T-DNA tagging and characterisation of a novel meristem-specific promoter from tobacco

We have evaluated T-DNA mediated plant promoter tagging, with a left-border-linked promoterless firefly luciferase (luc) construct, as a strategy for the isolation of novel plant promoters. In a population of approximately 300 transformed tobacco plants, IO lines showed LUC activity, including novel tissue-specific and developmental patterns of expression. One line, showing LUC activity only in the shoot and root apical meristems, was further characterised. Inverse PCR was used to amplify a 1.5 kb fragment of plant DNA flanking the single-copy T-DNA insertion in this line. With the exception of a 249 bp highly repetitive element, this sequence is present as a single copy in the tobacco genome, and is not homologous to any previously characterised DNA sequences. Sequence analysis revealed the presence of several motifs that may be involved in transcriptional regulation. Transgenic tobacco plants transformed with a transcriptional fusion of this putative promoter sequence to the beta-glucuronidase (uidA) reporter gene, showed GUS activity confined to the shoot tip and mature pollen. This promoter may be useful to direct the expression of genes controlling the transition to flowering, or genes to reduce losses due to pests and stresses damaging plant apical meristems.

Mucosal immunisation with papillomavirus virus-like particles elicits systemic and mucosal immunity in mice

It has been shown previously that recombinant virus-like particles (VLPs) of papillomavirus can induce VLP-specific humoral and cellular immune responses following parenteral administration. To test whether mucosal administration of bovine papillomavirus type 1 (BPV1) VLPs could produce mucosal as well as systemic immune responses to VLPs, 50 mu g chimeric BPV1 VLPs containing an HPV16 E7 CTL epitope (BPVL1/E7 VLP) was administered intranasally to mice. After two immunisations, L1-specific serum IgG and IgA were observed. L1-specific IgG and IgA were also found in respiratory and vaginal secretions. Both serum and mucosal antibody inhibited papillomavirus VLP-induced agglutination of RBC, indicating that the antibody induced by mucosal immunisation may recognize conformational determinants associated with virus neutralisation. For comparison, VLPs were given intramuscularly, and systemic and mucosal immune responses were generally comparable following systemic or mucosal delivery. However, intranasal administration of VLP induced significantly higher local IgA response in lung, suggesting that mucosally delivered HPV VLP may be more effective for mediating local mucosal immune responses. Intranasal immunisation with HPV6b L1 VLP produced VLP-specific T proliferative responses in splenocytes, and immunisation with BPVL1 VLP containing an HPV16 E7 CTL epitope induced E7-specific CTL responses. We conclude that immunisation with papillomavirus VLPs via mucosal and intramuscular routes, without adjuvant, can elicit specific antibody at mucosal surfaces and also systemic VLP epitope specific T cell responses. These findings suggest that mucosally delivered VLPs may offer an alternative HPV VLP vaccine strategy for inducing protective humoral immunity to anogenital HPV infection, together with cell-mediated immune responses to eliminate any cells which become infected. (C) 1998 Academic Press.

Amphilactams A-D: Novel nematocides from southern Australian marine sponges of the genus Amphimedon

Bioassay-directed fractionation of the ethanol extracts of two Amphimedon spp. collected during trawling operations in the Great Australian Eight yielded four new macrocyclic lactone/lactams, amphilactams A-D (1-4). The amphilactams possess potent in vitro nematocidal properties, and their structures were assigned on the basis of detailed spectroscopic analysis and comparison with synthetic model compounds. The amphilactams feature both carbon skeletons and an enamino lactone/lactam moiety unprecedented in the natural products literature.

The solution structure of the N-terminal zinc finger of GATA-1 reveals a specific binding face for the transcriptional co-factor FOG

Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional H-1/N-15 NMR spectroscopy, and we use H-1/N-15 spin relation measurements to investigate its backbone dynamics. The structure consists of two distorted beta-hairpins and a single alpha-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C-4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NE Notably, this face is well removed from the putative DNA-binding face of the NE an observation which is suggestive of simultaneous roles for the NF; that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions.

A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation

The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMR alpha) and a common signal-transducing beta-subunit (hpc) that is shared with the interleukin-3 and -5 receptors, We have previously identified a constitutively active extracellular point mutant of hpc, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287), This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMR alpha (mGMR alpha) subunit, since introduction of mGMR alpha, but not hGMR alpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence, Experiments utilizing mouse/human chimeric GMR alpha subunits indicated that the species specificity lies in the extracellular domain of GMRa. Importantly, the requirement for mGMR alpha correlated with the ability of I374N (but not wild-type hpc) to constitutively associate with mGMRa. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMR alpha surface expression. Taken together, these findings suggest a critical role for association with GMR alpha in the constitutive activity of I374N.

