Quantification of nitrotyrosine in nitrated proteins

For kinetic studies of protein nitration reactions, we have developed a method for the quantification of nitrotyrosine residues in protein molecules by liquid chromatography coupled to a diode array detector of ultraviolet-visible absorption. Nitrated bovine serum albumin (BSA) and nitrated ovalbumin (OVA) were synthesized and used as standards for the determination of the protein nitration degree (ND), which is defined as the average number of nitrotyrosine residues divided by the total number of tyrosine residues in a protein molecule. The obtained calibration curves of the ratio of chromatographic peak areas of absorbance at 357 and at 280 nm vs. nitration degree are nearly the same for BSA and OVA (relative deviations <5%). They are near-linear at low ND (< 0.1) and can be described by a second-order polynomial fit up to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\hbox{ND}} = 0.5\left( {{R^2} > 0.99} \right)$$\end{document}. A change of chromatographic column led to changes in absolute peak areas but not in the peak area ratios and related calibration functions, which confirms the robustness of the analytical method. First results of laboratory experiments confirm that the method is applicable for the investigation of the reaction kinetics of protein nitration. The main advantage over alternative methods is that nitration degrees can be efficiently determined without hydrolysis or digestion of the investigated protein molecules

Comorbidity in patients with diabetes mellitus: impact on medical health care utilization

BACKGROUND: Comorbidity has been shown to intensify health care utilization and to increase medical care costs for patients with diabetes. However, most studies have been focused on one health care service, mainly hospital care, or limited their analyses to one additional comorbid disease, or the data were based on self-reported questionnaires instead of health care registration data. The purpose of this study is to estimate the effects a broad spectrum of of comorbidities on the type and volume of medical health care utilization of patients with diabetes. METHODS: By linking general practice and hospital based registrations in the Netherlands, data on comorbidity and health care utilization of patients with diabetes (n = 7,499) were obtained. Comorbidity was defined as diabetes-related comorbiiabetes-related comorbidity. Multilevel regression analyses were applied to estimate the effects of comorbidity on health care utilization. RESULTS: Our results show that both diabetes-related and non diabetes-related comorbidity increase the use of medical care substantially in patients with diabetes. Having both diabeterelated and non diabetes-related comorbidity incrases the demand for health care even more. Differences in health care utilization patterns were observed between the comorbidities. CONCLUSION: Non diabetes-related comorbidity increases the health care demand as much as diabetes-related comorbidity. Current single-disease approach of integrated diabetes care should be extended with additional care modules, which must be generic and include multiple diseases in order to meet the complex health care demands of patients with diabetes in the future

Impact of Cardiovascular Risk Factors on Carotid Intima-Media Thickness and Degree of Severity: A Cross-Sectional Study

OBJECTIVE: Age, hypertension, dyslipidemia and diabetes are common cardiovascular risk factors (CVRFs) that contribute to the development of atherosclerosis in cardiovascular system including carotid artery disease. However, the impact of these risk factors on the increased carotid intima-media thickness (cIMT) and degree of carotid severity remains to be further clarified. This study aims to evaluate the relationship between CVRFs and degree of carotid severity and cIMT in high-risk subjects. METHODS: Four thousand and three hundred ninety-four subjects with one or more risk factors were retrospectively reviewed in this study. Patients were divided into different groups based on age, the type and quantity of CVRFs. cIMT and degree of carotid artery stenosis were measured and analyzed based on carotid ultrasound imaging with findings compared to the CVRFs to determine the correlation between these variables. RESULTS: Aging was significantly associated with degree of severity (P &lt; 0.05) and cIMT was significantly increased with age (P &lt; 0.05). Individual CVRF analysis shows that hypertension was more related to the degree of severity than dyslipidemia and diabetes with corresponding abnormal cIMT rates being 79.39%, 72.98% and 32.37%, respectively. The prevalence of carotid atherosclerosis were 20.06%, 22.88% and 28.63%, respectively corresponding to patients with zero, one and more than one chronic diseases. The percentage of abnormal cIMT in hypertensive patient group with dyslipidemia is significantly higher than the other groups (P&lt; 0.05). CONCLUSIONS: This study shows a direct correlation between the degree of carotid severity and cIMT and cardiovascular risk factors, especially with age and hypertension. Carotid atherosclerosis is closely related to the number of cardiovascular risk factors

CD1a expression in psoriatic skin following treatment with propylthiouracil, an antithyroid thioureylene

