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At least 973 records · Page 54Linked to original sources
PubMed2000

The p44S10 locus, encoding a subunit of the proteasome regulatory particle, is amplified during progression of cutaneous malignant melanoma.

Gene amplification is frequently present in human tumors, although specific target genes relevant to many amplified loci remain unidentified. An expression cloning assay enabled identification of a candidate oncogene derived from human chromosome 3p14.1. The cDNA retrieved from morphologically transformed cells contained the full-length protein coding region and detected an abundant transcript in the same cells. Sequence analysis revealed identity with the wild-type sequence of p44S10, a highly conserved subunit of the 26S proteasome that exhibits similarity to the Arabidopsis fus6/cop11 family of signaling molecules. p44S10 gene copy number and mRNA expression were increased in association with segmental 1.8 - 11-fold chromosomal gains in cutaneous malignant melanoma cell lines (5/13; 40%) and tumors (2/40; 5%), and in breast cancer MCF-7 cells. Likewise, malignant progression of human radial growth phase WM35 melanoma cells was associated with amplification and increased expression of endogenous p44S10, and increased expression of p44S10 was sufficient to induce proliferation of WM35 cells in vivo. The results demonstrate segmental copy number gains within chromosome 3p in cutaneous malignant melanoma and suggest that deregulation of a proteasome regulatory particle subunit may contribute to the malignant phenotype.

Adenosine Triphosphatases↗
PubMed2004

Interleukin 24 is induced by the RET/PTC3 oncoprotein and is an autocrine growth factor for epithelial cells.

Thyroid cancers, like hematological malignancies, are commonly associated with chromosomal translocations leading to the formation of fusion proteins. Through altered signaling by fusion proteins, cell death and survival pathways are disrupted and the physiological balance of cell-cell communication may be lost. A consequence of this disruption is the release of factors by stressed cells that alert the host. One type of host response is leukocytic infiltration that may develop into chronic inflammation or autoimmune disease. Although inflammation can be associated with neoplastic tissue, the mechanism driving this process is largely unknown. Therefore, to address the mechanism of cancer inflammation we investigated the effects of an oncogene in a murine model system. A comprehensive genetic analysis revealed several soluble factors that were induced by RET/papillary thyroid carcinoma (PTC)3 gene expression including several proinflammatory cytokines, chemokines and immunologically relevant costimulatory molecules. Following a large genetic screen using RP3-expressing thyroid cells, we identified a highly abundant transcript and later identified it as interleukin 24 (Il24), a cytokine with diverse tumor suppressor and inflammatory activities. We show that RET/PTC3 induces Il24 expression in rat thyrocytes and that this expression is dependent on the signaling properties of its tyrosine kinase. Likewise, RET/PTC3 induces large amounts of Il24 following expression in murine thyrocytes, but its expression is dramatically reduced in poorly differentiated carcinomas, a finding that parallels the loss of RET/PTC3 expression. Consistent with its behavior as a tumor suppressor, the loss of Il24 coincided with the loss of RET/PTC3 in poorly differentiated mouse tumors. A functional role of Il24 in the autocrine growth/survival of RET/PTC3-expressing thyroid cells was identified helping to support its role in cellular transformation. These data suggest that the induction of Il24 by oncogenes may support tumor growth at the early stages of cancer.

Animals↗
PubMed1991

Molecular cloning of a human cannabinoid receptor which is also expressed in testis.

