bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of H2N3 influenza A viruses from swine in the United States
Authors: Ma Wenjun , Vincent Amy L. , Gramer Marie R. , Brockwell Christy B. , Lager Kelly M. , Janke Bruce H. , Gauger Phillip C. , Patnayak Devi P. , Webby Richard J. , Richt Jürgen A.
Published: December 2007

Although viruses of each of the 16 influenza A HA subtypes are potential human pathogens, only viruses of the H1, H2, and H3 subtype are known to have been successfully established in humans. H2 influenza viruses have been absent from human circulation since 1968, and as such they pose a substantial human pandemic risk. In this report, we isolate and characterize genetically similar avian/swine virus reassortant H2N3 influenza A viruses isolated from diseased swine from two farms in the United States. These viruses contained leucine at position 226 of the H2 protein, which has been associated with increased binding affinity to the mammalian α2,6Gal-linked sialic acid virus receptor. Correspondingly, the H2N3 viruses were able to cause disease in experimentally infected swine and mice without prior adaptation. In addition, the swine H2N3 virus was infectious and highly transmissible in swine and ferrets. Taken together, these findings suggest that the H2N3 virus has undergone some adaptation to the mammalian host and that their spread should be very closely monitored.
MET
amplification occurs with or without
T790M
mutations in
EGFR
mutant lung tumors with acquired resistance to gefitinib or erlotinib
Authors: Bean James , Brennan Cameron , Shih Jin-Yuan , Riely Gregory , Viale Agnes , Wang Lu , Chitale Dhananjay , Motoi Noriko , Szoke Janos , Broderick Stephen , Balak Marissa , Chang Wen-Cheng , Yu Chong-Jen , Gazdar Adi , Pass Harvey , Rusch Valerie , Gerald William , Huang Shiu-Feng , Yang Pan-Chyr , Miller Vincent , Ladanyi Marc , Yang Chih-Hsin , Pao William
Published: December 2007

In human lung adenocarcinomas harboring
EGFR
mutations, a second-site point mutation that substitutes methionine for threonine at position 790 (T790M) is associated with approximately half of cases of acquired resistance to the EGFR kinase inhibitors, gefitinib and erlotinib. To identify other potential mechanisms that contribute to disease progression, we used array-based comparative genomic hybridization (aCGH) to compare genomic profiles of
EGFR
mutant tumors from untreated patients with those from patients with acquired resistance. Among three loci demonstrating recurrent copy number alterations (CNAs) specific to the acquired resistance set, one contained the
MET
proto-oncogene. Collectively, analysis of tumor samples from multiple independent patient cohorts revealed that
MET
was amplified in tumors from 9 of 43 (21%) patients with acquired resistance but in only two tumors from 62 untreated patients (3%) (
P
= 0.007, Fisher's Exact test). Among 10 resistant tumors from the nine patients with
MET
amplification, 4 also harbored the

EGFR
T790M

mutation. We also found that an existing
EGFR
mutant lung adenocarcinoma cell line, NCI-H820, harbors
MET
amplification in addition to a drug-sensitive
EGFR
mutation and the
T790M
change. Growth inhibition studies demonstrate that these cells are resistant to both erlotinib and an irreversible EGFR inhibitor (CL-387,785) but sensitive to a multikinase inhibitor (XL880) with potent activity against MET. Taken together, these data suggest that
MET
amplification occurs independently of

EGFR
T790M

mutations and that MET may be a clinically relevant therapeutic target for some patients with acquired resistance to gefitinib or erlotinib.
Impact of the Implementation of Telemanagement on a Disease Management Program in an Elderly Heart Failure Cohort
Authors: Gambetta Miguel , Dunn Patrick , Nelson Dawn , Herron Bobbi , Arena Ross
Published: December 2007

The purpose of the present investigation is to examine the impact of a telemanagement component on an outpatient disease management program in patients with heart failure (HF). A total of 282 patients in whom HF was diagnosed and who were enrolled in an outpatient HF program were included in this analysis. One hundred fifty‐eight patients additionally participated in a self‐directed telemanagement component. The remaining 124 patients received care at an HF clinic but declined telemanagement. During the 7‐month tracking period, 19 patients in the HF clinic plus telemanagement group and 53 patients in the HF clinic only group were hospitalized for cardiac reasons (log rank, 36.0; P<.001). The HF clinic only group had a significantly higher risk for hospitalization (hazard ratio, 4.0; 95% confidence interval, 2.4–6.7; P<.001). The results of the present study indicate that telemanagement is an important component of a disease management program in patients with HF.
Can Intensive Statin Therapy Halt the Progression of Atherosclerosis? Recent Evidence and Potential Implications for Patient Management
Authors: Kushner Pamela R.
Published: December 2007

A number of studies using various imaging techniques have demonstrated that intensive lipid lowering with statins can halt or delay the progression of atherosclerosis and even, in some cases, lead to plaque regression. Improvements in atheroma burden with intensive statin therapy appear to be related not just to decreasing low‐density lipoprotein cholesterol but also to anti‐inflammatory and antiproliferative effects. Clinical trial results also suggest that achieving low‐density lipoprotein cholesterol levels even lower than those currently recommended can produce improved clinical outcomes across a range of patient types. Given this body of evidence, it appears appropriate to use intensive statin therapy to treat dyslipidemic patients at high risk for coronary heart disease.
A muscle‐specific actin gene from the Mediterranean fruit fly, Ceratitis capitata
Authors: He M. , Haymer D. S.
Published: December 2007

