HX600, a synthetic agonist for RXR-Nurr1 heterodimer complex, prevents ischemia-induced neuronal damage.

Ischemic stroke is amongst the leading causes of death and disabilities. The available treatments are suitable for only a fraction of patients and thus novel therapies are urgently needed. Blockage of one of the cerebral arteries leads to massive and persisting inflammatory reaction contributing to the nearby neuronal damage. Targeting the detrimental pathways of neuroinflammation has been suggested to be beneficial in conditions of ischemic stroke. Nuclear receptor 4A-family (NR4A) member Nurr1 has been shown to be a potent modulator of harmful inflammatory reactions, yet the role of Nurr1 in cerebral stroke remains unknown. Here we show for the first time that an agonist for the dimeric transcription factor Nurr1/retinoid X receptor (RXR), HX600, reduces microglia expressed proinflammatory mediators and prevents inflammation induced neuronal death in in vitro co-culture model of neurons and microglia. Importantly, HX600 was protective in a mouse model of permanent middle cerebral artery occlusion and alleviated the stroke induced motor deficits. Along with the anti-inflammatory capacity of HX600 in vitro, treatment of ischemic mice with HX600 reduced ischemia induced Iba-1, p38 and TREM2 immunoreactivities, protected endogenous microglia from ischemia induced death and prevented leukocyte infiltration. These anti-inflammatory functions were associated with reduced levels of brain lysophosphatidylcholines (lysoPCs) and acylcarnitines, metabolites related to proinflammatory events. These data demonstrate that HX600 driven Nurr1 activation is beneficial in ischemic stroke and propose that targeting Nurr1 is a novel candidate for conditions involving neuroinflammatory component.

Transplacental transfer of melamine.

OBJECTIVE: To characterize transplacental transfer of melamine and related mechanisms as well as toxicity using human placental perfusion and cultured cells. METHODS: Transfer and toxicity were analyzed in 4-h perfusions with 10 µM or 1 mM melamine, or 10 µM melamine with 10 nM cyanuric acid (CYA). Efflux transporters were studied in accumulation assay and toxicity in BeWo cells by MTT assay. RESULTS: Of added melamine 34-45% was transferred to fetal circulation and CYA made no difference. Histology, hCG production, and PLAP activity indicated functionality of placental tissue with no grave toxicity. Highest concentration of melamine used (2 mM) with CYA and long treatment time decreased viability of BeWo cells. Inhibitors of ABCB1, ABCG2, ABCC2 did not affect the accumulation of melamine in cells. CONCLUSION: Melamine goes through human term placenta with no contribution of efflux transporters. Toxicity of melamine is low in placental tissue and BeWo cells.

Assessment of bisphosphonate activity in vitro.

Bisphosphonates are a class of drugs developed over the past three decades for the treatment of metabolic bone diseases with high bone turnover, such as Paget's disease, tumor associated osteolysis and osteoporosis. The exceptional pharmacokinetic profile of bisphosphonates makes them very suitable and safe drugs for the treatment of bone diseases, because, by conventional administration, osseous tissue and bone resorbing osteoclasts are the targets for these drugs as a result of the very high affinity of bisphosphonates for bone mineral. Several recent studies have demonstrated, however, that bisphosphonates decrease tumor burden in bone in rodent models of myeloma and metastatic bone disease, with suggestions of antitumor effects also in patients. Although decreased tumor burden could be a consequence of inhibition of bone resorption, there is increasing evidence that bisphosphonates might also have direct effects on tumor cell in vivo, since effects on tumors outside of skeleton or at doses not inhibiting bone resorption have been reported. Recent studies also suggest that bisphosphonates have inhibitory effect also on endothelial cell function and angiogenesis in tumor tissue. These findings suggest that the target cells for bisphosphonates as well as their molecular mechanism of action may be more diverse and complex than realized so far. This review highlights the main methodologies used to monitor the action of BPs in vitro cell models, with a special emphasis on the detection of BP-induced ATP-analoques by mass spectrometry. In addition, cell death monitoring, immunomodulatory effects and inhibition of growth/proliferation are described.

Degradation of coeliac disease-inducing rye secalin by germinating cereal enzymes: diminishing toxic effects in intestinal epithelial cells.

