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The role of tetraspanin CD81 in malignant transformation is best studied in colorectal cancer, and it appears that other transcripts beside the fully coding mRNA may also be dysregulated in malignant cells. Recent data from a comprehensive pan-cancer transcriptome analysis demonstrated differential activity of two alternative CD81 gene promoters in malignant versus nonmalignant gut mucosa. The promoter active in gut mucosa gives rise to transcripts CD81-203 and CD81-213, while the promoter active in colon and rectal cancer gives rise to transcripts CD81-205 and CD81-215. Our study aimed to explore the biomarker potential of the transcripts from the alternative CD81 gene promoters in colon cancer, as well as to investigate their structure and potential function using in silico tools. The analysis of the transcripts' expression in several colon cell lines cultivated in 2D and 3D and a set of colon cancer and healthy gut mucosa samples by qPCR and RNA sequencing suggested their low expression and stromal origin. Expression patterns in tumor and nontumor tissue along with in silico data suppose that the transcript CD81-215 may be a noncoding RNA of stromal origin with possible involvement in signaling related to malignant transformation.

More than 50 lysosomal storage diseases (LSDs) are associated with lysosomal dysfunctions with the frequency of 1:5,000 live births. As a result of missing enzyme activity, the lysosome dysfunction accumulates undegraded or partially degraded molecules, affecting the entire body. Most of them are life-threatening diseases where patients could die within the first or second decade of life. Approximately 20 LSDs have the approved treatments, which do not provide the cure for the disorder. Therefore, the delivery of missing genes through gene therapy is a promising approach for LSDs. Over the years, ex vivo lentiviral-mediated gene therapy for LSDs has been approached using different strategies. Several clinical trials for LSDs are under investigation.Ex vivo lentiviral-mediated gene therapy needs optimization in dose, time of delivery, and promoter-driven expression. Choosing suitable promoters seems to be one of the important factors for the effective expression of the dysfunctional enzyme. This review summarizes the research on therapy for LSDs that has used different lentiviral vectors, emphasizing gene promoters.

More than 50 lysosomal storage diseases (LSDs) are associated with lysosomal dysfunctions with the frequency of 1:5,000 live births. As a result of missing enzyme activity, the lysosome dysfunction accumulates undegraded or partially degraded molecules, affecting the entire body. Most of them are life-threatening diseases where patients could die within the first or second decade of life. Approximately 20 LSDs have the approved treatments, which do not provide the cure for the disorder. Therefore, the delivery of missing genes through gene therapy is a promising approach for LSDs. Over the years, ex vivo lentiviral-mediated gene therapy for LSDs has been approached using different strategies. Several clinical trials for LSDs are under investigation.Ex vivo lentiviral-mediated gene therapy needs optimization in dose, time of delivery, and promoter-driven expression. Choosing suitable promoters seems to be one of the important factors for the effective expression of the dysfunctional enzyme. This review summarizes the research on therapy for LSDs that has used different lentiviral vectors, emphasizing gene promoters.

Insulin receptor (IR) signaling is central to normal metabolic control and dysregulated in prevalent chronic diseases. IR binds insulin at the cell surface and transduces rapid signaling via cytoplasmic kinases. However, mechanisms mediating long-term effects of insulin remain unclear. Here, we show that IR associates with RNA polymerase II in the nucleus, with striking enrichment at promoters genome-wide. The target genes were highly enriched for insulin-related functions including lipid metabolism and protein synthesis and diseases including diabetes, neurodegeneration, and cancer. IR chromatin binding was increased by insulin and impaired in an insulin-resistant disease model. Promoter binding by IR was mediated by coregulator host cell factor-1 (HCF-1) and transcription factors, revealing an HCF-1-dependent pathway for gene regulation by insulin. These results show that IR interacts with transcriptional machinery at promoters and identify a pathway regulating genes linked to insulin's effects in physiology and disease.

