Chemical synthesis, antibacterial activity and conformation of diptericin, an 82-mer peptide originally isolated from insects

The small amounts of antibacterial peptides that can be isolated from insects do not allow detailed studies of their range of activity, side-chain sugar requirements, or their conformation, factors that frequently play roles in the mode of action. In this paper, we report the solid-phase step-by-step synthesis of diptericin, an 82-mer peptide, originally isolated from Phormia terranovae. The unglycosylated peptide was purified to homogeneity by conventional reversed-phase high performance liquid chromatography, and its activity spectrum was compared to that Of synthetic unglycosylated drosocin, which shares strong sequence homology with diptericin's N-terminal domain. Diptericin appeared to have antibacterial activity:for only a limited number of Gram-negative bacteria. Diptericin's submicromolar potency against Escherichia coli strains indicated that, in a manner similar to drosocin, the presence of the carbohydrate side chain is not,necessary to kill bacteria. Neither the N-terminal, drosocin-analog fragment, nor the C-terminal, glycine-rich attacin-analog region was active against any of the bacterial strains studied, regardless of whether the Gal-GalNAc disaccharide units were attached. This suggested that the active site of diptericin fell outside the drosocin or attacin homology domains. In addition, the conformation of diptericin did not seem to play a role in the antibacterial activity, as was demonstrated by the complete lack of ordered structure by two-dimensional nuclear magnetic resonance spectroscopy and circular dichroism. Diptericin completely killed bacteria within I h, considerably faster than drosocin and the attacins; unlike some other, fast-acting antibacterial peptides, diptericin did not lyse normal mammalian cells. Taken together, these data suggest diptericin does not belong to any known class of antibacterial peptides.

Computational chemistry on Fujitsu vector-parallel processors: Development and performance of applications software

In this and a preceding paper, we provide an introduction to the Fujitsu VPP range of vector-parallel supercomputers and to some of the computational chemistry software available for the VPP. Here, we consider the implementation and performance of seven popular chemistry application packages. The codes discussed range from classical molecular dynamics to semiempirical and ab initio quantum chemistry. All have evolved from sequential codes, and have typically been parallelised using a replicated data approach. As such they are well suited to the large-memory/fast-processor architecture of the VPP. For one code, CASTEP, a distributed-memory data-driven parallelisation scheme is presented. (C) 2000 Published by Elsevier Science B.V. All rights reserved.

Lentiviral vector-mediated tyro sinase-related protein 2 gene transfer to dendritic cells for the therapy of melanoma

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs), which play a vital role in primary immune responses. Introducing genes into DCs will allow constitutive expression of the encoded proteins and thus prolong the presentation of the antigens derived therefrom. In addition, multiple and unidentified epitopes encoded by the entire tumor-associated antigen (TAA) gene may enhance T cell activation. This study demonstrated that an HIV-1-based lentiviral vector conferred efficient gene transfer to DCs. The transgene, murine tyrosinase-related protein 2 (mTRP-2), encodes a clinically relevant melanoma-associated antigen (MAA), which has been found to be a tumor rejection antigen for B16 melanoma. The transfer and proper processing of mTRP-2 in DCs, in terms of RNA transcription activity and protein expression, were verified by RT-PCR and specific antibody, respectively. Administration of mTRP-2 gene-modified DCs (DC-HR'CmT2) to C57BL/6 mice evoked strong protection against tumor challenge, for which the presence of CD4(+) and CD8(+) cells during both the priming and challenge phase was essential. In a therapy model, our results showed that four of seven mice with preestablished tumor remained tumor free for 80 days after therapeutic vaccination. Given the results shown in this study, mTRP-2 gene transfer to DCs provides a potential therapeutic strategy for the management of melanoma, especially in the early stage of the disease.

A preliminary phylogeny of the scale insects (Hemiptera : Sternorrhyncha : Coccoidea) based on nuclear small-subunit ribosomal DNA

Scale insects (Hemiptera: Sternorrhyncha: Coccoidea) are a speciose and morphologically specialized group of plant-feeding bugs in which evolutionary relationships and thus higher classification are controversial. Sequences derived from nuclear small-subunit ribosomal DNA were used to generate a preliminary molecular phylogeny for the Coccoidea based on 39 species representing 14 putative families. Monophyly of the archaeococcoids (comprising Ortheziidae, Margarodidae sensu lato, and Phenacoleachia) was equivocal, whereas monophyly of the neococcoids was supported. Putoidae, represented by Puto yuccae, was found to be outside the remainder of the neococcoid clade. These data are consistent with a single origin (in the ancestor of the neococcoid clade) of a chromosome system involving paternal genome elimination in males. Pseudococcidae (mealybugs) appear to be sister to the rest of the neococcoids and there are indications that Coccidae (soft scales) and Kerriidae (lac scales) are sister taxa. The Eriococcidae (felt scales) was not recovered as a monophyletic group and the eriococcid genus Eriococcus sensu lato was polyphyletic. (C) 2002 Elsevier Science (USA). All rights reserved.