Currently the only treatment for coeliac disease is a lifelong gluten-free diet excluding food products containing wheat, rye and barley. There is, however, only scarce evidence as to harmful effects of rye in coeliac disease. To confirm the assumption that rye should be excluded from the coeliac patient's diet, we now sought to establish whether rye secalin activates toxic reactions in vitro in intestinal epithelial cell models as extensively as wheat gliadin. Further, we investigated the efficacy of germinating cereal enzymes from oat, wheat and barley to hydrolyse secalin into short fragments and whether secalin-induced harmful effects can be reduced by such pretreatment. In the current study, secalin elicited toxic reactions in intestinal Caco-2 epithelial cells similarly to gliadin: it induced epithelial cell layer permeability, tight junctional protein occludin and ZO-1 distortion and actin reorganization. In high-performance liquid chromatography and mass spectroscopy (HPLC-MS), germinating barley enzymes provided the most efficient degradation of secalin and gliadin peptides and was thus selected for further in vitro analysis. After germinating barley enzyme pretreatment, all toxic reactions induced by secalin were ameliorated. We conclude that germinating enzymes from barley are particularly efficient in the degradation of rye secalin. In future, these enzymes might be utilized as a novel medical treatment for coeliac disease or in food processing in order to develop high-quality coeliac-safe food products.

Acetaminophen bioactivation by human cytochrome P450 enzymes and animal microsomes.

Acetaminophen is a widely used analgesic antipyretic agent. When used at low doses, it is a safe drug, but at higher doses it can cause acute hepatic necrosis in humans and experimental animals. The key mechanism in the hepatotoxicity is cytochrome P450 (CYP)-catalysed formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) that is capable of binding to cellular macromolecules and in that way an LC/MS liquid chromatography/mass spectrometry (LC/MS) method was developed to measure NAPQI formation by trapping it to reduced glutathione. This method was used to determine the bioactivation of acetaminophen at two concentrations: 50 microM therapeutic and 1 mM toxic by using nine human recombinant CYP enzymes: CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4; and with different microsomes from experimental animals. At the toxic concentration the formation of NAPQI-glutathione was highest with CYP3A4 followed by CYP2E1, CYP1A2, and CYP2D6. At the therapeutic concentration, CYP3A4 had also the highest bioactivation capacity. In a comparison of the enzyme kinetics, CYP3A4 was the most efficient CYP with the lowest K(m) value 130 microM (95% confidence interval = 63-210 microM). Dexamethasone-induced rat liver microsomes had the most effective bioactivation capacity at therapeutic and toxic acetaminophen concentrations. This study suggests that CYP3A4 is the major CYP enzyme form catalysing acetaminophen oxidation to NAPQI in human liver.

Nicotine metabolism and urinary elimination in mouse: in vitro and in vivo.

This study aimed at elucidating the in vivo metabolism of nicotine both with and without inhibitors of nicotine metabolism. Second, the role of mouse CYP2A5 in nicotine oxidation in vitro was studied as such information is needed to assess whether the mouse is a suitable model for studying chemical inhibitors of the human CYP2A6. The oxidation of nicotine to cotinine was measured and the ability of various inhibitors to modify this reaction was determined. Nicotine and various inhibitors were co-administered to CD2F1 mice, and nicotine and urinary levels of nicotine and four metabolites were determined. In mouse liver microsomes anti-CYP2A5 antibody and known chemical inhibitors of the CYP2A5 enzyme blocked cotinine formation by 85-100%, depending on the pre-treatment of the mice. The amount of trans-3-hydroxycotine was five times higher than cotinine N-oxide, and ten times higher than nicotine N-1-oxide and cotinine. Methoxsalen, an irreversible inhibitor of CYP2A5, significantly reduced the metabolic elimination of nicotine in vivo, but the reversible inhibitors had no effect. It is concluded that the metabolism of nicotine in mouse is very similar to that in man and, therefore, that the mouse is a suitable model for testing novel chemical inhibitors of human CYP2A6.

Animal-derived lipocalin allergens exhibit immunoglobulin E cross-reactivity.

