The limited immunomodulatory effects of escharectomy on the kinetics of endotoxin, cytokines, and adhesion molecules in major burns

Escharectomy has been shown to improve the survival rates and the outcomes in burns. This observational study was conducted to assess the role of escharectomy on the inflammatory mediators in major burns. Seventeen ASA physical status II or status III adult surviving major burn patients were recruited. When the escharectomy was scheduled, a series of blood samples was obtained at -3 and -1 days preoperation, and +1 and +3 postoperation. The changing levels of endotoxin, cytokines, and adhesion molecules were measured with a quantitative sandwich immunoassay. Extensive escharectomy did not appear to have any significant impact on the levels of tumor necrosis factor alpha, interleukin-10, soluble intracellular adhesion molecule-1 and soluble vascular adhesion molecule-1. Meanwhile, endotoxin and E-selectin were significantly decreased after escharectomy. Escharectomy appeared to have a limited immunomodulatory effect on the inflammatory mediators in systemic inflammatory responses induced by major burns. This is probably related to the timing and extent of surgery, and the complex nature of burn-related inflammation.     

Non-classic characteristics define prominent DNase activities from the intestine and other tissues of Haemonchus contortus

The anthelmintic fenbendazole (FBZ) induces nuclear DNA fragmentation (DF) in intestinal cells of Haemonchus contortus. The DNA fragments had 3'-OH, which suggests involvement of a neutral DNase. To identify candidate DNase(s) involved, DNase activity in H. contortus intestine and other worm fractions was characterized relative to classic DNases I (neutral) and II (acidic). Seven distinct DNase activities were identified and had Mrs of 34, 36, 37 or 38.5 kDa on zymographic analysis. The different activities were distinguished according to pH requirement, sensitivity to 10 mM EDTA and worm compartment. Activities of intestinal DNases at 34, 36 and 38.5 kDa were sensitive to EDTA at pH 5.0 and 7.0. Sensitivity to EDTA at pH 5.0 was unexpected compared to classic acidic DNase II activity, suggesting unusual properties of these DNases. In whole worms, however, the activities at 36 and 38.5 kDa were relatively insensitive to EDTA, indicating predominance of DNases that are distinct from the intestine. The activity at 37 kDa in excretory/secretory products had an acidic pH requirement and was insensitive to EDTA, resembling classic acidic DNase activity. Under conditions of pH 5.0 and 7.0, intestinal DNases produced 3'-ends that could be labeled by terminal deoxynucleotidyl transferase, indicating presence of 3'-OH. The labeling of 3'-ends at pH 5.0, again, was unexpected for acidic DNase activity. These results and several other activities suggest that multiple H. contortus DNases have characteristics distinct from the classic mammalian DNases I and II. Treatment of H. contortus with FBZ did not induce any detectable DNase activities distinct from normal intestine, although relative activities of intestinal DNases appear to have been altered by this treatment.     

The cooperative binding of large ligands to a one-dimensional lattice: the steric hindrance effect

The cooperative binding of monomeric ligands to a long lattice of a linear polymer with complete or partial steric hindrance is treated using a matrix method. Results and typical calculations of the model are represented. Non-saturated cooperative binding as well as two-step (biphasic) binding isotherms can be interpreted by the steric hindrance model. This is applicable to the analysis of the binding of surfactants to polymer. The usefulness and the limitation also are discussed.     

Alterations in hepatic metabolism of sulfur-containing amino acids induced by ethanol in rats

Summary.  Alterations in hepatic metabolism of S-amino acids were monitored over one week in male rats treated with a single dose of ethanol (3 g/kg, ip). Methionine and S-adenosylhomocysteine concentrations were increased rapidly, but S-adenosylmethionine, cysteine, and glutathione (GSH) decreased following ethanol administration. Activities of methionine adenosyltransferase, cystathionine γ-lyase and cystathionine β-synthase were all inhibited. γ-Glutamylcysteine synthetase activity was increased from t = 8 hr, but GSH level did not return to control for 24 hr. Hepatic hypotaurine and taurine levels were elevated immediately, but reduced below control in 18 hr. Changes in serum and urinary taurine levels were consistent with results observed in liver. Cysteine dioxygenase activity was increased rapidly, but declined from t = 24 hr. The results show that a single dose of ethanol induces profound changes in hepatic S-amino acid metabolism, some of which persist for several days. Ethanol not only inhibits the cysteine synthesis but suppresses the cysteine availability further by enhancing its irreversible catabolism to taurine, which would play a significant role in the depletion of hepatic GSH. Received April 26, 2002 Accepted June 12, 2002 Published online October 14, 2002 Authors' address: Young C. Kim, Ph.D., Professor of Toxicology, College of Pharmacy, Seoul National University, San 56-1 Shinrim-Dong, Kwanak-Ku, Seoul, Korea, Fax: +82-2-872-1795, E-mail: youckim@snu.ac.kr Abbreviations: CβS, cystathionine β-synthase; CDC, cysteine sulfinate decarboxylase; CDO, cysteine dioxygenase; CγL, cystathionine γ-lyase; GCS, γ-Glutamylcysteine synthetase; GSH, glutathione; MAT, methionine adenosyltransferase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.     

