In susceptible mice, Leishmania major induce very rapid interleukin-4 production by CD4+ T cells which are NK1.1-.

Susceptibility of BALB/c mice to infection with Leishmania major is associated with a T helper type 2 (Th2) response. Since interleukin-4 (IL-4) is critically required early for Th2 cell development, the kinetics of IL-4 mRNA expression was compared in susceptible and resistant mice during the first days of infection. In contrast to resistant mice, susceptible mice exhibited a peak of IL-4 mRNA in their spleens 90 min after i.v. injection of parasites and in lymph nodes 16 h after s.c. injection. IL-12 and interferon-gamma (IFN-gamma) down-regulated this early peak of IL-4 mRNA; the effect of IL-12 was IFN-gamma dependent. Treatment of resistant C57BL/6 mice with anti-IFN-gamma allowed the expression of this early IL-4 response to L. major. The increased IL-4 mRNA expression occurred in V beta 8, 7, 2- CD4+ cells in BALB/c mice and NK1.1- CD4+ cells in anti-IFN-gamma treated C57BL/6 mice. These results show that the NK1.1+ CD4+ cells, responsible for the rapid burst of IL-4 production after i.v. injection of anti-CD3, do not contribute to the early IL-4 response to L. major.

Expression of the proto-oncogene c-fos in three-dimensional fetal brain cell cultures and the lack of correlation with maturation-inducing stimuli.

Previous work has shown that aggregating fetal brain cell cultures are able to attain a highly differentiated state, and that their development is greatly enhanced by growth and/or differentiation factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and the protein kinase C-activating tumor promoter mezerein. The present study shows that in these 3-dimensional cultures the peptide growth factors EGF and bFGF as well as mezerein are able to induce the expression of the proto-oncogene c-fos. This induction was rapid and transient, in good agreement with observations reported from a wide variety of cell types in vitro. The maximal levels of c-fos mRNA found after stimulation were low in immature cultures and increased greatly as maturation progressed. Of the three factors tested, mezerein was the most potent inducer of c-fos. In contrast to the peptide growth factors EGF and bFGF which were found to induce c-fos only in glial cells, mezerein was stimulatory in glial cells as well as in neurons. A similar cell type specificity has been observed previously for the maturation-enhancing response in immature aggregate cultures. However, in the present study no correlation was found between the degree of c-fos induction and the extent of the maturation-enhancing stimulation. Immature cultures known to be most sensitive and responsive to these maturation-enhancing agents required relatively high doses of peptide growth factors for the induction of c-fos, and the maximal levels of c-fos mRNA elicited were much lower than those in differentiated cultures which did not show any long-term response to these stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3

Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of alpha- and gamma-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCD(cl1) cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC

Overexpression of a mutant form of TGFBI/BIGH3 induces retinal degeneration in transgenic mice.

PURPOSE: Despite ubiquitous expression of the keratoepithelin (KE) protein encoded by the transforming growth factor beta induced/beta induced gene human clone 3 (TGFBI/BIGH3) gene, corneal dystrophies are restricted to the cornea, and no other tissues are affected. We investigated the role of TGFBI/BIGH3 in Groenouw corneal dystrophies by generating transgenic mice overexpressing TGFBI/BIGH3 containing the R555W mutation. METHODS: Transgenic animals expressing the Groenouw mutation of human TGFBI/BIGH3 were generated using lentiviral vectors. The line expressed TGFBI/BIGH3 containing the R555W mutation under the control of the phosphoglycerate kinase (PGK) promoter. Expression of the transgene was monitored by Southern and western blotting and by RT-PCR. Electroretinogram analysis was performed and four mice were subjected to complete necroscopy. RESULTS: Transgene expression was observed in different organs although without specific expression in the cornea. The overall morphology of the transgenic animals was not severely affected by KE overexpression. However, we observed an age-dependent retinal degeneration both functionally and histologically. Female-specific follicular hyperplasia in the spleen and increased levels of lipofuscin in the adrenal gland were also seen in transgenic animals. CONCLUSIONS: Cellular degeneration in the retina of transgenic animals suggest that perturbation of the transforming growth factor beta (TGFbeta) family regulation may affect photoreceptor survival and may induce possible accelerated aging in several tissues. No corneal phenotype could be observed, probably due to the lack of transgene expression in this tissue.

RNA pentaloop structures as effective targets of regulators belonging to the RsmA/CsrA protein family.

