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At least 1,045 records · Page 58Linked to original sources
PubMed2006

Laser microdissection of plant tissue: what you see is what you get.

Laser microdissection (LM) utilizes a cutting or harvesting laser to isolate specific cells from histological sections; the process is guided by microscopy. This provides a means of removing selected cells from complex tissues, based only on their identification by microscopic appearance, location, or staining properties (e.g., immunohistochemistry, reporter gene expression, etc.). Cells isolated by LM can be a source of cell-specific DNA, RNA, protein or metabolites for subsequent evaluation of DNA modifications, transcript/protein/metabolite profiling, or other cell-specific properties that would be averaged with those of neighboring cell types during analysis of undissected complex tissues. Plants are particularly amenable to the application of LM; the highly regular tissue organization and stable cell walls of plants facilitate the visual identification of most cell types even in unstained tissue sections. Plant cells isolated by LM have been the starting point for a variety of genomic and metabolite studies of specific cell types.

Chromosomes, Plant↗
PubMed1998

Perinatal determinants of adult cardiovascular disease and cancer.

Interest in perinatal factors, especially birthweight, as determinants of adult-onset diseases has been steadily growing. Low birthweight has been associated with increased risk of cardiovascular disease, and high birthweight has been linked to higher risk of breast and possibly other cancers. Most mechanistic hypotheses that have been advanced to explain the empirical evidence linking perinatal conditions to adult-life disease in humans have invoked modulation of physiological processes by exogenous factors or poorly specified "programming" during fetal life. A form of programming that has a strong biological foundation and has recently been suggested for further study is genomic imprinting, which involves non-permanent DNA modifications and allows for influences that span three generations. The subject of the perinatal origin of adult-onset disease has profound implications and a large and reliable body of evidence needs to be assembled for biological theories to be validly evaluated against.

Adult↗
PubMed2002

Androgen receptor (AR) coregulators: an overview.

The biological action of androgens is mediated through the androgen receptor (AR). Androgen-bound AR functions as a transcription factor to regulate genes involved in an array of physiological processes, most notably male sexual differentiation and maturation, and the maintenance of spermatogenesis. The transcriptional activity of AR is affected by coregulators that influence a number of functional properties of AR, including ligand selectivity and DNA binding capacity. As the promoter of target genes, coregulators participate in DNA modification, either directly through modification of histones or indirectly by the recruitment of chromatin-modifying complexes, as well as functioning in the recruitment of the basal transcriptional machinery. Aberrant coregulator activity due to mutation or altered expression levels may be a contributing factor in the progression of diseases related to AR activity, such as prostate cancer. AR demonstrates distinct differences in its interaction with coregulators from other steroid receptors due to differences in the functional interaction between AR domains, possibly resulting in alterations in the dynamic interactions between coregulator complexes.

Animals↗
PubMed2003

The eukaryotic genome: a system regulated at different hierarchical levels.

Eukaryotic gene expression can be viewed within a conceptual framework in which regulatory mechanisms are integrated at three hierarchical levels. The first is the sequence level, i.e. the linear organization of transcription units and regulatory sequences. Here, developmentally co-regulated genes seem to be organized in clusters in the genome, which constitute individual functional units. The second is the chromatin level, which allows switching between different functional states. Switching between a state that suppresses transcription and one that is permissive for gene activity probably occurs at the level of the gene cluster, involving changes in chromatin structure that are controlled by the interplay between histone modification, DNA methylation, and a variety of repressive and activating mechanisms. This regulatory level is combined with control mechanisms that switch individual genes in the cluster on and off, depending on the properties of the promoter. The third level is the nuclear level, which includes the dynamic 3D spatial organization of the genome inside the cell nucleus. The nucleus is structurally and functionally compartmentalized and epigenetic regulation of gene expression may involve repositioning of loci in the nucleus through changes in large-scale chromatin structure.

Animals↗
PubMed2001

Insulin glargine: the first clinically useful extended-action insulin analogue.

