Sequence analysis and expression of a virus-like particle protein,VLP2, from the parasitic wasp Venturia canescens

Endoparasitoid wasps produce maternal protein secretions, which are transported into the body of insect hosts at oviposition to regulate host physiology for successful development of their offspring. Venturia canescens calyx fluid contains so-called virus-like particles (VLPs) that are essential for immune evasion of the developing parasitoid inside the host. VLPs consist of four major proteins. In this paper, we describe the isolation and molecular cloning of a gene (vlp2) that is a constituent of VLPs and discuss its possible role in VLP structure and function.

Isolation and characterization of a neprilysin-like protein from Venturia canescens virus-like particles

Maternal protein secretions from endoparasitoid wasps are evolutionary adaptations to regulate host physiology as part of an extended wasp phenotype. Virus-like particles (VLPs) produced in the calyx region of Venturia canescens wasps are involved in immune evasion of the developing parasitoid inside the host. In contrast to polydnaviruses (PDVs), VcVLPs are devoid of any nucleic acids. To understand the role of these particles in the regulation of host physiology and phylogenetic relationship between VLPs and PDVs, it is essential to identify particle proteins. In this paper, we describe the isolation and molecular cloning of a neprilysin-like gene (VcNEP) coding for a 94 kDa VcVLP protein and discuss its possible role in host regulation.

Virus or not? Phylogenetics of polydnaviruses and their wasp carriers

Our current, still limited, understanding of the comparative biology and evolution of polydnaviruses (PDVs) is reviewed, especially in the context of the possible origins of these parasitoid viruses and of their coevolution with carrier wasps. A hypothetical scenario of evolution of PDVs from ascovirus (or ascovirus-like) ancestors is presented, with examples of apparent extant transitional forms. PDVs appear, in the case of bracoviruses, to show phylogenetic relationships that mirror those of their wasp carriers: with ichno-viruses, the picture is less clear. Ongoing sequencing studies of entire PDV genomes from diverse wasp species are likely to greatly contribute to our understanding of PDV evolution. (C) 2003 Elsevier Science Ltd. All rights reserved.

Targeting c-Myb expression in human disease

c-Myb is a transcription factor employed in the haematopoietic system and gastrointestinal tract to regulate the exquisite balance between cell division, differentiation and survival. In its absence, these tissues either fail to form, or show aberrant biology. Mice lacking a functional c-myb gene die in utero by day 15 of development. When inappropriately expressed, as is common in leukaemia and epithelial cancers of the breast, colon and gastro-oesophagus, c-Myb appears to activate gene targets of key importance to cancer progression and metastasis. These genes include cyclooxygenase-2 (COX-2), Bcl-2, Bcl-X-L and c-Myc, which influence diverse processes such as angiogenesis, proliferation and apoptosis. The clinical potential for blocking c-Myb expression in malignancies is based upon strong preclinical data and some trial-based evidence. The modest clinical experience to date has been with haematopoietic malignancies, but other disease classes may be amenable to similar interventions. The frontline agents to achieve this are nuclease-resistant oligodeoxynucleotides (ODNs), which are proving to be acceptable therapeutic reagents in terms of tolerable toxicities and delivery. Nevertheless, further effort must be focused on improving their efficacy, eliminating non-specific toxicity and optimising delivery. Optimisation issues aside, it would appear that anti-c-Myb therapies will be used with most success when combined with other agents, some of which will be established cytotoxic and differentiation-inducing drugs. This review will explore the future strategic use of ODNs in vivo, focusing on a wide spectrum of diseases, including several beyond the haematopoietic malignancies, in which c-Myb appears to play a role.

Cooperation of cytokine signaling with chimeric transcription factors in leukemogenesis: PML-retinoic acid receptor alpha blocks growth factor-mediated differentiation

We utilized a mouse model of acute promyelocytic leukemia (APL) to investigate how aberrant activation of cytokine signaling pathways interacts with chimeric transcription factors to generate acute myeloid leukemia. Expression in mice of the APL-associated fusion, PML-RARA, initially has only modest effects on myelopoiesis. Whereas treatment of control animals with interleukin-3 (IL-3) resulted in expanded myelopoiesis without a block in differentiation, PML-RARA abrogated differentiation that normally characterizes the response to IL-3. Retroviral transduction of bone marrow with an IL-3-expressing retrovirus revealed that IL-3 and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) combined to generate a lethal leukemia-like syndrome in

Modulation of cytochrome P450 monooxygenase by cadmium chloride in the mouse liver: Involvement of transcription factor Nrf2.

