Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.

Gene replacement with the human BRCA1 locus: tissue specific expression and rescue of embryonic lethality in mice

We have generated transgenic mice that harbor a 140 kb genomic fragment of the human BRCA1 locus (TgN.BRCA1(GEN)). We find that the transgene directs appropriate expression of human BRCA1 transcripts in multiple mouse tissues, and that human BRCA1 protein is expressed and stabilized following exposure to DIVA damage, Such mice are completely normal, with no overt signs of BRCA1 toxicity commonly observed when BRCA1 is expressed from heterologous promoters. Most importantly, however, the transgene rescues the otherwise lethal phenotype associated with the targeted hypomorphic allele (Brca1(Delta exIISA)). Brca1(-/-); TgN.BRCA1(GEN) bigenic animals develop normally and can be maintained as a distinct line. These results show that a 140 kb fragment of chromosome 17 contains all elements necessary for the correct expression, localization, and function of the BRCA1 protein, Further, the model provides evidence that function and regulation of the human BRCA1 gene can be studied and manipulated in a genetically tractable mammalian system.

Analysis of Mouse Keratin 6a Regulatory Sequences in Transgenic Mice Reveals Constitutive, Tissue-Specific Expression by a Keratin 6a Minigene

The analysis of keratin 6 expression is complicated by the presence of multiple isoforms that are expressed constitutively in a number of internal stratified epithelia, in palmoplantar epidermis, and in the companion cell layer of the hair follicle. In addition, keratin 6 expression is inducible in interfollicular epidermis and the outer root sheath of the follicle, in response to wounding stimuli, phorbol esters, or retinoic acid. In order to establish the critical regions involved in the regulation of keratin 6a (the dominant isoform in mice), we generated transgenic mice with two different-sized mouse keratin 6a constructs containing either 1.3 kb or 0.12 kb of 5' flanking sequence linked to the lacZ reporter gene. Both constructs also contained the first intron and the 3' flanking sequence of mouse keratin 6a. Ectopic expression of either transgene was not observed. Double-label immunofluorescence analyses demonstrated expression of the reporter gene in keratin 6 expressing tissues, including the hair follicle, tongue, footpad, and nail bed, showing that both transgenes retained keratinocyte-specific expression. Quantitative analysis of beta -galactosidase activity verified that both the 1.3 and 0.12 kb keratin 6a promoter constructs produced similar levels of the reporter. Notably, both constructs were constitutively expressed in the outer root sheath and interfollicular epidermis in the absence of any activating stimulus, suggesting that they lack the regulatory elements that normally silence transcription in these cells. This study has revealed that a keratin 6a minigene contains critical cis elements that mediate tissue-specific expression and that the elements regulating keratin 6 induction lie distal to the 1.3 kb promoter region.

Tissue-specific expression of head-to-tail cyclized miniproteins in Violaceae and structure determination of the root cyclotide Viola hederacea root cyclotide1

The plant cyclotides are a family of 28 to 37 amino acid miniproteins characterized by their head-to-tail cyclized peptide backbone and six absolutely conserved Cys residues arranged in a cystine knot motif: two disulfide bonds and the connecting backbone segments form a loop that is penetrated by the third disulfide bond. This knotted disulfide arrangement, together with the cyclic peptide backbone, renders the cyclotides extremely stable against enzymatic digest as well as thermal degradation, making them interesting targets for both pharmaceutical and agrochemical applications. We have examined the expression patterns of these fascinating peptides in various Viola species (Violaceae). All tissue types examined contained complex mixtures of cyclotides, with individual profiles differing significantly. We provide evidence for at least 57 novel cyclotides present in a single Viola species (Viola hederacea). Furthermore, we have isolated one cyclotide expressed only in underground parts of V, hederacea and characterized its primary and three-dimensional structure. We propose that cyclotides constitute a new family of plant defense peptides, which might constitute an even larger and, in their biological function, more diverse family than the well-known plant defensins.

