Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro

Ketamine, a phencyclidine derivative, is used for induction of anesthesia, as an anesthetic drug for short term surgical interventions and in subanesthetic doses for postoperative pain relief. Ketamine undergoes extensive hepatic first-pass metabolism. Enantioselective capillary electrophoresis with multiple isomer sulfated -cyclodextrin as chiral selector was used to identify cytochrome P450 enzymes involved in hepatic ketamine and norketamine biotransformation in vitro. The N-demethylation of ketamine to norketamine and subsequently the biotransformation of norketamine to other metabolites were studied via analysis of alkaline extracts of in vitro incubations of racemic ketamine and racemic norketamine with nine recombinantly expressed human cytochrome P450 enzymes and human liver microsomes. Norketamine was formed by CYP3A4, CYP2C19, CYP2B6, CYP2A6, CYP2D6 and CYP2C9, whereas CYP2B6 and CYP2A6 were identified to be the only enzymes which enable the hydroxylation of norketamine. The latter two enzymes produced metabolic patterns similar to those found in incubations with human liver microsomes. The kinetic data of ketamine N-demethylation with CYP3A4 and CYP2B6 were best described with the Michaelis-Menten model and the Hill equation, respectively. This is the first study elucidating the individual enzymes responsible for hydroxylation of norketamine. The obtained data suggest that in vitro biotransformation of ketamine and norketamine is stereoselective.

Vasculitides associated with IgG antineutrophil cytoplasmic autoantibodies in childhood

Immunoglobulin (Ig)G antineutrophil cytoplasmic autoantibodies are causally associated with necrotizing vasculitides that are characterized immunopathologically by little or no deposition of immunoreactants, such as Wegener granulomatosis, microscopic polyangiitis, Churg-Strauss angiitis, "renal-limited" vasculitis and a number of drug-induced vasculitides. Clinical routine testing targets the antigens myeloperoxidase and proteinase 3. However, in all of the conditions mentioned, the renal histopathologic findings are indistinguishable. Churg-Strauss angiitis (characterized by necrotizing vasculitis, granulomatous inflammation and tissue eosinophilia), Wegener granulomatosis (characterized by necrotizing vasculitis and granulomatous inflammation) and microscopic polyangiitis (characterized by necrotizing vasculitis) often present with fever, weight loss and a multisystem involvement (ear, nose, throat, lung, eyes, peripheral nerve and heart). Fifty years ago these conditions were very often fatal within 6 months of diagnosis. The introduction of corticosteroids and cyclophosphamide has resulted in a dramatic clinical benefit. Patients who develop treatment-related morbidity can be switched from cyclophosphamide to azathioprine after achieving remission. In patients with less severe disease, methotrexate achieves remission with a success rate similar to that of cyclophosphamide. Plasma exchange, in association with immunosuppression, is likely to be a beneficial therapy for patients with severe kidney disease or pulmonary hemorrhage.

High-level cytoplasmic cyclin D1 expression in lymph node metastases from prostate cancer independently predicts early biochemical failure and death in surgically treated patients

To test the prognostic significance of cyclin D1 in nodal-positive prostate cancer.

Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery

Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs). GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut) forms of T. brucei. Three regulatory elements common to the 3' UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE) is unique to the GPEET 3' UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A) RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3' UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A) binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are also consistent with stage-specific regulation of translation in trypanosomes, but most likely in the context of initiation.

Dietary intervention induces flow of changes within biomarkers of lipids, inflammation, liver enzymes, and glycemic control

To determine how changes in lipids, liver enzymes, and inflammatory and glycemia markers intercorrelate during prolonged dietary intervention in obese participants with or without type 2 diabetes (T2D).

Inhibition of cytochrome P450 enzymes involved in ketamine metabolism by use of liver microsomes and specific cytochrome P450 enzymes from horses, dogs, and humans

To identify and characterize cytochrome P450 enzymes (CYPs) responsible for the metabolism of racemic ketamine in 3 mammalian species in vitro by use of chemical inhibitors and antibodies.

