Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cells--a mechanism of immune evasion?

The CD95 (APO-1/Fas) system is an important mediator of T-cell cytotoxicity. We investigated this system in 22 hepatocellular carcinomas (HCCs) from patients. All HCCs had partially or completely lost the expression of the CD95 receptor constitutively expressed by normal liver cells and might thus evade CD95-mediated killing. We also considered a new mechanism of immune evasion, namely, the active destruction of T-lymphocytes by tumor cells expressing CD95 ligand (CD95L). CD95L messenger RNA and protein could be detected in the HCCs. In coculture experiments, HepG2 hepatoblastoma cells, expressing CD95L mRNA after treatment with cytostatic drugs, killed CD95+ Jurkat lymphocytes. Our data suggest that tumor cells can evade immune attack by down-regulation of the CD95 receptor and killing of lymphocytes through expression of CD95L.

Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo.

The pro-inflammatory cytokine interleukin (IL)-6 (refs. 1-5) can bind to cells lacking the IL-6 receptor (IL-6R) when it forms a complex with the soluble IL-6R (sIL-6R) (trans signaling). Here, we have assessed the contribution of this system to the increased resistance of mucosal T cells against apoptosis in Crohn disease (CD), a chronic inflammatory disease of the gastrointestinal tract. A neutralizing antibody against IL-6R suppressed established experimental colitis in various animal models of CD mediated by type 1 T-helper cells, by inducing apoptosis of lamina propria T cells. Similarly, specific neutralization of sIL-6R in vivo by a newly designed gp130-Fc fusion protein caused suppression of colitis activity and induction of apoptosis, indicating that sIL-6R prevents mucosal T-cell apoptosis. In patients with CD, mucosal T cells showed strong evidence for IL-6 trans signaling, with activation of signal transducer and activator of transcription 3, bcl-2 and bcl-xl. Blockade of IL-6 trans signaling caused T-cell apoptosis, indicating that the IL-6-sIL-6R system mediates the resistance of T cells to apoptosis in CD. These data indicate that a pathway of T-cell activation driven by IL-6-sIL-6R contributes to the perpetuation of chronic intestinal inflammation. Specific targeting of this pathway may be a promising new approach for the treatment of CD.

Expanding ecological assessment by integrating microorganisms into routine freshwater biomonitoring.

Bioindication has become an indispensable part of water quality monitoring in most countries of the world, with the presence and abundance of bioindicator taxa, mostly multicellular eukaryotes, used for biotic indices. In contrast, microbes (bacteria, archaea and protists) are seldom used as bioindicators in routine assessments, although they have been recognized for their importance in environmental processes. Recently, the use of molecular methods has revealed unexpected diversity within known functional groups and novel metabolic pathways that are particularly important in energy and nutrient cycling. In various habitats, microbial communities respond to eutrophication, metals, and natural or anthropogenic organic pollutants through changes in diversity and function. In this review, we evaluated the common trends in these changes, documenting that they have value as bioindicators and can be used not only for monitoring but also for improving our understanding of the major processes in lotic and lentic environments. Current knowledge provides a solid foundation for exploiting microbial taxa, community structures and diversity, as well as functional genes, in novel monitoring programs. These microbial community measures can also be combined into biotic indices, improving the resolution of individual bioindicators. Here, we assess particular molecular approaches complemented by advanced bioinformatic analysis, as these are the most promising with respect to detailed bioindication value. We conclude that microbial community dynamics are a missing link important for our understanding of rapid changes in the structure and function of aquatic ecosystems, and should be addressed in the future environmental monitoring of freshwater ecosystems.

The Drosophila lethal(2)giant larvae tumor suppressor protein is a component of the cytoskeleton.

