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C Murgia

Publications and source records attributed to C Murgia.

At least 19 recordsLinked to original sources
PubMed2006

Zinc and its specific transporters as potential targets in airway disease.

The dietary group IIb metal zinc (Zn) plays essential housekeeping roles in cellular metabolism and gene expression. It regulates a number of cellular processes including mitosis, apoptosis, secretion and signal transduction as well as critical events in physiological processes as diverse as insulin release, T cell cytokine production, wound healing, vision and neurotransmission. Critical to these processes are the mechanisms that regulate Zn homeostasis in cells and tissues. The proteins that control Zn uptake and compartmentalization are rapidly being identified and characterized. Recently, the first images of sub-cellular pools of Zn in airway epithelium have been obtained. This review discusses what we currently know about Zn in the airways, both in the normal and inflamed states, and then considers how we might target Zn metabolism by developing strategies to monitor and manipulate airway Zn levels in airway disease.

Absorption↗
PubMed2001

Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary carcinoma, a unique animal model for human bronchioalveolar carcinoma. We previously isolated a JSRV proviral clone and showed that it was both infectious and oncogenic. Thus JSRV is necessary and sufficient for the development of ovine pulmonary carcinoma, but no data are available on the mechanisms of transformation. Inspection of the JSRV genome reveals standard retroviral genes, but no evidence for a viral oncogene. However, an alternate ORF in pol (orf-x) might be a candidate for a transforming gene. We tested whether the JSRV genome might encode a transforming gene by transfecting an expression plasmid for JSRV [pCMVJS21, driven by the cytomegalovirus (CMV) immediate early promoter] into mouse NIH 3T3 cells. Foci of transformed cells appeared in the transfected cultures 2-3 weeks posttransfection; cloned transformants showed anchorage independence for growth, and they expressed JSRV RNA. These results indicate that the JRSV genome contains information with direct transforming potential for NIH 3T3 cells. Transfection of a mutated version of pCMVJS21 in which the orf-x protein was terminated by two stop codons also gave transformed foci. Thus, orf-x was eliminated as the candidate transforming gene. In addition, another derivative of pCMVJS21 (pCMVJS21DeltaGP) in which the gag, pol (and orf-x) coding sequences were deleted also gave transformed foci. These results indicate that the envelope gene carries the transforming potential. This is an unusual example of a native retroviral structural protein with transformation potential.

Animals↗
PubMed2001

A phosphatidylinositol 3-kinase docking site in the cytoplasmic tail of the Jaagsiekte sheep retrovirus transmembrane protein is essential for envelope-induced transformation of NIH 3T3 cells.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer of sheep known as ovine pulmonary carcinoma. Recently, we have found that the expression of the JSRV envelope (Env) is sufficient to transform mouse NIH 3T3 cells in classical transformation assays. To further investigate the mechanisms of JSRV oncogenesis, we generated a series of envelope chimeras between JSRV and the JSRV-related endogenous retroviruses of sheep (enJSRVs) and assessed them in transformation assays. Chimeras containing the exogenous JSRV SU region and the enJSRV TM region were unable to transform NIH 3T3 cells. Additional chimeras containing only the carboxy-terminal portion of TM (a region that we previously identified as VR3) of the endogenous envelope with SU and the remaining portion of TM from the exogenous JSRV were also unable to transform NIH 3T3 cells. The VR3 region includes the putative membrane-spanning region and cytoplasmic tail of the JSRV TM glycoprotein; this suggested that the cytoplasmic tail of the JSRV Env mediates transformation, possibly via a cell signaling mechanism. Mutations Y590 and M593 in the cytoplasmic tail of the JSRV envelope were sufficient to inhibit the transforming abilities of these constructs. Y590 and M593 are part of a Y-X-X-M motif that is recognized by the phosphatidylinositol 3-kinase (PI-3K). PI-3K initiates a cell signaling pathway that inhibits apoptosis and is required for a number of mitogens during the G(1)-to-S-phase transition of the cell cycle. PI-3K activates Akt by phosphorylation of threonine 308 and serine 473. We detected by Western blot analysis phosphorylated Akt in serum-starved MP1 cells (NIH 3T3 cells transformed by JSRV) but not in the parental NIH 3T3 cells. These data indicate that the cytoplasmic tail of the JSRV TM is necessary for cell transformation and suggest a new mechanism of retroviral transformation. In addition, the ability to dissociate the function of the JSRV envelope to mediate viral entry from its transforming capacity has direct relevance for the design of JSRV-based vectors that target the differentiated epithelial cells of the lungs.

