Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides

Protein carbonyl detection has been commonly used to analyze the degree of damage to proteins under oxidative stress conditions. Most laboratories rely on derivatization of carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this approach, Western blot and immunodetection are no longer needed, shortening the procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy hydrazide allows easy matching of the spots of interest and those obtained by general fluorescent protein staining methods, which facilitates excising target proteins from the gels and identifying them. This method is effective for detecting protein carbonylation in samples of proteins submitted to metal-catalyzed oxidation "in vitro" and assessing the effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast cells.     

Decreased agonist-stimulated mitochondrial ATP production caused by a pathological reduction in endoplasmic reticulum calcium content in human complex I deficiency

Although a large number of mutations causing malfunction of complex I (NADH:ubiquinone oxidoreductase) of the OXPHOS system is now known, their cell biological consequences remain obscure. We previously showed that the bradykinin (Bk)-induced increase in mitochondrial [ATP] ([ATP](M)) is significantly reduced in primary skin fibroblasts from a patient with an isolated complex I deficiency. The present work addresses the mechanism(s) underlying this impaired response. Luminometry of fibroblasts from 6 healthy subjects and 14 genetically characterized patients expressing mitochondria targeted luciferase revealed that the Bk-induced increase in [ATP](M) was significantly, but to a variable degree, decreased in 10 patients. The same variation was observed for the increases in mitochondrial [Ca(2+)] ([Ca(2+)](M)), measured with mitochondria targeted aequorin, and cytosolic [Ca(2+)] ([Ca(2+)](C)), measured with fura-2, and for the Ca(2+) content of the endoplasmic reticulum (ER), calculated from the increase in [Ca(2+)](C) evoked by thapsigargin, an inhibitor of the ER Ca(2+) ATPase. Regression analysis revealed that the increase in [ATP](M) was directly proportional to the increases in [Ca(2+)](C) and [Ca(2+)](M) and to the ER Ca(2+) content. Our findings provide evidence that a pathological reduction in ER Ca(2+) content is the direct cause of the impaired Bk-induced increase in [ATP](M) in human complex I deficiency.     

6-Mercaptopurine reduces cytokine and Muc5ac expression involving inhibition of NFκB activation in airway epithelial cells

Background

Mucus hypersecretion and excessive cytokine synthesis is associated with many of the pathologic features of chronic airway diseases such as asthma. 6-Mercaptopurine (6-MP) is an immunosuppressive drug that is widely used in several inflammatory disorders. Although 6-MP has been used to treat asthma, its function and mechanism of action in airway epithelial cells is unknown.

Methods

Confluent NCI-H292 and MLE-12 epithelial cells were pretreated with 6-MP followed by stimulation with TNFα or PMA. mRNA levels of cytokines and mucins were measured by RT-PCR. Western blot analysis was performed to assess the phosphorylation of IκBα and luciferase assays were performed using an NFκB reporter plasmid to determine NFκB activity. Periodic Acid Schiff staining was used to assess the production of mucus.

Results

6-MP displayed no effect on cell viability up to a concentration of 15 μM. RT-PCR analysis showed that 6-MP significantly reduces TNFα- and PMA-induced expression of several proinflammatory cytokines in NCI-H292 and MLE-12 cells. Consistent with this, we demonstrated that 6-MP strongly inhibits TNFα-induced phosphorylation of IκBα and thus attenuates NFκB luciferase reporter activity. In addition, 6-MP decreases Rac1 activity in MLE-12 cells. 6-MP down-regulates gene expression of the mucin Muc5ac, but not Muc2, through inhibition of activation of the NFκB pathway. Furthermore, PMA- and TNFα-induced mucus production, as visualized by Periodic Acid Schiff (PAS) staining, is decreased by 6-MP.

Conclusions

Our data demonstrate that 6-MP inhibits Muc5ac gene expression and mucus production in airway epithelial cells through inhibition of the NFκB pathway, and 6-MP may represent a novel therapeutic target for mucus hypersecretion in airway diseases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0236-0) contains supplementary material, which is available to authorized users.     