Phylogeny of the hard ticks (Ixodidae) inferred from 18S rRNA indicates that the genus Aponomma is paraphyletic

We examined the phylogeny of ticks (Acari:Parasitiformes:Ixodida) and their closest known mite relatives (Acari:Parasitiformes:Mesostigmata and Holothyrida) using 18S rRNA sequences. In our analyses, we included sequences from 36 taxa. Sequences for 13 hard ticks (Family Ixodidae), 5 soft ticks (Family Argasidae), and 2 mesostigmatid mites were obtained from the GenBank database and we generated sequences for 15 hard ticks and 1 holothyrid mite. Ten of these tick species were endemic to Australia. Our analyses indicated that the suborder Holothyrida is more closely related to Ixodida than to Mesostigmata, the group used as outgroup in earlier molecular studies. This finding is consistent with Lehtinen's (1991) hypothesis that the Holothyrida rather than the Mesostigmata is the sister-group to the Ixodida. Within the hard ticks the genus Aponomma and thus the family Amblyomminae were paraphyletic. Taxonomic revision of these taxa is needed. The genus Amblyomma was paraphyletic without the inclusion of typical Aponomma species (Ap. latum and Ap. fimbriatum). There was a basal divergence between endemic Australian and other species in both the Metastriata and the Prostriata divisions of the hard ticks. (C) 1999 Academic Press.

Brain region-specific studies of the excitatory behavioral effects of morphine-3-glucuronide

This study was designed to determine in rats whether morphine-3-glucuronide (M3G) produces its neuro-excitatory effects most potently in the ventral hippocampus (as has been reported previously for subanalgesic doses of opioid peptides). Guide cannulae were implanted into one of seven regions of the rat brain: lateral ventricle; ventral, CA1 and CA2-CA3 regions of the hippocampus; amygdala; striatum or cortex. After a 7 day recovery period, rats received intracerebral injections of (i) M3G (1.1 or 11 nmol) (ii) DADLE ([D-Ala(2),D-Leu(5)]enkephalin), (45 nmol, positive controls) or (iii) vehicle (deionised water), and behavioral excitation was quantified over 80 min. High-dose M3G (11 nmol) evoked behavioral excitation in all brain regions but the onset, severity and duration of these effects varied considerably among brain regions. By contrast, low-dose M3G (1.1 nmol) evoked excitatory behaviors only when administered into the ventral hippocampus and the amygdala, with the most potent effects being observed in the ventral hippocampus. Prior administration of the nonselective opioid antagonists, naloxone and beta-funaltrexamine into the ventral hippocampus, markedly attenuated low-dose M3G's excitatory effects but did not significantly alter levels of excitation evoked by high-dose M3G. Naloxone given 10 min after M3G (1.1 or 11 nmol) did not significantly attenuate behavioral excitation. Thus, M3G's excitatory behavioral effects occur most potently in the ventral hippocampus as reported previously for subanalgesic doses of opioid peptides, and appear to be mediated through at least two mechanisms, one possibly involving excitatory opioid receptors and the other, non-opioid receptors.

T cell-mediated and non-specific inflammatory mechanisms contribute to the skin pathology of HPV 16 E6E7 transgenic mice

One of three lines of mice transgenic for the E6 and E7 genes of human papillomavirus type 16 (HPV16) expressed from an alpha A-crystallin promoter also expresses the transgene ectopically in the skin. This line, designated alpha ACE6E7#19, develops skin disease from 3 months of age, characterised by epidermal hyperplasia and eventual skin loss. Administration of complete Freund's adjuvant (CFA) to alpha ACE6E7#19 mice, but not to nontransgenic littermate controls, induced local epidermal hyperplasia which was histologically similar to the spontaneously arising skin pathology. Local application of 2,4-dinitrochlorobenzene (DNCB) to DNCB-sensitised aACE6E7#19 mice, but not DNCB-sensitised controls, also induced hyperplasia. Treatment with cyclosporin A (CsA) or systemic depletion of CD4+ cells significantly reduced the incidence of skin disease. These data suggest that local inflammation, and cytokines produced by T helper cells, contribute to the induction of hyperplastic skin disease in alpha ACE6E7#19 mice. Spontaneous skin disease with similar histological appearance, frequency, age of onset and severity in alpha ACE6E7#19 mice was observed in scid-/- aACE6E7#19 mice, despite immune paresis. Antigen-specific immune responses and T-cell cytokines a re therefore not necessary for the induction of skin disease. We propose that epidermal hyperplasia associated with HPV16 E6 and E7 expression in skin is accelerated by local secretion of pro-inflammatory cytokines, whose production can be enhanced by activated CD4+ T cells.

