Differential Expression Of Many Genes Research Articles

Retinoic acid pathway activity in wilms tumors and characterization of biological responses in vitro

BackgroundWilms tumor (WT) is one of the most common malignancies in childhood. With current therapy protocols up to 90% of patients can be cured, but there is still a need to improve therapy for patients with aggressive WT and to reduce treatment intensity where possible. Prior data suggested a deregulation of the retinoic acid (RA) signaling pathway in high-risk WT, but its mode of action remained unclear.ResultsThe association of retinoid signaling and clinical parameters could be validated in a large independent tumor set, but its relevance in primary nephrectomy tumors from very young children may be different. Reduced RA pathway activity and MYCN overexpression were found in high risk tumors as opposed to tumors with low/intermediate risk, suggesting a beneficial impact of RA especially on advanced WT. To search for possible modes of action of retinoids as novel therapeutic options, primary tumor cell cultures were treated in vitro with all-trans-RA (ATRA), 9cis-RA, fenretinide and combinations of retinoids and a histone deacetylase (HDAC) inhibitor. Genes deregulated in high risk tumors showed opposite changes upon treatment suggesting a positive effect of retinoids. 6/7 primary cultures tested reduced proliferation, irrespective of prior RA signaling levels. The only variant culture was derived from mesoblastic nephroma, a distinct childhood kidney neoplasm. Retinoid/HDAC inhibitor combinations provided no synergistic effect. ATRA and 9cis-RA induced morphological changes suggestive of differentiation, while fenretinide induced apoptosis in several cultures tested. Microarray analysis of ATRA treated WT cells revealed differential expression of many genes involved in extracellular matrix formation and osteogenic, neuronal or muscle differentiation. The effects documented appear to be reversible upon drug withdrawal, however.ConclusionsAltered retinoic acid signaling has been validated especially in high risk Wilms tumors. In vitro testing of primary tumor cultures provided clear evidence of a potential utility of retinoids in Wilms tumor treatment based on the analysis of gene expression, proliferation, differentiation and apoptosis.

Altered Levels of Histone Deacetylase OsHDT1 Affect Differential Gene Expression Patterns in Hybrid Rice

Hybrids between different inbred varieties display novel patterns of gene expression resulted from parental variation in allelic nucleotide sequences. To study the function of chromatin regulators in hybrid gene expression, the histone deacetylase gene OsHDT1 whose expression displayed a circadian rhythm was over-expressed or inactivated by RNAi in an elite rice parent. Increased OsHDT1 expression did not affect plant growth in the parent but led to early flowering in the hybrid. Nonadditive up-regulation of key flowering time genes was found to be related to flowering time of the hybrid. Over-expression of OsHDT1 repressed the nonadditive expression of the key flowering repressors in the hybrid (i.e. OsGI and Hd1) inducing early flowering. Analysis of histone acetylation suggested that OsHDT1 over-expression might promote deacetylation on OsGI and Hd1 chromatin during the peak expression phase. High throughput differential gene expression analysis revealed that altered OsHDT1 levels affected nonadditive expression of many genes in the hybrid. These data demonstrate that nonadditive gene expression was involved in flowering time control in the hybrid rice and that OsHDT1 level was important for nonadditive or differential expression of many genes including the flowering time genes, suggesting that OsHDT1 may be involved in epigenetic control of parental genome interaction for differential gene expression.

Differential expression of immune and stress genes in the skin of Atlantic cod ( Gadus morhua)

The present study describes the transcriptional profiles of selected immune and stress genes with putative important roles in the cutaneous immune defense of Atlantic cod ( Gadus morhua). In addition it shows differential expression of many genes at the dorsal and ventral sides of fish, in general having the highest expression at the latter side. Genes related to antibacterial activity, antiviral response, cytokine production, glucose transport, stress response and anti-apoptotic activity were monitored and bactericidal/permeability-increasing protein/lipopolysaccharide-binding protein ( BPI-LBP), g-type lysozyme, transferrin, metallothionein, fortilin, interferon regulatory factor-1 ( IRF-1), a CC chemokine isoform, interleukin-8 ( IL-8), glucose transport ( GLUT) -1, -3 and -4, Cu,Zn-superoxide dismutase ( Cu,Zn-SOD), catalase and hsp 70 showed significantly higher expression at the ventral side. Further g-type lysozyme, metallothionein, fortilin, IRF-1, interferon γ, interleukin-1β ( IL-1β), GLUT-3 and -4, catalase and anti apoptotic gene Bcl-X1 were highly expressed in adult cod skin. Therefore fish skin can be considered an immunological active site, especially at the ventral side of Atlantic cod.

Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii

BackgroundSphingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. Thus far, however, little is known about the adaptive strategies used by Sphingomonas bacteria to respond to changes in water potential. To improve our understanding, strain RW1 was perturbed with either the cell-permeating solute sodium chloride or the non-permeating solute polyethylene glycol with a molecular weight of 8000 (PEG8000). These solutes are assumed to simulate the solute and matric components of the total water potential, respectively. The responses to these perturbations were then assessed and compared using a combination of growth assays, transcriptome profiling, and membrane fatty acid analyses.ResultsUnder conditions producing a similar decrease in water potential but without effect on growth rate, there was only a limited shared response to perturbation with sodium chloride or PEG8000. This shared response included the increased expression of genes involved with trehalose and exopolysaccharide biosynthesis and the reduced expression of genes involved with flagella biosynthesis. Mostly, the responses to perturbation with sodium chloride or PEG8000 were very different. Only sodium chloride triggered the increased expression of two ECF-type RNA polymerase sigma factors and the differential expression of many genes involved with outer membrane and amino acid metabolism. In contrast, only PEG8000 triggered the increased expression of a heat shock-type RNA polymerase sigma factor along with many genes involved with protein turnover and repair. Membrane fatty acid analyses further corroborated these differences. The degree of saturation of membrane fatty acids increased after perturbation with sodium chloride but had the opposite effect and decreased after perturbation with PEG8000.ConclusionsA combination of growth assays, transcriptome profiling, and membrane fatty acid analyses revealed that permeating and non-permeating solutes trigger different adaptive responses in strain RW1, suggesting these solutes affect cells in fundamentally different ways. Future work is now needed that connects these responses with the responses observed in more realistic scenarios of soil desiccation.

Aging induces a distinct gene expression program in mouse islets

The role of aging in the pathogenesis of type 2 diabetes remains poorly understood. In the past adult β-cells were assumed to undergo frequent turnover. However, we find that β-cell turnover declines to very low levels in middle-aged mice. We therefore hypothesized that aged islets could exhibit a distinct gene expression program. We compared gene expression in islets from young mice to islets from aged mice under basal conditions. Aging was associated with differential expression of many genes in islets, including mRNAs encoding for chromatin remodeling components, RNA binding proteins, and pancreatic endocrine transcription factors. We previously observed that cell cycle entry of β-cells is severely restricted by middle age, with minimal of β-cell proliferation in response to regenerative stimuli such as 50% partial pancreatectomy. To characterize the effect of age in adaptive β-cell proliferation, we measured gene expression in islets from young mice after pancreatectomy. As expected, partial pancreatectomy induced differential expression of many genes, including those encoding Reg (regenerating) proteins. Surprisingly, partial pancreatectomy also induced expression of Reg genes in islets from aged mice, which have greatly reduced capacity for adaptive β-cell proliferation. However, there was little overlap (besides the Reg genes) in between the partial pancreatectomy induced islet genes in young mice versus old mice. Thus, partial pancreatectomy does not induce the same gene expression program in young mice vs old mice. Taken together, our results reveal that aged islets exhibit a unique gene expression signature that could contribute to the limited regenerative capacity of mature β-cells.