Tumor suppressor genes act as recessive determinants of cancer. In Drosophila these genes play a role in normal development and are essential for regulating cell growth and differentiation. Mutations in the gene, lethal(2)giant larvae, l(2)gl, besides causing malignant tumors in the brain and imaginal discs, generate developmental defects in a number of other tissues. Much of the uncertainty regarding the function of the l(2)gl gene product, p127, results from a lack of knowledge as to the precise location of this protein in the cell. We have investigated the cellular and subcellular localization of p127, using confocal and electron microscopy as well as biochemical and cell fractionation procedures. Our analyses indicate that p127 is located entirely within the cell in both the cytoplasm and bound to the inner face of lateral cell membranes in regions of cell junctions. On the membrane, p127 can form large aggregates which are resistant to solubilization by nonionic detergents, indicating that p127 is participating in a cytoskeletal matrix. These findings suggest that the changes in cell shape and the loss of apical-basal polarity observed in tumorous tissues are a direct result of alterations in the cytoskeleton organization caused by l(2)gl inactivation and also suggest that p127 is involved in a cytoskeletal-based intercellular communication system directing cell differentiation.

The overgrown hematopoietic organs-31 tumor suppressor gene of Drosophila encodes an Importin-like protein accumulating in the nucleus at the onset of mitosis.

The tumor suppressor gene overgrown hematopoietic organs-31 (oho31) of Drosophila encodes a protein with extensive homology to the Importin protein of Xenopus (50% identity), the related yeast SRP1 protein, and the mammalian hSRP1 and RCH1 proteins. A strong reduction in the expression of oho31 by a P element inserted in the 5' untranslated region of the oho31 transcript or a complete inactivation of oho31 by imprecise P element excision leads to malignant development of the hematopoietic organs and the genital disc, as shown by their growth autonomy in transplantation assays. We have cloned the oho31 gene of Drosophila melanogaster and determined its nucleotide sequence. The gene encodes a phosphoprotein of 522 amino acids made of three domains: a central hydrophobic domain of eight repeats of 42-44 amino acids each, displaying similarity to the arm motif found in junctional and nucleopore complex proteins, and flanked by two hydrophilic NH2- and COOH-terminal domains. Immunostaining revealed that the OHO31 protein is supplied maternally and rapidly degraded during the first 13 nuclear divisions. Thereafter, the OHO31 protein is predominantly expressed, albeit at reduced levels, in proliferating tissues. During the interphase of early embryonic cell cycles, the OHO31 protein is present in the cytoplasm and massively accumulates in the nucleus at the onset of mitosis in late interphase and prophase. The nuclear import of OHO31 is, however, less pronounced during later developmental stages. These results suggest that, similar to Importin, OHO31 may act as a cytosolic factor in nuclear transport. Moreover, the cell cycle-dependent accumulation of OHO31 in the nucleus indicates that this protein may be required for critical nuclear reactions occurring at the onset of mitosis.

The Drosophila lethal(2)giant larvae tumor suppressor protein forms homo-oligomers and is associated with nonmuscle myosin II heavy chain.

Inactivation of the Drosophila lethal(2)giant larvae (l(2)gl) gene causes malignant tumors in the brain and the imaginal discs and produces developmental abnormalities in other tissues, including the germline, the ring gland and the salivary glands. Our investigations into the l(2)gl function have revealed that the gene product, or p127 protein, acts as a cytoskeletal protein distributed in both the cytoplasm and on the inner face of lateral cell membranes in a number of tissues throughout development. To determine whether p127 can form oligomers or can stably interact with other proteins we have analyzed the structure of the cytosolic form of p127. Using gel filtration and immunoaffinity chromatography we found that p127 is consistently recovered as high molecular weight complexes that contain predominantly p127 and at least ten additional proteins. Blot overlay assays indicated that p127 can form homo-oligomers and the use of a series of chimaeric proteins made of segments of p127 fused to protein A, which alone behaves as a monomer, showed that p127 contains at least three distinct domains contributing to its homo-oligomerization. Among the proteins separated from the immuno-purified p127 complexes or isolated by virtue of their affinity to p127, we identified one of the proteins by microsequencing as nonmuscle myosin II heavy chain. Further blot overlay assay showed that p127 can directly interact with nonmuscle myosin II. These findings confirm that p127 is a component of a cytoskeletal network including myosin and suggest that the neoplastic transformation resulting from l(2)gl gene inactivation may be caused by the partial disruption of this network.

aPKCzeta cortical loading is associated with Lgl cytoplasmic release and tumor growth in Drosophila and human epithelia.

