Biomedical subjects

Kenneth S Ramos

Publications and source records attributed to Kenneth S Ramos.

At least 19 recordsLinked to original sources

PubMed2026

Exploiting DNA damage tolerance for precision oncology.

Unresolved DNA lesions trigger replication stress, forcing cancer cells to hijack DNA damage tolerance (DDT) networks, specifically translesion synthesis (TLS) and template switching, to sustain replication. While DDT prevents lethal fork collapse, error-prone TLS drives mutagenesis, tumor evolution, chemoresistance and radioresistance. Proliferating cell nuclear antigen post-translational modifications dynamically govern pathway selection. Cancer cells exploit this plasticity, creating actionable vulnerabilities such as postreplicative single-stranded DNA gaps. Emerging inhibitors targeting TLS polymerases, upstream regulators such as ubiquitin-specific peptidase 1 (USP1), and critical protein-protein interactions offer unprecedented opportunities for precision oncology. By integrating DDT inhibition with biomarkers such as homologous recombination deficiency and tumor mutational burden, we can drive synthetic lethality, sensitize tumors to genotoxic agents, suppress treatment-induced mutagenesis, and potentially enhance responses to immunotherapy.

DDT↗

PubMed2006

A stochastic model of gene transcription: an application to L1 retrotransposition events.

A simplified mathematical model of gene transcription is presented based on a system of coupled chemical reactions and a corresponding set of stochastic equations similar to those used in enzyme kinetics theory. The quasi-stationary distribution for the model is derived and its usefulness illustrated with an example of model parameters estimation using sparse time course data on L1 retrotransposon expression kinetics. The issue of model validation is also discussed and a simple validation procedure for the estimated model is devised. The procedure compares model predicted values with the laboratory data via the standard Bayesian techniques with the help of modern Markov-Chain Monte-Carlo methodology.

Animals↗

PubMed2006

Sequence analysis of RNase MRP RNA reveals its origination from eukaryotic RNase P RNA.

RNase MRP is a eukaryote-specific endoribonuclease that generates RNA primers for mitochondrial DNA replication and processes precursor rRNA. RNase P is a ubiquitous endoribonuclease that cleaves precursor tRNA transcripts to produce their mature 5' termini. We found extensive sequence homology of catalytic domains and specificity domains between their RNA subunits in many organisms. In Candida glabrata, the internal loop of helix P3 is 100% conserved between MRP and P RNAs. The helix P8 of MRP RNA from microsporidia Encephalitozoon cuniculi is identical to that of P RNA. Sequence homology can be widely spread over the whole molecule of MRP RNA and P RNA, such as those from Dictyostelium discoideum. These conserved nucleotides between the MRP and P RNAs strongly support the hypothesis that the MRP RNA is derived from the P RNA molecule in early eukaryote evolution.

Base Sequence↗

PubMed2006

Activation of human long interspersed nuclear element 1 retrotransposition by benzo(a)pyrene, an ubiquitous environmental carcinogen.

Long interspersed nuclear elements [LINE-1 (L1)] are abundant retrotransposons in mammalian genomes that remain silent under most conditions. Cellular stress signals activate L1, but the molecular mechanisms controlling L1 activation remain unclear. Evidence is presented here that benzo(a)pyrene (BaP), an environmental hydrocarbon metabolized by mammalian cytochrome P450s to reactive carcinogenic intermediates, increases L1 retrotransposition in HeLa cells. Increased retrotransposition is mediated by up-regulation of L1 RNA levels, increased L1 cDNA synthesis, and stable genomic integration. Activation of L1 is dependent on the ability of BaP to cause DNA damage because it is absent in HeLa cells challenged with nongenotoxic hydrocarbon carcinogens. Thus, the mutations and genomic instability observed in human populations exposed to genotoxic environmental hydrocarbons may involve epigenetic activation of mobile elements dispersed throughout the human genome.

Benzo(a)pyrene↗

PubMed2006

Modulation of biological regulatory networks during nephrogenesis.