BACKGROUND: Although knowledge of the IgE cross-reactivity between allergens is important for understanding the mechanisms of allergy, the regulation of the allergic immune response and the development of efficient modes of allergen immunotherapy, the cross-reactivity of animal allergens is poorly known. OBJECTIVE: The aim of this study was to characterize IgE cross-reactivities between lipocalin proteins, including five animal-derived lipocalin allergens and one human endogenous lipocalin, tear lipocalin (TL). METHODS: The recombinant proteins were validated by chromatography and mass spectrometry. The IgE-binding capacity of the allergens was confirmed by IgE. immunoblotting and IgE immunoblot inhibition. IgE ELISA was performed with sera from 42 atopic patients and 21 control subjects. The IgE cross-reactivities between the lipocalin proteins were determined by ELISA inhibition. RESULTS: ELISA inhibition revealed IgE cross-reactivities between Can f 1 and human TL, between Can f 1 and Can f 2, and between Equ c 1 and Mus m 1. Low levels of IgE to human TL were found in the sera of seven dog-allergic patients of whom six were IgE-positive for Can f 1. CONCLUSION: Several lipocalins exhibited IgE cross-reactivity, probably due to the sequential identity of the proteins and also due to similarities in their three-dimensional structures. The clinical significance of the findings needs to be elucidated. Low-level IgE cross-reactivity can play a role in regulating immune response to lipocalin allergens.

Effects of glucosamine sulfate on intracellular UDP-hexosamine and UDP-glucuronic acid levels in bovine primary chondrocytes.

OBJECTIVE: To analyze the effects of exogenously added glucose (Glc), glucosamine (GlcN) and glucosamine sulfate (GS) on the intracellular UDP-hexoses (UDP-Hex), UDP-N-acetylhexosamines (UDP-HexN) and UDP-glucuronic acid (UDP-GlcA) levels in bovine primary chondrocytes. METHODS: Chondrocytes were incubated with different concentrations of Glc, GlcN and GS either in high- or low-glucose DMEM for up to 120min to analyze the intracellular levels of UDP-Hex, UDP-GlcA and UDP-HexN by a reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry analysis. Glycosaminoglycan (GAG) synthesis rate and aggrecan mRNA expression levels were quantified using (35)S-sulfate incorporation assay and quantitative real-time RT-PCR, respectively. The cells were cultivated for 2 days or 8 days before UDP-sugar analysis. RESULTS: Levels of UDP-HexN and UDP-GlcA were unchanged at 10microM concentration of GS in low-glucose DMEM, while addition of 1mM GlcN or GS in low-glucose DMEM for 10min increased UDP-HexN level. The highest intracellular level of UDP-HexN was reached at 30min after addition of 1mM GS to the cells. The intracellular contents of UDP-HexN and UDP-GlcA related to UDP-Hex were higher after prolonged cultivation of chondrocytes for 8 days compared with 2-day-old cultures. Aggrecan mRNA expression and GAG synthesis remained at control level after the cells were treated with 10, 100microM or 1mM of GS for 24h. CONCLUSION: Physiologically relevant level of GS could not increase the intracellular UDP-HexN and UDP-GlcA levels in bovine primary chondrocyte, while longer-time culture itself appeared to increase the intracellular UDP-HexN and UDP-GlcA levels.

In vitro evaluation of the effect of cyclodextrin complexation on pulmonary deposition of a peptide, cyclosporin A.

The effect of hydroxypropyl-alpha-cyclodextrin (HP-alpha-CD) complexation on in vitro pulmonary deposition of a cyclic peptide cyclosporin A (CsA) was studied. In addition, the effect of storage (32 days, 40 degrees C, 75% RH) on CsA/HP-alpha-CD complexes was studied. The complexation of CsA with CDs was evaluated by a phase-solubility method. Solid CsA/HP-alpha-CD complexes were prepared by freeze drying. Three inhalation formulations were prepared: CsA/lactose reference formulation (LF) (drug:carrier 1:364, w/w), CsA/HP-alpha-CD complex formulation (CDF) (drug:CD 1:269, w/w) and CsA/HP-alpha-CD complex/lactose formulation (CDLF) (complex:carrier 100:114, w/w). The inhalation studies were performed in vitro using Andersen Sampler (Ph. Eur.) and Taifun multi-dose dry powder inhalers (DPIs). Before the storage, the respirable fraction value (RF%) of CsA was 19.8+/-0.7%, 33.0+/-7.0% and 34.6+/-1.1% (mean+/-S.D., n=4 x 20) with LF, CDF and CDLF, respectively. When exposed to moisture (storage in a permeable polystyrene tube), the RF% values of CsA from formulations containing CsA/HP-alpha-CD complexes were lower than before the storage. However, when stored in the Taifun DPI, the RF% value of CsA from any of the formulations did not decrease. In conclusion, an acceptable RF% value of a peptide CsA from freeze-dried, simply micronized CsA/HP-alpha-CD complex powder was achieved before and after storage in the DPI.

