Is nitrotyrosine generated in human erythrocytes in circulation?

Nitrotyrosine is considered a stable biomarker of reactive nitrogen species, including nitrogen dioxide (NO2) and peroxynitrous acid (ONOOH) in biomaterials. There are inconsistent observations on the detection of free and protein-associated nitrotyrosine in normal human plasma. Human erythrocytes, differentiated from erythrocyte precursor cells in the bone marrow, circulating in the body for an average of 120 d, and finally removed by spleen macrophages, may be exposed to reactive nitrogen species. In the present study, membrane proteins and hemoglobin from the senescent erythrocyte population were compared with those from young erythrocytes separated from the same individuals in their nitrotyrosine presence using newly prepared rabbit polyclonal anti-nitrotyrosine-ribonuclease A and anti-nitro(N-butoxycarbonyl)tyrosine-bovine serum albumin antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the membranes and hemoglobin, and subsequent Western blot analysis, showed that these antibodies only slightly bind to the bands of the proteins from both young and senescent erythrocytes, whereas these antibodies definitely bind to the protein bands of membranes and hemoglobin nitrated by NO2 or ONOOH in vitro. This result indicates that nitrotyrosine is not detected in the membrane proteins and hemoglobin in human normal erythrocytes in circulation. However, this does not conclude that erythrocytes are not exposed to reactive nitrogen species in the circulation.     

Clomipramine N-demethylation metabolism in human liver microsomes

目的：研究ＣＹＰ４５０选择性抑制剂对体外氯米帕明（Ｃｌｏ）Ｎ去甲基代谢的影响．方法：应用米氏方程计算肝微粒体中ＣｌｏＮ去甲基代谢的动力学参数，比较加入抑制剂前后这些参数的改变．结果：Ｋｍ１，Ｋｍ２，Ｖｍａｘ１，Ｖｍａｘ２，Ｖｍａｘ１／Ｋｍ１和Ｖｍａｘ２／Ｋｍ２分别为（０１１±００６），（２４±１１）μｍｏｌ·Ｌ－１，（１１４±４７），（４２８±１８８）ｎｍｏｌ·ｇ－１·ｍｉｎ－１，（１８±１６）和（００１９±０００５）Ｌ·ｇ－１·ｍｉｎ－１．后四个参数的个体差异分别可达２５，７３，３４和１８倍．二硫卡钠（Ｄｉｔ）、美芬妥英（Ｍｅｐ）、呋拉茶碱（Ｆｕｒ）、醋竹桃霉素（Ｔｒｏ）和Ｆｕｒ＋Ｔｒｏ的抑制率分别为２７０％，３２９％，６３９％，６６４％和７８３％．Ｔｒｏ和Ｆｕｒ的ＩＣ５０分别为４２１和２７７μｍｏｌ·Ｌ－１．结论：人肝微粒体中存在高、低亲合力ＣｌｏＮ脱甲基酶．在低浓度下，ＣｌｏＮ去甲基代谢主要由ＣＹＰ３Ａ４和ＣＹＰ１Ａ２催化，其次由ＣＹＰ２Ｃ１９介导．     

Metabolism of α-thujone in human hepatic preparations in vitro

1. This study aims to characterize the metabolism of α-thujone in human liver preparations in vitro and to identify the role of cytochrome P450 (CYP) and possibly other enzymes catalyzing α-thujone biotransformations.

2. With a liquid chromatography–mass spectrometry (LC-MS) method developed for measuring α-thujone and four potential metabolites, it was demonstrated that human liver microsomes produced two major (7- and 4-hydroxy-thujone) and two minor (2-hydroxy-thujone and carvacrol) metabolites. Glutathione and cysteine conjugates were detected in human liver homogenates, but not quantified. No glucuronide or sulphate conjugates were detected. Major hydroxylations accounted for more than 90% of the primary microsomal metabolism of α-thujone.

3. Screening of α-thujone metabolism with CYP recombinant enzymes indicated that CYP2A6 was principally responsible for the major 7- and 4-hydroxylation reactions, although CYP3A4 and CYP2B6 participated to a lesser extent and CYP3A4 and CYP2B6 catalyzed minor 2-hydroxylation. Based on the intrinsic efficiencies of different recombinant CYP enzymes and average abundances of these enzymes in human liver microsomes, CYP2A6 was calculated to be the most active enzyme in human liver microsomes, responsible for 70–80% of the metabolism on average.

