Structure and distribution of N-glycans on the S-7-allele stylar self-incompatibility ribonuclease of Nicotiana alata

S-RNases are the stylar products of the self-incompatibility (S)-locus in solanaceous plants (including Nicotiana alata), and as such, are involved in the prevention of self-pollination. All cDNA sequences of S-RNase products of functional S-alleles contain potential N-glycosylation sites, with one site being conserved in all cases, suggesting that N-glycosylation is important in self-incompatibility. In this study, we report on the structure and localization of the N-glycans on the S-7-allele RNase of N, alata, A total of nine N-glycans, belonging to the high-mannose- and xylosylated hybrid-classes, were identified and characterized by a combination of electrospray-ionization mass-spectrometry (ESI-MS), H-1-NMR spectroscopy, and methylation analyses. The glycosylation pattern of individual glycosylation sites was determined by ESI-MS of the glycans released from isolated chymotryptic glycopeptides, All three N-glycosylation sites showed microheterogeneity and each had a unique complement of N-glycans, The N-glycosylation pattern of the S-7-RNase is significantly different to those of the S-1- and S-2-RNases.

A comparison of lipopolysaccharide-induced hepatitis in ethanol-fed Wistar and Lewis rats

Elevated concentrations of plasma proinflammatory cytokines have been detected in patients with alcoholic hepatitis (AH) and in a model of lipopolysaccharide-induced hepatitis in ethanol-fed Wistar rats. These cytokines have been implicated in the pathogenesis of the liver damage. Considering the likely involvement of the immune system in AH, and the frequent use of Lewis rats in autoimmune disease models, Lewis rats were examined in the model to determine whether they would more closely mimic the immune status of a chronic alcoholic and be a preferable strain for use in future experiments. Lipopolysaccharide-induced hepatic tumor necrosis factor-cu, interleukin-1 alpha, interleukin-1 beta, and interleukin-6 mRNA expression was examined in both rat strains. The overall pattern of histological (panlobular piecemeal necrosis) and biochemical liver damage (plasma ALT levels), and cytokine expression was similar in both strains. Thus, it would appear that, despite the known susceptibility of Lewis rats to autoimmune phenomena, they do not respond to the experimental regime significantly better than Wistar rats. This study confirms that unknown mediators are contributing to the liver damage seen in this model and possibly in AH.

Dysregulated hematopoiesis and a progressive neurological disorder induced by expression of an activated form of the human common beta chain in transgenic mice

Previously we described activating mutations of h beta(c), the common signaling subunit of the receptors for the hematopoietic and inflammatory cytokines, GM-CSF, IL-3, and IL-5. The activated mutant, h beta(c)FI Delta, is able to confer growth factor-independent proliferation on the murine myeloid cell line FDC-P1, and on primary committed myeloid progenitors. We have used this activating mutation to study the effects of chronic cytokine receptor stimulation. Transgenic mice were produced carrying the h beta(c)FI Delta cDNA linked to the constitutive promoter derived from the phosphoglycerate kinase gene, PGK-1. Transgene expression was demonstrated in several tissues and functional activity of the mutant receptor was confirmed in hematopoietic tissues by the presence of granulocyte macrophage and macrophage colony-forming cells (CFU-GM and CFU-M) in the absence of added cytokines. All transgenic mice display a myeloproliferative disorder characterized by splenomegaly, erythrocytosis, and granulocytic and megakaryocytic hyperplasia. This disorder resembles the human disease polycythemia vera, suggesting that activating mutations in h beta(c) may play a role in the pathogenesis of this myeloproliferative disorder. In addition, these transgenic mice develop a sporadic, progressive neurological disease and display bilateral, symmetrical foci of necrosis in the white matter of brain stem associated with an accumulation of macrophages. Thus, chronic h beta(c) activation has the potential to contribute to pathological events in the central nervous system.

