Heme oxygenase-1 in tissue pathology: the Yin and Yang.
Explore the source record for details and available documents.
Acute Kidney Injury↗
Biomedical subjects
A K Roy
Publications and source records attributed to A K Roy.
Role of redox-regulated transcription factors in inflammation, aging and age-related diseases.
A progressive rise of oxidative stress due to the altered redox homeostasis appears to be one of the hallmarks of the aging process. Reactive oxygen species (ROS) also serve as signaling agents for inflammation, a systemic defensive reaction against microbial pathogens and other foreign bodies. Changes in the pattern of gene expression through ROS-sensitive transcription factors give rise to both aging and inflammation phenotypes. Chronic oxidative stress and inflammatory reaction also lead to many age-associated diseases such as atherosclerosis and arthritis. Transcription factors that are directly influenced by ROS and proinflammatory cytokines include nuclear factor kappa B (NF-kappaB), activator protein 1 (AP-1), specificity protein 1 (Sp1), peroxisome proliferator-activated receptors (PPARs) and other members of the nuclear receptor superfamily. Here we describe the basic components of the intracellular redox control machinery and their dysregulation with age leading to altered transcription factor function and age-associated pathophysiology.
Aging↗
Role of clinical, radiological, and neurophysiological changes in predicting the outcome of tuberculous meningitis: a multivariable analysis.
OBJECTIVES: The role of EEG and evoked potentials has not been evaluated in predicting the prognosis of tuberculous (TB) meningitis. The present study was aimed at evaluating the prognostic significance of clinical, radiological, and neurophysiological variables using multi-variable analysis. METHODS: Patients with TB meningitis diagnosed on the basis of clinical, radiological, and CSF criteria have been prospectively evaluated. All the patients were subjected to a detailed neurological evaluation. The outcome was defined 6 months after starting treatment on the basis of the Barthel index (BI) score into poor (BI <12) and good recovery (BI> or =12). Death was included in the poor recovery group for statistical analysis. Thirteen clinical (age, sex, seizure, focal weakness, stage of meningitis, Glasgow coma scale score, methyl prednisolone therapy), CT (infarction, hydrocephalus, tuberculoma) and neurophysiological (EEG, motor and somatosensory evoked potentials) variables were evaluated employing single variable logistic regression followed by multivariable logistic regression analysis. The best set of predictors were obtained by stepdown logistic regression analysis. RESULTS: Fifty four patients were included in the present study. Their age ranged between 5 and 62 years, 11 were children younger than 12 years and 14 were female. Nine patients were in stage I meningitis, 12 in stage II, and 33 in stage III. On single variable logistic regression analysis the significant predictors of 6 months outcome of TB meningitis included focal weakness, Glasgow coma scale (GCS), motor evoked potential (MEP) and somatosensory evoked potential (SEP). On multivariable analysis the best set of predictors comprised focal weakness, GCS, and SEP. CONCLUSIONS: In patients with TB meningitis focal weakness, GCS, and SEP are the best predictors of 6 month outcome.
Adolescent↗
Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells.
An expression construct containing the cDNA encoding a modified aequorea green fluorescent protein (GFP) ligated to the 5'-end of the rat androgen receptor (AR) cDNA (GFP-AR) was used to study the intracellular dynamics of the receptor movement in living cells. In three different cell lines, ie. PC3, HeLa, and COS1, unliganded GFP-AR was seen mostly in the cytoplasm and rapidly (within 15-60 min) moved to the nuclear compartment after androgen treatment. Upon androgen withdrawal, the labeled AR migrated back to the cytoplasmic compartment and maintained its ability to reenter the nucleus on subsequent exposure to androgen. Under the condition of inhibited protein synthesis by cycloheximide (50 microg/ml), at least four rounds of receptor recycling after androgen treatment and withdrawal were recorded. Two nonandrogenic hormones, 17beta-estradiol and progesterone at higher concentrations (10(-7)/10(-6) M), were able to both transactivate the AR-responsive promoter and translocate the GFP-AR into the nucleus. Similarly, antiandrogenic ligands, cyproterone acetate and casodex, were also capable of translocating the cytoplasmic AR into the nucleus albeit at a slower rate than the androgen 5alpha-dihydrotestosterone (DHT). All AR ligands with transactivation potential, including the mixed agonist/antagonist cyproterone acetate, caused translocation of the GFP-AR into a subnuclear compartment indicated by its punctate intranuclear distribution. However, translocation caused by casodex, a pure antagonist, resulted in a homogeneous nuclear distribution. Subsequent exposure of the casodex-treated cell to DHT rapidly (15-30 min) altered the homogeneous to punctate distribution of the already translocated nuclear AR. When transported into the nucleus either by casodex or by DHT, GFP-AR was resistant to 2 M NaCl extraction, indicating that the homogeneously distributed AR is also associated with the nuclear matrix. Taken together, these results demonstrate that AR requires ligand activation for its nuclear translocation where occupancy by only agonists and partial agonists can direct it to a potentially functional subnuclear location and that one receptor molecule can undertake multiple rounds of hormonal signaling; this indicates that ligand dissociation/inactivation rather than receptor degradation may play a critical role in terminating hormone action.