Assessment of recombinant dog allergens Can f 1 and Can f 2 for the diagnosis of dog allergy.

BACKGROUND: The use of recombinant allergens for the diagnosis and immunotherapy of allergy may offer several advantages over allergen extracts. OBJECTIVE: To produce recombinant dog allergens Can f 1 and Can f 2 in Pichia pastoris yeast and to assess their suitability for the diagnosis of dog allergy. METHODS: Clinically diagnosed dog-allergic patients' and healthy non-atopic dog owners' reactivities against recombinant Can f 1 and Can f 2 and commercial dog epithelial extract were studied by a panel of methods including skin prick test (SPT), ELISA and IgE immunoblotting. RESULTS: Recombinant Can f 1 and Can f 2 were found immunologically functional: they bound dog-allergic patients' IgE in immunoblotting and inhibited specifically the binding of IgE to their natural counterparts in the dog allergen extract. Moreover, patients' IgE reactivity in immunoblotting to natural Can f 1 and their SPT with the recombinant allergen were perfectly concordant (phi coefficient 1.0, P<0.001). The concordance was slightly lower with recombinant Can f 2 (phi coefficient 0.92, P<0.001). A lower number of dog-allergic patients, 52%, reacted against Can f 1 than previously reported. About one-third of the patients reacted to Can f 2. In immunoblotting, the highest prevalence of reactivity, 60%, was directed to an 18 kDa component. Aminoterminal sequencing showed this to be a previously unidentified allergenic protein. CONCLUSIONS: The recombinant allergens can be used reliably to identify Can f 1 and Can f 2-sensitized individuals. However, the two allergens are insufficient as reagents for diagnosing dog allergy.

Determination of long-chain fatty acid acyl-coenzyme A compounds using liquid chromatography-electrospray ionization tandem mass spectrometry.

Acyl-CoAs have important role in fat and glucose metabolism of the cells. In this study we have developed an on-line HPLC-ESI-MS/MS method for determination of long-chain acyl-CoA compounds in rat liver samples. Six long-chain acyl-CoAs (C16:0, C16:1, C18:0, C18:1, C20:0 and C20:4) were separated with a C4 reversed-phase column using triethylamine acetate and acetonitrile gradient. Negative electrospray ionization is very suitable for acyl-CoA compounds and excellent MS/MS spectra for long-chain acyl-CoAs can be obtained. MS/MS method with an ion trap mass spectrometer makes it possible to identify and quantitate individual acyl-CoAs simultaneously. The method proved to be sensitive enough for determination of all compounds of interest using 0.4-0.7 g of tissue and was validated in the range of 0.1-15.0 pmol/microl.

Liquid chromatography/atmospheric pressure ionization-mass spectrometry in drug metabolism studies.

The study of the metabolic fate of drugs is an essential and important part of the drug development process. The analysis of metabolites is a challenging task and several different analytical methods have been used in these studies. However, after the introduction of the atmospheric pressure ionization (API) technique, electrospray and atmospheric pressure chemical ionization, liquid chromatography/mass spectrometry (LC/MS) has become an important and widely used method in the analysis of metabolites owing to its superior specificity, sensitivity and efficiency. In this paper the feasibility of LC/API-MS techniques in the identification, structure characterization and quantitation of drug metabolites is reviewed. Sample preparation, LC techniques, isotope labeling, suitability of different MS techniques, such as tandem mass spectrometry, and high-resolution MS in drug metabolite analysis, are summarized and discussed. Automation of data acquisition and interpretation, special techniques and possible future trends are also the topics of the review.

Removal of common Fusarium toxins in vitro by strains of Lactobacillus and Propionibacterium.

