Transition-state analysis of AMP deaminase

The transition state of the allosteric AMP deaminase from Saccharomyces cerevisiae has been characterized by 14C and 15N Vmax/Km heavy-atom kinetic isotope effects. The primary 6-14C isotope effect was measured with [6-14C]AMP, and the 6-15N primary isotope effect was measured by isotope ratio mass spectrometry using the natural abundance of 15N in AMP and by using 15N release from ATP as a slow substrate. Isotope effects for AMP as the substrate were measured in the presence and absence of ATP as an allosteric activator and GTP as an allosteric inhibitor. Kinetic isotope effects with [6-14C]AMP were 1.030 +/- 0.003, 1.038 +/- 0.004, and 1.042 +/- 0.003 in the absence of effectors and in the presence of ATP and GTP, respectively. Isotope effects for [6-15N]AMP averaged 1.010 +/- 0.002. Allosteric activation increased the 15N isotope effect to 1.016 +/- 0.003. A primary 15N kinetic isotope effect with ATP, which has a Vmax/Km 10(-6) that for AMP, was 1.013 +/- 0.001. The presence of D2O as solvent caused a marginally significant decrease in the [6-15N]AMP kinetic isotope effect from 1.011 +/- 0.001 to 1.007 +/- 0.002. Previous studies have established that the solvent D2O effect is inverse (0.34) for slow substrates with two or more protons transferred prior to transition state formation and remains inverse (0.79) with AMP as substrate [Merkler, D. J., & Schramm, V. L. (1993) Biochemistry 32, 5792-5799]. Bond vibrational analysis was used to identify transition states for AMP deaminase that are consistent with all kinetic isotope effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free radical centers in isolated rat heart tissue in a normal state, in ischemia, and reperfusion

EPR spectroscopy was used to measure paramagnetic species in rat hearts freeze-clamped during control perfusion by the Neely procedure, after 25 min of normothermic global ischemia or 20 min of total reperfusion with oxygenated perfusate. The analysis of spectral and relaxation parameters measured at -40 degrees C showed that in all three cases free radicals in heart tissue were semiquinones of CoQ10 and flavins. Ischemia increased the amount of free radical species (mostly flavosemiquinones) in myocardium about two times, the beginning of reflow of perfusate resulted in decrease of the intensity of the EPR signal to an initial level. The saturation curves were different for control, ischemic and reoxygenated postischemic samples, and they demonstrated the heterogeneity of free radical centers in cardiac mitochondria.     

The 4‐acetylantroquinonol B isolated from mycelium of Antrodia cinnamomea inhibits proliferation of hepatoma cells

BACKGROUND: Antrodia cinnamomea is known for its antihepatoma activity, yet the identity of its active compound was unclear. In this study, a 5‐ton fermenter was used to prepare sufficient mycelium of A. cinnamomea for active compound isolation and identification. RESULTS: Using antiproliferative activity toward HepG2 cells as guidance in the isolation process, 4‐acetylantroquinonol B was purified and identified to be the major bioactive compound of A. cinnamomea cultivated by submerged fermentation. The median effective doses (EC₅₀) of 4‐acetylantroquinonol B for HepG2 cells were 0.10 ± 0.00 and 0.08 ± 0.00 µg mL^?1 for 72 and 96 h treatments, respectively. The selective indices of 4‐acetylantroquinonol B were 100 and 125 for 72 and 96 h treatments, respectively, indicating that this compound had high selective activity for hepatoma cells. CONCLUSION: 4‐Acetylantroquinonol B is the major antihepatoma constituent of Antrodia cinnamomea mycelium produced by submerged fermentation. Copyright © 2010 Society of Chemical Industry     

Antimicrobial Effects of Allyl Isothiocyanate and Modified Atmosphere on Pseduomonas Aeruginosa in Fresh Catfish Fillet under Abuse Temperatures