Plant responses to cold: transcriptome analysis of wheat

Temperature and light are important environmental stimuli that have a profound influence on the growth and development of plants. Wheat varieties can be divided on the basis of whether they require an extended period of cold to flower (vernalization). Varieties that have a requirement for vernalization also tend to be winter hardy and are able to withstand quite extreme subzero temperatures. This capacity, however, is not constitutive and plants require a period of exposure to low, non-freezing temperatures to acquire freezing tolerance: this process is referred to as cold acclimation. Cold acclimation and the acquisition of freezing tolerance require the orchestration of many different, seemingly disparate physiological and biochemical changes. These changes are, at least in part, mediated through the differential expression of many genes. Some of these genes code for effector molecules that participate directly to alleviate stress. Others code for proteins involved in signal transduction or transcription factors that control the expression of further banks of genes. In this review, we provide an overview of some of the main features of cold acclimation with particular focus on transcriptome reprogramming. In doing so, we highlight some of the important differences between cold-hardy and cold-sensitive varieties. An understanding of these processes is of great potential importance because cold and freezing stress are major limiting factors for growing crop plants and periodically account for significant losses in plant productivity.

Gene Expression Profiling Indicates the Roles of Host Oxidative Stress, Apoptosis, Lipid Metabolism, and Intracellular Transport Genes in the Replication of Hepatitis C Virus

Hepatitis C virus (HCV) is a leading cause of chronic liver disease. The identification and characterization of key host cellular factors that play a role in the HCV replication cycle are important for the understanding of disease pathogenesis and the identification of novel antiviral therapeutic targets. Gene expression profiling of JFH-1-infected Huh7 cells by microarray analysis was performed to identify host cellular genes that are transcriptionally regulated by infection. The expression of host genes involved in cellular defense mechanisms (apoptosis, proliferation, and antioxidant responses), cellular metabolism (lipid and protein metabolism), and intracellular transport (vesicle trafficking and cytoskeleton regulation) was significantly altered by HCV infection. The gene expression patterns identified provide insight into the potential mechanisms that contribute to HCV-associated pathogenesis. These include an increase in proinflammatory and proapoptotic signaling and a decrease in the antioxidant response pathways of the infected cell. To investigate whether any of the host genes regulated by infection were required by HCV during replication, small interfering RNA (siRNA) silencing of host gene expression in HCV-infected cells was performed. Decreasing the expression of host genes involved in lipid metabolism (TXNIP and CYP1A1 genes) and intracellular transport (RAB33b and ABLIM3 genes) reduced the replication and secretion of HCV, indicating that they may be important factors for the virus replication cycle. These results show that major changes in the expression of many different genes in target cells may be crucial in determining the outcome of HCV infection.

MicroRNA 21: response to hormonal therapies and regulatory function in leiomyoma, transformed leiomyoma and leiomyosarcoma cells

Aberrant expression of microRNAs (miRNAs), including miR-21, and alteration of their target genes stability have been associated with cellular transformation and tumorigenesis. We investigated the expression, regulation and function of miR-21 in leiomyomas which develop from myometrial cellular transformation. The results indicated that miR-21 is over-expressed in leiomyomas with specific elevation during the secretory phase of the menstrual cycle and in women who received Depo-Provera and oral contraceptives, but reduced due to GnRHa therapy (P < 0.05). Bioinformatic analysis of microarray gene expression profiles previously obtained from the above cohorts, and myometrial smooth muscle cells (MSMC) and leiomyoma smooth muscle cells (LSMC) treated with GnRHa, transforming growth factor (TGF)-β and TGF-β receptor type II (TGF-βRII) antisense oligomer, indicated that a number of miR-21-predicted target genes were co-expressed and differentially regulated in these cohorts. Gain- and loss-of-function of miR-21 in MSMC, LSMC, transformed LSMC and leiomyosarcoma cell line (SKLM-S1) resulted in differential expression of many genes, including some of the miR-21-predicted/validated target genes, PTEN, PDCD4 and E2F1, and TGF-βRII, in a cell-specific manner. Gain-of miR-21 function in MSMC and LSMC reduced TGF-β-induced expression of fibromodulin and TGF-β-induced factor (P < 0.05), and moderately altered the rate of cell growth and caspase-3/7 activity in these cells. We concluded that miR-21 is aberrantly expressed and hormonally regulated in leiomyomas where, through functional interaction with ovarian steroids and the TGF-β signaling pathway, either directly or indirectly regulates a number of genes whose products are critical in leiomyoma growth and regression as well as their potential cellular transformation.

