Quantitation of protein S-glutathionylation by liquid chromatography–tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

Protein S-glutathionylation is a posttranslational modification that links oxidative stimuli to reversible changes in cellular function. Protein–glutathione mixed disulfide (PSSG) is commonly quantified by reduction of the disulfide and detection of the resultant glutathione species. This methodology is susceptible to contamination by free unreacted cellular glutathione (GSH) species, which are present in 1000-fold greater concentration. A liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based method was developed for quantification of glutathione and glutathione disulfide (GSSG), which was used for the determination of PSSG in biological samples. Analysis of rat liver samples demonstrated that GSH and GSSG coprecipitated with proteins similar to the range for PSSG in the sample. The use of [¹³C₂,⁵N]GSH and [¹³C₄,⁵N₂]GSSG validated these results and demonstrated that the release of GSH from PSSG did not occur during sample preparation and analysis. These data demonstrate that GSH and GSSG contamination must be accounted for when determining PSSG content in cellular/tissue preparations. A protocol for rinsing samples to remove the adventitious glutathione species is demonstrated. The fragmentation patterns for glutathione were determined by high-resolution mass spectrometry, and candidate ions for detection of PSSG on protein and protein fragments were identified.     

Glutathione metabolism of the erythrocyte. The enzymic cleavage of glutathione–haemoglobin preparations by glutathione reductase

A complex of haemoglobin and GSH was prepared by incubating haemoglobin with GSH and acetylphenylhydrazine. GSH could be released from the crude preparation by incubation with NADPH. However, when the haemoglobin preparation was separated from glutathione reductase by DEAE-Sephadex chromatography, NADPH no longer released GSH. Rather, the addition of a combination of either partially purified human erythrocyte or crystalline glutathione reductase and NADPH was required to release GSH from the haemoglobin-GSH complex. This complex is commonly believed to represent a mixed disulphide of GSH and the cysteine-beta-93 thiol group. This interpretation was supported by the finding that prior alkylation of available haemoglobin thiol groups prevented the formation of the complex. By using haemoglobin-[(35)S]GSH complex as a substrate, it was shown that GSH itself released the radioactivity from the complex only very slowly. In contrast, the release of [(35)S]GSH was very rapid in the presence of NADPH and glutathione reductase. This suggests that the cleavage of the haemoglobin-GSH complex is not mediated by GSH with cyclic reduction of GSSG formed, but rather proceeds enzymically through glutathione reductase.     

MLPA: A prenatal diagnostic tool for the study of congenital heart defects?

Congenital heart defects (CHD) represent the most common birth defects, so they are not a rare finding when performing routine ultrasound examinations during pregnancy. Once chromosome abnormalities have been excluded in a fetus with a CHD, chromosome 22q11.2 deletion is usually investigated by FISH, as it is the most frequent microdeletion syndrome and is generally associated with cardiac malformations. If 22q11.2 microdeletion is ruled out, the etiology of the CHD remains generally unexplained, making familial genetic counseling difficult. To evaluate the usefulness of Multiplex Ligation-dependent Probe Amplification (MLPA) kits designed for the study of 22q11.2 and other genomic regions previously associated with syndromic CHD, we performed MLPA in 55 pregnancies with fetuses presenting CHD, normal karyotype and negative FISH results for 22q11.2 microdeletion, which constitutes the largest prenatal series reported. Definitive MLPA results were obtained in 50 pregnancies, and in this setting such MLPA kits did not detect any imbalance. On the other hand, to compare FISH and MLPA techniques for the study of 22q11.2 microdeletions, we performed MLPA in 4 pregnancies known to have 22q11.2 deletions (by FISH). All four 22q11.2 microdeletions were also detected by MLPA, which corroborates that it is a reliable technique for the diagnosis and characterization of 22q11.2 deletions. Finally, we evaluated the possibility of replacing conventional FISH by MLPA for the prenatal diagnosis of CHD, comparing the diagnostic potential, results delivery times, repetition and failure rates and cost of both techniques, and concluded that FISH should still be the technique of choice for the prenatal diagnosis of fetuses with CHD.     

A plea for the ‘de-migranticization' of research on migration and integration1

ABSTRACT

Migration and integration research has been institutionalized over the last few decades. However, an increasing number of voices has been calling for more reflexivity, criticizing the nation-state- and ethnicity-centred epistemology that often informs this discipline. Consistently with this line of reasoning, I argue that migration and integration research originates in a historically institutionalized nation-state migration apparatus and is thus entangled with a particular normalization discourse. Therefore, this field of study contributes to reproducing the categories of this particular migration apparatus. This entanglement poses some serious dilemmas for this research tradition, dilemmas that ask for further consideration and possible solutions. My main proposition is to ‘de-migranticize' migration and integration research. I outline possible ways of doing so and discuss the consequences of such a strategy for the future of migration and integration studies.     