The effects of allyl isothiocyanate (AIT; 18 and 36 μg/L) in vapor phase, modified atmosphere (MA; 49% CO₂, 0.5% O₂, and 50.5% N₂), and their combinations on the growth behavior of Pseduomonas aeruginosa in fresh catfish fillet at different abuse temperatures (8, 15, and 20 °C) were evaluated in this study. Lag phase, maximum growth rate, and shelf life were used as parameters to analyze the antimicrobial effects. Both gaseous AIT and MA alone inhibited the growth potential of P. aeruginosa effectively, prolonging the shelf life by 1.5 to 3.4 times compared to the control at abuse temperatures between 8 and 20 °C. The synergistic effect was observed at 8 °C, extending the shelf life of fresh catfish by more than 6.5 times (≥ 550 h). In addition, the maximum growth rate decreased with decreasing storage temperature, but it was not significantly influenced by the addition of AIT or MA. Hence, the combination of AIT and MA may be used as an effective antimicrobial system to reduce the microbial risks due to temperature abuse and to improve the shelf life of fresh catfish fillet. The proper combination of AIT and MA may be further optimized for industrial applications.     

Antimicrobial Effects of Vapor Phase Thymol,Modified Atmosphere,and Their Combination against Salmonella spp. on Raw Shrimp

Salmonella contamination on raw shrimp is a big food safety concern in the United States currently. This research evaluated the inhibition effects of vapor phase thymol, modified atmosphere (MA), and their combination against Salmonella spp. on raw shrimp. Growth profiles of a Salmonella spp. cocktail (6 strains), inoculated onto the surface of raw shrimp, treated with vapor phase thymol at 3 levels (0, 0.8, and 1.6 mg/L), or MA (59.5% CO₂ + 39.5% N₂ + 1% O₂), both alone and in combination, at 3 temperatures (8, 12, and 16 ºC), were determined. Lag time and maximum growth rate of Salmonella spp. under each treatment were obtained using Baranyi and Roberts models. Results indicated that both vapor phase thymol and MA treatments alone inhibited the growth potential of Salmonella spp. effectively, extending the lag time by 10% to 100% and reducing the maximum growth rate by 14% to 71% compared with controlled samples at experimental temperatures (8, 12, and 16 ºC). Combination treatments of vapor phase thymol and MA exhibited greater inhibition effectiveness than each individual treatment and a synergistic antimicrobial effectiveness could be observed on the lag time extension. To the maximum, at 12 ºC, lag time of Salmonella spp. was extended 59.6% more by the combination treatment of 0.8 mg/L thymol + MA (36.97 h) than those effects combined from 0.8 mg/L thymol treatment and MA treatment alone (23.16 h in total). This combination strategy could be potentially utilized for Salmonella inhibition during the long distance and temperature‐abused raw shrimp import process. Practical Application The vapor phase thymol + MA combination strategy could be potentially applied in temperature‐abused and long‐distance raw shrimp import process to retard the Salmonella spp. growth, therefore reducing its import rejection rate as well as enhancing its food safety level to the consumers’ concerns.     

Kinetics of thermal destruction of Salmonella in ground chicken containing trans-cinnamaldehyde and carvacrol

We investigated the heat resistance of an eight-strain cocktail of Salmonella serovars in chicken supplemented with trans cinnamaldehyde (0 to 1.0%, wt/wt) and carvacrol (0 to 1.0%, wt/wt). Inoculated meat was packaged in bags that were completely immersed in a circulating water bath and held at 55 to 71°C for predetermined lengths of time. The recovery medium was tryptic soy agar supplemented with 0.6% yeast extract and 1% sodium pyruvate. D-values in chicken, determined by linear regression, were 17.45, 2.89, 0.75, and 0.29 min at 55, 60, 65, and 71°C, respectively (z = 9.02°C). Using a survival model for nonlinear survival curves, D-values in chicken ranged from 13.52 min (D(1), major population) and 51.99 min (D(2), heat-resistant subpopulation) at 55°C to 0.15 min (D(1)) and 1.49 min (D(2)) at 71°C. When the Salmonella cocktail was in chicken supplemented with 0.1 to 1.0% trans-cinnamaldehyde or carvacrol, D-values calculated by both approaches were consistently less at all temperatures. This observation suggests that the addition of natural antimicrobials to chicken renders Salmonella serovars more sensitive to the lethal effect of heat. Thermal death times from this study will be beneficial to the food industry in designing hazard analysis and critical control point plans to effectively eliminate Salmonella contamination in chicken products used in this study.     