The binding of divalent metal ions to polyelectrolytes in mixed counterion systems. II. Dextransulfate-Mg2+ and dextransulfate-Ca2+ in solutions containing added NaCl or KCl

Measurements of magnesium and calcium ion activities in solutions of the polyelectrolyte dextransulfate, with added sodium chloride or potassium chloride are presented. A two wavelength dye spectrophotometric method is used. Dextransulfate concentrations Cp (expressed as moles sulfate ion/litre) vary between 0.001 and 0.007, total ionic strengths between 0.005 and 0.08 mole/XXX. Divalent metal ion concentrations are varied between 0 and 1.2 Cp. The results for the metal ion activities are expressed in the form of parameters theta2 = C2/Cp (C(2bp) = bound divalent metal ion concentration) and K2 = theta2/(C2-C2b). For each divalent/univalent counterion pair the values obtained for theta2 and K2 as a function of C2,Cp, and ionic strength are compared to predictions of the "two variable theory" developed for these mixed counterion systems by Manning. This comparison shows that the observed decrease in theta2 with increasing ionic strength at fixed C2 and Cp is generally well predicted by the two variable theory. The extent of divalent ion binding at a given C2, Cp, and ionic strength is largest for the Ca/Na counterion combination, and lowest for the Mg/K combination.     

Transference numbers, polyion mobilities, and charge fractions in aqueous solutions of lithium, sodium, and potassium dextransulfate

Results of Hittorf type transference number measurements are reported for aqueous solutions of lithium, sodium, and potassium dextransulfate (DS) in the concentration range 0.008-0.09 moles of sulfate groups per liter. The results for the polyion transference number are combined with equivalent conductance measurements reported earlier to calculate the polyion equivalent conductance, and the polyion charge fraction based on Wall's method. Our results show that the transference number of this high charge density polyion is larger than unity over the entire concentration range studied, and decreases monotonously with increasing concentration. The calculated charge fraction f of the polyion increases with increasing concentration, and at any concentration/decreases in the order LiDS > NaDS > KDS. A comparison between the conclusions derived from these transport experiments and from both mean and single ion activity measurements in dextransulfate solutions shows the considerable uncertainties involved in either of these methods, and emphasizes the need for the application of a variety of techniques, including spectroscopic techniques, to determine differential ion binding by polyions.     

Galactomannan detection in bronchoalveolar lavage fluid

Prompt diagnosis of invasive pulmonary aspergillosis (IPA) remains a challenge. Galactomannan (GM) assay in serum has been incorporated into diagnostic criteria for IPA, but its performance varies depending on the population in which the test is used. GM assay on bronchoalveolar lavage (BAL) fluid aims to improve upon the test by applying it directly on samples from the target organ. The studies that have examined the utility of BAL-GM are a heterogeneous group, but the results are intriguing, especially in patients who are at risk for IPA from causes other than hematologic malignancy and neutropenia. BAL-GM had sensitivities ranging from 60% to 100% in this group, often far exceeding the performance of serum GM assay. The test shows promise as a useful adjunctive diagnostic modality in the diagnosis of IPA.     

S1219 residue of 53BP1 is phosphorylated by ATM kinase upon DNA damage and required for proper execution of DNA damage response

B lymphocyte receptor-mediated apoptosis is associated with increased expression of the BimL isoform of Bim. The mechanisms involved in the regulation of BimL protein expression are still unknown. We report that BimL expression following BCR activation is not associated with a specific increase of BimL mRNA but rather to the intron retention structure of the BimEL mRNA. Indeed, expression of a BimEL cDNA leads in Hela cells leads to the production of both BimEL and BimL proteins. Mutation of the intron-splicing GT sequence present in the exon 3 results in the production of only BimEL protein. Ectopic expression of BimEL cDNA resulted in a large increase of BimL expression upon BCR-stimulation, whereas cells transfected with the GT/AA mutated form of BimEL only produced BimEL proteins upon BCR-activation. These data showed that BimL expression induced by BCR activation may result from the splicing of BimEL mRNA independently of Bim promoter regulation.     

Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z

The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.