A cDNA clone encoding a receptor protein which presents all the characteristics of a guanine-nucleotide-binding protein (G-protein)-coupled receptor was isolated from a human brain stem cDNA library. The probe used (HGMP08) was a 600 bp DNA fragment amplified by a low-stringency PCR, using human genomic DNA as template and degenerate oligonucleotide primers corresponding to conserved sequences amongst the known G-protein-coupled receptors. The deduced amino acid sequence encodes a protein of 472 residues which shares 97.3% identity with the rat cannabinoid receptor cloned recently [Matsuda, Lolait, Brownstein, Young & Bronner (1990) Nature (London) 346, 561-564]. Abundant transcripts were detected in the brain, as expected, but lower amounts were also found in the testis. The same probe was used to screen a human testis cDNA library. The cDNA clones obtained were partially sequenced, demonstrating the identity of the cannabinoid receptors expressed in both tissues. Specific binding of the synthetic cannabinoid ligand [3H]CP55940 was observed on membranes from Cos-7 cells transfected with the recombinant receptor clone. In stably transfected CHO-K1 cell lines, cannabinoid agonists mediated a dose-dependent and stereoselective inhibition of forskolin-induced cyclic AMP accumulation. The ability to express the human cannabinoid receptor in mammalian cells should help in developing more selective drugs, and should facilitate the search for the endogenous cannabinoid ligand(s).

Amino Acid Sequence↗
PubMed1994

Activation of Ito cells involves regulation of AP-1 binding proteins and induction of type I collagen gene expression.

Activation of liver Ito cells is characterized by increased proliferation, fibrogenesis, loss of cellular retinoid and change of cell-shape. Here, we have described fundamental differences between freshly isolated Ito cells (FIC) and long-term cultured Ito cells (LTIC). This process of activation correlates with the absence of expression of Pro alpha 1(I) gene in FIC. LTIC expressed abundant transcripts of Pro alpha 1(I) gene. Nuclear run-off experiments showed the inability of FIC to support Pro alpha 1(I) RNA transcription while LTIC transcribed it greater than 5-fold as compared with FIC. Transforming growth factor beta (TGF beta)-treated LTIC had a preferential increase in the rate of Pro alpha 1(I) gene transcription as compared with control LTIC. A human collagen type I promoter-enhancer construct (pCOL-KT) [Thompson, Simkevich, Holness, Kang and Raghow (1991) J. Biol. Chem. 266, 2549-2556] was readily expressed in LTIC but failed to be expressed in FIC. Furthermore, TGF beta treatment of LTIC resulted in an increased expression of pCOL-KT. The deletion of an activator protein-1 (AP-1) binding site (+598 to +604) in the 360 bp enhancer region of pCOL-KT (S360) caused decreased expression of the CAT reporter gene, suggesting that this bonafide AP-1 site can, at least in part, mediate the transactivation effect of TGF beta. Using DNAase I protection, we demonstrate a single foot-print located at +590 to +625 in the S360 fragment; nuclear extracts prepared from TGF beta-treated LTIC exhibited greater activity of these AP-1 binding proteins. Gel mobility assays corroborated and extended the footprinting observation. No AP-1-binding activity was found in the nuclear extracts of FIC. Double-stranded oligonucleotides containing the consensus AP-1 motif were able to compete out the binding; consensus NF-1 motif oligonucleotides failed to do so. The preincubation of nuclear extracts from control and TGF beta-treated LTIC with antibodies against c-jun and c-fos rendered a reduced binding of AP-1 proteins to the target S360 fragment.

Animals↗
PubMed1995

Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase.

A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed.

Adenylyl Cyclases↗
PubMed2003

A new paradigm for drug discovery: integrating clinical, genetic, genomic and molecular phenotype data to identify drug targets.

Application of statistical genetics approaches to variations in mRNA transcript abundances in segregating populations can be used to identify genes and pathways associated with common human diseases. The combination of this genetic information with gene expression and clinical trait data can also be used to identify subtypes of a disease and the genetic loci specific to each subtype. Here we highlight results from some of our recent work in this area and further explore the many possibilities that exist in employing a more comprehensive genetics and functional genomics approach to the functional annotation of genomes, and in applying such methods to the validation of targets for complex traits in the drug discovery process.

Animals↗
PubMed2001

Dominant lesional T cell receptor rearrangements persist in relapsing psoriasis but are absent from nonlesional skin: evidence for a stable antigen-specific pathogenic T cell response in psoriasis vulgaris.