In the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens CHA0, the dimeric RNA-binding proteins RsmA and RsmE, which belong to the vast bacterial RsmA/CsrA family, effectively repress translation of target mRNAs containing a typical recognition sequence near the translation start site. Three small RNAs (RsmX, RsmY, RsmZ) with clustered recognition sequences can sequester RsmA and RsmE and thereby relieve translational repression. According to a previously established structural model, the RsmE protein makes optimal contacts with an RNA sequence 5'- (A)/(U)CANGGANG(U)/(A)-3', in which the central ribonucleotides form a hexaloop. Here, we questioned the relevance of the hexaloop structure in target RNAs. We found that two predicted pentaloop structures, AGGGA (in pltA mRNA encoding a pyoluteorin biosynthetic enzyme) and AAGGA (in mutated pltA mRNA), allowed effective interaction with the RsmE protein in vivo. By contrast, ACGGA and AUGGA were poor targets. Isothermal titration calorimetry measurements confirmed the strong binding of RsmE to the AGGGA pentaloop structure in an RNA oligomer. Modeling studies highlighted the crucial role of the second ribonucleotide in the loop structure. In conclusion, a refined structural model of RsmE-RNA interaction accommodates certain pentaloop RNAs among the preferred hexaloop RNAs.

Thiamine-auxotrophic mutants of Pseudomonas fluorescens CHA0 are defective in cell-cell signaling and biocontrol factor expression.

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 x 10(-8) M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.

Superantigen-reactive CD4+ T cells are required to stimulate B cells after infection with mouse mammary tumor virus.

Superantigens are defined by their ability to stimulate a large fraction of T cells via interaction with the T cell receptor (TCR) V beta domain. Endogenous superantigens, classically termed minor lymphocyte-stimulating (Mls) antigens, were recently identified as products of open reading frames (ORF) in integrated proviral copies of mouse mammary tumor virus (MMTV). We have described an infectious MMTV homologue of the classical endogenous superantigen Mls-1a (Mtv-7). The ORF molecules of both the endogenous Mtv-7 and the infectious MMTV(SW) interact with T cells expressing the TCR V beta 6, 7, 8.1, and 9 domains. Furthermore, the COOH termini of their ORF molecules, thought to confer TCR specificity, are very similar. Since successful transport of MMTV from the site of infection in the gut to the mammary gland depends on a functional immune system, we were interested in determining the early events after and requirements for MMTV infection. We show that MMTV(SW) infection induces a massive response of V beta 6+ CDC4+ T cells, which interact with the viral ORF. Concomitantly, we observed a B cell response and differentiation that depends on both the presence and stimulation of the superantigen-reactive T cells. Furthermore, we show that B cells are the main target of the initial MMTV infection as judged by the presence of the reverse-transcribed viral genome and ORF transcripts. Thus, we suggest that MMTV infection of B cells leads to ORF-mediated B-T cell interaction, which maintains and possibly amplifies viral infection.

Clinicopathologic and molecular analysis of a choroidal pigmented schwannoma in the context of a PTEN hamartoma tumor syndrome.

PURPOSE: To report the first case of choroidal schwannoma in a patient affected by PTEN hamartoma tumor syndrome (PHTS) and investigate the molecular involvement of the phosphatase and tensin homolog (PTEN) and neurofibromin 2 (NF2) genes in this rare intraocular tumor. DESIGN: Observational case report. PARTICIPANT: A 10-year-old girl diagnosed with PHTS. METHODS: The enucleated specimen underwent histologic, immunohistochemical, and transmission electronic microscopy. The expression of PTEN and NF2 and their protein products were evaluated by reverse transcription-polymerase chain reaction and immunohistochemistry. Somatic mutations of PTEN and NF2, as well as allelic loss, were investigated by direct sequencing of DNA extracted from the tumor. PTEN epigenetic silencing was investigated by pyrosequencing. MAIN OUTCOME MEASURES: Histopathologic and molecular characterization of a choroidal pigmented schwannoma. RESULTS: Histopathologic, immunohistochemical, and electron microscopic analysis demonstrated features consistent with a pigmented cellular schwannoma of the choroid. We found no loss of heterozygosity at the genomic level for the PTEN germline mutation and no promoter hypermethylation or other somatic intragenic mutations. However, we observed an approximate 40% reduction of PTEN expression at both the mRNA and the protein level, indicating that the tumor was nonetheless functionally deficient for PTEN. Although DNA sequencing of NF2 failed to identify any pathologic variants, its expression was abolished within the tumor. CONCLUSIONS: We report the first description of a pigmented choroidal schwannoma in the context of a PHTS. This rare tumor showed a unique combination of reduction of PTEN and absence of NF2 expression.

Characterization of the enzyme involved in the processing of big endothelin-1 in human lung epithelial cells.

