Acute metal stress in Populus tremula×P. alba (717‐1B4 genotype): Leaf and cambial proteome changes induced by cadmium2+

The comprehension of metal homeostasis in plants requires the identification of molecular markers linked to stress tolerance. Proteomic changes in leaves and cambial zone of Populus tremula×P. alba (717‐1B4 genotype) were analyzed after 61 days of exposure to cadmium (Cd) 360 mg/kg soil dry weight in pot‐soil cultures. The treatment led to an acute Cd stress with a reduction of growth and photosynthesis. Cd stress induced changes in the display of 120 spots for leaf tissue and 153 spots for the cambial zone. It involved a reduced photosynthesis, resulting in a profound reorganisation of carbon and carbohydrate metabolisms in both tissues. Cambial cells underwent stress from the Cd actually present inside the tissue but also a deprivation of photosynthates caused by leaf stress. An important tissue specificity of the response was observed, according to the differences in cell structures and functions.     

镉胁迫下三个萝卜栽培种蛋白质变化的双向电泳比较研究

在0.5mmol·L^-1 Cd²⁺处理时,小五缨(XWY)的发芽率下降至92%,并且随着Cd²⁺浓度的增加,发芽率逐渐降低,在1和5mmol·L^-1 Cd²⁺浓度处理时,发芽率分别降至83%和67%。象牙白(XYB)则在5mmol·L^-1 Cd²⁺浓度时,发芽率下降。卫青(WQ)在0.05mmol·L^-1 Cd²⁺浓度时,发芽率已降至83%, 1和5mmol·L^-1时则下降至58%。幼苗生长也明显受Cd²⁺处理影响,在0.05mmol·L^-1 Cd²⁺处理时, 3种栽培萝卜的幼苗生长均受到明显影响,并且随Cd²⁺浓度的增加,生长受抑加重。从发芽率和幼苗生长两种实验结果看,卫青对Cd²⁺最为敏感。双向电泳结果表明, Cd²⁺处理后3种栽培萝卜幼苗中蛋白质组分有明显变化。小五缨中, 0.1 mmol·L^-1Cd²⁺处理后,有5个蛋白质点消失, 15个新的蛋白质点被诱导产生。象牙白中, 2个蛋白质点消失, 1个蛋白质点含量减少, 13个新的蛋白质点被诱导产生。卫青中, 12个新的蛋白质点诱导产生,但没有发现蛋白质点消失现象。Cd²⁺处理后, 3种栽培萝卜中,蛋白质合成的变化与幼苗生长受抑存在明显相关性,这一实验结果对于探讨萝卜Cd²⁺害的生化机理是有重要意义的。     

Protein changes in abalone foot muscle from three geographical populations of Haliotis diversicolor based on proteomic approach

Using two‐dimensional gel electrophoresis, the foot muscle proteome of three geographical populations of Haliotis diversicolor were examined, with a total of 922 ± 21 protein spots detected in the Japanese population (JJ), 904 ± 25.6 in the Taiwanese population (TT), and 936 ± 16.2 in the Vietnamese population (VV). Of these, 254 spots showed differential expression and 85 protein spots percentage volumes varied more than twofold. Both “genotype” and “spot” analysis of variance approaches significantly showed differences among the three populations. Hierarchical clustering analysis showed that TT and VV clustered together followed by clustering with JJ, which is consistent with their geographical location. Following matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, 30 differentially expressed proteins involved in major biological processes including energy production and storage and stress response were identified. Of these proteins, proteins pertaining to muscle contraction and muscle protein regulation showed highest expression levels in VV samples. Proteins involved in energy production and storage, including ATP synthase beta subunit, fructose‐1,6‐bisphosphate aldolase, arginine kinase, enolase, triosephosphate isomerase, and tauropine dehydrogenase, showed diverse expression patterns among the three populations. For stress‐responsive proteins, the expression of heat shock protein 70 was JJ > VV > TT. The expression pattern of Cu/Zn‐superoxide dismutase was JJ > VV > TT. Overall, these results may aid in the detection of new differentially expressed proteins within three different abalone populations.     

Early changes in the liver-soluble proteome from mice fed a nonalcoholic steatohepatitis inducing diet

Despite the increasing incidence of nonalcoholic steatohepatitis (NASH) with the rise in lifestyle-related diseases such as the metabolic syndrome, little is known about the changes in the liver proteome that precede the onset of inflammation and fibrosis. Here, we investigated early changes in the liver-soluble proteome of female C57BL/6N mice fed an NASH-inducing diet by 2D-DIGE and nano-HPLC-MS/MS. In parallel, histology and measurements of hepatic content of triglycerides, cholesterol and intermediates of the methionine cycle were performed. Hepatic steatosis manifested itself after 2 days of feeding, albeit significant changes in the liver-soluble proteome were not evident before day 10 in the absence of inflammatory or fibrotic signs. Proteomic alterations affected mainly energy and amino acid metabolism, detoxification processes, urea cycle, and the one-carbon/S-adenosylmethionine pathways. Additionally, intermediates of relevant affected pathways were quantified from liver tissue, confirming the findings from the proteomic analysis.     

