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D Lembo

Publications and source records attributed to D Lembo.

32 records · Page 2Linked to original sources
PubMed1994

Characterization of nuclear factors involved in 202 gene induction by IFN-alpha, viruses or dsRNA in murine leukemia cells.

When treated with IFN-alpha, L1210 leukemia cells express high levels of the mouse 202 gene mRNA after a few hours. Three tandem copies of a 43 bp fragment (GAbox) homologous to the IFN-stimulatable response element (ISRE), located in the 5'-flanking region of the 202 gene, were linked to the reporter CAT gene and transiently transfected into L1210 cells. The data suggest that the GA box is sufficient to confer transcriptional inducibility upon IFN stimulation. Binding assays, using the labeled GA box as a probe, demonstrated the presence of a retarded complex, designated GAbfl, in the nuclear extracts of L1210 cells treated with IFN-alpha. This complex is absent in the extracts of L1210 cells treated with ssRNA viruses or synthetic dsRNA. Moreover, photoaffinity cross-linking experiments revealed that GAbfl contains a protein of about 50 kDa. Altogether these results demonstrate that antiviral state induction by IFN-alpha in L1210 cells is preceded by GAbfl binding to the ISRE of the IFN-inducible genes.

Animals↗
PubMed1994

Reduction of spontaneous autoimmune diabetes in diabetes-prone BB rats with the novel immunosuppressant fusidic acid. Effect on T-cell proliferation and production of interferon-gamma.

Diabetes-prone (DP) BB rats spontaneously develop a hyperglycaemic condition which closely resembles human insulin-dependent diabetes mellitus (IDDM), both in terms of clinical and histological features. The incidence of IDDM was significantly reduced when these animals were treated with 2 or 4 mg fusidic acid (FA)/day i.m. from day 30 to day 120 of age. In addition, the mean insulitis score was significantly diminished in the animals treated with FA compared to both vehicle-treated and untreated controls. Finally, 2 mg/day of FA i.m. prevented cell proliferation and interferon-gamma secretion from peripheral blood mononuclear cells upon ex vivo stimulation with concanavalin A. The capacity of FA to substantially reduce the incidence of autoimmune diabetes in a well-known animal model of human IDDM supports previous observations regarding the immunosuppressive properties of FA and its potential use in the treatment of human autoimmune diabetes.

Age Factors↗
PubMed1993

Effect of interferon-alpha on immediate early gene expression of murine cytomegalovirus.

Interferon-alpha (IFN-alpha) significantly reduced the replication of murine cytomegalovirus (MCMV) in mouse embryo fibroblasts derived from the susceptible mouse strain C3H/HeJ. When infectious virus production was measured, a strong decrease in virus titer was observed in IFN-treated cells at a multiplicity of infection (moi) of 1 and 0.5 pfu/cell. Analysis of virus-specified mRNAs by Northern blot assay revealed that IFN-alpha had a significant effect on the expression of viral mRNAs at 48h. In particular, the mRNAs of the major immediate early (IE) transcription units, IE1, IE2, and IE3, were impaired by IFN-alpha. In addition, decrease of IE1 mRNA synthesis was accompanied by a reduction of the major IE product (pp89), as revealed by Western blot assay. These results suggest that IFN-alpha may inhibit MCMV replication by directly impairing IE gene transcription.

Animals↗
PubMed1993

Segmental neurofibromatosis with only macular lesions.

A 16-year-old girl had a three-year history of many café au lait spots and freckles in a dermatomal distribution. The diagnosis of segmental neurofibromatosis (NF-5) was made on the basis of the clinical features, distribution of the lesions, and absence of systemic involvement. Only a few cases in the literature describe NF-5 with only macular lesions. An accurate clinical examination is important to identify this unusual presentation.

Adolescent↗
PubMed1992

Regulation of the 202 gene expression by interferons in L929 cells.