Myocardial Metastases on 6-[18F] fluoro-L-DOPA PET/CT: A Retrospective Analysis of 116 Serotonin Producing Neuroendocrine Tumour Patients

Purpose

This study evaluates the prevalence of cardiac metastases in patients with serotonin producing neuroendocrine tumours (NET), examined with ¹⁸F-FDOPA PET/CT, and the relationship of these metastases to the presence of carcinoid heart disease (CHD) based on echocardiography.

Background

CHD occurs in patients with serotonin producing NET. The diagnostic method of choice remains echocardiography. The precise prevalence of cardiac metastases is unknown given the limitations of standard technologies. Nuclear medicine modalities have the potential to visualize metastases of NET.

Methods

All patients who underwent ¹⁸F-FDOPA PET/CT because of serotonin producing NET between November 2009 and May 2012 were retrospectively analyzed. The presence of cardiac metastasis was defined as myocardial tracer accumulation higher than the surrounding physiological myocardial uptake. Laboratory tests and transthoracic echocardiography (TTE) results were digitally collected.

Results

116 patients (62 male) underwent ¹⁸F-FDOPA PET/CT, mean age was 61±13 years. TTE was performed in 79 patients. Cardiac metastases were present in 15 patients, of which 10 patients also underwent TTE. One patient had both cardiac metastasis (only on ¹⁸F-FDOPA PET/CT) and echocardiographic signs of CHD. There were no differences in echocardiographic parameters for CHD between patients with and without cardiac metastases. TTE in none of the 79 patients showed cardiac metastases.

Conclusion

The prevalence of cardiac metastases detected with ¹⁸F-FDOPA PET/CT in this study is 13%. ¹⁸F-FDOPA PET/CT can visualize cardiac metastases in serotonin producing NET patients. There appears to be no relationship between the presence of cardiac metastases and TTE parameters of CHD.     

Nanog overcomes reprogramming barriers and induces pluripotency in minimal conditions

Induced pluripotency requires the expression of defined factors and culture conditions that support the self-renewal of embryonic stem (ES) cells. Small molecule inhibition of MAP kinase (MEK) and glycogen synthase kinase 3 (GSK3) with LIF (2i/LIF) provides an optimal culture environment for mouse ES cells and promotes transition to naive pluripotency in partially reprogrammed (pre-iPS) cells. Here we show that 2i/LIF treatment in clonal lines of pre-iPS cells results in the activation of endogenous Nanog and rapid downregulation of retroviral Oct4 expression. Nanog enables somatic cell reprogramming in serum-free medium supplemented with LIF, a culture condition which does not support induced pluripotency or the self-renewal of ES cells, and is sufficient to reprogram epiblast-derived stem cells to naive pluripotency in serum-free medium alone. Nanog also enhances reprogramming in cooperation with kinase inhibition or 5-aza-cytidine, a small molecule inhibitor of DNA methylation. These results highlight the capacity of Nanog to overcome multiple barriers to reprogramming and reveal a synergy between Nanog and chemical inhibitors that promote reprogramming. We conclude that Nanog induces pluripotency in minimal conditions. This provides a strategy for imposing naive pluripotency in mammalian cells independently of species-specific culture requirements.     

Successive cambia development in Avicennia marina (Forssk.) Vierh. is not climatically driven in the seasonal climate at Gazi Bay, Kenya

This study is intended to provide early access to recent findings on the formation of the successive cambia of Avicennia marina (Forssk.) Vierh. in Kenya. The non-annual character of the growth layers was demonstrated by using three trees from a cambial marking experiment and three trees from a plantation of known age. The respective number of growth layers produced during one year was on average a half and three. Considering 28 stem disks of trees at three study sites, differing in local site conditions, growth layer development was shown to be strongly correlated with stem diameter (R²=0.84, p<0.0001, n=31). However, an additional influence of the site conditions was also demonstrated (homogeneity-of-slopes model test: F=54.72, p<0.0001, n=28). With increasing salinity and/or inundation class the width of the growth layers decreased. The significance of these variations in growth layer width offer interesting perspectives. The larger proportion of xylem in comparison with phloem in trees with wide as opposed to narrow growth layers may provide extra mechanical strength. On the other hand, the larger fraction of living tissue (phloem and parenchyma) in trees with thin growth layers may be beneficial for the water balance of the tree. Next to the non-annual nature of the growth layers and their networking pattern, more than one cambium was found to be simultaneously active. We conclude that classical dendrochronological methods (ring width measurements) should not be applied to A. marina (from Kenya).     

