The structural basis of species differences in cytochrome P450 2B-mediated hydroxylation of 2,2',3,3',6,6'-hexachlorobiphenyl (236HCB) was evaluated by using 14 site-directed mutants of cytochrome P450 2B1 and three point mutants of 2B11 expressed in Escherichia coli. To facilitate metabolite identification, seven possible products, including three hydroxylated and four dihydroxylated hexachlorobiphenyls, were synthesized by direct functionalization of precursors and Ullmann and crossed Ullmann reactions. HPLC and GC/MS analysis and comparison with authentic standards revealed that 2B1, 2B11, and all their mutants produced 4, 5-dihydroxy-236HCB and 5-hydroxy-236HCB, while 2B11 L363V and 2B1 I114V mutants also catalyzed hydroxylation at the 4-position. The amount of products formed by 2B1 mutants I114V, F206L, L209A, T302S, V363A, V363L, V367A, I477A, I477L, G478S, I480A, and I480L was smaller than that of the wild type. I477V exhibited unaltered 236HCB metabolism, and I480V produced twice as much dihydroxy product as the wild type. For 2B11, substitution of Val-114 or Asp-290 with Ile decreased the product yields. Replacement of Leu-363 with Val dramatically altered the profile of 236HCB metabolites. In addition to an increase in the overall level of hydroxylation, the mutant mainly catalyzed hydroxylation at the 4-position. Incubation of P450 2B1 with 5-hydroxy-236HCB produced 4,5-dihydroxy-236HCB, which indicates that 4,5-dihydroxy-236HCB may be formed by a direct hydroxylation of 5-hydroxy-236HCB. The findings from this study demonstrate the importance of residues 114, 206, 209, 302, 363, 367, 477, 478, and 480 in 2B1 and 114, 290, and 363 in 2B11 for 236HCB metabolism.

The model of Norden was used to induce osteomyelitis in the left tibia of New Zealand White rabbits. Twenty-one days following inoculation, the animals had primary debridement and then were randomized into one of three treatment groups. Group I received no additional treatment; in Group II, plain hydroxyapatite beads were packed into the defect; and in Group III, gentamicin crobefat-loaded hydroxyapatite beads were packed into the defect. The animals were observed for 40 days after the primary debridement and then were killed. The intensity of infection was determined by swab cultures and quantitative bacterial cultures of the debrided material. At primary debridement, all of the animals in each group were equally infected. At the time of secondary debridement, only the animals in Group III had a statistically significant reduction in infection (p < 0.001). In this study, we demonstrated that an antibiotic-loaded osteoinductive ceramic bead can effectively eliminate bacteria from an osteomyelitic cavity.

The reaction of peroxynitrite with gamma-tocopherol (gamma-TH) in a methanol/potassium phosphate buffer solution results in the formation of four major products, which were identified as 2,7,8-trimethyl-2-(4,8,12-trimethyldecyl)-5-nitro-6-chromanol++ + (NGT), 2,7,8-trimethyl-2-(4,8,12-trimethyldecyl)-5,6-chromaquinone (tocored), and two diastereomers of 8a-(hydroxy)-gamma-tocopherone. NGT was the major product formed in these reactions, and its formation was modestly increased by increasing amounts of Fe(3+)-EDTA. Tocored and NGT also were formed when gamma-TH was exposed to 3-morpholinosydnonimine (SIN-1), a compound that decomposes to form peroxynitrite. When gamma-TH reacted with the nitrating agent NO2+BF4- in acetonitrile or methanol/potassium phosphate buffer, NGT and tocored also were formed, but the major product detected was gamma-tocopherol quinone (gamma-TQ). This product was not detected in reactions involving peroxynitrite. Oxidation of gamma-TH by peroxynitrite involves nitration and electron transfer reactions. Since the product distribution in oxidations with NO2+BF4- differed substantially from that in oxidations with peroxynitrite and SIN-1, NO2+ appeared not to be the principal species involved in NGT formation. Nitration of gamma-TH may involve either peroxynitrite or some peroxynitrite-derived oxidant other than NO2+. Because of its stability and formation as a novel product of the reaction between gamma-TH with peroxynitrite, NGT may be a useful in vivo marker for peroxynitrite interactions with lipid structures that contain gamma-TH.

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was ϳ31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3 and 5 nucleoside monophosphates at pH 6. Calculated K m s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 mol of P i /s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.Clostridium perfringens is a ubiquitous gram-positive, endospore-forming anaerobic bacterium. While found in soil, the organism is often a constituent of the intestinal flora and is capable of causing severe gastrointestinal and histotoxic infections in humans and animals. As a species, C. perfringens is a very heterogeneous group of organisms with respect to metabolic by-products produced in vitro and pathogenic potential (25). Although the literature is replete with descriptions of the immunological, bio-and physicochemical attributes of C. perfringens-encoded toxins, as well as molecular and morphological details of sporulation, detailed analysis of other constituent enzymes, including those involved in acquisition and regulation of inorganic phosphate, has received relatively little attention. This lack of information is notable. A growing cadre of investigators (1, 7) has recognized the clinical utility of acid phosphatase as a biochemical marker for the identification of C. perfringens isolated from water and other sources and its use in the differentiation of C. perfringens from ϳ95% of all currently recognized clostridial species in the genus (42).Acid phosphatases (EC 3.1.3.2) are ubiquitous and catalyze, at an acidic pH, the transfer of phosphoryl groups from phosphomonoesters to water (48). These enzymes play essential roles in the generation, acquisition, and mobilization of inorganic phosphate and critical roles in phosphoryl relay systems intimately involved in signal transduction pathways in both prokaryotes and eukaryotes. A subgroup of phosphatases has been designated nonspecific acid phosphatases (NSAPs) by Thaller and coworkers (45) and includes those bacterial polyspecific phosphatases that are secreted across the cytoplasmic membrane and exhibit optimum catalytic activity at an acidic pH (41). NSAPs are recognized as a distinct group of phosphatases and categorized into three classes (46). The class C enzyme...

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Condyloma acuminata or genital warts are caused by human papilloma virus (HPV). Ongoing proliferation of HPV in patients with congenital or acquired immunodeficiency states leads to the development of rapidly progressive and sometimes locally invasive tumor with or without dysplasia. Aggressive treatment, diagnostic immuno-typing, and follow-up are necessary in patients with ongoing immunosuppression. We report a case of giant ano-genital condylomatosis due to HPV types 6 and 11 in a patient with chronic myeloid leukemia after allogeneic bone marrow transplantation. The tumor was successfully treated with surgical excision and local application of 5% imiquimod cream.