Mesenchymal-to-epithelial transition is an important biological event during the course of renal cell differentiation as condensing mesenchyme gives rise to tubuloepithelial structures. Wilms' tumor suppressor gene (Wt1) has been identified as a master regulator of the complex genetic events that mediate mesenchymal transdifferentiation. Evidence is summarized here showing that the tightly regulated series of genetic and biochemical events during nephrogenesis is disrupted by superactivation of aryl hydrocarbon receptor (Ahr) by benzo(a)pyrene (BaP), a ubiquitous polycyclic aromatic hydrocarbon and renal carcinogen (Falahatpisheh and Ramos, 2003). Nephron formation is inhibited by BaP, a response that involves inhibition of metanephric cell differentiation and shifts in the relative abundance of Wt1 splice variants. A systems biology paradigm that combined approaches from genomics, transcriptomics, and bioinformatics revealed that the global response of murine metanephric cultures to BaP involves downregulation of Ahr and disruption of downstream targets of Wt1. Discrete networks of genetic regulation were resolved using Boolean idealizations and included genes involved in renal cell differentiation and metabolic control. This work has established a role for Ahr in renal cell differentiation and kidney development and resolved putative molecular interactions between Ahr and Wt1.

Animals↗

PubMed2006

Transcriptional profiling and functional genomics reveal a role for AHR transcription factor in nephrogenesis.

Transcriptional profiling and functional genomics experiments using E11.5 metanephros organ cultures from Ahr(-/-) and Ahr(+/+) have shown that aryl hydrocarbon receptor (AHR) transcription factor is involved in the regulation of mesenchymal-to-epithelial transition (MET) during nephrogenesis. This response is mediated by alterations in the post-transcriptional control of Wilms' tumor suppressor (Wt1) gene and Wt1 splicing. In this article, biologically relevant gene predictor sets of the nephrogenic response were calculated for target genes of interest. The predictability of the gene set for each target was quantified by the coefficient of determination which provided a good criterion for identification of predictor sets that define the complex gene-gene interactions co-regulated by Ahr and Wt1. A subset of the signature genes was found to be co-regulated by Ahr and Wt1 and was responsible for shifts in renal cell transdifferentiation.

Animals↗

PubMed2005

Modulation of alpha7-integrin-mediated adhesion and expression by platelet-derived growth factor in vascular smooth muscle cells.

We showed previously that the expression of alpha(7)-integrin in aortic vascular smooth muscle cells (VSMC) is enhanced in a rat model of atherosclerosis. In the present study, we investigated the effects of platelet-derived growth factor (PDGF) on alpha(7)-integrin expression and VSMC adhesion and migration. Expression of the alpha(7)-integrin gene was determined by real-time RT-PCR, whereas protein levels were determined by fluorescence-activated cell sorting analysis. PDGF increased alpha(7) cell surface protein expression (12 and 24 h: 3.3 +/- 0.8- and 3.6 +/- 0.4-fold, P < 0.05 vs. control) and mRNA levels (24 h: 3.1-fold, P < 0.05 vs. control) in a time-dependent manner. Actinomycin D and cycloheximide attenuated PDGF-induced increases in alpha(7)-integrin, indicating the involvement of de novo mRNA and protein synthesis. Treatment with the MAPK inhibitors PD-98059, SP-600125, and SB-203580 attenuated PDGF-induced increases in mRNA. In contrast, PD-98059 and SP-600125, but not SB-203580, attenuated PDGF-induced increases in cell surface protein levels. PDGF-treated VSMC adhered to laminin more efficiently (42 +/- 6% increase, P < 0.01), and this increase was partially inhibited by anti-alpha(7)-integrin function-blocking antibody. However, PDGF did not alter migration on laminin, and there was no effect of the anti-alpha(7)-integrin function-blocking antibody on basal or PDGF-stimulated migration. Immunofluorescence imaging revealed an increase in alpha(7)-integrin distribution along the stress fibers. Together, these observations indicate that PDGF enhances alpha(7)-integrin expression in VSMC and promotes alpha(7)-integrin-mediated adhesion to laminin.

Animals↗

PubMed2005

Disruption of glomerular cell-cell and cell-matrix interactions in hydrocarbon nephropathy.

Environmental chemicals play an etiological role in greater than 50% of idiopathic glomerular diseases. The present studies were conducted to define mechanisms of renal cell-specific hydrocarbon injury. Female rats were given 10 mg/kg benzo(a)pyrene (BaP) once a week for 16 wk. Progressive elevations in total urinary protein, protein/creatinine ratios, and microalbuminuria were observed in rats treated with BaP for up to 16 wk. The nephropathic response involved early reductions in mesangial cell numbers and fibronectin levels by 8 wk, coupled to transient increases in podocyte cellularity. Changes in podocyte numbers subsided by 16 wk and correlated with rebound increases in mesangial cell numbers and fibronectin levels, along with increased alpha-smooth muscle actin and Cu/Zn superoxide dismutase and fusion of podocyte foot processes. In culture, mesangial cells were more sensitive than podocytes to hydrocarbon injury and expressed higher levels of inducible aryl hydrocarbon hydroxylase activity. Naïve mesangial cells exerted a strong inhibitory influence on podocyte proliferation under both direct and indirect coculture conditions, and this response involved a mesangial cell-derived matrix that selectively inhibited podocyte proliferation. These findings indicate that hydrocarbon nephropathy in rats involves disruption of glomerular cell-cell and cell-matrix interactions mediated by deposition of a mesangial cell-derived growth-inhibitory matrix that regulates podocyte proliferation.