In a previous study we reported that clonally expanded T cell receptor beta-chain rearrangements characterized the T cell receptor usage in skin lesions of psoriasis vulgaris and indicated antigen-specific T cell selection. To assess the relevance of clonal T cell expansion for disease progression, we now determined if select clonal T cell receptor rearrangements persisted over time and were present in nonlesional skin. Sequential biopsies were taken from psoriatic skin lesions of two patients. V-D-J junctional regions of T cell receptor beta-chain variable region gene families 2, 3, 6, 13S1, and BV17 were cloned and sequenced, as these particular BV gene families are preferentially selected in psoriatic skin lesions. The lesional T cell receptor rearrangements were compared with the T cell receptor usage in nonlesional skin and in blood. Several T cell receptor beta-chain rearrangements with high transcript frequency in the first lesional biopsy were again found in sequential lesional biopsies taken as much as 3 y later from psoriasis relapses. Only T cell receptor beta-chain rearrangements with low transcript abundance showed variability in that several clones appeared for the first time or disappeared. Although nonlesional skin also exhibited a restricted T cell receptor usage with clonal T cell receptor rearrangements, the T cell receptor usage in lesional and nonlesional skin differed nearly completely. The select lesional recurrence of identical T cell receptor rearrangements reveals that inflammation in psoriasis involves the same clonally expanded T cell populations and the same antigens over prolonged periods of time. It hereby suggests that specifically recruited and locally expanded T cell clones are permanently involved in psoriatic inflammation and may play a crucial part in disease perpetuation.

Epitopes↗
PubMed1998

A member of the hsp70 gene family from the Mediterranean fruit fly, Ceratitis capitata.

A gene from the Mediterranean fruit fly (Medfly) Ceratitis capitata, belonging to the hsp70 family of genes, has been cloned and characterized. This gene appears to be an example of a heat shock cognate (hsc) gene based on a variety of structural and functional criteria. The heat shock cognate genes serve critical roles in chaperoning of cellular proteins, and they are expressed in a more constitutive manner compared to the inducible heat shock genes. We determined the sequence of the entire coding region of this gene (designated Cerhsc1) together with 5' and 3' flanking regions. The coding region exhibits specific structural features that are characteristic of the subclass of heat shock genes producing proteins localized to the cytosol. The 5' regulatory region of Cerhsc1 contains onlythe minimal array of canonical heatshock element sequences, a phenomenon commonly seen in heat shock cognates. Abundant transcripts are detected from this gene in adult tissue compared to the larval stage. In larval tissue, heat shock appears to produce only a slight increase in the level of transcripts detected. Based on overall structure and function, this Medfly gene appears to be an ortholog of heat shock cognate gene 4 (Drohsc4) of Drosophila melanogaster.

Amino Acid Sequence↗
PubMed2001

Expression and induction of three family 4 cytochrome P450 (CYP4)* genes identified from insecticide-resistant and susceptible western corn rootworms, Diabrotica virgifera virgifera.

We have previously determined that cytochrome P450-based oxidation is involved in resistance to the insecticides methyl parathion and carbaryl in geographically distinct Nebraska western corn rootworm populations. Three new family 4 cytochrome P450 (CYP4) gene fragments (CYP4AJ1, CYP4G18 and CYP4AK1) were cloned and sequenced from insecticide-resistant and -susceptible western corn rootworms. Insecticide bioassays indicated the resistant population employed in this study was significantly resistant to the insecticides methyl parathion and carbaryl. CYP4AJ1 and CYP4G18 were cloned from both genomic PCR and RT-PCR products, although only CYP4AJ1 contains an intronic region. Alignments of inferred amino acid sequences with other homologous insect CYP4 genes indicates a high degree of similarity. Northern analysis concurrently employing mixed probes representing each of the three rootworm CYP4 fragments identified increased mRNA transcript signals (i) in resistant rootworms and (ii) following induction by the P450 inducer pentamethyl benzene. These results support our previous documentation of P450-based insecticide resistance and suggest increased CYP4 transcript abundance can serve as a molecular resistance-associated marker.