A characterization of an actin gene isolated from the genome of the Mediterranean fruit fly, Ceratitis capitata, including the complete sequencing of the coding, 3′ and 5′ flanking regions of this gene and a partial cDNA was carried out. The partial cDNA was derived from the 3′ untranslated region of the actin gene described here, and has been used to identify this gene uniquely. The DNA sequence data presented here, together with the pattern of expression exhibited by this gene during development, strongly support the interpretation that this is a muscle‐specific actin gene. Peaks of expression are seen in tissues and during temporal phases of development where muscle differentiation is occurring. The derived protein sequence of the Medfly actin gene shows the highest degrees of similarity, 98.4 and 96.6% respectively, with the two muscle‐specific actin genes 79B and 88F from D. melanogaster. The Medfly actin gene also has a single intervening sequence, and an intron is found at the same position in the 79B and 88F actin genes. In the coding region at the DNA level, 17.2 and 16.4% nucleotide differences, respectively, are observed between the Medfly actin gene and these same two D. melanogaster actin genes. The disparity between the amino acid and nucleotide comparisons can be explained, in part, by a high level of synonymous changes in the DNA sequence. In addition, despite the many similarities, codon usage appears to be very different between the actin genes of these species.
Distribution of split 5.8S ribosomal RNA in Diptera
Authors: Shimada T.
Published: December 2007

Although the 5.8S ribosomal RNA (rRNA) of most eucaryotes consists of 155–170 nucleotides, in two dipterous species the 5.8S rRNA consists of two pieces, 123 and 30 nucleotides in length. The distribution of split 5.8S rRNA was studied in other Diptera and the most closely related order Siphonaptera to learn the origin of split 5.8S rRNA. Four species of mosquitoes, Culex tritaeniorhynchus, Culex pipiens molestus, Aedes albopictus, Anopheles sp. (Culicidae) had a single 5.8S rRNA consisting of˜154 nucleotides. A flea Ctenocephalides felis (Siphonaptera: Pulicidae) also had a single RNA of ˜158 nucleotides. A crane fly (Tipulidae), a midge Orthocladius akamusi (Chironomidae), a robber fly (Asilidae), and a house fly Musca domestica (Muscidae), on the other hand, had divided 5.8S rRNA as did a fruit fly Drosophila melanogaster (Drosophilidae) and a darkwinged fungus gnat Sciara coprophila (Sciaridae). Three hypotheses are proposed on the relationship between the evolution of the 5.8S rRNA and the phylogeny of Diptera.
Expression of the chloramphenicol acetyltransferase gene in Aedes albopictus (C6/36) cells using a non‐infectious Sindbis virus expression vector
Authors: Olson K. E. , Carlson J. O. , Beaty B. J.
Published: December 2007

Genomic RNA was transcribed in vitro from the non‐infectious Sindbis (SIN) virus expression vector (pTRCAT) and introduced into C6/36 (Aedes albopictus) cells by liposome‐mediated transfections. The chloramphenicol acetyltransferase (CAT) polypeptide expressed within cells was detected by an indirect immunofluorescent assay directly into 24‐well polystyrene tissue culture plates. Approximately 1 in 1000 C6/36 cells showed fluorescence when the transfection was optimized. CAT enzyme activity was also assayed; in C6/36 cells CAT expression was detected as early as 8 h after transfection, peaked at 24 h, and could still be detected at 7 days. At 24 h post‐transfection each transfected C6/36 cell was calculated to express 1.3 × 107 CAT polypeptides.
Plasmid modifications in a tick‐borne pathogen, Borrelia burgdorferi, cocultured with tick cells
Authors: Munderloh U. G. , Park Y.‐J. , Dioh J. M. , Fallon A. M. , Kurtti T. J.
Published: December 2007

We describe an in vitro system that will facilitate molecular analysis of the association between Lyme disease spirochetes and vector cells. We cocultured Borrelia burgdorferi continuously with two tick cell lines, RAE25 (from Rhipicephalus appendiculatus) and IDE8 (from Ixodes scapularis). A clone isolated after twenty‐two passages with RAE25 cells had lost the largest (49 kb) plasmid, and probes containing information normally encoded on it, including genes for two surface proteins, hybridized to smaller plasmids. Spirochetes maintained with IDE 8 cells showed a new 43 kb plasmid that hybridized to a probe made from the 49 kb plasmid. After reisolation from hamsters, these spirochetes carried a large plasmid (100 kb) that hybridized with the 49 kb plasmid. These changes may illustrate a plasticity that enables B. burgdorferi to adapt to different environments.
The salivary glands of the vector mosquito, Aedes aegypti, express a novel member of the amylase gene family
Authors: Grossman G. L. , James A. A.
Published: December 2007

Several cDNA clones with similarity to α‐amylases have been characterized from a library made from adult female salivary gland RNA isolated from the vector mosquito, Aedes aegypti. The corresponding gene, designated Amylase I (Amy I), is expressed specifically in the proximal‐lateral lobes of the adult female salivary gland, a pattern overlapping that of another gene, Mal I, involved in carbohydrate metabolism. The deduced amino acid sequence of Amy I indicates that this gene encodes a protein, approximate Mr= 81,500, that appears to be a novel member of the amylase gene family. The mosquito protein contains a putative signal peptide for secretion and several consensus sites for asparagine‐linked glycosylation. The Amy I protein shows significant similarity to invertebrate and vertebrate amylases including the conservation of four reactive and substrate binding sites. However, the amino‐terminal region of the Amy‐I protein is unique to the mosquito. Similarity with the Drosophila melanogaster protein is evident only after the first 260 amino acids in the mosquito sequence. The identification of this gene and its expression pattern adds to the observed relationship between spatial‐specific gene expression in the female salivary glands and the specific feeding mode of the adult mosquito.
Gene structure of Bombyx mori larval serum protein (BmLSP)
Authors: Fujiwara Y. , Yamashita O.
Published: December 2007