Type I and II interferons (IFNs) stimulate the expression of the 202 and 2'-5' oligoadenylate synthetase (OASE) genes in L929, NIH 3T3 and LM-TK- fibroblastic cell lines. In two other cell lines, B16 melanoma and F9 teratocarcinoma, these cytokines induce OASE but not the 202 mRNA. In L929 cells, IFN-alpha induces the 202 mRNA at concentrations between 10 and 10(3) units/ml. To achieve maximal induction of the 202 mRNA, continuous exposure of L929 cells to IFN-alpha is necessary, whereas 30 minutes of exposure are sufficient to trigger maximal upregulation of the OASE transcript. The induction of the 202 mRNA is the consequence of both transcriptional and post-transcriptional events. Cycloheximide, a known inhibitor of protein synthesis, does not block the induction of 202 mRNA by IFN-alpha, demonstrating that new protein synthesis is not required for this effect. Protein kinase C, arachidonic acid metabolism via the cyclooxygenase or the lipoxygenase pathways and cAMP are not involved as second messengers in the induction of the 202 mRNA by IFN-alpha in L929 cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1991

Interferon action: binding of viral RNA to the 40-kilodalton 2'-5'-oligoadenylate synthetase in interferon-treated HeLa cells infected with encephalomyocarditis virus.

The 40-kDa 2'-5'-oligoadenylate [(2'-5') (A)n] synthetase isoenzyme was proven to be a mediator of the inhibition of encephalomyocarditis virus (EMCV) replication by interferon (IFN). When activated by double-stranded RNA, this enzyme converts ATP into 2'-5'-oligoadenylate [(2'-5') (A)n], and (2'-5') (A)n was found to accumulate in IFN-treated, EMCV-infected cells. The only known function of (2'-5') (A)n is the activation of RNase L, a latent RNase, and this was also implicated in the inhibition of EMCV replication. Intermediates or side products in EMCV RNA replication, presumed to be partially double stranded, were shown to activate (2'-5') (A)n synthetase in vitro. These findings served as the basis of the long-standing hypothesis that the activator of (2'-5') (A)n synthetase in IFN-treated, EMCV-infected cells is the viral RNA. To test this hypothesis, we have generated a polyclonal rabbit antiserum to the human 40-kDa (2'-5') (A)n synthetase. The antiserum immunoprecipitated, from IFN-treated HeLa cells that had been infected with EMCV, the 40-kDa (2'-5') (A)n synthetase protein in complex with both strands of EMCV RNA. The immunoprecipitate was active in (2'-5') (A)n synthesis even without addition of double-stranded RNA, whereas the immunoprecipitate from IFN-treated, uninfected cells was not. These and other results demonstrate that in IFN-treated, EMCV-infected cells, viral RNA is bound to the (2'-5') (A)n synthetase and suggest that the agent activating the (2'-5') (A)n synthetase is the bound viral RNA.

2',5'-Oligoadenylate Synthetase↗
PubMedDate not supplied

The Ifi 200 genes: an emerging family of IFN-inducible genes.

The biological activities of interferons (IFNs) are mediated by IFN-induced proteins. One family is encoded by several structurally related genes located on murine chromosome 1 (Ifi 200 cluster) and three homologous genes (MNDA, IFI 16 and AIM2) located on human chromosome 1 as well, within a linkage group highly conserved between mouse and human. All the proteins of this family contain at least one copy of a conserved 200 amino acid domain, in addition to other regions that are different or missing among the various family members. Conservation of the 200 amino acid segment, therefore, may be responsible for a common function, while individually expressed domains may afford other tissue- or cell-specific functions. The data available demonstrate that at least two members of the Ifi 200 protein family, p202 and p204, inhibit cell proliferation in vitro. Moreover, high constitutive levels of p204 expression impair normal embryo development in transgenic animals. Here, we will review the principal features of murine and human proteins belonging to this family and their function in the cell growth-regulatory activities mediated by IFNs.

Animals↗
PubMedDate not supplied

Molecular cloning of interferon-gamma inducible genes from a murine pre-B cell leukemia.

In the present study, a pre-B cell leukemia L1210-C7, representing a very early stage of the B lineage, was used to characterize the molecular mechanisms exploited by IFN-gamma to modulate B cell activity. A cDNA library was prepared with poly (A)+ RNA from cells stimulated with IFN-gamma and three cDNAs clones complementary to IFN-gamma inducible mRNAs were isolated by differential screening. Of these, the 9.5 cDNA hybridized to a 2.4 kb mRNA not homologous with previously cloned IFN-gamma inducible mRNAs. Furthermore, when compared with RNAs obtained from cells of different origins (fibroblasts and T cells) the 9.5 mRNA appeared to be increased only in cells belonging to the B lineage. Taken as a whole, these results demonstrate that in leukemic pre-B cells IFN-gamma induces the expression of a gene that could be employed as specific cell activation marker.

Animals↗
PubMedDate not supplied

The murine IFN-gamma receptor does not transduce the activation signal to a murine promoter in human cells.

