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At least 1,315 records · Page 73Linked to original sources
PubMed1995

Ontogeny of membrane cofactor protein: phenotypic divergence in the fetal heart.

Human adult cells are protected from complement-induced damage in part by membrane cofactor protein (MCP, CD46). To examine fetal characteristics which might influence autoantibody-mediated diseases acquired in utero, such as heart block in neonatal lupus, the tissue expression of MCP was studied. Using a high ratio of acrylamide:bisacrylamide, immunoblots of tissues from six fetuses (aged 19-24 weeks) probed with rabbit anti-MCP antibodies revealed a band at 60 KD in addition to the known 65 KD and 55 KD isoforms which comprise the codominant allelic system of MCP. Five fetuses expressed the most common MCP polymorphism (predominance of the 65 KD isoform, upper band alpha-phenotype) in the kidney, spleen, liver and lung. In contrast, all hearts from these five fetuses demonstrated a different pattern in which there was a marked decrease in the intensity of the 65 KD band and accentuation of the lower molecular weight bands. In a sixth fetus, which expressed the second most common polymorphism (equal expression of the 65 KD and 55 KD MCP isoforms, alpha beta-phenotype), the heart was similar to the other tissues. These studies confirm the expression of MCP in early gestational life. Preferential expression of the MCP beta-isoform in the majority of fetal hearts irrespective of the phenotype of other organs, suggests tissue-specific RNA splicing or post-translational modification which may relate to autoantibody-mediated injury in diseases such as neonatal lupus.

Antigens, CD↗
PubMed2006

Epigenetic regulation of stem cell differentiation.

Stem cells undergo extensive self-renewal and have the capacity to differentiate along multiple cell lineages. Progression from stem cells into differentiated progeny requires long-lasting changes in gene expression. Epigenetic mechanisms, including DNA methylation, histone modifications, and non-coding RNA-mediated regulatory events, are essential to controlling the heritable cellular memory of gene expression during development. Recent studies on cell fate specification of embryonic and adult stem cells/progenitors have highlighted a general and critical role for dynamic epigenetic regulation in stem cell self-renewal and differentiation.

Adult↗
PubMed2005

Telomerase: is it the future diagnostic and prognostic tool in human cancer?

A number of methods exist to detect levels of telomerase activity and the presence of telomerase subunits in a variety of tissues. As telomerase activation seems to be an important step in tumorigenesis, accurate detection of the presence and activity of the enzyme and its subunits is vital. The original method of detecting telomerase activity was developed by Kim and coworkers in 1994, and was termed the telomeric repeat amplification protocol. This assay led to a staggering increase in the number of telomerase-associated publications in scientific journals (85 publications from 1974-1994, 5063 publications from 1994-2004). A number of methods have been described to detect telomeres and to measure their length, with the standard measurement of telomere length performed using a modification of the Southern blot protocol. RNA in situ hybridization can be performed to detect levels of the RNA component of telomerase, and standard in situ hybridization and immunohistochemistry can be applied to examine expression levels and localization of the catalytic subunit of the enzyme. Reverse transcriptase PCR has also been applied to assess expression levels of the telomerase components in various tissues. This review provides a synopsis of telomeres, telomerase, telomerase and cancer, and finally, methods for the detection of telomerase in cancer.

Animals↗
PubMed2003

Novel phosphoramidite building blocks in synthesis and applications toward modified oligonucleotides.