Diagnostic value of biopsies in identifying cytoplasmic antineutrophil cytoplasmic antibody-negative localized Wegener's granulomatosis presenting primarily with sinonasal disease

A substantial proportion of Wegener's disease (WG) patients present with localized disease of the upper airways, i.e., sinonasal and other ear/nose/throat (ENT) symptoms. Because of the oligosymptomatic presentation a timely diagnosis of this potentially fatal disease is challenging. This study evaluates diagnostic peculiarities between WG in its localized and generalized form of the disease.

Regional mRNA expression of key gluconeogenic enzymes in the liver of dairy cows

Liver tissue was collected from eight random dairy cows at a slaughterhouse to test if gene expression of pyruvate carboxylase (PC), mitochondrial phosphoenolpyruvate carboxykinase (PEPCKm) and cytosolic phosphoenolpyruvate carboxykinase (PEPCKc) is different at different locations in the liver. Obtained liver samples were analysed for mRNA expression levels of PC, PEPCKc and PEPCKm and subjected to the MIXED procedure of SAS to test for the sampled locations with cow liver as repeated subject. Additionally, the general linear model procedure (GLM) for analysis of variance was applied to test for significant differences for mRNA abundance of PEPCKm, PEPCKc and bPC between the livers. In conclusion, this study demonstrated that mRNA abundance of PC, PEPCKc and PEPCKm is not different between locations in the liver but may differ between individual cows.

Metformin inhibits human androgen production by regulating steroidogenic enzymes HSD3B2 and CYP17A1 and complex I activity of the respiratory chain

Metformin is treatment of choice for the metabolic consequences seen in polycystic ovary syndrome for its insulin-sensitizing and androgen-lowering properties. Yet, the mechanism of action remains unclear. Two potential targets for metformin regulating steroid and glucose metabolism are AMP-activated protein kinase (AMPK) signaling and the complex I of the mitochondrial respiratory chain. Androgen biosynthesis requires steroid enzymes 17α-Hydroxylase/17,20 lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2), which are overexpressed in ovarian cells of polycystic ovary syndrome women. Therefore, we aimed to understand how metformin modulates androgen production using NCI-H295R cells as an established model of steroidogenesis. Similar to in vivo situation, metformin inhibited androgen production in NCI cells by decreasing HSD3B2 expression and CYP17A1 and HSD3B2 activities. The effect of metformin on androgen production was dose dependent and subject to the presence of organic cation transporters, establishing an important role of organic cation transporters for metformin's action. Metformin did not affect AMPK, ERK1/2, or atypical protein kinase C signaling. By contrast, metformin inhibited complex I of the respiratory chain in mitochondria. Similar to metformin, direct inhibition of complex I by rotenone also inhibited HSD3B2 activity. In conclusion, metformin inhibits androgen production by mechanisms targeting HSD3B2 and CYP17-lyase. This regulation involves inhibition of mitochondrial complex I but appears to be independent of AMPK signaling.

Inbreeding Alters Activities of the Stress-Related Enzymes Chitinases and β-1,3-Glucanases

Pathogenesis-related proteins, chitinases (CHT) and β-1,3-glucanases (GLU), are stress proteins up-regulated as response to extrinsic environmental stress in plants. It is unknown whether these PR proteins are also influenced by inbreeding, which has been suggested to constitute intrinsic genetic stress, and which is also known to affect the ability of plants to cope with environmental stress. We investigated activities of CHT and GLU in response to inbreeding in plants from 13 Ragged Robin (Lychnis flos-cuculi) populations. We also studied whether activities of these enzymes were associated with levels of herbivore damage and pathogen infection in the populations from which the plants originated. We found an increase in pathogenesis-related protein activity in inbred plants from five out of the 13 investigated populations, which suggests that these proteins may play a role in how plants respond to intrinsic genetic stress brought about by inbreeding in some populations depending on the allele frequencies of loci affecting the expression of CHT and the past levels of inbreeding. More importantly, we found that CHT activities were higher in plants from populations with higher levels of herbivore or pathogen damage, but inbreeding reduced CHT activity in these populations disrupting the increased activities of this resistance-related enzyme in populations where high resistance is beneficial. These results provide novel information on the effects of plant inbreeding on plant–enemy interactions on a biochemical level.