4. Inhibition screening indicated that α-thujone inhibited both CYP2A6 and CYP2B6, with 50% inhibitory concentration values of 15.4 and 17.5 µM, respectively.

     

Metabolism of α-thujone in human hepatic preparations in vitro

This study aims to characterize the metabolism of α-thujone in human liver preparations in vitro and to identify the role of cytochrome P450 (CYP) and possibly other enzymes catalyzing α-thujone biotransformations. With a liquid chromatography-mass spectrometry (LC-MS) method developed for measuring α-thujone and four potential metabolites, it was demonstrated that human liver microsomes produced two major (7- and 4-hydroxy-thujone) and two minor (2-hydroxy-thujone and carvacrol) metabolites. Glutathione and cysteine conjugates were detected in human liver homogenates, but not quantified. No glucuronide or sulphate conjugates were detected. Major hydroxylations accounted for more than 90% of the primary microsomal metabolism of α-thujone. Screening of α-thujone metabolism with CYP recombinant enzymes indicated that CYP2A6 was principally responsible for the major 7- and 4-hydroxylation reactions, although CYP3A4 and CYP2B6 participated to a lesser extent and CYP3A4 and CYP2B6 catalyzed minor 2-hydroxylation. Based on the intrinsic efficiencies of different recombinant CYP enzymes and average abundances of these enzymes in human liver microsomes, CYP2A6 was calculated to be the most active enzyme in human liver microsomes, responsible for 70-80% of the metabolism on average. Inhibition screening indicated that α-thujone inhibited both CYP2A6 and CYP2B6, with 50% inhibitory concentration values of 15.4 and 17.5 μM, respectively.     

Nitric oxide synthase in the human airway epithelium

ＮｉｔｒｉｃｏｘｉｄｅｓｙｎｔｈａｓｅｉｎｔｈｅｈｕｍａｎａｉｒｗａｙｅｐｉｔｈｅｌｉｕｍＲａｅｄＡＤＷＥＩＫ１，ＦｕｈｕａＨＧＵＯ１，２，ＫｏｈｓａｋｕＵＥＴＡＮＩ１，２，ＳｅｒｐｉｌＣＥＲＺＵＲＵＭ１，２（１ＤｅｐａｒｔｍｅｎｔｓｏｆＰｕｌｍｏｎ...     

Effect of selected antioxidants in β-cyfluthrin-induced oxidative stress in human erythrocytes in vitro

β-Cyfluthrin is one of the most widely used type II pyrethroid in agriculture. The aim of this study was to examine (1) the possibility of β-cyfluthrin to induce oxidative stress in human erythrocytes in vitro and its effect on catalase (CAT) and superoxide dismutase (SOD) activities as well as (2) the role of melatonin (MEL; 2 mM), its precursor – N-acetylserotonin (NAS; 1 mM), quercetin (Q; 80 μM) and rutin (R; 80 μM) in alleviating the cytotoxic effects of β-cyfluthrin. Erythrocytes were divided into portions. The first portion was incubated for 4 h at 37 °C with different concentrations (0, 43, 215, 1075 ppm) of β-cyfluthrin. The other portions were preincubated with selected antioxidant, respectively for 30 min and followed by β-cyfluthrin incubation for 4 h. Malondialdehyde (MDA) concentrations, CAT and SOD activities, as well as haemolysis percentage (H) were measured in all treatment portions of erythrocytes.It could be concluded that the in vitro toxicity of β-cyfluthrin may be associated with oxidative stress. Significant reduction in the activities of CAT was observed at all β-cyfluthrin concentrations, while SOD activities were significantly decreased only in erythrocytes incubated with the highest β-cyfluthrin concentration. SOD activity of the non-pretreated erythrocytes exposed to the lowest dose of β-cyfluthrin was significantly greater when compared to comparably β-cyfluthrin-exposed antioxidant pretreated cells. The highest concentration of β-cyfluthrin has caused over 35% haemolysis, and the lowest concentration about 15%. MEL pretreatment had no effect on H and MDA induction by β-cyfluthrin. NAS, Q and R reduced H and MDA level, but could not prevent induction of these parameters.Compared to other antioxidants NAS appeared to maintain better the CAT activity at control levels for all doses of β-cyfluthrin. Pretreatment with Q was found to protect against the decrease in SOD activity induced by β-cyfluthrin.     