Direct evidence for inter-organ transport of glutathione and that the non-filtration renal mechanism for glutathione utilization involves γ-glutamyl transpeptidase

A direct examination of the inter-organ cycle of glutathione metabolism was made by determining glutathione levels in plasma obtained from various blood vessels of the rat. High levels of GSH were found in hepatic vein plasma, relative to arterial and systemic venous levels, reflecting translocation of GSH from the liver to the plasma. Renal vein plasma has a level that is 20% of arterial plasma indicating that the kidney removes glutathione from plasma not only by glomerular filtration (which can account for 20–30% of the glutathione removed), but also by a non-filtration mechanism. Inhibitors of γ-glutamyl transpeptidase decrease the fraction of glutathione removed by the kidney to a value approaching that filtered, indicating that the non-filtration mechanism involves γ-glutamyl transpeptidase.     

Applying GPS to the study of primate ecology: A useful tool?

Data on the spatiotemporal distribution of resources can be collected and plotted using GPS (global positioning system) and GIS (geographical information system) technologies. By combining such data with information on foraging and ranging behavior of nonhuman primates, one can analyze the influence of resource distribution on social organization and group cohesion. We investigated the abilities of a three-channel GPS receiver to collect location data under varying canopy densities in both temperate and tropical forests. Eighty randomly selected points were sampled in a beech–maple forest in northeast Ohio, USA; 65 points also were sampled at several tropical forests in Costa Rica and Trinidad. At each point we attempted to obtain a GPS position fix; we also determined the speed of satellite acquisition and measured canopy density using a spherical densiometer. The ability to obtain a reading differed greatly between the two forest types (χ² = 53.79, P < 0.001). Ninety-seven percent of all attempts were successful in the temperate forest, whereas only a 34% acquisition rate was obtained in the tropical forests. Logistic regression showed that the probability of obtaining a reading in Neotropical forests was 75% but only when canopy cover was less than 20%. Thus, these minimal-channel GPS units may be of limited utility for behavioral ecologists working in closed-canopy Neotropical forests. Am. J. Primatol. 46:167–172, 1998. © 1998 Wiley-Liss, Inc.     

A SPOT on the chromatin landscape? Histone peptide arrays as a tool for epigenetic research

Post-translational modifications of histones serve as docking sites and signals for effector proteins and chromatin-remodeling enzymes, thereby influencing many fundamental cellular processes. Nevertheless, there are huge gaps in the knowledge of which proteins read and write the 'histone code'. Several techniques have been used to decipher complex histone-modification patterns. However, none is entirely satisfactory owing to the inherent limitations of in vitro studies of histones, such as deficits in the knowledge of the proteins involved, and the associated difficulties in the consistent and quantitative generation of histone marks. An alternative technique that could prove to be a useful tool in the study of the histone code is the use of synthetic peptide arrays (SPOT blot analysis) as a screening approach to characterize macromolecules that interact with specific covalent modifications of histone tails.     

Enzymic and immunochemical properties of lysozyme. XIII. Accurate delineation of the reactive site around the disulfide 6-127 by immunochemical study of β-propiolactone lysozyme derivative and of synthetic disulfide peptides

In previous reports from this laboratory it was shown that an antigenic reactive site resides around the sequences 6–13 and 126–128 linked by the disulfide 6–127. The present work provides a strong support for the location of the reactive site by an independent approach. It also determines accurately the boundaries of the reactive site.