In recent years modified oligonucleotides (oligos) have become an area of increased research activity. These synthetic biopolymers have long been used as molecular probes to understand better the interaction between proteins and nucleic acids at molecular level, which in fact is responsible for all aspects of cellular or viral gene expression. Modified oligos have also been used as therapeutic and diagnostic agents. For example, the antisense oligos are used to block gene expression by binding especially with complimentary sequences of target mRNA. Also, over the past decade efforts are made to modify the native phosphodiester linkages to improve nuclease resistance and membrane permeation of naturally occurring oligonucleotide for therapeutic applications. Therefore, design and synthesis of modified oligonucleotides for a wide range of applications are of interest for bioorganic chemists and biologists. The advent of the phosphoramidite chemistry has accelerated the development of oligos with modified nucleobases, phosphate-protecting groups and modified sugars. In this review, we have presented the recently reported nucleoside based phosphoramidites with modified base, sugar or backbone units and their subsequent conversion into modified DNA and RNA analogs. The effect of such modification on structural, thermodynamic, and hybridization properties of the modified oligos and their duplex with natural complementary oligos have also been discussed for some cases. Similarly, we have highlighted the role of modified nucleoside phosphoramidite building blocks in synthesis of oligos modified with optically or electrochemically active molecules. Nucleoside phosphoramidites or small oligos immobilized over solid supports through different linkages or groups and their biological applications has also been mentioned.

Amides↗
PubMed1999

The cartilage-specific (V+C)- fibronectin isoform exists primarily in homodimeric and monomeric configurations.

Fibronectin is an extracellular-matrix glycoprotein encoded by a single gene, but with significant protein heterogeneity introduced through alternative RNA splicing and post-translational modifications. The (V+C)(-) splice variant, in which nucleotides encoding protein segments III-15 and I-10 are deleted along with the entire variable region, is unique in that expression is restricted to cartilaginous tissues. All known fibronectin splice variants retain the two C-terminal cysteine residues essential for dimerization, but cellular and/or structural constraints appear to influence homo- and heterodimerization patterns. Dimerization patterns of the (V+C)(-) isoform were studied under native conditions within canine articular cartilage and experimentally in COS-7, NIH-3T3 and CHO-K1 cell cultures. In all systems, (V+C)(-) fibronectin secretion was predominantly in a homodimeric configuration. Lower levels of (V+C)(-) monomers were also present. Heterodimers of (V+C)(-) with V(+),C(+) (V120) isoforms were not detected. Heterodimers of (V+C)(-) with V(-),C(+) (V0) subunits were detected only at low levels. Functional properties may differ significantly among monomers, homodimers and heterodimers. The unique dimerization pattern of (V+C)(-) fibronectin is consistent with this isoform having specialized functional properties in situ that are important for either the structural organization and biomechanical properties of cartilage matrix or regulation of a chondrocytic phenotype.

Animals↗
PubMedDate not supplied

Primary HIV infection: molecular approaches in diagnosis and monitoring.

OBJECTIVE: The aim of this study was to evaluate, in patients with primary HIV infection (PHI), the modification of HIV molecular parameters (HIV, RNA, and DNA) induced by highly active antiretroviral therapy (HAART) in peripheral blood mononuclear cells (PBMC) and in lymphoid tissue (LNMC). METHODS: Nineteen patients with primary HIV infection, 4 women and 15 men with an average age of 35 years (range 27-62), were included in this study. Ten patients received 4 drugs: zidovudine plus lamivudine plus saquinavir plus ritonavir, 7 patients received 3 drugs: zidovudine plus lamivudine plus saquinavir and 2 patients received a different combination of 3 drugs: zidovudine plus lamivudine plus indinavir. As control group we included 8 patients who had been enrolled in a placebo-controlled trial of zidovudine between 1991 and 1995: four received placebo and 4 were treated with zidovudine alone. Peripheral blood samples and lymphoid tissue obtained by echo-driven fine needle biopsies were drawn to monitor molecular HIV parameters. A quantitative in house PCR method in the HIV gag region was used to monitor viral DNA burden and the NASBA system for viremia. RESULTS: A certain heterogeneity in the baseline values of HIV, DNA, and RNA was observed. Early HAART determined a rapid recovery of the CD4 cell number with normalisation of the CD4/CD8 ratio in most patients. HIV-RNA levels dropped to undetectable levels after a few months of therapy and HIV-DNA was consistently reduced although it never reached undetectable levels. Lymph-node biopsies were well tolerated due to the non-invasive sampling, however an optimisation of the method is needed to improve cell recovery. In the valuable samples the amount of HIV DNA recovered is comparable to that from peripheral blood samples, both at baseline and at follow-up.

