The gene (crc) responsible for catabolite repression control in Pseudomonas aeruginosa has been cloned and sequenced. Flanking the crc gene are genes encoding orotate phosphoribosyl transferase (pyrE) and RNase PH (rph). New crc mutants were constructed by disruption of the wild-type crc gene. The crc gene encodes an open reading frame of 259 amino acids with homology to the apurinic/apyrimidinic endonuclease family of DNA repair enzymes. However, crc mutants do not have a DNA repair phenotype, nor can the crc gene complement Escherichia coli DNA repair-deficient strains. The crc gene product was overexpressed in both P. aeruginosa and in E. coli, and the Crc protein was purified from both. The purified Crc proteins show neither apurinic/ apyrimidinic endonuclease nor exonuclease activity. Antibody to the purified Crc protein reacted with proteins of similar size in crude extracts from Pseudomonas putida and Pseudomonas fluorescens, suggesting a common mechanism of catabolite repression in these three species.The genus Pseudomonas is noteworthy for its diversity in habitat and physiology. Some Pseudomonas strains are able to use over 100 organic compounds as the sole or principal source of carbon. Pseudomonas aeruginosa can utilize at least 80 different organic compounds (41). A mechanism of catabolite repression control exists in these organisms (18) which prevents them from wasting energy maintaining the enzymes for all these catabolic pathways and ensures the preferential utilization of the most efficient source of carbon and energy. Such a regulatory mechanism has also been identified in the enteric bacteria (20) and in Bacillus spp. (6).In the enteric bacteria, the molecular mechanism of catabolite repression control involves a catabolite activator protein (Cap) which, when bound to cyclic AMP (cAMP), interacts with promoter regions of regulated genes to facilitate the binding of RNA polymerase, thereby initiating transcription (20). In the presence of glucose, the cAMP pool is lowered, Cap is not bound, and the regulated genes are not transcribed (i.e., they are repressed). The effect of glucose on cAMP pools is mediated by components of the phosphoenolpyruvate phosphotransferase system which also serve to activate adenylate cyclase (33). Since the initial identification of these regulatory components, catabolite repression has proven to be a global mechanism in the enteric bacteria, affecting at least 28 separate promoters which regulate biosynthetic as well as catabolic operons (7). It also has been found to act as a negative regulator as well as a positive regulator (1,7,23,24).In Bacillus spp. catabolite repression is not mediated by glucose, nor is cAMP involved (6,37,40). Genetic evidence has implicated the catabolite control protein (CcpA), a member of the GalR family of repressor proteins (6), Hpr, a component of the phosphoenolpyruvate phosphotransferase system (9), and a cis-acting DNA sequence (CRE) (15,48). Recent work has shown that HPr specifically phosphorylated at Ser-46 forms a complex wit...

Organophosphate (OP) anticholinesterases cause excess acetylcholine leading to seizures which, if prolonged, result in neuronal damage in the rodent brain. Novel substituted phenoxyalkyl pyridinium oximes have previously shown evidence of penetrating the rat blood-brain barrier (BBB) in in vivo tests with a sarin surrogate (nitrophenyl isopropyl methylphosphonate, NIMP) or the active metabolite of the insecticide parathion, paraoxon (PXN), by reducing the time to cessation of seizure-like behaviors and accumulation of glial fibrillary acidic protein, whereas 2-PAM did not. The neuroprotective ability of our lead oximes (15, 20, and 55) was tested using NeuN, Nissl, and Fluoro-Jade B staining in the rat hippocampus. Following lethal-level subcutaneous challenge with NIMP or PXN, rats were intramuscularly administered a novel oxime or 2-PAM plus atropine and euthanized at 4 days. There were statistically significant increases in the median damage scores of the NeuN-stained NIMP, NIMP/2-PAM, and NIMP/Oxime 15 groups compared with the control whereas the scores of the NIMP/Oxime 20 and NIMP/ Oxime 55 were not significantly different from the control. The same pattern of statistical significance was observed with PXN. Nissl staining provided a similar pattern, but without statistical differences. Fluoro-Jade B indicated neuroprotection from PXN with novel oximes but not with 2-PAM. The longer blood residence times of Oximes 20 and 55 compared with Oxime 15 might have contributed to their greater efficacy. These results suggest that novel oximes 20 and 55 were able to penetrate the BBB and attenuate neuronal damage after NIMP and PXN exposure, indicating potential broad-spectrum usefulness.

In August and November 2010 we collected and examined peripheral blood and tissues from three species of Gulf of Mexico fish. Findings were compared to non-exposed control fish. The leukocyte counts of exposed alligator gar were not significantly different from controls, while exposed Gulf killifish and sea trout had significantly decreased lymphocyte counts. Liver ethoxyresorufin-O-deethylase (EROD) values from sea trout were significantly greater than control sea trout EROD values, suggesting poly aromatic hydrocarbon exposure. Splenic melano-macrophage centers (MMCs) from exposed sea trout and Gulf killifish showed a significant increase in number compared to non-exposed fish. Sea trout splenic MMCs were also significantly greater in size. These findings suggest that Gulf fish sampled were exposed to crude oil from the Macondo well and were in a lymphopenic or immuno-compromised state.