XPC polymorphisms play a role in tissue-specific carcinogenesis: a meta-analysis

XPC participates in the initial recognition of DNA damage during the DNA nucleotide excision repair process in global genomic repair. Polymorphisms in XPC gene have been analyzed in case-control studies to assess the cancer risk attributed to these variants, but results are conflicting. To clarify the impact of XPC polymorphisms in cancer risk, we performed a meta-analysis that included 33 published case-control studies. Polymorphisms analyzed were Lys939Gln and Ala499Val. The overall summary odds ratio (OR) for the associations of the 939Gln/Gln genotype with risk of cancer was 1.01 (95% confidence interval (95% CI): 0.94-1.09), but there were statistically significant associations for lung cancer, observed for the recessive genetic model (Lys/Lys + Lys/Gln vs Gln/Gln), (OR 1.30; 95% CI: 1.113-1.53), whereas for breast cancer a reduced but nonsignificant risk was observed for the same model (OR 0.87; 95% CI: 0.74-1.01). The results for Ala499Val showed a significant overall increase in cancer risk (OR 1.15; 95% CI: 1.02-1.31), and for bladder cancer in both the simple genetic model (Ala/Ala vs Val/Val) (OR 1.30; 95% CI: 1.04-1.61) and the recessive genetic model (Ala/Ala + Ala/Val vs Val/Val) (OR 1.32; 95% CI: 1.06-1.63). Our meta-analysis supports that polymorphisms in XPC may represent low-penetrance susceptibility gene variants for breast, bladder, head and neck, and lung cancer. XPC is a good candidate for large-scale epidemiological case-control studies that may lead to improvement in the management of highly prevalent cancers.

Application of recombinant antibody library for screening specific antigens in a bovine sperm cell subpopulation

Antibody phage display libraries are a useful tool in proteomic analyses. This study evaluated an antibody recombinant library for identification of sex-specific proteins on the sperm cell surface. The Griffin.1 library was used to produce phage antibodies capable of recognizing membrane proteins from Nelore sperm cells. After producing soluble monoclonal scFv, clones were screened on Simental sperm cells by flow cytometry and those that bound to 40-60% of cells were selected. These clones were re-analyzed using Nelore sperm cells and all clones bound to 40-60% of cells. Positive clones were submitted to a binding assay against male and female bovine leukocytes by flow cytometry and one clone preferentially bound to male cells. The results indicate that phage display antibodies are an alternative method for identification of molecules markers on sperm cells. (C) 2007 Elsevier B.V. All rights reserved.

Tissue-specific induction of SOCS gene expression by PRL

The mechanisms whereby tissue sensitivity to PRL is controlled are not well understood. Here we report that expression of mRNA and protein for members of the SOCS/CIS/JAB family of cytokine signaling inhibitors is increased by PRL administration in ovary and adrenal gland of the lactating rat deprived of circulating PRL and pups for 24 h but not in mammary gland. Moreover, suckling increases SOCS mRNA in the ovary but not in the mammary gland of pup-deprived rats. Deprivation of PRL and pups for 48 h allows the mammary gland to induce SOCS genes in response to PRL administration, and this is associated with a decrease in basal SOCS-3 mRNA and protein expression to the level seen in other tissues, suggesting that SOCS-3 induced refractoriness related to filling of the gland. In reporter assays, SOCS-1, SOCS-3, and CIS, but not SOCS-2, are able to inhibit transactivation of the STAT 5-responsive beta -lactoglobulin promoter in transient transfection assays. Moreover, suckling results in loss of ovarian and adrenal responsiveness to PRL administered 2 h after commencement of suckling, as determined by STAT 5 gel shift assay. Immunohistochemistry was used to localize the cellular sites of SOCS-3 and CIS protein expression in the ovary and adrenal gland. We propose that induced SOCS-1, SOCS-3, and CIS are actively involved in the cellular inhibitory feedback response to physiological PRL surges in the corpus luteum and adrenal cortex during lactation, but after pup withdrawal, the mammary gland is rendered unresponsive to PRL by increased levels of SOCS-3.

Tyrosinase-Cre mice for tissue-specific gene ablation in neural crest and neuroepithelial-derived tissues

This study describes the derivation of two new lines of transgenic mice that express Cre recombinase under the control of tyrosinase transcriptional elements. To determine the suitability of the Tyrosinase-Cre transgene for tissue-specific gene ablation studies, a fate map of Cre expression domains was determined using the Z/AP reporter strain. It was shown that Cre-expressing cells contribute to a wide array of neural crest and neuroepithelial-derived lineages. The melanocytes of the harderian gland and eye choroid, sympathetic cephalic ganglia, leptomeninges of the telencephalon, as well as cranial nerves (V), (VII), and (IX) are derived either fully or partly from Cre-expressing cephalic crest. The cells contributing to the cranial nerves were the first to exhibit Cre expression at E10.5 as they were migrating into the branchial arches. The melanocytes, chromaffin cells of the adrenal medulla, and dorsal root ganglia are derived from trunk neural crest that either express Cre or were derived from Cre-expressing precursors. An array of brain tissue including the basal forebrain, hippocampus, olfactory bulb, and the granule cell layer of the lateral cerebellum, as well as the retinal pigmented epithelium and glia of the optic nerve originate from Cre-expressing neuroepithelial cells. (C) 2003 Wiley-Liss, Inc.