Modulation of the cytochrome P450 (CYP) monooxygenase system and haem oxygenase by cadmium was investigated in male, adult DBA/2J mice treated with a single dose (16 Amol/kg body weight, i.p.) of cadmium chloride (CdCl2), at various time points. Total CYP content of liver microsomes decreased significantly (P < 0.05) at 12, 18, and 24 hours (22%, 47%, and 56%, respectively) after treatment. In contrast, progressive increases of hepatic coumarin 7-hydroxylase (COH) activity (indicative of CYP2A5 activity) were observed at 8 hrs (2-fold), 12 hrs (3-fold), and 7-fold at 18 and 24 hrs. Simultaneously, haem oxygenase activity increased significantly at 4 hours and continued to increase progressively to more than 50-fold compared to control. Liver CYP2A5 mRNA levels increased maximally 12 hours after treatment and decreased to almost half 6 hours later, while western blot analysis showed 2- and 3- fold increase in CYP2A5 apoprotein at 12 and 24 hours. The CYP2A5 mRNA levels in the liver increased after Cd treatment in Nrf2 +/+ but not in Nrf2 / mouse. This study demonstrates that hepatic haem oxygenase and CYP2A5 are upregulated by cadmium. The upregulation of haem oxygenase precedes that of CYP2A5. The strong upregulation of the CYP2A5 both at mRNA and enzyme activity levels, with a simultaneous decrease in the total CYP concentration suggest an unusual mode of regulation of CYP2A5 in response to cadmium exposure, amongst the CYP enzymes. The observed increase in the mRNA but not in protein levels after maximal induction may suggest involvement of post-transcriptional mechanisms in the regulation. Upregulation of CYP2A5 by cadmium in the Nrf2 +/+ mice but not in the Nrf2 / mice indicates a role for this transcription factor in the regulation.

PETAL LOSS, a trihelix transcription factor gene, regulates perianth architecture in the Arabidopsis flower

Perianth development is specifically disrupted in mutants of the PETAL LOSS (PTL) gene, particularly petal initiation and orientation. We have cloned PTL and show that it encodes a plant-specific trihelix transcription factor, one of a family previously known only as regulators of light-controlled genes. PTL transcripts were detected in the early-developing flower, in four zones between the initiating sepals and in their developing margins. Strong misexpression of PTL in a range of tissues universally results in inhibition of growth, indicating that its normal role is to suppress growth between initiating sepals, ensuring that they remain separate. Consistent with this, sepals are sometimes fused in ptl single mutants, but much more frequently in double mutants with either of the organ boundary genes cup-shaped cotyledon1 or 2. Expression of PTL within the newly arising sepals is apparently prevented by the PINOID auxin-response gene. Surprisingly, PTL expression could not be detected in petals during the early stages of their development, so petal defects associated with PTL loss of function may be indirect, perhaps involving disruption to signalling processes caused by overgrowth in the region. PTL-driven reporter gene expression was also detected at later stages in the margins of expanding sepals, petals and stamens, and in the leaf margins; thus, PTL may redundantly dampen lateral outgrowth of these organs, helping define their final shape.

Improved method for counting virus and virus like particles

An improved method for counting virus and virus like particles by electron microscopy (EM) was developed. The procedure involves the determination of the absolute concentration of pure or semi-pure particles once deposited evenly on EM grids using either centrifugation or antibody capture techniques. The counting of particles was done with a Microfiche unit which enlarged approximately 50 x the image of particles on a developed negative film which had been taken at a relatively low magnification (2500 x) by EM. Initially, latex particles of a known concentration were counted using this approach, to prove the accuracy of the technique. The latex particles were deposited evenly on an EM grid using centrifugation (Modified Beckmen EM-90 Airfuge technique). Subsequently, recombinant Bluetongue virus (BTV) core-like particles (CLPs) captured by a Monoclonal antibody using a hovel sample loading method were counted by the Microfiche unit method and by a direct EM method. Comparison of the simplified counting method developed with a conventional method, showed good agreement. The method is simple, accurate, rapid, and reproducible when used with either pure particles or with particles from crude cell culture extracts.

