Phenotypic analysis of genes whose mRNA accumulation is dependent on calcineurin in Aspergillus fumigatus

Calcineurin plays an important role in the control of cell morphology and virulence in fungi. Calcineurin is a serine/threonine-specific protein phosphatase heterodimer consisting of a catalytic subunit A and a regulatory subunit B. A mutant of Aspergillus fumigatus lacking the calcineurin A (calA) catalytic subunit exhibited defective hyphal morphology related to apical extension and branching growth, which resulted in drastically decreased filamentation. Here, we investigated which pathways are influenced by A. fumigatus calcineurin during proliferation by comparatively determining the transcriptional profile of A. fumigatus wild type and Delta calA mutant strains. Our results showed that the mitochondrial copy number is reduced in the Delta calA mutant strain, and the mutant has increased alternative oxidase (aoxA) mRNA accumulation and activity. Furthermore, we identified four genes that encode transcription factors that have increased mRNA expression in the Delta calA mutant. Deletion mutants for these transcription factors had reduced susceptibility to itraconazole, caspofungin, and sodium dodecyl sulfate (SDS). (C) 2009 Elsevier Inc. All rights reserved.

Differential expression of immunomodulatory galectin-1 in peripheral leukocytes and adult tissues and its cytosolic organization in striated muscle

Galectin-1 (Gal-1) is important in immune function and muscle regeneration, but its expression and localization in adult tissues and primary leukocytes remain unclear. To address this, we generated a specific monoclonal antibody against Gal-1, termed alpha hGal-1, and defined a sequential peptide epitope that it recognizes, which is preserved in human and porcine Gal-1, but not in murine Gal-1. Using alpha hGal-1, we found that Gal-1 is expressed in a wide range of porcine tissues, including striated muscle, liver, lung, brain, kidney, spleen, and intestine. In most types of cells, Gal-1 exhibits diffuse cytosolic expression, but in cells within the splenic red pulp, Gal-1 showed both cytosolic and nuclear localization. Gal-1 was also expressed in arterial walls and exhibited prominent cytosolic and nuclear staining in cultured human endothelial cells. However, human peripheral leukocytes and promyelocytic HL60 cells lack detectable Gal-1 and also showed very low levels of Gal-1 mRNA. In striking contrast, Gal-1 exhibited an organized cytosolic staining pattern within striated muscle tissue of cardiac and skeletal muscle and colocalized with sarcomeric actin on I bands. These results provide insights into previously defined roles for Gal-1 in inflammation, immune regulation and muscle biology.

The expression of messenger RNAs coding for growth factors, their receptors, and eph-class receptor tyrosine kinases in normal and ototoxically damaged chick cochleae

Messenger RNAs coding for growth factors and receptor tyrosine kinases were measured by quantitative competitive and by semi-quantitative reverse-transcription polymerase chain reaction in whole and dissected chick inner ears. The fibroblast growth factor (FGF) receptor 1 chick embryonic kinase (CEK) 1 was expressed in all structures examined (otocyst, hatchling whole cochlea, cochlear nerve ganglion, and cochlear and vestibular sensory epithelia), although slightly more heavily in the otocyst. The related fibroblast growth factor receptors CEK 2 and 3 were preferentially expressed in the nerve ganglion and in the vestibular sensory epithelium, respectively. FGF 1 mRNA was low in early development, increasing to mature levels at around embryonic age 11 days, while FGF2, mRNA was expressed at constant levels at all ages. In response to ototoxic damage, FGF1 mRNA levels were increased in the early damaged cochlear sensory epithelium. Immunohistochemistry for CEK1 showed that normal hair cells expressed the receptor heavily on the hair cell stereocilia, while with early damage, CEK1 came to be expressed heavily on the apical surfaces of the supporting cells. In normal chicks, the CEK4 and CEK8 eph-class receptor tyrosine kinases were expressed relatively heavily by the cochlear nerve ganglion, and CEK10 was expressed relatively heavily by the cochlear hair cell sensory epithelium. The results suggest that the FGF system may be involved in the response of the cochlear epithelium to ototoxic damage. The eph-class receptor tyrosine kinase CEK10 may be involved in cell interactions in the cochlear sensory epithelium, while CEK4 and CEK8 may play a role in the cochlear innervation.