This study was conducted to examine the ability of selected strains of Lactobacillus and Propionibacterium to remove common Fusarium toxins, trichothecenes, from liquid media. The trichothecenes studied were deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), nivalenol (NIV), fusarenon (FX), diacetoxyscirpenol (DAS), T-2 toxin (T-2) and HT-2 toxin (HT-2). The Lactobacillus rhamnosus strain GG (LGG), Lactobacillus rhamnosus strain LC-705 (LC-705) and Propionibacterium freudenreichii ssp. shermanii JS (PJS) were incubated in PBS buffer containing 20 microg toxin ml(-1) for 1h at 37 degrees C, and after centrifugation the concentration of the toxins was measured in the supernatant fraction. Both viable and heat-killed forms of LGG and PJS were more efficient than LC-705 in removing the toxins from the liquid media. LGG and PJS removed four of the seven tested toxins (the removal varying from 18 to 93%) and LC-705 two toxins (10-64%). Of the toxins, 3-AcDON was not removed by any of the bacteria; HT-2 was removed by the non-viable LGG and also slightly by non-viable LC-705; DAS was removed by all three bacteria tested. Binding is postulated as the possible mechanism of the removal, since no difference was observed between the ability of viable and heat-killed bacteria in removing the trichothecenes, and no degradation products of the toxins were detected by gas chromatography (GC)-mass spectrometry (MS) analysis. It is concluded that significant differences exist in the ability of the bacteria to bind trichothecenes in vitro.

Two new variants of the lipocalin allergen Bos d 2.

Allergens from various sources have been shown to comprise several isoforms. In the present study, a series of chromatographic steps was carried out to separate the lipocalin allergen Bos d 2 isoforms present in cow dander. Subsequent HPLC-MS-MS analyses revealed two new Bos d 2 variants. In one of the proteins, tyrosine (Y83) was substituted by aspartic acid, and in the other protein valine (V102) was replaced by alanine. We propose the three Bos d 2 variants be named as Bos d 2.0101 (previously sequenced Bos d 2), Bos d 2.0102 and Bos d 2.0103. Our results suggest that molecular polymorphism is a common property among lipocalin allergens. Since allergen isoforms may show variation in their IgE binding and/or T-cell reactivity, all of the many allergen forms should be taken into account when planning preparations for immunotherapy.

The molecular mechanism of action of the antiresorptive and antiinflammatory drug clodronate: evidence for the formation in vivo of a metabolite that inhibits bone resorption and causes osteoclast and macrophage apoptosis.

OBJECTIVE: The primary aims of this study were to determine whether clodronate and liposome-encapsulated clodronate are metabolized to adenosine 5'-(beta,gamma-dichloromethylene) triphosphate (AppCCl2p) by osteoclasts and macrophages in vivo, and to determine whether intracellular accumulation of this metabolite accounts for the antiresorptive and antimacrophage effects of clodronate. To compare the mechanism of action of clodronate and alendronate, effects on protein prenylation in osteoclasts and macrophages in vivo were also assessed. METHODS: High-performance liquid chroma-tography-mass spectrometry was used to determine whether rabbit osteoclasts (purified ex vivo with immunomagnetic beads) metabolize clodronate, and whether rat peritoneal macrophages metabolize liposome-encapsulated clodronate, following in vivo administration. The effects of clodronate and AppCCl2p on bone resorption, osteoclast number, and apoptosis in vitro were compared. Using an antibody to the unprenylated form of RaplA, effects on protein prenylation were assessed by Western blot analysis of osteoclast and peritoneal macrophage lysates from bisphosphonate-treated animals. RESULTS: AppCCl2p could be detected in extracts from osteoclasts purified from clodronate-treated rabbits. Intracellular accumulation of AppCCl2p caused a reduction in the number of osteoclasts, increased osteoclast apoptosis, and inhibited bone resorption in vitro. These effects were indistinguishable from those of clodronate. Liposome-encapsulated clodronate was also metabolized to AppCCl2p by rat peritoneal macrophages in vivo. Liposome-encapsulated clodronate caused an increase in peritoneal macrophage apoptosis in ex vivo cultures that was indistinguishable from the increase in apoptosis caused by liposome-encapsulated AppCCl2p. Unlike alendronate, clodronate and its metabolite did not affect prenylation of the small GTPase RaplA in osteoclasts or macrophages in vivo. CONCLUSION: These results provide the first direct evidence that the antiinflammatory and antiresorptive effects of clodronate on macrophages and osteoclasts in vivo occur via the intracellular formation of AppCCl2p.

