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Biomedical subjects

N M Maraldi

Publications and source records attributed to N M Maraldi.

At least 19 recordsLinked to original sources
PubMed2001

pRb2/p130 and p107 control cell growth by multiple strategies and in association with different compartments within the nucleus.

It has been recently reported that retinoblastoma family proteins suppress cell growth by regulating not only E2F-dependent mRNA transcription but also rRNA and tRNA transcription and, through HDAC1 recruitment, chromatin packaging. In the present study we report data showing that these various control strategies are correlated, at least in part, with nuclear compartmentalization of retinoblastoma proteins. In a first series of experiments, we showed that pRb2/p130 and p107 are not evenly distributed within the nucleus and that cell cycle-dependent binding with E2F4 changes also as a function of their subnuclear localization. Namely, in the nucleoplasm pRb2/p130-E2F4 complexes are more numerous during G0/G1 while in the nucleolus they increase in S phase. Partially different functions for p107 are suggested since p107-E2F4 complexes in the nucleoplasm are more numerous is S phase with respect to G0/G1 and no cell cycle change is observed in the nucleolus. In a second series of experiments we showed that pRb2/p130, p107, E2F4, and pRb2/p130-HDAC1 complexes are all inner nuclear matrix-associated proteins and localize to sites different from pRb/p105 ones. We provide further evidence of multiple and partially distinct retinoblastoma protein family functional roles during cell cycle. Moreover, our data support emerging evidence for functional interrelationships between nuclear structure and gene expression.

Cell Compartmentation↗
PubMed2001

Nuclear alterations in autosomal-dominant Emery-Dreifuss muscular dystrophy.

Electron microscopy study of muscle biopsies from patients with autosomal-dominant Emery-Dreifuss muscular dystrophy revealed nuclear alterations in about 10% of the preserved muscle fibers. The major findings consisted of peripheral heterochromatin loss or detachment from the nuclear envelope, and of interchromatin texture alterations. These abnormalities are similar to those reported in an animal model of the disease and to those found in the X-linked form of Emery-Dreifuss muscular dystrophy. These results suggest that an abnormal ultrastructural arrangement of the nuclear periphery is a common feature in the known forms of Emery-Dreifuss muscular dystrophy, and that several proteins of the nuclear scaffold are necessary in muscle cells to maintain the nuclear structural/functional integrity and a normal muscle cell metabolism.

Adult↗
PubMed2001

Basic fibroblast growth factor enhances in vitro mineralization of rat bone marrow stromal cells grown on non-woven hyaluronic acid based polymer scaffold.

A biodegradable non-woven hyaluronic acid polymer scaffold (Hyaff 11) was analysed in vitro as a carrier vehicle for differentiation and mineralization of rat bone marrow stromal cells (BMSC). BMSC were grown on Hyaff 11 in a mineralizing medium in the presence/absence of basic fibroblast growth factor (bFGF). Osteoblastic differentiation was investigated by light and electron microscopy analysing the expression of osteogenic markers: calcium, alkaline phosphatase (AP), osteopontin (OP), bone sialoprotein (BSP) and collagen type 1. We also measured proliferation, AP activity and mRNA expression of AP and osteocalcin (OC). Electron microscopy and Toluidine-blue staining demonstrated that bFGF accelerated (day 20 vs. day 40) and increased mineralization. With bFGF, calcium, OP and BSP were strongly enhanced at day 40, whereas AP decreased. Our in vitro results demonstrate that Hyaff 11 is a useful vehicle for growth, differentiation and mineralization of rat BMSC, and that it permits bone development.

Alkaline Phosphatase↗
PubMed2001

Searching the point of no return in Helicobacter pylori life: necrosis and/or programmed death?

AIMS: Ultrastructural and molecular studies to support the hypothesis of programmed cell death in Helicobacter pylori were conducted. METHODS AND RESULTS: Evidence of programmed death in H. pylori is provided through electron microscopic detection and cytochemical labelling of electrondense bodies (EDB), containing packaged DNA in coccoid cells, resembling micronuclei of apoptotic eukaryotic cells. This morphological evidence is also supported by DNA cleavage in homogeneous fragments of about 100 base pairs. Programmed cell death was observed in H. pylori cultures at 37 degrees C, with a maximum of 37.5% of EDB coccoid cells after 7 days. The non-permissive temperature of 4 degrees C anticipated this process, with 40% of EDB coccoid forms within 3 days, and it remained substantially unaffected during the observation time of 14 days. CONCLUSION: In these experiments, deprivation of nutrients and a non-permissive temperature acted as a powerful trigger for programmed cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: Helicobacter pylori bacterial populations, under stressing stimuli, can respond with programmed cell suicide as a means of species preservation.

Apoptosis↗
PubMed2001

Increased activity and nuclear localisation of inositol lipid signal transduction enzymes in rat hepatoma cells.