Biosynthesis of active endothelin-1 (ET-1) implies an enzymatic processing of the inactive precursor Big ET-1 (1-39) into the mature, 21 amino acid peptide. The aim of this study was to characterize in airway and alveolar epithelial cells the enzymes responsible for this activation. BEAS-2B and A549 cells, which both produce ET-1, were studied in vitro as models for bronchiolar and alveolar cells, respectively. Both cell lines were able to convert exogenously added Big ET-1 (0.1 microM) into ET-1, suggesting a cell surface or an extracellular processing. The conversion was inhibited by phosphoramidon in both cell lines with an IC50 approximately 1 microM, but not by thiorphan, a specific inhibitor of neutral endopeptidase 24.11 (NEP). The endogenous production of serum-stimulated BEAS-2B and A549 cells was not inhibited by thiorphan, and phosphoramidon showed inhibition only at high concentration (>100 microM). Western blotting following electrophoresis in reducing conditions demonstrated a protein of MR 110 corresponding to the ECE-1 monomer in both BEAS-2B and A549 cells, as well as in whole lung extracts. By RT-PCR we revealed the mRNA encoding for the ECE-1b and/or -1c subtype, but not ECE-1a, in both cell lines. We conclude that BEAS-2B and A549 cells are able to process either endogenous or exogenous Big ET-1 by ECE-1 and that isoforms 1b and 1c could be involved in this processing with no significant role of NEP.

Antiapoptotic role of PPARbeta in keratinocytes via transcriptional control of the Akt1 signaling pathway.

Apoptosis, differentiation, and proliferation are cellular responses which play a pivotal role in wound healing. During this process PPARbeta translates inflammatory signals into prompt keratinocyte responses. We show herein that PPARbeta modulates Akt1 activation via transcriptional upregulation of ILK and PDK1, revealing a mechanism for the control of Akt1 signaling. The resulting higher Akt1 activity leads to increased keratinocyte survival following growth factor deprivation or anoikis. PPARbeta also potentiates NF-kappaB activity and MMP-9 production, which can regulate keratinocyte migration. Together, these results provide a molecular mechanism by which PPARbeta protects keratinocytes against apoptosis and may contribute to the process of skin wound closure.

Four functionally distinct populations of human effector-memory CD8+ T lymphocytes.

In humans, the pathways of memory and effector T cell differentiation remain poorly defined. We have dissected the functional properties of ex vivo effector-memory (EM) CD45RA-CCR7- T lymphocytes present within the circulating CD8+ T cell pool of healthy individuals. Our studies show that EM T cells are heterogeneous and are subdivided based on differential CD27 and CD28 expression into four subsets. EM(1) (CD27+CD28+) and EM(4) (CD27-CD28+) T cells express low levels of effector mediators such as granzyme B and perforin and high levels of CD127/IL-7Ralpha. EM(1) cells also have a relatively short replicative history and display strong ex vivo telomerase activity. Therefore, these cells are closely related to central-memory (CD45RA-CCR7+) cells. In contrast, EM(2) (CD27+CD28-) and EM(3) (CD27-CD28-) cells express mediators characteristic of effector cells, whereby EM(3) cells display stronger ex vivo cytolytic activity and have experienced larger numbers of cell divisions, thus resembling differentiated effector (CD45RA+CCR7-) cells. These data indicate that progressive up-regulation of cytolytic activity and stepwise loss of CCR7, CD28, and CD27 both characterize CD8+ T cell differentiation. Finally, memory CD8+ T cells not only include central-memory cells but also EM(1) cells, which differ in CCR7 expression and may therefore confer memory functions in lymphoid and peripheral tissues, respectively.

Differential transgene expression profiles in rat brain, using rAAV2/1 vectors with tetracycline-inducible and cytomegalovirus promoters.

The biodistribution of transgene expression in the CNS after localized stereotaxic vector delivery is an important issue for the safety of gene therapy for neurological diseases. The cellular specificity of transgene expression from rAAV2/1 vectors (recombinant adeno-associated viral vectors pseudotyped with viral capsids from serotype 1) using the tetracycline-inducible (TetON) expression cassette in comparison with the cytomegalovirus (CMV) promoter was investigated in the rat nigrostriatal pathway. After intrastriatal injection, although green fluorescent protein (GFP) was expressed mainly in neurons with both vectors, the relative proportions of DARPP-32-positive projection neurons and parvalbumin-positive interneurons were, respectively, 13:1 and 2:1 for the CMV and TetON vectors. DARP32-positive neurons projecting to the globus pallidus were strongly GFP positive with both vectors, whereas those projecting to the substantia nigra pars reticulata (SNpr) were efficiently labeled by the CMV vector but poorly by the TetON vector. Numerous GFP-positive cells were evidenced in the subventricular zone with both vectors. However, in the olfactory bulb (OB), GFP-positive neurons were observed with the CMV vector but not the TetON vector. We conclude that the absence of significant amounts of transgene product in distant regions (SN and OB) constitutes a safety advantage of the AAV2/1-TetON vector for striatal gene therapy. Midbrain injections resulted in selective GFP expression in tyrosine hydroxylase-positive neurons by the TetON vector whereas with the CMV vector, GFP-positive cells covered a widespread area of the midbrain. The biodistribution of GFP protein corresponded to that of the transcripts and not of the viral genomes. We conclude that the rAAV2/1-TetON vector constitutes an interesting tool for specific transgene expression in midbrain dopaminergic neurons.

