Transcription factors interact with RNA to regulate genes.

Transcription factors (TFs) orchestrate the gene expression programs that define each cell's identity. The canonical TF accomplishes this with two domains, one that binds specific DNA sequences and the other that binds protein coactivators or corepressors. We find that at least half of TFs also bind RNA, doing so through a previously unrecognized domain with sequence and functional features analogous to the arginine-rich motif of the HIV transcriptional activator Tat. RNA binding contributes to TF function by promoting the dynamic association between DNA, RNA, and TF on chromatin. TF-RNA interactions are a conserved feature important for vertebrate development and disrupted in disease. We propose that the ability to bind DNA, RNA, and protein is a general property of many TFs and is fundamental to their gene regulatory function.

The dynamic clustering of insulin receptor underlies its signaling and is disrupted in insulin resistance.

Insulin receptor (IR) signaling is central to normal metabolic control and is dysregulated in metabolic diseases such as type 2 diabetes. We report here that IR is incorporated into dynamic clusters at the plasma membrane, in the cytoplasm and in the nucleus of human hepatocytes and adipocytes. Insulin stimulation promotes further incorporation of IR into these dynamic clusters in insulin-sensitive cells but not in insulin-resistant cells, where both IR accumulation and dynamic behavior are reduced. Treatment of insulin-resistant cells with metformin, a first-line drug used to treat type 2 diabetes, can rescue IR accumulation and the dynamic behavior of these clusters. This rescue is associated with metformin's role in reducing reactive oxygen species that interfere with normal dynamics. These results indicate that changes in the physico-mechanical features of IR clusters contribute to insulin resistance and have implications for improved therapeutic approaches.

Mediator Condensates Localize Signaling Factors to Key Cell Identity Genes.

The gene expression programs that define the identity of each cell are controlled by master transcription factors (TFs) that bind cell-type-specific enhancers, as well as signaling factors, which bring extracellular stimuli to these enhancers. Recent studies have revealed that master TFs form phase-separated condensates with the Mediator coactivator at super-enhancers. Here, we present evidence that signaling factors for the WNT, TGF-ß, and JAK/STAT pathways use their intrinsically disordered regions (IDRs) to enter and concentrate in Mediator condensates at super-enhancers. We show that the WNT coactivator ß-catenin interacts both with components of condensates and DNA-binding factors to selectively occupy super-enhancer-associated genes. We propose that the cell-type specificity of the response to signaling is mediated in part by the IDRs of the signaling factors, which cause these factors to partition into condensates established by the master TFs and Mediator at genes with prominent roles in cell identity.

Ambroxol alleviates hepatic ischemia reperfusion injury by antioxidant and antiapoptotic pathways.

BACKGROUND: Hepatic ischemia/reperfusion (HI/R) injury is a common pathologic process caused by many clinical settings, such as liver resection, liver transplantation, hypovolemic shock, and trauma. The use of ambroxol, which acts as a mucolytic agent, provides antioxidant and anti-inflammatory effects. METHODS: A rat model of HI/R was induced by clamping the hepatic artery, the hepatoportal vein, and the bile duct with a vascular clamp for 30 minutes followed by reperfusion for 6 hours under anesthesia. The sham group underwent laparotomy without hepatic ischemia. The ambroxol group was injected into the tail vein in the ambroxol group 5 minutes before HI/R at one dose of 20 mg/kg, 80 mg/kg, or 140 mg/kg. The control group underwent the same procedure as the ambroxol group but with administration of physiological saline. Liver injury was evaluated by biochemical and histopathological examinations. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed in serum samples. Superoxide dismutase (SOD), catalase (CAT), malondiadehyde (MDA), and glutathione (GSH) were spectrophotometrically measured. Furthermore, caspase-3, Bcl-2 and Bax expression as well as the level of c-Jun N-terminal kinases (JNK) we estimated activation. RESULTS: Wistar rats that received 20, 80 mg or 140 mg of ambroxol displayed reduced HI/R injury compared with controls. Use of ambroxol reduced the histologic injury and significantly decreased serum ALT and AST levels. In addition, ambroxol enhanced the activity of hepatic tissue SOD and CAT, increasing GSH but decreasing MDA tissue contents. In the ambroxol group, Bcl-2 expression was increased and Bax and caspase-3 decreased compared with the controls. Furthermore, ambroxol reduced levels of phosphorylated JNK (P < .05). CONCLUSION: These results indicated that ambroxol attenuated rat HI/R through upregulation of intracellular antioxidant and anti-apoptotic signaling pathways.