BACKGROUND: The antithyroid thioureylenes, propylthiouracil (PTU) and methimazole (MMI), are effective in the treatment of patients with plaque psoriasis. The mechanism of action of the drugs in psoriasis is unknown. Since the drugs reduce circulating IL-12 levels in patients with Graves' hyperthyroidism, the effect of propylthiouracil on CD1a expression in psoriatic lesions was examined in biopsy samples of patients with plaque psoriasis. CD1a is a marker of differentiated skin antigen presenting cells (APC, Langerhans cells). Langerhans cells and skin monocyte/macrophages are the source of IL-12, a key cytokine involved in the events that lead to formation of the psoriatic plaque. METHODS: Biopsy specimens were obtained from six patients with plaque psoriasis who were treated with 300 mg propylthiouracil (PTU) daily for three months. Clinical response to PTU as assessed by PASI scores, histological changes after treatment, and CD1a expression in lesional skin before and after treatment were studied. RESULTS: Despite significant improvement in clinical and histological parameters the expression of CD1a staining cells in the epidermis did not decline with propylthiouracil treatment. CONCLUSIONS: It appears that the beneficial effect of propylthiouracil in psoriasis is mediated by mechanisms other than by depletion of skin antigen-presenting cells

Enhanced tonic GABAA inhibition in typical absence epilepsy

The cellular mechanisms underlying typical absence seizures, which characterize various idiopathic generalized epilepsies, are not fully understood, but impaired GABAergic inhibition remains an attractive hypothesis. In contrast, we show here that extrasynaptic GABAA receptor–dependent ‘tonic’ inhibition is increased in thalamocortical neurons from diverse genetic and pharmacological models of absence seizures. Increased tonic inhibition is due to compromised GABA uptake by the GABA transporter GAT–1 in the genetic models tested, and GAT–1 is critical in governing seizure genesis. Extrasynaptic GABAA receptors are a requirement for seizures in two of the best characterized models of absence epilepsy, and the selective activation of thalamic extrasynaptic GABAA receptors is sufficient to elicit both electrographic and behavioural correlates of seizures in normal animals. These results identify an apparently common cellular pathology in typical absence seizures that may have epileptogenic significance, and highlight novel therapeutic targets for the treatment of absence epilepsy.peer-reviewe

Infection efficiency of Phaeoisariopsis personata and the influence of different wetness patterns on germ-tube growth of the pathogen

Controlled environment studies with P. personata [Mycosphaerella berkeleyi], the causal agent of late leaf spot disease of groundnut, showed that infection is enhanced if leaves are exposed to alternate wet and dry periods (intermittent wetness) compared with continuous wetness. Detailed investigations to elucidate this phenomenon revealed more germ tubes per conidium and more branching of germ tubes with intermittent wetness than with continuous wetness. With intermittent wetness there was clear evidence of tropic growth of germ tubes and branches towards stomata and subsequent penetration. With continuous wetness, germ tube growth did not appear to be directional and germ tubes commonly passed over the stomatal guard cells, therefore leading to relatively few stomatal penetrations. For both wetness regimes, stomatal penetrations continued to increase with increased leaf wetness for at least 6 d after inoculation and there was a linear relationship between the number of stomatal penetrations and the number of resultant lesions. Infection efficiency was markedly increased when the spore load was reduced to 0.1 conidia/cm² (c. 1 spore/leaflet)

Comparative molecular biological analysis of membrane transport genes in organisms

Comparative analyses of membrane transport genes revealed many differences in the features of transport homeostasis in eight diverse organisms, ranging from bacteria to animals and plants. In bacteria, membrane-transport systems depend mainly on single genes encoding proteins involved in an ATP-dependent pump and secondary transport proteins that use H+ as a co-transport molecule. Animals are especially divergent in their channel genes, and plants have larger numbers of P-type ATPase and secondary active transporters than do other organisms. The secondary transporter genes have diverged evolutionarily in both animals and plants for different co-transporter molecules. Animals use Na+ ions for the formation of concentration gradients across plasma membranes, dependent on secondary active transporters and on membrane voltages that in turn are dependent on ion transport regulation systems. Plants use H+ ions pooled in vacuoles and the apoplast to transport various substances; these proton gradients are also dependent on secondary active transporters. We also compared the numbers of membrane transporter genes in Arabidopsis and rice. Although many transporter genes are similar in these plants, Arabidopsis has a more diverse array of genes for multi-efflux transport and for response to stress signals, and rice has more secondary transporter genes for carbohydrate and nutrient transport