Epigenetics is coming to the fore as a key process which underpins health. In particular emerging experimental evidence has associated alterations to DNA methylation status with healthspan and aging. Mammalian DNA methylation status is maintained by an intricate array of biochemical and molecular processes. It can be argued changes to these fundamental cellular processes ultimately drive the formation of aberrant DNA methylation patterns, which are a hallmark of diseases, such as cancer, Alzheimer’s disease and cardiovascular disease. In recent years mathematical models have been used as effective tools to help advance our understanding of the dynamics which underpin DNA methylation. In this paper we present linear and nonlinear models which encapsulate the dynamics of the molecular mechanisms which define DNA methylation. Applying a recently developed Bayesian algorithm for parameter estimation and model selection, we are able to estimate distributions of parameters which include nominal parameter values. Using limited noisy observations, the method also identified which methylation model the observations originated from, signaling that our method has practical applications in identifying what models best match the biological data for DNA methylation.

[EN] Pectin methylesterases (PMEs) are a family of enzymes involved in plant reproductive processes such as pollen development and pollen tube growth. We have isolated and characterized PsPMEP, a pea (Pisum sativum L.) pollen-specific gene that encodes a protein with homology to PMEs. Sequence analysis showed that PsPMEP belongs to group 2 PMEs, which are characterized by the presence of a processable amino-terminal PME inhibitor domain followed by the catalytic PME domain. Moreover, PsPMEP contains several motifs highly conserved among PMEs with the essential amino acid residues involved in enzyme substrate binding and catalysis. Northern blot and in situ hybridization analyses showed that PsPMEP is expressed in pollen grains from 4 days before anthesis till anther dehiscence and in pollinated carpels. In the PsPMEP promoter region, we have identified several conserved cis-regulatory elements that have been associated with gene pollen-specific expression. Expression analysis of PsPMEP promoter fused to the uidA reporter gene in Arabidopsis thaliana plants showed a similar expression pattern when compared with pea, indicating that this promoter is also functional in a non-leguminous plant. GUS expression was detected in mature pollen grains, during pollen germination, during pollen tube elongation along the transmitting tract, and when the pollen tube reaches the embryo sac in the ovule.

[EN] Pectin methylesterases (PMEs) are a family of enzymes involved in plant reproductive processes such as pollen development and pollen tube growth. We have isolated and characterized PsPMEP, a pea (Pisum sativum L.) pollen-specific gene that encodes a protein with homology to PMEs. Sequence analysis showed that PsPMEP belongs to group 2 PMEs, which are characterized by the presence of a processable amino-terminal PME inhibitor domain followed by the catalytic PME domain. Moreover, PsPMEP contains several motifs highly conserved among PMEs with the essential amino acid residues involved in enzyme substrate binding and catalysis. Northern blot and in situ hybridization analyses showed that PsPMEP is expressed in pollen grains from 4 days before anthesis till anther dehiscence and in pollinated carpels. In the PsPMEP promoter region, we have identified several conserved cis-regulatory elements that have been associated with gene pollen-specific expression. Expression analysis of PsPMEP promoter fused to the uidA reporter gene in Arabidopsis thaliana plants showed a similar expression pattern when compared with pea, indicating that this promoter is also functional in a non-leguminous plant. GUS expression was detected in mature pollen grains, during pollen germination, during pollen tube elongation along the transmitting tract, and when the pollen tube reaches the embryo sac in the ovule.

[EN] In this contribution, a design of a synthetic calibration genetic circuit to characterize the relative strength of different sensing promoters is proposed and its specifications and performance are analyzed via an effective mathematical model. Our calibrator device possesses certain novel and useful features like modularity (and thus the possibility of being used in many different biological contexts), simplicity, being based on a single cell, high sensitivity and fast response. To uncover the critical model parameters and the corresponding parameter domain at which the calibrator performance will be optimal, a sensitivity analysis of the model parameters was carried out over a given range of sensing protein concentrations (acting as input). Our analysis suggests that the half saturation constants for repression, sensing and difference in binding cooperativity (Hill coefficients) for repression are the key to the performance of the proposed device. They furthermore are determinant for the sensing speed of the device, showing that it is possible to produce detectable differences in the repression protein concentrations and in turn in the corresponding fluorescence in less than two hours. This analysis paves the way for the design, experimental construction and validation of a new family of functional genetic circuits for the purpose of calibrating promoters.