MuIFN-gamma receptor cDNA has been stably transfected in the human WISH cell line. A stable transfectant, denominated WISH-C2, expressed 46;000 receptors/cell whose affinity was similar to that observed on murine cells (Kd = 2.6 x 10(-9) M). When WISH-C2 cells were treated with Hu- or MuIFN-gamma, antiviral and antiproliferative activities were observed with HuIFN-gamma only. These findings suggest that other species-specific components associated to the binding site are required for the signal transduction. To overcome the complexity of measuring biological functions that very likely involve more than one regulatory and structural gene, we set up for the first time a system where a reporter gene driven by a murine promoter was used to directly evaluate the interaction of MuIFN-gamma receptor with an inducible promoter through the missing transducer factor.

Animals↗
PubMedDate not supplied

[Early diagnosis of HCMV infections in patients undergoing transplantation of the kidney].

HCMV infection is a major cause of morbidity and mortality following kidney transplantation. Clinical diagnosis is difficult, and rapid and sensitive diagnostic methods are needed since antiviral therapy is available. One hundred-forty-five consecutive kidney-transplanted patients were studied during a period of three months after transplantation. For laboratory diagnosis of HCMV infection, we looked for the presence of pp-65 antigen in polymorphonuclear leukocytes, HCMV-DNA and IgM. Demonstration of HCMV pp-65 antigen by immunofluorescence and HCMV DNA by PCR in leukocytes were efficient methods for early diagnosis of infection.

Antigens, Viral↗
PubMedDate not supplied

[Protein p53 inhibits the activity of the enhancer of the immediate-early genes of murine cytomegalovirus].

The protein encoded by the tumor suppressor gene p53 can complex and functionally interact with cytomegalovirus proteins produced during the immediate-early phase of infection. The functions of these complex are unclear but there is some evidence to suggest that binding of p53 to these viral proteins may inactivate p53 functions. Recent reports have shown that p53 is involved in regulation of transcription. In this study we have considered the possibility that p53 may regulate transcription of cytomegalovirus immediate early genes which play a crucial role for virus replication. Here we report that experiments in which NIH 3T3 cells were cotransfected with a p53 expression plasmid together with a reporter gene linked to the mouse cytomegalovirus immediate-early enhancer/promoter revealed that wild type p53 could efficiently reduce the transcriptional activity of this viral regulatory sequence. By contrast expression of a mutated p53 correlated with a much smaller reduction of transcription. Deletion mutants analysis of the enhancer revealed that repression of transcription by p53 requires a minimal promoter containing an SP1 consensus sequence and a TATA box.

3T3 Cells↗
PubMedDate not supplied

[Serum stimulates the transcriptional activity of the enhancer of the immediate-early genes of the murine cytomegalovirus through p21 ras].

The analysis of the MCMV IE enhancer revealed the presence of many putative binding sites for the transcription factors AP-1 and NFkB. Previous studies suggested that such factors represent a final target for the metabolic cascade triggered by serum and growth factors. On these basis we wanted to verify if serum stimulates the transcriptional activity of the MCMV IE enhancer through p21ras and AP-1 and NFkB according to the actual model of transduction of the mitogenic signal. Our data demonstrate that serum stimulates the MCMV IE enhancer through a pathway in which the p21ras is involded, as demonstrated by using the dominant inhibitory mutant ras(Asn 17). Moreover deletion mutant analysis of the enhancer showed that the serum responsive region lies between nucleotides -1280 and -285 and contains a high concentration of putative AP-1 and NFkB binding sites.

3T3 Cells↗
PubMedDate not supplied

Constitutive expression of the interferon-inducible protein p202 in NIH 3T3 cells affects cell cycle progression.

p202 is a protein expressed in murine cells after Interferon treatment. Although the function of p202 is still basically unknown, its ability to bind the hypophosphorylated form of the retinoblastoma protein pRb suggests a possible role in the control of cell proliferation. To investigate the role of p202 we have generated several cell clones of NIH 3T3 fibroblasts that constitutively express p202. Here we show that proliferation of quiescent cells on stimulation by serum addition is strongly inhibited by constitutive p202 expression. Moreover, when growth arrested cells are stimulated to proliferate, expression of p202 inhibits G0/G1 progression into the S phase and the cells accumulate with a DNA content that is equivalent to cells arrested in the G0/G1 phase of the cell cycle. Taken together, these studies suggest that p202 may play a negative role in growth regulation.

3T3 Cells↗