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At least 451 records · Page 25Linked to original sources
PubMed2001

Quantitative cytochemical mapping of mitochondrial enzymes in rat cerebella.

Mitochondrial metabolic competence, defined as the organelle's capacity to provide adequate amounts of ATP in due time, appears to constitute an important determinant in several biological processes and pathological conditions. Thus, the assessment of the metabolic efficiency of the mitochondrial population in a given tissue area or cellular compartment may provide clues to identifying alterations of the cellular bioenergetic machinery, which may constitute a predisposing condition leading to impaired organ and system functions. In the cerebellar cortex of adult rats, the activities of the enzymes cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were, respectively, evidenced by means of the diaminobenzidine and copper ferrocyanide preferential cytochemical techniques. At the electron microscope, the activities of these two key molecules of the respiratory chain were clearly visualised as dark precipitates at the inner mitochondrial membrane sites where COX and SDH are located. By means of the disector method, unbiased mitochondrial samplings were carried out to measure: the number of mitochondria/microm(3) of tissue (numeric density: Nv); the mitochondrial volume fraction/microm(3) of tissue (volume density: Vv) and the average mitochondrial volume (V) both on COX- and SDH-positive organelles in the cerebellar glomeruli and Purkinje cells, respectively. The ratio R (total area of the precipitates due either to COX or SDH activity within the single mitochondrion/area of the same organelle) was also evaluated to get information on the enzyme activity related to mitochondrial size.The documented accumulation of mutant mitochondrial DNA particularly in postmitotic cells results in a marked heteroplasmy (mixtures of normal and mutated genomes) at mitochondrial and cellular levels, thus the cellular potential for energy production is demanded to a mosaic of organelles with different functional capabilities. Assessment of the mitochondrial mosaic outline by means of quantitative cytochemistry of key enzymes of the respiratory chain, such as COX and SDH, may allow for the morphofunctional metabolic mapping of mitochondrial efficiency in discrete cellular or tissue compartments.

Animals↗
PubMed2001

Correlation between p21(waf1) and p16(INK4a) expression in hepatocellular carcinoma.

Background: The cyclin dependent kinase p21(waf1) plays a crucial role in the regulation of cell cycle. The family of p53 proteins has the ability to induce p21(waf1), whereas p16(INK4a) modulates post-transcriptionally the expression of p21(waf1). Methods: Total 36 hepatocellular carcinomas (HCCs) and 24 paired adjacent liver tissues were evaluated for the following: (1) expression of p21(waf1) and p16(INK4a); (2) that of p21(waf1), p73 and p63 mRNAs; (3) genomic mutations and the loss of heterozygosity of p73 and p53; and (4) frequency of methylation in the 5'CpG promoter region of p16(INK4a). Results: In HCCs compared with the adjacent non-cancerous liver tissues, the expression of p21(waf1) and p16(INK4a) was reduced. Indeed, p21(waf1) was not detected in 36% (8/22) of HCCs in spite of the presence of p21(waf1) mRNA: among them, mutations of p53 gene were found in 50%, whereas a lack of p16(INK4a) expression in all of them. p21(waf1) and p16(INK4a) were reduced in proportion to the degree of methylation in p16(INK4a) gene. p73 did not mutated, and p63 did not expressed in HCCs. Conclusion: Methylation status of p16(INK4a) gene will play a part for reducing constitutive expression of p16(INK4a) and of p21(waf1) coordinately in HCCs.

Journal Article↗
PubMed2004

Genotoxicity of melanoidin fractions derived from a standard glucose/glycine model.

Genotoxic compounds can act at various levels in the cell (causing gene, chromosome, or genome mutations), necessitating the use of a range of genotoxicity assays designed to detect these different types of mutations. The production of melanoidins during the processing and cooking of foods is associated with changes in their nutritional character, and the discovery of mutagenic substances in pyrolyzed protein and amino acids has raised concern about the safety of these foods. The aim of this work was to test melanoidin fractions in three different in vitro assays (Ames test, Vitotox test, and micronucleus test). These melanoidin fractions were produced from the condensation of glucose with glycine and their separation was conducted by dialysis. The crude reaction mixture (before dialysis) and both the LMW and HMW fractions obtained by dialysis showed no genotoxicity in these assays, despite being tested at concentrations much higher than those naturally found in food products. The LMW fraction, however, showed toxicity at these high concentrations. The volatile fraction produced in this reaction showed genotoxicity only in the Vitotox test, at high concentrations.