Atypical protein kinase C (aPKC) and Lethal giant larvae (Lgl) regulate apical-basal polarity in Drosophila and mammalian epithelia. At the apical domain, aPKC phosphorylates and displaces Lgl that, in turn, maintains aPKC inactive at the basolateral region. The mutual exclusion of these two proteins seems to be crucial for the correct epithelial structure and function. Here we show that a cortical aPKC loading induces Lgl cytoplasmic release and massive overgrowth in Drosophila imaginal epithelia, whereas a cytoplasmic expression does not alter proliferation and epithelial overall structure. As two aPKC isoforms (iota and zeta) exist in humans and we previously showed that Drosophila Lgl is the functional homologue of the Human giant larvae-1 (Hugl-1) protein, we argued if the same mechanism of mutual exclusion could be impaired in human epithelial disorders and investigated aPKCiota, aPKCzeta and Hugl-1 localization in cancers deriving from ovarian surface epithelium. Both in mucinous and serous histotypes, aPKCzeta showed an apical-to-cortical redistribution and Hugl-1 showed a membrane-to-cytoplasm release, perfectly recapitulating the Drosophila model. Although several recent works support a causative role for aPKCiota overexpression in human carcinomas, our results suggest a key role for aPKCzeta in apical-basal polarity loosening, a mechanism that seems to be driven by changes in protein localization rather than in protein abundance.

The transcription factor ZEB1 (deltaEF1) promotes tumour cell dedifferentiation by repressing master regulators of epithelial polarity.

Epithelial to mesenchymal transition (EMT) is implicated in the progression of primary tumours towards metastasis and is likely caused by a pathological activation of transcription factors regulating EMT in embryonic development. To analyse EMT-causing pathways in tumourigenesis, we identified transcriptional targets of the E-cadherin repressor ZEB1 in invasive human cancer cells. We show that ZEB1 repressed multiple key determinants of epithelial differentiation and cell-cell adhesion, including the cell polarity genes Crumbs3, HUGL2 and Pals1-associated tight junction protein. ZEB1 associated with their endogenous promoters in vivo, and strongly repressed promotor activities in reporter assays. ZEB1 downregulation in undifferentiated cancer cells by RNA interference was sufficient to upregulate expression of these cell polarity genes on the RNA and protein level, to re-establish epithelial features and to impair cell motility in vitro. In human colorectal cancer, ZEB1 expression was limited to the tumour-host interface and was accompanied by loss of intercellular adhesion and tumour cell invasion. In invasive ductal and lobular breast cancer, upregulation of ZEB1 was stringently coupled to cancer cell dedifferentiation. Our data show that ZEB1 represents a key player in pathologic EMTs associated with tumour progression.

Peripheral blood dendritic cells are phenotypically and functionally intact in chronic hepatitis B virus (HBV) infection.

Persistence of hepatitis B virus (HBV) infection is associated with reduced anti-viral T cell responses. Impaired dendritic cell (DC) function was suggested as the cause of reduced T cell stimulation in chronic HBV carriers. Thus, we compared myeloid (mDC) and plasmacytoid DC (pDC) from chronic HBV carriers and controls. Frequency and phenotype of isolated DC were analysed by fluorescence activated cell sorter staining, DC function by mixed lymphocyte reaction, cytokine bead array, intracellular cytokine staining, enzyme-linked immunosorbent assay and enzyme-linked immunospot. Expression of HBV DNA and mRNA was studied by polymerase chain reaction (PCR). Circulating total DC, mDC or pDC were not reduced in chronic HBV carriers. Isolated mDC and pDC from chronic HBV carriers exhibited similar expression of co-stimulatory molecules and alloreactive T helper cell stimulation as control DC, whether tested directly ex vivo or after in vitro maturation. Secretion of pro- and anti-inflammatory cytokines by CD40 or Toll-like receptor ligand-stimulated patient DC was intact, as was human leucocyte antigen A2-restricted HBV-specific cytotoxic lymphocyte stimulation. Although both DC populations contained viral DNA, viral mRNA was undetectable by reverse transcription-PCR, arguing against viral replication in DC. We found no quantitative, phenotypic or functional impairment of mDC or pDC in chronic hepatitis B, whether studied ex vivo or after in vitro maturation.

