Replication of bovine papillomavirus type 1 (BPV-1) DNA in Saccharomyces cerevisiae following infection with BPV-1 virions

Saccharomyces cerevisiae protoplasts exposed to bovine papillomavirus type 1 (BPV-1) virions demonstrated uptake of virions on electron microscopy. S. cerevisiae cells looked larger after exposure to BPV-1 virions, and cell wall regeneration was delayed. Southern blot hybridization of Hirt DNA from cells exposed to BPV-1 virions demonstrated BPV-1 DNA, which could be detected over 80 days of culture and at least 13 rounds of division. Two-dimensional gel analysis of Hirt DNA showed replicative intermediates, confirming that the BPV-1 genome was replicating within S. cerevisiae. Nicked circle, linear, and supercoiled BPV-1 DNA species were observed in Hirt DNA preparations from S. cerevisiae cells infected for over 50 days, and restriction digestion showed fragments hybridizing to BPV-1 in accord with the predicted restriction map for circular BPV-1 episomes. These data suggest that BPV-1 can infect S. cerevisiae and that BPV-1 episomes can replicate in the infected S. cerevisiae cells.

Saccharomyces cerevisiae is permissive for replication of bovine papillomavirus type 1

We recently demonstrated that Saccharomyces cerevisiae protoplasts can take up bovine papillomavirus type 1 (BPV1) virions and that viral episomal DNA is replicated after uptake. Here we demonstrate that BPV virus-like particles are assembled in infected S. cerevisiae cultures from newly synthesized capsid proteins and also package newly synthesized DNA, including full-length and truncated viral DNA and S. cerevisiae-derived DNA. Virus particles prepared in S. cerevisiae are able to convey packaged DNA to Cos1 cells and to transform C127 cells. Infectivity was blocked by antisera to BPV1 L1 but not antisera to BPV1 E4. We conclude that S. cerevisiae is permissive for the replication of BPV1 virus.

Isolation (from a basal cell carcinoma) of a functionally distinct fibroblast-like cell type that overexpresses Ptch

In this study we report on the isolation and characterization of a nonepithelial, nontumorigenic cell type (BCC1) derived from a basal cell carcinoma from a patient. The BCC1 cells share many characteristics with dermal fibroblasts, such as the expression of vimentin, lack of expression of cytokeratins, and insensitivity to agents that cause growth inhibition and differentiation of epithelial cells; however, significant differences between BCC1 cells and fibroblasts also exist. For example, BCC1 cells are stimulated to undergo DNA synthesis in response to interferon-gamma, whereas dermal fibroblasts are not. More over, BCC1 cells overexpress the basal cell carcinoma-specific genes ptch and ptch2 . These data indicate that basal cell carcinomas are associated with a functionally distinct population of fibroblast-like cells that overexpress known tumor-specific markers (ptch and ptch2 ).

Induction of immune tolerance by dendritic cells: implications for preventative and therapeutic immunotherapy of autoimmune disease

Dendritic cells (DC) have a key role in controlling the immune response, by determining the outcome of antigen presentation to T cells. Through costimulatory molecules and other factors, DC are involved in the maintenance of peripheral tolerance through modulation of the immune response. This modulation occurs both constitutively, and in inflammation, in order to prevent autoimmunity and to control established immune responses. Dendritic cell control of immune responses may be mediated through cytokine or cell-contact dependent mechanisms. The molecular and cellular basis of these controls is being understood at an increasingly more complex level. This understanding is reaching a level at which DC-based therapies for the induction of immune regulation in autoimmunity can be tested in vivo. This review outlines the current state of knowledge of DC in immune tolerance, and proposes how DC might control both T cell responses, and themselves, to prevent autoimmunity and maintain peripheral tolerance.

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV Proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles. At times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in 'conventional' HLA A*0201 transgenic (A2.1K(b) ) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.

Human immunodeficiency virus type 1 reverse transcription is stimulated by Tat from other lentiviruses

The tat gene is required by HIV-1 for efficient reverse transcription and this function of Tat can be distinguished from its role in transcription by RNA polymerase II using tat point mutations that abrogate each function independently The mechanism of Tat's role in reverse transcription, however, is not known, nor is it known whether this role is conserved among trans-activating factors in other retroviruses. Here we examine the abilities of heterologous viral trans-activating proteins from jembrana disease virus (jTat), HIV-2 (Tat2), and equine infectious anemia virus (eTat) to substitute for HIV-1 Tat (Tat1) and restore reverse transcription in HIV-1 carrying an inactivated tat gene. Natural endogenous reverse transcription assays showed that trans-activators from some retroviruses (Tat2 and jTat, but not eTat) could substitute for Tat1 in complementation of HIV-1 reverse transcription. Finally, we show that Y47 is critical for Tat1 to function in reverse transcription, but not HIV-1 gene expression. We mutated the homologous position in jTat to H62Y and found it did not improve its ability to stimulate reverse transcription, but an H62A mutation did inhibit jTat complementation. These data highlight the finding that the role of Tat in reverse transcription is not related to trans-activation and demonstrate that other tat genes conserve this function. (C) 2002 Elsevier Science (USA).

