNOVIC Borivoj , WOSCHOLSKI Rudiger , DOWNWARD Julian , LARIJANI Banafshé
Published: May 2003

To be able to detect in situ changes in protein conformation without perturbing the physiological environment would be a major step forward in understanding the precise mechanism occurring in protein interaction. We have developed a novel approach to monitoring conformational changes of proteins in intact cells. A double-labelled fluorescent green fluorescent protein–yellow fluorescent protein (GFP–YFP) fusion protein has been constructed, allowing the exploitation of enhanced-acceptor-fluorescence (EAF)-induced fluorescence resonance energy transfer (FRET). Additionally, a novel fusion partner, YFPdark, has been designed to act as a sterically hindered control for EAF-FRET. Any conformational changes will cause a variation in FRET, which, in turn, is detected by fluorescence lifetime imaging microscopy (‘FLIM’). Protein kinase B (PKB)/Akt, a key component of phosphoinositide 3-kinase-mediated signalling, was selected for this purpose. Although conformational changes in PKB/Akt consequent to lipid binding and phosphorylation have been proposed in models, its behaviour in intact cells has not been tractable. We report here that platelet-derived-growth-factor (‘PDGF’) stimulation of NIH3T3 cells expressing the GFP–Akt–YFP construct resulted in a loss of FRET at the plasma membrane and hence a change in PKB/Akt conformation. We also show that the GFP–Akt–YFP construct conserves fully its functional integrity. This novel approach of monitoring the in situ conformational changes has broad application for other members of the AGC kinase superfamily and other proteins.
Protein conformation: through a lens, darkly
Authors: INSALL Robert
Published: May 2003

This commentary describes the study by Calleja and co-workers, who have used fluorescence resonance energy transfer between two fluorescent proteins fused to one protein kinase (protein kinase B/Akt) to reveal conformational changes in a functional protein. The findings of their study offer an intriguing insight into the behaviour of an enzyme that is essential for multiple aspects of mammalian signalling.
Transposable element (TE) display and rapid detection of TE insertion polymorphism in the Anopheles gambiae species complex
Authors: Biedler J. , Qi Y. , Holligan D. , Della Torre A. , Wessler S. , Tu Z.
Published: May 2003

Transposable element (TE) display was shown to be a highly specific and reproducible method of detecting the insertion sites of TEs in individuals of the African malaria mosquito, Anopheles gambiae, and its sibling species, A. arabiensis. Relatively high levels of insertion polymorphism were observed during the TE display of several families of miniature inverted‐repeat TEs (MITEs) that have variable copy numbers. The genomic locations of selected insertion sites were identified by matching the sequences of their corresponding bands in a TE display gel to specific regions of the draft A. gambiae genome assembly. We discuss different scenarios in which TE display will provide powerful dominant and co‐dominant genetic markers to study the behaviour of TEs in A. gambiae populations and to illustrate the complex population genetics of this intriguing disease vector. We suggest that TE display can also provide tools for a phylogenetic analysis of the A. gambiae complex.
B96Bom encodes a Bombyx mori tyramine receptor negatively coupled to adenylate cyclase
Authors: Ohta H. , Utsumi T. , Ozoe Y.
Published: May 2003

A cDNA encoding a biogenic amine receptor (B96Bom) was isolated from silkworm (Bombyx mori) larvae, and the ligand response of the receptor stably expressed in HEK‐293 cells was examined. Tyramine (TA) at 0.1–100 µm reduced forskolin (10 µm)‐stimulated intracellular cAMP levels by approximately 40%. The inhibitory effect of TA at 1 µm was abolished by yohimbine and chlorpromazine (each 10 µm). Although octopamine (OA) also reduced the cAMP levels, the potency was at least two orders of magnitude lower than that of TA. Furthermore, unlabelled TA (IC50 = 5.2 nm) inhibited specific [3H]TA binding to the membranes of B96Bom‐transfected HEK‐293 cells more potently than did OA (IC50 = 1.4 µm) and dopamine (IC50 = 1.7 µm). Taken together with the result of phylogenetic analysis, these findings indicate that the B96Bom receptor is a B. mori TA receptor, which is negatively coupled to adenylate cyclase. The use of this expression system should facilitate physiological studies of TA receptors as well as structure–activity studies of TA receptor ligands.
Molecular analysis of the serine/threonine kinase Akt and its expression in the mosquito Aedes aegypti
Authors: Riehle M. A. , Brown M. R.
Published: May 2003