Both CD4(+) and CD8(+) lymphocytes reduce the severity of tissue lesions in murine systemic candidiasis, and CD4(+) cells also demonstrate strain-specific immunopathological effects

The role of T lymphocytes in host responses to sublethal systemic infection with Candida albicans was evaluated by mAb depletion of CD4(+) and CD8(+) cells from BALB/c and CBA/CaH mice, which develop mild and severe tissue damage, respectively. Depletion of CD4(+) lymphocytes from BALB/c mice markedly increased tissue damage, but did not alter the course of infection. In CBA/CaH mice, depletion of CD4+ cells abrogated tissue destruction in both brain and kidney at day 4 after infection, and significantly decreased fungal colonization in the brain. However, the severity of tissue lesions increased relative to controls from day 8 onwards. A small increase in tissue damage was evident in both mouse strains after depletion of CD8(+) cells. There were no major differences between days 4 end 8 after infection in cDNA cytokine profiles of CD4(+) lymphocytes from either BALB/c or CBA/CaH mice. After passive transfer into infected syngeneic recipients, spleen cells from infected CBA/CaH mice markedly increased tissue damage when compared to controls, and also caused a significant increase in fungal colonization in the brain. A similar transfer in BALB/c mice increased the number of inflammatory cells in and around the lesions, but had no effect on the fungal burden in brain and kidney. The data demonstrate that both CD4(+) and CD8(+) lymphocytes contribute to the reduction of tissue damage after systemic infection with C. albicans, and that the development and expression of CD4(+) lymphocyte effector function is influenced by the genetic background of the mouse.

Miscibility and specific interactions in blends of poly(4-vinylphenol) and poly(2-ethoxyethyl methacrylate)

The properties of the hydrogen-bonded polymer blends of poly(4-vinylphenol) and poly(2-ethoxyethyl methacrylate) are presented. Spectroscopic techniques such as C-13 solid-state NMR and FT-IR are used to probe specific interactions of the blends at various compositions. Spectral features from both techniques revealed that site-specific interactions are present, consistent with a significant degree of mixing of the blend components. Changes in chemical shift and line shape of the phenolic carbon and carbonyl resonances in the C-13 CPMAS spectra of the blends as a function of composition are interpreted as resulting from changes in the relative intensities of two closely overlapped signals. A quantitative measure of hydrogen-bonded carbonyl groups using C-13 NMR has been obtained which agreed well with the results from FT-IR analyses. It is also shown that C-13 NMR can be used to measure the fraction of hydroxyl groups associated with carbonyl groups, which was not possible previously using FT-IR due to extensive overlapping of bands in the hydroxyl stretching region. The results of measurements of H-1 T-1 and 1H T-1 rho indicate that PVPh and PEEMA are intimately mixed on a scale less than 2-3 nm.

The genus Deretrema Linton, 1910 (Digenea : Zoogonidae) from southern Great Barrier Reef fishes, with a description of Deretrema woolcockae n. sp.

Two species of Deretrema (Zoogonidae) are reported from labrid fishes from the Great Barrier Reef. D. nahaense Yamaguti, 1942 is recorded from the gall-bladders of the labrids Thalassoma hardwicke (Bennett), T. jansenii (Bleeker), T. lunare (Linnaeus) and T. lutescens (Lay & Bennett). This species is recognised, despite having been formerly synonymised with D. pacificum Yamaguti, 1942. In addition to morphological distinction, D. nahaense appears to have strict host-specificity for the genus Thalassoma. D. woolcockae n.sp. is described from the gall-bladder of Hemigymnus fasciatus (Bloch). The new species is close to D. acutum Pritchard, 1963 and D. plotosi Yamaguti, 1940, but differs slightly in the distribution of the vitelline follicles, the sucker-ratio and the position of the cirrus-sac. In addition, this species also appears to have a distinct host-specificity, being restricted to one labrid species.

Season-specific directional movement in migratory Australian butterflies

Large numbers of adults of certain species of butterfly flying in an apparently 'purposeful' manner are often noted by entomologists and the general public. Occasionally, these are recorded in the literature. Using these records we summarise information regarding the direction of movement in Australian butterflies and test whether there are consistent patterns that could account for known seasonal shifts in geographical range. The data were analysed using contingency tables and directionality statistics. Vanessa itea, Vanessa kershawi, Danaus plexippus, Danaus chrysippus and Badamia exclamationis flew predominately south in the spring-summer and north in the autumn-winter. Tirumala hamata has a strong southern component to its flight in spring but, as in Euploea core, appears non-directional in the autumn. For many supposedly known migratory species, the number of literature records are few, particularly in one season (mainly autumn). Thus, for Appias paulina, four of seven records were south in the spring-summer, as were six of nine records for Catopsilia pomona, and three of five for Zizina labradus. For Belenois java, flight records were only available for the spring and these showed geographical differences; predominantly north-west in northern Australia (Queensland) and south-west in southern Australia (Victoria, New South Wales). There were too few records for Papilio demoleus in the literature (four only) to draw any conclusions. Major exceptions to the seasonal trend of south in the spring and north in the autumn were Junonia villida, which showed a predominant north-westward direction in both seasons, and Eurema smilax, with a predominant southern or western flight in both seasons. We discuss these species specific trends in migration direction in relation to seasonal shifts in suitable habitat conditions, possible cues used in orientation and in timing changes in direction.

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.