ISSR-PCR: tool for discrimination and genetic structure analysis of Plutella xylostella populations native to different geographical areas.

The diamondback moth (DBM), Plutella xylostella (L.) is considered as the most destructive pest of Brassicaceae crops world-wide. Its migratory capacities and development of insecticide resistance in many populations leads to more difficulties for population management. To control movement of populations and apparitions of resistance carried by resistant migrant individuals, populations must be identified using genetic markers. Here, seven different ISSR markers have been tested as a tool for population discrimination and genetic variations among 19 DBM populations from Canada, USA, Brazil, Martinique Island, France, Romania, Austria, Uzbekistan, Egypt, Benin, South Africa, Réunion Island, Hong Kong, Laos, Japan and four localities in Australia were assessed. Two classification methods were tested and compared: a common method of genetic distance analyses and a novel method based on an advanced statistical method of the Artificial Neural Networks' family, the Self-Organizing Map (SOM). The 188 loci selected revealed a very high variability between populations with a total polymorphism of 100% and a global coefficient of gene differentiation estimated by the Nei's index (Gst) of 0.238. Nevertheless, the largest part of variability was expressed among individuals within populations (AMOVA: 73.71% and mean polymorphism of 94% within populations). Genetic differentiation among the DBM populations did not reflect geographical distances between them. The two classification methods have given excellent results with less than 1.3% of misclassified individuals. The origin of the high genetic differentiation and efficiency of the two classification methods are discussed.

Genetic differentiation among various populations of the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae).

Genetic variation among 14 populations of Plutella xylostella (Linnaeus) from USA (Geneva, New York), Brazil (Brasilia), Japan (Okayama), The Philippines (Caragan de Oyo), Uzbekistan (Tashkent), France (Montpellier), Benin (Cotonou), South Africa (Johannesburg), Réunion Island (Montvert), and five localities in Australia (Adelaide, Brisbane, Mareeba, Melbourne, Sydney) were assessed by analysis of allozyme frequencies at seven polymorphic loci. Most of the populations were not in Hardy-Weinberg equilibrium and had a deficit in heterozygotes. The global differentiation among populations was estimated by the fixation index (Fst) at 0.103 for the 14 populations and at 0.047 when populations from Australia and Japan, which differed most and had a strong genetic structure, were excluded from the analysis. By contrast, the populations from Benin (West Africa) and Brazil (South America) were very similar to each other. Genetic differentiation among the populations was not correlated with geographical distance.

Expression of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor monocistronic and bicistronic transcripts in primary effusion lymphomas.

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) encodes a G protein-coupled receptor (vGPCR) in open reading frame (ORF) 74, which is homologous to human chemokine receptors. KSHV vGPCR is constitutively active and induces VEGF-mediated angiogenesis. Previous studies have shown that ORF 74 is transcribed as part of a bicistronic message containing ORF K14 upstream of ORF 74, with an early lytic pattern of expression. We have now extended these studies by analyzing three different KSHV-positive primary effusion lymphoma (PEL) cell lines and three PEL clinical samples. In addition, we have identified another less abundant monocistronic transcript containing only ORF 74. Both transcripts were identified at low but similar levels in two PEL clinical samples. We evaluated the degree of sequence and functional conservation of ORF74 in three additional PELs and two KS clinical specimens, demonstrating complete identity at the amino acid level among all isolates. While it is expressed as an early lytic transcript in PEL cell lines, in primary clinical PEL samples transcription of KSHV vGPCR can be readily detected.

Inhibition of constitutive signaling of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor by protein kinases in mammalian cells in culture.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, which is consistently present in tissues of patients with Kaposi's sarcoma and primary effusion lymphomas, contains a gene that encodes a G protein-coupled receptor (KSHV-GPCR). We recently showed that KSHV-GPCR exhibits constitutive signaling via activation of phosphoinositide-specific phospholipase C and stimulates cell proliferation and transformation. In this study, we determined whether normal cellular mechanisms could inhibit constitutive signaling by KSHV-GPCR and thereby KSHV-GPCR-stimulated proliferation. We show that coexpression of GPCR-specific kinases (GRKs) and activation of protein kinase C inhibit constitutive signaling by KSHV-GPCR in COS-1 monkey kidney cells and in mouse NIH 3T3 cells. Moreover, GRK-5 but not GRK-2 inhibits KSHV-GPCR-stimulated proliferation of rodent fibroblasts. These data provide evidence that cell regulatory pathways of receptor desensitization may be therapeutic targets in human diseases involving constitutively active receptors.