Insulin glargine is a new extended-action insulin analogue, created by recombinant DNA modification of human insulin. Extension of the C-terminal of the B-chain with two arginine residues and the substitution of glycine for asparagine at position A-21 increases the isoelectric point, resulting in precipitation of the insulin at the injection site and a protracted absorption. Pharmacodynamic studies have demonstrated a prolonged metabolic profile without a pronounced peak and with a duration of action of 20 - 30 h. In clinical studies in people with Type 1 and Type 2 diabetes, insulin glargine has demonstrated improved pre-breakfast blood glucose control and a reduction in the frequency of hypoglycaemia, especially nocturnal hypoglycaemia, in comparison with neutral protamine hagedorn (NPH) insulin. In addition, 24h glycaemic control in Type 2 diabetes and treatment satisfaction may also be improved. However, whilst appearing achievable, insulin glargine has not yet demonstrated the ability to improve HbA(1c), though this may relate to inexperience in the use of the new compound. In order to fully exploit its metabolic advantages, it appears vital that the dose of insulin glargine should be titrated to achieve aggressive pre-breakfast blood glucose targets beyond those achievable with NPH in the absence of nocturnal hypoglycaemia. Insulin glargine appears to be a promising new addition to the insulin family and with increased experience in its use, especially in combination with rapid-acting insulin analogues, its full benefits may be realised. The use of insulin glargine with a rapid-acting insulin analogue brings us the closest we have ever been to providing the physiological insulin replacement that has long been awaited.

Animals↗
PubMedDate not supplied

Methylation of a CpG island within the uroplakin Ib promoter: a possible mechanism for loss of uroplakin Ib expression in bladder carcinoma.

Uroplakin Ib is a structural protein on the surface of urothelial cells. Expression of uroplakin Ib mRNA is reduced or absent in many transitional cell carcinomas (TCCs) but molecular mechanisms underlying loss of expression remain to be determined. Analysis of the uroplakin Ib promoter identified a weak CpG island spanning the proximal promoter, exon 1, and the beginning of intron 1. This study examined the hypothesis that methylation of this CpG island regulates uroplakin Ib expression. Uroplakin Ib mRNA levels were determined by reverse transcription polymerase chain reaction and CpG methylation was assessed by bisulfite modification of DNA, PCR, and sequencing. A correlation was demonstrated in 15 TCC lines between uroplakin Ib mRNA expression and lack of CpG methylation. In support of a regulatory role for methylation, incubating uroplakin Ib-negative lines with 5-aza-2'-deoxycytidine reactivated uroplakin Ib mRNA expression. A trend between uroplakin Ib mRNA expression and CpG methylation was also observed in normal urothelium and bladder carcinomas. In particular, loss of uroplakin Ib expression correlated with methylation of a putative Sp1/NFkappaB binding motif. The data are consistent with the hypothesis that methylation of specific sites within the uroplakin Ib promoter may be an important factor in the loss of uroplakin Ib expression in TCCs.

Azacitidine↗
PubMed1995

Mechanisms of oxidant stress-induced acute tissue injury.

During the last 25 years, a large body of experimental evidence has accumulated from pharmacological intervention studies that suggests an important role for reactive oxygen species in numerous pathophysiological processes. While a variety of chemical mechanisms of reactive oxygen-induced damage to lipids, proteins, and DNA is fairly well understood, the molecular pathology of oxidant stress-induced tissue injury in vivo remains unclear in most cases. Recent advances indicate that the direct destructive potential of reactive oxygen in vivo is limited by the extensive detoxification capacity of most cells and may be restricted to a small fraction of cells exposed to a locally high oxidant stress. However, reactive oxygen species can participate in recruitment of inflammatory cells by upregulation of adhesion molecules and generation of chemotactic factors, and are necessary for protease-mediated cell injury in vivo. Reactive oxygen species can also scavenge other biologically active molecules (e.g., nitric oxide), thereby modulating indirectly their effector cells. In addition to the discussed effects relevant for acute injury, other oxidant stress-induced mechanisms (e.g., DNA modifications) may be relevant in chronic disease states. A solid mechanistic understanding of the role of reactive oxygen species in the overall pathophysiology is critical for providing a rationale for antioxidant therapy and the targeted development of new antioxidant drugs.