A fluorescently labeled dendronized polymer-enzyme conjugate carrying multiple copies of two different types of active enzymes

A hybrid structure of a synthetic dendronized polymer, two different types of enzymes (superoxide dismutase and horseradish peroxidase), and a fluorescent dye (fluorescein) was synthesized. Thereby, a single polymer chain carried multiple copies of the two enzymes and the fluorescein. The entire attachment chemistry is based on UV/vis-quantifiable bis-aryl hydrazone bond formation that allows direct quantification of bound molecules: 60 superoxide dismutase, 120 horseradish peroxidase, and 20 fluorescein molecules on an average polymer chain of 2000 repeating units. To obtain other enzyme ratios the experimental conditions were altered accordingly. Moreover, it could be shown that both enzymes remained fully active and catalyzed a two-step cascade reaction.

Inhibition of indoleamine 2,3-dioxygenase in mixed lymphocyte reaction affects glucose influx and enzymes involved in aerobic glycolysis and glutaminolysis in alloreactive T-cells

Indoleamine 2,3-dioxygenase (IDO) suppresses adaptive immunity. T-cell proliferation and differentiation to effector cells require increased glucose consumption, aerobic glycolysis and glutaminolysis. The effect of IDO on the above metabolic pathways was evaluated in alloreactive T-cells. Mixed lymphocyte reaction (MLR) in the presence or not of the IDO inhibitor, 1-DL-methyl-tryptophane (1-MT), was used. In MLRs, 1-MT decreased tryptophan consumption, increased cell proliferation, glucose influx and lactate production, whereas it decreased tricarboxylic acid cycle activity. In T-cells, from the two pathways that could sense tryptophan depletion, i.e. general control nonrepressed 2 (GCN2) kinase and mammalian target of rapamycin complex 1, 1-MT reduced only the activity of the GCN2 kinase. Additionally 1-MT treatment of MLRs altered the expression and/or the phosphorylation state of glucose transporter-1 and of key enzymes involved in glucose metabolism and glutaminolysis in alloreactive T-cells in a way that favors glucose influx, aerobic glycolysis and glutaminolysis. Thus in alloreactive T-cells, IDO through activation of the GCN2 kinase, decreases glucose influx and alters key enzymes involved in metabolism, decreasing aerobic glycolysis and glutaminolysis. Acting in such a way, IDO could be considered as a constraining factor for alloreactive T-cell proliferation and differentiation to effector T-cell subtypes.

Perinuclear antineutrophilic cytoplasmic antibody and response to treatment in diarrheic dogs with food responsive disease or inflammatory bowel disease

The goal of this study was to investigate the correlation between perinuclear antineutrophilic cytoplasmic antibody (pANCA) and clinical scores before and after treatment in diarrheic dogs with food-responsive disease (FRD) or inflammatory bowel disease (IBD). pANCA serology was evaluated prospectively by indirect immunofluorescence in 65 dogs with signs of gastrointestinal disease, and if positive, pANCA antibody titers were determined. Thirty-nine dogs with FRD responded to a novel diet, and 26 dogs with IBD were treated with corticosteroids. The severity of clinical signs was scored by means of a canine IBD activity index (CIBDAI). At initial examination, a significantly (P = .002) higher percentage of dogs were pANCA-positive in the FRD group (62%) compared with the IBD group (23%). pANCA titers were significantly higher (P = .003) before treatment in the FRD group (median titer 100) compared with the IBD group (median titer 1). However, there was no difference in pANCA titers between the groups after respective treatments because dogs in the IBD group had a significant increase in pANCA titer after treatment. The CIBDAI score decreased significantly (P < .001) after treatment in both groups (74% moderate to severe in FRD dogs before versus 8% after treatment; 85% moderate to severe in IBD dogs before versus 32% after treatment). There was no correlation between pANCA status in FRD or IBD dogs before treatment and scores for CIBDAI, endoscopy, or histopathology before or after treatment, except for the endoscopic duodenal score in dogs with FRD after treatment (P = .03). A positive pANCA test before therapy may aid in the diagnosis of FRD.