Hydrolysis of homocysteine thiolactone results in the formation of Protein-Cys-S-S-homocysteinylation

An increased level of homocysteine, a reactive thiol amino acid, is associated with several complex disorders and is an independent risk factor for cardiovascular disease. A majority (>80%) of circulating homocysteine is protein bound. Homocysteine exclusively binds to protein cysteine residues via thiol disulfide exchange reaction, the mechanism of which has been reported. In contrast, homocysteine thiolactone, the cyclic thioester of homocysteine, is believed to exclusively bind to the primary amine group of lysine residue leading to N-homocysteinylation of proteins and hence studies on binding of homocysteine thiolactone to proteins thus far have only focused on N-homocysteinylation. Although it is known that homocysteine thiolactone can hydrolyze to homocysteine at physiological pH, surprisingly the extent of S-homocysteinylation during the exposure of homocysteine thiolactone with proteins has never been looked into. In this study, we clearly show that the hydrolysis of homocysteine thiolactone is pH dependent, and at physiological pH, 1 mM homocysteine thiolactone is hydrolysed to ~0.71 mM homocysteine within 24 h. Using albumin, we also show that incubation of HTL with albumin leads to a greater proportion of S-homocysteinylation (0.41 mol/mol of albumin) than N-homocysteinylation (0.14 mol/mol of albumin). S-homocysteinylation at Cys³⁴ of HSA on treatment with homocysteine thiolactone was confirmed using LC-MS. Further, contrary to earlier reports, our results indicate that there is no cross talk between the cysteine attached to Cys³⁴ of albumin and homocysteine attached to lysine residues.     

Predictors of major infections in systemic lupus erythematosus

Introduction  

Infections commonly complicate the course of systemic lupus erythematosus (SLE). Our aim is to investigate the clinical predictors of major infections in patients with SLE.     

Clinical and Biochemical Correlates of Serum L-Ergothioneine Concentrations in Community-Dwelling Middle-Aged and Older Adults

Background

Despite the increasing interest towards the biological role of L-ergothioneine, little is known about the serum concentrations of this unusual aminothiol in older adults. We addressed this issue in a representative sample of community-dwelling middle-aged and older adults.

Methods

Body mass index, estimated glomerular filtration rate, serum concentrations of L-ergothioneine, taurine, homocysteine, cysteine, glutathione, cysteinylglycine, and glutamylcysteine were evaluated in 439 subjects (age 55–85 years) randomly selected from the Hunter Community Study.

Results

Median L-ergothioneine concentration in the entire cohort was 1.01 IQR 0.78–1.33 µmol/L. Concentrations were not affected by gender (P = 0.41) or by presence of chronic medical conditions (P = 0.15). By considering only healthy subjects, we defined a reference interval for L-ergothioneine serum concentrations from 0.36 (90% CI 0.31–0.44) to 3.08 (90% CI 2.45–3.76) µmol/L. Using stepwise multiple linear regression analysis L-ergothioneine was negatively correlated with age (rpartial = −0.15; P = 0.0018) and with glutamylcysteine concentrations (rpartial = −0.13; P = 0.0063).

Conclusions

A thorough analysis of serum L-ergothioneine concentrations was performed in a large group of community-dwelling middle-aged and older adults. Reference intervals were established. Age and glutamylcysteine were independently negatively associated with L-ergothioneine serum concentration.     

Regulatory T cells in health and disease

Regulatory T (Treg) cells have emerged as a central control point in the modulation of various immune responses, including autoimmune responses and immunity to transplants, allergens, tumors, and infectious microbes. The immune system has evolved complex processes to ensure tolerance to autoantigens while preserving the potential to mount and maintain life-long humoral and cellular immune responses against invading pathogens. In this review, we summarize research showing that naturally occurring (nTreg) and induced Treg (iTreg) cell subsets, and in particular CD4+Foxp3+ Treg cells, are critical in the control of immune responses in rodents and humans. We also discuss the cellular and molecular factors that affect CD4+Foxp3+ Treg cell development, homeostasis, and function and consequential immunity to self and non-self antigens. Recent studies have shed light in our understanding of the development of novel methods of autoimmune disease prevention and treatment via enhancing and re-establishing Treg-mediated dominant control over self-reactive T cells in animal models and humans.     