Amino Acid Sequence↗
PubMed2000

Occurrence of three PII-like signal transmitter proteins in the diazotrophic proteobacterium Azoarcus sp. BH72.

PII-like signal transmitter proteins are involved in the regulation of ammonium assimilation and nitrogen fixation. We report the identification of three PII-like proteins in the diazotrophic, endophytic proteobacterium Azoarcus sp. BH72, encoded by glnB (monocistronically transcribed) or in the glnKamtB and glnYamtY operons. Phylogenetic analysis revealed that glnB, glnK and glnY represent distinct lineages within the Proteobacteria. A combined approach of two-dimensional gel electrophoresis, Western blotting with paralogue-specific antibodies, N-terminal sequencing and marker exchange mutagenesis allowed us to analyse PII protein expression of Azoarcus sp. BH72 in vivo. GlnK and GlnB were present on all nitrogen sources. Knock-out mutant analysis revealed that GlnB was the only detectable PII protein in a glnK- background, whereas GlnY was only present in a glnK/glnB- double mutant. Nitrogen limitation enhanced transcript abundance of glnK strongly, glnY moderately and glnB not at all in wild-type, glnB-/glnK- or glnK- backgrounds respectively. Phenotypic characterization of knock-out mutants revealed that, unlike in other Proteobacteria, neither glnK nor glnB were essential for nitrogen fixation. As the growth of a double mutant was drastically impaired only on minimal media, both proteins are probably involved in the control of ammonium and nitrate assimilation. The PII-like proteins differed from each other in details of N-sensing. They were covalently modified by uridylylation upon nitrogen limitation, as shown by mass spectrometry; however, the modification patterns in relation to the supplied nitrogen source differed. The novel paralogue GlnY was unusual, as it only occurred in the uridylylated state in vivo and thus lacked a deuridylylation response to nitrogen excess.

Amino Acid Sequence↗
PubMed1996

Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato.

The tomato ACC oxidase gene family is comprised of three members designated AC01, AC02 and AC03. These are highly homologous throughout the protein coding regions but do show a degree of sequence divergence within the 3' untranslated regions. These regions have been cloned and used as gene-specific probes to analyse the differential expression of the tomato ACC oxidase gene family in various tissues at different stages of development. Results indicate that all three genes are transcriptionally active and display a high degree of inducibility in a number of organs at various stages of the life cycle. Both AC01 and Ac03 transcripts accumulate during the senescence of leaves, fruit and flowers. In addition, it appears that AC01 is wound-inducible in leaves. All three ACC oxidase genes are expressed during flower development, with each showing a temporally distinct pattern of accumulation. In addition, the ACC oxidase transcripts are also spatially regulated throughout flower development; AC01 is predominantly expressed in the petals and the stigma and style, AC02 expression is mainly restricted to tissues associated with the anther cone whereas AC03 transcripts accumulate in all of the floral organs examined apart from the sepals. ACC oxidase enzyme assays and Western blot analysis indicate that both enzyme activity and ACC oxidase protein increase with transcript abundance in several tissues. The physiological role of the differential expression of the ACC oxidase gene family, in relation to the regulation of ethylene synthesis, during these various developmental processes is discussed.

Aging↗
PubMed2000

Induction of dwarfism in transgenic Solanum dulcamara by over-expression of a gibberellin 20-oxidase cDNA from pumpkin.