• 1.1. The two methionine residues in lysozyme were carboxyethylated by reaction with β-propiolactone. The electrophoretically homogeneous derivative had no other modified amino acids and showed no conformational changes, relative to native lysozyme, as determined by ORD and CD measurements. However, it exhibited a slight increase in disulfide reducibility relative to native lysozyme and its lytic activity was about half that of native lysozyme, probably as a result of the slight conformational change. On the other hand, the antigenic reactivity of the derivative was equal to that of native lysozyme with several goat and rabbit antisera to lysozyme. It was therefore concluded that methionines 12 and 105 were not parts of antigenic reactive sites in native lysozyme.
• 2.2. Eleven peptides, corresponding to various sequences on the two sides of the disulfide 6–127 (i.e. two groups of peptides) were synthesized, purified and characterized. One group (A) of peptides comprised sequences 3–14, 5–14, 6–14, 5–13, 5–12 and an analog of sequence 5–14 in which methionine 12 is replaced by glycine. The second group (B) of peptides comprised sequences 125–129, 125–128, 126–128, 127–128, and 125–127. From groups A and B, nine disulfide-containing peptides (see Fig. 2) were synthesized, purified, characterized and their immunochemical interactions with antisera to native lysozyme studied. Towards each of the antisera studied here, Phe-3, Gly-4, Arg-5, Arg-125 and Leu-129 were not essential parts of the reactive site. On the other hand, Arg-14, Lys-13, Gly-126 and with some antisera Arg-128 were each critical for the reactivity of the site. Peptides from group A alone or group B alone did not inhibit the reaction of lysozyme with its antisera, confirming our previous findings that the integrity of the disulfide bond is essential for bringing the two distant (in sequence) parts of the site together. Finally, replacement of Met-12 by glycine did not influence the immunochemical reactivity of the site, confirming the above conclusion that neither of the two methionine residues takes part in interaction of lysozyme with its antibodies. An accurate delineation of the antigenic reactive site is, therefore derived here and its shape in the three-dimensional structure of native lysozyme is described.

     

Changes in cigarette smoking behavior among breast cancer and unaffected women – A prospective study in the MARIE cohort

BackgroundSmoking cessation after a cancer diagnosis can reduce adverse cancer treatment outcomes. Whether a breast cancer diagnosis, a cancer commonly seen as unrelated to smoking cigarettes, motivates changes in smoking behavior is not fully understood. We aimed to compare long-term changes at three follow-up times of cigarette smoking behavior in women with breast cancer and baseline age- and region-matched unaffected women.MethodsWe used longitudinal data from the population-based case-control study MARIE (Mamma Carcinoma Risk Factor Investigation). Women with breast cancer (N = 3813) and unaffected women (N = 7341) aged 50–74 years were recruited from 2002 to 2005. Analyses on changes in smoking were based on data from those who also completed follow-up 1 in 2009–2012, follow-up 2 in 2014–2016 and follow-up 3 in 2020. Multinomial logistic regression for changes (quitting, stable, or start smoking) adjusted for age, study region, education, comorbidities, living situation, and follow-up time, was applied to examine the associations between breast cancer status and changes in smoking behavior.ResultsWomen with breast cancer had significantly higher odds than unaffected women of quitting smoking (OR = 1.38, 95 % CI: 1.01–1.89) and lower odds of returning to smoking (OR = 0.29, 95 % CI: 0.09–0.94) at follow-up 1, but were more likely to start or return to smoking at follow-up 2 (OR = 2.11, 95 % CI 1.08–4.15). No significant group differences were found for changes in smoking behavior at follow-up 3.ConclusionOur findings indicate that short-term changes in smoking behavior can be attributed to a breast cancer diagnosis, but that over time the effect diminishes and changes in smoking no longer differ between breast cancer and breast cancer-free women. To support smoking cessation and to prevent relapse, guidelines to address smoking in cancer care, as well as comprehensive tobacco treatment services, are needed.     

What is the function of mitochondrial networks? A theoretical assessment of hypotheses and proposal for future research

Mitochondria can change their shape from discrete isolated organelles to a large continuous reticulum. The cellular advantages underlying these fused networks are still incompletely understood. In this paper, we describe and compare hypotheses regarding the function of mitochondrial networks. We use mathematical and physical tools both to investigate existing hypotheses and to generate new ones, and we suggest experimental and modelling strategies. Among the novel insights we underline from this work are the possibilities that (i) selective mitophagy is not required for quality control because selective fusion is sufficient; (ii) increased connectivity may have non‐linear effects on the diffusion rate of proteins; and (iii) fused networks can act to dampen biochemical fluctuations. We hope to convey to the reader that quantitative approaches can drive advances in the understanding of the physiological advantage of these morphological changes.

     

A double blind,randomized, placebo controlled study of the efficacy and safety of 5-Loxin<Superscript>®</Superscript>for treatment of osteoarthritis of the knee

Introduction

5-Loxin^® is a novel Boswellia serrata extract enriched with 30% 3-O-acetyl-11-keto-beta-boswellic acid (AKBA), which exhibits potential anti-inflammatory properties by inhibiting the 5-lipoxygenase enzyme. A 90-day, double-blind, randomized, placebo-controlled study was conducted to evaluate the efficacy and safety of 5-Loxin^® in the treatment of osteoarthritis (OA) of the knee.