Ecological regulation of development: Induction of marine invertebrate metamorphosis

In the marine environment a wide range of invertebrates have a pelagobenthic lifecycle that includes planktonic larval and benthic adult phases. Transition between these morphologically and ecologically distinct phases typically occurs when the developmentally competent larva comes into contact with a species-specific environmental cue. This cue acts as a morphogenetic signal that induces the completion of the postlarval/juvenile/adult developmental program at metamorphosis. The development of competence often occurs hours to days after the larva is morphologically mature. In the non-feeding - lecithotrophic - larvae of the ascidian Herdmania curvata and the gastropod mollusc Haliotis asinina, gene expression patterns in pre-competent and competent stages are markedly different, reflecting the different developmental states of these larval stages. For example, the expression of Hemps, an EGF-like signalling peptide required for the induction of Herdmania metamorphosis, increases in competent larvae. Induction of settlement and metamorphosis results in further changes in developmental gene expression, which apparently is necessary for the complete transformation of the larval body plan into the adult form.

A genomewide search for type 2 diabetes-susceptibility genes in indigenous Australians

The prevalence of type 2 diabetes among Australian residents is 7.5%; however, prevalence rates up to six times higher have been reported for indigenous Australian communities. Epidemiological evidence implicates genetic factors in the susceptibility of indigenous Australians to type 2 diabetes and supports the hypothesis of the thrifty genotype, but, to date, the nature of the genetic predisposition is unknown. We have ascertained clinical details from a community of indigenous Australian descent in North Stradbroke Island, Queensland. In this population, the phenotype is characterized by severe insulin resistance. We have conducted a genomewide scan, at an average resolution of 10 cM, for type 2 diabetes-susceptibility genes in a large multigeneration pedigree from this community. Parametric linkage analysis undertaken using FASTLINK version 4.1p yielded a maximum two-point LOD score of +2.97 at marker D2S2345. Multipoint analysis yielded a peak LOD score of +3.9

The role of homeotic genes in determining the segmental pattern of chordotonal organs in Drosophila

The homeotic genes are instrumental in establishing segment-specific characteristics. In Drosophila embryos there is ample evidence that the homeotic genes are involved in establishing the differences in the pattern of sense organs between segments. The chordotonal organs are compound sense organs made up of several stretch receptive sensilla. A set of serially homologous chordotonal organs, Ich3 in the 1(st) thoracic segment, dch3 in the 2(nd) and 3(rd) thoracic segments and Ich5 in abdominal segments 1 to 7, is composed of different numbers of sensilla with different positions and orientations. Here we examine this set of sense organs and a companion set, vchA/B and vch 1, in the wild type and mutants for Sex combs reduced, Antennapedia, Ultrabithorax, and abdominal-A, using immunostaining. Mutant phenotypes indicate that Ultrabithorax and abdominal-A in particular influence the formation of these sense organs. Differential expression of abdominal-A and Ultrabithorax within compartments of individual parasegments can precisely modulate the types of sense organs that will arise from a segment.

Interaction of temperature and vegetative flush maturity influences shoot structure and development of lychee (Litchi chinensis Sonn.)

Potted lychee trees (cv. Tai so) of varying vegetative flush maturity were grown under a range of temperature regimes and monitored for subsequent shoot structure and development. A combination of low temperature (15/17 or 18/13 degreesC day/night) and high vegetative flush maturity was necessary for floral initiation to occur, Exposure to high temperatures (28/23 degreesC) invariably resulted in the production of vegetative shoots, irrespective of flush maturity. Strong floral initiation was marked by the emergence of terminal particles and accompanying axillary particles. A decrea,;e in vegetative flush maturity or increase in temperature (e.g. 23/18 degreesC) resulted in a decrease in axillary shoot formation and the production of several intermediate shoot structures. These included leafy particles, stunted particles, partially emerged buds and non-emergent swollen buds, often produced on the same tree. At 23/18 degreesC, closer synchronisation of initial flush maturity was required for the production of a consistent shoot-type. Trees with synchronised mature flushes (I-2) at 23/18 degreesC resulted in the production of swollen terminal buds. Healthy trees were maintained in this state for at least 11 months. These results indicate that both temperature and flush maturity can influence subsequent shoot structure of lychee. In the absence of either a strong floral temperature (18/13 degreesC) or strong vegetative temperature (28/23 degreesC), slight differences in initial flush maturity have gteater impact on the type of emerging shoot formed. (C) 2002 Elsevier Science B.V. All rights reserved.

Callus induction and plant regeneration from broccoli (Brassica oleracea var. italica) for transformation

We evaluated the efficiency of callus induction and plantlet regeneration from hypocotyl explants of broccoli (Brassica oleracea var. italica). The cultivars were ‘Marathon’, ‘Greenbelt’, and ‘Shogun’. Transformation success was not affected by the presence of tobacco feeder-cell layers on the culture media. The frequency of shoot regeneration was greater from 10-d-old hypocotyls than from 14-d-old hypocotyls. Both ‘Marathon’ and ‘Greenbelt’ had higher potentials for tissue regeneration than did ‘Shogun’. We found that for transformation selection, the optimum concentration was either 50 mg/L kanamycin or 100 mg/L genetkin.

