Inactivation of DNA polymerase I (Klenow fragment) by adenosine 2',3'-epoxide 5'-triphosphate: evidence for the formation of a tight-binding inhibitor

The suicidal inactivation of Escherichia coli DNA polymerase I by epoxy-ATP has been previously reported (Abboud et al., 1978). We have examined in detail the mechanism of this inactivation utilizing a synthetic DNA template-primer of defined sequence. Epoxy-ATP inactivates the large fragment of DNA polymerase I (the Klenow fragment) in a time- and concentration-dependent manner (KI = 21 microM; kinact = 0.021 s-1). Concomitant with inactivation is the incorporation of epoxy-AMP into the primer strand. The elongated DNA duplex directly inhibits the polymerase activity of the enzyme (no time dependence) and is resistant to degradation by the 3'----5' exonuclease and pyrophosphorylase activities of the enzyme. Inactivation of the enzyme results from slow (4 X 10(-4) s-1) dissociation of the intact epoxy-terminated template-primer from the enzyme and is thus characterized as a tight-binding inhibition. Surprisingly, while the polymerase activity of the enzyme is completely suppressed by epoxy-ATP, the 3'----5' exonuclease activity remains intact. The data presented demonstrate that even though the polymerase site is occupied with duplex DNA, the enzyme can bind a second DNA duplex and carry out exonucleolytic cleavage.     

Elimination of precipitates in oil red O fat stain by adding dextrin.

Addition of dextrin in the final 60% isopropanol of the Lillie-Ashburn super-saturated oil red O isopropanol technic moderately intensified the stain and decreased our required staining interval. Precipitates were decreased and the diluted solution remained usable into the second week. A saturated 60% isopropanol oil red O solution contained 33 mg/100 ml. Without dextrin the fresh supersaturated solution contains 40 mg, after 3 days 25 mg. With dextrin the fresh solution contained 130 mg dye, the 10-day-old one 100 mg/100 ml.     

Elimination of Precipitates in Oil Red O Fat Stain by Adding Dextrin

Addition of dextrin in the final 60% isopropanol of the Lillie-Ashburu supersaturated oil red O isopropanol technic moderately intensified the stain and decreased our required staining interval. Precipitates were decreased and the diluted solution remained usable into the second week.

A saturated 60% isopropanol oil red O solution contained 33 mg/100 ml. Without dextrin the fresh supersaturated solution contains 40 mg, after 3 days 25 mg. With dextrin the fresh solution contained 130 mg dye, the 10-day-old one 100 mg/100 ml.     

Structure-mechanism relationships in hemoproteins. Oxygenations catalyzed by chloroperoxidase and horseradish peroxidase

Chloroperoxidase and H2O2 oxidize styrene to styrene oxide and phenylacetaldehyde but not benzaldehyde. The epoxide oxygen is shown by studies with H2(18)O2 to derive quantitatively from the peroxide. The epoxidation of trans-[1-2H]styrene by chloroperoxidase proceeds without detectable loss of stereochemistry, as does the epoxidation of styrene by rat liver cytochrome P-450, although much more phenylacetaldehyde is produced by chloroperoxidase than cytochrome P-450. Chloroperoxidase and cytochrome P-450 thus oxidize styrene by closely related oxygen-transfer mechanisms. Horseradish peroxidase does not oxidize styrene but does oxidize 2,4,6-trimethylphenol to 2,6-dimethyl-4-hydroxymethylphenol. The new hydroxyl group is partially labeled in incubations with H2(18)O but not H2(18)O2. The hydroxyl group thus appears to be introduced by addition of oxygen to the benzylic radical and water to the quinone methide intermediate but not by a cytochrome P-450-like oxene transfer mechanism. The results support the thesis that substrates primarily or exclusively react with the heme edge of horseradish peroxidase but are able to react with the ferryl oxygen of chloroperoxidase.     