Viral myocarditis induced by Coxsackievirus B3 in A.BY/SnJ mice: Analysis of changes in the myocardial proteome

Enteroviral myocarditis displays highly diverse clinical phenotypes ranging from mild dyspnoea or chest pain to cardiogenic shock and death. Despite detailed studies of the virus life cycle in vitro and in vivo, the molecular interplay between host and virus in disease progression is largely unresolved. Murine models of Coxsackievirus B3 (CVB3)‐induced myocarditis well mimic the human disease patterns and can thus be explored to study mechanisms leading from acute to chronic myocarditis. Here, we present a 2‐D gel‐based proteomic survey of the changes in the murine cardiac proteome that occurs following infection with CVB3. In total, 136 distinct proteins were affected. Proteins, which are involved in immunity and defense and protein metabolism/modification displayed pronounced changes in intensity not only during acute but also at later stages of CVB3 myocarditis. Proteins involved in maintenance of cell structure and associated proteins were particularly influenced in the acute phase of myocarditis, whereas reduction of levels of metabolic enzymes was observed in chronic myocarditis. Studies about changes in protein intensities were complemented by an analysis of protein phosphorylation that revealed infection‐associated changes in the phosphorylation of myosin binding protein C, atrial and ventricular isoforms of myosin regulatory light chain 2, desmin, and Rab GDP dissociation inhibitor beta‐2.     

Regulation of amino acid/carnitine transporter B 0,+ (ATB 0,+) in astrocytes by protein kinase C: independent effects on raft and non-raft transporter subpopulations

Neutral and basic amino acid transporter B(0,+) belongs to a Na,Cl-dependent superfamily of proteins transporting neurotransmitters, amino acids and osmolytes, known to be regulated by protein kinase C (PKC). The present study demonstrates an increased phosphorylation of B(0,+) on serine moiety after treatment of rat astrocytes with phorbol 12-myristate 13-acetate, a process correlated with an augmented activity of l-leucine transport and an enhanced presence of the transporter at the cell surface. After solubilization with Triton X-100 and sucrose gradient centrifugation, B(0,+) was detected in non-raft as well as in detergent-resistant raft fractions under control conditions, while phorbol 12-myristate 13-acetate treatment resulted in a complete disappearance of the transporter from the raft fraction. B(0,+) was observed to interact with caveolin-1 and flotillin-1 (reggie-2) proteins, the markers of detergent-resistant microdomains of plasma membrane. As verified in immunocytochemistry and immunoprecipitation experiments, modification of PKC activity did not affect these interactions. It is proposed that PKC reveals different effects on raft and non-raft subpopulations of B(0,+). Phorbol ester treatment results in trafficking of the transporter from the intracellular pool to non-raft microdomains and increased activity, while B(0,+) present in raft microdomains undergoes either internalization or is transferred laterally to non-raft domains.     

Long‐term ethanol consumption promotes changes in β‐defensin isoform gene expression and induces structural changes and oxidative DNA damage to the epididymis of rats

Chronic alcohol ingestion causes sexual dysfunction, impairs sperm motility and fertility, and changes semen quality. Considering the key role of epididymis in sperm development, the aim of the present study was to evaluate the effects of long‐term ethanol consumption on epididymis changes, including alterations in β‐defensin isoform gene expression, oxidative stress, and pathological changes, such as cell proliferation and fibrosis in the epididymis of rats. In this study, male Wistar rats were equally divided into control and ethanol (4.5 g/kg BW) groups. After six weeks of treatment, the results revealed the proliferation of epididymis cells, fibrosis in the epididymis tissue, and a significant rise in the level of 8‐OHdG and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the ethanol group, compared with the control group. Moreover, the ethanol group showed an increase in the gene expression of epididymal β‐defensin isoforms 15 and 21 and a reduction in the gene expression of β‐defensin isoforms 27 and 30, compared with the controls. These findings indicate that ethanol‐induced epididymal damage and sperm abnormalities might be partly associated with changes in β‐defensin isoforms and epididymal structure, mediated by the increased activities of 8‐OHdG and NADPH oxidase.     