Fibroblast growth factor receptor expression in outer hair cells of rat cochlea

FIBROBLAST growth factors (FGFs) are critical for normal development of the organ of Corti, and may also protect hair cells from ototoxic damage. Four different fibroblast growth factors are known, three of which have different splice variants in the extracellular immunoglobin-like (Ig) III FGF-binding domain, giving different patterns of sensitivity to the different FGFs. Analysis of a cDNA library of rat outer hair cells by the polymerase chain reaction, using isoform specific primers, showed expression only of FGF receptor 3, splice variant IIIc. This allows us to predict the pattern of sensitivity to applied FGFs, may be useful in targeting outer hair cells selectively during an FGF-based strategy for cochlear therapy. (C) 1998 Lippincott Williams & Wilkins.

The common tetratricopeptide repeat acceptor site for steroid receptor-associated immunophilins and Hop is located in the dimerization domain of hsp90

Structurally related tetratricopeptide repeat motifs in steroid receptor-associated immunophilins and the STI1 homolog, Hop, mediate the interaction with a common cellular target, hsp90, We have identified the binding domain in hsp90 for cyclophilin 40 (CyP40) using a two-hybrid system screen of a mouse cDNA library. All isolated clones encoded the intact carboxyl terminus of hsp90 and overlapped with a common region corresponding to amino acids 558-724 of murine hsp84, The interaction was confirmed in vitro with bacterially expressed CyP40 and deletion mutants of hsp90 beta and was delineated further to a 124-residue COOH-terminal segment of hsp90, Deletion of the conserved MEEVD sequence at the extreme carboxyl terminus of hsp90 precludes interaction with CyP40, signifying an important role for this motif in hsp90 function. We show that CyP40 and Hop display similar interaction profiles with hsp90 truncation mutants and present evidence for the direct competition of Hop and FK506-binding protein 52 with CyP40 for binding to the hsp90 COOH-terminal region. Our results are consistent with a common tetratricopeptide repeat interaction site for Hop and steroid receptor associated immunophilins within a discrete COOH-terminal domain of hsp90. This region of hsp90 mediates ATP-independent chaperone activity, overlaps the hsp90 dimerization domain, and includes structural elements important for steroid receptor interaction.

Increased expression of mitochondrial genes in human alcoholic brain revealed by differential display

Polymerase chain reaction (PCR)-based differential display was used to screen for alterations in gene expression in the mesolimbic system of the human alcoholic brain. Total RNA was extracted from the nucleus accumbens of five alcoholic and five control brains. A selected subpopulation of mRNA was reverse-transcribed to cDNA and amplified by PCR. A differentially expressed cDNA fragment was recovered, cloned, and sequenced. Full sequence analysis of this 467 bp fragment revealed 98.2% homology with the human mitochondrial 12S rRNA gene. Dot-blot analysis showed increased expression of this gem in nucleus accumbens and hippocampus, but not in the superior frontal cortex, primary motor cortex, caudate, and pallidus/putamen In a total of eight human alcoholic brains, compared with seven control brains. A similar increased expression was observed by dot-blot analysis, using RNA from the cerebral cortex of rats chronically treated with alcohol vapor. Hybridization of a 16S rRNA oligonucleotide probe indicated that the expression of both rRNAs genes was significantly increased in nucleus accumbens. These results indicate that chronic alcohol consumption induces alteration in expression of mitochondrial genes in selected brain regions. The altered gene expression may reflect mitochondrial dysfunction In the alcohol-affected brain.

Sustained gene expression in transplanted skin fibroblasts in rats

Retrovirus-mediated gene transfer into adult skin fibroblasts has provided measurable amounts of therapeutic proteins in animal models. However, the major problem emerging from these experiments was a limited time of vector encoded gene expression once transduced cells were engrafted We hypothesized that sustained transduced gene expression in quiescent fibroblasts in vivo might be obtained by using a fibronectin (Fn) promoter. Fibronectin plays a key role in cell adhesion, migration and wound healing and is up-regulated in quiescent fibroblasts. Retroviral vectors containing human adenosine deaminase (ADA) cDNA linked to rat fibronectin promoter (LNFnA) or viral LTR promoter (LASN) were compared for their ability to express ADA from transduced primary rat skin fibroblasts in vivo. Skin grafts formed from fibroblasts transduced with LNFnA showed strong human ADA enzyme activity from 1 week to 3 months. In contrast, skin grafts containing LASN-transduced fibroblasts tested positive for human ADA for weeks 1 and 2, were faintly positive at week 3 and showed no human ADA expression at 1, 2 and 3 months. Thus, a fibronectin promoter provided sustained transduced gene expression at high levels for at least 3 months in transplanted rat skin fibroblasts, perhaps permitting the targeting of this tissue for human gene therapy.