The influence of pyridostigmine administration on human neuromuscular functions—Studies in healthy human subjects

Pyridostigmine has a protective effect against organophosphate poisoning when given in a dosage of 30 mg three times daily causing 20–40% cholinesterase inhibition. To test its safety in the human neuromuscular system, a double-blind study on 35 subjects divided into two matched groups was performed. One group was treated with pyridostigmine in a dose of 30 mg three times daily and the other group was treated similarly with placebo, both for a 10-day period. The resultant average cholinesterase inhibition in the treatment group was 23%. Muscle strength and endurance were tested before, during (on the 8th day), and after treatment. Electrodiagnostic studies, including nerve conduction, electromyography, and response to repetitive stimulation, were carried out on four subjects of the treatment group and on two subjects of the placebo group, both before and during treatment (eighth day). Isometric handgrip strength, isokinetic elbow flexor, and extensor strength did not differ between groups as a result of the treatment. Knee flexor and extensor isokinetic strength showed a small (but statistically significant) trend to improve more during placebo treatment, whereas knee extensor endurance decreased slightly in the placebo group. Both these effects are probably due to large fluctuations in performance of the placebo group, whereas the treatment group performance was quite constant. They probably do not represent any adverse effect of pyridostigmine. No electrophysiological changes were found in any of the subjects during treatment. We conclude that pyridostigmine does not cause any significant neuromuscular effect in healthy subjects when taken in a dosage of 90 mg daily for 8 days, causing 20–30% inhibition of cholinesterase.     

Thioredoxin: friend or foe in human disease?

Thioredoxin (Trx), a small, ubiquitous thiol [sulfydryl (-SH)] protein, is one of the most important regulators of reduction-oxidation (redox) balance and, thus, redox-controlled cell functions. Although Trx was discovered 40 years ago in bacteria, the number and diversity of processes that Trx influences in human cells have only been appreciated recently. Processes influenced by Trx include the control of cellular redox balance, the promotion of cell growth, the inhibition of apoptosis and the modulation of inflammation. Not surprisingly, the role of Trx in a wide range of human diseases and conditions, including cancer, viral disease, ischaemia-reperfusion injury, cardiac conditions, aging, premature birth and newborn physiology, is subject to intense investigation. However, whether Trx contributes to or prevents the pathology of a particular condition is not always clear. In this article, we review the role of Trx in human disease and relate this to its redox activity and biological properties, and discuss the development and use of therapies that either inhibit or augment Trx activity.     

Kynurenic acid in human saliva--does it influence oral microflora?

Kynurenic acid (KYNA) is an endogenous antagonist of alpha7 nicotinic receptors and all ionotropic glutamate receptors. Its neuroprotective activity has been suggested. In this study, the presence of KYNAin human saliva and its potential bactericidal role was investigated. KYNAwas found in all samples of human saliva with mean concentration of 3.4 nM. The concentration of KYNA in saliva obtained from patients with odontogenic abscesses was 3.5 times higher than in healthy subjects. We have shown that the human gingival fibroblasts produce KYNAand an inflammatory stimulant, lipopolysaccharide, enhanced its synthesis in vitro. The bactericidal effect of KYNA was also presented. We hypothesize that KYNA may contribute to the control of oral microflora.     

Oral administration of human recombinant interferon-α2 in rats

The absorption of human recombinant interferon-α₂ (IFNα₂) from the oropharynx has been investigated in the rat. We have shown that small amounts of interferon can be absorbed and measured in blood. The absorption of IFNα₂ dissolved in saline was not improved by the addition of different promoters. The practical usefulness of a small absorption of interferon has been discussed.     

