Chemotaxis-mediated biodegradation of cyclic nitramine explosives RDX, HMX, and CL-20 by Clostridium sp. EDB2

Cyclic nitramine explosives, RDX, HMX, and CL-20 are hydrophobic pollutants with very little aqueous solubility. In sediment and soil environments, they are often attached to solid surfaces and/or trapped in pores and distribute heterogeneously in aqueous environments. For efficient bioremediation of these explosives, the microorganism(s) must access them by chemotaxis ability. In the present study, we isolated an obligate anaerobic bacterium Clostridium sp. strain EDB2 from a marine sediment. Strain EDB2, motile with numerous peritrichous flagella, demonstrated chemotactic response towards RDX, HMX, CL-20, and NO(2)(-). The three explosives were biotransformed by strain EDB2 via N-denitration with concomitant release of NO(2)(-). Biotransformation rates of RDX, HMX, and CL-20 by the resting cells of strain EDB2 were 1.8+/-0.2, 1.1+/-0.1, and 2.6+/-0.2nmol h(-1)mgwet biomass(-1) (mean+/-SD; n=3), respectively. We found that commonly seen RDX metabolites such as TNX, methylenedinitramine, and 4-nitro-2,4-diazabutanal neither produced NO(2)(-) during reaction with strain EDB2 nor they elicited chemotaxis response in strain EDB2. The above data suggested that NO(2)(-) released from explosives during their biotransformation might have elicited chemotaxis response in the bacterium. Biodegradation and chemotactic ability of strain EDB2 renders it useful in accelerating the bioremediation of explosives under in situ conditions.     

Fluorinated aminoglycosides and their mechanistic implication for aminoglycoside 3'-phosphotransferases from Gram-negative bacteria

Aminoglycoside 3'-phosphotransferases [APH(3')s] are important bacterial resistance enzymes for aminoglycoside antibiotics. These enzymes phosphorylate the 3'-hydroxyl of these antibiotics, a reaction that inactivates the drug. A series of experiments were carried out to shed light on the details of the turnover chemistry by these enzymes. Quench-flow pre-steady-state kinetic analyses of the reactions of Gram-negative APH(3') types Ia and IIa with kanamycin A, neamine, and their respective difluorinated analogues 4'-deoxy-4',4'-difluorokanamycin A and 4'-deoxy-4',4'-difluoroneamine were carried out, in conjunction with measurements of thio effect and viscosity studies. The fluorinated analogues were shown to be severely impaired as substrates for these enzymes. The magnitude of the effect of the impairment of the fluorinated substrates was in the same range as when the D198A mutant APH(3')-Ia was studied with nonfluorinated substrates. Residue 198 is the proposed active site base that promotes the aminoglycoside hydroxyl for phosphorylation. These findings collectively argue that the Gram-negative APH(3')s show significant nucleophilic participation in the transition state for the phosphate transfer reaction.     

Polyphenols of mature plant,seedling and tissue cultures of Theobroma cacao

The major flavone in mature cocoa leaves is isovitexin, with smaller amounts of vitexin and 7-O-glucosides of apigenin, luteolin and chrysoeriol. F     

Metabolite production during transformation of 2,4,6-trinitrotoluene (TNT) by a mixed culture acclimated and maintained on crude oil-containing media

Metabolites formed during 2,4,6-trinitrotoluene (TNT) removal by a mixed bacterial culture (acclimated and maintained on crude oil-containing medium and capable of high rates of TNT removal) were characterized. In resting cell experiments in the absence of glucose, 46.2 mg/l TNT were removed in 171 h (87.5% removal), with a combined total formation of 7.7 mg/l amino-4,6-dinitrotoluene (ADNT) and 0.3 mg/l 4,4-azoxytetranitrotoluene and 2,4-azoxytetranitrotoluene, leaving 70% of the initial TNT unaccounted for. In the presence of glucose, resting cells removed 45.4 mg/l TNT in 49 h (95.5% removal), with 9.1 mg/l ADNT and 2.4 mg/l azoxy compounds being produced, leaving 70.3% of the TNT unaccounted for. Growing cells (glucose present) were capable of removing 44.2 mg/l TNT within 21 h (97.9% removal), with the concomitant formation of 1.8 mg/l ADNTs and 2.2 mg/l azoxy compounds. Denitrated TNT in the form of 2,6-dinitrotoluene was also produced in growing cells with a maximum amount of 1.31 mg/l after 28 h, followed by a slight decrease with time, leaving 88.5% of the initial TNT unaccounted for after 171 h. Radiolabeled ¹⁴C-TNT studies revealed 4.14% mineralization after an incubation period of 163 days with growing cells.     