The evolution of gene expression levels in mammalian organs.

Changes in gene expression are thought to underlie many of the phenotypic differences between species. However, large-scale analyses of gene expression evolution were until recently prevented by technological limitations. Here we report the sequencing of polyadenylated RNA from six organs across ten species that represent all major mammalian lineages (placentals, marsupials and monotremes) and birds (the evolutionary outgroup), with the goal of understanding the dynamics of mammalian transcriptome evolution. We show that the rate of gene expression evolution varies among organs, lineages and chromosomes, owing to differences in selective pressures: transcriptome change was slow in nervous tissues and rapid in testes, slower in rodents than in apes and monotremes, and rapid for the X chromosome right after its formation. Although gene expression evolution in mammals was strongly shaped by purifying selection, we identify numerous potentially selectively driven expression switches, which occurred at different rates across lineages and tissues and which probably contributed to the specific organ biology of various mammals.

Transcript profiling suggests that differential metabolic adaptation of mice to a high fat diet is associated with changes in liver to muscle lipid fluxes.

Genetically homogenous C57Bl/6 mice display differential metabolic adaptation when fed a high fat diet for 9 months. Most become obese and diabetic, but a significant fraction remains lean and diabetic or lean and non-diabetic. Here, we performed microarray analysis of "metabolic" transcripts expressed in liver and hindlimb muscles to evaluate: (i) whether expressed transcript patterns could indicate changes in metabolic pathways associated with the different phenotypes, (ii) how these changes differed from the early metabolic adaptation to short term high fat feeding, and (iii) whether gene classifiers could be established that were characteristic of each metabolic phenotype. Our data indicate that obesity/diabetes was associated with preserved hepatic lipogenic gene expression and increased plasma levels of very low density lipoprotein and, in muscle, with an increase in lipoprotein lipase gene expression. This suggests increased muscle fatty acid uptake, which may favor insulin resistance. In contrast, the lean mice showed a strong reduction in the expression of hepatic lipogenic genes, in particular of Scd-1, a gene linked to sensitivity to diet-induced obesity; the lean and non-diabetic mice presented an additional increased expression of eNos in liver. After 1 week of high fat feeding the liver gene expression pattern was distinct from that seen at 9 months in any of the three mouse groups, thus indicating progressive establishment of the different phenotypes. Strikingly, development of the obese phenotype involved re-expression of Scd-1 and other lipogenic genes. Finally, gene classifiers could be established that were characteristic of each metabolic phenotype. Together, these data suggest that epigenetic mechanisms influence gene expression patterns and metabolic fates.

Requirement of glucose metabolism for regulation of glucose transporter type 2 (GLUT2) gene expression in liver.

Previous studies have shown that glucose increases the glucose transporter (GLUT2) mRNA expression in the liver in vivo and in vitro. Here we report an analysis of the effects of glucose metabolism on GLUT2 gene expression. GLUT2 mRNA accumulation by glucose was not due to stabilization of its transcript but rather was a direct effect on gene transcription. A proximal fragment of the 5' regulatory region of the mouse GLUT2 gene linked to a reporter gene was transiently transfected into liver GLUT2-expressing cells. Glucose stimulated reporter gene expression in these cells, suggesting that glucose-responsive elements were included within the proximal region of the promoter. A dose-dependent effect of glucose on GLUT2 expression was observed over 10 mM glucose irrespective of the hexokinase isozyme (glucokinase K(m) 16 mM; hexokinase I K(m) 0.01 mM) present in the cell type used. This suggests that the correlation between extracellular glucose and GLUT2 mRNA concentrations is simply a reflection of an activation of glucose metabolism. The mediators and the mechanism responsible for this response remain to be determined. In conclusion, glucose metabolism is required for the proper induction of the GLUT2 gene in the liver and this effect is transcriptionally regulated.