A critical evaluation of methods for the reconstruction of tissue-specific models

Under the framework of constraint based modeling, genome-scale metabolic models (GSMMs) have been used for several tasks, such as metabolic engineering and phenotype prediction. More recently, their application in health related research has spanned drug discovery, biomarker identification and host-pathogen interactions, targeting diseases such as cancer, Alzheimer, obesity or diabetes. In the last years, the development of novel techniques for genome sequencing and other high-throughput methods, together with advances in Bioinformatics, allowed the reconstruction of GSMMs for human cells. Considering the diversity of cell types and tissues present in the human body, it is imperative to develop tissue-specific metabolic models. Methods to automatically generate these models, based on generic human metabolic models and a plethora of omics data, have been proposed. However, their results have not yet been adequately and critically evaluated and compared. This work presents a survey of the most important tissue or cell type specific metabolic model reconstruction methods, which use literature, transcriptomics, proteomics and metabolomics data, together with a global template model. As a case study, we analyzed the consistency between several omics data sources and reconstructed distinct metabolic models of hepatocytes using different methods and data sources as inputs. The results show that omics data sources have a poor overlapping and, in some cases, are even contradictory. Additionally, the hepatocyte metabolic models generated are in many cases not able to perform metabolic functions known to be present in the liver tissue. We conclude that reliable methods for a priori omics data integration are required to support the reconstruction of complex models of human cells.

Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion.

Cell invasion targets specific tissues in physiological placental implantation and pathological metastasis, which raises questions about how this process is controlled. We compare dermis and endometrium capacities to support trophoblast invasion, using matching sets of human primary fibroblasts in a coculture assay with human placental explants. Substituting endometrium, the natural trophoblast target, with dermis dramatically reduces trophoblast interstitial invasion. Our data reveal that endometrium expresses a higher rate of the fibronectin (FN) extra type III domain A+ (EDA+) splicing isoform, which displays stronger matrix incorporation capacity. We demonstrate that the high FN content of the endometrium matrix, and not specifically the EDA domain, supports trophoblast invasion by showing that forced incorporation of plasma FN (EDA-) promotes efficient trophoblast invasion. We further show that the serine/arginine-rich protein serine/arginine-rich splicing factor 1 (SRSF1) is more highly expressed in endometrium and, using RNA interference, that it is involved in the higher EDA exon inclusion rate in endometrium. Our data therefore show a mechanism by which tissues can be distinguished, for their capacity to support invasion, by their different rates of EDA inclusion, linked to their SRSF1 protein levels. In the broader context of cancer pathology, the results suggest that SRSF1 might play a central role not only in the tumor cells, but also in the surrounding stroma.

Comparative analysis of human and mouse expression data illuminates tissue-specific evolutionary patterns of miRNAs.

MicroRNAs (miRNAs) constitute an important class of gene regulators. While models have been proposed to explain their appearance and expansion, the validation of these models has been difficult due to the lack of comparative studies. Here, we analyze miRNA evolutionary patterns in two mammals, human and mouse, in relation to the age of miRNA families. In this comparative framework, we confirm some predictions of previously advanced models of miRNA evolution, e.g. that miRNAs arise more frequently de novo than by duplication, or that the number of protein-coding gene targeted by miRNAs decreases with evolutionary time. We also corroborate that miRNAs display an increase in expression level with evolutionary time, however we show that this relation is largely tissue-dependent, and especially low in embryonic or nervous tissues. We identify a bias of tag-sequencing techniques regarding the assessment of breadth of expression, leading us, contrary to predictions, to find more tissue-specific expression of older miRNAs. Together, our results refine the models used so far to depict the evolution of miRNA genes. They underline the role of tissue-specific selective forces on the evolution of miRNAs, as well as the potential co-evolution patterns between miRNAs and the protein-coding genes they target.

Hypertension increases connexin43 in a tissue-specific manner.

BACKGROUND: Connexin43 (Cx43), a membrane protein involved in the control of cell-to-cell communication, is thought to play a role in the contractility of the vascular wall and in the electrical coupling of cardiac myocytes. The aim of this study was to investigate the effects of experimental hypertension on Cx43 expression in rat aorta and heart. METHODS AND RESULTS: Rats were made hypertensive after one renal artery was clipped (two kidney, one-clip renal model) or after the administration of deoxycorticosterone and salt (DOCA-salt model). After 4 weeks, all rats showed a similar increase in intra-arterial mean blood pressure and in the thickness of both the aortic wall and the heart. Northern blot analysis of aorta mRNA and immunolabeling for Cx43 showed that hypertensive rats expressed twice as much Cx43 in aorta as the control animals. In contrast, no difference in Cx43 mRNA or in the immunolabeled protein was observed in heart. CONCLUSIONS: The results show that rats exhibiting a similar degree of blood pressure elevation, as the result of different mechanisms, feature a comparable increase in Cx43 gene expression, which was observed in the aortic but not in the cardiac muscle. These data suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.

Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25

Background: The ubiquitin-dependent protein degradation pathway is essential for the proteolysis of intracellular proteins and peptides. Deubiquitinating enzymes constitute a complex protein family involved in a multitude of cellular processes. The ubiquitin-specific proteases (UBP) are a group of enzymes whose predicted function is to reverse the ubiquitinating reaction by removing ubiquitin from a large variety of substrates. We have lately reported the characterization of human USP25, a specific-ubiquitin protease gene at 21q11.2, with a specific pattern of expression in murine fetal brains and adult testis. Results: Database homology searches at the DNA and protein levels and cDNA library screenings led to the identification of a new UBP member in the human genome, named USP28, at 11q23. This novel gene showed preferential expression in heart and muscle. Moreover, cDNA, expressed sequence tag and RT-PCR analyses provided evidence for alternatively spliced products and tissue-specific isoforms. Concerning function, USP25 overexpression in Down syndrome fetal brains was shown by real-time PCR. Conclusions: On the basis of the genomic and protein sequence as well as the functional data, USP28 and USP25 establish a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity. The overexpression of USP25 in Down syndrome fetal brains supports the gene-dosage effects suggested for other UBP members related to aneuploidy syndromes.

Integrative molecular bioinformatics study of human adrenocortical tumors : microRNA, tissue-specific target prediction, and pathway analysis.

MicroRNAs (miRs) are involved in the pathogenesis of several neoplasms; however, there are no data on their expression patterns and possible roles in adrenocortical tumors. Our objective was to study adrenocortical tumors by an integrative bioinformatics analysis involving miR and transcriptomics profiling, pathway analysis, and a novel, tissue-specific miR target prediction approach. Thirty-six tissue samples including normal adrenocortical tissues, benign adenomas, and adrenocortical carcinomas (ACC) were studied by simultaneous miR and mRNA profiling. A novel data-processing software was used to identify all predicted miR-mRNA interactions retrieved from PicTar, TargetScan, and miRBase. Tissue-specific target prediction was achieved by filtering out mRNAs with undetectable expression and searching for mRNA targets with inverse expression alterations as their regulatory miRs. Target sets and significant microarray data were subjected to Ingenuity Pathway Analysis. Six miRs with significantly different expression were found. miR-184 and miR-503 showed significantly higher, whereas miR-511 and miR-214 showed significantly lower expression in ACCs than in other groups. Expression of miR-210 was significantly lower in cortisol-secreting adenomas than in ACCs. By calculating the difference between dCT(miR-511) and dCT(miR-503) (delta cycle threshold), ACCs could be distinguished from benign adenomas with high sensitivity and specificity. Pathway analysis revealed the possible involvement of G2/M checkpoint damage in ACC pathogenesis. To our knowledge, this is the first report describing miR expression patterns and pathway analysis in sporadic adrenocortical tumors. miR biomarkers may be helpful for the diagnosis of adrenocortical malignancy. This tissue-specific target prediction approach may be used in other tumors too.

Tissue-specific FLAGELLIN-SENSING 2 (FLS2) expression in roots restores immune responses in Arabidopsis fls2 mutants.

The flagellin receptor of Arabidopsis, At-FLAGELLIN SENSING 2 (FLS2), has become a model for mechanistic and functional studies on plant immune receptors. Responses to flagellin or its active epitope flagellin 22 (flg22) have been extensively studied in Arabidopsis leaves. However, the perception of microbe-associated molecular patterns (MAMPs) and the immune responses in roots are poorly understood. Here, we show that isolated root tissue is able to induce pattern-triggered immunity (PTI) responses upon flg22 perception, in contrast to elf18 (the active epitope of elongation factor thermo unstable (EF-Tu)). Making use of fls2 mutant plants and tissue-specific promoters, we generated transgenic Arabidopsis lines expressing FLS2 only in certain root tissues. This allowed us to study the spatial requirements for flg22 responses in the root. Remarkably, the intensity of the immune responses did not always correlate with the expression level of the FLS2 receptor, but depended on the expressing tissue, supporting the idea that MAMP perception and sensitivity in different tissues contribute to a proper balance of defense responses according to the expected exposure to elicitors. In summary, we conclude that each investigated root tissue is able to perceive flg22 if FLS2 is present and that tissue identity is a major element of MAMP perception in roots.