[EN] In this contribution, a design of a synthetic calibration genetic circuit to characterize the relative strength of different sensing promoters is proposed and its specifications and performance are analyzed via an effective mathematical model. Our calibrator device possesses certain novel and useful features like modularity (and thus the possibility of being used in many different biological contexts), simplicity, being based on a single cell, high sensitivity and fast response. To uncover the critical model parameters and the corresponding parameter domain at which the calibrator performance will be optimal, a sensitivity analysis of the model parameters was carried out over a given range of sensing protein concentrations (acting as input). Our analysis suggests that the half saturation constants for repression, sensing and difference in binding cooperativity (Hill coefficients) for repression are the key to the performance of the proposed device. They furthermore are determinant for the sensing speed of the device, showing that it is possible to produce detectable differences in the repression protein concentrations and in turn in the corresponding fluorescence in less than two hours. This analysis paves the way for the design, experimental construction and validation of a new family of functional genetic circuits for the purpose of calibrating promoters.

Diabetic nephropathy (DN) is one of the major complications of type 2 diabetes and is associated with coronary disease. Nephrin, a protein mainly expressed in glomeruli, is decreased in DN and other kidney diseases. Since insulin levels are misregulated in type 2 diabetes, a possible connection between DN and its decreased nephrin expression could be the presence of regulatory elements responsive to insulin in the nephrin gene (NPHS1) promoter region. In this work, using bioinformatic tools, we identified a purine-rich GAGA element in the nephrin gene promoter and conducted a genomic study in search of the presence of polymorphisms in this element and its possible association with DN in type 2 diabetic patients. We amplified and sequenced a 514 bp promoter region of 100 individuals and found no genetic variants in the purine-rich GAGA-box of the nephrin gene promoter between groups of patients with diabetes type 2 with and without renal and coronary complications, control patients without diabetes and healthy controls

Glucose stimulates the transcription of the glucagon receptor gene in hepatocytes and in pancreatic β cells. We recently identified a glucose response element in the immediate upstream non-coding region of the rat glucagon receptor gene. We previously showed that this DNA element is centered on a palindromic sequence of 19 nucleotides (termed as G box), containing two E boxes separated by three nucleotides. In the present study, we further characterized the DNA sequence requirements for the glucose induced expression of the rat glucagon receptor gene. Transfection study realized in the insulin-producing INS-1 cells revealed that a fragment of 49 nucleotides, centered on the G box, bears all the features required for the glucose activation. Mutations performed in the 5′-E box totally abolished the glucose responsiveness, whereas mutations or deletion of the 3′-E box only had a limited effect. Deletions performed upstream from the G box revealed that an accessory factor binding site, located in the region just upstream from the G box, is required for full stimulation by glucose. Finally, by using G box based probes in gel shift experiments, we demonstrated that USF1/USF2 transcription factors are part of the proteinic complex that binds to the glucose response element of the rat glucagon receptor gene promoter. In conclusion, in contrast to many other glucose regulated genes, only the 5′-E box appears to be a crucial DNA element for the glucose transcriptional effect. However, an accessory factor binding site located in the region just upstream from the G box is required for a complete stimulation by glucose. © 2002 Elsevier Science B.V. All rights reserved., info:eu-repo/semantics/published

The glucagon receptor gene is a member of a gene family, the expression of which is strongly upregulated by glucose. This review deals with the structure of both the glucagon receptor gene and its promoter. Attention is focused on the glucose regulatory element that we discovered in the promoter of this gene. Regulation by glucose of genes implicated in glucose homeostasis represents one mechanism contributing to the control of fuel utilization. Its deficiency or imbalance could potentially lead to or participate in pathological situations such as diabetes mellitus. On the other hand, the regulatory element of the glucagon receptor gene promoter could be used as a tool for the glucose-regulated expression of other genes. Indeed, an analysis of the glucagon receptor gene promoter demonstrated that only a short fragment of the genomic DNA, easy to subclone, contains all required elements for activation by glucose. Its potential use for gene therapy is also considered, therefore, in this report., SCOPUS: sh.j, info:eu-repo/semantics/published