Androgen Antagonists↗
Regulation of androgen action.
Explore the source record for details and available documents.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
Ribozyme-mediated cleavage of the estrogen receptor messenger RNA and inhibition of receptor function in target cells.
Estrogen receptor (ER) functions as a ligand-activated transcription factor for estrogen-regulated genes. Because of the critical role of the ER in the proliferation of certain estrogen-dependent cancer cell types such as the mammary tumor, inhibitors of estrogen action at the level of receptor function are of major clinical interest. Here we describe developments of two ribozymes that can selectively degrade the human ER mRNA and inhibit trans-activation of an artificial promoter containing the estrogen response element. Two ribozymes, designated RZ-1 and RZ-2, cleave the human ER alpha mRNA at nucleotide positions +956 and +889, respectively. These cleavage sites lie within the coding sequence for the DNA-binding domain of the receptor protein. Both RZ-1 and RZ-2 were also effective in inhibiting the progression of quiescent MCF-7 breast cancer cells to the S phase of the cell cycle after their exposure to 17beta-estradiol (10(-9) M). These results provide a new avenue for inhibition of estrogen action by selective mRNA degradation with its potential therapeutic application through targeted gene delivery vectors.
Animals↗
Negative regulation of the androgen receptor gene promoter by NFI and an adjacently located multiprotein-binding site.
The upstream promoter of the rat androgen receptor (AR) gene contains a strong negative regulatory region located at the -388 to -340 nucleotide position. The distal part (-388/-373) of this regulatory region binds NFI, a ubiquitous transcription factor, while the proximal portion (-372/-340) contains an overlapping binding site for two nuclear proteins. This composite regulatory region (-388/-340) was initially defined by deoxyribonuclease I footprinting as the continuous stretch of a nuclease-protected site. NFI specificity of the distal portion (-388/-373) of the footprint was established through cross-competition in electrophoretic mobility shift assay (EMSA) using the well characterized NFI element of the adenovirus major late promoter and by immunoreactivity to the NFI antibody. EMSA with oligonucleotide duplexes corresponding to the proximal domain (-372/-340) indicated multiple retarded bands with at least two major DNA-protein complexes. Further analysis with truncated oligonucleotide duplexes showed that these two major proteins bind to this domain in an overlapping manner. Within this overlapping area, the position spanning -359 to -347 is essential for the formation of either of these two complexes. Substitution of four G with T residues in the overlapping area totally abolished all protein binding at the downstream -372/-340 site. Point mutations that abolish specific binding at either the NFI or immediately downstream multiprotein-binding site caused about a 10-fold increase in AR promoter activity in transfected HepG2 cells. Double mutation involving both the NFI and proximal overlapping protein-binding sites failed to cause any additional increase in promoter function. From these results we conclude that the AR promoter contains a composite negative regulatory region at -388/-340, and the repressor function may involve a coordinate interaction between NFI and at least two other nuclear factors.
Animals↗
Tissue-specific and androgen-repressible regulation of the rat dehydroepiandrosterone sulfotransferase gene promoter.