Anti-neoplastic agent thymoquinone induces degradation of α and β tubulin proteins in human cancer cells without affecting their level in normal human fibroblasts

The microtubule-targeting agents derived from natural products, such as vinca-alkaloids and taxanes are an important family of efficient anti-cancer drugs with therapeutic benefits in both haematological and solid tumors. These drugs interfere with the assembly of microtubules of α/β tubulin heterodimers without altering their expression level. The aim of the present study was to investigate the effect of thymoquinone (TQ), a natural product present in black cumin seed oil known to exhibit putative anti-cancer activities, on α/β tubulin expression in human astrocytoma cells (cell line U87, solid tumor model) and in Jurkat cells (T lymphoblastic leukaemia cells). TQ induced a concentration- and time-dependent degradation of α/β tubulin in both cancer cell types. This degradation was associated with the up-regulation of the tumor suppressor p73 with subsequent induction of apoptosis. Interestingly, TQ had no effect on α/β tubulin protein expression in normal human fibroblast cells, which were used as a non-cancerous cell model. These data indicate that TQ exerts a selective effect towards α/β tubulin in cancer cells. In conclusion, the present findings indicate that TQ is a novel anti-microtubule drug which targets the level of α/β tubulin proteins in cancer cells. Furthermore, they highlight the interest of developing anti-cancer therapies that target directly tubulin rather than microtubules dynamics.     

Melanocortin MC₁ receptor in human genetics and model systems

The melanocortin MC(1) receptor is a G-protein coupled receptor expressed in the melanocytes of the skin and hair and is known for its key role in the regulation of human pigmentation. Melanocortin MC(1) receptor activation after ultraviolet radiation exposure results in a switch from the red/yellow pheomelanin to the brown/black eumelanin pigment synthesis within cutaneous melanocytes; this pigment is then transferred to the surrounding keratinocytes of the skin. The increase in melanin maturation and uptake results in tanning of the skin, providing a physical protection of skin cells from ultraviolet radiation induced DNA damage. Melanocortin MC(1) receptor polymorphism is widespread within the Caucasian population and some variant alleles are associated with red hair colour, fair skin, poor tanning and increased risk of skin cancer. Here we will discuss the use of mouse coat colour models, human genetic association studies, and in vitro cell culture studies to determine the complex functions of the melanocortin MC(1) receptor and the molecular mechanisms underlying the association between melanocortin MC(1) receptor variant alleles and the red hair colour phenotype. Recent research indicates that melanocortin MC(1) receptor has many non-pigmentary functions, and that the increased risk of skin cancer conferred by melanocortin MC(1) receptor variant alleles is to some extent independent of pigmentation phenotypes. The use of new transgenic mouse models, the study of novel melanocortin MC(1) receptor response genes and the use of more advanced human skin models such as 3D skin reconstruction may provide key elements in understanding the pharmacogenetics of human melanocortin MC(1) receptor polymorphism.     

Endocannabinoids modulate human blood–brain barrier permeability in vitro

Background and Purpose

Endocannabinoids alter permeability at various epithelial barriers, and cannabinoid receptors and endocannabinoid levels are elevated by stroke, with potential neuroprotective effects. We therefore explored the role of endocannabinoids in modulating blood–brain barrier (BBB) permeability in normal conditions and in an ischaemia/reperfusion model.

Experimental Approach

Human brain microvascular endothelial cell and astrocyte co-cultures modelled the BBB. Ischaemia was modelled by oxygen-glucose deprivation (OGD) and permeability was measured by transepithelial electrical resistance. Endocannabinoids or endocannabinoid-like compounds were assessed for their ability to modulate baseline permeability or OGD-induced hyperpermeability. Target sites of action were investigated using receptor antagonists and subsequently identified with real-time PCR.

Key Results

Anandamide (10 μM) and oleoylethanolamide (OEA, 10 μM) decreased BBB permeability (i.e. increased resistance). This was mediated by cannabinoid CB₂ receptors, transient receptor potential vanilloid 1 (TRPV1) channels, calcitonin gene-regulated peptide (CGRP) receptor (anandamide only) and PPARα (OEA only). Application of OEA, palmitoylethanolamide (both PPARα mediated) or virodhamine (all 10 μM) decreased the OGD-induced increase in permeability during reperfusion. 2-Arachidonoyl glycerol, noladin ether and oleamide did not affect BBB permeability in normal or OGD conditions. N-arachidonoyl-dopamine increased permeability through a cytotoxic mechanism. PPARα and γ, CB₁ receptors, TRPV1 channels and CGRP receptors were expressed in both cell types, but mRNA for CB₂ receptors was only present in astrocytes.

Conclusion and Implication

The endocannabinoids may play an important modulatory role in normal BBB physiology, and also afford protection to the BBB during ischaemic stroke, through a number of target sites.     