The gall-inducing habit has evolved multiple times among the eriococcid scale insects (Sternorrhyncha : Coccoidea : Eriococcidae)

The habit of inducing plant galls has evolved multiple times among insects but most species diversity occurs in only a few groups, such as gall midges and gall wasps. This phylogenetic clustering may reflect adaptive radiations in insect groups in which the trait has evolved. Alternatively, multiple independent origins of galling may suggest a selective advantage to the habit. We use DNA sequence data to examine the origins of galling among the most speciose group of gall-inducing scale insects, the eriococcids. We determine that the galling habit has evolved multiple times, including four times in Australian taxa, suggesting that there has been a selective advantage to galling in Australia. Additionally, although most gall-inducing eriococcid species occur on Myrtaceae, we found that lineages feeding on Myrtaceae are no more likely to have evolved the galling habit than those feeding on other plant groups. However, most gall-inducing species-richness is clustered in only two clades (Apiomorpha and Lachnodius + Opisthoscelis), all of which occur exclusively on Eucalyptus s.s. The Eriococcidae and the large genus Eriococcus were determined to be non-monophyletic and each will require revision. (C) 2004 The Linnean Society of London.

Transfer of an autonomously replicating vector between vegetatively incompatible biotypes of Colletotrichum gloeosporioides

Previous research has indicated that biotypes A and B of Colletotrichum gloeosporioides that infect Stylosanthes spp. in Australia are asexual and vegetatively incompatible. Selectable marker genes conferring resistance either to hygromycin or phleomycin were introduced into isolates of these biotypes. Vectors conferring resistance to hygromycin and carrying telomeric sequences from Fusarium oxysporum replicated autonomously in C. gloeosporioides and gave frequencies of transformation 100-times higher than vectors that integrated into the genome. Monoconidial colonies resistant to both antibiotics were recovered when hygromycin-resistant biotype-A transformants carrying an autonomously replicating vector were paired in culture with a phleomycin-resistant biotype-B transformant carrying integrative vector sequences. Molecular analysis of double antibiotic-resistant progeny indicated that they contained the autonomous vector in a biotype-B genetic background, Results indicate that transfer of the autonomous vector had occurred from biotype A to biotype B, demonstrating the potential for transfer of genetic information between these biotypes.

Spiroacetals in insects

Spiroacetals, cryptic ketodiols showing a hydroxyl group at both sides of a carbonyl whithin reachable distances are very widespread in nature. A group of 30 different structures, not including stereoisomers, represent volatile, less polar constituents of insect secretions. Five different systems were identified: 1,6-dioxaspirol[4.4]nonanes, 1,6-dioxaspiro[4.5]decanes, 1,6-dioxaspiro[4.6]undecanes, 1,7-dioxaspiro[5.5] undecanes, and 1,7-dioxaspiro[5.6]dodecanes. Some spiroacetals are insect pheromones: (2S,5R)-2-ethyl-1,6-dioxaspiro[4.4]nonane, chalcogran, 1, is a key component of the male produced aggregation pheromone of the spruce bark beetle, Pityogenes cha2cographus. In contrast, (5S,7S)-7-methyl-1,6-dioxaspiro[4.5]decane, 2, conophthorin, acts as a repellent or spacer in several bark beetles. Racemic 1,7-diosaspiro[5.5]undecane, olean, 5, is the female produced sex pheromone of the olive fly, Bactrocera (Dacus) oleae. The most widespread spiroacetal is 2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, 8. Tt often forms a mixture of (E,E)- and (E,Z)-isomers, the (E,E)-isomer showing (2S,6R,8S)-configuration. In the solitary bee, Andrena wilkella, it serves as an aggregation pheromone. Present knowledge on structures and distribution of volatile spiroacetals is comprehensively compiled. Stereochemical aspects and mass spectrometric fragmentation patterns are discussed in detail to facilitate identifications of hitherto unknown compounds. Synthetic approaches to spiroacetals are classified and reviewed. Last but not least, facts and speculations on the biosynthesis of volatile spiroacetals are presented.

A DNA fingerprinting procedure for ultra high-throughput genetic analysis of insects

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular Information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability In three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced similar to 50 scoreable polymorphic DNA markers, between individuals of three Independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from Individual DNA samples that had been combined to create the bulked samples.