Dehydroepiandrosterone sulfotransferase (Std) catalyzes sulfonation of androgenic steroids and certain aromatic procarcinogens. In rats, this enzyme is selectively expressed in the liver, and its expression is strongly repressed by androgens. DNase I footprinting and electrophoretic mobility shift analyses revealed two hepatocyte nuclear factor-1 (HNF1), three CCAAT/enhancer-binding protein (C/EBP), and one consensus palindromic thyroid hormone response elements within the first 215 base pairs (bp) of the promoter sequence of rat Std. This promoter is normally inactive in fibroblast-derived NIH 3T3 cells. However, overexpression of HNF1 and C/EBP resulted in synergistic activation of the Std promoter in this cell type, indicating essential roles of these two trans-regulators in liver-selective expression of the rat Std gene. On the other hand, point mutations at any one of five cis elements proximal to the -215 bp region markedly reduced reporter gene expression, suggesting that all of these sites are important for overall promoter function. Androgenic repression of the Std gene in rat liver can be recapitulated in androgen receptor (AR)-negative HepG2 hepatoma cells after cotransfection with an AR expression plasmid. Functional assay of a nested set of 5'-deleted promoters mapped the negative androgen response region between positions -235 and -310. Antibody supershift and oligonucleotide competition identified three OCT-1 and two C/EBP elements between bp -231 and -292. An additional OCT-1 site was found to overlap with a C/EBP element at the -262/-252 position. Mutational inactivation of any one of five cis elements within the -231/-292 region abolished negative androgen response. However, none of these cis elements showed DNase I protection by recombinant AR in footprinting assay, suggesting the absence of a direct AR-DNA interaction. Thus, these studies on rat Std promoter function indicate that (i) HNF1 and C/EBP are responsible for liver specificity of the rat Std gene; (ii) androgenic repression of the gene requires the presence of all of the OCT-1 and C/EBP elements between positions -231 and -292; and (iii) AR may exert its negative regulatory effect indirectly through transcriptional interference of OCT-1 and C/EBP rather than through a direct DNA-AR interaction.
3T3 Cells↗
Inhibition of androgen action by dehydroepiandrosterone sulfotransferase transfected in PC-3 prostate cancer cells.
Age-dependent loss of androgen sensitivity of the rat liver is associated with a marked increase in dehydroepiandrosterone/hydroxysteroid sulfotransferase (rStd) activity. Sulfonated steroid hormones are known to be ineffective in binding receptor proteins. These observations suggest that intracellular androgen sulfonation can physiologically influence androgen action. We have examined the inhibitory effect of rStd on androgen action in the human prostate cancer-derived PC-3 cells transfected with the rat androgen receptor (AR) expression plasmid and two androgen-responsive promoter reporter constructs (murine mammary tumor long-terminal repeat ligated to chloramphenicol acetyltransferase (CAT) gene and rat probasin androgen response element (ARE) ligated to firefly luciferase (LUC) gene). These transfected cells were dependent on 5alpha-dihydrotestosterone (DHT) for the activation of both reporter genes and showed about a 200- and a 800-fold increase of CAT and LUC activity, respectively, at 10(-10) M DHT over the no-hormone control. Expression of the sulfonating enzyme in this cell transfection system via the rStd expression plasmid caused a dose-dependent decline in the reporter activity with approximately 90% inhibition of androgen action at a rStd:AR plasmid ratio of 100. From these results we conclude that irrespective of a high level of AR, changes in the Std expression can markedly alter the androgen sensitivity of target cells.
Adenocarcinoma↗
A rapid and reliable PCR-based assay for gene transmission and sex determination in newborn transgenic mice.
This article describes a reliable and rapid method for simultaneous detection of a transgene and sex determination in the newborn mouse pups by PCR using three sets of primers in a single reaction. One set of sense/antisense primers is used to amplify the experimental transgene (androgen receptor gene in this case), the second set for the mouse Y-chromosome-specific SRY gene, and the third set for the beta subunit of the thyroid stimulating hormone (TSH beta), an internal control. This procedure allowed us to promptly analyze pups born from transgenic founders carrying the androgen receptor transgene and, at the same time, establish the sex of the animals. The method is simple, rapid and highly reproducible.
Animals↗
Catalytic cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme.