A key component of the insulin‐signalling pathway, the protein kinase Akt, was identified and cloned as a cDNA from ovaries of the mosquito Aedes aegypti. An ortholog gene was found in the Anopheles gambiae genome database, and like other Akts, both mosquito Akts possess pleckstrin homology domains for membrane binding and a serine/threonine kinase domain. When Ae. aegypti ovaries were treated with bovine insulin in vitro, a putative Akt was threonine‐phosphorylated, as expected for Akts. AaegAKT was only expressed in embryos for the first 6 h after oviposition and in ovaries before and during a gonotrophic cycle.
An endothelin‐converting enzyme homologue in the locust, Locusta migratoria: functional activity, molecular cloning and tissue distribution
Authors: Macours N. , Poels J. , Hens K. , Luciani N. , De Loof A. , Huybrechts R.
Published: May 2003

Endothelin‐converting enzyme is the key enzyme in the process of endothelin production. Endothelin is a peptide that plays an important role in vasoconstriction and the development of neural crest‐derived cells in vertebrates. Activity assays performed on membrane extracts from Locusta migratoria brain revealed the existence of a protease activity responsible for the formation of mature endothelin‐1 from its precursor, big endothelin. Cloning experiments led to a cDNA sequence (Lom ECE) with an open reading frame of 727 amino acid residues displaying all the characteristic ECE features. A comparison of ECE activity levels among different tissues of the locust showed a high enzyme activity in the gonads and midgut. RT‐PCR experiments showed a wide tissue distribution of Lom ECE mRNA, with transcription being most abundant in brain tissue.
The pyrethroid knock‐down resistance gene in the Anopheles gambiae complex in Mali and further indication of incipient speciation within An. gambiae s.s.
Authors: Fanello C. , Petrarca V. , Della Torre A. , Santolamazza F. , Dolo G. , Coulibaly M. , Alloueche A. , Curtis C. F. , Touré Y. T. , Coluzzi M.
Published: May 2003

In Mali the Anopheles gambiae complex consists of An. arabiensis and Mopti, Savanna and Bamako chromosomal forms of An. gambiae s.s. Previous chromosomal data suggests a complete reproductive isolation among these forms. Sequence analysis of rDNA regions led to the characterization of two molecular forms of An. gambiae, named M‐form and S‐form, which in Mali correspond to Mopti and to Savanna/Bamako, respectively, while it has failed so far to show any molecular difference between Savanna and Bamako. The population structure of An. gambiae s.l. was analysed in three villages in the Bamako and Sikasso areas of Mali and the frequency of pyrethroid resistance of the knock‐down resistance (kdr) type was calculated. The results show that the kdr allele is associated only with the Savanna form populations and absent in sympatric and synchronous populations of Bamako, Mopti and An. arabiensis. This is the first molecular indication of barriers to gene flow between the Bamako and Savanna chromosomal forms. Moreover, analyses of specimens collected in the Bamako area in 1987 show that the kdr allele was already present in the Savanna population at that time, and that the frequency of this allele has gradually increased since then.
Identification of regulatory regions in the DNA puff BhC4‐1 promoter
Authors: Monesi N. , Basso L. R. , Paçó‐Larson M. L.
Published: May 2003

The mechanisms that control DNA puff BhC4‐1 expression in the salivary gland of sciarid late larvae have been shown to be conserved in Drosophila. By analysing Drosophila transformed with constructs carrying progressive deletions of the BhC4‐1 promoter fragment (−3314/+40) fused to the lacZ reporter gene we show that the elements required for the correct BhC4‐1‐lacZ developmental regulation in prepupal salivary glands are contained in a 226 bp fragment (−186/+40). Also, interestingly, this study identified a 67 bp fragment (−253/−187) that activates BhC4‐1‐lacZ expression specifically in the ring gland.
Identification and localization of the nicotinic acetylcholine receptor alpha3 mRNA in the brain of the honeybee, Apis mellifera
Authors: Thany S. H. , Lenaers G. , Crozatier M. , Armengaud C. , Gauthier M.
Published: May 2003