A global proteomics approach was applied to model the hepatic response elicited by the toxicologically well-characterized xenobiotic phenobarbital (PB), a prototypical inducer of hepatic xenobiotic metabolizing enzymes and a well-known nongenotoxic liver carcinogen in rats. Differential detergent fractionation two-dimensional liquid chromatography electrospray ionization tandem mass spectrometry and systems biology modeling were used to identify alterations in toxicologically relevant hepatic molecular functions and biological processes in the livers of rats following a 5-day exposure to PB at 80 mg/kg/day or a vehicle control. Of the 3342 proteins identified, expression of 121 (3.6% of the total proteins) was significantly increased and 127 (3.8%) significantly decreased in the PB group compared to controls. The greatest increase was seen for cytochrome P450 (CYP) 2B2 (167-fold). All proteins with statistically significant differences from control were then analyzed using both Gene Ontology (GO) and Ingenuity Pathways Analysis (IPA, 5.0 IPA-Tox) for cellular location, function, network connectivity, and possible disease processes, especially as they relate to CYP-mediated metabolism and nongenotoxic carcinogenesis mechanisms. The GO results suggested that PB's mechanism of nongenotoxic carcinogenesis involves both increased xenobiotic metabolism, especially induction of the 2B subfamily of CYP enzymes, and increased cell cycle activity. Apoptosis, however, also increased, perhaps, as an attempt to counter the rising cancer threat. Of the IPA-mapped proteins, 41 have functions which are procarcinogenic and 14 anticarcinogenic according to the hypothesized nongenotoxic mechanism of imbalance between apoptosis and cellular proliferation. Twenty-two additional IPA nodes can be classified as procarcinogenic by the competing theory of increased metabolism resulting in the formation of reactive oxygen species. Since the systems biology modeling corresponded well to PB effects previously elucidated via more traditional methods, the global proteomic approach is proposed as a new screening methodology that can be incorporated into future toxicological studies.

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Paraoxonase (PON1) is a calcium dependent enzyme that is capable of hydrolyzing organophosphate anticholinesterases. PON1 activity is present in most mammals and previous research established that PON1 activity differs depending on the species. These studies mainly used the organophosphate substrate paraoxon, the active metabolite of the insecticide parathion. Using serum PON1 from different mammalian species, we compared the hydrolysis of paraoxon with the hydrolysis of the active metabolites (oxons) of two additional organophosphorus insecticides, methyl parathion and chlorpyrifos. Paraoxon hydrolysis was greater than that of methyl paraoxon, but the level of activity between species displayed a similar pattern. Regardless of the species tested, the hydrolysis of chlorpyrifos-oxon was significantly greater than that of paraoxon or methyl paraoxon. These data indicate that chlorpyrifos-oxon is a better substrate for PON1 regardless of the species. The pattern of species differences in PON1 activity varied with the change in substrate to chlorpyrifos-oxon from paraoxon or methyl paraoxon. For example, the sex difference observed here and reported elsewhere in the literature for rat PON1 hydrolysis of paraoxon was not present when chlorpyrifos-oxon was the substrate.

Traditionally, the liver has been considered a homogeneous organ, but literature suggests that the cytochromes P450 are differentially distributed among the hepatocytes and that the pattern of this distribution is altered by various xenobiotics. In this study, the CYP2B1/2 messenger RNA (mRNA) in the hepatocytes was compared following treatment of rats with either of two inducers, phenobarbital (PB), or dieldrin. Adult male Sprague-Dawley-derived rats were treated with either ip PB in saline at 80 mg/kg/day for 5 days or dieldrin in corn oil by oral gavage at 2.5 mg/kg/day for 13 days. Control rats received ip saline or po corn oil for the same time. Laser capture microdissection (LCM) followed by duplex quantitative real-time reverse transcriptase PCR was used to measure the CYP2B1/2 mRNA produced in bands of hepatocytes isolated from three locations along the sinusoidal path. The amounts of mRNA present in whole liver subsamples were also analyzed. CYP2B1/2 enzyme activity was determined by assaying 16beta-hydroxytestosterone formation. Whole liver mRNA samples exhibited significant induction in CYP2B1/2 transcript levels: sixfold for PB and 2200-fold for dieldrin. All the LCM band samples also showed significant fold induction in CYP2B1/2 mRNA compared to controls. Dieldrin caused marked increases in CYP2B1/2 mRNA levels in the direction of blood flow through the acinus: periportal, 300-fold; midzonal, 600-fold; and centrilobular, 1700-fold. A different pattern of induction was observed in the PB-treated rats: periportal, 1800-fold; midzonal, 8800-fold; and centrilobular, 1600-fold. The present study indicates the differences in spatial responses that can be exhibited within the liver following exposure to various xenobiotics. It also indicates the importance of examining xenobiotic metabolism in the liver in light of its nonhomogeneous, zoned microenvironment.

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