GETTING TO THE HEART OF EXERCISE

Cardiovascular disease is unlikely to be a concern for the many amazing athletes at this year's summer Olympics. This is because exercise capacity is related to heart function, and a strong healthy heart is important for pumping blood around the body during exercise. Being a good athlete and having a strong heart depends a lot on genetics, which can contribute half of the variation seen in human exercise capacity. Some other animal species have also evolved the ability to perform astounding feats of athleticism, often with very little training. However, in humans or animals from the wild it can be very difficult to separate the genetic basis for exercise capacity, heart function and cardiovascular disease from the effects of training and environment. Anja Bye and colleagues from the Norwegian University of Science and Technology therefore decided to examine this topic in rats artificially selected for running endurance.Artificial selection is a way of mimicking natural selection in the lab. Every generation, individual rats with the best and worst running endurance were selected and bred together, generating lines with high and low capacities for running long distances. The authors knew that hearts from the athletic line could pump more blood than those from the unathletic line, because their hearts were bigger and their heart muscle fibres could contract more. They also knew that the low-capacity runners had risk factors for cardiovascular disease, like high blood pressure and elevated lipid levels in the blood. To better understand the underlying molecular basis for these differences, Bye and colleagues compared gene expression in the hearts of these high- and low-capacity rat lines.By measuring the expression of 28,000 genes with gene chips, small microarray slides that quantify the expression of many different genes at once, the authors found that 1540 genes were differentially expressed between high- and low-capacity runners. Many genes were expressed at higher levels in the athletic line, which could explain how their hearts perform better than those of the unathletic line. Contraction and calcium signalling genes were more highly expressed, potentially explaining how their heart muscle fibres are better at contracting. Expression of fat metabolism genes was also enhanced in the high-capacity line, allowing lipids to fuel the high metabolism of their hearts for prolonged periods. In contrast, genes important for carbohydrate metabolism and transport were more highly expressed in the unathletic line, similar to what happens in the hearts of humans with heart disease. Expression of genes involved in cell stress and pathological growth signalling was also higher in the low-capacity line, possibly because the hearts of low-capacity runners aren't getting enough oxygen but are constantly pumping blood at a higher pressure.The results of this study suggest that there is a switch in gene expression between athletes and couch potatoes, from a pattern supporting lipid metabolism and heart muscle contractility to one that indicates an unhealthy heart. The authors have shown that many complex molecular changes contribute to the evolution of athleticism, and have generated novel hypotheses about the links between exercise capacity and heart disease. Although many unanswered questions remain, Bye and colleagues have brought us one step closer to understanding just how much heart it takes to get to the podium!

Role of the DksA-Like Protein in the Pathogenesis and Diverse Metabolic Activity of Campylobacter jejuni

DksA is well known for its regulatory role in the transcription of rRNA and genes involved in amino acid synthesis in many bacteria. DksA has also been reported to control expression of virulence genes in pathogenic bacteria. Here, we elucidated the roles of a DksA-like protein (CJJ81176_0160, Cj0125c) in the pathogenesis of Campylobacter jejuni. As in other bacteria, transcription of stable RNA was repressed by the DksA-like protein under stress conditions in C. jejuni. Transcriptomic and proteomic analyses of C. jejuni 81-176 and an isogenic mutant lacking the DksA-like protein showed differential expression of many genes involved in amino acid metabolism, iron-related metabolism, and other metabolic reactions. Also, the C. jejuni DksA-like protein mutant exhibited a decreased ability to invade intestinal cells and induce release of interleukin-8 from intestinal cells. These results suggest that the DksA-like protein plays an important regulatory role in diverse metabolic events and the virulence of C. jejuni.