The nicotinic acetylcholine receptors are ligand‐gated ion channels responsible for rapid neurotransmission and are target sites for pesticides in insects. In the honeybee Apis mellifera, pharmacological and electrophysiological studies have shown that different nicotinic acetylcholine receptor subtypes may exist in the brain. Here, we have identified a honeybee cDNA that encodes a 537 amino acid protein with features typical of nicotinic acetylcholine receptor α subunit, and sequence homology to human α3. In situ hybridization on cryosections shows that the Apisα3 mRNA is differently expressed in larvae and adult. In larvae, Apisα3 mRNA expression is restricted to the suboesophageal ganglia. In adult, it is further expressed in the optic lobes, the dorsal lobes, the antennal lobes and the calyces of mushroom bodies. Together our results suggest that Apisα3 shows a controlled expression pattern during development.
The scarlet eye colour gene of the tephritid fruit fly: Bactrocera tryoni and the nature of two eye colour mutations
Authors: Zhao J. T. , Bennett C. L. , Stewart G. J. , Frommer M. , Raphael K. A.
Published: May 2003

A homologue of the Drosophila melanogaster eye‐colour gene, scarlet (st), has been isolated from the genome of the tephritid fruit fly, Bactrocera tryoni. The comparison of the B. tryoni and D. melanogaster scarlet gene shows 71.2% and 79.3% sequence identity at the DNA and the derived amino acid level, respectively. Two allelic eye‐colour mutations of B. tryoni, orange‐eyes and lemon‐eyes, have been recovered and found to be colocalized with the st gene. The st gene sequence in the two mutant strains has been examined for DNA sequence changes and expression levels.
Quantitative expression analysis of a Glyptapanteles indiensis polydnavirus protein tyrosine phosphatase gene in its natural lepidopteran host, Lymantria dispar
Authors: Chen Y. P. , Taylor P. B. , Shapiro M. , Gundersen‐Rindal D. E.
Published: May 2003

In the present study, expression of a newly identified Glyptapanteles indiensis polydnavirus (GiPDV) gene encoding a putative protein tyrosine phosphatase (PDVPTP) was monitored in vivo in the parasitized host, L. dispar, using one step RT‐PCR. Expression levels of the PDVPTP transcript were also evaluated in various host tissues at different times post parasitization (pp) using RT quantitative competitive PCR (RT‐qcPCR). Expression levels varied, with the most abundant transcript detected in host haemolymph 2 h pp. The high expression level in host haemolymph at an early stage of parasitization suggested a potential role for viral PDVPTP in disruption of the host immune system and protection of the endoparasitoid egg from encapsulation. Additionally, the PDVPTP gene or its homolog(s) mapped to more than one GiPDV genomic DNA segment, which may account for its increased level of expression in the absence of virus replication.
Variations in mitochondrial DNA and gene transcription in freezing‐tolerant larvae of Eurosta solidaginis (Diptera: Tephritidae) and Gynaephora groenlandica (Lepidoptera: Lymantriidae)
Authors: Levin D. B. , Danks H. V. , Barber S. A.
Published: May 2003

Respiration, mitochondrial (mt)DNA content, and mitochondrial‐specific RNA expression in fat body cells from active and cold‐adapted larvae of the goldenrod gall fly, Eurosta solidaginis, and the Arctic woolly bear caterpillar, Gynaephora groenlandica, were compared. Reduced amounts of mtDNA were observed in cold‐adapted larvae of both E. solidaginis and G. groenlandica collected in fall or winter, compared with summer‐collected larvae. mtDNA increased to levels similar to those of summer‐collected larvae after incubation at 10 °C or 15 °C for 5 h. Mitochondrial‐specific RNAs (COI and 16S) were observed in fat body cells of both active and cold‐adapted E. solidaginis larvae. Our results suggest that mitochondrial proteins required for respiration may be restored rapidly from stable RNAs present in overwintering larvae.
Identification of a tachykinin‐related neuropeptide from the honeybee brain using direct MALDI‐TOF MS and its gene expression in worker, queen and drone heads
Authors: Takeuchi H. , Yasuda A. , Yasuda‐Kamatani Y. , Kubo T. , Nakajima T.
Published: May 2003

Using a combination of MALDI‐TOF and on‐line capillary HPLC/Q‐Tof mass spectroscopy, we identified and determined the amino acid sequence of a novel neuropeptide in the brain of the honeybee Apis mellifera L., termed AmTRP peptide (Apis mellifera tachykinin‐related peptide), related to insect tachykinin. A cDNA for a prepro‐protein (prepro‐AmTRP) of AmTRP was isolated and determined to encode seven AmTRPs 1–7. Northern blot analysis indicated that the prepro‐AmTRP gene is expressed differentially in the nurse bee, forager, queen and drone heads. Strong expression was detected in the queen and forager heads, while weak and almost no significant expression was detected in the nurse and drone heads, respectively. These results suggest that AmTRP peptide functions as a neuromodulator and/or hormone, associated with sex‐specific or age/division of labour‐selective behaviour and/or physiology of the honeybees.
RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum
Authors: Aljamali M. N. , Bior A. D. , Sauer J. R. , Essenberg R. C.
Published: May 2003