Dialysis↗
PubMed2000

Disorders related to mitochondrial membranes: pathology of the respiratory chain and neurodegeneration.

Faulty oxidative phosphorylation (OXPHOS) is observed in a number of mitochondrial disorders, and may be associated with single or multiple defects of the five complexes of the respiratory chain. From the genetic standpoint, the respiratory chain is unique as it is formed by means of the complementation of two separate genetic systems: the nuclear genome and the mitochondrial genome. The nuclear genome encodes most of the protein subunits of the respiratory complexes and most of the mtDNA replication and expression systems, whereas the mitochondrial genome encodes only 13 OXPHOS subunits and some RNA components of the mitochondrial translation apparatus. Accordingly, mitochondrial disorders due to defects in oxidative phosphorylation include both Mendelian-inherited and cytoplasmic-inherited diseases. Although our knowledge of these diseases has grown at an impressive rate in the past few years, it is worth noting that, as a result of complementation in the respiratory chain of nuclear-encoded and mitochondrially-encoded polypeptides, it is often difficult to establish a precise relationship between genomic mutations and biochemical phenotypes, or to distinguish pathogenic mutations from polymorphic variants in gene sequences. This review deals with human diseases due to mutations of the structural components of the respiratory chain, particularly focusing on the recently identified disorders caused either by mutations in nuclear genes encoding subunits of the subcomplexes or by mutations in nuclear genes affecting the functional efficiency, homeostasis, and assembly of respiratory chain subcomplexes. To better focus on OXPHOS genotype-phenotype correlations, mutations of the mtDNA-encoded structural genes are also discussed.

ATPases Associated with Diverse Cellular Activitie↗
PubMedDate not supplied

The role of neutral sphingomyelinase in interleukin-1beta signal transduction in mouse cerebral cortex cells.

The cytokine interleukin-1 (IL-1) is an important mediator of neuroimmune interactions, though it has not been established precisely how the IL-lbeta signal is transmitted in nerve cells. This study demonstrates the involvement of the sphingomyelin cascade in IL-1beta signal transduction in the P2 membrane fraction of the mouse cerebral cortex. The key role of the membrane enzyme neutral sphingomyelinase in initiating the sphingomyelin signal transduction pathway for this cytokine is supported. The stimulating activity of IL-1beta on sphingomyelinase activity in the P2 fraction of the cerebral cortex was found to be dose-dependent. Studies using this membrane fraction from mice lacking the IL-1 type I receptor due to genomic mutations, along with studies using an IL-1 receptor antagonist. yielded data showing that IL-1beta binding with the type I receptor is a necessary event for activation of neutral sphingomyelinase. The results obtained here lead to the conclusion that the action of IL-1beta in the CNS is mediated by the IL-1 type I receptor and activation of neutral sphingomyelinase as the initiating enzyme of the sphingomyelin cascade.

Animals↗
PubMed2004

Dpo4 is hindered in extending a G.T mismatch by a reverse wobble.

The ability or inability of a DNA polymerase to extend a mispair directly affects the establishment of genomic mutations. We report here kinetic analyses of the ability of Dpo4, a Y-family polymerase from Sulfolobus solfataricus, to extend from all mispairs opposite a template G or T. Dpo4 is equally inefficient at extending these mispairs, which include, surprisingly, a G.T mispair expected to conform closely to Watson-Crick geometry. To elucidate the basis of this, we solved the structure of Dpo4 bound to G.T-mispaired primer template in the presence of an incoming nucleotide. As a control, we also determined the structure of Dpo4 bound to a matched A-T base pair at the primer terminus. The structures offer a basis for the low efficiency of Dpo4 in extending a G.T mispair: a reverse wobble that deflects the primer 3'-OH away from the incoming nucleotide.

Bacterial Proteins↗
PubMed2003

Aberrant caspase-activated DNase (CAD) transcripts in human hepatoma cells.

