Arachidonic acid epoxidation: epoxyeicosatrienoic acids are endogenous constituents of rat liver

Epoxyeicosatrienoic acids have been isolated and purified from the livers of male rats. They were identified by gas chromatography-mass spectrometric techniques. These results expand the list of in vivo-produced eicosanoids. Their documented in vitro biological activities suggest a role for them in cell and tissue homeostasis.     

Oligomerization controls in tissue-specific manner ligand binding of native, affinity-purified p42(IP4)/centaurin alpha1 and cytohesins-proteins with high affinity for the messengers D-inositol 1,3,4,5-tetrakisphosphate/phosphatidylinositol 3,4,5-trisphosphate

Several distinct receptor proteins for the second messengers Ins(1,3,4,5)P(4) and PtdIns(3,4,5)P(3) are already known, such as the brain-specific p42(IP4), which we have previously cloned from different species, and cytohesins. However, it is still unclear whether proteins interacting with phosphoinositide and inositolpolyphosphate second messengers are regulated differently in different tissues. Here, we investigated these native proteins for comparison also from rat lung cytosol and purified them by PtdIns(3,4,5)P(3) affinity chromatography. Proteins selectively binding Ins(1,3,4,5)P(4) with high affinity also showed high affinity and specificity towards PtdIns(3,4,5)P(3). In lung cytosol, two prominent protein bands were found in the eluate from a PtdIns(3,4,5)P(3) affinity column. We identified these proteins by mass spectrometry as the cytohesin family of Arf guanosine nucleotide exchange factors (cytohesin 1, ARNO, GRP-1) and as Bruton's tyrosine kinase. Western blot analysis indicated that p42(IP4) was present in lung only at very low concentrations. Applying the affinity purification scheme established for rat lung cytosol to cytosol from rat brain, however, yielded only p42(IP4). We identified cytohesins in rat brain by Western blotting and PCR, but cytohesins surprisingly did not bind to the PtdIns(3,4,5)P(3)-affinity column. Gel filtration experiments of brain cytosol revealed that brain cytohesins are bound to large molecular weight complexes (150 to more than 500 kDa). Thus, we hypothesize that this finding explains why brain cytohesins apparently do not bind the inositolphosphate ligand. In lung cytosol, on the other hand, cytohesins occur as dimers. Gel filtration also showed that p42(IP4) in brain cytosol occurs as a monomer. Thus, oligomerization (homomeric or heteromeric) of InsP(4)/PtdInsP(3) binding proteins can modulate their function in a tissue-dependent manner because it can modify their ability to interact with the ligands.     

Comparison of Fc N-Glycosylation of Pharmaceutical Products of Intravenous Immunoglobulin G

Intravenous immunoglobulin (IVIg) products from different pharmaceutical companies vary in composition, in part because of the selected blood donors and production process. N-glycosylation of the Fc-portion of IgG varies between blood donors and may influence both the side-effects and therapeutic effectiveness of IVIg. At present, the variation in Fc N-glycosylation between IVIg products has not been defined. Utilizing mass spectrometry, we performed relative quantitation of the Fc N-glycosylation of IgG, assessing a total of 154 unique lot numbers of IVIg. Seven products showed comparable Fc N-glycosylation, with only one product differing from the others in all glycosylation features (galactosylation, sialylation, fucosylation and bisecting N-acetylglucosamine). However, the mean difference did not exceed 3%. Within product variation was present to a minor degree, but largely indistinguishable from analytical variation. In conclusion, we expect that the minor variation in Fc N-glycosylation between IVIg products has a small effect, if any, on the biological activity.     