Adult↗
PubMed1995

[Identification of the breakpoint-flanking markers on chromosomes 1 and 17 of a constitutional translocation T(1;17)(P36;Q12-21) in a patient with neuroblastoma].

Neuroblastoma is a childhood cancer which originates in the embryonic tissue of the developing sympathetic neural crest. In 1972, Dr. A. Knudson hypothesised a similar 'two-hit mutation' model for the origin of neuroblastoma as for retinoblastoma and Wilms tumor. In this model, malignant cell growth is caused by mutations of both alleles of a tumor suppressor gene. In hereditary tumors, a germinal mutation is present in all cells of the individual, a mutation of the remaining allele by a somatic hit causes loss of gene function. Sporadic tumors result from two somatic mutations of a tumor suppressor gene involving both alleles within the same cell. The occurrence of patients with a constitutional chromosomal deletion syndrome in association with tumor facilitated the cloning of a retinoblastoma gene and of a Wilms tumor suppressor gene. In neuroblastoma, cytogenetic and molecular studies suggest the existence of a neuroblastoma (suppressor) gene at chromosome 1, at subband 1p36. A constitutional chromosomal deletion syndrome was not known for neuroblastoma. We described a constitutional chromosome translocation t(1;17)(p36.31-21; q11.2-12) in a patient with neuroblastoma. We hypothesised that this translocation, involving the chromosomal band 1p36, predisposed the patient to neuroblastoma development by disturbance of a gene located at the translocation breakpoint. Consequently, identification of the breakpoint flanking markers can be an important step towards the identification and cloning of a neuroblastoma suppressor gene. Radioactive in situ hybridization methods were first applied on the patient's fibroblasts. Soon it became evident that cells with better growth characteristics were needed and that the availability of sufficient patient material was essential. Therefore a somatic cell fusion experiment was performed between the patient's fibroblasts and a thymidine kinase-deficient Chinese hamster cell line. Somatic cell hybrid clones were selected on the presence of the derivative human chromosomes 1 and 17, and of the normal homologues. With the use of fluorescence in situ hybridisation (FISH), the position of chromosome 1 and chromosome 17 markers respective to the breakpoints was determined on chromosome metaphases of the hybrid cell lines containing the human derivative chromosomes. The pronatriodilatine (PND) and the adenovirus 12 modification site (A12M2) were identified as distal and proximal 'single copy' flanking markers of the chromosome 1 breakpoint, respectively. The chromosomal break occurred in a highly repetitive region containing an adenovirus modification site and genes encoding transfer RNA and small U1-RNA genes. The breakpoint on chromosome 17 is located in a region with as proximal boundary the distal part of the neurofibromatosis 1 (NF1) gene locus and as distal flanking marker the SCYA7 locus, encoding the monocyte chemotactic protein-3. Southern blot analysis showed no rearrangements of hybrid DNA using single copy probes for the four flanking markers. Identification of the four breakpoint flanking markers on chromosomes 1 and 17 constitutes a pivotal step for the cloning of the translocation breakpoints and for the identification of a presumed neuroblastoma suppressor gene.

Abdominal Neoplasms↗
PubMed1997

[Idiopathic CD4+ T-lymphocytopenia terminating in Burkitt's lymphoma].

A 33-year-old male was admitted because of severe neutropenia (55/microliter) in 1985. A diagnosis of autoimmune neutropenia was not made. CD4+ T-lymphocyte count was 128/microliter and CD4/CD8, 0.1, though the titers of immunoglobulins were in normal range. Nine times examinations of CD4+ T-lymphocyte count resulted in 5 times lower counts than 300/microliter between 1985 and 1993. His HIV (type 1 and type 2) studies were negative. He suffered from EB virus infection in 1987. The titers of VCA-IgG and EA-IgG had been elevated for half a year after that. He was operated polyp-like tumors in bilateral nasal cavities on February in 1994. The diagnosis was Burkitt's lymphoma that was also demonstrated by in situ hybridization using EB virus RNA. He was treated with a modification of COPBLAM III, but he died on June in 1994. Autopsy revealed tumor cell infiltrations in multiple organs. We suspect that he developed Burkitt's lymphoma 82 months later, when he had complicated EB virus infection in condition of idiopathic CD4+ T-lymphocytopenia caused by unknown origin.

