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PubMed2025

Multi-Omics Platforms Reveal Synergistic Intestinal Toxicity in Tilapia from Acute Co-Exposure to Polystyrene Microplastics, Sulfamethoxazole, and BDE153.

Polystyrene microplastic (MP) and its co-existing contaminants may exert different toxic effects on its surrounding aquatic organisms. In order to detect the intestinal harmful responses, tilapia were subjected to exposure with 75 nm of MPs, 100 ng·L-1 of sulfamethoxazole (SMZ), 5 ng·L-1 of BDE153, and combinations thereof over periods of 2, 4, and 8 days. Enzymatic assays, transcriptomics, proteomics, and metabolomics were employed to evaluate intestinal histopathological effects. Results showed that significant reductions were observed in ATP, ROS, SOD, EROD, lipid metabolism-related enzymes, pro-inflammatory cytokines (TNFα and IL-1β), and apoptosis marker caspase 3 across all groups at day 8. Histological evaluation revealed diminished goblet cell density, with distinct vacuole formation in the BDE153+MPs group. KEGG pathway analysis highlighted disruptions in endocytosis, MAPK signaling, phagosome formation, and actin cytoskeleton regulation. Proteomic findings indicated notable enrichment in endocytosis (decreased sorting nexin-2; increased Si:dkey-13a21.4), MAPK/PPAR signaling, protein processing in the endoplasmic reticulum (Sec61 subunit gamma), and cytoskeletal modulation (reduced fibronectin; elevated activation peptide fragment 1), with or without SMZ and BDE153. Metabolomic profiling showed significant alterations in ABC transporters, aminoacyl-tRNA biosynthesis, protein digestion and absorption, and linoleic acid metabolism. In summary, these findings suggest that BDE153 and MPs synergistically exacerbate intestinal damage and gene/protein expression over time, while SMZ appears to exert an antagonistic, mitigating effect.

Animals↗
PubMed2006

Identification of memory B cells using a novel transgenic mouse model.

Memory B cells help to protect the host from invading pathogens by maintaining persistent levels of Ag-specific serum Ab and generating rapid Ab responses upon re-exposure to Ag. Unambiguous identification of memory B cells has been a major obstacle to furthering our knowledge concerning both the development of B cell memory and secondary Ab responses due to an absence of specific cell surface markers. Germinal centers (GCs) are thought to be the major site of Ig hypermutation and Ag-driven selection of memory B cells. To develop a model that would identify GC-derived memory B cells, we generated transgenic mice that expressed cre recombinase in a GC-specific fashion. Interbreeding these mice with the cre-reporter strain, ROSA26R, produced progeny in which beta-galactosidase (beta-gal) was permanently expressed in B cells of the GC-memory pathway. Analysis following immunization with (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma globulin showed that long-lived beta-gal+ B cells exclusively contained somatically mutated lambda1 V regions and were capable of producing Ag-specific Ab-forming cell (AFC) responses that were >100-fold higher than those afforded by beta-gal- B cells following adoptive transfer to naive hosts. Secondary challenge of immune mice showed that only approximately 20% of secondary AFCs expressed beta-gal. Interestingly, we found that somatic hypermutation of rearranged lambda1 V regions within secondary AFCs showed a strong correlation with beta-gal expression, suggesting that nonmutated B cells contribute significantly to secondary Ab responses. This model should provide useful insights into memory B cell development, maintenance, and differentiation following immunization or pathogenic infection.

Animals↗
PubMed2001

Modelling production of extracellular polymeric substances in a Pseudomonas aeruginosa chemostat culture.

Formation of extracellular polymeric substances (EPSs) by mucoid Pseudomonas aeruginosa was investigated using literature data from chemostat cultures. The data were used to calibrate a product formation regime allowing substrate sufficient and endogenous EPS formation. Yield coefficients for both formation conditions were elucidated based on metabolic pathway analysis. Growth and non-growth related specific formation rates of 0.18 g CEPS/g Ccell/h and 0.03 1/h were estimated, respectively. The exogenous and endogenous EPS yield was found to be 0.77 g CEPS/g Cglu and 0.79 g CEPS/g Ccell, respectively. Being structurally equivalent to comprehensive maintenance models, this model allows for non-growth related product formation, showing the same quality of fit as previous models restricted to exogenous EPS precursors.

Biofilms↗
PubMed2004

[Saccharomyces cerevisiae: an efficient tool and model system for anticancer research].

