Identification of an 11T allele in the polypyrimidine tract of intron 8 of the CFTR gene.

PURPOSE: Most of the kits or reagents available for testing for mutations in the cystic fibrosis transmembrane conductance regulator gene include testing for the 5/7/9T polypyrimidine tract, but these methods only screen for three variants in this region: 5T, 7T, and 9T. Although such commercial products may not have been designed to screen for rare alleles of the polypyrimidine tract, we demonstrate that at least one of them (Tag-It Cystic Fibrosis Kit, Tm Bioscience, Toronto, Ontario, Canada) has enough sensitivity to differentiate samples with rare alleles by describing how this product allowed us to detect a previously uncharacterized 11T allele. METHODS: A total of 139 banked and anonymized clinical samples from carrier adults and children with cystic fibrosis (The Hospital for Sick Children, Toronto, Canada) were tested and analyzed using the Tag-It Cystic Fibrosis Kit. RESULTS: Two samples displayed allelic ratios for the polypyrimidine tract that were significantly different from the other samples and did not correspond to values expected to be seen for samples with 5T, 7T, or 9T alleles. Further tests with sequencing and an extended Tag-It assay confirmed the presence of an 11T allele. CONCLUSION: Although commercial products used in cystic fibrosis testing may not have been designed to screen for rare alleles of the polypyrimidine tract, we demonstrated that at least the Tag-It assay may have enough sensitivity to differentiate samples with such rare alleles, which can then be further analyzed for clarification.     

Interleukin-1 gene expression in rabbit vascular tissue in vivo.

Cultured human vascular endothelial and smooth muscle cells express interleukin-1 (IL-1) genes when exposed to bacterial lipopolysaccharides (LPS) and a variety of inflammatory mediators. Local production of IL-1 may contribute to the pathogenesis of various vascular diseases. Therefore the ability of intact vascular tissue to accumulate IL-1 mRNA and synthesize de novo biologically active IL-1 protein was examined. Escherichia coli LPS (10 micrograms/kg) was administered intravenously to adult rabbits and total RNA was isolated from aortic tissue at various times after LPS injection. In saline-injected rabbits, RNA extracted from the thoracic aorta contained little or no IL-1 message detected by Northern analysis using IL-1 alpha and beta cDNA probes cloned from an LPS-stimulated rabbit splenic macrophage library. Lipopolysaccharide treatment promptly induced transient accumulation of mRNA for IL-1 alpha and IL-1 beta within the aorta (maximal 1-hour after injection). Short-term organoid cultures of rabbit aorta exposed to LPS in vitro synthesized immunoprecipitable IL-1 alpha protein. Extracts of aortic tissue excised 1.5 to 3.0 hours after intravenous LPS administration contained immunoreactive and biologically active IL-1 alpha. Anti-rabbit IL-1 alpha antibody neutralized the biologic activity (more than 90%). Microscopic and immunohistochemical studies did not disclose adherent or infiltrating macrophages in rabbit aorta at the time of maximal IL-1 mRNA accumulation after LPS administration (1.5 hours), indicating that intrinsic vascular wall cells rather than mononuclear phagocytes probably account for the IL-1 activity induced by LPS. In addition, aortic tissue from rabbits fed an atherogenic diet showed an enhanced ability to accumulate IL-1 alpha and beta mRNA and produce immunodetectable protein in response to LPS administration. These studies demonstrate inducible IL-1 gene expression in rabbit vascular tissue in vivo and support a local role for this cytokine in vascular pathophysiology.     