To understand the molecular mechanisms of the larval‐specific transcription of Bombyx mori larval serum protein (BmLSP), we isolated a clone of the BmLSP gene from a genomic library and sequenced a 3.5‐kb fragment. An intron was found in the 5′ noncoding region of the BmLSP gene. A putative transcription start point was determined by primer extension analysis. Genomic Southern hybridization showed that there is one copy of the BmLSP gene in a haploid genome. A database search revealed that the BmLSP gene has presumptive repetitive sequences found in other B. mori genes, the sequence homologous to ecdysone‐responsive elements and a heptamer sequence found in storage protein genes.
Sequence analysis of a mosquito ribosomal protein rpL8 gene and its upstream regulatory region
Authors: Lan Que , Fallon A. M.
Published: December 2007

The gene encoding Aedes albopictus ribosomal protein L8 was isolated using a cDNA probe. Based on the deduced amino acid sequence, rpL8 has a mass of 28,605 Da, a pl of 11.97, and contains 9.6% Arg and 11.9% Lys. The rpL8 gene spans 1229 nucleotides, and contains three exons measuring 73, 150, and 648 nucleotides. The first intron is 293 nucleotides long and interrupts an 85‐nucleotide untranslated leader sequence. The AUG codon is located 12 nucleotides downstream of the 5′‐end of the second exon. Separating the second and third exons is a 65‐nucleotide intron. The major transcription initiation site, identified by primer extension and polymerase stop reactions, mapped 378 nucleotides upstream from the AUG start codon; minor initiation sites were also detected. The DNA sequence upstream of the rpL8 gene was T‐rich, but conventional TATA and CAAT boxes were absent. This is the first molecular analysis of a mosquito ribosomal protein gene.
Genomic subtractive hybridization to isolate species‐specific DNA sequences in insects
Authors: Clapp J. P. , McKee R. A. , Allen‐Williams L. , Hopley J. G. , Slater R. J.
Published: December 2007

Selective enrichment has been used in a number of instances for the isolation of species‐specific sequences in prokaryotes. This paper reports the successful application of the technique to insects. Genomic probes were derived to the target species D. funebris and D. simulans. The method involves the biotinylation of non‐target ‘driver’ DNA prepared from the closely related species D. melanogaster and its hybridization to homologous sequences in the target DNA. Hybrid molecules were removed from the reaction by incubation with streptavidin followed by phenol extraction, leaving a preparation enriched for target fragments. All DNA fragments isolated in the D. funebris experiments proved to be specific to that species. Five out of twenty‐four fragments screened in the D. simulans experiments were specific when screened with homologous DNA and genomic DNA from its sibling species, D. melanogaster.
Polymorphic cDNAs encode for the methionine‐rich storage protein from Manduca sexta
Authors: Wang Xiao‐Yu , Frohlich D. R. , Wells M. A.
Published: December 2007

By cDNA cloning and sequencing we have shown that Manduca sexta larvae produce three very closely related methionine‐rich storage proteins, MMR1, MMR2 and MMR3. Out of 2256 nucleotides in the coding region, the cDNAs differ by at most twenty‐one bases and this leads to a single amino acid difference between MMR1 and MMR2, and between MMR2 and MMR3, whereas MMR1 and MMR3 differ by two amino acids. Using both distance and parsimony methods, similarities between the M. sexta and Bombyx mori methionine‐rich and arylphorin storage proteins were examined. Homologous proteins from the two species tend to be more closely related than are the two classes of storage proteins in a single species.
Isolation, sequencing and characterization of two cDNA clones coding for trypsin‐like enzymes from the midgut of Aedes aegypti
Authors: Kalhok. S. E. , Tabak L M. , Prosser D. E. , Brook W. , Downe A. E. R. , White B. N.
Published: December 2007

In order to understand the regulation of trypsin genes by the blood meal, we constructed a cDNA library from mRNA isolated from midguts of blood‐fed female Aedes aegypti. The library was screened with a Dros‐ophila melanogaster trypsin‐like gene; twelve cDNAs were isolated and sequenced. Two clones were 846 bp and 788 bp long with 762 bp and 716 bp open reading frames, respectively. The cDNAs were identified as coding for serine proteases by the presence of conserved serine, histidine and aspartic acid residues; the presence of an aspartate residue at position 176 suggests that the clones were derived from trypsin‐like gene transcripts rather than chymotrypsin or other serine proteases. One of the clones contained a 5 untranslated region and coding regions for putative signal and activation peptides, suggesting that the product is secreted as an inactive zymogen and processed by autoactivation. Southern analysis of genomic DNA suggests that trypsin is encoded by a multi‐gene family in A. aegypti.
Nucleotide sequence of an adult‐specific cuticular protein gene from the beetle Tenebrio molitor: effects of 20‐hydroxyecdysone on mRNA accumulation
Authors: Bouhin H. , Braquart C. , Charles J.‐P. , Quennedey B. , Delachambre J.
Published: December 2007