Increased hepatic iron concentration in nonalcoholic steatohepatitis is associated with increased fibrosis

Background & Aims: Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that occasionally progresses to cirrhosis but usually has a benign course. The aim of this study was to investigate the role of the hemochromatosis mutation Cys282Tyr in development of the mild hepatic iron overload found in some patients with NASH and its association with hepatic damage in these patients. Methods: Fifty-one patients with NASH were studied. The presence of the Cys282Tyr mutation was tested in all patients, and the data were analyzed with respect to the histological grade of steatosis, inflammation, Perls' staining, hepatic iron concentration (HIC), and serum iron indices. Results: Thirty-one percent of patients with NASH were either homozygous or heterozygous for the Cys282Tyr mutation. This mutation was significantly associated with Perls' stain grade (P < 0.005), HIC (P < 0.005), and transferrin saturation percentage (P < 0.005) but not with serum ferritin levels. Linear regression analysis showed that increased hepatic iron (Perls' stain or HIC) had the greatest association with the severity of fibrosis (P < 0.0001). Conclusions: The Cys282Tyr mutation is responsible for most of the mild iron overload found in NASH and thus has a significant association with hepatic damage in these patients. Heterozygosity for the hemochromatosis gene mutation therefore cannot always be considered benign.

Localisation of aryl sulfotransferase expression in human tissues using hybridisation histochemistry and immunohistochemistry

To date, the laboratory has cloned seven unique human sulfotransferases; five aryl sulfotransferases (HAST1, HAST2, HAST3, HAST4 and HAST4v), an estrogen sulfotransferase and a dehydroepiandrosterone sulfotransferase. The cellular distribution of human aryl sulfotransferases in human hepatic and extrahepatic tissues has been determined using the techniques of hybridization histochemistry and immunohistochemistry. Human aryl sulfotransferase expression was detected in liver, epithelial cells of the gastrointestinal mucosal layer, epithelial cells lining bronchioles and in mammary duct epithelial cells. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

hnRNP A2 selectively binds the cytoplasmic transport sequence of myelin basic protein mRNA

Segregation of mRNAs in the cytoplasm of polar cells has been demonstrated for proteins involved in Xenopus and Drosophila oogenesis, and for some proteins in somatic cells. It is assumed that vectorial transport of the messages is generally responsible for this localization. The mRNA encoding the basic protein of central nervous system myelin is selectively transported to the distal ends of the processes of oligodendrocytes, where it is anchored to the myelin membrane and translated. This transport is dependent on a 21-nucleotide cis-acting segment of the 3'-untranslated region (RTS). Proteins that bind to this cis-acting segment have now been isolated from extracts of rat brain. A group of six 35-42-kDa proteins bind to a 35-base oligoribonucleotide incorporating the RTS, but not to several oligoribonucleotides with the same composition but randomized sequences, thus establishing specificity for the base sequence in the RTS. The most abundant of these proteins has been identified, by Edman sequencing of tryptic peptides and mass spectroscopy, as heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a 36-kDa member of a family of proteins that are primarily, but not solely, intranuclear. This protein was most abundant in samples from rat brain and testis, with lower amounts in other tissues. It was separated from the other polypeptides by using reverse-phase HPLC and shown to retain preferential association with the RTS. In cultured oligodendrocytes, hnRNP A2 was demonstrated by confocal microscopy to be distributed throughout the nucleus, cell soma, and processes.

Acute intermittent porphyria: alternative splicing of hydroxymethylbilane synthase mRNA excludes exons 3 and 12

The hydroxymethylbilane synthase (HMBS) mRNAs from 44 control individuals and 30 patients suffering from acute intermittent porphyria (AIP), were screened for length differences by reverse transcriptase polymerase chain reaction (RT-PCR) and any abnormalities were characterized by direct sequencing. Examination of the mRNAs extracted from the peripheral blood lymphocytes of the samples revealed varying degrees of alternative splicing, involving the removal of exons 3 and 12. Approximately 10-50% of the mRNA molecules were affected, despite the absence of genomic splice site mutations or any major deviance from consensus splice sequence values. The preliminary data obtained from this study suggest that this event is a normal occurrence in peripheral blood lymphocytes, and may not be associated with the molecular pathology responsible for AIP. (C) 1998 Academic Press Limited.