The gibberellin (GA) 20-oxidase (CmGA20ox1) from immature pumpkin seed produces predominantly inactive tricarboxylic acid GAs. We expressed CmGA20ox1 under the control of the CaMV 35S promoter in Solanum dulcamara to assess the usefulness of this gene for reducing GA content in transgenic plants. All transgenic plants obtained were semi-dwarfs with smaller, deep-green leaves and highly pigmented stems compared to the wild-type. Such transformants flowered earlier than the wild-type plants and produced more fruit and more seeds per fruit. The transgene was efficiently expressed, producing high levels of CmGA20ox1 transcript and protein. Furthermore, the concentration of GA(1) was reduced in leaves of the transformants to approximately 20% or less of that in the wild-type and to about 40% or less in stems. The concentrations of other 13-hydroxylated GAs were also reduced, except for the tricarboxylic acid, GA(17), which accumulated in the transformants due to 13-hydroxylation of GA(25). By contrast, the concentrations of non-13-hydroxylated GAs, GA(4) and GA(34), were not consistently reduced, indicating that the effect of expressing the pumpkin gene may not be predictable. Transcript abundance for a native GA 20-oxidase gene was higher in the leaves and stems of S. dulcamara transformed with the pumpkin gene than in wild-type, reflecting the feedback control of 20-oxidase gene expression that serves as a homeostatic mechanism for GAs.

Base Sequence↗
PubMed1999

Cysteine biosynthesis in Chlamydomonas reinhardtii. Molecular cloning and regulation of O-acetylserine(thiol)lyase.

A cDNA, Cys1ACr, encoding an isoform of O-acetylserine(thiol) lyase has been isolated from Chlamydomonas reinhardtii, using a PCR-based approach. The inclusion of dimethylsulfoxide in the PCR reaction has been demonstrated to be essential for the correct amplification of C. reinhardtii templates with complex secondary structures caused by a high G + C content. The deduced amino acid sequence exhibited highest similarity with plant O-acetylserine(thiol)lyase isoforms, indicating that the C. reinhardtii enzyme was structurally more similar to higher plant O-acetylserine(thiol)lyase than to the corresponding prokaryotic enzymes. The N-terminal extension present in Cys1ACr showed several characteristics of an organellar transit peptide, with a length typical for C. reinhardtii. Southern blot analysis suggested that the C. reinhardtii genome may contain a single copy of the organellar O-acetylserine(thiol)lyase gene. O-acetylserine(thiol)lyase activity was strongly induced by sulfur-deficient conditions (up to sevenfold the level observed in a sulfur-repleted cell culture) and required the presence of a nitrogen source. Northern blot analysis showed a different pattern of regulation of Cys1ACr to that observed at the activity level. To obtain an increase of transcript abundance a longer period of sulfur limitation was required, reaching a maximum level of approximately threefold Cys1ACr mRNA when compared with the level of a sulfate-grown culture.

Amino Acid Sequence↗
PubMed2004

Leaf senescence is delayed in maize expressing the Agrobacterium IPT gene under the control of a novel maize senescence-enhanced promoter.

We have genetically modified maize plants to delay leaf senescence. A senescence-enhanced promoter from maize (P(SEE1)) was used to drive expression of the Agrobacterium cytokinin biosynthesis gene IPT in senescing leaf tissue. Three maize lines expressing IPT from P(SEE1), Sg1, Sg2 and Sg3, were analysed in detail, representing mild, intermediate and extreme expression, respectively, of the delayed-senescence phenotype. Backcross populations segregating for the presence or absence of the P(SEE1Xba)IPTNOS transgene also simultaneously segregated for the senescence phenotype. At the time of ear leaf emergence, individuals of lines Sg1 and Sg2 segregating for the presence of the transgene carried about three fewer senescing leaves than control (transgene-minus) segregants, and IPT transcript levels were higher in leaves at incipient senescence than in young leaves. Leaves of transgenic Sg3 plants were significantly greener than controls and progressed directly from fully green to bleached and dead without an intervening yellowing phase. IPT transcript abundance in this line was not related to the initiation of senescence. Extended greenness was accompanied by a delay in the loss of photosynthetic capacity with leaf age. The delayed-senescence trait was associated with relatively minor changes in morphology and development. The phenotype was particularly emphasized in plants grown in low soil nitrogen. The reduced ability of the extreme transgenic line Sg3 to recycle internal nitrogen from senescing lower leaves accounted for significant chlorosis in emerging younger leaves when plants were grown in low nutrient conditions. This study demonstrates that the agronomically important delayed-senescence ('stay-green') trait can be engineered into a monocot crop, and is the first example outside Arabidopsis of senescence modification using a homologous senescence-enhanced promoter.