Asbestos-induced MKP-3 expression augments TNF-alpha gene expression in human monocytes

TNF-alpha is associated with the development of interstitial fibrosis. We have demonstrated that the p38 mitogen-activated protein (MAP) kinase regulates TNF-alpha expression in monocytes exposed to asbestos. In this report, we asked if extracellular signal-regulated kinase (ERK) was also involved in TNF-alpha expression in monocytes exposed to asbestos. We found that p38 and ERK were differentially activated in alveolar macrophages obtained from patients with asbestosis compared with normal subjects. More specifically, p38 was constitutively active and ERK activation was suppressed. Since the upstream pathway leading to ERK was intact, we hypothesized that an ERK-specific phosphatase was, in part, responsible for the decreased ERK activity. We evaluated whether the dual specificity phosphatase MAP kinase phosphatase (MKP)-3, which is highly expressed in the lung and specifically dephosphorylates ERK, was increased after exposure to asbestos. We found that MKP-3 increased after exposure to asbestos, and its expression was regulated by p38. We found that p38 and ERK negatively regulated one another, and MKP-3 had a role in this differential activation. We also found that p38 was a positive regulator and ERK was a negative regulator of TNF-alpha gene expression. Cells overexpressing MKP-3 had a significant increase in TNF-alpha gene expression, suggesting than an environment favoring p38 MAP kinase activation is necessary for TNF-alpha production in monocytes exposed to asbestos. Taken together, these data demonstrate that the p38 MAP kinase down-regulates ERK via activation of MKP-3 in human monocytes exposed to asbestos to enhance TNF-alpha gene expression.     

An intron 1 regulatory region from the murine adenosine deaminase gene can activate heterologous promoters for ubiquitous expression in transgenic mice

Ubiquitously expressed genes contain regulatory features which allow expression in virtually all cell types. In an effort to understand the molecular basis for this regulatory feature, the chromatin structure of the murine adenosine deaminase gene was examined by DNase I digestion in nuclei of several tissues. The promoter contained a strong hypersensitive site in all tissues examined, including those with very high and very low levels of ADA expression. Transgenic mouse studies revealed that a 3.3 kbEcoRI (3.3 EE) fragment from intron I was required to generate a strong promoter DNase I hypersensitive site, and to produce ubiquitous expression. The 3.3EE fragment also contained a thymic enhancer activity which mapped to sequences conserved with the human ADA gene T-lymphocyte enhancer. Mutational analysis indicated that ubiquitous expression was not dependent on the presence of a functional thymic enhancer. Both the thymic enhancer and the ubiquitous activator within the 3.3EE fragment functioned with heterologous promoters in transgenic mice. Present address: Department of Pediatries, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.     

载脂蛋白AⅠ基因启动子和内含子1中的单核苷酸多态性对其表达的影响

目的构建含ApoAⅠ基因调控区和荧光素酶报告基因的真核表达载体，探讨ApoAⅠ基因启动子区域-75bp G→A置换及第1内含子＋83bp C→T置换对ApoAⅠ表达的影响。方法采用PCR方法扩增人染色体中含ApoAⅠ基因-145～＋289bp的DNA片段，筛选出含ApoAⅠAA／CC基因型（-75bp变异，＋83bp无变异）、GG／TT基因型（-75bp无变异，＋83bp变异）及GG／CC（-75bp和＋83bp均无变异）的DNA片段，构建含以上3个基因片段的pUC重组载体，经SacⅠ和BglⅡ酶切后，再将不同的DNA片段连接到含荧光素酶报告基因的pGL2载体中，获得多个重组克隆。采用阳离子脂质体转染法将携带萤火虫荧光素酶报告基因的重组质粒和携带海肾荧光素酶报告基因的对照质粒共转染HepG2细胞，经48h培养，用化学发光法测定细胞裂解液中荧光素酶的活性，以反映ApoAⅠ的表达水平。结果质粒重组获得3个pGL2-ApoAⅠ-L（-2500～＋289bp）长片段质粒和3个pGL2-ApoAⅠ-S（-145～＋289bp）短片段质粒，分别含AA／CC、GG／TT及GG／CC3种基因型。荧光素酶报告基因活性显示：ApoAⅠAA／CC或GG／TT基因型携带者引起荧光素酶相对活性明显低于GG／CC基因型者。结论基因启动子ApoAⅠ-75bp G→A置换和＋83bp C→T置换对ApoAⅠ转录活性起抑制作用，这可能是-75A和＋83T等位基因携带者高密度脂蛋白血浆水平降低的原因。     

LI-cadherin gene expression during mouse intestinal development.