Adult↗
PubMed2006

Improving RNA interference in mammalian cells by 4'-thio-modified small interfering RNA (siRNA): effect on siRNA activity and nuclease stability when used in combination with 2'-O-alkyl modifications.

A systematic structure-activity relationship study of 4'-thioribose containing small interfering RNAs (siRNAs) has led to the identification of highly potent and stable antisense constructs. To enable this optimization effort for both in vitro and in vivo applications, we have significantly improved the yields of 4'-thioribonucleosides by using a chirally pure (R)-sulfoxide precursor. siRNA duplexes containing strategically placed regions of 4'-thio-RNA were synthesized and evaluated for RNA interference activity and plasma stability. Stretches of 4'-thio-RNA were well tolerated in both the antisense and sense strands. However, optimization of both the number and placement of 4'-thioribonucleosides was necessary for maximal potency. These optimized siRNAs were generally equipotent or superior to native siRNAs and exhibited increased thermal and plasma stability. Furthermore, significant improvements in siRNA activity and plasma stability were achieved by judicious combination of 4'-thioribose with 2'-O-methyl and 2'-O-methoxyethyl modifications. These optimized 4'-thio-siRNAs may be valuable for developing stable siRNAs for therapeutic applications.

Animals↗
PubMed2025

RNA Sequencing Protocols for Short-Read Sequencing.

RNA sequencing (RNA-seq) methodologies allow the discovery of novel variants and transcripts. These comprise three general steps: (1) capture of RNA species of interest, (2) conversion of RNA to complementary DNA (cDNA), and (3) modification of cDNA to fit the sequencing platform. Here we describe four different library preparation protocols for short-read sequencing: cDNA synthesis with poly(A) selection, library preparation with ribosomal depletion, and cDNA synthesis with SMART® (Switching Mechanism at 5' end of RNA Template) technology for low and Pico inputs.

Gene Library↗
PubMed2006

Isolation of a site-specifically modified RNA from an unmodified transcript.

Natural RNAs contain many base modifications that have specific biological functions. The ability to functionally dissect individual modifications is facilitated by the identification and cloning of enzymes responsible for these modifications, but is hindered by the difficulty of isolating site-specifically modified RNAs away from unmodified transcripts. Using the m1G37 and m1A58 methyl modifications of tRNA as two examples, we demonstrate that non-pairing base modifications protect RNAs against the DNA-directed RNase H cleavage. This provide a new approach to obtain homogeneous RNAs with site-specific base modifications that are suitable for biochemical and functional studies.

Base Sequence↗
PubMed2002

Ribosomal RNA pseudouridines and pseudouridine synthases.

Pseudouridines are found in virtually all ribosomal RNAs but their function is unknown. There are four to eight times more pseudouridines in eukaryotes than in eubacteria. Mapping 19 Haloarcula marismortui pseudouridines on the three-dimensional 50S subunit does not show clustering. In bacteria, specific enzymes choose the site of pseudouridine formation. In eukaryotes, and probably also in archaea, selection and modification is done by a guide RNA-protein complex. No unique specific role for ribosomal pseudouridines has been identified. We propose that pseudouridine's function is as a molecular glue to stabilize required RNA conformations that would otherwise be too flexible.

Animals↗
PubMed2001

Hybridization of 2'-ribose modified mixed-sequence oligonucleotides: thermodynamic and kinetic studies.