The accumulation of transcripts from two adult‐specific cuticular genes (ACP‐20 and ACP‐22) is shown to be modified after addition of exogenous 20‐hydroxyecdysone. In the continuous presence of high levels of the hormone, the expression of ACP‐20 gene is significantly weaker than that of untreated controls, while ACP‐22 expression is 2.5‐fold increased. During active synthesis of the ACP messages, a 0.5 µ20‐hydroxyecdysone injection causes a rapid 2‐fold increase in ACP‐22 mRNA and is not able to repress ACP‐20 mRNA accumulation. We conclude that these genes whose transcripts appear in an almost coordinated manner in epidermal cells during the moulting cycle are regulated by ecdysteroids in a different way. In order to undertake a functional dissection of the promoter regions of ACP‐22 gene, we have isolated and sequenced a genomic clone. The sequence similarities with other cuticular protein genes are discussed.
An amplified mosquito dihydrofolate reductase gene: ampiicon size and chromosomal distribution
Authors: Shotkoski F. A. , Fallon A. M.
Published: December 2007

The dihydrofolate reductase ampiicon in metho‐trexate‐resistant mosquito cells provides an amplified gene in insects that can be compared directly to the corresponding amplified locus in mammalian cells. A cloned Aedes albopictus dihydrofolate reductase gene was used as a probe to examine the structure of dihydrofolate reductase alleles in sensitive and resistant cells. In wild type cells, two distinct alleles could be distinguished by restriction fragment length polymorphisms, one of which was amplified in methotrexate‐resistant cells. Subsequent to amplification, an additional polymorphism at a ten base‐pair XmrA recognition site indicated that the amplified mosquito gene is subject to genetic changes similar to those that have been described in amplified genes from mammalian cells. Pulsed‐field gel electrophoresis was used to determine that the minimum size of the mosquito dihydrofolate reductase ampiicon was 140 kilobases; ethidium bromide staining patterns suggested a size of at least 233 kilobases. Dihydrofolate reductase probes hybridized to distinct locations in five of the thirteen chromosomes in Mtx‐5011‐128 cells, indicating that the amplified DNA probably occurs as tandem direct or inverted repeats.
Baculovirus immediate early gene promoter based expression vectors for transient and stable transformation of insect cells
Authors: Vulsteke V. , Janssen I. , Broeck J. Vanden , Loot A. De , Huybrechts R.
Published: December 2007

A recombinant plasmid vector was constructed in which the bacterial LacZ gene was placed under the control of a Bombyx mori baculovirus early promoter. The vector proved to be active in transfected cultured dipteran and lepidopteran cells. Co‐transfection carried out with this recombinant plasmid vector and a plasmid containing the hygromycin phosphotransferase gene followed by selection with the antibiotic hygromycin B, resulted in stable transformation of cultured Drosophila melanogaster Schneider 2 cells. Southern blot analysis of the host cell's genomic DNA in combination with chromosomal in situ hybridization demonstrated that multiple copies of both plasmids were integrated in the host cell's genome.
Phylogeny of the Nasonia species complex (Hymenoptera: Pteromalidae) inferred from an internal transcribed spacer (ITS2) and 28S rDNA sequences
Authors: Campbell B. C. , Steffen‐Campbell J. D. , Werren J. H.
Published: December 2007

The phylogeny of the cryptic species complex of wasps in the genus Nasonia was inferred by analysis of nucleotide sequences of an rDNA internal transcribed spacer (ITS2) and the D2 region of 28S rDNA. Phylogenetic analysis showed that N. vitripennis descended from a theoretical common ancestor with that of a lineage that diverged into N. longicornis and N. giraulti. Differences in the ITS2 regions clearly distinguished two strains of N. giraulti. Another member of the Dibrachys Group, Trichomalopsis dubius, was placed outside of the Nasonia complex. The D2 region had a base substitution rate =2 times slower than the ITS2 region and was used to resolve the phylogenetic affiliation of an eulophid, Melittobia digitata, to the pteromalids. Tree topology of the Nasonia complex was congruent with the phylogeny of a cluster of Wolbachia bacteria which infect these insects. The possible role of these bacteria in driving speciation is discussed.
Isolation of non‐LTR retrotransposon reverse transcriptase‐like sequences from phlebotomine sandflies
Authors: Booth D. R. , Ready P. D. , Smith D. F.
Published: December 2007

Reverse transcriptase‐like sequences (RTs) with amino acid motifs characteristic of non‐LTR retro‐transposons have been isolated from several medically important phlebotomine sandfly species. These sequences were amplified using the polymerase chain reaction (PCR) with primers based on conserved amino acid motifs present in previously described insect RTs. A further set of RTs were amplified using primers based on the conserved regions identified in phlebotomine RTs. The average similarity of the phlebotomine RTs to the Drosophila I, F and R1Dm elements was 28–29% between the closest primers used. Phlebotomine RTs were 31–62% similar to each other, the most dissimilar sequences coming from the same species. Several amino acid residues were invariant in the ten phlebotomine RTs, including motif Q(F/Y)GF, conserved in other non‐LTR retrotransposons, but not in retrovirus or LTR retrotransposon RTs. The remarkable conservation of this distinctive domain of non‐LTR retrotransposon RTs suggests it has a vital and possibly unique role in the mode of reverse transcription of this class of transposon.
Expression of polydnavirus genes from the parasitoid wasp Cotesia kariyai m two noctuid hosts
Authors: Hayakawa Y. , Yazaki K. , Yamanaka A. , Tanaka T.
Published: December 2007