Androgen receptor (AR) plays a key role in cell growth both in the normal prostate and in prostate cancer. Androgen ablation and prolonged antiandrogen therapy can give rise to AR-dependent prostate tumors, which nonetheless can grow in the androgen-deprived milieu. Here we describe the ribozyme approach to selectively degrading the AR mRNA and thereby inhibiting AR function. A trans-acting hammerhead ribozyme was designed to cleave the rat AR mRNA at the position +1827/ 1828, a region predicted to be minimally involved in generating stable secondary structures. Using AR mRNA fragments as substrates, it was established that this ribozyme can specifically cleave the RNA target in a sequence-specific manner. Kinetic experiments determined a Km for the substrate of 77 nM and a kcat/Km value of 1.8 x 10(7) M(-1) x min(-1), suggesting a catalytic efficiency similar to that of protein enzymes such as the relatively nonspecific ribonuclease A and a sequence-specific endonuclease EcoRI. Transient cotransfections of prostate-derived PC3 cells with three plasmids, an AR-inducible chloramphenicol acetyltransferase (CAT) reporter, an AR expression vector, and a ribozyme expression vector, showed that the ribozyme was capable of reducing the functional activity of AR. At an equimolar ratio of the AR expression plasmid to ribozyme expression plasmid, androgen-inducible CAT activity was inhibited 70%. Similar extents of inhibition were also observed at the cellular mRNA level using ribonuclease protection assays, indicating that the ribozyme functioned as an AR mRNA cleaving enzyme in cellulo. Immunocytochemical examination revealed a decline of AR immunoreactivity in ribozyme-transfected cells. In addition, no morphologically detectable cellular abnormalities were associated with ribozyme expression, indicating the absence of deleterious side effects. These results offer a new avenue for the control of AR function and cell growth, especially in the case of androgen-resistant, but AR-dependent, prostate cancer cells.
Animals↗
Therapeutic potential and mechanism of action of oligonucleotides and ribozymes.
Specific inactivation of gene expression is an attractive approach for rational drug design to combat degenerative diseases and infectious agents. Oligonucleotide-directed triple-helix formation at cis-acting elements of gene promoters, short oligonucleotides containing base sequences that are complementary to the messenger RNA (antisense oligos), and RNA enzymes (ribozymes) that specifically cleave messenger RNA molecules are currently being used both as experimental tools and as therapeutic agents. Mechanisms of action of various oligonucleotide-based drugs, recent developments in the drug-delivery approaches, and future potentials are discussed in this review.
Gene Expression↗
Functional role of a conformationally flexible homopurine/homopyrimidine domain of the androgen receptor gene promoter interacting with Sp1 and a pyrimidine single strand DNA-binding protein.
The androgen receptor (AR) gene promoter does not contain the TATA or CAAT box, but it contains a long (approximately 90-bp) homopurine/homopyrimidine (pur/ pyr) stretch immediately upstream of the Sp1-binding GC box site. This pur-pyr stretch is conserved at the same proximal position in the rat, mouse, and human AR gene promoters. Mutation of this region results in a 3-fold decline in promoter activity, indicating an important regulatory function. Examination of the conformational state of the AR pur/pyr region with the single-strand-specific S1 nuclease showed that it is capable of forming a non-B DNA structure involving unpaired single strands. Fine mapping of the S1-sensitive site revealed an unsymmetric cleavage pattern indicative of an intramolecular triple helical H-form DNA conformation. Electrophoretic mobility shift analyses showed that the pur/pyr region of the AR promoter can bind a novel pyrimidine single-strand-specific protein (ssPyrBF) and also a double-strand DNA-binding protein. Both oligonucleotide cross-competition and antibody supershift experiments established that the double-strand binding protein is equivalent to Sp1. Deoxyribonuclease I (DNase I) footprinting analysis showed multiple Sp1-binding to the pur/pyr site and a weaker Sp1 interaction to this region compared with the adjacently located GC box, where Sp1 functions to recruit the TFIID complex. These results suggest that the pur/pyr domain of the AR gene can serve to attract additional Sp1 molecules when it exists in the double-stranded B-DNA conformation. However, binding of ssPyrBF and the resultant stabilization of the non-B DNA structure is expected to prevent its interaction with Sp1. We speculate that in the TATA-less AR gene promoter, multiple weak Sp1 sites at the pur/pyr region adjacent to the GC box can provide a readily available source of this transcription factor to the functional GC box, thereby facilitating the assembly of the initiation complex.
Animals↗
Transcription factors and aging.
Explore the source record for details and available documents.
Aging↗
Structural relationships among members of the mammalian sulfotransferase gene family.
Sulfotransferases constitute a superfamily of related enzymes that play critical roles in the regulation of steroid hormone action, neurotransmitter function, detoxification, and carcinogenesis. Understanding the functional relationships among these enzymes has so far been difficult due to their overlapping substrate specificities. To help clarify these relationships, we conducted a thorough and comprehensive molecular phylogenetic analysis of 25 different mammalian sulfotransferase cDNA and gene (St) sequences using maximum parsimony and distance matrix methods. This analysis suggested five distinct gene families: an alcohol/androgen/hydroxysteroid/dehydroepiandrosterone (Std) family, an aryl/minoxidil/phenol (Stp) family, an estrone/estrogen (Ste) family, a thyroid hormone family (St1b1), and a family (St1c1) defined so far only on the basis of its specificity for the carcinogen N-hydroxy-2-acetylaminofluorene. New insights obtained through this study include (1) a bootstrap analysis supporting the reliability of family subgroupings, (2) identification of an insertion that appears to be characteristic of the St1b1 and Stlc1 families, (3) identification of sequences likely to represent paralogs of multigene families, and (4) identification of species likely to contain, or not contain, orthologous multigene families and thus their specialized functions.