Position effects due to chromosome breakpoints that map approximately 900 Kb upstream and approximately 1.3 Mb downstream of SOX9 in two patients with campomelic dysplasia

Campomelic dysplasia (CD) is a semilethal skeletal malformation syndrome with or without XY sex reversal. In addition to the multiple mutations found within the sex-determining region Y-related high-mobility group box gene (SOX9) on 17q24.3, several chromosome anomalies (translocations, inversions, and deletions) with breakpoints scattered over 1 Mb upstream of SOX9 have been described. Here, we present a balanced translocation, t(4;17)(q28.3;q24.3), segregating in a family with a mild acampomelic CD with Robin sequence. Both chromosome breakpoints have been identified by fluorescence in situ hybridization and have been sequenced using a somatic cell hybrid. The 17q24.3 breakpoint maps approximately 900 kb upstream of SOX9, which is within the same bacterial artificial chromosome clone as the breakpoints of two other reported patients with mild CD. We also report a prenatal identification of acampomelic CD with male-to-female sex reversal in a fetus with a de novo balanced complex karyotype, 46,XY,t(4;7;8;17)(4qter-->4p15.1::17q25.1-->17qter;7qter-->7p15.3::4p15.1-->4pter;8pter-->8q12.1::7p15.3-->7pter;17pter-->17q25.1::8q12.1-->8qter). Surprisingly, the 17q breakpoint maps approximately 1.3 Mb downstream of SOX9, making this the longest-range position effect found in the field of human genetics and the first report of a patient with CD with the chromosome breakpoint mapping 3' of SOX9. By using the Regulatory Potential score in conjunction with analysis of the rearrangement breakpoints, we identified a candidate upstream cis-regulatory element, SOX9cre1. We provide evidence that this 1.1-kb evolutionarily conserved element and the downstream breakpoint region colocalize with SOX9 in the interphase nucleus, despite being located 1.1 Mb upstream and 1.3 Mb downstream of it, respectively. The potential molecular mechanism responsible for the position effect is discussed.     

Enhanced antinociception by intracerebroventricularly and intrathecally-administered orexin A and B (hypocretin-1 and -2) in mice

Orexins are neuropeptides located exclusively in neurons of the lateral hypothalamic area, which send projections to most monoaminergic nuclei, such as noradrenergic locus coeruleus, dopaminergic ventral tegmental areas, and histaminergic tuberomammillary nuclei. The present work was carried out to examine the role of orexins in nociception in mice. C57BL/6 mice were administered with orexin A and B intracerebroventricularly (i.c.v.), intrathecally (i.t.) and subcutaneously (s.c.) to reveal the sites of action of these peptides and to examine the pain thresholds using four kinds of nociceptive tasks. Orexins showed antinociceptive effects in all four types of assays for thermal (hot-plate, tail-flick, paw-withdrawal), mechanical (tail-pressure), chemical (formalin, capsaicin and abdominal stretch) nociceptions and nociceptin-induced behavioral responses, when administered i.c.v. or i.t., whereas the s.c. administration was ineffective. The antinociceptive effects of orexin A were more remarkable than those of orexin B. The i.c.v. administration of orexin A was as effective as, or more potent than the i.t. administration. The effects of orexin A were completely blocked by adenosine type 1 receptor antagonists, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and theophylline, but not by naloxone, suggesting a possible involvement of the adenosine-containing neurons and/or the adenosine pathway in these orexin actions. The i.c.v. administration of nociceptin had no significant effects on orexin expression in the brain and spinal cord. The present findings suggest that orexins have an antinociceptive role in at least four different types of pains, probably acting on both the brain and spinal cord.     

Micro-quantitation of lipids in serum-free cell culture media: a critical aspect is the minimization of interference from medium components and chemical reagents

Lipids (fatty acids) at a concentration range of 10-100 microg/L are essential components included in most serum-free cell culture medium formulations. A gas chromatography/mass spectrometry (GC/MS) method for the micro-quantitation of lipids, determined as fatty acid methyl esters (FAMEs), in complex serum-free cell culture media was developed. The interference of derivatizing reagents, extraction solvents and medium additives in the micro-quantitation of lipids was also examined. The results show that the concentration of fatty acids such as palmitic and stearic acids detected in derivatizing reagents or extraction solvents was in the range of 10-230 microg/L. Tween-80, a surfactant and medium additive, produced nearly 20 FAMEs alone when methylated using a derivatizing agent. Moreover, the surfactant Pluronic F-68, a medium additive, interfered in the FAME recovery. Procedures, which include use of low volumetric ratio of reagent to medium and precipitation of the above surfactants, were developed to minimize background FAMEs to levels which do not significantly affect the quantitation of medium lipids and to diminish the interference caused by Pluronic F-68. Fatty acid concentrations in several complex serum-free culture media were quantitated by this method and were very close to the values indicated in their formulations.     

Novel oral transforming growth factor-β signaling inhibitor potently inhibits postsurgical adhesion band formation

Here, we have investigated the therapeutic potency of EW-7197, a transforming growth factor-β type I receptor kinase inhibitor, against postsurgical adhesion band formation. Our results showed that this pharmacological inhibitor prevented the frequency and the stability of adhesion bands in mice model. We have also shown that downregulation of proinflammatory cytokines, reduce submucosal edema, attenuation of proinflammatory cell infiltration, inhibition of oxidative stress, decrease in excessive collagen deposition, and suppression of profibrotic genes at the site of surgery are some of the mechanisms by which EW-7197 elicits its protective responses against adhesion band formation. These results clearly suggest that EW-7197 has novel therapeutic properties against postsurgical adhesion band formation with clinically translational potential of inhibiting key pathological responses of inflammation and fibrosis in postsurgery patients.