Identification of dysregulation of atrial proteins in rats with chronic obstructive apnea using two‐dimensional polyacrylamide gel electrophoresis and mass spectrometry

Obstructive sleep apnea (OSA) affects an estimated 20% of adults worldwide and has been associated with electrical and structural abnormalities of the atria, although the molecular mechanisms are not well understood. Here, we used two‐dimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS) to investigate the proteins that are dysregulated in the atria from severe and moderate apnea when compared to control. We found enzymes involved in the glycolysis, beta‐oxidation, electron transport chain and Krebs cycle to be down‐regulated. The data suggested that the dysregulated proteins may play a role in atrial pathology developing via chronic obstructive apnea and hypoxia. Our results are consistent with our previous 1D‐PAGE and nanoLC‐MS/MS study (Channaveerappa et al, J Cell Mol Med. 2017), where we found that some aerobic and anaerobic glycolytic and Krebs cycle enzymes were down‐regulated, suggesting that apnea may be a result of paucity of oxygen and production of ATP and reducing equivalents (NADH). The 2D‐PAGE study not only complements our current study, but also advances our understanding of the OSA. The complete mass spectrometry data are available via ProteomeXchange with identifier PXD011181.     

Identification of toxicological biomarkers of di(2‐ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up‐ or down‐regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.     

Spatio-temporal changes in germination and radical elongation of barley seeds tracked by proteome analysis of dissected embryo, aleurone layer, and endosperm tissues

Germination of barley is accompanied by changes in water-soluble seed proteins. 2-DE was used to describe spatio-temporal proteome differences in dissected seed tissues associated with germination and the subsequent radicle elongation. Protein identification by MS enabled assignment of proteins and functions to the seed embryo, aleurone, and endosperm. Abundance in 2-DE patterns was monitored for 48 different proteins appearing in 79 gel spots at 8 time-points up to 72 h post imbibition (PI). In embryo, a beta-type proteasome subunit and a heat shock protein 70 fragment were among the earliest proteins to appear (at 4 h PI). Other early changes were observed that affected spots containing desiccation stress-associated late embryogenesis abundant and abscisic acid (ABA)-induced proteins. From 12 h PI proteins characteristic for desiccation stress disappeared rapidly, as did a putative embryonic protein and an ABA-induced protein, suggesting that these proteins are also involved in desiccation stress. Several redox-related proteins differed in spatio-temporal patterns at the end of germination and onset of radicle elongation. Notably, ascorbate peroxidase that was observed only in the embryo, increased in abundance at 36 h PI. The surprisingly early changes seen in the protein profiles already 4 h after imbibition indicate that germination is programmed during seed maturation.     

Comparative proteomic profile of rat sciatic nerve and gastrocnemius muscle tissues in ageing by 2‐D DIGE

Ageing induces a progressive morphological change and functional decline in muscles and in nerves. Light and electron microscopy, 2‐D DIGE and MS, were applied to profile the qualitative and quantitative differences in the proteome and morphology of rat gastrocnemius muscle and sciatic nerve, in healthy 22‐month‐old rats. At muscle level, morphological changes are associated to fibre atrophy accompanied by myofibrillar loss and degeneration, disappearance of sarcomeres and sarcoplasmic reticulum dilatation, internal migration of nuclei, longitudinal fibre splitting, increment of subsarcolemmal mitochondria aggregates and increment of lipofuscin granules. Sciatic nerve shows myelin abnormalities like enfoldings, invaginations, onion bulbs, breakdowns and side axonal atrophy. Proteomic analysis identified changes correlated to morphological abnormalities in metabolic, contractile and cytoskeletal proteins, deregulation of iron homeostasis, change of Ca²⁺ balance and stress response proteins, accompanied by a deregulation of myelin membrane adhesion protein and proteins regulating the neuronal caliber. By comparing proteomic results from the two tissues, 16 protein isoforms showed the same up and down regulation trend suggesting that there are changes implying a general process which may act as a signal event of degeneration. Only β enolase and tropomyosin 1α were differentially expressed in the tissues.     

α‐MSH activates immediate defense responses to UV‐induced oxidative stress in human melanocytes

Exposure of cultured human melanocytes to ultraviolet radiation (UV) results in DNA damage. In melanoma, UV‐signature mutations resulting from unrepaired photoproducts are rare, suggesting the possible involvement of oxidative DNA damage in melanocyte malignant transformation. Here we present data demonstrating immediate dose‐dependent generation of hydrogen peroxide in UV‐irradiated melanocytes, which correlated directly with a decrease in catalase activity. Pretreatment of melanocytes with α‐melanocortin (α‐MSH) reduced the UV‐induced generation of 7,8‐dihydro‐8‐oxyguanine (8‐oxodG), a major form of oxidative DNA damage. Pretreatment with α‐MSH also increased the protein levels of catalase and ferritin. The effect of α‐MSH on 8‐oxodG induction was mediated by activation of the melanocortin 1 receptor (MC1R), as it was absent in melanocytes expressing loss‐of‐function MC1R, and blocked by concomitant treatment with an analog of agouti signaling protein (ASIP), ASIP‐YY. This study provides unequivocal evidence for induction of oxidative DNA damage by UV in human melanocytes and reduction of this damage by α‐MSH. Our data unravel some mechanisms by which α‐MSH protects melanocytes from oxidative DNA damage, which partially explain the strong association of loss‐of‐function MC1R with melanoma.     