DNA purified from polydnavirus particles isolated from the parasitoid wasp Cotesia kariyai contained double‐stranded closed circular molecules which were polydisperse in molecular weight. 2 days after viral injection into the host armyworm larvae viral DNA was detected in all tested larval tissues, including haemocytes, fat body, nerve cord and brain. Viral transcripts were also observed in all the tissues of virus‐injected larvae. The most specific tissue was haemocytes because more viral DNA and RNA was detected than in the other tissues. Viral transcripts were not detected in haemocytes of virus‐injected larvae of common cutworm. The present data suggest that C. kariyai virus (CkV) gene expression occurs species‐specifically, although viral DNA can be detected in ail tested tissues of the habitual host armyworm.
An estimate of the number of serine protease genes expressed in sheep blowfly larvae (Lucilia cuprina)
Authors: Elvin C. M. , Vuocolo T. , Smith W. J. M. , Eisemann C. H. , Riddles P. W.
Published: December 2007

A large and diverse family of serine protease genes was identified in first‐instar larval cDNA of the sheep blowfly (Lucilia cuprina). This complex repertoire of genes was identified via a PCR approach using highly degenerate primers based on structurally conserved regions which surround the active site His and Ser residues found in all serine proteases. PCR products from entire first‐instar larval cDNA, or from third‐instar larval salivary glands or cardia, generated using a microscale RT‐PCR method, were cloned into a plas‐mid vector. Comparison of the restriction fragment patterns of PCR products generated from the three different sources suggests a highly diverse tissue‐specific pattern of serine protease expression in this organism. Detailed analysis of the restriction fragment patterns of sixty‐nine randomly selected clones from entire first‐instar larvae revealed forty‐nine different classes of PCR product. Maximum likelihood analysis of these data indicate that between 125 and 220 different serine protease genes are expressed in first‐instar larvae of L. cuprina. DNA sequence analysis of ten randomly‐selected clones, derived from the three tissue sources, indicated that all ten encoded serine protease gene fragments. A frequently occurring PCR product, generated from both first‐instar total cDNA and third‐instar cardia cDNA, showed 73% amino acid identity to a digestive protease expressed in Droso‐phila melanogaster larval gut cells.
Molecular identification and phylogeny of parasitic wasp species (Hymenoptera: Trichogrammatidae) by mitochondrial DNA RFLP and RAPD markers
Authors: Vanlerberghe‐Masutti F.
Published: December 2007

Mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) were tested for their ability to discriminate between seven species of minute parasitic wasps belonging to the genus Trichogramma. They proved to be reliable species‐diagnostic molecular markers. Pairwise comparisons of the mtDNA restriction maps revealed considerable differentiation among the seven species. Percentage of common restriction sites ranged from 30% to 83%. Phylogenetic analyses performed either on the mtDNA nucleotide distance matrix or on the matrix of the restriction site‐state generated a tree congruent with those based on allozymes and morphology.

RAPD procedures also revealed species‐specific banding patterns and seem promising for a rapid and easy identification of Trichogramma species. Moreover, for some Trichogramma species, RAPD banding patterns might be informative of the phylogenetic related ness.
Bombyx gene promoter analysis in transplanted silk gland transformed by particle delivery system
Authors: Horard B. , Mangé A. , Pelissier B. , Couble P.
Published: December 2007

In most, if not all non‐Drosophila insect systems, the study of gene regulation relies on in vitro analyses. With the aim of providing an in vivo assay, we developed an original protocol using the Bombyx mori silk gland, by which the functioning of cloned gene promoters can be efficiently measured. This consists of introducing DNA‐coated tungsten particles into isolated silk glands by ballistic methods, according to a modified protocol in which the DNA is maintained in a hydrated state. The treated organ is then grafted into the haemolymph cavity of a host larva before analysis of transgene expression. In these reconstituted physiological conditions more than half of the cells express the transfected gene construct, and expression is both promoter‐dependent and cell type‐specific.
Identification by rDNA‐PCR of Anopheles bwambae, a geothermal spring species of the An. gambiae complex
Authors: Townson H. , Onapa A. W.
Published: December 2007

The identification of the malaria vector Anopheles bwambaeby rDNA‐PCR is described. PCR primers that amplify a region of the intergenic spacer of rDN A of An. gambiae s.s. produce two diagnostic PCR products of 690 bp and 390 bp with An. bwambae.
Functional expression of insecticide‐resistant GABA receptors from the mosquito Aedes aegypti
Authors: Shotkoski F. , Lee H.‐J. , Zhang H.‐G. , Jackson M. B. , Ffrench‐Constant R. H.
Published: December 2007

We are interested in cloning insecticide resistance genes from vector mosquitos for use as selectable markers in their genetic transformation. As a first step towards this goal, we here report the functional homo‐muitimeric expression of a γ‐aminobutyric acid (GABA) receptor subunit gene, Resistance to dieldrin (Rdl), from the yellow fever mosquito Aedes aegypti in baculovirus‐infected insect cell lines. Replacement of alanine296 with a serine leads to approximately 100‐fold insensitivity to picrotoxin as previously observed in Drosophila. This shows not only that the mosquito GABA receptor cDNA is functional but also that it can be simply mutated to resistance. Strategies for incorporation of this cDNA into a minigene for the genetic transformation of mosquitoes are discussed.
of the aphid Schlechtendalia chinensis
Authors: Lai C.‐Y. , Baumann P. , Moran N. A.
Published: December 2007