Animals↗
PubMed1999

Transcription factors as targets of anticancer drugs.

Several general and gene- and cell-selective transcription factors are required for specific transcription to occur. Many of them exert their functions through specific contacts either in the promoter region or at distant sequences regulating the initiation. These contacts may be altered by anticancer drugs which form non-covalent complexes with DNA. Covalent modifications of DNA by alkylating agents may prevent transcription factors from recognizing their specific sequences or may constitute multiple "unnatural" binding sites in DNA which attract the factors thus decreasing their availability in the cell. The anticancer drug-transcription factor interplay which is based on specific interactions with DNA may contribute to pharmacological properties of the former and provide a basis for the search for new drugs.

Animals↗
PubMed2000

Ionizing radiation-induced death in bovine lens epithelial cells: mechanisms and influence of irradiation dose rate.

We recently reported, in a series of patients receiving total body irradiation before transplant, an influence of dose rate (DR) on cataract formation. The aim of our present in vitro study was to investigate the influence of DR and the mechanisms of lens cell death in a bovine model. After a single fraction of 10 Gy, delivered using low (0.05 Gy/min) or high (2 Gy/min) DR (LDR and HDR, respectively), cells were incubated in media supplemented with two different fetal calf serum (FCS) concentrations (1% and 10%). Cell proliferation was evaluated using Hoechst 33342 (HO) probe and cell viability, with neutral red probe. These fluorimetric assays used a cold light cytofluorimeter. After HO assay, stained cells were examined with fluorescence microscopy to evaluate the nuclear changes related to apoptosis. Global comparison of the mean HO fluorescent values observed with LDR/controls (c) vs. HDR/c revealed a significant difference only after 96 hr (P = 0.036). In 1% FCS conditions, the difference between HDR/c and LDR/c was also statistically significant at 96 hr (P = 0.04). Pairwise multiple comparison using values observed in 1% FCS conditions after 96 hr incubation showed significant difference between HDR vs. c (P = 0.001) and HDR vs. LDR (P = 0.007). This difference, in terms of fluorescence, was correlated to the proportion of cells with nuclear apoptotic morphology. In contrast, cell viability was not influenced by DR whatever the FCS concentration used, from 24 to 96 hr after irradiation. We conclude that our fluorimetric methodology is adapted to evaluate intracellular DNA modifications and cell viability after x-ray irradiation. We observed that a single fraction of 10 Gy induces in vitro lens epithelial cell apoptosis, which is influenced by DR. In humans, HDR is considered more cataractogenic than LDR. Thus, we speculate that lens cell apoptosis could be one of the major mechanisms of radiation-induced cataract. Further investigations are necessary to study the other possible mechanisms of cataractogenesis. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 138-144 (2000).

Animals↗
PubMed1998

Research on the Diversity of PLA(2) Gene from Agkistrodon halys Pallas.

We used degenerate primers to amplify phospholipase A(2)(PLA(2)) gene by RT-PCR from venom total RNA of Agkistrodon halys Pallas, and screened with B-PLA(2) gene as probe. Finally we isolated three cDNAs containing A-PLA(2) and two other genes showing similar characteristic structure--Asn(49)-PLA(2) and BA-PLA(2). Their complete sequence was determined by bidirectional sequencing and their amino acid sequence was deduced. The amino acid sequence of A-PLA(2) deduced from the cDNA agreed with that determined by protein sequencing except for four residues; Asn(49 )-PLA(2) and B-PLA(2) are very alike (up to 95%), but the Asp(49)-PLA(2) of B-PLA(2) involved in the enzyme reaction is replaced by Asn(49) of Asn(49)-PLA(2). Therefore, it possibly affects the enzyme reaction of Asn(49)-PLA(2). The N-terminus sequence of BA-PLA(2) is highly homologous to B-PLA(2), but its C-terminus sequence is almost the same as A-PLA(2). The successful cloning of these isoenzyme genes not only discloses the structure diversity of PLA(2)(namely DNA modification and recombination), but also provides excellent material for the study of structure-function relationship in PLA(2).