G-protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator.

The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) is a gamma-2 herpesvirus that is implicated in the pathogenesis of Kaposi's sarcoma and of primary effusion B-cell lymphomas (PELs). KSHV infects malignant and progenitor cells of Kaposi's sarcoma and PEL, it encodes putative oncogenes and genes that may cause Kaposi's sarcoma pathogenesis by stimulating angiogenesis. The G-protein-coupled receptor encoded by an open reading frame (ORF 74) of KSHV is expressed in Kaposi's sarcoma lesions and in PEL and stimulates signalling pathways linked to cell proliferation in a constitutive (agonist-independent) way. Here we show that signalling by this KSHV G-protein-coupled receptor leads to cell transformation and tumorigenicity, and induces a switch to an angiogenic phenotype mediated by vascular endothelial growth factor, an angiogenesis and Kaposi's-spindle-cell growth factor. We find that this receptor can activate two protein kinases, JNK/SAPK and p38MAPK, by triggering signalling cascades like those induced by inflammatory cytokines that are angiogenesis activators and mitogens for Kaposi's sarcoma cells and B cells. We conclude that the KSHV G-protein-coupled receptor is a viral oncogene that can exploit cell signalling pathways to induce transformation and angiogenesis in KSHV-mediated oncogenesis.

Constitutively signaling G-protein-coupled receptors and human disease.

Dysregulation of G-protein-coupled receptor (GPCR) function has been shown to be associated with a growing number of human diseases. In some diseases, mutation of an endogenous GPCR causes the receptor to lose the ability to bind agonist or signal (;loss of function' mutation), whereas another mutation causes the receptor to be in an active state in the absence of agonist (;gain of function' mutation), leading to ;constitutive signaling activity'. A number of constitutively active GPCRs are tumorigenic in vitro and in animal models, and cause syndromes of hyperfunction and/or tumors in humans. The recent characterization of a constitutively active GPCR in the genome of a disease-associated, human herpesvirus provides a potential novel mechanism for viral tumorigenesis.

Human herpesvirus KSHV encodes a constitutively active G-protein-coupled receptor linked to cell proliferation.

Kaposi's sarcoma-associated herpesvirus (KSHV, also known as human herpesvirus 8, or HHV 8) is a virus that is consistently present in Kaposi's sarcoma and in primary-effusion (body-cavity-based) lymphomas, malignancies that occur frequently, but not exclusively, in AIDS patients. KSHV is a gamma herpesvirus with homology to herpesvirus Saimiri and Epstein-Barr virus, both of which can transform lymphocytes. Cloning of a KSHV genome fragment revealed the presence of an open reading frame encoding a putative G-protein-coupled receptor that is homologous to a G-protein-coupled receptor encoded by herpesvirus Saimiri and to human interleukin-8 receptors. Here we show that the KSHV G-protein-coupled receptor is a bona fide signalling receptor which has constitutive (agonist-independent) activity in the phosphoinositide-inositoltrisphosphate-protein kinase C pathway. Furthermore, the KSHV G-protein-coupled receptor stimulates cellular proliferation, making it a candidate viral oncogene.

Establishment and characterization of a primary effusion (body cavity-based) lymphoma cell line (BC-3) harboring kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in the absence of Epstein-Barr virus.

The recently identified Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), has been found to be consistently associated with an unusual subset of acquired immunodeficiency syndrome-related lymphomas, the so-called body cavity-based lymphomas (BCBL) or primary effusion lymphomas (PEL). These lymphomas are characterized by a unique spectrum of morphologic and molecular characteristics, and grow as lymphomatous effusions without an identifiable contiguous tumor mass. Until now, efforts to delineate the role of KSHV in the pathogenesis of PELs have been hampered by the lack of appropriate model systems and the concomitant presence of Epstein-Barr virus (EBV) in nearly all cases examined, and in all previously established cell lines. We now report the establishment and characterization of a novel PEL cell line, BC-3, which is KSHV+ by polymerase chain reaction (PCR) but EBV- as assessed by a variety of methods including PCR for EBER, EBNA-2, and EBNA-3C. This cell line was established from a lymphomatous effusion obtained from an HIV- patient, and has immunophenotypic and molecular features consistent with the diagnosis of PEL, including an indeterminate immunophenotype with a B-cell immunogenotype and lack of c-myc proto-oncogene rearrangements. Pulsed-field gel electrophoresis shows an intact KSHV genome of about 170 kb both in the cell line and in the viral isolate, whereas herpesvirus-like capsids are visible by electron microscopy. Consequently, the BC-3 cell line represents an invaluable tool as a source of KSHV, for both the evaluation of the pathogenic potential of this virus and the mechanistic characterization of its role in the development of Kaposi's sarcoma and malignant lymphoma.