Two DNA fragments (3941 and 7152 base pairs) from the procaryotic endosymbiont (Buchnera) of the aphid Schlechtendalia chinensis were cloned and sequenced. The smaller fragment contained trpEG and the larger fragment contained trpDC(F)BA, genes coding for enzymes of the tryptophan biosynthetic pathway which convert chorismate to tryptophan. Both of these gene clusters were present as one copy on the endosymbiont chromosome and probably constitute two transcription units. The deduced amino acid sequences of the proteins was 51–61% identical to the corresponding proteins were previously studied in Buchnera of the aphid Schizaphis graminum. In this endosymbiont, trpEG is amplified and located on a plasmid, whereas, in the endosymbiont of S. chinensis, as in other eubacteria, trpEG occurs as a single copy on the bacterial chromosome. Implications of these findings for the evolution of this mutualistic association are discussed.
Analysis of mitochondrial DNA and development of PCR‐based diagnostic molecular markers for Mediterranean fruit fly (Ceratitis capitata) populations
Authors: Gasparich G. E. , Sheppard W. S. , Han H.‐Y. , McPheron B. A. , Steck G. J.
Published: December 2007

A 2.99 kb mtDNA fragment containing two variable restriction endonuclease sites (EcoRV and Xbal) was subcloned and sequenced from the Mediterranean fruit fly (Ceratitis capitata). This fragment represents approximately one‐fifth of the entire mitochondrial sequence. The sequence was aligned with the comparable region from Drosophila yakuba and Anopheles gambiae, resulting in 81.8% and 76.7% identity at the nucleotide level, and 77% and 67.7% identity, respectively, at the amino acid level. The sequenced region includes the complete genes for NADH dehydrogenase 4, NADH dehydrogenase 4L, NADH dehydrogenase 6, and transfer RNAs for proline, threonine and histidine, and part of the genes for NADH dehydrogenase 5 and cytochrome b. Oligonucleotide primers were designed to asymmetrically bracket each of two variable restriction endonuclease sites to allow PCR amplification and subsequent restriction endonuclease analysis of individual fly samples.
The Simulium damnosum species complex: phylogenetic analysis and molecular identification based upon mitochondrially encoded gene sequences
Authors: Tang J. , Toè L. , Back C. , Zimmerman P. A. , Pruess K. , Unnasch T. R.
Published: December 2007

The DNA sequence of portions of the 16s rRNA and the NADH dehydrogenase subunit 4 (ND4) genes were used to determine phylogenetic relationships in the Simulium damnosum s.l. species complex. Results suggested that at least two major clades existed in the S. damnosum species complex, and that members of the S. damnosum s.l. species complex were not closely related to North American Simulium species. The sequence variability of the ND4 gene was exploited to develop a method to distinguish the sibling species of the S. damnosum s.l. species complex, based on directed heteroduplex analysis of PCR products derived from the ND4 gene. This method was capable of classifying the six sibling species into at least five groups.
Unusually high frequency of (CT)n and (GT)n microsatellite loci in a yellowjacket wasp, Vespula rufa (L.) (Hymenoptera: Vespidae)
Authors: Thorén P. A. , Paxton R. J. , Estoup A.
Published: December 2007

A high number of (GT)n and (CT)n microsatellites were isolated from a partial genomic library of a primitively eusocial wasp, Vespula rufa. On average, (GT)n and (CT)n repeats occur in the genome approximately every 8 kb and 2.5 kb respectively. This is the highest density of dinucleotide microsatellites reported in the literature. Predominant categories of repeats are imperfect and compound repeats for (GT)n microsatellites and perfect repeats for (CT)n microsatellites. Comparisons with other Hymenoptera and with mammalian species showed significant differences in the frequency distribution of these categories of repeats. Many (CT)n and (GT)n repeats are surrounded by various types of microsatellites, suggesting an associative distribution of short tandem repeats. A high level of intracolonial polymorphism is demonstrated with one microsatellite locus, showing that multiple mating of the queen occurs in V. rufa.
Cloning and characterization of the white gene from Anopheles gambiae
Authors: Besansky N. J. , Bedell J. A. , Benedict M. Q. , Mukabayire O. , Hilfiker D. , Collins F. H.
Published: December 2007

A 14 kb region of genomic DN A containing the X‐linked Anopheles gambiae eye colour gene, white, was cloned and sequenced. Genomic clones containing distinct white+ alleles were polymorphic for the insertion of a small transposable element in intron 3, and differed at 1% of nucleotide positions compared. Sequence was also determined from a rare 2914 bp cDNA. Comparison of cDNA and genomic sequences established an intron‐exon structure distinct from Drosophila white. Despite a common trend in Anopheles and Drosophila of weak codon bias given low levels of gene expression, codon usage by Anopheles gambiae white was strongly biased. Overall amino acid identity between the predicted mosquito and fruitfly proteins was 64%, but dropped to 14% at the amino terminus. To correlate phenotypically white‐eyed strains of A. gambiae with structural lesions in white, five available strains were analysed by PCR and Southern blotting. Although these strains carried allelic mutations, independently generated by gamma radiation (three strains) or spontaneous events (two strains), no white lesions were detected. Significantly, another non‐allelic X‐linked mutation, causing an identical white‐eyed phenotype, has been correlated with a structural defect in the cloned white gene (Benedict et al., 1995). Taken together, these observations suggest that the white‐eyed mutants analysed in the present study carry mutations in a second eye colour gene and are most likely white+.
PCR‐based detection of Wolbachia, cytoplasmic incompatibility microorganisms, infected in natural populations of Laodelphax striatellus (Homoptera: Delphacidae) in central Japan: has the distribution of Wolbachia spread recently?
Authors: Hoshizaki S. , Shimada T.
Published: December 2007