The gene of caspase-activated DNase (CAD), the key enzyme for nucleosome cleavage during apoptosis, is mapped at chromosome 1p36, a region usually associated with hemizygous deletions in human cancers, particularly in hepatoma (HCC). It is tempting to speculate that CAD plays a tumour-suppressive role in hepatocarcinogenesis. To address this, we examined the CAD transcripts in six human HCC cell lines, one liver tissue from a non-HCC subject, and peripheral blood leukocytes (PBL) from three healthy individuals. Alternatively spliced CAD transcripts with fusion of exon 1 to exon 7 were isolated in most of the examined samples including HCC cells and normal controls. However, relatively abundant alternatively spliced CAD transcripts with fusion of exon 2 to exon 6 or 7, in which the corresponding domain directing CAD interaction with ICAD was preserved, were found only in poorly differentiated Mahlavu and SK-Hep1 cells. Interestingly, an abnormal CAD transcript with its exon 3 replaced by a truncated transposable Alu repeat was isolated in Hep3B cells, indicative of the implication of an Alu-mediated genomic mutation. Moreover, mis-sense mutations in the CAD genes were identified in all six HCC cell lines. Upon UV-induced apoptosis, DNA fragmentation efficiency was found to be intact, partially reduced and remarkably reduced in Huh7 and J328, Hep3B and HepG2, and Mahlavu cells, respectively. That mutations and aberrantly spliced transcripts for the CAD gene are frequently present in human HCC cells, especially in poorly differentiated HCC cells, suggests a significant role of CAD in human hepatocarcinogenesis.

Alternative Splicing↗
PubMed2004

The peptide nucleic acids (PNAs), powerful tools for molecular genetics and cytogenetics.

Peptide nucleic acids (PNAs) are synthetic mimics of DNA in which the deoxyribose phosphate backbone is replaced by a pseudo-peptide polymer to which the nucleobases are linked. PNAs hybridize with complementary DNAs or RNAs with remarkably high affinity and specificity, essentially because of their uncharged and flexible polyamide backbone. The unique physico-chemical properties of PNAs have led to the development of a variety of research assays, and over the last few years, the use of PNAs has proven their powerful usefulness in molecular biology procedures and diagnostic assays. The more recent applications of PNA involve their use as molecular hybridization probes. Thus, several sensitive and robust PNA-dependent methods have been designed for developing antigene and anticancer drugs, modulating PCR reactions, detecting genomic mutation or labelling chromosomes in situ.

Cytogenetics↗
PubMed2003

Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene.

Integration of Hepatitis B Virus (HBV) DNA into liver cell DNA has been well established, but its implication in liver carcinogenesis is still being debated. In particular, insertion of the viral genome into cellular genes has been viewed as a rare event. By using HBV-Alu PCR, we have now isolated, from nine hepatocellular carcinomas, nine HBV-DNA integration sites showing that the viral genome mutates key regulatory cellular genes: neurotropic tyrosin receptor kinase 2 (NTRK2) gene, IL-1R-associated kinase 2 (IRAK2) gene, p42 mitogen-activated protein kinase 1 (p42MAPK1) gene, inositol 1,4,5-triphosphate receptor type 2 (IP3R2) gene, inositol 1,4,5-triphosphate receptor (IP3R) type 1 (IP3R1) gene, alpha 2,3 sialyltransferase (ST3GAL VI or SITA) gene, thyroid hormone uncoupling protein (TRUP) gene, EMX2-like gene, and human telomerase reverse transcriptase (hTERT) gene. This result brings to 15 the total number of genes targeted by HBV in a study of 22 human liver cancers. Overall, we found that both the inositol 1,4,5-triphosphate receptor gene and the telomerase gene were targeted by HBV in two different tumors. Thus, HBV frequently targets cellular genes involved in cell signalling and some of them may be preferential targets of the viral integration.

Calcium Channels↗
PubMed2003

A novel technique for the examination of skin biopsies by laser capture microdissection.

BACKGROUND: Skin is an inherently heterogeneous tissue, thus the procurement of pure cell populations is critical for the accurate correlation of a molecular profile to a particular cell type or histological location. Laser Capture Microdissection (LCM) permits the efficient procurement of cells and mapping of genetic changes from histologically prepared samples. METHODS: This paper describes a robust LCM protocol established in our laboratory for the extraction of high quality DNA which sequenced from 100% of microdissected samples without the need for cloning. The unique properties of skin, in particular its strong intercellular adhesive forces, have dictated a significant modification to the normal procedure of tissue preparation to ensure reliable cell procurement. RESULTS: Using the methods outlined below we were able to precisely map the pattern of genomic mutations in our target gene of interest in normal skin, actinic keratosis and squamous cell carcinoma. CONCLUSIONS: The capability to select pure cell populations from the skin will revolutionise our ability to understand the processes involved in cutaneous tumourigenesis.