Cancer risk assessment for 1,3-butadiene: dose-response modeling from an epidemiological perspective

The dose-response assessment of the association between 1,3-butadiene (BD) and leukemia mortality among workers in the North American synthetic rubber industry is explored. Analyses are based on the most recent University of Alabama at Birmingham epidemiological study and exposure estimation. The U.S. EPA Science Advisory Board recommendations of using the most recent data and giving consideration to peak exposures to BD have been followed. If cumulative BD ppm-years is to be used as the predictor of the leukemia rate ratio, then the performance of that predictor is statistically significantly improved if the slope in the predictor is estimated with age and the cumulative number of BD peaks (where a BD peak is any exposure, regardless of duration, to a BD concentration above 100 ppm) added as categorical covariates. After age and the cumulative number of BD peaks are incorporated as categorical covariates in the Poisson regression model, the estimated concentration (EC(001)) corresponding to an excess risk of 0.001 as a result of continuous environmental exposure is 11.2 ppm; however, the estimated slope for BD cumulative ppm-years in the linear rate ratio for leukemia used to derive this EC(001) is not statistically significantly different from zero. Sensitivity analyses using alternative models indicate either essentially no risk or estimated EC(001) values of 9 and 77 ppm. Analyses suggesting the absence of a statistically significant low-dose risk versus cumulative BD ppm-years are presented. Sensitivity analyses of other malignant neoplasms of lymphatic and hematopoietic tissue (specifically, lymphoid and myeloid neoplasms) resulted in conclusions about the dose-response modeling methodology that were supportive of the methodology used for leukemia.     

The evaluation of the oxidative state of native-LDL: three methods compared

LDL-oxidation is considered a contributing factor to the development of atherosclerotic lesions. However, to utilise the oxidative state of LDL as a marker of cardiovascular risk, reliable analytical methods for its detection must be defined. We have compared three methods for their capacity to evaluate the difference in the oxidation state of isolated LDL subjected to either dialysis (D-LDL) or gel filtration (F-LDL) to remove EDTA. Their susceptibility to oxidation promoted by Cu(2+) was monitored by following the time course of conjugated diene (CD) and lipid hydroperoxide (ROOH) accumulation. The relative electrophoretic mobility (REM) of the same LDL samples was evaluated by capillary electrophoresis. As measured by all three methods, F-LDL are less prone to oxidation than D-LDL when added with CuSO(4). REM of F-LDL and D-LDL significantly differs already before the addition of the metal catalyst, whereas CD and ROOH contents become significantly different only after it. Besides confirming that a rapid centrifugation followed by gel filtration is a more convenient procedure than dialysis to remove EDTA during LDL isolation, our study suggests the REM of isolated-LDL as the biochemical marker of choice in the evaluation of its oxidative state.     

Immunohistochemical detection of [corrected] TrkB in the enteric nervous system of the small intestine in pigeon (Columba livia)

The presence and cell localization of TrkB, the main receptor for the neurotrophins (NTs), was investigated immunohistochemically in the small intestine of adult pigeons, with special reference to the enteric nervous system (ENS). Several neuronal (neurofilament proteins and PGP 9.5) and glial cell (S100 protein) markers were studied in parallel. TrkB immunoreactivity (TrkB-IR) was found to be restricted to immunohistochemically-identified glial cells present in the enteric plexuses, and to Schwann cells forming the perivascular plexus. Also, TrkB-IR was detected in enterochromaffin cells and in unidentified dendritic cells within the gut-associated lymphoid tissue. The present results demonstrate that as for mammals, TrkB in the ENS is restricted to the glial cells. The possible function of the TrkB ligands, however, remains to be established.     

Optimization of ascorbic and uric acid separation in human plasma by free zone capillary electrophoresis ultraviolet detection

In this paper we propose a new fast free zone capillary electrophoresis method for the simultaneous determination of ascorbic acid (AA) and uric acid (UA) in human plasma. We investigated the effect of analytical parameters, such as concentration and pH of borate running buffer, cartridge temperature, and sample treatment, on resolution, migration times, corrected peak areas, and efficiency. A good separation was achieved using a 60.2-cmx75-microm uncoated silica capillary and 100 mmol/L sodium borate buffer, pH 8, when metaphosphoric acid was employed as protein precipitant, in less than 4 min. These conditions gave a good reproducibility of migration times (CV 0.35 and 0.34%) and peak areas (CV 3.2 and 3.1%) for ascorbate and urate, respectively. The limit of detection was 0.5mg/L for both analytes when the detection was performed at 254 nm for AA and at 292 nm for UA. We compared the present method with a validated capillary electrophoresis assay by measuring plasma urate and ascorbate in 32 normal subjects and the obtained data were analyzed by the Passing and Bablok regression.