Identification and characterisation of a cytochrome P450 gene and processed pseudogene from an arachnid: the cattle tick, Boophilus microplus

We isolated and sequenced the first known cytochrome P450 gene and pseudogene from an arachnid, the cattle tick, Boophilus microplus. Bath the gene and pseudogene belong to the family CYP4, but a new subfamily, CYP4W, had to be created for these genes because they are substantially different to other CYP4 genes. The gene, CPP4W1, has greatest homology with CYP4C1 from a cockroach, Blaberus discoidalis. The predicted molecular weight of the protein encoded by CYP4W1 (63 KDa) is greater than that of the other CYP4 genes. The pseudogene, CYP4W1P, is probably a processed pseudogene derived from the functional gene CYP4W1. This is only the third CYP processed pseudogene to be identified. The pseudogene is 98% identical to the functional gene, CYP4W1, therefore we hypothesise that this pseudogene evolved recently from the functional gene. The CYP4 genes from arthropods have diverged from each other more than those of mammals; consequently the phylogeny of the arthropod genes could not be resolved. (C) 1999 Elsevier Science Ltd. All rights reserved.

Molecular characterization of Drosophila C virus isolates

Reverse transcription coupled with polymerase chain reaction and restriction enzyme analysis was used to characterize 12 Drosophila C virus isolates from geographically different regions. A 1.2-kb fragment was amplified from cDNA and profiles from digestion with 20 restriction enzymes were generated. Analysis of the restriction fragment data gave estimates of nucleotide divergence of 0-10% between isolates. The isolates were grouped on the basis of genetic distance estimates derived from the restriction data. For the isolates from which a single genotype could be purified, a geographical pattern in the distribution of viral genotypes was identified. The 4 Moroccan isolates were very closely related to each other, differing in only 1 restriction profile. The 2 Australian isolates were each other's closest relatives, as were the 2 isolates first recovered in France. The PCR-RFLP technique used in this study has provided us with a simple procedure which can be used to characterize DCV isolates. A single enzyme, Tag I, generated 5 distinct and diagnostic restriction fragment patterns, which allowed easy assignment of isolates to one of the five viral genotypes identified in this study. (C) 1999 Academic Press.

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.

Comparison of acetylcholinesterase genes from cattle ticks

We describe the isolation and characterisation of two putatively new acetylcholinesterase genes from the African cattle ticks Boophilus decoloratus and Rhipicephalus appendiculatus. The nucleotide sequences of these genes had 93% homology to each other and 95% and 91% identity, respectively, to the acetylcholinesterase gene from an Australian strain of another cattle tick, Boophilus microplus. Translation of the nucleotide sequences revealed putative amino acids that are essential for acetylcholinesterase activity: the active site serine, and the histidine and glutamate residues that associate with this serine to form the catalytic triad. All known acetylcholinesterases have three sets of cysteines that form disulfide bonds; however, the acetylcholinesterase genes of these three species of ticks encode only two sets of cysteines. Acetylcholinesterases of B. microplus from South Africa, Zimbabwe, Kenya and Mexico had 98-99% identity with acetylcholinesterase from B. microplus from Australia, whereas acetylcholinesterase from B. microplus from Indonesia was identical to that from Australia. Preliminary phylogenetic analyses surprisingly indicate that the acetylcholinesterases of ticks are closer phylogenetically to acetylcholinesterases of vertebrates than they are to those of other arthropods. (C) 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Acute susceptibility of aged mice to infection with Candida albicans