RNA interference (RNAi), a gene silencing process, has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned putative Amblyomma americanum histamine binding protein (HBP) to test the significance of using this methodology in the assessment of the function and importance of gene products in ectoparasitic ticks. The female salivary glands incubated in vitro with HBP dsRNA had a significantly lower histamine binding ability. In addition, the injection of HBP dsRNA into the unfed females led both to a reduced histamine binding ability in the isolated salivary glands and to an aberrant tick feeding pattern or host response. Molecular data demonstrated less expression of the HBP mRNA in the RNAi group. Taken together, these results suggest that RNAi might be an important tool for assessing the significance of tick salivary gland secreted proteins modulating responses at the tick–host interface.
Examination of cytotoxicity and mutagenicity of AH26 and AH Plus sealers
Authors: Miletić I. , Jukić S. , Anić I. , Željezić D. , Garaj‐Vrhovac Osmak V., M.
Published: May 2003

Aim  To study in vitro the cytotoxic and mutagenic effects of AH26 and AH Plus.

Methodology  Cytotoxic effects on Chinese hamster V79 cells were determined by counting viable cells following incubation with eluations of AH26 and AH Plus. In one set of experiments, the materials were mixed, set for 1 h and then eluted with dimethyl sulphoxide (DMSO) for 1 h, 24 h and 7 days. In the other set, AH26 and AH Plus were mixed and set for 1 h, 24 h and 7 days in physiological saline then crushed and eluted in DMSO for 24 h. The cytotoxic effects of these eluates were evaluated. Three concentrations were chosen to examine the mutagenic effects of AH26 and AH Plus: 5.57, 16.7 and 55.7 μg mL−1. The structural chromosomal aberration analysis and micronucleus test were performed on human lymphocytes according to standard procedures.

Results  Dose–response curves of cell survival were obtained. Both materials were shown to be cytotoxic in doses larger than 55.7 μg mL−1, except for AH26, after 7 days setting time. AH Plus was also shown to be toxic in concentrations of 16.7 μg mL−1, except after 7 days setting time. Neither AH26 nor AH Plus induced a significant increase of chromosomal aberrations or micronuclei induction at any setting time or concentration.

Conclusion  There was no mutagenicity found for AH26 and AH Plus on human lymphocytes in highly controlled conditions in vitro.
Light and scanning electron microscopic evaluation of Glyde™ File Prep in smear layer removal
Authors: Lim T. S. , Wee T. Y. , Choi M. Y. , Koh W. C. , Sae‐Lim V.
Published: May 2003

Aim  To evaluate the effectiveness of Glyde™ File Prep used in conjunction with sodium hypochlorite irrigation in the removal of smear layer produced during root canal instrumentation.

Methodology  Thirty‐nine extracted human teeth with single root canals were used. Access cavities were prepared and the teeth divided into three groups of 13 teeth each. Each group was treated by one of the three different regimes of irrigation and conditioning during root canal instrumentation. Group A: 0.5 mL of 1% NaOCl irrigation after each file size with an additional final irrigation of 10 mL 1% NaOCl; group B: 0.5 mL of 1% NaOCl irrigation after each file size with an additional final irrigation of 10 mL 17% EDTA; group C: Glyde™ File Prep coated on each instrumentation file used in conjunction with 0.5 mL 1% NaOCl irrigation after each file size and an additional final irrigation of 10 mL 1% NaOCl. The teeth were then longitudinally grooved and sectioned. Root canal cleanliness was evaluated with the aid of a Nikon light microscope (×40 and ×100) and scanning electron microscope (×1000 and ×3000). The debris scores obtained at three canal regions were compared statistically within the same group and among different groups using repeated measurements of analysis of variance (anova) with Bonferroni adjustments and anova with posthoc Tukey HSD, respectively.

Results  The canals treated with EDTA and Glyde™ File Prep were significantly cleaner than those treated with NaOCl alone. The apical region of the root canals generally displayed more residual smear layer, but the difference was not significant.

Conclusions  Used in conjunction with NaOCl irrigation, Glyde™ File Prep was effective in removing smear layer produced during root canal instrumentation.
Endodontic treatment performed by Flemish dentists. Part 2. Canal filling and decision making for referrals and treatment of apical periodontitis
Authors: Hommez G. M. G. , De Moor R. J. G. , Braem M.
Published: May 2003

Aim  To gather information on root‐canal treatment carried out by dentists working in Flanders (Belgium).