Scale up of a viscous fungal fermentation: application of scale-up criteria with regime analysis and operating boundary conditions.

The scale up of the novel, pharmaceutically important pneumocandin (B(0)), from the filamentous fungus Glarea lozoyensis was successfully completed from pilot scale (0.07, 0.8, and 19 m(3)) to production scale (57 m(3)). This was accomplished, despite dissimilar reactor geometry, employing a combination of scale-up criteria, process sensitivity studies, and regime analysis using characteristic time constants for both oxygen mass transfer and bulk mixing. Dissolved oxygen tension, separated from the influence of agitation by gas blending at the 0.07 m(3)-scale, had a marked influence on the concentrations of pneumocandin analogs with different levels of hydroxylation, and these concentrations were used as an indicator of bulk mixing upon scale up. The profound impact of dissolved oxygen tension (DOT) (low and high levels) on analog formation dictated the use of constant DOT, at 80% air saturation, as a scale-up criterion. As a result k(L)a, Oxygen uptake rate (OUR) and hence the OTR were held constant, which were effectively conserved across the scales, while the use of other criterion such as P(g)/V(L), or mixing time were less effective. Production scale (57 m(3)) mixing times were found to be faster than those at 19 m(3) due to a difference in liquid height/tank diameter ratio (H(L)/D(T)). Regime analysis at 19 and 57 m(3) for bulk mixing (t(c)) and oxygen transfer (1/k(L)a) showed that oxygen transfer was the rate-limiting step for this highly shear thinning fermentation, providing additional support for the choice of scale-up criterion.

Combined global localization analysis and transcriptome data identify genes that are directly coregulated by Adr1 and Cat8.

In Saccharomyces cerevisiae, glucose depletion causes a profound alteration in metabolism, mediated in part by global transcriptional changes. Many of the transcription factors that regulate these changes act combinatorially. We have analyzed combinatorial regulation by Adr1 and Cat8, two transcription factors that act during glucose depletion, by combining genome-wide expression and genome-wide binding data. We identified 32 genes that are directly activated by Adr1, 28 genes that are directly activated by Cat8, and 14 genes that are directly regulated by both. Our analysis also uncovered promoters that Adr1 binds but does not regulate and promoters that are indirectly regulated by Cat8, stressing the advantage of combining global expression and global localization analysis to find directly regulated targets. At most of the coregulated promoters, the in vivo binding of one factor is independent of the other, but Adr1 is required for optimal Cat8 binding at two promoters with a poor match to the Cat8 binding consensus. In addition, Cat8 is required for Adr1 binding at promoters where Adr1 is not required for transcription. These data provide a comprehensive analysis of the direct, indirect, and combinatorial requirements for these two global transcription factors.

Antioxidant and lysosomotropic properties of acridine-propranolol: protection against oxidative endothelial cell injury.

The antioxidant and lysosomotropic properties of a fluorescent analogue of propranolol, 9-amino-acridine-propranolol (9-AAP) were compared to those of propranolol. Using isolated microsomal membranes exposed to a superoxide and hydroxyl radical generating system, 9-AAP was found to be at least 10-fold more potent than propranolol (and about 50% as potent as vitamin E) in inhibiting lipid peroxidation. In cultured endothelial cells, 9-AAP afforded moderate protective effect against acute loss of glutathione but potent cytoprotective activity against free radical-mediated loss of viability/survival. Intracellular localization of 9-AAP was examined by fluorescent microscopy and compared with two known fluorescent lysosomal markers: acridine orange and Lysosensor. All three agents appeared to localize to similar peri-nuclear vesicles, presumably lysosomes or pre-lysosomes. Lysosensor fluorescence was not observable in the presence of 9-AAP, foreclosing the possibility of a direct dual labeling experiment. We employed the pH sensitivity of acridine orange to determine if it labels the same vesicles as 9-AAP. When the endothelial cells were preloaded with acridine orange, washed and resuspended in buffer containing 9-AAP, the dark orange-labeled vesicles observed with acridine orange alone became increasingly lighter with time. Since the fluorescence of acridine orange is altered by pH change, this spectral shift in fluorescence emission is consistent with the indication that added propranolol (or the analog) leads to lysosomal alkalization. In conclusion, 9-AAP is both a strong antioxidant and a lysosomotropic agent that is remarkably insensitive to photobleaching. These properties may contribute to the enhanced endothelial cytoprotective effects against free radical-induced injury.