Methods

Seventy-five OA patients were included in the study. The patients received either 100 mg (n = 25) or 250 mg (n = 25) of 5-Loxin^® daily or a placebo (n = 25) for 90 days. Each patient was evaluated for pain and physical functions by using the standard tools (visual analog scale, Lequesne''s Functional Index, and Western Ontario and McMaster Universities Osteoarthritis Index) at the baseline (day 0), and at days 7, 30, 60 and 90. Additionally, the cartilage degrading enzyme matrix metalloproteinase-3 was also evaluated in synovial fluid from OA patients. Measurement of a battery of biochemical parameters in serum and haematological parameters, and urine analysis were performed to evaluate the safety of 5-Loxin^® in OA patients.

Results

Seventy patients completed the study. At the end of the study, both doses of 5-Loxin^® conferred clinically and statistically significant improvements in pain scores and physical function scores in OA patients. Interestingly, significant improvements in pain score and functional ability were recorded in the treatment group supplemented with 250 mg 5-Loxin^® as early as 7 days after the start of treatment. Corroborating the improvements in pain scores in treatment groups, we also noted significant reduction in synovial fluid matrix metalloproteinase-3. In comparison with placebo, the safety parameters were almost unchanged in the treatment groups.

Conclusion

5-Loxin^® reduces pain and improves physical functioning significantly in OA patients; and it is safe for human consumption. 5-Loxin^® may exert its beneficial effects by controlling inflammatory responses through reducing proinflammatory modulators, and it may improve joint health by reducing the enzymatic degradation of cartilage in OA patients.

Trail Registration

(Clinical trial registration number: ISRCTN05212803.)     

"So, what is an embryo?" A comparative study of the views of those asked to donate embryos for hESC research in the UK and Switzerland

The moral status of the human embryo has gained much attention in debates over the acceptability, or otherwise, of human embryonic stem cell research. Far less attention has been paid to the suppliers of those embryos: people who have undergone IVF treatment to produce embryos to assist them to have a baby. It is sociologically and ethically important to understand their views and experiences of being asked to donate embryos for research if we are to fully understand the wider social and regulatory aspects of hESC science. This paper reports on parallel studies investigating these issues in the UK and in Switzerland. The studies reveal the inextricable entangling of the social and moral status of embryos. Since donors participate in different discursive domains and contexts (public, clinic, family) that shape their perception of "what" an embryo is, their views of embryos embody conflicting ideas and ambivalences.     

Virulence and RAPD data—A tool to study the evolutionary trends of Colletotrichum lindemuthianum virulences in the North Western Himalayan region of India

Abstract

Bean anthracnose pathogen (Colletotrichum lindemuthianum) known to display high pathogenic variability, also explains the existence of large number of races in Himachal Pradesh. An evolution model based on virulence data of 29 C. lindemuthianum races and RAPD patterns revealed the existence of four evolutionary groups (EG I – EG IV) in Himachal Pradesh, accommodating 12, 14, 2 and 1 races, respectively. Some races viz., 935, 643, 529, 647 and 613, opted more than two evolutionary routes and races like 598, 707, 935, 631, 639, 615, 115 and 119 harbouring more than six virulence genes may pose a threat to bean cultivation in this part of the world as they can break many resistance genes present in the locally grown beans. However, two exotic accessions G 2333 and AB 136 resistant to all the Indian pathotypes could be exploited as resistance donors in developing anthracnose resistant cultivars suitable for cultivation in this region.     

Is dynamic thiol/disulfide homeostasis associated with the prognosis of myelodysplastic syndrome?