Allergological implication of the quaternary hexameric structure of the cockroach allergen Per a 3.

BACKGROUND: Cockroach allergens play a very important role in allergic diseases, especially asthma. The major allergen of the American cockroach (Periplaneta americana), Per a 3, naturally occurs as isoforms of hexamers. OBJECTIVE: The aim of this study was to investigate whether the hexameric structures of Per a 3 influence their allergenicity and immunogenicity. METHODS: Therefore, we compared the different effects of native hexamers and dissociated monomers of cockroach haemolymph (HL), containing almost only Per a 3 proteins (HL-Per a 3), on proliferation and T-helper type 1 (Th1)/Th2 cytokine production of human CD4(+) T cells in co-culture with allergen-pulsed monocyte-derived autologous dendritic cells (DC) as well as the leukotriene release of basophils. RESULTS: In P. americana-sensitized and non-sensitized donors the HL-Per a 3 monomers were internalized faster by immature DC and induced higher proliferation and IFN-gamma production than the hexamers. While in non-sensitized donors IL-4 and IL-5 as well as IL-10 production were also increased after stimulation with monomeric HL-Per a 3-pulsed DC, Th2 cytokine and IL-10 production were only enhanced in P. americana-sensitized donors using hexameric HL-Per a 3-pulsed DC. Furthermore, in the leukotriene release assay the monomers were less effective than the hexamers. CONCLUSION: Our data indicate that the quaternary structure can influence both allergenicity and immunogenicity, also depending on the sensitization status. The monomeric variant of Per a 3 allergens could be a possible candidate for a specific immunotherapy because the IgE-mediated allergic reaction and the Th2-inducing capacity are diminished while the Th1-inducing capacity is retained.

Determination of 12 commonly found compounds in DUID cases in whole blood using fully automated supported liquid extraction and UHPLC-MS/MS.

A high-throughput UHPLC-MS/MS method for the most frequently found compounds; tetrahydrocannabinol (THC), amphetamine, methamphetamine, MDMA, clonazepam, diazepam, nordiazepam, oxazepam, alprazolam, nitrazepam, morphine, and codeine, in driving under the influence of drugs (DUID) cases in whole blood, is presented. Automated sample preparation by 96-well supported liquid extraction (SLE) plates with ethyl acetate + heptane (80 + 20, v/v) as organic solvent was carried out on a Freedom Evo 200 platform from Tecan. An aliquot of 100 µL whole blood was used. Sample preparation time for 96 samples was 1.5 h. Compounds were separated with gradient elution on a C18 column (50 × 2.1 mm, 1.7 µm) with a mobile phase consisting of 5 mM pH 10.2 ammonium formate and methanol. The run time was 4.5 min and 1 µL was injected on an Acquity UPLC I-Class system with a Xevo TQS tandem-quadrupole mass spectrometer in multiple-reaction monitoring mode (MRM) from Waters. Isotope labelled, 13C, internal standards (ISs) were used for all compounds except for alprazolam and morphine, which had deuterated analogs. Quantification was carried out with calibrators without whole blood matrix. Full validation was carried out according to international guidelines, and a new approach for evaluation of process efficiency (PE) has been presented. Linear or quadratic weighted (1/x) calibration curves were used with R2 ≥ 0.999. The method showed satisfactory deviations ±16% when compared to the existing methods, and satisfactory agreement with proficiency testing control samples (z-score -1.6 to 1.8, n = 16 samples). The precision, estimated as the relative standard deviation (RSD) of the concentration difference between results from two independent analyses of authentic whole blood samples, was ≤7.2% in antemortem and ≤9.3% in postmortem samples. Recovery was ≥85% for all the compounds, except morphine ≥62% and THC ≥ 50%. PE was satisfactory for all the compounds with low variation in IS response, RSD ≤ 16% (THC 27%) in antemortem samples and ≤34% (THC 66%) in postmortem samples. To the best of our knowledge, this is the first automated 96-well SLE UHPLC-MS/MS method developed for the simultaneous determination of these 12 compounds in whole blood covering the concentration ranges found in forensic samples. The method has been used in routine work during the last ten months, analysing about 9900 antemortem and 1000 postmortem whole blood samples, and has proven to be robust and reliable.