The effect of aging on host resistance to systemic candidosis was assessed by monitoring the course of infection in 16-month-old CBA/CaH mice (aged non-immune) and in a comparable group that had been infected with a sublethal dose of Candida albicans at 6 weeks of age (aged immune). Aged non-immune mice showed rapid progression of the disease, with a marked increase in the number of mycelia in the brain and kidney, and early morbidity, Foci of myocardial necrosis were evident, but inflammatory cells were sparse. The histological picture in the aged immune mice was similar to that in the aged non-immune group, although fewer mycelial aggregates were seen. Both groups of aged mice showed a significantly lower fungal burden in the brain on day 1 of infection, but on day 4, colony counts increased significantly in the aged non-immune mice, Comparison of cytokine gene expression in the infected brains showed that the relative amount of interferon-gamma and tumour necrosis factor-alpha cDNA were similar in all three groups. Interleukin-6 was elevated in both infected non-immune and uninfected aged mice. Aged immune mice showed no morbidity after challenge, and both colonisation and tissue damage were reduced in comparison with the aged non-immune animals.

Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein (P160) 1A (MYBBP1A) to 17p13.3

We have previously isolated and characterized murine MYB binding protein (p160) 1a, a protein that specifically interacts with the leucine zipper motif within the negative regulatory domain of the c-Myb proto-oncoprotein, We now describe the molecular cloning of the human MYBBP1A cDNA and chromosomal localization to 17p13.3 by fluorescence in situ hybridization analysis, Given the likely presence of a tumor suppressor gene (or genes) within this region of chromosome 17, the position of MYBBP1A was further mapped by radiation hybrid analysis and was found to lie between markers D17S1828 and D17S938. A P1 artificial chromosome clone containing the 5' region of MYBBP1A was isolated and indicates a physical linkage between MYBBP1A and the 15-lipoxygenase gene (ALOX15), A novel, polymorphic (CA)(25) dinucleotide repeat was also isolated from this PAC and may serve as a useful marker for MYBBP1A and this region of chromosome 17. (C) 1999 Academic Press.

Differential localization and regulation of death and decoy receptors for TNF-related apoptosis-inducing ligand (TRAIL) in human melanoma cells

Induction of apoptosis in cells by TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, is believed to be regulated by expression of two death-inducing and two inhibitory (decoy) receptors on the cell surface. In previous studies we found no correlation between expression of decoy receptors and susceptibility of human melanoma cells to TRAIL-induced apoptosis, In view of this, we studied the localization of the receptors in melanoma cells by confocal microscopy to better understand their function. We show that the death receptors TRAIL-R1 and R2 are located in the trans-Golgi network, whereas the inhibitory receptors TRAIL-R3 and -R4 are located in the nucleus. After exposure to TRAIL, TRAIL-R1 and -R2 are internalized into endosomes, whereas TRAIL-R3 and -R4 undergo relocation from the nucleus to the cytoplasm and cell membranes. This movement of decoy receptors was dependent on signals from TRAIL-R1 and -R2, as shown by blocking experiments with Abs to TRAIL-R1 and -R2, The location of TRAIL-R1, -R3, and -R4 in melanoma cells transfected with cDNA for these receptors was similar to that in nontransfected cells, Transfection of TRAIL-R3 and -R4 increased resistance of the melanoma lines to TRAIL-induced apoptosis even in melanoma lines that naturally expressed these receptors. These results indicate that abnormalities in decoy receptor location or function may contribute to sensitivity of melanoma to TRAIL-induced apoptosis and suggest that further studies are needed on the functional significance of their nuclear location and TRAIL-induced movement within cell.

Identification and validation of candidate Myb target genes

While a considerable number of candidate Myb target genes have been reported to date, most of these are likely to play little or no role in transformation by myb oncogenes. Here we have used a conditionally myb-transformed myeloid cell line (ERMYB) to further examine Myb regulation of one candidate target gene-c-myc-that has the potential to affect cell proliferation. It was found that the major influence on c-myc expression was the presence of cytokine (GM-CSF) rather than Myb activity. We also describe the application of PCR-based subtractive hybridization and low-density cDNA array screening, in conjunction with the ERMYB line, to the identification of additional Myb target genes. Preliminary identification of a number of candidates is reported; these include myeloperoxidase, which is known to have essential Myb-binding sites in its regulatory region. (C) 2001 Academic Press.