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.

mRNA differential display of 2-amino-1-methyl-6-phenylinlidazo[4,5-b]pyridine-induced rat mammary gland tumors

The mRNA differential display technique was used to compare mRNAs between normal mammary gland and turner-derived epithelial cells from female Sprague-Dawley rat mammary gland tumors induced by the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by a high-fat diet (23.5% corn oil). Two genes, beta-casein and transferrin, were identified as differentially expressed. The expression of these genes was examined across a bank of rat mammary gland tumors derived from animals on a low-fat diet (5% corn oil) or the high-fat diet. Carcinomas had over a 10- and 50-fold lower expression of beta-casein and transferrin, respectively than normal mammary gland. In addition, carcinomas from animals on the high-fat diet showed on average a 5-fold higher expression of beta-casein, and transferrin than carcinomas from animals on the low-fat diet. The results indicate the process of mammary gland tumorigenesis alters the expression of certain genes in the mammary gland, and that the level of dietary fat further modulates the expression of these genes.

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.

Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma

Past studies have shown that apoptosis mediated by TNF-related apoptosis-inducing ligand (TRAIL) is regulated by the expression of two death receptors [TRAIL receptor 1 (TRAIL-RI) and TRAIL-R2] and two decoy receptors (TRAIL-R3 and TRAIL-R4) that inhibit apoptosis, In previous studies, me have shown that TRAIL but not other members of the tumor necrosis factor family induce apoptosis in approximately two-thirds of melanoma cell lines. Here, we examined whether the expression of TRAIL-R at the mRNA and protein level in a panel of 28 melanoma cell lines and melanocytes correlated with their sensitivity to TRAIL-induced apoptosis, We report that at least three factors appear to underlie the variability in TRAIL-induced apoptosis. (a) Pour of nine cell lines that were insensitive to TRAIL-induced apoptosis failed to express death receptors, and in two instances, lines were devoid of all TRAIL-Rs. Southern analysis suggested this was due to loss of the genes for the death receptors, (b) Despite the presence of mRNA for the TRAIL-R, some of the lines failed to express TRAIL-R protein on their surface. This was evident for TRAIL-RI and more so for the TRAIL decoy receptors TRAIL-R3 and -R4, Studies on permeabilized cells revealed that the receptors were located within the cytoplasm and redistribution from the cytoplasm may represent a posttranslational control mechanism. (c) Surface expression of TRAIL-RI and -R2 (but not TRAIL-R3 and -R4) showed an overall correlation with TRAIL-induced apoptosis. However, certain melanoma cell lines and clones were relatively resistant to TRAIL-induced apoptosis despite the absence of decoy receptors and moderate levels of TRAIL-RI and -R2 expression. This may indicate the presence of inhibitors within the cells, but resistance to apoptosis could not be correlated with expression of the caspase inhibitor FLICE-inhibitory protein. mRNA for another TRAIL receptor, osteoprotegerin, was expressed in 22 of the melanoma lines but not on melanocytes. Its role in induction of apoptosis remains to be studied. These results appear to have important implications for future clinical studies on TRAIL.

Expression of the estrogen receptor-associated immunophilins, cyclophilin 40 and FKBP52, in breast cancer

The estrogen receptor alpha (ER alpha) is implicated in the development of breast cancer. The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with ER alpha and other steroid receptors in mutually exclusive heterocomplexes and may differentially modulate receptor activity. Since previous studies have not assessed the levels of these immunophilins in breast cancer, we examined 10 breast cancer cell lines for mRNA and protein expression of CyP40 and FKBP52 and for amplification of the CyP40 gene. In addition, 26 breast carcinomas, including seven with matched normal breast tissue, were examined for mRNA expression of both immunophilins. CyP40 and FKBP52 were ubiquitously expressed in breast cancer cell lines, but there were significant differences in their pattern of expression. FKBP52 protein levels were generally an order of magnitude greater than those for CyP40. FKBP52 mRNA expression correlated strongly with protein expression and was significantly higher in ER alpha-positive compared with ER alpha-negative cell lines. However, CyP40 mRNA expression did not correlate with protein expression, nor did expression of this immunophilin correlate with ER alpha status. Relatively high expression of CyP40 in one cell line (BT-20) could be attributed to amplification of the CyP40 gene. Both immunophilins were also ubiquitously expressed in breast carcinomas, and we demonstrate for the first time that both CyP40 and FKBP52 mRNA are overexpressed in breast tumors compared to matched normal breast controls. The overexpression of CyP40 and FKBP52, coupled with relative differences in their expression in tumors, may have important functional implications for ER alpha and other steroid receptors in breast cancer.