Expression of Hugl-1 is strongly reduced in malignant melanoma.

The human gene Hugl-1 (Llgl/Lgl1) has significant homology to the Drosophila tumor suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that is involved in maintaining cell polarity and epithelial integrity. We speculate that Hugl-1 might play a role in epithelial-mesenchymal transition (EMT) and that loss of Hugl-1 expression plays a role in the development or progression of malignant melanoma. Thus, we evaluated melanoma cell lines and tissue samples of malignant melanoma for loss of Hugl-1 transcription. We found that Hugl-1 was downregulated or lost in all cell lines and in most of the tumor samples analysed, and that these losses were associated with advanced stage of the disease. Reduced Hugl-1 expression occurred as early as in primary tumors detected by both immunohistochemical and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Functional assays with stable Hugl-1-transfected cell lines revealed that Hugl-1 expression increased cell adhesion and decreased cell migration. Further, downregulation of MMP2 and MMP14 (MT1-MMP) and re-expression of E-cadherin was found in the Hugl-1-expressing cell clones supporting a role of Hugl-1 in EMT. Our studies thus indicate that loss of Hugl-1 expression contributes to melanoma progression.

In-vivo confocal real-time mini-microscopy in animal models of human inflammatory and neoplastic diseases.

BACKGROUND AND STUDY AIMS: Although various improvements in tissue imaging modalities have recently been achieved, in-vivo molecular and subsurface imaging in the field of gastroenterology remains a technical challenge. In this study we evaluated a newly developed, handheld, miniaturized confocal laser microscopy probe for real-time in-vivo molecular and subsurface imaging in rodent models of human disease. MATERIALS AND METHODS: The minimicroscope uses a 488-nm, single line laser for fluorophore excitation. The optical slice thickness is 7 microm, the lateral resolution 0.7 microm. The range of the z-axis is 0-250 microm below the tissue surface. Imaging was performed using different fluorescent staining protocols; 5-carboxyfluorescein-labeled octreotate was synthesized for targeted molecular imaging. RESULTS: Cellular and subcellular details of the gastrointestinal tract could be visualized in vivo at high resolution. Confocal real-time microscopy allowed in-vivo identification of tumor vessels and liver metastases, as well as diagnosis of focal hepatic inflammation, necrosis, and associated perfusion anomalies. Somatostatin-receptor targeting permitted in-vivo molecular staining of AR42-J-induced carcinoma and pancreatic islet cells. CONCLUSIONS: Confocal mini-microscopy allows rapid in-vivo molecular and subsurface imaging of normal and pathological tissue in the gastrointestinal tract at high resolution. Because this technology is applicable to humans, it might impact on future in-vivo microsocpic and molecular diagnosis of diseases such as cancer and inflammation.

The l(2)gl homologue of Drosophila pseudoobscura suppresses tumorigenicity in transgenic Drosophila melanogaster.

Mutations in the tumour-suppressor gene lethal(2)giant larvae (l(2)gl) of Drosophila cause malignant transformation of the optic centres of the larval brain and the imaginal discs. We report the cloning and sequencing of the l(2)gl gene from Drosophila pseudoobscura. Comparison of this sequence with D. melanogaster reveals a significant sequence conservation within the l(2)gl protein-coding domain and a strong sequence divergence in the 5' promoter region and in the introns. The deduced amino acid sequence of the D. pseudoobscura l(2)gl protein shows 17.7% divergence from D. melanogaster. However, despite these evolutionary differences, the D. pseudoobscura l(2)gl gene can fully suppress tumorigenicity and restore a normal development in l(2)gl-deficient D. melanogaster flies, although the rescued animals display poor viability and fertility. Furthermore, in D. melanogaster transgenic flies, the D. pseudoobscura l(2)gl protein is produced at a similar level as the D. melanogaster l(2)gl protein and displays an identical spatial pattern of expression. This shows that the highly divergent cis-regulatory elements of the D. pseudoobscura transgene can be fully recognized in D. melanogaster and lead to the synthesis of a transgenic protein that has enough specificity conserved for replacing the tumour-suppressor function normally fulfilled by the D. melanogaster l(2)gl protein.