To elucidate the relationship between inositol lipid signal transduction and oncogenic transformation, the activity and subcellular distribution of phospholipase C isoforms and of phosphatidylinositol 3-kinase were analysed in Morris hepatoma cells, MH(1)C(1), with respect to normal rat liver cells. The results provide evidence of a gain of function of the enzymes involved in inositide signal transduction, the amount of which increased mainly at the nuclear level. Phospholipase C and phosphatidylinositol 3-kinase activities are significantly higher in rat hepatoma than in rat liver cells. Moreover, some phospholipase C isoforms are expressed at higher levels at the nuclear level; this is particularly evident in the case of the delta 1 isoform which is not expressed at the nuclear level in rat liver cells. Therefore, the autonomous nuclear signal transduction system, formerly reported as involved in the modulation of cell proliferation and differentiation, appears also affected in oncogenic transformation.

Animals↗
PubMed2000

Unusual laminin alpha2 processing in myoblasts from a patient with a novel variant of congenital muscular dystrophy.

We recently described a novel congenital muscular dystrophy (CMD) syndrome characterized by mental retardation, microcephaly, and partial merosin deficiency on muscle biopsy. Linkage analysis excluded involvement of the known CMD loci. We now report on a study performed on the differentiation of cultured myoblasts from one patient affected by this condition to evaluate the potential to form myotubes and merosin processing in these cells. The differentiation rate was comparable to controls and myotubes were stable in culture. Biochemical analysis showed the expected 80-kDa merosin subunit in myoblasts. However, a shifted 60-kDa protein was detected in myotubes. Matrix-metalloproteinases (MMPs) zymography showed increased gelatinolytic activity, and immunoblotting identified an increased amount of membrane-type 1 matrix-metalloproteinase in pathological myotube preparations. Our results show that these CMD-derived myotubes contain a low molecular weight merosin. They further suggest that an altered regulation of MMPs can be involved in basal lamina damage.

Cell Differentiation↗
PubMed2000

A serine 37 mutation associated with two missense mutations at highly conserved regions of p53 affect pro-apoptotic genes expression in a T-lymphoblastoid drug resistant cell line.

The p53 protein accumulates rapidly through post-transcriptional mechanisms following cellular exposure to DNA damaging agents and is also activated as a transcription factor leading to growth arrest or apoptosis. Phosphorylation of p53 occurs after DNA damage thereby modulating its activity and impeding the interaction of p53 with its negative regulator oncogene Mdm2. The serines 15 and 37 present in the amino terminal region of p53 are phosphorylated by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. In order to verify if specific p53 mutations occur in the multi-drug resistance phenotype, we analysed the p53 gene in two T-lymphoblastoid cell lines, CCRF-CEM and its multi-drug-resistant clone CCRF-CEM VLB100, selected for resistance to vinblastine sulfate and cross-resistant to other cytotoxic drugs. Both cell lines showed two heterozygous mutations in the DNA binding domain at codons 175 and 248. The multi-drug resistant cell line, CCRF-CEM VLB100, showed an additional mutation that involves the serine 37 whose phosphorylation is important to modulate the protein activity in response to DNA damage. The effects of these mutations on p53 transactivation capacity were evaluated. The activity of p53 on pro-apoptotic genes expression in response to DNA damage induced by (-irradiation, was affected in the vinblastine (VLB) resistant cell line but not in CCRF-CEM sensitive cell line resulting in a much reduced apoptotic cell death of the multi-drug resistant cells.

Amino Acid Substitution↗
PubMed2000

Mutations in the retinoblastoma-related gene RB2/p130 in lung tumors and suppression of tumor growth in vivo by retrovirus-mediated gene transfer.

The retinoblastoma (Rb) family consists of the tumor suppressor pRb/p105 and related proteins p107 and pRb2/p130. Recent immunohistochemical studies of the retinoblastoma family of proteins in 235 specimens of lung cancer show the tightest inverse association between the histological grading in the most aggressive tumor types and pRb2/p130. This led us to study a panel of human lung cancers for mutations in the RB2/p130 gene. Mutations in the Rb-related gene RB2/p130 were detected in 11 of 14 (78.5%) primary lung tumors by single-strand conformation polymorphism and sequence analysis. A Moloney leukemia virus-based retroviral system was set up, and a comparable viral concentration of 1 x 10(7) infectious units/ml was obtained. Retrovirus-mediated delivery of wild-type RB2/p130 to the lung tumor cell line H23 potently inhibited tumorigenesis in vitro and in vivo, as shown by the dramatic growth arrest observed in a colony assay and the suppression of anchorage-independent growth potential and tumor formation in nude mice. The tumors transduced with the RB2/p130 retrovirus diminished in size after a single injection, and a 12-fold reduction in tumor growth after RB2/p130 transduction compared with the Pac-transduced tumors (92% reduction, P = 0.003) and lacZ-transduced tumors (93% reduction, P < 0.001) was found to be statistically significant. These findings provide the missing confirmation that RB2/p130 is a "bona fide" tumor suppressor gene and strengthen the hypothesis that it may be a candidate for cancer gene therapy for lung cancer.