Journal Article↗
PubMed1994

The effects of Alzheimer's disease, other dementias, and premortem course on beta-amyloid precursor protein messenger RNA in frontal cortex.

There are conflicting data regarding alterations in beta-amyloid precursor protein (APP) mRNAs in Alzheimer's disease (AD). This may be due partly to variables such as agonal state and choice of control group. We have used in situ hybridization histochemistry to study expression of APP mRNAs, with and without the domain encoding the Kunitz protease inhibitor, in a way that overcomes some of the limitations of the current data. Tissue from frontal cortex was collected at rapid autopsy from patients with AD or other cognitive impairments whose terminal phase was prospectively assessed. There were three main findings. Firstly, the amount of APP mRNAs correlated strongly with glutamate decarboxylase activity and was reduced in association with terminal pyrexia. These correlations suggest that agonal state affects APP mRNA and, therefore, that differences in premortem course may contribute to the varying changes in APP transcript abundance reported in AD. Secondly, a reduction of both forms of APP mRNA, normalized to polyadenylated mRNA, was found in AD compared with normal controls and with non-AD dementias. This supports findings that the APP-related pathology of AD is not due to overexpression of APP mRNA or an altered proportion of Kunitz protease inhibitor-containing isoforms. Thirdly, the amount of APP mRNA correlated inversely with that of heat-shock protein (hsx70) mRNA. This relationship was unexpected given current theories that APP expression occurs as part of a stress response, and suggests that other factors predominate in determining neocortical APP mRNA content in neurodegenerative disorders.

Aged↗
PubMed1994

Measurement of blood-brain barrier GLUT1 glucose transporter and actin mRNA by a quantitative polymerase chain reaction assay.

The expression of the blood-brain barrier GLUT1 glucose transporter is down-regulated in brain capillary endothelial cells in tissue culture. Consequently, the study of the regulation of this low-abundance transcript requires the isolation of poly(A)+ mRNA from relatively large numbers of brain endothelial cells in culture (approximately 10(7)). Therefore, in order to facilitate studies with smaller amounts of cells, we describe here a quantitative polymerase chain reaction (PCR) assay to measure the mRNA of GLUT1 and the mRNA of the housekeeping gene, actin, which is used as standard control. Bovine brain endothelial cells were grown as either a primary culture (EP cells) or as a brain endothelial cell line (ECL cells) in 25-mm 6-well cluster dishes, and total or poly(A)+ RNA was isolated. Following synthesis of cDNA with AMV reverse transcriptase and oligo(dT)18 primer, PCR was performed with sense and antisense primers for bovine GLUT1 and gamma-actin, respectively. Reactions were performed in the presence of 2.5 microCi of [alpha-32P]dCTP, and products were resolved in agarose gels and quantified by scanning densitometry of autoradiograms. A direct relationship between RNA-cDNA and PCR products was observed for GLUT1 after 30 cycles, and for actin after 15 PCR cycles. The method was reproducible within specified ranges of starting RNA-derived cDNA, and the intraassay coefficient of variation averaged 7.2 +/- 1.8%. The GLUT1/actin mRNA ratio was as follows: brain capillaries >> EP > ECL. In addition, it is demonstrated that tumor necrosis factor-alpha induced a three- to fourfold increase in the GLUT1/actin mRNA ratio in ECL cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1999

Leptin stimulates proliferation and TGF-beta expression in renal glomerular endothelial cells: potential role in glomerulosclerosis [seecomments].