Primary malignant fibrous histiocytoma of the lung. Report of a case with bronchial brushing cytologic features

BACKGROUND: Malignant fibrous histiocytoma (MFH) arising primarily in the lungs is rare, and a preoperative diagnosis, as well as a surgical planning, is very important because of the tumor's propensity for vascular invasion and its low incidence of lymph node metastasis. The correct preoperative diagnosis of thoracic MFH is not easy to establish because the small fragments from needle and transbronchial biopsies are often inadequate for a conclusive histologic analysis. A preoperative bronchial brushing cytology suggestion of the diagnosis of primary MFH of the lungs may be helpful in such cases. CASE: A 37-year-old male presented with a large, irregular mass in the inferior and middle lobes on chest roentgenography as well as on computed tomography. Two bronchoscopies were performed, with the diagnosis of undifferentiated large cell carcinoma. After surgical resection a more sophisticated pathologic analysis, including immunohistochemical and ultrastructural studies, revealed a primary MFH of the lungs. Revision of the bronchial brushing cytology disclosed many spindle-shaped cells with a "comet" configuration, strongly suggestive of MFH. CONCLUSION: The bronchial brushing cytology features of spindle-shaped and bizarre, multinucleated giant cells with a comet appearance may be the key to the cytomorphologic diagnosis of MFH.     

The immunomodulatory effect of topical cyclosporin A in atopic keratoconjunctivitis

PURPOSE: To perform a detailed examination of the immunomodulatory effects of topical cyclosporin A (CsA) in conjunctival tissue from patients with atopic keratoconjunctivitis (AKC). METHODS: Patients with active AKC were randomly allocated into two groups of four patients. For 3 months one group received 2% CsA drops, and the other group received placebo drops. Superior tarsal conjunctival biopsy specimens were harvested before and after treatment and examined by one- and two-color immunohistochemistry to compare leukocyte counts, HLA-DR+ and IL-2R+ cell counts, HLA-DR positivity of conjunctival epithelial cells, and counts of T cells expressing the cytokines interleukin (IL)-2, IL-3, IL-4, IL-5, and interferon (IFN)-gamma. RESULTS: Posttreatment values were significantly less than pretreatment values for the total number of leukocytes and in the numbers of CD3+ T cells, CD4+ cells, CD8+ cells, CD20+ B cells, neutrophils, and macrophages, and there was a decrease in the CD4-CD8 ratio (P = 0.03) in the CsA group. There was a reduction from before CsA treatment to after CsA-treatment in the numbers of HLA-DR+ and IL-2R+ cells (P = 0.03), but the reduction in the epithelial cell HLA-DR expression did not reach significance. The number of T cells staining for IL-3 and IL-5 was reduced, although not to statistical significance, but there was a significant reduction in the number of T cells expressing IL-2 and IFN-gamma (P = 0.03) after CsA treatment compared with initial values. There were no statistically significant differences between pretreatment and posttreatment values in the placebo group. There was a clinical improvement in the CsA group and a clinical worsening in the placebo group. CONCLUSIONS: The in vitro effects of CsA translate into a reduction in T cells, a normalization of the CD4-CD8 ratio, a decrease in T-cell activation, and a reduction in T-cell cytokine expression, especially IL-2 and IFN-gamma. The decrease in HLA-DR expression may be mediated by the change in IFN-gamma. There were fewer B cells but not fewer plasma cells after CsA and no change in IL-4 expression, suggesting minimal effects on type I hypersensitivity responses. There was no significant reduction in mast cell or eosinophil numbers, but direct effects of topical CsA on their function may play a role in the therapy of ocular allergic disease. These results show that the beneficial effects of topical CsA in AKC are accompanied by important changes in conjunctival immune cell profiles.