Amino Acid Sequence↗
Targeted overexpression of androgen receptor with a liver-specific promoter in transgenic mice.
The rodent liver displays marked age- and sex-dependent changes in androgen sensitivity due to the sexually dimorphic and temporally programmed expression of the androgen receptor (AR) gene. We have altered this normal phenotype by constitutive overexpression of the rat AR transgene in the mouse liver by targeting it via the human phenylalanine hydroxylase (hPAH) gene promoter. These transgenic animals in their heterozygous state produce an approximately 30-fold higher level of the AR in the liver as compared with the nontransgenic control. Androgen inactivation via sulfonation of the hormone by dehydroepiandrosterone sulfotransferase (DST), an androgen-repressible enzyme, also contributes to the age- and sex-dependent regulation of hepatic androgen sensitivity. DST has a broad range of substrate specificity and is responsible for the age- and sex-specific activation of certain polycyclic aromatic hepatocarcinogens as well, by converting them to electrophilic sulfonated derivatives. In the transgenic female, the hepatic expression of DST was approximately 4-fold lower than in normal females, a level comparable to that in normal males. The hPAH-AR mice will serve as a valuable model for studying the sex- and age-invariant expression of liver-specific genes, particularly those involved in the activation of environmental hepatocarcinogens such as the aromatic hydrocarbons.
Animals↗
Age-dependent expression of the androgen receptor gene in the prostate and its implication in glandular differentiation and hyperplasia.
The senescence phenotype is the product of both cumulative physical damages during the life span and a species-specific genetic program. The genetic program of aging appears to have co-evolved with the sexual mode of reproduction. The same developmental processes that prepare the animal for maximum vitality and reproductive competence during young adulthood, if allowed to continue, can be detrimental during old age. Androgen receptor-mediated development and growth of the prostate gland is an example of such "antagonistic pleiotropy." The prostate gland is composed of two major groups of cells: the epithelial and stromal. Among the epithelial type, the columnar cells on the luminal surface produce the prostatic secretions, and the basal cells are presumed to serve as progenitors of the columnar cells. Within the stromal cell population, fibroblastic and smooth muscle cells are thought to produce growth factors that support the development and function of the epithelial cells. Both epithelial and stromal cells are dependent on androgens. In this study, we have examined age-dependent expression of the androgen receptor gene in the prostatic tissues of rats and dogs. Unlike the rat, in which the prostatic growth ceases after sexual maturation, the dog prostate continues to grow during aging. Similar to the dog, the antagonistic pleiotropy of the prostatic growth in the human causes the pathological condition of benign prostatic hyperplasia (BPH), the major health problem in old men. Quantitation of the androgen receptor (AR) mRNA in the total prostate extracts from young and old animals by the reverse transcriptase-polymerase chain reaction (RT-PCR) method showed about a 30% decline in AR mRNA in the 24-month-old rat prostate, as compared to the prostate of 3-month-old young adult animals. However, no significant difference in AR mRNA contents between 1-year-old and 10-year-old dog prostates was observed. In situ immunostaining for the androgen receptor protein revealed that in the case of rat, developmental maturation during the first month of life is associated with an increase in AR immunoreactivity in the luminal columnar epithelium, with a concomitant loss of immunoreactivity in the basal cells. Furthermore, with aging, there was a marked increase in the proportion of AR-negative basal cells in comparison to luminal columnar cells. Surprisingly, in both young adult (approximately 1-year-old) and old (approximately 10-year-old) dogs, most of the AR immunoreactivity was localized in the fibroblastic stromal cells rather than in the epithelial cells. Based on these observations and the existing literature, we propose that normally, in most mammalian species, an age-dependent decline in the conversion of basal to columnar epithelial cells after sexual maturation serves as a stop signal for the prostate growth. However, in certain species, such as the dog, robust AR expression in the stromal cells overrides this regulatory blockage and leads to prostatic hyperplasia in old age.
Aging↗