Does hepatomegaly alter iron-dependent oxidative effects in human plasma?

A four-fold increase in the iron content of normal subjects was detected in plasma after 5 hours of iron administration. Iron supplementation had a surprisingly erratic effect on four patients with hepatomegaly secondary to heart insufficiency, since the increase in the iron content in the plasma after iron-dextran administration was either within the control range or significantly lower, independently of the initial values. Thiobarbituric acid reactive substances (TBARS) content was 2.5 +/- 0.2 and 4.3 +/- 0.4 microM for control and hepatomegalic subjects, respectively. The TBARS basal level was increased by iron supplementation. The difference between TBARS content in hepatomegalic and control subjects, after 5 hours of iron administration, was increased by 50% as compared to the difference in the basal content of TBARS. alpha-Tocopherol (alpha-T) content in plasma from subjects with hepatomegaly showed a significant decrease (-41%) as compared to control subjects. No significant difference over the basal level of alpha-T was measured after 5 hours of iron administration in any subject. The data presented here suggest that abnormal liver condition affects iron-dependent oxidative stress in plasma. Moreover, alpha-T does not seem to be the main antioxidant to control iron-dependent oxidative stress in plasma.     

Enzyme kinetic analysis and inhibition of amitriptyline N-demethylation in human liver microsomes in vitro

采用６例人的肝微粒体应用酶促动力学分析和抑制研究阐明参与阿米替林（ＡＴ）Ｎ－去甲基代谢的ＣＹＰ４５０的种类及性质．去甲替林（ＮＴ）生成的酶促动力学数据符合一两酶模型，其中高亲和力酶具有Ｍｉｃｈａｅｌｉｓ－Ｍｅｎｔｅｎ动力学特征，而低亲和力酶则具有底物别构激活的特性．当ＡＴ为２μｍｏｌ·Ｌ－１时Ｓ－美芬妥英和呋喃茶碱均可使ＮＴ生成被抑制达５０％左右；较高浓度的酮康唑也可使ＮＴ生成明显受到抑制，但醋竹桃霉素几乎对此没有影响；而当ＡＴ为１００μｍｏｌ·Ｌ－１时，酮康唑和醋竹桃霉素均是ＮＴ生成的强抑制剂．结果提示：当底物的浓度较低时，ＣＹＰ１Ａ２和ＣＹＰ２Ｃ１９是催化ＡＴ体外人肝微粒体中Ｎ－去甲基代谢的主要ＣＹＰ４５０酶类；当底物浓度较高时，由于底物别构激活的特性，ＣＹＰ３Ａ４逐渐占据主导地位．     

Characteristics of transient outward K~ current in human atrial cardiomyocytes

目的：研究人的心房肌细胞瞬间外向钾电流（Ｉｔｏ）的特性．方法：采用膜片箝全细胞记录法观察人心房肌通道电流的变化，在用ＣｄＣｌ２０１ｍｍｏｌ·Ｌ－１阻断钙电流的情况下，细胞膜去极化引出瞬间外向钾电流（Ｉｔｏ）．结果：Ｉｔｏ为电压依赖的电流，迅速激活，迅速失活．４ＡＰ（４氨基吡啶）１０ｍｍｏｌ·Ｌ－１（选择性的Ｉｔｏ的阻断剂）能完全阻断此电流．其ＩＣ５０值为０６７ｍｍｏｌ·Ｌ－１．４ＡＰ１ｍｍｏｌ·Ｌ－１能使Ｉｔｏ的激活曲线明显向正电位移动，使Ｉｔｏ的激活电压提高，且较难激活Ｉｔｏ．结论：在人心房肌细胞，Ｉｔｏ是一种主要的钾电流，它可被４ＡＰ阻断     

Pain-like behaviours in animals - how human are they?

The use of genetically manipulated animals in conjunction with classical physiological and biochemical measurement has unravelled many pathological changes in animal models of chronic pain that bear some striking similarities to those described in several chronic pain conditions in humans. In this article, I highlight several limitations in the validation of animal models of chronic pain and the methods that are used for assessing pain-like behaviours in these models. Alternative methods for assessing pain and stress in animals, which might better reflect the diverse symptomotology of chronic pain in humans, are proposed.