Human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus is a new transmissible virus that infects B cells.

Herpesviral DNA fragments isolated from AIDS-associated Kaposi's sarcoma (KS) tissue (KSHV-DNA) share homology with two lymphotropic oncogenic gamma-herpesviruses, Epstein-Barr virus and Herpesvirus saimiri, and are present in the lesions of more than 95% of HIV and non-HIV-associated forms of KS, AIDS-related body cavity-based lymphomas, and AIDS-related multicentric Castleman's disease. Here we show that BC-1, a KSHV-DNA-positive, body cavity-based lymphoma cell line, produces infective herpesviral particles carrying a linear 270-kb genome that specifically transmits KSHV-DNA to CD19+ B cells. Transmission of KSHV-DNA is dependent upon a biologically active, replicating virus, since it is blocked by UV irradiation and foscarnet, an inhibitor of viral DNA-polymerase. This study represents the first isolation and transmission of the human herpesvirus-8/KS-associated herpesvirus.

Characterization of transforming growth factor-beta 1 induced apoptosis in normal human B cells and lymphoma B cell lines.

Transforming growth factor beta 1 (TGF beta 1) has been shown to inhibit growth stimulation in normal human B cells as well as in Epstein Barr virus (EBV)-negative Burkitt's lymphoma (BL) cell lines. The mechanisms for this potent growth inhibition are not completely defined. Here we show that a number of EBV-negative lymphoma B cell lines (BL-41, Ramos and CAPA-2), when exposed in vitro to TGF beta 1, undergo apoptosis. Maximum apoptosis was observed at 48 h following TGF beta 1 treatment, with no apparent effect on the expression of c-myc and bcl-2 proteins. Similar induction of apoptosis was observed when these lymphoma cell lines were treated with aphidicolin, a DNA synthesis inhibitor. In contrast, various preparations (14 out of 17) of normal human tonsilar B cells showed no significant apoptosis, although both TGF beta 1 and aphidicolin inhibited anti-mu/IL-4 induced DNA synthesis in all preparations. Furthermore, another TGF beta 1 sensitive EBV-negative BL cell line, CA46, exhibited no apoptosis in response to TGF beta 1 and aphidicolin, corroborating the findings in normal human B cells. Taken together, these data support the hypothesis that exposure to TGF beta 1, which results in cell cycle arrest and DNA synthesis inhibition, may not be obligatory or sufficient for the induction of apoptosis. Rather, induction of apoptosis or lack of it may be intrinsically determined by an interplay between extracellular and intracellular regulators of cellular growth.

Latent membrane protein-1 induces cyclin D2 expression, pRb hyperphosphorylation, and loss of TGF-beta 1-mediated growth inhibition in EBV-positive B cells.

The normal cell cycle is regulated by several molecules, such as the tumor-suppressor protein pRb, the G1 cyclins, the cyclin-dependent kinases, and their inhibitors. These regulators are targeted by negative growth regulatory signals, such as that provided by TGF-beta. Here, we show that the presence of either wild-type EBV or its transforming latent membrane protein-1 (LMP-1) results in the loss of TGF-beta 1-mediated growth inhibition in human B cells. Chemical cross-linking with 125I-labeled TGF-beta 1 showed an essentially normal TGF-beta receptor profile in EBV-positive and EBV-negative Burkitt's lymphoma cell lines, and these receptors were shown to be functional in transducing signals, as evidenced by the TGF-beta 1-mediated modulation of junB gene expression. However, TGF-beta 1 did not induce dephosphorylation of pRb in EBV (or LMP-1)-positive cells as opposed to EBV-negative cells, suggesting a dichotomy in the TGF-beta 1 signaling pathway leading to separable gene regulatory and growth inhibitory responses. Furthermore, LMP-1 was found to induce the expression of cyclin D2; normal B cells or EBV-negative Burkitt's lymphoma cells do not express D-type cyclins. Taken together, these data point to a potential mechanism underlying EBV-mediated B cell transformation whereby constitutive induction of key cell cycle regulators by LMP-1 can lead to pRb hyperphosphorylation and uncontrolled cell proliferation.