Adenocarcinoma↗
PubMed2000

Genetic alterations disrupting the nuclear localization of the retinoblastoma-related gene RB2/p130 in human tumor cell lines and primary tumors.

The prototypic tumor suppressor gene, the retinoblastoma gene (RB/ p105), is mutated in a variety of human tumors. However, to date, mutational data on retinoblastoma family members p107 and RB2/p130 in tumors is lacking. We studied the expression of pRb2/p130 by immunocytochemistry and Western blot analysis in a panel of human osteosarcoma and lymphoid cell lines. Only the lymphoid cell lines showed an abnormal cytoplasmic localization of pRb2/p130, suggesting possible alterations within the region of nuclear localization signaling. We screened these cell lines for genetic alterations of the RB2/p130 gene in the region of the putative bipartite nuclear localization signal (NLS). This region is highly homologous with that of the RB/p105 gene. In addition, we screened four primary Burkitt's lymphomas for genetic alterations in the RB2/p130 gene. Naturally occurring mutations, which disrupt the putative bipartite NLS, were found in lymphoma cell lines and primary tumors, but not in the osteosarcoma cell lines, where normal nuclear localization of the protein was detectable. Site-directed mutagenesis and transfection assay using NLS mutants displayed markedly reduced biological activity as measured by flow cytometric analysis. This study clearly describes RB2/ p130 as an important target for mutations and subsequent inactivation in lymphoma pathogenesis, thus validating that RB2/p130 is a classical tumor suppressor gene.

Amino Acid Substitution↗
PubMed2000

Hepatitis C virus infection and myositis: a polymerase chain reaction study.

Muscle biopsy tissue from a patient with chronic hepatitis, who was hepatitis C virus (HCV) positive and showed slight weakness of the right arm and leg associated with increased serum creatine kinase levels, was studied using immunocytochemical and polymerase chain reaction (PCR) techniques. Muscle biopsy showed changes compatible with an inflammatory myopathy. Immunohistochemical studies included the use of monoclonal antibodies against human T lymphocytes, macrophages, immunoglobulins, major histocompatibility complex class I molecules (MHC-I), and the neoantigens of the terminal C5b-9 complement membrane attack complex (MAC). In addition to confirming the potential importance of cytotoxic T cells and MHC-I antigen expression in inducing muscle pathology, we demonstrated MAC deposition and the presence of HCV-RNA in the muscle of our patient, suggesting that direct involvement of the virus leading to complement activation might be important in inducing muscle damage.

Adult↗
PubMed2000

Emerin presence in platelets.

Emerin is an almost ubiquitous protein which is abnormal in X-linked Emery-Dreifuss muscular dystrophy (EMD), a syndrome characterized by muscle weakness, joint contractures and cardiac arrhythmia. Emerin is localized in the cells at the nuclear rim and its function is still unknown. In some models, emerin has also been described in the cytoplasm; however, its presence outside the nucleus is still matter of debate. We report the presence of emerin in circulating normal human platelets and its absence in platelets from X-linked EMD patients. Since platelets are cytoplasmic fragments derived from megakaryocytes, the presence of emerin in platelets confirms cytoplasmic localization of this protein, probably related to specific functions. We found also that emerin is present in the cytoplasm of megakaryocytes, while it is absent in circulating granulocytes.

Blood Platelets↗
PubMed2000

pUL25 immunolocalization in human cytomegalovirus-infected and gene-transfected cells.

By means of confocal and electron microscope immunocytochemistry we have studied the localization of a recently described structural protein (pUL25) of human cytomegalovirus, in both infected cells and in cells transiently transfected with UL25. pUL25 localization in infected cells was observed in typical cytoplasmic structures characterized by a very electrondense texture previously reported to accumulate other tegument proteins. At the virion level pUL25 seems to localize at the interface between the tegument and the capsid of both intracytoplasmic and extracellular virions. In UL-25-transfected cells, pUL25 has been found in characteristic para-crystalline cytoplasmic aggregates, suggesting its intrinsic ability to aggregate in a regular subunit pattern.

Animals↗
PubMed2000

Genetic alterations of the retinoblastoma-related gene RB2/p130 identify different pathogenetic mechanisms in and among Burkitt's lymphoma subtypes.

