Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination

Development of CD8 alpha beta CTL epitope-based vaccines requires an effective strategy capable of co-delivering large numbers of CTL epitopes, Here we describe a DNA plasmid encoding a polyepitope or polytope protein, which contained multiple contiguous minimal murine CTL epitopes, Mice vaccinated with this plasmid made MHC-restricted CTL responses to each of the epitopes, and protective CTL were demonstrated in recombinant vaccinia virus, influenza virus, and tumor challenge models, CTL responses generated by polytope DNA plasmid vaccination lasted for 1 yr, could be enhanced by co-delivering a gene for granulocyte-macrophage CSF, and appeared to be induced in the absence of CD4 T cell-mediated help, The ability to deliver large numbers of CTL epitopes using relatively small polytope constructs and DNA vaccination technology should find application in the design of human epitope-based CTL vaccines, in particular in vaccines against EBV, HIV, and certain cancers.

Targeting of a cholecystokinin-DNA complex to pancreatic cells in vitro and in vivo

The carboxy terminal octapeptide of cholecystokinin (CCK8) is a hormone that binds high affinity receptors in a number of tissues including pancreas and pancreatic tumours. As part of our studies to develop effective gene therapy for the treatment of pancreatic cancers, we have investigated various gene delivery systems that depend on CCK8 receptor targeting. In this paper,we describe the synthesis of a CCK8-DNA complex designed to deliver foreign DNA to cholecystokinin receptor-positive cells. CCK8 was ligated to avidin and then complexed to linearis biotinylated DNA (pSV-CAT). The uptake of P-32-labelled CCK8-DNA complex by rat pancreatic acini was linear with time over 4 h with 65-70% of uptake inhibited by 100 nM CCK8. The complex appeared to be internalised since it could not be removed by acid wash. When administered intra-arterially, the complex was rapidly removed from the circulation with no evidence of targeted delivery to the pancreas, However, following a single intraperitoneal dose, the pancreas accumulated-5- 8% of the total administered complex by 24 h. These results suggest that peptide-dependent gene delivery to CCK receptor positive cells in vivo is feasible but, when administered directly into the circulation, diffusional barriers across the endothelium may limit distribution to peripheral tissues. Intraperitoneal administration therefore may be a useful alternative for targeting the pancreas.

Microsatellite instability and loss of heterozygosity at DNA mismatch repair gene loci occurs during hepatic carcinogenesis

DNA mismatch repair is an important mechanism involved in maintaining the fidelity of genomic DNA. Defective DNA mismatch repair is implicated in a variety of gastrointestinal and other turners; however, its role in hepatocellular carcinoma (HCC) has not been assessed. Formalin-fixed, paraffin-embedded archival pathology tissues from 46 primary liver tumors were studied by microdissection and microsatellite analysis of extracted DNA to assess the degree of microsatellite instability, a marker of defective mismatch repair, and to determine the extent and timing of allelic loss of two DNA mismatch repair genes, human Mut S homologue-2 (hMSH2) and human Mut L homologue-1 (hMLH1), and the tumor suppressor genes adenomatous polyposis coli gene (APC), p53, and DPC4. Microsatellite instability was detected in 16 of the tumors (34.8%). Loss of heterozygosity at microsatellites linked to the DNA mismatch repair genes, hMSH2 and/or hMLH1, was found in 9 cases (19.6%), usually in association with microsatellite instability. Importantly, the pattern of allelic loss was uniform in 8 of these 9 tumors, suggesting that clonal loss had occurred. Moreover, loss at these loci also occurred in nonmalignant tissue adjacent to 4 of these tumors, where it was associated with marked allelic heterogeneity. There was relatively infrequent loss of APC, p53, or DPC4 loci that appeared unrelated to loss of hMSH2 or hMLH1 gene loci. Loss of heterozygosity at hMSH2 and/or hMLH1 gene loci, and the associated microsatellite instability in premalignant hepatic tissues suggests a possible causal role in hepatic carcinogenesis in a subset of hepatomas.