Identification of confounders in the assessment of the relationship between lead exposure and child development.

PURPOSE: To explore the best approach to identify and adjust for confounders in epidemiologic practice. METHODS: In the Port Pirie cohort study, the selection of covariates was based on both a priori and an empirical consideration. In an assessment of the relationship between exposure to environmental lead and child development, change-in-estimate (CE) and significance testing (ST) criteria were compared in identifying potential confounders. The Pearson correlation coefficients were used to evaluate the potential for collinearity between pairs of major quantitative covariates. In multivariate analyses, the effects of confounding factors were assessed with multiple linear regression models. RESULTS: The nature and number of covariates selected varied with different confounder selection criteria and different cutoffs. Four covariates (i.e., quality of home environment, socioeconomic status (SES), maternal intelligence, and parental smoking behaviour) met the conventional CE criterion (> or =10%), whereas 14 variables met the ST criterion (p < or = 0.25). However, the magnitude of the relationship between blood lead concentration and children's IQ differed slightly after adjustment for confounding, using either the CE (partial regression coefficient: -4.4; 95% confidence interval (CI): -0.5 to -8.3) or ST criterion (-4.3; 95% CI: -0.2 to -8.4). CONCLUSIONS: Identification and selection of confounding factors need to be viewed cautiously in epidemiologic studies. Either the CE (e.g., > or = 10%) or ST (e.g., p < or = 0.25) criterion may be implemented in identification of a potential confounder if a study sample is sufficiently large, and both the methods are subject to arbitrariness of selecting a cut-off point. In this study, the CE criterion (i.e., > or = 10%) appears to be more stringent than the ST method (i.e., p < or = 0.25) in the identification of confounders. However, the ST rule cannot be used to determine the trueness of confounding because it cannot reflect the causal relationship between the confounder and outcome. This study shows the complexities one can expect to encounter in the identification of and adjustment for confounders.

Inhibition of recombinant human mitochondrial aldehyde dehydrogenase by two intermediate metabolites of disulfiram.

Disulfiram is used in aversion therapy for alcoholism. S-Methyl-N,N-diethylthiocarbamate (MeDTC) sulfoxide, a potent inhibitor of the target enzyme mitochondrial aldehyde dehydrogenase (ALDH2), is thought to be the principal active metabolite of disulfiram in vivo. We examined the effects on recombinant human ALDH2 of two intermediate metabolites of disulfiram, S-methyl-N,N-diethyldithiocarbamate (MeDDC) sulfoxide and MeDDC sulfine. MeDDC sulfoxide was a potent inhibitor of ALDH2 with an IC50 of 2.2 +/- 0.5 microM (mean +/- SD, N = 4) after preincubation with enzyme for 30 min. MeDDC sulfine was a relatively weak inhibitor of ALDH2 under the same conditions with an IC50 value of 62 +/- 14 microM. The inhibition of ALDH2 by both compounds was irreversible and did not require the cofactor NAD. The latter finding demonstrates that inactivation of ALDH2 is independent of the dehydrogenase activity of the enzyme. GSH blocked almost completely the inhibition by 20 microM of MeDDC sulfoxide and greatly diminished the inhibition by 200 microM of MeDDC sulfine. Inactivation by MeDDC sulfoxide was time dependent. MeDTC sulfoxide was a more potent inhibitor of recombinant human ALDH2 (IC50 = 1.4 +/- 0.3 microM after preincubation for 15 min) than either of the intermediate metabolites, and its inhibition was unaffected by GSH. Our results suggest that these newer intermediate metabolites of disulfiram, especially the more potent MeDTC sulfoxide, have the potential to inhibit the target enzyme ALDH2 in patients receiving disulfiram. However, until the significance of the interactions of the inhibitors with GSH is more fully understood, the contribution of MeDDC sulfine and MeDDC sulfoxide to the pharmacological effects of disulfiram in vivo is uncertain.