BACKGROUND: Leptin inhibits food intake and increases energy expenditure. Although the kidney expresses abundant transcripts of the short form of the leptin receptor (Ob-Ra), a role for this hormone in renal function remains unclear. Because individuals with massive obesity who may exhibit increased leptin serum concentrations develop renal glomerulosclerosis, we studied whether leptin can influence renal growth and profibrogenic processes. METHODS: The effects of recombinant leptin on proliferation and synthesis of transforming growth factor-beta1 (TGF-beta1) was investigated in cultured glomerular endothelial cells of the rat (GERs) and syngeneic mesangial cells. Furthermore, leptin receptor expression and potential signal transduction pathways were evaluated in GERs. In addition, leptin was also infused for different time periods (72 hr and 3 weeks) into naive rats. RESULTS: Recombinant mouse leptin induced proliferation of GERs, but not of syngeneic mesangial cells. Coincubation with angiotensin II and leptin exerts additive proliferative effects in GERs. An antileptin-receptor antibody totally abolished this proliferation but did not influence serum-induced proliferation. GER expressed high affinity receptors of the Ob-Ra type (Kd, 4 nM; Bmax, 9700 receptors/cell). Leptin also stimulated phosphorylation of STAT1alpha, and kinase inhibitors attenuated proliferation, suggesting a pivotal role of phosphorylation in this process. Incubation of GERs with leptin also induced mRNA expression of TGF-beta1 and enhanced secretion of this profibrogenic cytokine. Short-term leptin infusion (72 hr) into naive rats induced a significant proliferation, mainly restricted to glomerular endothelial cells, and enhanced glomerular TGF-beta1 mRNA levels. In rats continuously infused for three weeks with leptin, glomerular TGF-beta1 expression was still enhanced, and an additional increase in glomerular collagen type IV mRNA and protein expression was detected. These animals revealed an increase in proteinuria compared with control-infused rats. CONCLUSION: Our findings are the first in vitro and in vivo demonstration that leptin is a renal growth and profibrogenic factor. These results may be an important contribution to our understanding of how leptin can contribute to renal damage, characterized by endocapillary proliferation and subsequent development of glomerulosclerosis, in pathophysiological situations with high circulating levels such as in diabetics or obese individuals. Although the effects of leptin itself are moderate, growth-promoting and profibrogenic effects may be enhanced in concert with other factors such as angiotensin II.

Animals↗
PubMed2005

Transcriptome analysis of ozone-responsive genes in leaves of European beech (Fagus sylvatica L.).

Suppression subtractive hybridization (SSH) was performed to isolate cDNAs representing genes that are differentially expressed in leaves of Fagus sylvatica upon ozone exposure. 1248 expressed sequence tags (ESTs) were obtained from 2 subtractive libraries containing early and late ozone-responsive genes. Sequences of 1139 clones (91 %) matched the EBI/NCBI database entries. For 578 clones, no putative function could be assigned. Most abundant transcripts were O-methyltransferases, representing 7 % of all sequenced clones. ESTs were organized into 12 functional categories according to the MIPS database. Among them, 12 % (early)/15 % (late) were associated with disease and defence, 19/11 % with cell structure, 4/10 % with signal transduction, and 9/6 % with transcription. The expression pattern of selected ESTs (ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit [rbcS], WRKY-type transcription factor, ultraviolet-B-repressible protein, aquaporine, glutathione S-transferase, catalase, caffeic acid O-methyltransferase, and pathogenesis-related protein 1 [PR1]) was analysed by quantitative real-time RT-PCR (qRT-PCR) which confirmed changed transcript levels upon ozone treatment of European beech saplings. The ESTs characterized will contribute to a better understanding of forest tree genomics and also to a comparison of ozone-responsive genes in woody and herbaceous plants.

Europe↗