Inhibition of HPV-16 L1 expression from L1 cDNAs correlates with the presence of hnRNP A1 binding sites in the L1 coding region

The human papillomavirus type 16 (HPV-16) L1 capsid protein is very poorly expressed from cDNA expression plasmids transiently transfected into mammalian cells. The results described herein demonstrate that inhibition of HPV-16 L1 expression from L1 cDNAs correlates with the presence of splicing regulatory sequences in the L1 coding region. This inhibitory effect correlates with the binding of hnRNP A1 to the RNA elements. Similar to unutilised splice sites that may retain mRNAs in the nucleus, regulatory splicing RNA elements may also inhibit gene expression in the absence of splicing. The results presented here explain the inefficient expression of HPV-16 L1 protein from the wild type L1 cDNA expression plasmids in mammalian cells. These results may be of general interest since alteration of RNA sequences to prevent unwanted RNA-protein interactions may increase expression of many different genes in transient transfections or after plasmid uptake in DNA vaccination approaches.

Protective effects of l-selenomethionine on space radiation induced changes in gene expression

Ionizing radiation can produce adverse biological effects in astronauts during space travel. Of particular concern are the types of radiation from highly energetic, heavy, charged particles known as HZE particles. The aims of our studies are to characterize HZE particle radiation induced biological effects and evaluate the effects of L-selenomethionine (SeM) on these adverse biological effects. In this study, microarray technology was used to measure HZE radiation induced changes in gene expression, as well as to evaluate modulation of these changes by SeM. Human thyroid epithelial cells (HTori-3) were irradiated (1 GeV/n iron ions) in the presence or in the absence of 5 microM SeM. At 6 h post-irradiation, all cells were harvested for RNA isolation. Gene Chip U133Av2 from Affymetrix was used for the analysis of gene expression, and ANOVA and EASE were used for a determination of the genes and biological processes whose differential expression is statistically significant. Results of this microarray study indicate that exposure to small doses of radiation from HZE particles, 10 and 20 cGy from iron ions, induces statistically significant differential expression of 196 and 610 genes, respectively. In the presence of SeM, differential expression of 77 out of 196 genes (exposure to 10 cGy) and 336 out of 610 genes (exposure to 20 cGy) is abolished. In the presence or in the absence of SeM, radiation from HZE particles induces differential expression of genes whose products have roles in the induction of G1/S arrest during the mitotic cell cycle, as well as heat shock proteins. Some of the genes, whose expressions were affected by radiation from HZE particles and were unchanged in irradiated cells treated with SeM, have been shown to have altered expression levels in cancer cells. The conclusions of this report are that radiation from HZE particles can induce differential expression of many genes, some of which are known to play roles in the same processes that have been shown to be activated in cells exposed to radiation from photons (like cell cycle arrest in G1/S), and that supplementation with SeM abolishes HZE particle-induced differential expression of many genes. Understanding the roles that these genes play in the radiation-induced transformation of cells may help to decipher the origins of radiation-induced cancer.

Molecular orchestration of the hepatic circadian symphony

A recent study reveals multiple levels of regulation of the circadian expression of liver proteins.

Microarray analysis of gene expression in metastatic gastric cancer cells after incubation with the methylation inhibitor 5-aza-2′-deoxycytidine

While the exact mechanisms involved in cancer metastasis are not fully clarified, the altered expression of many different genes has been reported. Hypermethylation of the promoters of cancer-related genes is often associated with their inactivation during tumorigenesis and may also be involved in metastasis. Here we used cDNA microarrays to examine the different gene expression profiles of a primary gastric adenocarcinoma cell line RF1 and its derivative metastasis subline RF48. Compared with RF1, 49 genes were down-regulated and 8 genes were up-regulated in RF48. After treatment of RF48 cells with a DNA methylation inhibitor, 5-aza-2'-deoxycytidine, 101 genes were up-regulated and 1 gene was down-regulated in treated RF48 when compared with untreated RF48. Comparing gene expression patterns of untreated RF1, untreated RF48 and treated RF48 cells showed 5 genes expressed in RF1 but silenced in RF48, which were reactivated after 5-aza-2'-deoxycytidine treatment. Two of those 5 genes have CpG islands within their promoter regions, suggesting that those genes activated by 5-aza-2'-deoxycytidine may result from the direct inhibition of promoter methylation. In conclusion, using global gene expression analysis together with inhibition of DNA methylation, we demonstrate that hypermethylation of the promoters of certain cancer-related genes may play a role in cancer metastasis.