Phosphorylation of the C-terminal sites of human p53 reduces non-sequence-specific DNA binding as modeled with synthetic peptides

Phosphorylation of the tumor suppressor p53 is generally thought to modify the properties of the protein in four of its five independent domains. We used synthetic peptides to directly study the effects of phosphorylation on the non-sequence-specific DNA binding and conformation of the C-terminal, basic domain. The peptides corresponded to amino acids 361-393 and were either nonphosphorylated or phosphorylated at the protein kinase C (PKC) site, Ser378, or the casein kinase II (CKII) site, Ser392, or bis-phosphorylated on both the PKC and the CKII sites. A fluorescence polarization analysis revealed that either the recombinant p53 protein or the synthetic peptides bound to two unrelated target DNA fragments. Phosphorylation of the peptide at the PKC or the CKII sites clearly decreased DNA binding, and addition of a second phosphate group almost completely abolished binding. Circular dichroism spectroscopy showed that the peptides assumed identical unordered structures in aqueous solutions. The unmodified peptide, unlike the Ser378 phosphorylated peptide, changed conformation in the presence of DNA. The inherent ability of the peptides to form an alpha-helix could be detected when circular dichroism and nuclear magnetic resonance spectra were: taken in trifluoroethanol-water mixtures. A single or double phosphorylation destabilized the helix around the phosphorylated Ser378 residue but stabilized the helix downstream in the sequence.

A DNA-based demonstration of a three-host life-cycle for the Bivesiculidae (Platyhelminthes : Digenea)

Immature bivesiculid trematodes collected from the intestine of Thalassoma lunare (Labridae) are shown to be morphologically consistent with adults of Bivesicula claviformis from Epinephelus fasciatus (Serranidae). In addition, the immature bivesiculids have the same sequence for the second internal transcribed spacer of the ribosomal DNA. Comparison with three other species of Bivesiculidae showed differences of between 23% and 30%. These results show that bivesiculids may have three-host life-cycles in addition to the two-host life-cycles that have been demonstrated previously. The three-host life-cycle enables bivesiculids to infect large carnivorous fishes. (C) 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved.

Inference of phylogeny and taxonomy within the Didymozoidae (Digenea) from the second internal transcribed spacer (ITS2) of ribosomal DNA

DNA sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) were determined for 11 species from four genera of Didymozoinae (Indodidymozoon, Helicodidymozoon, Rhopalotrema and Neometadidymozoon) and a species of the Lecithasteridae, Lecithaster stellatus. Sequences were used to test the validity of species recognised on morphological criteria and to infer phylogenetic relationships. Sequences of the 11 didymozoids differed by 0.5% to 19%. Our phylogenetic analyses: (i) indicate that species in the genera Helicodidymozoon and Rhopalotrema are a monophyletic group; (ii) support separation of the genus Helicodidymozoon from the genera Indodidymozoon and Neometadidymozoon; and (iii) support recognition of Rhopalotrema as a genus distinct from Neometadidymozoon. We found the gonochoristic species, I. pearsoni and I. suttiei, to be genetically similar to the hermaphroditic species in the genus Indodidymozoon and found no evidence to indicate that they belong in a separate genus.

Dehydromonocrotaline generates sequence-selective N-7 guanine alkylation and heat and alkali stable multiple fragment DNA crosslinks

Monocrotaline is a pyrrolizidine alkaloid known to cause toxicity in humans and animals. Its mechanism of biological action is still unclear although DNA crosslinking has been suggested to a play a role in its activity. In this study we found that an active metabolite of monocrotaline, dehydromonocrotaline (DHM), alkylates guanines at the N7 position of DNA with a preference for 5'-GG and 5'-GA sequences; In addition, it generates piperidine- and heat-resistant multiple DNA crosslinks, as confirmed by electrophoresis and electron microscopy. On the basis of these findings, we propose that DHM undergoes rapid polymerization to a structure which is able to crosslink several fragments of DNA.

Evidence of multiple transcription initiation and termination sites within the rDNA intergenic spacer and rRNA readthrough transcription in the urochordate Herdmania curvata

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H, curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

Gene and genotypic diversity of Phytophthora cinnamomi in South Africa and Australia revealed by DNA polymorphisms

Phytophthora cinnamomi isolates from South Africa and Australia were compared to assess genetic differentiation between the two populations. These two populations were analysed for levels of phenotypic diversity using random amplified polymorphic DNAs (RAPDs) and gene and genotypic diversity using restriction fragment length polymorphisms (RFLPs). Sixteen RAPD markers from four decanucleotide Operon primers and 34 RFLP alleles from 15 putative loci were used. A few isolates from Papua New Guinea known to posses alleles different from Australian isolates were also included for comparative purposes. South African and Australian P. cinnamomi populations were almost identical with an extremely low level of genetic distance between them (D-m = 0.003). Common features for the two populations include shared alleles, low levels of phenotypic/genotypic diversity, high clonality, and low observed and expected levels of heterozygosity. Furthermore, relatively high levels of genetic differentiation between mating type populations (D-m South Africa = 0.020 and D-m Australia = 0.025 respectively), negative fixation indices, and significant deviations from Hardy-Weinberg equilibrium, all provided evidence for the lack of frequent sexual reproduction in both populations. The data strongly suggest that both the South African and Australian P. cinnamomi populations are introduced.

Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR

Cylindrospermopsis raciborskii is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of C. raciborskii were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of rpoC1, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional rpoC1 gene sequences obtained for a range of cyanobacteria highlighted clustering of C. raciborskii with other heterocyst-producing cyanobacteria (orders Nostocales and Stigonematales). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among C. raciborskii isolates than did rpoC1 gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the rpoC1 gene unique to C. raciborskii was developed for the specific identification of C. raciborskii from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with C, raciborskii. This method provides an accurate alternative to traditional morphological identification of C. raciborskii.

Activation of the human diaphragm during a repetitive postural task

1. The co-ordination between respiratory and postural functions of the diaphragm was investigated during repetitive upper Limb movement. It was hypothesised that diaphragm activity would occur either tonically or phasically in association with the forces from each movement and that this activity would combine with phasic respiratory activity. 2. Movements of the upper limb and ribcage were measured while standing subjects performed repetitive upper limb movements 'as fast as possible'. Electromyographic (EMG) recordings of the costal diaphragm were made using intramuscular electrodes in four subjects. Surface electrodes were placed over the deltoid and erector spinae muscles. 3. In contrast to standing at rest, diaphragm activity was present throughout expiration at 78 +/- 17% (mean +/- S.D.) of its peak inspiratory magnitude during repeated upper limb movement. 4. Bursts of deltoid and erector spinae EMG activity occurred at the Limb movement frequency (similar to 2.9 Hz). Although the majority of diaphragm EMG power was at the respiratory frequency (similar to 0.4 Hz), a peak was also present at the movement frequency. This finding was corroborated by averaged EMG activity triggered from upper limb movement. In addition, diaphragm EMG activity was coherent with ribcage motion at the respiratory frequency and with upper limb movement at the movement frequency. 5. The diaphragm response was similar when movement was performed while sitting. In addition, when subjects moved with increasing frequency the peak upper limb acceleration correlated with diaphragm EMG amplitude. These findings support the argument that diaphragm contraction is related to trunk control. 6. The results indicate that activity of human phrenic motoneurones is organised such that it contributes to both posture and respiration during a task which repetitively challenges trunk posture.

Characterization of the sequential non-covalent and covalent interactions of the antitumour antibiotic hedamycin with double stranded DNA by NMR spectroscopy

Hedamycin, a member of the pluramycin class of antitumour antibiotics, consists of a planar anthrapyrantrione chromophore to which is attached two aminosugar rings at one end and a bisepoxide-containing sidechain at the other end, Binding to double-stranded DNA is known to involve both reversible and non-reversible modes of interaction. As a part of studies directed towards elucidating the structural basis for the observed 5'-pyGT-3' sequence selectivity of hedamycin, we conducted one-dimensional NMR titration experiments at low temperature using the hexadeoxyribonucleotide duplexes d(CACGTG)(2) and d(CGTACG)(2). Spectral changes which occurred during these titrations are consistent with hedamycin initially forming a reversible complex in slow exchange on the NMR timescale and binding through intercalation of the chromophore. Monitoring of this reversible complex over a period of hours revealed a second type of spectral change which corresponds with formation of a non-reversible complex. Copyright (C) 1999 John Wiley & Sons, Ltd.

Chromosomal fragile site FRA16D and DNA instability in cancer

It has been proposed that common aphidicolin-inducible fragile sites, in general, predispose to specific chromosomal breakage associated with deletion, amplification, and/or translocation in certain forms of cancer. Although this appears to be the case for the fragile site FRA3B and may be the case for FRA7G, it is not Set clear whether this association is a general property of this class of fragile site. The major aim of the present study was to determine whether the FRA16D chromosomal fragile site locus has a role to play in predisposing DNA sequences within and adjacent to the fragile site to DNA instability (such as deletion or translocation), which could lead to or be associated with neoplasia. We report the localization of FRA16D within a contig of cloned DNA and demonstrate that this fragile site coincides with a region of homozygous deletion in a gastric adenocarcinoma cell line and is bracketed by translocation breakpoints in multiple myeloma, as reported previously (Chesi, M., et al., Blood, 91: 4457-4463, 1998), Therefore, given similar findings at the FRA3B and FRA7G fragile sites, it is likely that common aphidicolin-inducible fragile sites exhibit the general property of localized DNA instability in cancer cells.