Animals↗

PubMed2005

Modulation of alpha4 integrin mRNA levels is coupled to deficits in vasomotor function in rat arterioles by allylamine.

Allylamine, a selective cardiovascular toxin that induces oxidative stress, is known to alter expression of extracellular matrix and cell adhesion proteins that are central to arterial remodeling. Our goals were to determine whether AAM treatment in rats modulates integrin/matrix-dependent arteriolar function, and to what extent integrin expression correlated to these alterations. Integrins are transmembrane proteins that facilitate mechanical and molecular signaling between the extracellular matrix and cytoskeleton, and so are suitable candidates for involvement in phenotypic and functional alterations of smooth muscle in response to oxidative stress. Arg-Gly-Asp (RGD) and Leu-Asp-Val (LDV), two integrin-binding motifs found in ECM proteins such as collagens and fibronectin, are known to interact with integrins alphavbeta3 and alpha4beta1, respectively. Previously, we found that RGD containing peptides induce vasodilation through alphavbeta3, while LDV containing peptides induce vasoconstriction through alpha4beta1 of normal rat cremasteric arterioles. In allylamine-treated rats (AAM), the vasomotor response to LDV, but not RGD, was attenuated in a dose-dependent manner. To determine whether changes in integrin subunit mRNA levels correlated with these functional changes, we performed reverse transcription and Real-time PCR for alpha4 and beta3 integrin subunits on RNA isolated from single, first-order cremasteric arterioles. AAM treatment caused a dose-dependent decrease in alpha4 mRNA expression, but not beta3 mRNA expression, suggesting that the changes in vasomotor activity to LDV peptides may be attributable in part to reduced alpha4 expression upon exposure to AAM. These data are supported by similar decreases in alpha4integrin cell surface protein expression in cultured vascular smooth muscle cells treated either in vivo and in vitro with AAM.

Allylamine↗

PubMed2005

Differential profiles of copper-induced ROS generation in human neuroblastoma and astrocytoma cells.

To determine neuronal and glial responses to copper (Cu) elevation in the CNS, human neuroblastoma and astrocytoma cells were used to compare their responses to Cu in terms of reactive oxygen species (ROS) generation and expression of enzymes responsible for anti-oxidation. Astrocytoma cells, not neuroblastoma cells, were responsive to Cu and Cu elevation was associated with ROS generation. Intracellular Cu levels as determined by inductively coupled plasma-mass spectrometry (ICP-MS), and expression levels of copper-transporting ATPase (ATP7A) and human copper transporter 1 (hCtr1) as detected by quantitative reverse transcription-polymerase chain reaction (RT-PCR), were comparable in both cell lines. Differences in Cu-induced ROS between two cell lines paralleled superoxide dismutase (SOD)-catalase expression as detected by Western blot analysis. Copper,zinc-SOD (Cu,Zn-SOD) and catalase protein levels were upregulated by Cu in neuroblastoma cells while Cu,Zn-SOD was down-regulated by Cu and catalase level was not changed in astrocytoma cells. Manganese-SOD (Mn-SOD) was not responsive to Cu in either cell line. Furthermore, 78-kDa glucose-regulated protein aggregation and upregulation were observed in Cu-treated astrocytoma cells, but not neuroblastoma cells. These data suggest that neurons use the SOD-catalase system to scavenge Cu-induced ROS while glia rely on the endoplasmic reticulum stress response to compensate for the reduction of ROS scavenging capacity.

Adenosine Triphosphatases↗

PubMed2005

The involvement of copper transporter in lead-induced oxidative stress in astroglia.

Lead (Pb), depositing primarily in astroglia in the brain, is a well-known neurotoxicant and a risk factor for neurologic disorders. Pb has been reported to induce oxidative stress by probably the disturbance of copper (Cu) homeostasis in astroglia. Thus, we hypothesized that Pb-induced oxidative stress is initiated by interfering with Cu transporter in astroglia. In this study, we observed Pb-induced oxidative stress as indicated by reactive oxygen species (ROS) augmentation and GRP78 and GRP94 protein induction, and it was parallel to Cu accumulation intracellularly by Pb. To further address Cu transporter as a potential Pb target, a heavy metal-binding (HMB) domain of Cu-transporting ATPase (Atp7a) was overexpressed and purified. Evidence showed that one molecule of HMB chelated 11 Pb ions or seven Cu ions and that Pb competed with Cu for binding to HMB. These findings suggest that Pb-induced oxidative stress results from the impairment of Cu metabolism by Pb targeting of Atp7a.