The cellular uptake and metabolism of clodronate in RAW 264 macrophages.

PURPOSE: Non-nitrogen-containing bisphosphonates, such as clodronate (dichloromethylene bisphosphonate), appear to act as prodrugs, their active form being the AppCp-type analogues of ATP. To further elucidate this, we examined the cellular uptake of clodronate and intracellular accumulation of the metabolite of clodronate (AppCCl2p) in RAW 264 macrophages, the influence of clodronate metabolism on the intracellular ATP concentration, and the time course of clodronate metabolism and the effects of clodronate on cytokine secretion from macrophages. METHODS: The cellular uptake of clodronate was measured using 14C-labeled clodronate. AppCCl2p was determined in cell extracts by using an ion-pairing HPLC-ESI-MS. The cytokine concentrations in the culture supernatants were measured with time-resolved fluoroimmunoassay. Intracellular ATP concentration was measured with a luminometer using a luciferin-luciferase assay. RESULTS: Of the clodronate internalized by macrophages in vitro, 30-55% is metabolized to AppCCl2p, which accumulates to high intracellular concentrations during the first 12 h of exposure. This accumulation does not affect the ATP levels in the cells. The time course of metabolite appearance in the cells and the inhibition of cytokine secretion were very similar. CONCLUSIONS: These results strongly support the idea that clodronate acts as a prodrug, the active form being its intracellular AppCCl2p metabolite.

Kinetic characteristics of norcocaine N-hydroxylation in mouse and human liver microsomes: involvement of CYP enzymes.

The first step in the oxidative metabolism of cocaine is N-demethylation to norcocaine, which is further N-hydroxylated to more toxic N-hydroxynorcocaine. In this study we examined the kinetics of norcocaine N-hydroxylation mediated by cytochrome P450 (CYP) in mouse and human liver microsomes. N-hydroxynorcocaine was identified by analytical HPLC-MS after incubation of norcocaine with mouse liver microsomes in the presence of NADPH. In mouse liver microsomes, there was no apparent difference in Km values for norcocaine N-hydroxylation between male and female microsomes, while the Vmax rate was approximately two times higher in female than in male microsomes (34+/-10 v. 16+/-4 pmol/min per mg protein). The Km value for norcocaine N-hydroxylation in human liver microsomes was approximately three times higher than that observed in comparable incubations using mouse liver microsomes, whereas the Vmax rate was ten times lower. Both cocaine and norcocaine induced type I difference spectra upon interaction with CYP in mouse liver microsomes. In contrast, in human microsomes both type I and type II spectra were recorded. In the 0.01 to 1 mM concentration range, cocaine and norcocaine inhibited mouse microsomal testosterone 6alpha-, 7alpha- and 16alpha-hydroxylation reactions by 20% to 30%. Testosterone 6beta- and 15alpha-hydroxylations were blocked by 60% and 50%, respectively, by 1 mM norcocaine, while only 40% inhibition was obtained with 1 mM cocaine. Coumarin 7-hydroxylation and pentoxyresorufin O-deethylation were inhibited by 50% by 1 and 0.4 mM norcocaine, respectively. In contrast, 10 and 2 mM cocaine, respectively, were needed to obtain the same degrees of inhibition. In human liver microsomes, 1 mM norcocaine and cocaine blocked testosterone 6beta-hydroxylase by 60% and 40%, respectively. Coumarin 7-hydroxylation was inhibited by only 30% by norcocaine (5.4 mM) and cocaine (10 mM). Norcocaine N-hydroxylation in mouse and human liver microsomes was blocked by 30% and 60%, respectively, by alpha-naphthoflavone (0.1 mM). The reaction was inhibited by 30-40% by metyrapone, cimetidine and gestodene at a concentration of 1 mM in mouse microsomes, while in human microsomes, 70% inhibition was obtained with 1 mM metyrapone and cimetidine. Taken together, these results indicate that (1) norcocaine N-hydroxylation is at least partly a CYP-mediated reaction, (2) the rate of reaction is considerably lower in human liver microsomes than in mouse liver microsomes and (3) several CYP subfamilies including 1A, 2A, 3A and possibly 2B may contribute to the formation of N-hydroxynorcocaine.