A human homologue of the Drosophila tumour suppressor gene l(2)gl maps to 17p11.2-12 and codes for a cytoskeletal protein that associates with nonmuscle myosin II heavy chain.

Inactivation of the tumour suppressor gene lethal(2) giant larvae (D-lgl) of Drosophila leads to malignant transformation of the presumptive adult optic centers in the larval brain and tumours of the imaginal discs. These malignancies result from the disorganization of a cytoskeletal network in which the D-LGL protein participates. Here we describe the isolation of a cDNA encoding the human homologue to the D-lgl gene designated as hugl. The hugl cDNA detects a locus spanning at least 25 kilobases (kb) in human chromosome band 17p11.2-12, which is centromeric to the p53 gene and recognizes a 4.5 kb RNA transcript. The hugl gene is expressed in brain, kidney and muscle but is barely seen in heart and placenta. Sequence analysis of the hugl cDNA demonstrates a long open reading frame, which has the potential to encode a protein of 1057 amino acids with a predicted molecular weight of 115 kDaltons (kD). To further substantiate and identify the HUGL protein, we have prepared polyclonal rabbit antibodies against synthetic peptides corresponding to the amino and carboxyl termini of the conceptual translation product of the hugl gene. The affinity-purified anti-HUGL antibodies recognize a single protein with an apparent molecular weight of approximately 115 kD. Similar to the Drosophila protein, HUGL is part of a cytoskeletal network and, is associated with nonmuscle myosin II heavy chain and a kinase that specifically phosphorylates HUGL at serine residues.

Homo-oligomerization domains in the lethal(2)giant larvae tumor suppressor protein, p127 of Drosophila.

The p127 tumor suppressor protein encoded by the lethal(2)giant larvae, l(2)gl, gene of Drosophila melanogaster forms high molecular mass complexes consisting predominantly of p127 molecules. To determine whether p127 can self-assemble in the absence of other binding factors, we analyzed the size of in vitro synthesized p127 by gel filtration and found that p127 is always recovered in a high molecular mass form, demonstrating that p127 can oligomerize on its own. Previous studies have revealed that p127 may contain three homo-oligomerization domains. To more accurately delineate these domains, we have generated a series of 32 chimaeric proteins made of defined portions of p127 fused to protein A, which behaves as a monomeric protein, and determined the level of oligomerization of the fused proteins. This study allowed us to map three discrete homo-oligomerization domains, each of approximately 50 amino acid residues in length. These domains, designated as HD-I, HD-II and HD-III, are located between amino acid residues 160 and 204, 247 and 298, and 706 and 749, respectively. Further analysis showed that the HD-I and HD-II domains can bind to themselves and to each other. We also mapped a domain in p127 between amino acid residues 377 and 438, which strongly reduces the degree of multimerization of chimaeric proteins containing HD-I and/or HD-II. Electron microscopy examination of negatively stained chimaeric proteins showed that protein A fused with either the domain HD-II or the domain HD-III forms discrete structures consistent with the formation of quaternary complexes, whereas protein A fused to a non-self binding domain of p127 appeared monomeric. Our results indicate that p127 alone is able to build quaternary structures forming a network with which other proteins associate. As revealed by the tumorous phenotype resulting from the inactivation of the l(2)gl gene, the organization of the p127 network and its association with other proteins play critical roles in the control of cell proliferation.

Casein kinase 2 binds and phosphorylates the nucleosome assembly protein-1 (NAP1) in Drosophila melanogaster.