3T3 Cells↗
PubMed2000

The long terminal repeat of Jaagsiekte sheep retrovirus is preferentially active in differentiated epithelial cells of the lungs.

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a contagious bronchioloalveolar carcinoma of sheep known as sheep pulmonary adenomatosis (SPA; ovine pulmonary carcinoma). JSRV is unique among retroviruses because it transforms the alveolar type II cells and the nonciliated bronchiolar cells (Clara cells) of the lungs; these cells are where JSRV is specifically expressed in both naturally and experimentally SPA-affected sheep. In this study, we investigated the cell specificity of JSRV expression. By transient-transfection assays of 23 different cell lines with a reporter plasmid driven by the JSRV long terminal repeat (LTR), pJS21-luc, we found that the JSRV LTR is preferentially active in cell lines derived from type II pneumocytes and Clara cells (MLE-15 and mtCC1-2 mouse cell lines). Reporter assays using progressive 5' deletions of pJS21-luc allowed us to establish that the JSRV enhancers are able to activate the JSRV proximal promoter in MLE-15 and mtCC1-2 cells, but they have very low activity in mouse cells of other lineages (e.g., NIH 3T3). The JSRV enhancers are able to activate heterologous promoters in both MLE-15 and 3T3 cells, although optimal activity is achieved in MLE-15 cells only with the homologous JSRV promoter. Thus, JSRV cell-specific LTR activity appears to result from an interaction between the enhancer elements and the JSRV proximal promoter elements. By mutation analysis, we established that an upstream NF-kappaB-like element appears to be responsible for approximately 50% of the JSRV LTR transcriptional activity in MLE-15 cells. Electrophoretic mobility shift assays showed evidence of a factor(s) that binds to this sequence. Antibody supershift experiments indicated that the factor(s) is not related to NF-kappaB component p50 or p52. This factor also appeared to be present in cells that do not support a high level of JSRV expression. Finally the JSRV(21) LTR contains putative enhancer binding motifs for transcription factors such as hepatocyte nuclear factor 3 (HNF-3) that are involved in lung-specific gene expression. Cotransfection experiments demonstrated that exogenous HNF-3 is able to enhance the expression of pJS21-luc in NIH 3T3 cells, which normally show minimal enhancer activity for the JSRV LTR.

3T3 Cells↗
PubMed2000

Molecular cloning and functional analysis of three type D endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus.

Integrated into the sheep genome are 15 to 20 copies of type D endogenous loci that are highly related to two exogenous oncogenic viruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). The exogenous viruses cause infectious neoplasms of the respiratory tract in small ruminants. In this study, we molecularly cloned three intact type D endogenous retroviruses of sheep (enJS56A1, enJS5F16, and enJS59A1; collectively called enJRSVs) and analyzed their genomic structures, their phylogenies with respect to their exogenous counterparts, their capacity to form viral particles, and the expression specificities of their long terminal repeats (LTRs). In addition, the pattern of expression of enJSRVs in vivo was studied by in situ hybridization. All of the three enJSRV proviruses had open reading frames for at least one of the structural genes. In particular, enJS56A1 had open reading frames for all structural genes, but it could not assemble viral particles when highly expressed in human 293T cells. We localized the defect for viral assembly in the first two-thirds of the gag gene by making a series of chimeras between enJS56A1 and the exogenous infectious molecular clone JSRV(21). Phylogenetic analysis distinguished five ovine type D retroviruses: enJSRV groups A and B, ENTV, and two exogenous JSRV groups (African versus United Kingdom/North America isolates). Transient transfection assays indicated that the LTRs of the three enJSRVs were not preferentially active in differentiated lung epithelial cells. This suggests that the pulmonary tropic JSRV developed from a type D retrovirus that did not have lung specificity. Consistent with this, in situ hybridization of a panel of normal ovine tissues revealed high expression of enJSRV mRNA in the luminal epithelium and glandular epithelium of the uterus; lower expression was localized in the lamina propria of the gut and in the bronchiolar epithelium of the lungs.