LI-cadherin (Liver-Intestine cadherin) is a member of a subclass (7-D cadherins) within the cadherin superfamily. Although its cellular function as a cell-cell adhesion molecule has been demonstrated in cell culture studies, its physiological function still needs to be explored in the intact organism. After isolating the cDNA for mouse LI-cadherin, we generated specific antibodies against the overexpressed protein and studied its expression pattern in adult mouse tissues and mouse embryos. The mouse LI-cadherin sequence is 91% identical to the sequence of rat LI-cadherin and exhibits the same structural features described for rat LI-cadherin. In mouse adult tissue, LI-cadherin is expressed in the intestine and in small amounts in the spleen. In contrast to rat, Mouse LI-cadherin was not expressed in liver. During mouse embryogenesis, LI-cadherin expression begins at embryonic day 12.5. With the exception of transient expression in the urogenital sinus and the common bile duct on day 13.5, LI-cadherin was found exclusively in the intestinal epithelium. Its expression coincides with the formation of intestinal villi, a developmental stage that includes major tissue remodeling, growth, and differentiation. LI-cadherin is expressed along the entire anterior-posterior axis of the developing intestine as well as along the entire villus axis once villi begin to form. LI-cadherin occupies all cell surfaces of the deeper layers of the epithelium, distributing to basolateral surfaces only in the cells of the outer epithelial layer. LI-cadherin was found to be always co-expressed with E-cadherin.     

A large deletion in the CFTR gene in CBAVD.

PURPOSE: Most cystic fibrosis mutation screening methods do not detect large exon deletions or duplications in the cystic fibrosis transmembrane regulator gene. We looked for such mutations in congenital bilateral absence of the vas deferens patients in whom routine screening assays had identified only one or no cystic fibrosis transmembrane regulator gene mutations. METHODS: DNA samples from 48 men with congenital bilateral absence of the vas deferens were tested for exonic deletions and duplications in the cystic fibrosis transmembrane regulator gene using a laboratory-developed semiquantitative fluorescent PCR assay. RESULTS: Semi-quantitative fluorescent PCR identified a large deletion in one (2%) of the 48 patients. This patient, previously characterized as carrying only the IVS8-5T mutation, was found to have a deletion of exons 22-24 of the cystic fibrosis transmembrane regulator gene. In a second patient with the IVS8-5T mutation, we identified a one-base pair insertion in exon 17b that disrupted the reading frame. CONCLUSIONS: Analysis of the cystic fibrosis transmembrane regulator gene for exon deletions and duplications should be included for complete study of CBAVD patients, especially those considering assisted reproduction.     

TNF-alpha augments the expression of RhoA in the rat bronchus.

While nonspecific airway hyperresponsiveness (AHR) is a central feature of allergic bronchial asthma, the mechanism underlying the development of AHR is not clearly understood. We have previously demonstrated in vitro hyperresponsiveness of bronchial smooth muscle to acetylcholine (ACh) in rats that were actively sensitized and repeatedly challenged with aerosolized antigen. It has also been demonstrated that the ACh-induced, RhoA-mediated Ca(2+) sensitization is markedly augmented concomitantly with an increased expression and activation of RhoA protein in the bronchial smooth muscle of the antigen-treated rats. In the present study, we have investigated whether TNF-alpha, a proinflammatory cytokine which is involved in bronchial asthma, causes upregulation of RhoA mRNA and protein in the rat bronchus. Treatment of rat bronchial smooth muscle preparations with TNF-alpha (300 ng/ml for 24 hr) significantly shifted the concentration-response curve to ACh upwards, but did not alter the response to high K(+), when compared to that of control tissues. Levels of RhoA mRNA and protein in the TNF-alpha-treated bronchus were significantly greater than those in the control group. In conclusion, it is suggested that the augmentation of the ACh-induced contractile response evoked by TNF-alpha might be mediated by an upregulation of RhoA in rat bronchial smooth muscle.     