The nucleosome assembly protein-1 (NAP1) was originally identified in HeLa cells as a factor facilitating the in vitro assembly of nucleosomes. However, in yeast cells NAP1 is required in the control of mitotic events induced by the Clb2/p34(CDC28). Here, we show that Drosophila NAP1 is a phosphoprotein that is associated with a kinase able to phosphorylate NAP1. By using an in-gel kinase assay we found that this kinase displays a molecular mass of 38 kDa. Following purification and peptide microsequencing, we identified the kinase phosphorylating NAP1 as the alpha subunit of casein kinase 2 (CK2). With the help of a series of NAP1 segments and synthetic peptides, we assigned the CK2 phosphorylation sites to residues Ser118, Thr120, and Ser284. Interestingly, Ser118 and Thr120 are located within a PEST domain, while Ser284 is adjacent to the nuclear localization signal. Substitution of the identified phosphoresidues by alanine was found to reduce considerably the ability of CK2 to phosphorylate NAP1. The enhanced ability of CK2 to phosphorylate phosphatase-treated NAP1 extracted from Drosophila embryos and the similar tryptic phospho-peptide pattern of in vivo labelled NAP1 and in vitro labelled NAP1 with CK2 indicate that NAP1 is a natural substrate of CK2. Further analysis revealed that both CK2alpha and beta subunits are associated with NAP1 but we found that only the catalytic alpha subunit establishes direct contact with NAP1 on two distinct domains of this protein. The location of CK2 phosphorylation sites in NAP1 suggests that their phosphorylation can contribute to a PEST-mediated protein degradation of NAP1 and the translocation of NAP1 between cytoplasm and nucleus.

Insertion of a copia element 5' to the Drosophila melanogaster alcohol dehydrogenase gene (adh) is associated with altered developmental and tissue-specific patterns of expression.

The Drosophila melanogaster alcohol dehydrogenase gene (adh) is under the control of two separate promoters (proximal and distal) which are preferentially utilized at the larval and adult life stages, respectively. A variant alcohol dehydrogenase allele (RI-42) isolated from a natural population contains a copia retroviral-like transposable element inserted 240 bp upstream from the distal (adult) adh transcriptional start site. Levels of adh transcripts in the RI-42 variant are reduced in tissues and at life stages where copia is actively expressed and are affected in trans- by mutant alleles at the suppressor-of-white-apricot (su(wa] and suppressor-of-forked (su(f] loci. These suppressor genes have no effect on adh expression in wild-type Drosophila.

Requirement of Drosophila I(2)gl function for survival of the germline cells and organization of the follicle cells in a columnar epithelium during oogenesis.

The lethal(2)giant larvae gene, or 1(2)gl, encodes a widely expressed cytoskeletal protein which acts in numerous biological processes during embryogenesis and oogenesis, including cell proliferation, and morphogenetic movements. Having identified the nucleotide change occurring in the l(2)gl(ts3) sequence, we produced by site-directed mutagenesis the identical change leading to the substitution of a serine by a phenylalanine at position 311 of p127l(2)gl and introduced the modified l(2)glF311 gene into l(2)gl flies. The transgene can fully rescue the development of l(2)gl flies raised at 22 degrees C but causes drastic effects on their development at 29 degrees C confirming the temperature sensitivity of the phenylalanine substitution at position 311. Fertility of females, albeit not of males, was strongly affected. Temperature-shift experiments and microscopic examination of ovaries showed that the mutation blocked egg chamber development at the onset of vitellogenesis (stages 8-9) with growth arrest of the oocyte, incomplete follicle cell migration over the oocyte associated with abnormal organization of the follicular epithelium, and apoptosis of the germline cells, as measured by TUNEL assays. By comparison to wildtype, we found that p127F311 is already reduced in amount at 22 degrees C and delocalized from the cytoskeletal matrix, albeit without affecting the apical localization of myosin II, a major partner of p127. At 29 degrees C, the level of p127F311 is even more reduced and the distribution of myosin-II becomes markedly altered at the apices of the follicle cells. These data indicate that during oogenesis p127 plays a critical function at the onset of vitellogenesis and regulates growth of the oocyte, follicle cell migration over the oocyte and their organization in a palisadic epithelium, as well as viability of the germline cells.

HIV-1 expression is posttranscriptionally repressed in Drosophila cells.

The long terminal repeats (LTRs) of the human immunodeficiency virus (HIV) the Rous sarcoma virus (RSV) and the copia Drosophila retrotransposon were compared in their capacity to direct expression of the bacterial cat (chloramphenicol acetyltransferase) gene in human, murine, and Drosophila cell lines. The results indicate that HIV and RSV LTR expression is post transcriptionally repressed in the Drosophila cells while copia LTR expression is post-transcriptionally repressed in the human and murine cells.