Cytoplasmic incompatibility is caused in various insects by intracellular infection with rickettsia‐like microorganisms of the genus Wolbachia. In Japan Laodelphax striatellus shows unidirectional cytoplasmic incompatibility between northeastern and southwestern populations. In this study, nine natural populations of L. striatellus collected from central Japan, including the geographic boundary between the two cytotype populations, were analysed for Wolbachia infection by PCR using primers specific to Wolbachia 16S rDNA. The geographic pattern of the infection rates of the southwestern (high) and the northeastern (low or zero) populations broadly resembled that of a previous study of incompatibility. In populations which originated from the boundary regions between the southwestern and northeastern populations, the infected and uninfected cytotypes coexisted. It is suggested that in some populations of L. striatellus, which formerly had been uninfected with Wolbachia, the infection property has changed to the infected. Based on our results, we conclude that the distribution of Wolbachia‐infected L. striatellus populations have spread northeasterly during the last 12 years.
A review of the use of ribosomal DNA (rDNA) to differentiate among cryptic Anopheles species
Authors: Collins F. H. , Paskewitz S. M.
Published: December 2007

Cryptic species complexes are groups of closely related species that are difficult or impossible to distinguish by morphological traits. These complexes are known from a wide variety of arthropods and are common among the well‐studied, medically‐important insects. For example, many of the anopheline vectors of malaria parasites are members of cryptic species complexes. Complexes typically include both vector and non‐vector species, and two or more member species are often found sympatrically. Until the late 1950s, only two such Anopheles complexes were known, the A. gambiae complex from Africa and the A. maculipennis complex from Europe. Today, dozens of Anopheles cryptic species complexes are recognized, and accumulating evidence suggests that most important malaria vectors are likely to be members of such complexes.

A variety of methods have been developed for identifying the species of individual specimens from these complexes, although until recently only those based on species‐specific allozymes and polytene chromosome inversions were widely used. The limitations inherent in these methods have been circumvented with DNA‐based procedures, which are especially useful because both sexes and all developmental stages can be identified, and DNA can be recovered from samples stored by a wide variety of simple methods. Several DNA‐based identification techniques have been developed, including hybridization assays based on species‐specific repeat sequences, and diagnostic PCR fragments produced either by the use of random PCR primers or by amplifying DNA with primers based on known species‐specific sequences. In this review we discuss the relative merits of different methods of cryptic species identification, with emphasis on the use of ribosomal DNA as a target for species‐diagnostic PCR assays.
Primary structure of ribosomal proteins S3 and S7 from Manduca sexta
Authors: Jiang H. , Wang Y. , Kanost M. R.
Published: December 2007

We have isolated from Manduca sexta full‐length cDNAs encoding proteins homologous to human ribosomal proteins S3 and S7. These are the first ribosomal protein sequences obtained from non‐Dipteran insects. M, sexta ribosomal protein S3 has a molecular mass of 26,715 Da. Ribosomal protein S7 has a mass 21,870 Da. Both are basic proteins, with abundant Lys and Arg residues that may interact with ribosomal RNA in the ribosome. Southern blot hybridization suggests the presence of single genes for both ribosomal proteins in the M. sexta genome. Alignments with other S3 and S7 sequences available in the database indicate regions of the ribosomal proteins that have been the most highly conserved in evolution and may point to important functional regions in the proteins. Ribosomal protein S3 appears to be more highly conserved than ribosomal protein S7. This may be due to greater constraints on the structure of S3 because of its dual functions in translation as a ribosomal protein and in DNA repair in the nucleus.
Identification and characterization of a RAPD‐PCR marker for distinguishing Asian and North American gypsy moths
Authors: Garner K. J. , Slavicek J. M.
Published: December 2007

The recent introduction of the Asian gypsy moth (Lymantria dispar L.) into North America has necessitated the development of genetic markers to distinguish Asian moths from the established North American population, which originated in Europe. We used RAPD‐PCR to identify a DNA length polymorphism that is diagnostic for the two moth strains. The polymorphism maps to an autosomal locus with codo‐minant Mendelian inheritance. DNA sequence analyses of the Asian and North American forms enabled development of locus‐specific primers so that this marker, designated FS‐1, will be useful for strain identification under varying conditions in different laboratories.
The sequence and structure of the meadow grasshopper (Chorthippus parallelus) mitochondrial srRNA, ND2, COl, COll ATPase8 and 9 tRNA genes
Authors: Szymura J. M. , Lunt D. H. , Hewitt G. M.
Published: December 2007

The nucleotide sequence of the mitochondrial ND2, COI, COll, ATPase8, srRNA and nine tRNA genes have been sequenced from two individuals of the meadow grasshopper Chorthippus parallelus. Comparisons are made to other insects for which the same regions are completely sequenced. Percentage A + T is found to be relatively low in C. parallelus though consistent with that of the other Orthopteran, Locusta migratoria. The relative number of substitutions observed in the different protein‐coding genes was analysed between pairs of insect species sharing different levels of relatedness. A clear change in this rate was observed between the within‐genus and between‐genera comparisons. This change is interpreted in terms of the functional constraints acting on these four different genes. The patterns seem to result from an early saturation of COl and COll genes with synonymous substitutions, and a tolerance of ND2 and ATPase8 function to high levels of amino acid replacements. This analysis highlights a need for further sequence studies and comparisons between taxa of different levels of divergence in order to understand the patterns of mtDNA evolution on which many evolutionary investigations are based.
Precise excision of TTAA‐specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera
Authors: Fraser M. J. , Clszczon T. , Elick T. , Bauser C.
Published: December 2007