Cerebral Activations Related to Writing and Drawing with Each Hand

BackgroundWriting is a sequential motor action based on sensorimotor integration in visuospatial and linguistic functional domains. To test the hypothesis of lateralized circuitry concerning spatial and language components involved in such action, we employed an fMRI paradigm including writing and drawing with each hand. In this way, writing-related contributions of dorsal and ventral premotor regions in each hemisphere were assessed, together with effects in wider distributed circuitry. Given a right-hemisphere dominance for spatial action, right dorsal premotor cortex dominance was expected in left-hand writing while dominance of the left ventral premotor cortex was expected during right-hand writing.MethodsSixteen healthy right-handed subjects were scanned during audition-guided writing of short sentences and simple figure drawing without visual feedback. Tapping with a pencil served as a basic control task for the two higher-order motor conditions. Activation differences were assessed with Statistical Parametric Mapping (SPM).ResultsWriting and drawing showed parietal-premotor and posterior inferior temporal activations in both hemispheres when compared to tapping. Drawing activations were rather symmetrical for each hand. Activations in left- and right-hand writing were left-hemisphere dominant, while right dorsal premotor activation only occurred in left-hand writing, supporting a spatial motor contribution of particularly the right hemisphere. Writing contrasted to drawing revealed left-sided activations in the dorsal and ventral premotor cortex, Broca’s area, pre-Supplementary Motor Area and posterior middle and inferior temporal gyri, without parietal activation.DiscussionThe audition-driven postero-inferior temporal activations indicated retrieval of virtual visual form characteristics in writing and drawing, with additional activation concerning word form in the left hemisphere. Similar parietal processing in writing and drawing pointed at a common mechanism by which such visually formatted information is used for subsequent sensorimotor integration along a dorsal visuomotor pathway. In this, the left posterior middle temporal gyrus subserves phonological-orthographical conversion, dissociating dorsal parietal-premotor circuitry from perisylvian circuitry including Broca''s area.     

People with multiple tattoos and/or piercings are not at increased risk for HBV or HCV in The Netherlands

Background

Although published results are inconsistent, it has been suggested that tattooing and piercing are risk factors for HBV and HCV infections. To examine whether tattooing and piercing do indeed increase the risk of infection, we conducted a study among people with multiple tattoos and/or piercings in the Netherlands who acquired their tattoos and piercings in the Netherlands and/or abroad.

Methods

Tattoo artists, piercers, and people with multiple tattoos and/or piercings were recruited at tattoo conventions, shops (N = 182), and a biannual survey at our STI-outpatient clinic (N = 252) in Amsterdam. Participants were interviewed and tested for anti-HBc and anti-HCV. Determinants of HBV and HCV infections were analysed using logistic regression analysis.

Results

The median number of tattoos and piercings was 5 (IQR 2–10) and 2 (IQR 2–4), respectively. Almost 40% acquired their tattoo of piercing abroad. In total, 18/434 (4.2%, 95%CI: 2.64%–6.46%) participants were anti-HBc positive and 1 was anti-HCV positive (0.2%, 95%CI: 0.01%–1.29%). Being anti-HBc positive was independently associated with older age (OR 1.68, 95%CI: 1.03–2.75 per 10 years older) and being born in an HBV-endemic country (OR 7.39, 95%CI: 2.77–19.7). Tattoo- and/or piercing-related variables, like having a tattoo or piercing in an HBV endemic country, surface percentage tattooed, number of tattoos and piercings etc., were not associated with either HBV or HCV.

Conclusions

We found no evidence for an increased HBV/HCV seroprevalence among persons with multiple tattoos and/or piercings, which might be due to the introduction of hygiene guidelines for tattoo and piercing shops in combination with the low observed prevalence of HBV/HCV in the general population. Tattoos and/or piercings, therefore, should not be considered risk factors for HBV/HCV in the Dutch population. These findings imply the importance of implementation of hygiene guidelines in other countries.