Amino Acid Sequence↗
PubMed1999

Cloning, expression, and vesicular localization of zinc transporter Dri 27/ZnT4 in intestinal tissue and cells.

We have identified the Dri 27 cDNA on the basis of its upregulated expression during rat intestinal development. It encodes a hydrophobic protein of 430 amino acids that shares significant homology with members of the mammalian zinc transporter family ZnT. The murine homologue of Dri 27 (named ZnT4) was recently associated with the mouse mutation "lethal milk." The primary sequence of Dri 27/ZnT4 displays features characteristic of polytopic membrane proteins. In this paper, we show that Dri 27/ZnT4 is localized in the membrane of intracellular vesicles, the majority of which concentrate in the basal cytoplasmic region of polarized enterocytes. A Dri 27/ZnT4 myc-tagged construct, transiently transfected in intestinal Caco-2 cells, partially colocalizes with the transferrin receptor and with the beta-subunits of the clathrin adaptor complexes AP-1 and AP-2 in a subpopulation of endosomal vesicles. By subcloning distinct portions of the protein in frame with glutathione-S-transferase, we also provide experimental evidence of their function as zinc-binding and protein-protein-interaction domains.

Amino Acid Sequence↗
PubMed1998

Cell cycle and adhesion defects in mice carrying a targeted deletion of the integrin beta4 cytoplasmic domain.

The cytoplasmic domain of the integrin beta4 subunit mediates both association with the hemidesmosomal cytoskeleton and recruitment of the signaling adaptor protein Shc. To examine the significance of these interactions during development, we have generated mice carrying a targeted deletion of the beta4 cytoplasmic domain. Analysis of homozygous mutant mice indicates that the tail-less alpha6beta4 binds efficiently to laminin 5, but is unable to integrate with the cytoskeleton. Accordingly, these mice display extensive epidermal detachment at birth and die immmediately thereafter from a syndrome resembling the human disease junctional epidermolysis bullosa with pyloric atresia (PA-JEB). In addition, we find a significant proliferative defect. Specifically, the number of precursor cells in the intestinal epithelium, which remains adherent to the basement membrane, and in intact areas of the skin is reduced, and post-mitotic enterocytes display increased levels of the cyclin-dependent kinase inhibitor p27(Kip). These findings indicate that the interactions mediated by the beta4 tail are crucial for stable adhesion of stratified epithelia to the basement membrane and for proper cell-cycle control in the proliferative compartments of both stratified and simple epithelia.

Animals↗
PubMed1997

The coupling of alpha6beta4 integrin to Ras-MAP kinase pathways mediated by Shc controls keratinocyte proliferation.

The signaling pathways linking integrins to nuclear events are incompletely understood. We have examined intracellular signaling by the alpha6beta4 integrin, a laminin receptor expressed in basal keratinocytes and other cells. Ligation of alpha6beta4 in primary human keratinocytes caused tyrosine phosphorylation of Shc, recruitment of Grb2, activation of Ras and stimulation of the MAP kinases Erk and Jnk. In contrast, ligation of the laminin- and collagen-binding integrins alpha3beta1 and alpha2beta1 did not cause these events. While the stimulation of Erk by alpha6beta4 was suppressed by dominant-negative Shc, Ras and RhoA, the activation of Jnk was inhibited by dominant-negative Ras and Rac1 and by the phosphoinositide 3-kinase inhibitor Wortmannin. Adhesion mediated by alpha6beta4 induced transcription from the Fos serum response element and promoted cell cycle progression in response to mitogens. In contrast, alpha3beta1- and alpha2beta1-dependent adhesion did not induce these events. These findings suggest that the coupling of alpha6beta4 integrin to the control of cell cycle progression mediated by Shc regulates the proliferation of basal keratinocytes and possibly other cells which are in contact with the basement membrane in vivo.

3T3 Cells↗
PubMed1995

Transcriptional regulation of the ezrin gene during rat intestinal development and epithelial differentiation.