Cellular and metabolic engineering. An overview.

Metabolic engineering is defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. Cellular engineering, a more inclusive term, is defined as the purposeful modification of cell properties using the same techniques. Examples of cellular and metabolic engineering are divided into five categories: 1. Improved production of chemicals already produced by the host organism; 2. Extended substrate range for growth and product formation; 3. Addition of new catabolic activities for degradation of toxic chemicals; 4. Production of chemicals new to the host organism; and 5. Modification of cell properties. Over 100 examples of cellular and metabolic engineering are summarized. Several molecular biological, analytical chemistry, and mathematical and computational tools of relevance to cellular and metabolic engineering are reviewed. The importance of host selection and gene selection is emphasized. Finally, some future directions and emerging areas are presented.

Isolation, characterization, and expression of mouse ICAM-2 complementary and genomic DNA.

Intercellular adhesion molecule-2 (ICAM-2), a cell surface glycoprotein, is a second counter-receptor for lymphocyte function-associated Ag-1 (LFA-1). We report here the isolation and characterization of the cDNA and the gene that encode murine ICAM-2 (Accession numbers X65493 and X65490, respectively). The deduced sequence of the cDNA has 60% amino acid identity with its human counterpart and has the same expression pattern in cells and tissues. Furthermore, COS cells transfected with mouse ICAM-2 complementary and genomic DNA bind to purified human LFA-1, demonstrating the conservation of the function of ICAM-2 as a ligand for LFA-1 and conservation across species of sequences that are critical for binding to human LFA-1. COS cells transfected with the ICAM-2 cDNA do not react with mAb PA3, previously suggested to define ICAM-2 in the mouse. The mouse ICAM-2 gene was isolated and its structural organization determined. The gene is present in a single copy in the mouse genome and contains four exons spanning about 5.0 kb of DNA. The exon/intron architecture correlates to the structural domains of the protein and resembles that of other Ig superfamily members. The gene for ICAM-2, which is constitutively expressed in endothelial cells, has several conserved sequence motifs in its promoter region, including a direct repeat, and lacks transcription factor-binding sites present in the ICAM-1 gene, which is inducible in endothelial cells.

Enhancement of 1,3-propanediol production by cofermentation in Escherichia coli expressing Klebsiella pneumoniae dha regulon genes.

1,3-Propanediol (1,3-PD) is an intermediate in chemical and polymer synthesis. We have previously expressed the genes of a biochemical pathway responsible for 1,3-PD production, the dha regulon of Klebsiella pneumoniae, in Escherichia coli. An analysis of the maximum theoretical yield of 1,3-PD from glycerol indicates that the yield can be improved by the cofermentation of sugars, provided that kinetic constraints are overcome. The yield of 1,3-PD from glycerol was improved from 0.46 mol/mol with glycerol alone to 0.63 mol/mol with glucose cofermentation and 0.55 mol/mol with xylose cofermentation. The engineered E. coli also provides a model system for the study of metabolic pathway engineering.

1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon.

The dha regulon in Klebsiella pneumoniae enables the organism to grow anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydroxyacetone and was screened for the production of 1,3-PD. The cosmid pTC1 (42.5 kb total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycerol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1,3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering.

Prosthetic synovitis: clinical and histologic characteristics.

Our study of 20 patients correlates the clinical picture of each patient with the pathology of synovial tissue obtained at the time of revision arthroplasty. While 12 patients had rheumatoid arthritis and 8 had osteoarthritis, the histopathology was identical. Additionally, while 15 of the 20 had an etiology for the revision, 5 patients were revised for pain alone with no explanation other than the synovitis. The characteristic histologic findings included lining cell hyperplasia, vascular congestion, giant cells, brightly birefringent high density polyethylene chards and cysts that represented ghosts of methyl methacrylate debris.