The major mechanisms of cell function, such as metabolic pathways, DNA repair and cell cycle control have been conserved throughout DNA evolution from yeast to human, despite the former lacks cancer-related features. The S. cerevisiae genome has been entirely sequenced and not only reveals sequence similarity to the human, but a number of proteins also have a conserved function. Owing to its genetic features, yeast has been used as a remarkable model and tool to study important enzymes that are also targets for anticancer drugs, particularly in the field of the pyrimidine salvage pathway. In this review, we describe the recent developments in using yeast for anticancer research, focusing on metabolic pathway analysis, systematic drugs screening with yeast mutants or prospects in gene therapy with yeast enzymes.

Antineoplastic Agents↗
PubMed2004

Pediatric pharmacokinetic data: implications for environmental risk assessment for children.

Pharmacology and toxicology share a common interest in pharmacokinetic data, especially as it is available in pediatric populations. These data have been critical to the clinical pharmacologist for many years in designing age-specific dosing regimens. Now they are being used increasingly by toxicologists to understand the ontogeny of physiologic parameters that may affect the metabolism and clearance of environmental toxicants. This article reviews a wide range of physiologic and metabolic factors that are present in utero and in early postnatal life and that can affect the internal dose of an absorbed chemical and its metabolites. It also presents a child/adult pharmacokinetic database that includes data for 45 therapeutic drugs organized into specific children's age groupings and clearance pathways. Analysis of these data suggests that substantial child/adult differences in metabolism and clearance are likely for a variety of drugs and environmental chemicals in the early postnatal period. These results are also relevant to in utero exposures, where metabolic systems are even more immature, but exposures are greatly modified by the maternal system and placental metabolism. The implications of these child/adult differences for assessing children's risks from environmental toxicants is discussed with special focus on physiologically based pharmacokinetic modeling strategies that could simulate children's abilities to metabolize and eliminate chemicals at various developmental stages.

Adolescent↗
PubMed2004

[Relevance of p53 protein and its mutations for novel strategies in cancer therapy].

Mutational inactivation of the p53 gene products is one of the most common genetic events that occur in human cancer confirming the central role of p53 as a tumour suppressor. The wild type p53 protein acts as a powerful transcription factor binding as many as 300 different promoter sequences in the genome. As a transcription factor, p53 can broadly alter patterns of specific gene expression leading to cell-cycle arrest and/or apoptosis. The transcriptional activity of the p53 protein is precisely regulated and its basal activity is enhanced in cells exposed to a wide variety of stress signals. This stress regulated transactivation function of p53 is driven by its sequence-specific DNA-binding domain and is co-ordinated by specific protein-protein interactions that can be modulated by covalent and non-covalent modifications. The mutant forms of p53 protein are defective in sequence specific DNA-binding because the missense mutation affects one or all of the assembly pathways. Analysis of p53 mutant has shown that different conformational classes of mutants exist and the reactivation of each class requires a different strategy depending on the mechanism of p53 protein misfolding. Potential reactivation of both wild type and mutant p53 protein in vivo raises our hopes of developing anti-cancer drugs useful in combination with sophisticated diagnostics for the treatment of cancer.

Apoptosis↗
PubMed2005

Global genomic approaches to the iron-regulated proteome.

Iron interacts with cells to regulate the proteome through complex effects on gene expression. In simple organisms such as bacteria and yeast, intra- and extra-cellular iron influences gene expression through defined signal transduction pathways. In higher organisms, effects are probably mediated at the transcriptional level through secondary effects of reactive oxygen species, while post-transcriptional effects operate through well-defined pathways involving iron-regulatory proteins. To investigate the impact of iron levels on gene expression and the proteome, approaches such as differential display and subtractive hybridization have the advantage of surveying the entire genome. However, they are technically demanding and have given way to microarray techniques. To date, numerous microarray experiments with various organisms have not yielded any definitive picture of the role of iron. Common themes throughout such studies are that both iron excess and iron depletion influence expression of proteins related to energy metabolism, cell proliferation, matrix structure, and the metabolism of iron itself. That no consistent set of genes is involved from one study to the next probably results both from the uncertainties inherent in the technique and the biological variability of the systems under study. We briefly describe two types of iron-dependent microarray experiments from our laboratory to examine major cellular targets of iron toxicity. Using Affymetrix oligonucleotide arrays with cardiac cells, we found several hundred genes whose mRNA levels were affected by iron, including an increase in several genes responding to oxidative stress and a decrease in several kinases and phosphatases. In a simpler experiment using a human liver cell line with a limited cDNA array, we targeted 13 genes affected by iron chelation. Metabolic pathway analysis shows links of 5 of these through phorbol ester responsiveness, and additional links through prostaglandin E2. We conclude that definitive understanding of the complex iron-regulated proteome requires global gene approaches and rigorous interlaboratory standardization.