Alterations to the protein profile of bladder carcinoma cell lines induced by plant extract MINA‐05 in vitro

Bladder cancer (BLCa) is a severe urological cancer of both men and women that commonly recurs and once invasive, is difficult to treat. MINA‐05 (CK Life Sciences Int'l, Hong Kong) is a derivative of complex botanical extracts, shown to reduce cellular proliferation of bladder and prostate carcinomas. We tested the effects of MINA‐05 against human BLCa cell sublines, B8, B8‐RSP‐GCK, B8‐RSP‐LN and C3, from a transitional cell carcinoma, grade IV, to determine the molecular targets of treatment by observing the cellular protein profile. Cells were acclimatised for 48 h then treated for 72 h with concentrations of MINA‐05 reflecting IC₅₀, IC₅₀ and 2×IC₅₀ (n = 3) or with vehicle, (0.5% DMSO). Dose‐dependant changes in protein abundance were detected and characterised using 2‐dimensional electrophoresis and MS. We identified 10 proteins that underwent changes in abundance, pI and/or molecular mass in response to treatment. MINA‐05 was shown to influence proteins across numerous functional classes including cytoskeletal proteins, energy metabolism proteins, protein degradation proteins and tumour suppressors, suggesting a global impact on these cell lines. This study implies that the ability of MINA‐05 to retard cellular proliferation is attributed to its ability to alter cell cycling, metabolism, protein degradation and the cancer cell environment.     

Effects of resveratrol on H2O2‐induced apoptosis and expression of SIRTs in H9c2 cells

Resveratrol, a polyphenol found in fruits, has been demonstrated to activate Sir2. Though many studies have demonstrated that resveratrol can activate SIRT1, whether it has effect on other sirtuins (SIRT2–7) are unknown. The present study shows that exposure of H9c2 cells to 50 µM H₂O₂ for 6 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry (FCM), but pretreatment of resveratrol (20 µM) eliminated H₂O₂‐induced apoptosis. Resveratrol also prevented H₂O₂‐induced caspase‐3 activation. Exposure of cells to resveratrol caused rapid activation of SIRT1,3,4,7. Sirtuin inhibitor, nicotinamide (20 mM) attenuated resveratrol's inhibitory effect on cell apoptosis and caspase‐3 activity. These results suggest that resveratrol protects cardiomyocytes from H₂O₂‐induced apoptosis by activating SIRT1,3,4,7. J. Cell. Biochem. 107: 741–747, 2009. © 2009 Wiley‐Liss, Inc.     

Differential regulation by fucoidan of IFN‐γ‐induced NO production in glial cells and macrophages

Fucoidan has shown numerous biological actions; however, the molecular bases of these actions have being issued. We examined the effect of fucoidan on NO production induced by IFN‐γ and the molecular mechanisms underlying these effects in two types of cells including glia (C6, BV‐2) and macrophages (RAW264.7, peritoneal primary cells). Fucoidan affected IFN‐γ‐induced NO and/or iNOS expression both in macrophages and glial cells but in a contrast way. Our data showed that in C6 glioma cells both JAK/STAT and p38 signaling positively regulated IFN‐γ‐induced iNOS, which were inhibited by fucoidan. In contrast, in RAW264.7 cells JAK/STAT is a positive regulator whereas p38 is a negative regulator of NO/iNOS production. In RAW264.7 cells, fucoidan enhanced p38 activation and induced TNF‐α production. We also confirmed the dual regulation of p38 in BV‐2 microglia and primary peritoneal macrophages. From these results, we suggest that fucoidan affects not only IFN‐γ‐induced NO/iNOS production differently in brain and peritoneal macrophages due to the different roles of p38 but the effects on TNF‐α production in the two cell types. These novel observations including selective and cell‐type specific effects of fucoidan on IFN‐γ‐mediated signaling and iNOS expression raise the possibility that it alters the sensitivity of cells to the p38 activation. J. Cell. Biochem. 111: 1337–1345, 2010. © 2010 Wiley‐Liss, Inc.