Polyinosinic acid and polycationic liposomes attenuate the hepatic clearance of circulating plasmid DNA

DNA that enters the circulation is rapidly cleared both by tissue uptake and by DNase-mediated degradation. In this study, we have examined the uptake of linear plasmid DNA in an isolated perfused liver model and following intra-arterial administration to rats. We found that the DNA was rapidly taken up by the isolated perfused liver without degradation. The single-pass extraction ratio was 0.76 +/- 0.05, the mean transit time was 15.3 +/- 3.6 s, and the volume of distribution was 0.29 +/- 0.07 ml/g. Hepatic uptake was saturable and was inhibited by polyinosinic acid or polycationic liposomes but not by condensation of the DNA with polylysine. When the linear plasmid DNA was administered in vivo, plasma half-life was 3.1 +/- 0.2 min, volume of distribution was 670 +/- 85 ml/kg, and clearance was 32 +/- 4 min. Coadministration of cationic liposomes decreased the volume of distribution to 180 +/- 28 ml/kg as well as the half-life (2.6 +/- 0.2 min). By contrast, polyinosinic acid significantly increased the circulating half-life (7.7 +/- 0.5 min), decreased the volume of distribution (95 +/- 17 ml/kg), and partially inhibited DNA degradation. When administered along with the liposomes and the polyinosinic acid, the distribution of plasmid-derived radioactivity decreased in the liver and increased in most other peripheral tissues. This study shows that pharmacological manipulation of the uptake and degradation of DNA can alter its distribution and clearance in vivo. These results may be useful in optimizing gene delivery procedures for in vivo gene therapy.

Estradiol-regulated expression of the immunophilins cyclophilin 40 and FKBP52 in MCF-7 breast cancer cells

The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with the unactivated estrogen receptor in mutually exclusive heterocomplexes and may differentially modulate receptor activity, We have recently shown that CyP40 and FKBP52 mRNA's are differentially elevated in breast carcinomas compared with normal breast tissue. Other studies suggest that such alterations ill the ratio of immunophilins might potentially influence steroid receptor function. Studies were therefore initiated to investigate the influence of estradiol on CyP40 and FKBP52 expression in MCF-7 breast cancer cells. Over a 24-h-treatment period with estradiol, CyP40 and FKBP52 mRNA expression was increased approximately five- and 14-fold, respectively. The corresponding protein levels were also elevated in comparison to controls. The antiestrogen, ICI 182,780, was an antagonist for CyP40 and FKBP52 mRNA induction. Cycloheximide treatment did not inhibit this increased immunophilin expression, suggesting that estradiol-mediated activation is independent off de novo protein synthesis. Treatment of MCF-7 cells with estradiol resulted in an increased half-life of both CyP40 and FKBP52 mRNA, as determined by actinomycin D studies. These results suggest that estradiol regulates CyP40 and FKBP52 mRNA expression through both transcriptional and posttranscriptional mechanisms. (C) 2001 Academic Press.

Expression of RANTES and CCR1 in oral lichen planus and association with mast cell migration

Background: T lymphocytes and mast cells infiltrate the lamina propria in oral lichen planus (OLP). Chemokines and their receptors are involved in T cell and mast cell migration and accumulation during the inflammatory process. Methods: In the present study, we investigated the role of RANTES and its receptors in OLP using immunohistochemistry, RT-PCR and an in vitro chemotaxis assay. Results: RANTES and CCR1 were expressed on T cells and mast cells in OLP, while OLP lesional T cell supernatants stimulated CCR1 mRNA expression in a human leukemia mast cell line (HMC-1). TNF-alpha stimulated CCR1, CCR4 and CCR5 mRNA expression in the same cell line. OLP lesional T cell supernatants stimulated HMC-1 migration, which was partly inhibited by anti-RANTES antibody. Conclusions: The present study shows, for the first time, the distribution of RANTES and CCR1 in OLR It is hypothesized that RANTES and CCR1 may play important roles in mast cell trafficking and related events in OLP.