Journal Article↗
PubMed2002

Dependence of histone modifications and gene expression on DNA hypermethylation in cancer.

We examined the relationship between aberrant DNA hypermethylation and key histone code components at a hypermethylated, silenced tumor suppressor gene promoter in human cancer. In lower eukaryotes, methylated H3-lysine 9 (methyl-H3-K9) determines DNA methylation and correlates with repressed gene transcription. Here we show that a zone of deacetylated histone H3 plus methyl-H3-K9 surrounds a hypermethylated, silenced hMLH1 promoter, which, when unmethylated and active, is embedded in methyl-H3-K4 and acetylated H3. Inhibiting DNA methyltransferases, but not histone deacetylases, leads first to promoter demethylation, second to gene reexpression, and finally to complete histone code reversal. Our findings suggest a new paradigm-DNA methylation may directly, or indirectly by inhibiting transcription, maintain key repressive elements of the histone code at a hypermethylated gene promoter in cancer.

Acetylation↗
PubMed2006

[Trans-acting short interfering RNAs].

RNA is now considered a key factor in the regulation of gene expression. There are several classes of small regulatory RNAs in plants, functioning in posttranscriptional gene silencing (PTGS) or epigenetic DNA modification. Trans-acting short interfering RNAs (tasiRNAs) form a class of small regulatory RNAs which has been distinguished only recently. To date, five genes encoding tasiRNAs have been identified in Arabidopsis thaliana. TasiRNAs derive from non-coding RNA precursors which are initially targeted for cleavage by a miRNA. Cleavage products are then converted into dsRNAs by a RNA dependent RNA polymerase and sequentially cleaved into 21-nt tasiRNAs. Like the majority of plant miRNAs, tasiRNAs regulate gene expression at the posttranscriptional level, guiding cleavage of ARF and PPR transcripts. Here, we briefly present tasiRNAs and speculate whether they form a homogeneous class of siRNAs.

Gene Expression Profiling↗
PubMed1990

Structure-function analysis of the human interferon gamma. The COOH terminus is not essential for functional activity.

The structure-function relationships for the human interferon gamma (HuIFN-gamma) were studied using recombinant variants that had various deletions at the carboxyl terminus. Four COOH-terminal deletion variants were constructed that contained the amino-terminal 122, 117, 111, and 106 amino acid residues. These variants were constructed by specific DNA modifications and were expressed in Escherichia coli. The deletion of 21 amino acid residues resulted in only 2- and 3-fold reduction in the antiviral and antiproliferative specific activities, respectively. Thus, the carboxyl-terminal 21 residues are not directly involved in the function of the HuIFN-gamma. The level of intracellular accumulation was also decreased by 3-5-fold. Further deletions of 26, 32, and 37 residues from the COOH terminus resulted in the lack of detectable activity as well as in 50-100-fold reduction in the level of accumulation in the bacterial cell. However, each of the modified plasmids was found to have comparable efficiency in directing the production of the respective variant molecules relative to the full-length HuIFN-gamma molecule in an in vitro transcription-translation assay. Thus, the failure of some of the deletion variant molecules to accumulate in the E. coli cell is likely due to their instability in vitro. The loss of the COOH-terminal 21 amino acid residues of HuIFN-gamma also resulted in a substantial reduction in the ability of the molecule to be renatured in vitro from the treatment with chaotrophic agents, a method frequently used to extract and purify recombinant polypeptides from E. coli host. The latter result may account for some earlier reports which inferred the involvement of the COOH terminus in the functions of the HuIFN-gamma molecule due to their failure to detect activity upon deletions of only 11-18 residues.

Amino Acid Sequence↗
PubMed1988

alpha-Amino-N-butyric acid stimulates fetal hemoglobin in the adult.