In this study, we characterize the thermodynamics of hybridization, binding kinetics and conformations of four ribose-modified (2'-fluoro, 2'-O-propyl, 2'-O-methoxyethyl and 2'-O-aminopropyl) decameric mixed-sequence oligonucleotides. Hybridization to the complementary non-modified DNA or RNA decamer was probed by fluorescence and circular-dichroism spectroscopy and compared to the same duplex formed between two non-modified strands. The thermal melting points of DNA-DNA duplexes were increased by 1.8, 2.2, 0.3 and 1.3 degrees C for each propyl, methoxyethyl, aminopropyl and fluoro modification, respectively. In the case of DNA-RNA duplexes, the melting points were increased by 3.1, 4.1 and 1.0 degrees C for each propyl, methoxyethyl and aminopropyl modification, respectively. The high stability of the duplexes formed with propyl-, methoxyethyl- and fluoro-modified oligonucleotides correlated with high preorganization in these single-strands. Despite higher thermodynamic duplex stability, hybridization kinetics to complementary DNA or RNA was slower for propyl- and methoxyethyl-modified oligonucleotides than for the non-modified control. In contrast, the positively-charged aminopropyl-modified oligonucleotide showed rapid binding to the complementary DNA or RNA.

Base Pairing↗
PubMed1978

Impairment of RNA synthesis and its recovery in angelicin photosensitized mammalian cells. A probe for DNA damage and repair.

The template activity of DNA for ribosomal RNA transcription has been investigated in monkey kidney CV-1 cells after angelicin photosensitization in order to monitor the induction of lesions in the DNA and their possible subsequent disappearance, i.e. repair. Separate confluent cultures were submitted to a single angelicin treatment at different time intervals before incubation with [3H]uridine. The labeled RNA prepared from whole cells was analysed by polyacrylamide-agarose gel electrophoresis. The results indicate that: Angelicin monoadditions on DNA constitute transcription-terminating lesions which depress overall RNA synthesis, give rise to shortened RNA chains and modify the expression of transcriptional linked genes. CV-1 cells are able to repair, at least partially, the induced transcription-terminating lesions and progressively recover RNA synthesis with a reversion of the initially observed modifications. The repair seems to be independent of semiconservative DNA synthesis since fluorodeoxyuridine does not affect the recovery of RNA transcription. The present work also confirms the arrangement of rRNA genes in tandem behind a common operator in the order 18--28 S as previously determined in the same cells by a radiological mapping technique and reinforces the potential applicability of transcription analysis to the study of repair processes operating on physically or chemically induced damage in DNA.

Cell Line↗
PubMedDate not supplied

Simultaneous sequencing of RNA by solid-phase chemical degradation using DE 81 anion-exchange paper.

A solid-phase chemical degradation method for simultaneous sequencing of RNA and RNA fragments has been developed using Whatman DE 81 anion-exchange paper as the support. The approach involves the following operations: (1) immobilization of the 3'-end labeled RNAs or RNA fragments on DE 81 paper; (2) washing; (3) modification reactions; (4) washing; (5) sorting of the paper segments; (6) aniline reaction; (7) lyophilization; (8) desorption of the RNA by TEAB; and (9) lyophilization.

Animals↗
PubMed1982

Universal structural features of prokaryotic and eukaryotic ribosomal 5S RNA derived from comparative analysis of their sequences.

An extensive comparative analysis of more than fifty available sequences of ribosomal 5S RNA has been made. Both for prokaryotic and eukaryotic 5S RNA a generalized secondary structure is presented which is similar to that suggested by Nishikawa and Takemura modified in few positions only. Both generalized secondary structures contain five main helical regions and a high base-pairing content of about 65 +/- 5%. The general structural architecture of prokaryotic and eukaryotic 5S RNA molecules appears to be very similar with minor modifications within particular subgroups of organisms. Conserved and semiconserved nucleotides are accumulated in the single stranded parts of 5S RNA. Functional importance was suggested for some of these regions; other short conserved nucleotide stretches may be involved in the folding of 5S RNA molecules. In particular, we propose a tertiary base-pairing interaction between the universal invariant GUA sequence (positions 76-78 and 75-77 in prokaryotic and eukaryotic 5S RNA, respectively) and the complementary conserved CPuU sequence (positions 38-40 and 36-38) in a parallel manner. A molecular model of the 5S RNA of human KB cells was constructed, which verifies the proposed tertiary interaction, probably stabilizing the two neighboured helices E and D and a stacking arrangement of the bases in the sequence positions 67-108 (and 70-106) in eukaryotic (and prokaryotic) 5S RNAs, respectively.