Methodology  A questionnaire reported in a previous study was also used to gather information on canal medicaments, canal filling, and in decision making for referrals and treatment of apical periodontitis.

Results  Calcium hydroxide as an interappointment dressing was used by 69.7% of the respondents. Approximately one‐third of the respondents did not use any intracanal medicament. Caustic products used for pulp tissue fixation were used by 66.8% of the respondents. Cavit® (48.2%) and glass‐ionomer (31.3%) were the temporary coronal‐filling materials used most often, followed by zinc oxide–eugenol and IRM®. Cold lateral condensation of gutta‐percha was the filling technique most used by the respondents (65.8%). Single‐cone gutta‐percha placement (16%), paste techniques (4.9%) and silver points (3.9%) were still used. Resin‐based sealers were used most often (88.6%). Paraformaldehyde containing sealers such as Endomethasone and N2 were used infrequently. Approximately half of the practitioners were satisfied with their canal‐filling technique, others felt that they could do better (43.0%); 0.7% were not satisfied. In cases with apical periodontitis, the size of periapical lesions and/or the presence of a root filling influenced the choice of endodontic treatment. The most common reasons for referral of endodontic cases were: retrieval of silver points, surgery, and post removal.

Conclusions  The results of this study indicate that techniques and methods used for canal medication and canal filling were acceptable for the majority of the respondents. Re‐treatment was underestimated as a treatment option.
Induction of interleukin‐6 gene expression by pro‐inflammatory cytokines and black‐pigmented Bacteroides in human pulp cell cultures
Authors: Yang L.‐C. , Tsai C.‐H. , Huang F.‐M. , Liu C.‐M. , Lai C.‐C. , Chang Y.‐C.
Published: May 2003

Aim  To investigate the effect of pro‐inflammatory cytokines and black‐pigmented Bacteroides on the expression of IL‐6 gene in human pulp fibroblasts.

Methodology  IL‐1α, tumour necrosis factor‐α (TNF‐α) and the supernatants of Porphyromonas endodontalis, P. gingivalis and Prevotella intermedia were used to evaluate IL‐6 gene expression in human pulp fibroblasts. The levels of mRNAs were measured by the quantitative reverse‐transcriptase polymerase chain reaction analysis.

Results  Investigations of the time dependence of IL‐6 mRNA expression in pro‐inflammatory cytokines‐treated cells revealed a rapid accumulation of the transcript after 2 h of exposure and remained elevated throughout the 24‐h incubation period. In addition, black‐pigmented Bacteroides also induced IL‐6 gene expression in human pulp fibroblasts.

Conclusions  Pro‐inflammatory cytokines and black‐pigmented Bacteroides may be involved in developing pulpal inflammation through the stimulation of IL‐6 production.
A comparative study of root canal preparation using FlexMaster and HERO 642 rotary Ni–Ti instruments
Authors: Hülsmann M. , Gressmann G. , Schäfers F.
Published: May 2003

Aim  To compare several parameters of root canal preparation using two different rotary nickel–titanium (Ni–Ti) instruments: FlexMaster (VDW, Munich, Germany) and HERO 642 (Micro‐Mega, Besançon, France).

Methodology  Fifty extracted human mandibular molars with root canal curvatures between 20 and 40° were embedded into a muffle system. All root canals were prepared to size 45 using a high‐torque motor with two different Ni–Ti instruments, FlexMaster and HERO 642. In both groups, irrigation was performed with 2 mL NaOCl (3%) after each instrument size. RC‐Prep (Premier, Philadelphia, USA) was used as a chelating agent with each instrument. The following parameters were evaluated: straightening of curved root canals, postoperative root canal diameter, working safety (file fractures, perforations, apical blockages, loss of working length), cleaning ability and working time. Statistical analysis was performed using the Wilcoxon's test (P < 0.05) for straightening and Fishers's exact test (P < 0.05) for comparison of cross‐sections, contact between pre‐ and postoperative diameter, root canal cleanliness and working time.