Omega-oxidation of 20-hydroxyeicosatetraenoic acid (20-HETE) in cerebral microvascular smooth muscle and endothelium by alcohol dehydrogenase 4

20-Carboxyeicosatetraenoic acid (20-COOH-AA) is a bioactive metabolite of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid that produces vasoconstriction in the cerebral circulation. We found that smooth muscle (MSMC) and endothelial (MEC) cultures obtained from mouse brain microvessels convert [3H]20-HETE to 20-COOH-AA, indicating that the cerebral vasculature can produce this metabolite. The [3H]20-COOH-AA accumulated primarily in the culture medium, together with additional radiolabeled metabolites identified as the chain-shortened dicarboxylic acids 18-COOH-18:4, 18-COOH-18:3, and 16-COOH-16:3. N-Heptylformamide, a potent inhibitor of alcohol dehydrogenase (ADH), decreased the conversion of [3H]20-HETE to 20-COOH-AA by the MSMC and MEC and also by isolated mouse brain microvessels. Purified mouse and human ADH4, human ADH3, and horse liver ADH1 efficiently oxidized 20-HETE, and ADH4 and ADH3 were detected in MSMC and MEC by Western blotting. N-Heptylformamide inhibited the oxidation of 20-HETE by mouse and human ADH4 but not by ADH3. These results demonstrated that cerebral microvessels convert 20-HETE to 20-COOH-AA and that ADH catalyzes the reaction. Although ADH4 and ADH3 are expressed in MSMC and MEC, the inhibition produced by N-heptylformamide suggests that ADH4 is primarily responsible for 20-COOH-AA formation in the cerebral microvasculature.     

Detection of the Influenza A(H1N1)pdm09 Virus Carrying the K-15E,P83S and Q293H Mutations in Patients Who Have Undergone Bone Marrow Transplant

The 2009 pandemic influenza A(H1N1)pdm09 virus emerged and caused considerable morbidity and mortality in the third world, especially in Brazil. Although circulating strains of A(H1N1)pdm09 are A/California/04/2009-like (CA-04-like) viruses, various studies have suggested that some mutations in the viral hemagglutinin (HA) may be associated with enhanced severity and fatality. This phenomenon is particularly challenging for immunocompromised individuals, such as those who have undergone bone marrow transplant (BMT), because they are more likely to display worse clinical outcomes to influenza infection than non-immunocompromised individuals. We studied the clinical and viral aspects of post-BMT patients with confirmed A(H1N1)pdm09 diagnosis in the largest cancer hospital in Brazil. We found a viral strain with K-15E, P83S and Q293H polymorphisms in the HA, which is presumably more virulent, in these individuals. Despite that, these patients showed only mild symptoms of infection. Our findings complement the discovery of mild cases of infection with the A(H1N1)pdm09 virus with the K-15E, P83S and Q293H mutations in Brazil and oppose other studies that have linked these changes with increased disease severity. These results could be important for a better comprehension of the impact of the pandemic influenza in the context of BMT.     

Indication Alerts Intercept Drug Name Confusion Errors during Computerized Entry of Medication Orders

Background

Confusion between similar drug names is a common cause of potentially harmful medication errors. Interventions to prevent these errors at the point of prescribing have had limited success. The purpose of this study is to measure whether indication alerts at the time of computerized physician order entry (CPOE) can intercept drug name confusion errors.

Methods and Findings

A retrospective observational study of alerts provided to prescribers in a public, tertiary hospital and ambulatory practice with medication orders placed using CPOE. Consecutive patients seen from April 2006 through February 2012 were eligible if a clinician received an indication alert during ordering. A total of 54,499 unique patients were included. The computerized decision support system prompted prescribers to enter indications when certain medications were ordered without a coded indication in the electronic problem list. Alerts required prescribers either to ignore them by clicking OK, to place a problem in the problem list, or to cancel the order. Main outcome was the proportion of indication alerts resulting in the interception of drug name confusion errors. Error interception was determined using an algorithm to identify instances in which an alert triggered, the initial medication order was not completed, and the same prescriber ordered a similar-sounding medication on the same patient within 5 minutes. Similarity was defined using standard text similarity measures. Two clinicians performed chart review of all cases to determine whether the first, non-completed medication order had a documented or non-documented, plausible indication for use. If either reviewer found a plausible indication, the case was not considered an error. We analyzed 127,458 alerts and identified 176 intercepted drug name confusion errors, an interception rate of 0.14±.01%.