Direct evidence for the expression of multiple endogenous retroviruses in the synovial compartment in rheumatoid arthritis

Objective. Circumstantial evidence links retroviruses (RVs) with human autoimmune diseases, The aim of the present study was to obtain direct evidence of RV gene expression in rheumatoid arthritis (RA). Methods. Synovial samples were obtained from patients with RA, patients with osteoarthritis (OA), and normal control subjects, Reverse transcription-polymerase chain reaction (RT-PCR) was performed using synovial RNA and primers to conserved sequences in the polymerase (pol) genes of known RVs. Results. PCR products (n = 857) were cloned and sequenced, Multiple pol transcripts, many with open reading frames, were expressed in every sample, Sequences were aligned and classified into 6 families (F1-F6) that contained 33 groups of known and unknown endogenous RVs (ERVs), each distinguished by a specific, deduced peptide motif, The frequency of sequences in each family was similar between RA, OA, and normal synovial tissue, but differed significantly in RA synovial fluid cells, F1 sequences (undefined, but related to murine and primate type C RVs) were lower in frequency, F2 (ERV-9-related), F4 (HERV-K-related), and F6 (HERV-L-related) sequences were higher in frequency, and F3 (RTVL-H-related) sequences were not detected, in the RA synovial fluid cells compared with the RA synovial tissues. Conclusion. Multiple ERVs are expressed in normal and diseased synovial compartments, but specific transcripts can be differentially expressed in RA.

Zipping up transcription factors: Rational design of anti-Jun and anti-Fos peptides

Various members of the bZip and bHLH-Zip families of eukaryotic transcription factors, including Jun, Fos, and Myc, have been identified as oncoproteins; mutation or deregulated expression of these proteins leads to certain types of cancer. These proteins can only bind to their cognate DNA enhancer sites following homodimerization, or heterodimerization with another family member, via their leucine zipper domain. Thus, a novel anticancer strategy would be to inhibit dimerization of these proteins, thereby blocking their DNA binding and transactivation functions. In this paper we show that it is possible to rationally design leucine zipper peptides that bind with high affinity to the leucine zipper dimerization domains of c-Jun and c-Fos, thus preventing the formation of functional c-Jun homodimers and c-Jun:c-Fos heterodimers; we refer to such peptides as superzippers (SZs). In vivo, c-Jun:SZ and c-Fos:SZ heterodimers should be nonfunctional as they lack one of the two basic domains that are essential for DNA binding. While the transport of a peptidic agent into cells often poses a severe obstacle to its therapeutic use, we show that a 46-residue leucine zipper peptide can be transported into HeLa cells by coupling it to a 17-residue carrier peptide from the Antennapedia homeodomain, thus paving the way for detailed studies of the therapeutic potential of superzipper peptides.

A systemic parvo-like virus in the freshwater crayfish Cherax destructor

Systemic Cowdry Type A inclusions (CAs) were observed in a moribund Cherax destructor collected at an aquaculture farm in South Australia. Inclusions were most common in the gills and were associated with multifocal necrosis of the main gill axis and lamellae. The hepatopancreas was necrotic; however, only one CA was observed in the interstitial tissues. CAs were associated with necrosis in the abdominal and gut musculature. CAs were also observed in the spongy connective tissues and the epicardium. Empty capsids (17.5 +/- 0.5 nm) and microfilaments were most commonly observed within these inclusions by transmission electron microscopy. Complete icosahedral viral particles (20.8 +/- 1.2 nm) were difficult to distinguish within the viroplasm, but were visualised better in aggregates between the viroplasm and the inner nuclear membrane. The nucleolus was closely associated with the developing viroplasm, and was hypertrophied and segregated into its fibrillar and granular components. The virus was named Cherax destructor systemic parvo-like virus (CdSPV) on the basis of its histopathology, cytopathology and morphology. CdSPV is the first systemic virus described in a freshwater crayfish.

Egr transcription factors in the nervous system

The Egr proteins, Egr-1, Egr-2, Egr-3 and Egr-4, are closely related members of a subclass of immediate early gene-encoded, inducible transcription factors. They share a highly homologous DNA-binding domain which recognises an identical DNA response element. In addition, they have several less-well conserved structural features in common. As immediate early proteins, the Egr transcription factors are rapidly induced by diverse extracellular stimuli within the nervous system in a discretely controlled manner. The basal expression of the Egr proteins in the developing and adult rat brain and the induction of Egr proteins by neurotransmitter analogue stimulation, physiological mimetic and brain injury paradigms is reviewed. We review evidence indicating that Egr proteins are subject to tight differential control through diverse mechanisms at several levels of regulation. These include transcriptional, translational and posttranslational (including glycosylation, phosphorylation and redox) mechanisms and protein-protein interaction. Ultimately the differentially co-ordinated Egr response may lead to discrete effects on target gene expression. Some of the known target genes of Egr proteins and functions of the Egr proteins in different cell types are also highlighted. Future directions for research into the control and function of the different Egr proteins are also explored. (C) 1997 Elsevier Science Ltd.