Evaluating short-term effects of omnivorous fish removal on water quality and zooplankton at a subtropical lake

We evaluated a biomanipulation program to test for short-term changes in water quality (chlorophyll a, Secchi depth, total phosphorus) and macrozooplankton biomass following partial removal of omnivorous gizzard shad Dorosoma cepedianum. The removal occurred at a eutrophic subtropical lake, and responses were compared to an unmanipulated control lake using a before-after-control-impact paired series analysis. The removal reduced the biomass of large (>300 mm) gizzard shad by 75% over 2 years via a subsidized commercial gill net fishery. However, the total population biomass of gizzard shad was reduced by approximately 32% from an average pre-manipulation biomass of 224 kg ha⁻¹ due to the size selectivity of the gear, which did not effectively capture small fish (<300 mm). No significant short-term changes in chlorophyll a concentration, Secchi depth, total phosphorus concentration or macrozooplankton biomass were detected following biomanipulation. The partial removal may have fallen short of the biomass reduction required to cause ecosystem responses. Our results suggest that moderate omnivore removals (i.e., <40% biomass reduction) will have little short-term benefits to these lakes, and future manipulations should use a less size-selective gear to achieve a larger total biomass reduction.     

Karyological studies inCoris julis (Pisces,Labridae)

In the present investigation the diploid number 2n=48 (NF=58) has been determined for females, primary males, and secondary males ofCoris julis from the Gulf of Palermo. Differentiated sex chromosomes have not been observed in the population under study.     

An informative Bayesian structural equation model to assess source-specific health effects of air pollution

A primary objective of current air pollution research is the assessment of health effects related to specific sources of air particles or particulate matter (PM). Quantifying source-specific risk is a challenge because most PM health studies do not directly observe the contributions of the pollution sources themselves. Instead, given knowledge of the chemical characteristics of known sources, investigators infer pollution source contributions via a source apportionment or multivariate receptor analysis applied to a large number of observed elemental concentrations. Although source apportionment methods are well established for exposure assessment, little work has been done to evaluate the appropriateness of characterizing unobservable sources thus in health effects analyses. In this article, we propose a structural equation framework to assess source-specific health effects using speciated elemental data. This approach corresponds to fitting a receptor model and the health outcome model jointly, such that inferences on the health effects account for the fact that uncertainty is associated with the source contributions. Since the structural equation model (SEM) typically involves a large number of parameters, for small-sample settings, we propose a fully Bayesian estimation approach that leverages historical exposure data from previous related exposure studies. We compare via simulation the performance of our approach in estimating source-specific health effects to that of 2 existing approaches, a tracer approach and a 2-stage approach. Simulation results suggest that the proposed informative Bayesian SEM is effective in eliminating the bias incurred by the 2 existing approaches, even when the number of exposures is limited. We employ the proposed methods in the analysis of a concentrator study investigating the association between ST-segment, a cardiovascular outcome, and major sources of Boston PM and discuss the implications of our findings with respect to the design of future PM concentrator studies.     

A novel leptin antagonist peptide inhibits breast cancer growth in vitro and in vivo

The role of the obesity cytokine leptin in breast cancer progression has raised interest in interfering with leptin's actions as a valuable therapeutic strategy. Leptin interacts with its receptor through three different binding sites: I–III. Site I is crucial for the formation of an active leptin–leptin receptor complex and in its subsequent activation. Amino acids 39‐42 (Leu‐Asp‐Phe‐Ile‐ LDFI) were shown to contribute to leptin binding site I and their mutations in alanine resulted in muteins acting as typical antagonists. We synthesized a small peptide based on the wild‐type sequence of leptin binding site I (LDFI) and evaluated its efficacy in antagonizing leptin actions in breast cancer using in vitro and in vivo experimental models. The peptide LDFI abolished the leptin‐induced anchorage‐dependent and ‐independent growth as well as the migration of ERα‐positive (MCF‐7) and ‐negative (SKBR3) breast cancer cells. These results were well correlated with a reduction in the phosphorylation levels of leptin downstream effectors, as JAK2/STAT3/AKT/MAPK. Importantly, the peptide LDFI reversed the leptin‐mediated up‐regulation of its gene expression, as an additional mechanism able to enhance the peptide antagonistic activity. The described effects were specific for leptin signalling, since the developed peptide was not able to antagonize the other growth factors' actions on signalling activation, proliferation and migration. Finally, we showed that the LDFI pegylated peptide markedly reduced breast tumour growth in xenograft models. The unmodified peptide LDFI acting as a full leptin antagonist could become an attractive option for breast cancer treatment, especially in obese women.     

Metabolic programming: fetal origins of obesity and metabolic syndrome in the adult

Exposure of the fetus to the intrauterine milieu can have profound effects on the health of the offspring in adulthood. Results of a series of studies demonstrate the powerful influence of the mother's metabolic state on whether the emerging adult develops obesity and hyperinsulinemia. Importantly, these attributes can be passed on to the next generation nongenetically and can be reversed and prevented.