Action of alkaline substances on 10-bromocamphor. II.
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Polyhalogenated compounds are often environmentally persistent and toxic to mammals. Microorganisms that metabolize these compounds can detoxify contaminated environments. Different biochemical mechanisms are used to metabolize polyhalogenated compounds, but few naturally occurring bacteria have this capability. A recombinant bacterium was constructed to metabolize polyhalogenated compounds to nonhalogenated end products. Seven genes were expressed in Pseudomonas putida G786 to biosynthesize cytochrome P450CAM and toluene dioxygenase. Cytochrome P450CAM catalyzed reductive dechlorinated reactions and toluene dioxygenase catalyzed oxidative dechlorination. With pentachloroethane, reductive dechlorination yielded trichloroethylene, which was further oxidized to formate and glyoxylate. The sequential action of cytochrome P450CAM and toluene dioxygenase with polyhalogenated compounds constitutes a novel engineered metabolic pathway.
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We report here the first measurement of near-infrared (NIR) vibrational circular dichroism (VCD) with Fourier transform (FT) instrumentation. The measurements were carried out using a commercial rapid-scan FT-IR VCD spectrometer modified for operation in the NIR region between 3750 and 6150 cm-1. Overtone and combination band VCD intensities, first reported by Keiderling and Stephens using dispersive VCD instrumentation in 1976, were reproduced with equivalent signal quality and spectral resolution using collection times of only a few minutes. These results offer substantial promise for the routine use of rapid-scan FT instrumentation for the measurement of overtone and combination band VCD spectra in the NIR region.
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Cytochrome P-450 isozymes represent a critical component of nature's spectrum of detoxification catalysts that could be exploited for bioremediation. The ethanol-inducible human cytochrome P-450 2E1 serves as a model eukaryotic P-450 that complements the bacterial P-450 cam in dehalogenation and detoxification of environmental pollutants. We explored the construction of novel chimeric P-450s using cytochrome P-450 camC and 2E1 genes. For construction of chimera 1 (478 amino acids, 55.14 kDa), 145 amino acids from the N-terminus of P-450 2E1 protein (493 amino acids, 56.84 kDa) were replaced with 130 amino acids from the N-terminus of P-450 camC protein (415 amino acids, 46.66 kDa). In chimera 2 (525 amino acids, 60.24 kDa) the strategy involves replacement of 28 amino acids in the C-terminus of chimera 1 with 75 amino acids from the C-terminus of P-450 camC gene. Homology models of both the chimeric proteins were developed using SWISS-MODEL based on the known crystal structure of cytochrome P-450 camC, BM-3, 1DT6A, and 2C17A. The models indicated that the proposed heme-binding site was intact, which is inevitable for catalytic activity of cytochrome P-450s. The expression of chimera 1 and 2 genes in Escherichia coli DH5alpha was evident from light-pink cell pellets, protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, and diagnostic carbon monoxide-difference spectra. Our studies show that strategies can be developed to exploit the natural diversity of the P-450 superfamily to generate chimeric biocatalysts that would provide new templates amenable to directed evolution.
Deficiencies of pulpotomy and pulpectomy treatment of primary teeth have promoted considerable laboratory and clinical research. Some of these findings and their implications are reviewed.
In this paper, a headspace solid-phase microextraction (HS-SPME) method was applied to analyse the volatile compounds in a traditional Chinese medicine (TCM), Rhioxma Curcumae Aeruginosae. SPME parameters such as fibers, extraction temperature, extraction time and desorption time were investigated. Thirty-five volatile compounds were separated and identified. Relative standard deviations (RSDs) were less than 8.4%, showing that the method has a good reproducibility. The volatile constituents were also analyzed by steam distillation (SD) and thirty-seven compounds were identified. The similar results obtained by the two methods showed that SPME is a good alternative for the analysis of volatile constituents in Rhioxma Curcumae Aeruginosae samples and it is a relatively simple, rapid and solvent-free method.
We have previously shown that skin reconstructed in vitro is a useful model to study the effects of UVB and UVA exposure. Wavelength-specific biological damage has been identified such as the formation of sunburn cells (SBC) and pyrimidine dimers after UVB irradiation and alterations of dermal fibroblasts after UVA exposure. These specific effects were selected to evaluate the protection afforded by two sunscreens after topical application on the skin surface. Simplified formulations having different absorption spectra but similar sun protection factors were used. One contained a classical UVB absorber, 2-ethylhexyl-p-methoxycinnamate. The other contained a broad-spectrum absorber called Mexoryl SX, characterized by its strong absorbing potency in the UVA range. Both filters were used at 5% in a simple water/oil vehicle. The evaluation of photoprotection on in vitro reconstructed skin revealed good efficiency for both preparations in preventing UVB-induced damage, as shown by SBC counting and pyrimidine dimer immunostaining. By contrast, only the Mexoryl SX-containing preparation was able to efficiently prevent UVA-specific damage such as dermal fibroblast disappearance. Our data further support the fact that skin reconstructed in vitro is a reliable system to evaluate the photoprotection provided by different sunscreens against specific UVB and UVA biological damage.
Since the use of antimicrobial agents is required in endodontic therapies, this study aimed at determining the minimum inhibitory concentrations (MICs) of chlorhexidine digluconate and paramonochlorophenol (PMC) against microorganisms commonly found in endodontic infections. Both agents were tested by agar dilution tests against Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Candida albicans, Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella denticola and Prevotella melaninogenica. The MIC of chlorhexidine ranged from 2.67 to 80.00 microg/ml, and the MIC of PMC from 46.67 to 213.33 microg/ml. The highest MIC value of PMC was detected for E. faecalis whereas E. coli was the most susceptible microorganism to this agent. The highest MIC values of chlorhexidine were observed for P. aeruginosa whereas E. coli and P. denticola were the most susceptible microorganisms to this agent. Since the MIC values observed are much lower than the concentrations currently used in the endodontic therapy, it is suggested that both agents are effective in reducing the microbiota in the root canal.
Acquired immunodeficiency syndrome (AIDS), a degenerative disease of the immune and central nervous systems, is an enormous world-wide health threat. No cure has been found, and research is aimed at developing chemotherapy against the causative agent, human immunodeficiency virus (HIV). Chemotherapy for AIDS has progressed steadily in the past decade. However, new, effective, and less toxic chemotherapeutic agents are still needed. Plants, particularly anti-infective or immunomodulating herbal medicines, can serve as sources of new active leads to be further developed as anti-AIDS drug candidates. A lot of structurally different natural coumarins were found to display potent anti-HIV activity and continued progress is anticipated in the discovery of new leads and in the development of these agents as potential anti-AIDS drug candidates. Recent studies based on the account of various coumarins from plant sources and their analogs--synthetic coumarins, indicate that some of them serve as potent nonnucleoside RT-inhibitors, another as inhibitors of HIV-integrase or HIV-protease. The current review demonstrates the variety of coumarins of natural plant origin and synthetic coumarins having unique mechanism of action to one of the most important stage of HIV replication (RT-inhibition). The merits of selecting potential anti-HIV agents to be used in rational combination drugs design and structure-activity relationships are discussed. The scientific community is looking actively for new drugs and combinations for treatment of HIV infection effective for first-line treatment, as well as against drug-resistant mutants.