The glucagon receptor gene is a member of a gene family, the expression of which is strongly upregulated by glucose. This review deals with the structure of both the glucagon receptor gene and its promoter. Attention is focused on the glucose regulatory element that we discovered in the promoter of this gene. Regulation by glucose of genes implicated in glucose homeostasis represents one mechanism contributing to the control of fuel utilization. Its deficiency or imbalance could potentially lead to or participate in pathological situations such as diabetes mellitus. On the other hand, the regulatory element of the glucagon receptor gene promoter could be used as a tool for the glucose-regulated expression of other genes. Indeed, an analysis of the glucagon receptor gene promoter demonstrated that only a short fragment of the genomic DNA, easy to subclone, contains all required elements for activation by glucose. Its potential use for gene therapy is also considered, therefore, in this report., SCOPUS: sh.j, info:eu-repo/semantics/published

The lrp gene of the extreme thermophilic archaeon Sulfolofus solfataricus, encoding a homologue of the eubacterial global leucine-responsive regulatory protein, was identified by DNA sequencing and sequence comparisons on a 6.9-kb genomic fragment cloned into Escherichia coli. The S. solfataricus Lrp subunit is a 155-aa polypeptide that bears between 24.5 and 29% sequence identity with eubacterial regulatory proteins of the Lrp/AsnC family and 30.6% and 25.8% with the archaeal homologues of respectively Methanococcus jannaschii and Pyrococcus furiosus. Transcription initiation from the strong S. solfataricus lrp promoter was analyzed by primer extension mapping. The abundance of the S. solfataricus lrp messenger strongly suggests that this protein might function in archaea as a global transcriptional regulater and genome organizer, as proposed for E. coli Lrp, rather than as a local, specific regulatory protein. Our findings suggest the presence of a eubacterial type of regulatory mechanism in archaea, a situation that is noteworthy indeed, since the transcriptional machinery of archaea is more closely related to that of eukaryotes, whereas these latter apparently do not possess a homologue of Lrp., SCOPUS: ar.j, info:eu-repo/semantics/published

The lrp gene of the extreme thermophilic archaeon Sulfolofus solfataricus, encoding a homologue of the eubacterial global leucine-responsive regulatory protein, was identified by DNA sequencing and sequence comparisons on a 6.9-kb genomic fragment cloned into Escherichia coli. The S. solfataricus Lrp subunit is a 155-aa polypeptide that bears between 24.5 and 29% sequence identity with eubacterial regulatory proteins of the Lrp/AsnC family and 30.6% and 25.8% with the archaeal homologues of respectively Methanococcus jannaschii and Pyrococcus furiosus. Transcription initiation from the strong S. solfataricus lrp promoter was analyzed by primer extension mapping. The abundance of the S. solfataricus lrp messenger strongly suggests that this protein might function in archaea as a global transcriptional regulater and genome organizer, as proposed for E. coli Lrp, rather than as a local, specific regulatory protein. Our findings suggest the presence of a eubacterial type of regulatory mechanism in archaea, a situation that is noteworthy indeed, since the transcriptional machinery of archaea is more closely related to that of eukaryotes, whereas these latter apparently do not possess a homologue of Lrp., SCOPUS: ar.j, info:eu-repo/semantics/published

The role of the ErbB3 protein in breast cancer cell proliferation was examined. In previous studies, we investigated the signal transduction mechanism of the ErbB3 protein, which cooperates with ErbB2 in the formation of a coreceptor for the growth factor heregulin. We demonstrated that the ErbB3 protein, although possessing a protein tyrosine kinase (PTK) homology domain, is in fact devoid of intrinsic PTK activity. In the second year of funding, we have demonstrated the critical dependence of heregulin signalling upon the PTK activity of ErbB2. To this end, the PTK domain of ErbB2 was mutated, such that its intrinsic kinase activity was abolished, and this mutant ErbB2 protein was expressed along with wild-type ErbB3 in cultured cells. Our results indicated that the ErbB2 mutation abolished heregulin signal transduction. We also characterized the interaction of ErbB3 with the p85 subunit of phosphatidylinositol 3-kinase, an enzyme known to be involved in heregulin signalling. By use of the yeast two-hybrid system, we mapped the sites of interaction between ErbB3 and p85. Finally, we have pursued the cloning of the heregulin gene promoter, in effort to understand the regulation of heregulin expression in breast cancer cells.