Adenosine Triphosphatases↗

PubMed2005

GRP78 compartmentalized redistribution in Pb-treated glia: role of GRP78 in lead-induced oxidative stress.

Glucose-regulated protein of 78 kDa (GRP78) is an endoplasmic reticulum (ER) molecular chaperone functioning in protein folding, assembly and trafficking. GRP78 also plays a role in protection against cytotoxicity and apoptosis induced by environmental insults. Our previous study showed that lead (Pb) directly targets GRP78 by binding to the protein and increasing GRP78 levels. In this study, the effect of Pb on compartmentalized distribution of GRP78 in living cells was examined. A rat GRP78-EGFP fusion protein and EGFP were transiently expressed in astrocytoma cells exposed to 5 microM Pb acetate or 50 microM CuSO4 and fluorescence signals were captured. Control cells expressing EGFP showed a homogeneous distribution of EGFP that was not changed by Pb or Cu treatment. Cells expressing GRP78-EGFP showed a compartmentalized, non-homogeneous distribution of GRP78-EGFP in the cytosol. The redistribution of GRP78-EGFP fluorescent bodies was observed in cells exposed to Pb for 10 h, but not 5 h. Redistribution was also observed in cells exposed to 50 microM Cu for 5 or 10 h. To assess GRP78 function, GRP78 was depleted with dsRNAi oligos in rat C6 glioma cells. GRP78 depletion significantly increased the sensitivity of cells to Pb exposure as indicated by the generation of reactive oxygen species (ROS). These data suggest that Pb directly targets GRP78 and induces its compartmentalized redistribution in living cells and that GRP78 plays a protective role in Pb neurotoxicity.

Animals↗

PubMed2005

In vitro models to evaluate acute and chronic injury to the heart and vascular systems.

Multiple in vitro model systems are currently available to evaluate structure and function relationships in the cardiovascular system as well as the system's response to injury. As the level of molecular sophistication continues to advance, so does the level of complexity of the analysis. One of the most daunting tasks faced by researchers interested in studying cardiovascular function and injury is the selection of the system or systems best suited to answer the particular question at hand. In order to successfully apply any given model system, the researcher must recognize the advantages and limitations in the system of choice. This review provides a listing of the historical and modern techniques used to study cardiovascular function and chemically-induced toxicity. With the growing number of new pharmaceuticals discovered each year, it is imperative to use experimental model systems that allow for identification of targets that participate in or mediate adverse outcomes. Clearly, in vitro analysis cannot replace in vivo experimentation, but the methods currently available allow for a reduction in the number of animals used for experimentation and a better understanding of the complexity associated with the injury response.

Animals↗

PubMed2005

Bioactivation of polycyclic aromatic hydrocarbon carcinogens within the vascular wall: implications for human atherogenesis.

Atherogenesis is a complex pathogenetic process involving a variety of structural and functional deficits within the arterial wall that culminate in the formation of fibrous atherosclerotic plaques. Cigarette smoking is potentially the most remediable contributor to cardiovascular mortality and morbidity. Among the 4000 plus chemicals present in tobacco and tobacco smoke, polycyclic aromatic hydrocarbons (PAHs) have been firmly implicated in the etiology of atherosclerosis in experimental model systems. However, the molecular mechanisms responsible for PAH-induced vascular injury are not well understood. In this review, we have focused on the mechanisms of bioactivation of PAHs in the vas-culature, and the possible role(s) of cytochrome P4501A and 1B enzymes in the formation of PAH-DNA adducts within the vessel wall, a phenomenon that may contribute to the development of atherosclerotic plaques in humans.

Animals↗

PubMed2005

A surprisingly large RNase P RNA in Candida glabrata.