Alterations of cell cycle-associated genes probably contribute to the pathogenesis of Burkitt's Lymphoma (BL), in addition to c-myc translocation. Mutations disrupting the nuclear localization signal of the retinoblastoma-related gene RB2/p130 have been documented recently in BL cell lines and primary tumors. Given the importance of the RB2/p130 gene in controlling cell growth, mutations of this gene may result in uncontrolled cell proliferation. We tested the expression and genomic organization of the RB2/p130 gene in relation to the proliferative features of a series of BL samples collected from the endemic and sporadic regions, regardless of whether the samples were acquired immune deficiency syndrome (AIDS)-related. The expression of the Rb2/p130, p107, and cell proliferation-related proteins (cyclin A and B) was determined by immunohistochemistry. The structures of exons 19 through 22 of the RB2/p130 gene, encoding for the B domain and C terminus, were analyzed by polymerase chain reaction (PCR) analysis and single-strand conformation polymorphism (SSCP) technique. The direct PCR products were sequenced to identify the actual mutations. Our results suggest that BL is composed of a mixture of molecular types with distinct genetic and phenotypic patterns, probably resulting from different pathogenetic mechanisms. In endemic BL, the RB2/p130 gene is mutated in most of the cases, and the protein is restricted to the cytoplasm. In AIDS-related BL, high levels of nuclear expression of the wild-type pRb2/p130, p107, and cell proliferation-related proteins were detected. This finding is in line with the molecular mechanisms observed in virus-linked oncogenesis. Sporadic BLs were mainly characterized by the low nuclear values of the wild-type pRb2/p130 and, conversely, the high values of p107. The increased cell proliferation due to different alterations of cell growth control by Rb-related proteins may be the first step in lymphomagenesis, during which additional genetic changes, including missense mutations of c-myc, may subsequently occur.

Adolescent↗
PubMed2000

Congenital muscular dystrophy associated with calf hypertrophy, microcephaly and severe mental retardation in three Italian families: evidence for a novel CMD syndrome.

We describe four Italian patients (aged 3, 4, 12, and 13 years ) affected by a novel autosomal form of recessive congenital muscular dystrophy. These patients were from three non-consanguineous families and presented an almost identical phenotype. This was characterized by hypotonia at birth, joint contractures associated with severe psychomotor retardation, absent speech, inability to walk and almost no interest in their surroundings. In addition, all patients had a striking enlargement of the calf and quadriceps muscles. Ophthalmologic examination revealed no structural ocular abnormalities in any of the children; one patient had severe myopia. In all cases a magnetic resonance imaging of the brain showed an abnormal posterior cranial fossa with enlargement of the cisterna magna and variable hypoplasia of the vermis of the cerebellum. Abnormality of the white matter was also present in all patients, in the form of patchy signal most evident in the periventricular areas. Serum CK was grossly elevated in all. The muscle biopsy from all cases showed dystrophic changes compatible with congenital muscular dystrophy. Immunofluorescence studies showed mild to moderate partial deficiency of laminin alpha 2 chain. Linkage analysis in the only informative family excluded the known loci for congenital muscular dystrophy, including laminin alpha 2 chain on chromosome 6q2, the Fukuyama congenital muscular dystrophy locus on 9q3 and the muscle-eye-brain disease on chromosome 1p3. We propose that this represent a novel severe variant of congenital muscular dystrophy, with associated central nervous system involvement.

Adolescent↗
PubMed2000

Inhibition of phosphoinositide 3-kinase impairs pre-commitment cell cycle traverse and prevents differentiation in erythroleukaemia cells.

During the early hours after exposure to differentiation inducing agents, Friend erythroleukaemia cells undergo alterations which commit them to cessation of growth and development of the characteristics of differentiation. Our current experiments have compared the expression and activity of phosphoinositide 3-kinase (PI 3-kinase) in control cells with cells undergoing differentiation which has been induced by dimethyl sulfoxide (DMSO). When the cultures were initiated with stationary phase cells and DMSO was added at the time of seeding, PI 3-kinase activity was stimulated in both treated and control cells during the first 3 h from seeding. This event appears to be a rate limiting step in commitment since pretreatment of cells with 10 microM LY294002 or down-regulation of p85 expression prior to adding DMSO completely prevents commitment to erythropoiesis. Accordingly, PI 3-kinase inhibition during the commitment period prevents DNA-binding of the transcription factor GATA-1, essential for erythroid differentiation. However, once cells are committed to differentiate, PI 3-kinase activity and expression dramatically decreases along with the differentiation programme, to become barely detectable after 96 h. Remarkably, LY294002 treatment leads to accumulation of cell in G1 phase and prevents DMSO-dependent cyclin D3 induction. Based on these data, we suggest that PI 3-kinase is rate limiting for the completion of the first round cycle of cell division required for initiation of erythrocytic differentiation. On the other hand, the late decrease of PI 3-kinase associated with the differentiation process seems to be part of the programmed shut off of genes not needed in mature erythrocytes.

Androstadienes↗