Differential regulation of stress proteins by high hydrostatic pressure, heat shock, and unbalanced calcium homeostasis in chondrocytic cells.

High hydrostatic pressure (HP) has recently been shown to increase cellular heat shock protein 70 (Hsp70) level in a specific way that does not involve transcriptional activation of the gene, but rather the stabilisation of the mRNA for Hsp70. In this study, we investigated whether there are other observable changes caused by HP stress, and compared them with those induced by certain other forms of stressors. A chondrocytic cell line T/C28a4 was exposed to 30 MPa continuous HP, heat shock at 43 degrees C, and increased cytosolic calcium concentration by the addition of sarco-endoplasmic reticulum Ca(2+) ATPase inhibitor thapsigargin (25 nM) or calcium ionophore A23187 (1 microM) in the cultures. The protein synthesis was studied by in vitro metabolic labelling followed by one- and two-dimensional polyacrylamide gel electrophoresis, and mass spectrometry was utilized to confirm the identity of the protein spots on two-dimensional gels. Continuous 30 MPa HP increased remarkably the relative labelling of Hsp70. Labelling of Hsp90 was also increased by 15-20%, although no clear change was evident at the protein level in Western blots. Elevated intracellular Ca(2+) concentration induced by thapsigargin and calcium ionophore A23187 increased mainly the synthesis of glucose-regulated protein 78 (Grp78/BiP), whereas Hsp70 and Hsp90 were decreased by the treatment. Heat shock was the strongest inducer of Hsp70 and Hsp90. This study further confirmed the induction of Hsp70 in chondrocytic cells exposed to high HP, but it also showed that calcium-mediated responses are unlikely to cause the stress response observed in the hydrostatically pressurized cells.

Analysis of an adenine nucleotide-containing metabolite of clodronate using ion pair high-performance liquid chromatography-electrospray ionisation mass spectrometry.

Clodronate belongs to the family of bisphosphonates, which are synthetic analogues of pyrophosphate. Bisphosphonates are widely used in the treatment of metabolic bone diseases. Some bisphosphonates, including clodronate, can be metabolized in cells into non-hydrolysable nucleotide analogues. In this paper, we describe a new method for extraction and quantitation of the clodronate metabolite in cell lysates by using ion-pairing HPLC method that is compatible with negative ion electrospray ionization mass spectrometry (ESI-MS). The method was used for detection of the metabolite of clodronate in extracts from RAW 264 macrophage cells after treatment with clodronate.

Enzymatic and permeation barrier of [D-Ala(2)]-Met-enkephalinamide in the anterior membranes of the albino rabbit eye.

Enzymatic and physical barrier properties of anterior ocular membranes were characterized. The permeation and metabolic degradation of [D-Ala(2)]-methionine enkephalinamide (DAMEA) in the albino rabbit cornea, conjunctiva and sclera were studied in vitro. DAMEA was administered with and without peptidase inhibitors bestatin (aminopeptidase inhibitor) and SCH 39370 (enkephalinase inhibitor). The modified Ussing chambers were used to study the peptide permeation and the samples were analyzed with a novel HPLC method using UV and EC detectors. Sclera was the most permeable membrane to DAMEA, while cornea was almost impermeable to DAMEA. Without inhibitors, the permeability coefficients of DAMEA were 2. 7x10(-8) cm/s, 3.1x10(-6) cm/s and 12.5x10(-6) cm/s in the cornea, conjunctiva and sclera, respectively. DAMEA was partly metabolized to tyrosine (Tyr) and tyrosine-D-alanine-glycine (Tyr-D-Ala-Gly). When inhibitors were co-administered with DAMEA, the corneal permeability of intact DAMEA increased 15 times, while conjunctival permeability increased 5.5 times and scleral permeability remained practically unaltered. The formation of metabolites decreased markedly, when the inhibitors were used. Interestingly, when the permeability of DAMEA was compared to permeabilities of polyethylene glycols in different membranes, the permeation was in the same range suggesting that DAMEA permeates through cornea via a paracellular pathway. Both enzymatic and physical barriers were more prominent in the cornea than in the conjunctiva and sclera. Non-corneal pathway of absorption and combined with inhibition of peptidases may be the most viable pathway for ocular peptide administration.