We have found an extremely large ribonuclease P (RNase P) RNA (RPR1) in the human pathogen Candida glabrata and verified that this molecule is expressed and present in the active enzyme complex of this hemiascomycete yeast. A structural alignment of the C. glabrata sequence with 36 other hemiascomycete RNase P RNAs (abbreviated as P RNAs) allows us to characterize the types of insertions. In addition, 15 P RNA sequences were newly characterized by searching in the recently sequenced genomes Candida albicans, C. glabrata, Debaryomyces hansenii, Eremothecium gossypii, Kluyveromyces lactis, Kluyveromyces waltii, Naumovia castellii, Saccharomyces kudriavzevii, Saccharomyces mikatae, and Yarrowia lipolytica; and by PCR amplification for other Candida species (Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida stellatoidea, and Candida tropicalis). The phylogenetic comparative analysis identifies a hemiascomycete secondary structure consensus that presents a conserved core in all species with variable insertions or deletions. The most significant variability is found in C. glabrata P RNA in which three insertions exceeding in total 700 nt are present in the Specificity domain. This P RNA is more than twice the length of any other homologous P RNAs known in the three domains of life and is eight times the size of the smallest. RNase P RNA, therefore, represents one of the most diversified noncoding RNAs in terms of size variation and structural diversity.

Ascomycota↗

PubMed2005

DNA sequence determinants of nuclear protein binding to the c-Ha-ras antioxidant/electrophile response element in vascular smooth muscle cells: identification of Nrf2 and heat shock protein 90 beta as heterocomplex components.

The antioxidant/electrophile response element (ARE/EpRE) is a cis-acting element involved in redox regulation of c-Ha-ras gene. Protein binding to the ARE/EpRE may be credited to deoxyribonucleic acid sequence; therefore, studies were conducted to evaluate the influence of internal and flanking regions to the 10-bp human c-Ha-ras ARE/EpRE core (hHaras10) on nuclear protein binding in oxidant-treated vascular smooth muscle cells. A protein doublet bound to an extended oligonucleotide comprising the ARE/EpRE core in genomic context (hHaras27), whereas a single complex bound to hHarasl0. Protein binding involved specific interactions of 25- and 23-kDa proteins with hHarasl0, and binding of 80-, 65-, and 55-kDa proteins to hHaras27. Competition assays with hNQO1 and rGSTA2 confirmed the specificity of deoxyribonucleic acid-protein interactions and indicated preferred binding of p25 and p23 to the c-Ha-ras ARE/EpRE. "NNN" sequences within the core afforded unique protein-binding profiles to the c-Ha-ras ARE/EpRE. In addition, Nrf2 and heat shock protein 90beta (p80) were identified as components of the c-Ha-ras ARE/EpRE heterocomplex. We conclude that both internal bases and flanking sequences regulate nuclear protein recruitment and complex assembly on the c-Ha-ras ARE/EpRE.

Genes, ras↗

PubMed2005

Atherosclerosis and cancer: flip sides of the neoplastic response in mammalian cells?

Scientific and medical evidence over the past 30 years has established striking parallels between atherosclerosis and cancer--pathogenetic relationships that cross the boundaries of fiction into the realm of reason. Both diseases in humans are characterized by uncontrolled regulation of cellular growth and differentiation and share many common genomic targets during the course of growth dysregulation. Such parallels can be reconciled if atherosclerotic plaques are viewed as neoplasms of smooth muscle origin.

Animals↗

PubMed2004

Calmodulin involvement in stress-activated nuclear localization of albumin in JB6 epithelial cells.

We report that albumin is translocated to the nucleus in response to oxidative stress. Prior measurements have demonstrated that in concert with known transcription factors albumin binds to an antioxidant response element, which controls the expression of glutathione S-transferase and other antioxidant enzymes that function to mediate adaptive cellular responses [Holderman, M. T., Miller, K. P., Dangott, L. J., and Ramos, K. S. (2002) Mol. Pharmacol. 61, 1174-1183]. To investigate the mechanisms underlying this adaptive cell response, we have identified linkages between calcium signaling and the nuclear translocation of albumin in JB6 epithelial cells. Under resting conditions, albumin and the calcium regulatory protein calmodulin (CaM) co-immunoprecipitate using antibodies against either protein, indicating a tight association. Calcium activation of CaM disrupts the association between CaM and albumin, suggesting that transient increases in cytosolic calcium levels function to mobilize intracellular albumin to facilitate its translocation into the nucleus. Likewise, nuclear translocation of albumin is induced by exposure of cells to hydrogen peroxide or a phorbol ester, indicating a functional linkage between reactive oxygen species, calcium, and PKC-signaling pathways. Inclusion of an antioxidant enzyme (i.e., superoxide dismutase) blocks nuclear translocation, suggesting that the oxidation of sensitive proteins functions to coordinate the adaptive cellular response. These results suggest that elevated calcium transients and associated increases in reactive oxygen species contribute to adaptive cellular responses through the mobilization and nuclear translocation of cellular albumin.

Albumins↗