Polarized intestinal epithelial cells are characterized by the presence of a brush border at their apical surface. The brush border cytoskeleton is assembled during cell differentiation and is composed of parallel actin bundles, held together by specific actin-binding proteins. Using specific cDNA probes we have studied the expression of the mRNAs encoding ezrin and moesin, two members of a class of proteins that connect the microvillar cytoskeleton to the plasma membrane, during the process of enterocyte maturation that occurs both in the embryonic and in the adult small intestine, along the crypt-villus axis. The steady state levels of ezrin mRNA were found to increase in the fetal gut epithelium between day 15 and day 20 of gestation and during the first week after birth, in parallel with the morphogenetic process that leads to cell polarization and brush border assembly. On the contrary, moesin mRNA is expressed at very low levels in the mature small intestine, with a sudden drop in transcription occurring at birth. In the continuously renewing epithelium of adult animals, ezrin mRNA levels are higher in the differentiated villus cells of the distal portions of the gastrointestinal tract and very low in undifferentiated crypt cells. These data demonstrate that the expression of the ezrin gene is regulated at the level of mRNA abundance during development and differentiation of the intestinal epithelium.

Amino Acid Sequence↗
PubMed1994

Subtractive hybridization cloning of novel genes differentially expressed during intestinal development.

Intestinal genes whose expression is regulated during development and differentiation were identified and cloned from a rat villi cDNA library using a subtracted cDNA probe. The isolated clones are transcribed in the fully differentiated intestinal epithelium 21 days after birth and absent or poorly expressed in the fetal gut at 15 days of gestation. Two of the DRI (differentially-expressed in rat intestine) genes are novel, while the others encode the microvillar protein ezrin and intracellular carrier proteins for retinol and fatty acids. Expression of the newly isolated DRI27 and DRI42 clones parallels epithelial differentiation during development and it is more pronounced in the distal portions of the small intestine. In situ hybridization experiments indicate that the DRI mRNAs are expressed in the differentiated cell types of the gut epithelium. Moreover, the expression of DRI27 and DRI42 is strongly related to the stage of epithelial differentiation during gut development. This relationship holds true also for the expression of DRI42 in other tissues. These clones will be a valuable tool to identify regulatory sequences and factors responsible for confining gene expression to the differentiated epithelial cell types in mammalian small intestine.

Animals↗
PubMed1994

First-trimester human trophoblast is class II major histocompatibility complex mRNA+/antigen.

Lack of expression of the polymorphic class I and class II MHC antigens in the cytotrophoblast is one of the major factors determining the privileged immunologic status of the placenta. In this report, we show that first-trimester human placental cytotrophoblast cells display moderate to strong expression of class II MHC (HLA-DR alpha and -DR beta) and Ii chain transcripts, apparently in absence of detectable class II antigens and Ii chain. In addition, DR alpha, DR beta, and Ii mRNAs, but not antigens, are consistently upregulated by IFN-gamma. Constitutive expression and upregulation of mRNAs are detectable in trophoblast cells kept in short term as well as prolonged (2-3 weeks) culture. These results are reminiscent of an analogous mRNA+/antigen- dissociation occurring, in the case of class I MHC gene products, in a subpopulation of first-trimester cytotrophoblast cells. Thus, analogous mechanisms prevent the expression of potentially hazardous class I and II allodeterminants at early stages of semiallogeneic pregnancy.

Blotting, Northern↗
PubMed1994

The prevalence of endometriosis in premenopausal women undergoing gynecological surgery.

Endometriosis is a disease of the female pelvic mesenchyme in which tissue with epithelial and stromal characteristics of the endometrium develops in a situation other than in the uterus. The aim of this study was to evaluate the prevalence of endometriosis in premenopausal women submitted to laparoscopy and/or laparotomy for infertility, chronic pelvic pain, benign ovarian cysts and uterine myomas. The prevalence of the disease was higher in patients with infertility (30.5%), chronic pelvic (45%) and benign ovarian cysts (43%) than in patients with uterine mvomas (8.5%).

Adolescent↗
PubMed1994

Shrinkage of pituitary PRL-secernent adenoma after short-term treatment with bromocriptine long-acting repeatable injections.