The effect of alpha-amino-N-butyric acid (alpha ABA) on fetal hemoglobin production in the adult was examined in vivo after being administered to normal and anemic baboons and in erythroid progenitor cell cultures. Infusion of alpha ABA for five days resulted in four- to fivefold increases in the level of F reticulocytes of normal or chronically anemic baboons. The induction of HbF by alpha ABA was strikingly enhanced by the administration of 5-azacytidine. The addition of alpha ABA in culture produced a concentration-related increase of HbF in baboon CFUe and e-cluster colonies. In addition to the induction of HbF, alpha ABA stimulated the growth of all classes of erythroid progenitors in vivo or in culture. The activation of gamma-globin gene expression by alpha ABA is attributed to an interaction between regulatory sites of globin chromatin modified by alpha ABA and the immature intracellular environment of the expanding erythropoiesis. The combination of chromatin modification, DNA methylation, and the immature intracellular environment of rapid erythroid regeneration may explain the synergistic induction of HbF by alpha ABA and 5-azacytidine.

Adult↗
PubMed1989

Molecular analysis of mutagenesis in E. coli.

In this paper we present a general strategy that is suitable for the analysis of the forward mutation spectrum induced by any physical or chemical mutagen or carcinogen. The assay is based upon the inactivation of the tetracycline resistance gene located on plasmid pBR322. Plasmid DNA is treated in vitro with the mutagen and transformed into the host bacteria of choice. Mutant clones are selected and analysed by sequencing. We present also two techniques that allow the determination of the DNA modification spectrum. The comparison for a given mutagen of the modification spectrum and the induced mutation spectrum permits the identification of hot spot sequences. Using different chemical mutagens (derivatives of the carcinogenic aromatic amide N-2-acetylaminofluorene, cis-platinum, etc.) this assay was found to be able to detect the different classes of mutagenic events: base substitutions, frameshifts, insertions and deletions. The advantages and limitations of this assay are discussed.

Escherichia coli↗
PubMed1989

[Utilization of genotoxicity tests for biological surveillance of personnel exposure].

Monitoring of those staff exposed to products or atmospheres containing carcinogenic substances may be carried out using genetic toxicology tests. These tests may highlight primary DNA modifications, genetic or chromosomal mutations, detect mutagens at the urinary level or assess a global effect, expressed by a genotoxic effect. All these tests must be used taking into account exposure kinetics, the substance in question and exposure conditions.

Humans↗
PubMed1989

Changes in activities of free radical detoxifying enzymes in kidneys of male Syrian hamsters treated with estradiol.

Target organ-specific estrogen-induced DNA adducts were previously shown to precede renal carcinogenesis in Syrian hamsters. Because estrogens induced these DNA modifications, but were not part of the adduct structure, free radical activation of endogenous electrophiles was postulated as a mechanism of tumor induction by estrogens. In the present study, the activities of enzymes which detoxify reactive intermediates were studied in liver and kidney of hamsters treated with estradiol for 1, 2, and 4 mo and in untreated controls. These studies were done to detect oxidative stress in the target organ of carcinogenesis. In the estrogen-exposed hamster kidney (1, 2, and 4 mo), activities of glutathione peroxidases I and II were significantly increased. The activity of catalase was decreased compared to those in untreated controls. In livers which are not the target organ of carcinogenesis, treatment of hamsters with estrogen for 1, 2, and 4 mo resulted in changes of activities of glutathione peroxidases I and II and catalase, which were opposite to the pattern found in the kidney. Activities of superoxide dismutase, glutathione reductase, glucose-6-phosphate dehydrogenase, gamma-glutamyl transpeptidase, and glutathione transferase in estradiol-treated hamster liver and kidney did not differ significantly from those in either liver or kidney of untreated age-matched controls. Fluorescent products of lipid peroxidation more than doubled in the kidney, but not in the liver of hamsters treated with estradiol for 1 mo. It is concluded that the increases in glutathione, in the activity of glutathione peroxidase, and in products of lipid peroxidation in the kidneys of hamsters treated chronically with estrogen all point towards elevated levels of oxidative stress.

Animals↗