Animals↗
PubMedDate not supplied

Comparative effects of DHEA and DHT on gene expression in human LNCaP prostate cancer cells.

BACKGROUND: DHEA is widely used as a dietary supplement in older men. Because DHEA can be converted to androgens or estrogens, such use may promote prostate cancer. In this study, the effects of DHEA were compared with those of DHT using gene expression array profiles in human LNCaP prostate cancer cells. MATERIALS AND METHODS: LNCaP cells were exposed to DHEA (300 nM), DHT (300 nM), or vehicle for 48 h, and mRNA expression was measured using Affymetrix HU-95 gene chips. Gene expression values were sorted in ascending order on the p-values corresponding to the extent of differential RNA expression between control and either hormone treatment. RESULTS: S100 calcium binding protein, neurotensin, 24-dehydrocholesterol reductase, and anterior-gradient 2 homologue were the four most differentially expressed genes (p-values all < 3 x 10(-5)). Nested tests of differential expression revealed lesser effects of DHEA versus DHT treatment (p < 0.01) for the S100 calcium binding protein and neurotensin genes. Microarray findings were confirmed by QRT-PCR. The top 83 genes exhibiting differential expression after DHEA or DHT were used for pathway analysis. DHT decreased expression of more genes involved in intercellular communication, signal transduction, nucleic acid binding and transport, and in structural components, such as myosin and golgin, than DHEA. CONCLUSION: These data revealed consistent, measurable changes in gene expression patterns following treatment of LNCaP prostate cancer cells with DHEA and DHT. Understanding the mechanisms of DHEA versus DHT actions in the prostate may help clarify the separate and interactive effects of androgenic and estrogenic actions in prostate cancer progression.

Adjuvants, Immunologic↗
PubMed1991

Expression and processing of mouse proopiomelanocortin in bovine adrenal chromaffin cells. A model system to study tissue-specific prohormone processing.

Many neuroendocrine precursor proteins, such as proopiomelanocortin (POMC), are cleaved in a tissue specific manner at distinct pairs of basic amino acids. Elucidating the specificity of the prohormone endoprotease(s) is essential to understanding cleavage specificity. However, isolation of these enzymes has been difficult, due to the inability to distinguish authentic maturation enzyme from the many other trypsin-like activities present in tissue homogenates. Recently, a "signature" of the insulin cell endoprotease(s) was defined in vivo by assessing the processing of a series of mutant cleavage sites in a model prohormone, mouse POMC (mPOMC) (Thorne, B. A., and Thomas, G. (1990) J. Biol. Chem. 265, 8436-8443. To investigate mechanisms of tissue-specific processing, we sought to identify the endoprotease signature of a cell having a processing phenotype distinct from insulinoma cells. In this report, the cleavage site specificity of the endoprotease(s) expressed in bovine adrenal chromaffin cells is examined. High levels of mPOMC (1.6 pmol/10(6) cells) were expressed in these cells using a vaccinia virus vector, and the precursor was targeted to the regulated secretory pathway. Analysis of POMC-derived peptides revealed that chromaffin cells processed the prohormone to a set of peptides highly similar to anterior pituitary corticotrophs, including adrenocorticotropin hormone (ACTH) and beta-lipotropin, gamma-lipotropin, and beta-endorphin. This processing contrasted with the pattern of cleavage site utilization in Rin m5F insulinoma cells, which more closely resembled that of the intermediate pituitary melanotrophs. However, the processing preference for the sequences of pairs of basic amino acids (as tested using the entire series of mutant cleavage sites; -LysArg- (native), -ArgArg-, -ArgLys-, -LysLys-, -HisArg-, -MetArg- at the ACTH/beta-lipotropin junction and -LysLys- (native), -LysArg-, -ArgArg-, -ArgLys- in beta-endorphin) was the same in both insulinoma and adrenal chromaffin cells, suggesting recognition and cleavage by similar enzymes in both cell types. The cell-specific processing of mPOMC may thus result from expression of a common core set of processing enzymes and factors unique to each cell type affecting the enzyme accessibility to precursor cleavage sites.