Animals↗
PubMed1999

Bcl-2, Bax and Bcl-x expression following kainic acid administration at convulsant doses in the rat.

Neuronal death was produced in the CA1 and CA3 areas of the hippocampus, amygdala, and piriform and entorhinal cortices after intraperitioneal administration of kainic acid at convulsant doses to adult rats. To assess the involvement of members of the Bcl-2 family in cell death or survival, immunohistochemistry, western and northern blotting to Bcl-2, Bcl-x and Bax, and in situ hybridization to Bax were examined at different time-points after kainic acid treatment. Members of the Bcl-2 family were expressed in the cytoplasm of pyramidal neurons in the hippocampus, and in a subset of neurons of the piriform and the entorhinal cortices, amygdala and neocortex in the normal adult brain. Dying neurons in the pyramidal cell layer of CA1 and CA3 areas, entorhinal and piriform cortices, and amygdala also expressed Bcl-2, Bax and Bcl-x following excitotoxicity, although many dying cells did not. In addition, a number of cells in the affected areas showed Bax immunoreactivity in their nuclei at 24-48 h following kainic acid administration, thus indicating Bax nuclear translocation in a subset of dying cells. Western blots disclosed no modifications in the intensity of the bands corresponding to Bcl-2, Bcl-x and Bax, between control and kainic acid-treated rats. No modifications in the intensity of the bcl-2 messenger RNA band on northern blots was observed in kainic acid-treated rats. However, a progressive increase in the intensity of the bax messenger RNA band was found in kainic acid-treated rats at 6 h, 12 h and 24 h following kainic acid administration. Interestingly, a slight increase in Bax immunoreactivity was observed in the cytoplasm of neurons of the dentate gyrus at 24-48 h, a feature which matches the increase of bax messenger RNA in the same area, as shown by in situ hybridization at 12-24 h following kainic acid injection. The present results suggest that cell death or survival does not correlate with modifications of Bcl-2, Bax and Bcl-x protein, and messenger RNA expression, but rather that kainic acid excitotoxicity is associated with Bax translocation to the nucleus in a subset of dying cells.

Animals↗
PubMed1981

Low-level binding of halothane metabolites to rat liver histones in vivo.

Binding of halothane metabolites to rat liver histones was investigated after in vivo administration of 14C-halothane. Animals were injected with either a mixture of triiodothyronine, glucagon and heparin (TGH) to stimulate liver growth or with saline as a control. Twenty-four hours later, animals were administered 14C-halothane and maintained at 8--10 per cent O2 for 6 hours. Detergent washed nuclei from liver homogenates were subfractionated to allow quantitative measurements of 14C-halothane binding to histones. Although our studies suggest that much of the previously reported binding of halothane metabolites to major cell fractions was a result of redistribution of endoplasmic reticulum components during isolation procedures, carefully controlled experiments demonstrated that the radioactivity associated with histones could not be due to residual microsomal lipid. Of the initial 132 mumol of 14C-halothane administered, 1.1 mumol remained as nonvolatile metabolites in the liver homogenate and 25 pmol were associated with purified histones. This corresponds to approximately one halothane moiety per 15,000 histone molecules. No significant binding to liver cell RNA or DNA was observed. With this low level of histone modification and lack of convincing evidence of halothane metabolite binding to hepatic DNA or RNA, it is unlikely that significant alteration of the genome occurs after exposure to halothane.

Alkylating Agents↗