Endotoxin stimulates expression of the murine urokinase receptor gene in vivo.

The regulation of urokinase receptor (u-PAR) gene expression during endotoxemia was studied in vivo with a murine model system. Northern blot analysis demonstrated relatively high levels of u-PAR mRNA in mouse placenta, with intermediate levels in lung and spleen and very low levels in heart and kidney. No u-PAR mRNA could be detected in liver, gut, thymus, brain, or skeletal muscle. Intraperitoneal injection of endotoxin (lipopolysaccharide) increased the steady-state levels of u-PAR mRNA in most tissues examined. The greatest induction (sevenfold) was observed in the lung at 1 hour after injection. The cellular localization of u-PAR mRNA was assessed by in situ hybridization. In control mice, u-PAR mRNA was detected primarily in alveolar macrophages of the lung and lymphocytes of the spleen and thymus, although a specific signal was also present in other cell types. In general, endothelial cells lacked detectable u-PAR mRNA. The induction of u-PAR mRNA by lipopolysaccharide was apparent within 30 minutes and was localized to tissue macrophages, lymphocytes, and endothelial cells lining arteries and veins. At later times (1 to 3 hours), specialized epithelial cells present in gastrointestinal tract, bile ducts, and uterus were also positive for u-PAR mRNA. Induction of u-PAR in vivo by lipopolysaccharide may facilitate the extravasation and migration of leukocytes during inflammation.     

The VpreB1 enhancer drives developmental stage-specific gene expression in vivo

In adult mice, the VpreB genes are expressed in bone marrow progenitor (pro-) and precursor (pre-) B cells. As part of the pre-B cell receptor, the proteins are crucial for the proliferation of these cells and consequently normal B lymphocyte development. Using cell lines, we identified a lineage- and developmental-stage-specific VpreB1 enhancer. Here, we analyze its specificity in vivo by generating transgenic mice in which expression of a reporter gene (human CD122) is regulated by the VpreB1 enhancer in the context of its own promoter. All transgenic lines expressed the reporter gene in the bone marrow in a copy number-independent manner, whereas expression levels were integration site-dependent. While the enhancer is not tissue specific, within the B cell lineage the expression pattern of human CD122 mimicked that of endogenous VpreB1. Thus, low levels were detected in pro-B cells, high levels in pre-BI and slightly lower levels in pre-BII cells; no expression was detected in immature/mature B cells. Furthermore, when in vitro cultured transgenic pre-B cells differentiated into immature B cells there was concomitant down-regulation of human CD122 and endogenous VpreB1. Thus the VpreB1 enhancer is sufficient to ensure developmental stage-specific expression of a reporter gene in B lymphocytes in vivo.     

STAT-1-mediated repression of monocyte interleukin-10 gene expression in vivo

There have been substantial advances in understanding the events that regulate gene expression at the cellular and molecular level; however, there has been limited progress integrating this information to understand how biological systems function in vivo. For example, the anti-inflammatory cytokine IL-10 is thought to down-regulate the effects of the pro-inflammatory cytokine IFN-gamma on monocyte activation following LPS stimulation. However, the often-postulated reciprocal regulation of IL-10 gene expression by IFN-gamma has not been studied in vivo. Here we demonstrate that the regulation of IL-10 gene expression has at least two phases following challenge with LPS or a gram-negative organism. In C57BL/6 mice, early IL-10 induction occurs independently of STAT-1, while a delayed active repression of IL-10 gene expression is critically dependent on STAT-1, but only partially dependent upon IFN-alpha/beta and IFN-gamma. This in vivo IL-10 production comes from blood monocytes, but not tissue macrophages, and cannot be reproduced in vitro. This study provides new insights into the regulation of IL-10 following challenge with a gram-negative organism, and highlights the importance of studying these cytokine regulatory pathways in vivo.