Transposon mutagenesis of baculoviruses provides an ideal experimental system for analysis of the movement of a unique family of mobile element identified from lepidopteran genomes. Members of this family of short‐inverted‐repeat elements are characterized by their extreme Specificity for TTAA target sites. This report describes the analysis of excision events for two representatives of this family, tagalong (formerly TFP3) and piggysac (formerly IFP2). These elements were tagged with a polyhedrin/lacZ reporter gene and inserted back into the virus genome either by homologous recombination or by transposition. Revertants were selected based on a white plaque phenotype. Both elements excise in a precise fashion from their positions in the baculovirus genome in either TN‐368 cells or IPLB‐SF21 AE cells. The precise excision of these elements in infected IPLB‐SF21 AE cells occurs in the absence of either tagalong or piggysac element encoded functions. The common characteristics of both insertion and excision for these elements provides further validation for their inclusion In a single family of unique transposons.
Polymerase chain reaction‐based identification of insecticide resistance genes and DNA methylation in the aphid Myzus persicae (Sulzer)
Authors: Field L. M. , Crlck S. E. , Devonshire A. L.
Published: December 2007

The ability of a peach‐potato aphid (Myzus persicae) to resist insecticides can depend on both the presence of amplified esterase genes and on differences in their expression. Here we report a polymerase chain reaction‐restriction enzyme (PCR‐REN) technique which can detect the presence of ampiifled esterase genes and distinguish between the two possible types of amplified gene (E4 and FE4) and a homologous fragment from susceptible aphids. The technique is quick and sensitive enough to be used on a fraction of an aphid or an individual embryo. Furthermore, it can be combined with a pre‐PCR digestion using a methylation‐sensitive enzyme (Hpail) to determine whether or not the esterase genes contain 5–methyicytosine, the presence or absence of which is known to correlate with changes in gene expression.
Mobility of hAT transposable elements in the Old World bollworm, Helicoverpa armigera
Authors: Plnkerton A. C. , O'Brochta D. A. , Atkinson P. W.
Published: December 2007

The P, hobo, Hermes, Minos and mariner transposable elements have been successfully used as gene vectors to achieve genetic transformation of Drosophila melamagaster. The hobo transposable element of D. melamagaster, a member of the hAT family of transposable elements, can also transpose In other dipteran species Including the house fly Musca domestica and the Oueensland fruit fly, Bactrocera tryonl. Another hAT element, Hermes, can also transpose In a wide range of insect species. We report here that both of these hAT elements are also capable of mobility and accurate transposition In the noctuid Helicoverpa armigera and thus may be able to be used as gene vectors that will enable the genetic transformation of this important pest species. Significantly, the ability of hobo to excise in this species is independent of the presence of an exogenous source of hobo transposase, which suggests that H. armigera may possess an endogenous hobe‐like transposable element.
Assessing patient satisfaction. Part 2. Findings: nursing, the hospital and patients' concerns
Authors: PONTIN DAVID J. T. , WEBB CHRISTINE
Published: December 2007

Part 1 of this article discussed the process of evaluating patient satisfaction by means of unstructured, in‐depth interviews.

In this part, the content of interviews will be presented and the analytic domains derived from the research will be described.

These are ‘nursing’, ‘the hospital’ and ‘patients’ concerns', and the categories which make up these domains will be discussed.
Establishing a framework for research: the example of needs assessment
Authors: COWLEY SARAH , BERGEN ANN , YOUNG KATE , KAVANAGH ANN
Published: December 2007

This paper explains the early planning stage of a study commissioned by the English National Board which will investigate the changing educational needs of community nurses with regard to needs assessment and quality of care in the context of the NHS and Community Care Act 1990.

Two focus groups, comprising 22 participants altogether, generated data which were used to augment and clarify issues explored in an initial literature review. Some of the methodological issues are explained.

Traditional community nursing approaches to needs assessment appeared to value process and integration, while the new legislation emphasizes the separate‐ness of assessment; there is a danger that it may be seen as a single event. The consumer views were both supportive and critical about each of the approaches; some important insights were gained, and a confident basis from which to launch the study identified.

The approach offers one possible way to clarify the starting point of a project when carrying out a standard literature review seems insufficient. This may occur with under‐researched or rapidly changing phenomena, or if a field of interest is the subject of multiple interpretations or lack of consensus.
Nursing the physically disabled in a general hospital ward
Authors: CONWAY KIM
Published: December 2007

Progress in medical treatment over the last three decades has meant that many more people survive either traumatic birth situations or diseases/injuries which would have previously resulted in death, and are leading normal lives with some form of physical disability.

Until quite recently, little attention has been given to their longer‐term health needs. The following article looks at the attitudes of nurses towards physically disabled patients, the experiences and attitudes of these patients, the interaction between nurse and patient, practical outcomes and future directions.

Literature on this subject is not extensive. More empirical research is required focusing on the impact of admission of such people on general hospital ward functioning, the experiences/insights of nursing staff caring for them and the reaction and comments of physically disabled patients.