RNA isolation for the study of gene expression in human embryos

Most information currently available on the human genome has been collected from adult tissues. As researchers continue to examine in vivo and in vitro development and its impact on infertility, there exists a need to study the genes that are expressed during the preimplantation stages. In the past, it has been difficult to collect this information due to the unavailability of sufficient numbers of human embryos for research. With the advent of new technologies such as microarray analysis, it is now possible to study the expression of many different genes from samples consisting of few cells, including preimplantation stage embryos.

Molecular basis for anaerobic growth of Saccharomyces cerevisiae on xylose, investigated by global gene expression and metabolic flux analysis.

Yeast xylose metabolism is generally considered to be restricted to respirative conditions because the two-step oxidoreductase reactions from xylose to xylulose impose an anaerobic redox imbalance. We have recently developed, however, a Saccharomyces cerevisiae strain that is at present the only known yeast capable of anaerobic growth on xylose alone. Using transcriptome analysis of aerobic chemostat cultures grown on xylose-glucose mixtures and xylose alone, as well as a combination of global gene expression and metabolic flux analysis of anaerobic chemostat cultures grown on xylose-glucose mixtures, we identified the distinguishing characteristics of this unique phenotype. First, the transcript levels and metabolic fluxes throughout central carbon metabolism were significantly higher than those in the parent strain, and they were most pronounced in the xylose-specific, pentose phosphate, and glycerol pathways. Second, differential expression of many genes involved in redox metabolism indicates that increased cytosolic NADPH formation and NADH consumption enable a higher flux through the two-step oxidoreductase reaction of xylose to xylulose in the mutant. Redox balancing is apparently still a problem in this strain, since anaerobic growth on xylose could be improved further by providing acetoin as an external NADH sink. This improved growth was accompanied by an increased ATP production rate and was not accompanied by higher rates of xylose uptake or cytosolic NADPH production. We concluded that anaerobic growth of the yeast on xylose is ultimately limited by the rate of ATP production and not by the redox balance per se, although the redox imbalance, in turn, limits ATP production.

Effect of power-frequency magnetic fields on genome-scale gene expression in Saccharomyces cerevisiae.

To estimate the effect of 50 Hz magnetic-field exposure on genome-wide gene expression, the yeast Saccharomyces cerevisiae was used as a model for eukaryotes. 2D PAGE (about 1,000 spots) for protein and cDNA microarray (about 5,900 genes) analysis for mRNA were performed. The cells were exposed to 50 Hz vertical magnetic fields at 10, 150 or 300 mT r.m.s. for 24 h. As positive controls, the cells were exposed to aerobic conditions, heat (40 degrees C) or minimal medium. The 2D PAGE and microarray analyses for the positive controls showed high-confidence differential expression of many genes including those for known or unknown proteins and mRNAs. For magnetic-field exposure, no high-confidence changes in expression were observed for proteins or genes that were related to heat-shock response, DNA repair, respiration, protein synthesis and the cell cycle. Principal component analysis showed no statistically significant difference in principal components, with only insignificant differences between the magnetic-field intensities studied. In contrast, the principal components for the positive controls were significantly different. The results indicate that a 50 Hz magnetic field below 300 mT did not act as a general stress factor like heat shock or DNA damage, as had been reported previously by others. This study failed to find a plausible differential gene expression that would point to a possible mechanism of an effect of magnetic fields. The findings provide no evidence that the magnetic-field exposure alters the fundamental mechanism of translation and transcription in eukaryotic cells.