Results  Both Ni–Ti systems maintained the curvature well: the mean degree of straightening was 0.6° for FlexMaster and 0.5° for HERO 642. One file was fractured with the FlexMaster system, but further procedural incidents were not recorded. Following preparation with FlexMaster, 18% of the root canals had a round diameter, 53% an oval diameter and 29% an irregular diameter; HERO 642 preparations resulted in a round diameter in 25%, oval shape in 47% and irregular cross‐sections in 28% of the cases. Mean working time was shorter for HERO 642 (66.0 s) than for FlexMaster (71.1 s). Cleanliness of the root canal walls was investigated under the SEM using 5‐score indices for debris and smear layer. For debris, HERO 642 and FlexMaster achieved 73 and 70% scores of 1 and 2, respectively. The results for smear layer were similar: HERO 642 and FlexMaster achieved 33 and 26% scores of 1 and 2, respectively. Significant differences between the two systems were not detected for any of the parameters evaluated.

Conclusions  Both systems respected original root canal curvature well and were safe. Both systems failed to remove debris and smear layer in the majority of the cases.
In vitro evaluation of apical microleakage following canal filling with a coated carrier system compared with lateral and thermomechanical gutta‐percha condensation techniques
Authors: Boussetta F. , Bal S. , Romeas A. , Boivin G. , Magloire H. , Farge P.
Published: May 2003

Aim  The apical sealing ability of a coated carrier system was evaluated in extracted human teeth and compared with lateral and thermomechanical condensation techniques using dye penetration.

Methodology  Sixty‐four extracted single‐rooted teeth were instrumented to an apical size 30 using 4% taper Hero 642™ instruments (Micromega, Besançon, France). The sample was divided into three experimental groups. Twenty teeth were randomly obturated with lateral condensation, 20 with thermomechanical compaction and 20 teeth with the Herofill™ Soft‐Core system. Four teeth were used as positive and negative controls. The teeth were covered with nail varnish up to 2 mm from the apical foramen and immersed in a 2% aqueous methylene blue dye solution for 1 week and then washed, dehydrated and embedded in resin. The apical 1 mm of each tooth was removed to reveal the apical limit of the preparation. Transverse sections of the teeth were taken at 500, 1000 and 1500 µm from this point and evaluated for apical leakage. Significant differences between the preparations were analysed with a semiautomatic analyser and the ratio of the dye‐penetrated surface to the total dentinal surface was calculated.

Results  Statistical analysis of the results demonstrated significantly less leakage for the Herofill™ Soft‐Core system compared to lateral condensation in terms of total mean dentinal surface and at the 500 µm level. No other differences were noted between Herofill™ Soft‐Core and thermomechanical or lateral condensation, either for the total mean value or at each level.

Conclusions  The Herofill™ Soft‐Core system was a reliable obturation system in the apical portion and compared favourably with other gutta‐percha filling techniques.
Endodontic treatment of developmental anomalies in posterior teeth: treatment of geminated/fused teeth – report of two cases
Authors: Tsesis I. , Steinbock N. , Rosenberg E. , Kaufman A. Y.
Published: May 2003

Aim  Gemination or fusion is a rare occurrence in the mandibular posterior teeth. Endodontic treatment of these teeth needs special care and attention to the bizarre anatomy. The aim of this article is to describe the problems encountered and the strategy in treating such cases.

Case report  Two cases of complex endodontic treatment of fused/geminated teeth are presented. The first is an 11‐year‐old girl with an anomalous ‘double’ first mandibular molar and premolar diagnosed as having necrotic pulp with chronic apical abscess of endodontic origin; the second is a 16‐year‐old boy with ‘double’ second and supernumerary mandibular molars, who was diagnosed with irreversible pulpitis. Both cases were treated successfully in multiple appointments. The common features and treatment modalities are discussed.

Key learning points 

Failure to diagnose fused/geminated teeth leads to misdiagnosis and a treatment plan that could cause permanent damage and tooth loss.

Generally, there is communication between root canal systems of fused/geminated teeth which should be treated as one entity.

Use of magnification is an important aid during treatment.
Review of the current status of tooth whitening with the walking bleach technique
Authors: Attin T. , Paqué F. , Ajam F. , Lennon Á. M.
Published: May 2003

Internal bleaching procedures such as the walking bleach technique can be used for whitening of discoloured root‐filled teeth. The walking bleach technique is performed by application of a paste consisting of sodium perborate‐(tetrahydrate) and distilled water (3% H2O2), respectively, in the pulp chamber. Following a critical review of the scientific literature, heating of the mixture is contra‐indicated as the risk of external cervical resorption and the formation of chemical radicals is increased by application of heat. An intracoronal dressing using 30% H2O2 should not be used in order to reduce the risk of inducing cervical resorption. This review provides advice based on the current literature and discusses how the walking bleach technique can lead to successful whitening of non‐vital root‐filled teeth without the risks of side‐effects.