Adrenal Medulla↗
PubMed1986

Mannose receptor-mediated uptake of ricin toxin and ricin A chain by macrophages. Multiple intracellular pathways for a chain translocation.

The role of the high mannose carbohydrate chains in the mechanism of action of ricin toxin was investigated. Ricin is taken up by two routes in macrophages, by binding to cell surface mannose receptors, or by binding of the ricin galactose receptor to cell surface glycoproteins. Removal of carbohydrate from ricin by periodate oxidation led to a large loss in toxicity via both routes of uptake by an effect on the B chain not due to a loss of galactose binding affinity. These data suggest that the carbohydrate chains of ricin B chain may be required for full toxicity. The pathway of uptake of ricin by the macrophage mannose receptor was found to differ in several respects from uptake via the galactose-specific pathway. Analysis of intoxication of macrophages by ricin in the presence of ammonium chloride suggested that mannose receptor bound ligand passes through acidic vesicles prior to translocation, unlike galactose bound ligand. Intoxication by ricin via galactose-specific uptake was potentiated by swainsonine but not by castanospermine, suggesting that ricin may be attacked by an endogenous mannosidase within the cell, and that ricin passes through either a lysosomal or a Golgi compartment prior to translocation.

Ammonium Chloride↗
PubMed1979

[Granulocyte defence against bacterial infection in diabetes mellitus with special reference to bactericidal activity].

Granulocytic defence against bacterial infection has been studied in a group of diabetic patients. With the skin chamber method markedly reduced leukocyte mobilization in vivo which was independent of metabolic compensation was found in all diabetic patients. Investigation of phagocytic function of Latex particles and bacterial killing of E. coli revealed firm dependence on metabolic regulation: in decompensated diabetes mellitus the two function tests were clearly pathological, whereas in the compensated metabolism there was no difference between normal individuals and diabetics. Thus, on granulocytic defence pathway analysis, this appears to confirm that reduction of infection depends on how diabetes is controlled.

Bacterial Infections↗
PubMed1996

Alanine scanning mutagenesis of conserved arginine/lysine-arginine/lysine-X-X-arginine/lysine G protein-activating motifs on m1 muscarinic acetylcholine receptors.

Alanine scanning mutagenesis of B-B-X-X-B motifis (where B is a basic residue and X is any nonbasic residue) in m1 muscarinic acetylcholine receptors was performed to determine the relative roles of basic amino acids in receptor coupling. This conserved motif is found in many G protein-coupled receptors and has been implicated in G protein activation. The KKAAR365 motif, located at the carboxyl-terminal third intracellular loop of m1 receptors, was mutated to AAAAA365, thereby generating a triple-substitution mutant devoid of ability to stimulate either phosphoinositide (PI) hydrolysis or cAMP accumulation. In contrast, a triple-alanine substitution of the KRTPR140 motif in the carboxyl-terminal second intracellular loop, yielding mutant AATPA140, had no effect on receptor coupling to the two independent second messenger pathways. Analysis of a series of single- and double-substitution mutants demonstrate that all three basic residues of the KKAAR365 motif participate in efficient m1 receptor coupling. The presence of second and third basic residues in this motif was absolutely critical for full agonist recognition of a high and low affinity state of the receptor. Mutation of either Lys362 or Lys365, but not-Lys361, abolished guanine nucleotide-dependent conversion of agonist affinity states and correlated with an inability of full agonists to fully activate PI hydrolysis. The different combinatorial double-substitution mutants also revealed that Lys365 was necessary but not sufficient, in the context of the KKAAR365 motif, for efficient receptor coupling. This residue cannot facilitate full agonist-stimulated Pl hydrolysis in the absence of both Lys361 and Lys362. In comparison, the critical residue Lys362 was both necessary and sufficient. Substitution of nearby basic residues Lys361 and Lys365 with alanine yielded mutant AKAAA365, which exhibited partial ability to couple PI hydrolysis after full agonist stimulation. Therefore, Lys365 seems to function in a hierarchal (interdependent) manner with nearby basic residues, whereas Lys361 and Lys362 can act independent of surrounding basic residues to facilitate partial m1 receptor coupling after full agonist stimulation. In contrast, all three residues must be present for stimulation of PI hydrolysis by a partial agonist.

Alanine↗
PubMed1997

Prediction of enzyme classification from protein sequence without the use of sequence similarity.