BackgroundThis study planned to investigate the relationship of dynamic thiol/disulfide homeostasis with the prognosis of myelodysplastic syndrome (MDS).Methods80 patients who had been diagnosed with MDS between 2012 and 2017 and who were older than 18 were included in the study together with 80 healthy control subjects. The MDS diagnosis was confirmed using bone marrow aspiration-biopsy immunostaining. Dynamic thiol/disulfide homeostasis and ischemia-modified albumin (IMA) levels were examined.ResultsThe average IMA (0.71±0.08 vs. 0.67±0.09; p=0.002), median disulfide (18.0 vs. 11.6; p<0.001), median disulfide/native thiol (6 vs. 3; p<0.001), and median disulfide/total thiol (5.4 vs. 2.9; p<0.001) were found higher in the MDS patients compared to control group, and the median hemoglobin, median white blood cell count, median neutrophil count, median lymphocyte count, average native thiol (290.7±48.5 vs. 371.5±103.8; p<0.001), average total thiol (328.2±48.9 vs. 393±105.5; p<0.001), and average native thiol/total thiol (%) (88.3±4.3 vs. 94.2±2.1; p<0.001) were found to below. Risk factors such as collagen tissue disease (HR:9.17; p=0.005), MDS-EB-1 (HR:10.14; p=0.032), MDS-EB-2 (HR:18.2; p=0.043), and disulfide/native thiol (HR:1.17; p=0.023) were found as the independent predictors anticipating progression to acute myeloid leukemia. In the Cox regression model, risk factors such as age (HR:1.05; p=0.002), MDS-EB-1 (HR:12.58; p<0.001), MDS-EB-2 (HR:5.75; p=0.033), disulfide/native thiol (HR:1.14; p=0.040), and hemoglobin (HR:0.64; p=0.007) were found as predictors anticipating for mortality.ConclusionsWe can argue that dynamic thiol/disulfide homeostasis could have significant effects on both the etiopathogenesis and the survival of patients with MDS, and it could be included in new prognostic scoring systems.     

Generation of retroviruses for the overexpression of cytosolic and mitochondrial glutathione reductase in macrophages in vivo

Retroviral gene transfer and bone marrow transplantation has been used by many investigators to study the role of macrophage proteins in different mouse models of human disease. While this approach is faster and less expensive than generating transgenic mice with macrophage-specific promoters and applicable to a wider array of mouse models, it has been hampered by two major drawbacks: labor-intensive cloning procedures involved in generating retroviral vectors for each gene of interest and low viral titers. Here we describe the construction of a MSCV-based retroviral vector that can serve as an acceptor vector for commercially available Cre-lox-compatible donor vectors. Using this new retroviral vector in combination with a FACS approach to enhance viral titers, we generated high-titer retroviruses carrying either EGFP-tagged cytosolic or EGFP-tagged mitochondria-targeted glutathione reductase. We show that the introduction of these constructs via retroviral gene transfer and bone marrow transplantation into atherosclerosis-prone LDL receptor-null mice results in the long-term increase in macrophage glutathione reductase activity.     

A study of French centenarians: are ACE and APOE associated with longevity?

Association study is the method of choice to identify genes involved in complex processes that result from the interaction of environmental and genetic factors. However, because of biases that increase the risk of false positive reports, preliminary positive conclusions have to be reproduced on other populations to be validated as firm conclusions. In 1994, certain alleles of two genes, APOE (Apolipoprotein E) and ACE (angiotensin converting enzyme), were reported to be more frequent in French centenarians, suggesting an association with such a complex polyfactorial process as longevity. Enlargement of the French centenarian cohort allows a new assessment of this hypothesis on 563 centenarians. In contrast to APOE, the ACE association was not confirmed. Retrospective analysis of the initial study revealed discrepancies that may in part explain this observation. Risk of reporting false positive associations is discussed and recommendations to set up a rigorous experimental design are proposed.     

A study of protein–carotenoid interactions in the astaxanthin-protein crustacyanin by absorption and Stark spectroscopy; evidence for the presence of three spectrally distinct species

Molecular mechanisms underlying the peculiar spectral properties of the carotenoid astaxanthin in α-crustacyanin, the blue carotenoprotein isolated from the exoskeleton of the lobster Homarus gammarus, were investigated by comparing the basic electrooptical parameters of astaxanthin free in vitro with those of astaxanthin in the complex. Absorption and electroabsorption (Stark effect) spectra were obtained for α-crustacyanin in low-temperature glasses to provide information about the molecular interactions that lead to the large bathochromic shift of the spectra resulting from this complexation. The low-temperature spectra reveal the presence of at least three spectral forms of α-crustacyanin, with vibronic (0–0) transitions at 14 000 cm⁻¹, 13 500 cm⁻¹ and 11 600 cm⁻¹ (corresponding to approximately 630, 660 and 780 nm, respectively, at room temperature) and with relative aboundance 85%, 10% and 5%. The longer wavelength absorbing species have not previously been detected. The changes in polarizability and in permanent dipole moments associated with the S₀→S₂ electronic transition for all these forms are about 1.5 times larger than for isolated astaxanthin. The results are discussed with reference to the symmetric polarization model for astaxanthin in α-crustacyanin.