The Clinical Relevance of the Expression of Several Multidrug-Resistant-Related Genes in Patients with Primary Acute Myeloid Leukemia

Multidrug resistance (MDR) is a complex phenomenon that includes the expression of many different genes regulating drug transport or metabolism, cellular repair or detoxification mechanisms. The co-expression of several genes could be at the basis of the resistant phenotype in vivo. In order to test a possible prognostic role of the expression and co-expression of several MDR-related genes (MDR1, topoisomerase IIα, topoisomerase IIβ, MRP, GSTπ, LRP), 35 patients affected by acute myeloid leukemia (AML) were tested by RT-PCR assays. In our series, topoisomerase IIβ was significantly co-expressed with MRP ( p=0.05), GSTπ (p=0.017) and LRP (p=0.005). GSTπ was co-expressed with LRP (p=0.03) and MRP (p=0.007); on the other hand, 53.8% of patients were LRP and MRP-positive (p=0.02). The PCR-positivity did not differ according to biological/clinical characteristics of patients, including age; this latter was the only parameter conditioning the response and overall survival. Neither the expression nor the co-expression of the tested genes was significantly correlated with the response to the induction treatment and long-term outcome.

Ability of E. coli Cyclic AMP Receptor Protein To Differentiate Cyclic Nucelotides: Effects of Single Site Mutations

Escherichia coli cyclic AMP receptor protein (CRP) is a global transcriptional regulator which controls the expression of many different genes. Although different cyclic nucleotides can bind to CRP with almost equal affinity, only in the presence of cAMP could wild-type CRP bind to specific DNA sequences. Molecular genetic studies have identified a class of mutants, CRP*, which either do not require exogenous cAMP for activation or can be activated by cGMP. Thus, these mutants might aid in identifying the structural elements that are involved in the modulation of CRP to correctly differentiate the messages embedded in cyclic nucleotides. In this in vitro study, five CRP* mutants, namely, D53H, S62F, G141Q, G141K, and L148R, were tested for their abilities to bind the lac promoter sequence and the effects of cyclic nucleotides in modulating DNA sequence recognition. For comparison, non-CRP* mutants K52N, T127L, H159L, and K52N/H159L were studied. cCMP and cGMP can replace cAMP as an allosteric effector in all of these CRP mutants except S62F and non-CRP* mutants. The D53H, G141Q, G141K, and L148R mutants exhibit significantly higher affinity for the lac promoter sequence than wild-type CRP while S62F and the non-CRP* mutants exhibit reduced affinity. To probe the pathway of communication, the energetics of subunit assembly in these mutants were monitored by sedimentation equilibrium, and the conformational states of these mutants were probed by proteolysis and accessibility of Cys178 to chemical modifications. Results from these studies imply that signals due to mutations are mostly transmitted through the subunit interface. Thus, residues in CRP outside of the cyclic nucleotide binding site modulate the ability of CRP to differentiate these three cyclic nucleotides through long-range communication. Furthermore, this study shows that CRP* mutations do not impart any unique properties to CRP except that the DNA binding constants are shifted to a regime of higher affinity.

Cellular metabolism and actions of 13-cis-retinoic acid

Retinoids (vitamin A and its derivatives) are potent substances for regulating the expression of many different genes within the body. The gene regulatory activities of retinoids are mediated primarily by the all-trans and 9-cis isomers of retinoic acid. Although 13-cis-retinoic acid (isotretinoin) does not have the potent gene regulatory activity of the other two isomers, it is an effective pharmacologic agent for treating a variety of dermatologic conditions. Because 13-cis-retinoic acid is also a naturally occurring retinoid that is present in the circulation, question is raised as to the biochemical mechanism(s) responsible for its pharmacologic efficacy. Some of this efficacy likely arises from the ability of 13-cis-retinoic acid to undergo isomerization to the significantly more active all-trans and 9-cis isomers; however, this does not account for all of the pharmacologic effects observed upon use of this retinoid. Some recent studies suggest that 13-cis-retinoic acid may act by inhibiting the actions of enzymes that are needed to metabolize steroids, while other recent studies indicate that 13-cis-retinoic acid acts through membrane receptors present on the surface of cells. At the present, it is not possible to rule out still other possible biochemical actions of 13-cis-retinoic acid in the body. It is clear, however, that if we are to fully understand the basis for the clinical efficacy of 13-cis-retinoic acid, a better understanding of such biochemical actions is needed. (J Am Acad Dermatol 2001;45:S129-35.)