Thiazolidinediones and type 2 diabetes: new drugs for an old disease

Thiazolidinediones are a new class of drugs for the treatment of type 2 diabetes, and act by improving insulin sensitivity in adipose tissue, liver and skeletal muscle. Rosiglitazone and pioglitazone are registered for use in monotherapy, and in combination with sulfonylureas and metformin. Pioglitazone is also licensed for use in combination with insulin. There is level II evidence that in patients with inadequate glycaemic control both drugs reduce the level of HbA(1c) and fasting plasma glucose (FPG) when used as monotherapy and in combination with sulfonylurea or metformin or insulin; and both drugs increase levels of HDL and LDL and lower free fatty acid levels, but only pioglitazone significantly lowers triglyceride levels. Both drugs lower fasting insulin and C-peptide levels. In monotherapy, they may be slightly less potent at reducing the level of HbA(1c) than sulfonylureas or metformin. The maximal effect of these agents may not be seen for 6-14 weeks after commencement. Both drugs are well tolerated but liver function must be checked at baseline every second month for the first year, and periodically thereafter. The drugs are currently contraindicated in patients with moderate to severe liver dysfunction and alanine aminotransferase levels more than 2.5 times normal, New York Heart Association III-IV cardiac status, pregnancy, lactation and in children. The main side effects include weight gain, oedema, and mild dilutional anaemia.

Control of dimethylsulfoxide reductase expression in Rhodobacter capsulatus: the role of carbon metabolites and the response regulators DorR and RegA

Regulation of the expression of dimethylsulfoxide (DMSO) reductase was investigated in the purple phototrophic bacterium Rhodobacter capsulatus. Under phototrophic, anaerobic conditions with malate as carbon source, DMSO caused an approximately 150-fold induction of DMSO reductase activity. The response regulator DorR was required for DMSO-dependent induction and also appeared to slightly repress DMSO reductase expression in the absence of substrate. Likewise, when pyruvate replaced malate as carbon source there was an induction of DMSO reductase activity in cells grown at low light intensity (16 W m(-2)) and again this induction was dependent on DorR. The level of DMSO reductase activity in aerobically grown cells was elevated when pyruvate replaced malate as carbon source. One possible explanation for this is that acetyl phosphate, produced from pyruvate, may activate expression of DMSO reductase by direct phosphorylation of DorR, leading to low levels of induction of dor gene expression in the absence of DMSO. A mutant lacking the global response regulator of photosynthesis gene expression, RegA, exhibited high levels of DMSO reductase in the absence of DMSO, when grown phototrophically with malate as carbon source. This suggests that phosphorylated RegA acts as a repressor of dor operon expression under these conditions. It has been proposed elsewhere that RegA-dependent expression is negatively regulated by the cytochrome cbb(3) oxidase. A cco mutant lacking cytochrome cbb(3) exhibited significantly higher levels of Phi[dorA::lacZ] activity in the presence of DMSO compared to wild-type cells and this is consistent with the above model. Pyruvate restored DMSO reductase expression in the regA mutant to the same pattern as found in wild-type cells. These data suggest that R. capsulatus contains a regulator of DMSO respiration that is distinct from DorR and RegA, is activated in the presence of pyruvate, and acts as a negative regulator of DMSO reductase expression.

Towards the clonal propagation of coconut

Past studies from our laboratory have shown that whole immature, or mature sliced, zygotic embryos are a very good starting explant for coconut somatic embryogenesis. The highest rate of somatic embryogenesis was obtained when certain polyamines were added into the culture medium as well as activated charcoal (AC) to absorb unwanted phenolics. These past studies also showed that the development and maturation of the somatic embryos produced could be improved by the addition of abscisic acid (ABA), alone or with one of several osmotically active agents, into the culture medium. In the present study this well characterised somatic embryogenic system for zygotic tissues is being modified and applied to somatic tissues. This recent approach should be a better method for the rapid production of clonal, true-to-type coconut palms. The present research approach is focused on young leaf section explants which have been found to be very responsive to callus production. Young leaf sections produced optimum callus when cultured on media containing 2,4-D (150 μM) and the amount produced could be increased by soaking the sections in sterile water (15 to 60 minutes) or ascorbic acid (15 to 30 minutes) prior to culturing. Further improvement in callus production, as well as a reduction in the time taken for callogenesis was obtained when casein hydrolysate and/or certain polyamines were added to the callus induction medium. The development of the somatic embryos was improved by using ABA and polyethylene glycol (PEG) in the maturation medium. Despite these initial successes in improving coconut somatic embryogenesis, further studies are now being considered to shorten the time to achieve somatic embryogenesis, to better germinate somatic embryos and to improve the rate of somatic seedling conversion into plantlets.