Biopsy↗
PubMed2002

Apoptosis mediated by the Fas system in the fulminant hepatitis by hepatitis B virus.

The pathogenesis of the fulminant hepatitis B is poorly understood and both viral factors and the hosts immune response play a role. Previous studies in liver tissues of patients with chronic hepatitis B showed overexpression of Fas antigen and this was correlated with the activity of the hepatitis. The present study was done to determine the role of Fas in fulminant hepatitis B and the virological characteristics of hepatitis B infection. We studied three patients with fulminant hepatitis B. HBV-DNA was detected by dot-blot hybridization and polymerase chain reaction. The S and C gene were sequenced. Levels of serum soluble Fas antigen were detected by enzymoimmunoassays procedure. Apoptosis was determined by the TUNEL technique. Fas antigen expression was evaluated by a immunoperoxidase method. Ten healthy subjects acted as controls. The three patients showed a high expression of Fas antigen particularly among infiltrating lymphocytes; in these areas we also found many cells with in situ DNA nick labelling signals in the nuclei of most viable hepatocytes. Serum levels of soluble Fas antigen were higher in patients with fulminant hepatitis B than in controls. No specific genome mutations of hepatitis B virus were found. These data suggest that the Fas system involved in the liver injury of patients with fulminant hepatitis B.

Adult↗
PubMed2002

Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation.

Transformation of plant cells by Agrobacterium tumefaciens involves both bacterial virulence proteins and host proteins. We have previously shown that the Arabidopsis thaliana gene H2A-1 (RAT5), which encodes histone H2A-1, is involved in T-DNA integration into the plant genome. Mutation of RAT5 results in a severely decreased frequency of transformation, whereas overexpression of RAT5 enhances the transformation frequency (Mysore et al., 2000b). We show here that the expression pattern of RAT5 correlates with plant root cells most susceptible to transformation. As opposed to a cyclin-GUS fusion gene whose expression is limited to meristematic tissues, the H2A-1 gene is expressed in many non-dividing cells. Under normal circumstances, the H2A-1 gene is expressed in the elongation zone of the root, the region that is most susceptible to Agrobacterium transformation. In addition, when roots are incubated on medium containing phytohormones, a concomitant shift in H2A-1 expression and transformation susceptibility to the root base is observed. Inoculation of root segments with a transfer-competent, but not a transformation-deficient Agrobacterium strain induces H2A-1 expression. Furthermore, pre-treatment of Arabidopsis root segments with phytohormones both induces H2A-1 expression and increases the frequency of Agrobacterium transformation. Our results suggest that the expression of the H2A-1 gene is both a marker for, and a predictor of, plant cells most susceptible to Agrobacterium transformation.

Agrobacterium tumefaciens↗
PubMed2003

Disordered hematopoiesis and myelodysplasia in the elderly.

Normal hematopoiesis constitutes the process of producing diverse, differentiated blood cell types in a manner related to physiological requirement. During aging, modulation of hematopoiesis becomes disordered, impairing the ability of older people to respond appropriately to the physiological demand for blood cell replacement triggered by stimuli such as blood loss or cytoreductive chemotherapy. This may contribute to the increase in the prevalence of anemia that is observed during aging. In addition, various age-related events, such as genomic mutations secondary to oxidative stress and impaired regulation of cytokine production, may contribute to or cause the emergence of abnormal clones of hematopoietic cells. Therefore, normal hematopoiesis is disrupted, and the hematopoietic system is populated with cells that are quantitatively and functionally deficient and are also subject to leukemic transformation. These defects in the production and maturation of the various differentiated blood cells are referred to as myelodysplastic syndromes. These syndromes are so tightly associated with aging that they are considered to be geriatric disorders; they can lead to anemia, neutropenia, and thrombocytopenia and to the development of acute nonlymphoblastic leukemia. Dysregulation of mechanisms controlling hematopoiesis is therefore an important characteristic of the hematopoietic system in the elderly, but the response of progenitor cells to humoral stimulators is preserved and accounts for the effectiveness of recombinant hematopoietic growth factors used as emerging treatment modalities for hematopoietic disorders in the elderly.

Aged↗
PubMed2001

Molecular characterization and sequence analysis of polyomavirus BKV-strain in a renal-allograft recipient.