We describe a novel approach for predicting the function of a protein from its amino-acid sequence. Given features that can be computed from the amino-acid sequence in a straightforward fashion (such as pI, molecular weight, and amino-acid composition), the technique allows us to answer questions such as: Is the protein an enzyme? If so, in which Enzyme Commission (EC) class does it belong? Our approach uses machine learning (ML) techniques to induce classifiers that predict the EC class of an enzyme from features extracted from its primary sequence. We report on a variety of experiments in which we explored the use of three different ML techniques in conjunction with training datasets derived from PDB and from Swiss-Prot. We also explored the use of several different feature sets. Our method is able to predict the first EC number of an enzyme with 74% accuracy (thereby assigning the enzyme to one of six broad categories of enzyme function), and to predict the second EC number of an enzyme with 68% accuracy (thereby assigning the enzyme to one of 57 subcategories of enzyme function). This technique could be a valuable complement to sequence-similarity searches and to pathway-analysis methods.

Algorithms↗
PubMedDate not supplied

Information processing for the analysis of metabolic pathways and inborn errors.

Methods of biotechnology allow the analysis of molecular structures and molecular processes. Different molecular database systems are available which allow worldwide data access. Methods and concepts of bioinformatics are important for the analysis of these molecular data, which represent complex regulatory networks. The detection of metabolic diseases is one application of information systems, which represents molecular and medical data and allows the stimulation of metabolic processes. In this paper we present the architecture of our molecular information system.

Database Management Systems↗
PubMed1999

Pathway alignment: application to the comparative analysis of glycolytic enzymes.

Comparative analysis of metabolic pathways in different genomes yields important information on their evolution, on pharmacological targets and on biotechnological applications. In this study on glycolysis, three alternative ways of comparing biochemical pathways are combined: (1) analysis and comparison of biochemical data, (2) pathway analysis based on the concept of elementary modes, and (3) a comparative genome analysis of 17 completely sequenced genomes. The analysis reveals a surprising plasticity of the glycolytic pathway. Isoenzymes in different species are identified and compared; deviations from the textbook standard are detailed. Several potential pharmacological targets and by-passes (such as the Entner-Doudoroff pathway) to glycolysis are examined and compared in the different species. Archaean, bacterial and parasite specific adaptations are identified and described.

Enzymes↗
PubMed1986

METAMOD: software for steady-state modelling and control analysis of metabolic pathways on the BBC microcomputer.

METAMOD, a BBC microcomputer-based software package for steady-state modelling and control analysis of model metabolic pathways, is described, The package consists of two programs. METADEF allows the user to define the pathway in terms of reactions, rate equations and initial concentrations of metabolites. METACAL uses one of two algorithms to calculate the steady-state concentrations and fluxes. One algorithm uses the current ratio of production and consumption rates of variable metabolites to adjust iteratively their concentrations in such a way that they converge towards the steady state. The other algorithm solves the roots of the system equations by means of a quasi-Newtonian procedure. Control analysis allows the calculation of elasticity, control and response coefficients, by means of finite difference approximation. METAMOD is interactive and easy to use, and suitable for teaching and research purposes.

Algorithms↗
PubMed1989

Quantitative analysis of outflow pathway of corpora cavernosa by pressure flow technique.

Using a pressure flow technique, quantitative analysis of the physiological characteristics of the outflow pathway of the corpus cavernosum was carried out in 19 male dogs weighing 7.5 to 23.0 kg. Pressure flow curves were made on dogs whose pelvic nerve was stimulated electrically and on dogs left unstimulated. When a cyclical change in saline perfusion rate was applied without nerve stimulation, the variable of the intracorporeal pressure showed a large hysteretic loop, indicating that the resistance of the outflow canals to flow was altered by the distension of the sinusoidal space. In dogs whose pelvic nerve was stimulated, the pressure flow curves shifted to the left side in comparison with the outward phase of the pressure flow curve of animals without pelvic nerve stimulation, and this curve piled on the returning phase. No hysteretic relation was observed between the outward and returning phase of the pressure flow curve with pelvic nerve stimulation, but in the detailed analysis, in which the % flow rate was used instead of actual flow rate of saline perfusion, a small hysteretic loop based on the difference of the elasticity of the outlet canals was found. The distension of the corpora cavernosa and the pelvic nerve electrostimulation probably act as the triggers of the same occlusive mechanism in the outflow pathway. The percentage decrease in the blood flow in the outflow canal of the corpus cavernosum induced by the distension of the sinusoidal space or by the pelvic nerve electrostimulation was 69.6 +/- 14.4% (mean +/- SD).

Animals↗