The efficacy, safety and tolerability of a single bromocriptine-LAR injection (50 mg) and of a 6 injection course at 28-day intervals, were evaluated respectively in 13 and in 9 hyperprolactinemic women with radiological signs of PRL-secreting pituitary adenoma. The long-lasting repeatable formulation of bromocriptine induced a rapid and prolonged hypoprolactinemic effect. Side effects related to central activity of the compound were observed only on the first day of compound administration in all subjects except one, whereas no modifications of cardiologic and haematologic parameters were observed. In one subject the occurrence of side effects was observed also during the 6 injection course of treatment. A significant shrinkage of pituitary adenoma was observed at the second CT scan performed in 7 of the 9 subjects treated for 6 months with bromocriptine-LAR. CT scan was not performed in one subject who achieved pregnancy after second bromocriptine-LAR injection, whereas unmodified size of pituitary microadenoma was found in one subject whose PRL secretion did not decrease during the treatment and who referred severe side effects.

Adolescent↗
PubMed1993

Metallothionein gene is expressed in developing rat intestine and is induced by zinc but not by corticosteroids.

The expression of metallothionein (MT) mRNA during perinatal development of rat intestine and its induction by zinc and corticosteroids were studied. Pregnant rats from d 17 to 22 of gestation and rats at 2, 4, 13 and 21 d of postnatal life were injected with saline solution (control) or with zinc (10 mg/kg body wt) or corticosteroids (1 mg/kg body wt). After 6 h, tissues were removed for analysis. Northern hybridization of polyA + RNA to 32P-MT-cDNA revealed that MT was expressed already at d 17 of fetal life, increased afterwards (reaching the maximal expression around birth) and decreased soon after until weaning. Metallothionein mRNA was markedly induced by zinc at d 18 of fetal life to a level that remained constant throughout postnatal life. Corticosteroids were ineffective in inducing MT gene expression during prenatal and postnatal development. In 21-d-old adrenalectomized rats the level of MT mRNA was similar to that of control rats of the same age and was not changed by hormone treatment. The results indicate that MT gene expression can be induced by zinc during fetal life and that its expression without exogenous inducers cannot be ascribed to circulating corticosteroids.

Adrenal Cortex Hormones↗
PubMed1991

[Embolism of the superior mesenteric artery. A clinical experience].

The Authors report their clinical experience in superior mesenteric artery embolism: 10 arterial embolisms (71%) collected from a series of 14 obstructions of the superior mesenteric artery. The main interval from the beginning of the symptomatology to hospital admission was 48 h. Laparotomy was performed in all ten patients; gangrenous bowel was resected in 2 and 2 had an embolectomy of the superior mesenteric artery without intestinal resection. The remaining 6 patients had laparotomy alone and died. The Authors emphasize the difficulty in recognizing the disease at an early stage and suggest to contemplate in patients at risk with a persistent abdominal pain, the possibility of a superior mesenteric artery embolism.

Aged↗
PubMed1990

[Complementary profundoplasty. Clinical experience].

The Authors report a 68% overall success rate in a three years follow-up study of 45 "complementary" profundoplasties. The key factors with a significant prognostic effect on the clinical surgical results are the early postoperative increase of the resting ankle pressure index (I.W.) and the angiographic preoperative patency of the tibial arteries. An early postoperative increase of I.W. has been always associated with good results whereas an unchanged postoperative I.W. has always been associated with poor results; an angiographic tibial run-off (one or two or three patient tibial arteries) has been associated with significant better clinical results (p less than 0.001).

Aged↗
PubMed1990

[Endarterectomy of the internal carotid. Clinical experience].

The authors report a clinical experience on 37 internal carotid endarterectomies of 33 patients classified as: group A (asymptomatic patients); 4 patients (13%), group B (RIA); 9 patients (27%), group C (stabilized neurological lesions); 19 patients (56%), group D (acute cerebral ischemia): 1 patient (3%). Sixteen thromboendarterectomies (43%) were carried on for the prophylaxis of neurological lesions, 20 (54%) to increase and balance the cerebral blood flow in patients with stable neurological lesions, 1 (3%) for acute cerebral ischemia. The overall operative mortality was 6%, the operative morbidity was 22%. The clinical follow-up concerned 26 patients: the overall mortality was 19%: in survivors the late results were excellent. The rate of postoperative stenosis was 18%: 80% of these arteries had been treated without a patch angioplasty.

Aged↗