The significance of polyomavirus (PV) infection was investigated in a 53-year-old patient who underwent renal transplantation and was treated with triple immunosuppressive therapy (tacrolimus, prednisone, and azathioprine). A renal biopsy taken because of the suspicion of acute rejection showed focal inflammatory interstitial infiltration, tubulitis, and tubular cell nuclear changes consistent with the hypothesis of viral infection. Both the tubular and decoy cells identified by means of urinalysis positively stained for anti-SV40 antibody. Polymerase chain reaction performed on the DNA extracted from renal tissue and isolated from urine showed the presence of an antigenic variant (AS) of the BKV archetype after sequence analysis of the transcription control region (TCR). On the basis of the diagnosis of BKV infection, immunosuppressive therapy was reduced. The patient's renal function improved and was still stable 8 months later when urinalysis showed only a few decoy cells, which were found to be infected by JC but not BK virus. These data suggest that only the BKV, probably favoured by immunosuppressive therapy (tacrolimus), causes renal damage. It is worth underlining that even small and sporadic viral genome mutations may lead to pathologic effects.

BK Virus↗
PubMed2006

Biological insights into the pathogenesis of cutaneous T-cell lymphomas (CTCL).

Mycosis fungoides and Sezary syndrome, collectively known as cutaneous T-cell lymphomas (CTCLs), are low-grade, indolent, clonal, non-Hodgkin's lymphomas consisting of CD4+ CD45RO+ T cells with a CLA+ CCR4+ skin-homing phenotype. There are several variants of primary CTCLs with differences in clinical behavior and prognosis. Currently, the precise etiologies of mycosis fungoides and Sezary syndrome are unknown. This article reviews our current understanding of the pathogenetic abnormalities involving genomic mutations, abnormal cDNA expression, and dysregulation of signaling pathways in CTCL.

Humans↗
PubMed2000

Molecular evaluation of the gonadotropin-releasing hormone receptor.

The gonadotropin-releasing hormone receptor (GnRHR) is a guanine nucleotide-binding protein-coupled receptor with a characteristic seven transmembrane domain motif. It transduces the hypothalamic message carried by the decapeptide gonadotropin-releasing hormone. At the gonadotrope cell surface the hormone binds to the receptor, leading to pituitary synthesis and secretion of gonadotropins. These glycoprotein hormones, in turn, modulate folliculogenesis and ovulation in the ovary. Failure of the GnRHR to respond appropriately to its cognate ligand has been demonstrated in humans. These patients have genomic mutations in the gene for the GnRHR and represent the first individuals with hypogonadotropic hypogonadism of autosomal inheritance. The mutant receptors are poorly expressed at the cell surface and have a diminished capacity to transduce the hypothalamic message efficiently. To date, no mutations have been identified that lead to constitutively active receptors and autonomous gonadotropin function. This article identifies the mutations in the GnRHR reported to date and reviews how these abnormal receptors help us to better understand the biology of this interesting molecule.

Female↗
PubMed2003

Subacute sclerosing panencephalitis in two brothers.

We report the occurrence of subacute sclerosing panencephalitis (SSPE) in two brothers two years after measles infection. The diagnosis was confirmed by compatible data from medical history, occurrence of autochthonic measles virus (MV) IgG production in the central nervous system (CNS), and pathognomonic EEG changes. Pathogenetically, SSPE is caused by a genome mutation of intracellularly persisting MV, causing viral nucleocapsides to accumulate in the brain cells. A specific predisposing immune defect is not known. The occurrence of two cases in one family is suggestive of a genetic predisposing factor.

Child↗
PubMed2006

New nucleic acid diagnostic tests in viral hepatitis.

The development of nucleic acid assays is of critical clinical importance for the diagnosis of viral hepatitis and the management of antiviral therapy. For both chronic hepatitis B and C, sensitive and quantitative assays are required for the monitoring of viral load. Better assays are being developed and their role in the management of antiviral treatment has been clearly demonstrated. Genotyping assays are critical for the decision of treatment strategies in hepatitis C, although in hepatitis B this issue is more debated. The detection of viral genome mutations for the diagnosis of hepatitis B virus resistance is critical with the use of nucleoside analogs. This may also prove useful for anti-hepatitis C virus therapy with the rapid development of specific inhibitors that may select for drug-resistant mutants.

Hepatitis B, Chronic↗