Conclusions

Indication alerts intercepted 1.4 drug name confusion errors per 1000 alerts. Institutions with CPOE should consider using indication prompts to intercept drug name confusion errors.     

Interferometric biosensor based on planar optical waveguide sensor chips for label-free detection of surface bound bioreactions

A label free optical biosensor based on a free-space Young interferometer configuration is presented. Commercial planar Ta(2) O(5) waveguides are used as sensing elements and allow the investigation of surface bound bioreactions like immunoreactions or biological affinity systems. Design criteria are discussed and a detailed characterization of the sensor performance is presented. The developed interferometer yields an effective refractive index resolution of 9 x 10(-9), corresponding to a surface coverage of approximately 13 fg/mm(2). The performance of the system is characterized by two different affinity systems: the antibody-antigen complex protein G-immunoglobulin G is used as a model system for monitoring reaction kinetics. Further measurements on a silanized surface show the formation of a streptavidin monolayer on a biotinylated surface.     

A novel activity of microsomal epoxide hydrolase: metabolism of the endocannabinoid 2-arachidonoylglycerol

Microsomal epoxide hydrolase (EPHX1, EC 3.3.2.9) is a highly abundant α/β-hydrolase enzyme that is known for its catalytical epoxide hydrolase activity. A wide range of EPHX1 functions have been demonstrated including xenobiotic metabolism; however, characterization of its endogenous substrates is limited. In this study, we present evidence that EPHX1 metabolizes the abundant endocannabinoid 2-arachidonoylglycerol (2-AG) to free arachidonic acid (AA) and glycerol. The EPHX1 metabolism of 2-AG was demonstrated using commercially available EPHX1 microsomes as well as PC-3 cells overexpressing EPHX1. Conversely, EPHX1 siRNA markedly reduced the EPHX1 expression and 2-AG metabolism in HepG2 cells and LNCaP cells. A selective EPHX1 inhibitor, 10-hydroxystearamide, inhibited 2-AG metabolism and hydrolysis of a well-known EPHX1 substrate, cis-stilbene oxide. Among the inhibitors studied, a serine hydrolase inhibitor, methoxy-arachidonyl fluorophosphate, was the most potent inhibitor of 2-AG metabolism by EPHX1 microsomes. These results demonstrate that 2-AG is an endogenous substrate for EPHX1, a potential role of EPHX1 in the endocannabinoid signaling and a new AA biosynthetic pathway.     

Integration of hypoxic dilation signaling pathways for skeletal muscle resistance arteries

Mediator contributions to hypoxic dilation of rat gracilis muscle resistance arteries were determined by measuring dilation, vascular smooth muscle hyperpolarization, and metabolite production after incremental hypoxia. Nitric oxide (NO) synthase inhibition abolished responses to mild hypoxia, whereas COX inhibition impaired responses to more severe hypoxia by 77%. Blocking 20-hydroxyeicosatetraenoic acid (20-HETE) impaired responses to moderate hypoxia. With only NO systems intact, responses were maintained with mild hypoxia (88% normal) mediated via K(Ca) channels. When only COX pathways were intact, responses to moderate-severe hypoxia were largely retained (79% of normal) mediated via K(ATP) channels. Vessel responses to moderate hypoxia were retained with only 20-HETE systems intact mediated via K(Ca) channels. NO production increased 5.6-fold with mild hypoxia; greater hypoxia was without further effect. With increased hypoxia, 20-HETE levels fell to 40% of control values. 6-keto-PGF(1alpha) levels were not altered with mild hypoxia, but increased 4.6-fold with severe hypoxia. These results suggest vascular reactivity to progressive hypoxia represents an integration of NO production (mild hypoxia), PGI(2) production (severe hypoxia), and reduced 20-HETE levels (moderate hypoxia).