Toxicity of extracellular secreted alpha-synuclein: Its role in nitrosative stress and neurodegeneration

It has been demonstrated that both oligomerisation and accumulation of α-synuclein (ASN) are the key molecular processes involved in the pathophysiology of neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and other synucleinopathies. Alterations of ASN expression and impairment of its degradation can lead to the formation of intracellular deposits of this protein, called Lewy bodies. Overexpressed or misfolded ASN could be secreted to the extracellular space. Today the prion-like transmission of ASN oligomers to neighbouring cells is believed to be responsible for protein modification and propagation of neurodegeneration in the brain. It was presented that oxidative/nitrosative stress may play a key role in ASN secretion and spread of ASN pathology. Moreover, ASN-evoked protein oxidation, nitration and nitrosylation lead to disturbances in synaptic transmission and cell death. The interaction of secreted ASN with other amyloidogenic proteins and its involvement in irreversible mitochondrial disturbances and oxidative stress were also described. A better understanding of the mechanisms of ASN secretion and dysfunction may help to explain the molecular mechanisms of neurodegeneration and may be the basis for the development of novel therapeutic strategies.     

Amelioration of cognitive impairment and neurodegeneration by catechin hydrate in rat model of streptozotocin-induced experimental dementia of Alzheimer’s type

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder resulting in cognitive decline and enhancement of oxidative loads in the brain. Flavonoids have been considered to exert human health benefits by anti-oxidant and anti-inflammatory properties. The present study is aimed to elucidate the neuroprotective effect of catechin hydrate (CH), a natural flavanoid with potential antioxidant and anti-inflammatory properties, on intracerebroventricular streptozotocin (ICV-STZ) induced neuronal loss and memory impairment. To test this hypothesis, male Wistar rats were pretreated with CH (10 and 20 mg/kg bwt) orally once daily for 21 days and then bilaterally injected with ICV-STZ (3 mg/kg bwt), while sham group rats receive the same volume of vehicle. After 2 weeks of ICV-STZ infusion, rats were tested for cognitive performance using Morris water maze (MWM) test and then sacrifice for biochemical and histopathological assays. CH was found to be successful in upregulating the antioxidant status and prevented the memory loss. The expression of choline acetyl transferase (ChAT) was decreased in ICV-STZ group and CH pretreatment increases the expression of ChAT. Moreover, inflammatory mediators like TNF-α, IL-1β levels and expression of iNOS were significantly attenuated by CH pretreatment. The study suggests that CH is effective in preventing memory loss, ameliorating the oxidative stress and might be beneficial for the treatment of sporadic dementia of Alzheimer’s type (SDAT).     

Plasma intermedin levels in patients with acute myocardial infarction

It has been shown that adrenomedullin (ADM) may function as a cardiovascular-regulatory peptide in humans. Intermedin (IMD) is a newly discovered peptide related to ADM and has a greater range of biological effects on the cardiovascular in animal experiments. The purpose of the study was to investigate the pathophysiological role of IMD in patients with acute myocardial infarction (AMI). The present study included twenty patients with acute ST-segment elevation myocardial infarction (STEMI), thirty-three with stable coronary heart disease (SCHD), and eighteen healthy controls. Plasma levels of IMD, malonaldehyde (MDA), and superoxide dismutase (SOD) and cardiac biomarkers were determined at one, two, four and seven days following AMI. Plasma IMD levels were significantly increased on day 1 in AMI patients when compared with SCHD subjects (P = 0.014), and reached a peak of 181.88 ± 9.47 pg/ml at 96 h. Plasma IMD concentrations were correlated with MDA and SOD. Furthermore, patients with severe lesions in their coronary arteries tended to have higher plasma IMD levels (P < 0.05) in AMI patients. A significant increase in plasma IMD following AMI may be associated with oxidative stress, and could be used as a marker to reflect the severity of the coronary stenosis.     

Glucocerebrosidase inhibition causes mitochondrial dysfunction and free radical damage

Mutations of the gene for glucocerebrosidase 1 (GBA) cause Gaucher disease (GD), an autosomal recessive lysosomal storage disorder. Individuals with homozygous or heterozygous (carrier) mutations of GBA have a significantly increased risk for the development of Parkinson’s disease (PD), with clinical and pathological features that mirror the sporadic disease. The mechanisms whereby GBA mutations induce dopaminergic cell death and Lewy body formation are unknown. There is evidence of mitochondrial dysfunction and oxidative stress in PD and so we have investigated the impact of glucocerebrosidase (GCase) inhibition on these parameters to determine if there may be a relationship of GBA loss-of-function mutations to the known pathogenetic pathways in PD. We have used exposure to a specific inhibitor (conduritol-β-epoxide, CβE) of GCase activity in a human dopaminergic cell line to identify the biochemical abnormalities that follow GCase inhibition. We show that GCase inhibition leads to decreased ADP phosphorylation, reduced mitochondrial membrane potential and increased free radical formation and damage, together with accumulation of alpha-synuclein. Taken together, inhibition of GCase by CβE induces abnormalities in mitochondrial function and oxidative stress in our cell culture model. We suggest that GBA mutations and reduced GCase activity may increase the risk for PD by inducing these same abnormalities in PD brain.     

The translation initiation factor 3 subunit eIF3K interacts with PML and associates with PML nuclear bodies

The promyelocytic leukemia protein (PML) is a tumor suppressor protein that regulates a variety of important cellular processes, including gene expression, DNA repair and cell fate decisions. Integral to its function is the ability of PML to form nuclear bodies (NBs) that serve as hubs for the interaction and modification of over 90 cellular proteins. There are seven canonical isoforms of PML, which encode diverse C-termini generated by alternative pre-mRNA splicing. Recruitment of specific cellular proteins to PML NBs is mediated by protein–protein interactions with individual PML isoforms. Using a yeast two-hybrid screen employing peptide sequences unique to PML isoform I (PML-I), we identified an interaction with the eukaryotic initiation factor 3 subunit K (eIF3K), and in the process identified a novel eIF3K isoform, which we term eIF3K-2. We further demonstrate that eIF3K and PML interact both in vitro via pull-down assays, as well as in vivo within human cells by co-immunoprecipitation and co-immunofluorescence. In addition, eIF3K isoform 2 (eIF3K-2) colocalizes to PML bodies, particularly those enriched in PML-I, while eIF3K isoform 1 associates poorly with PML NBs. Thus, we report eIF3K as the first known subunit of the eIF3 translation pre-initiation complex to interact directly with the PML protein, and provide data implicating alternative splicing of both PML and eIF3K as a possible regulatory mechanism for eIF3K localization at PML NBs.     

A thin‐walled polydimethylsiloxane bioreactor for high‐density hepatocyte sandwich culture

In vitro drug testing requires long‐term maintenance of hepatocyte liver specific functions. Hepatocytes cultured at a higher seeding density in a sandwich configuration exhibit an increased level of liver specific functions when compared to low density cultures due to the better cell to cell contacts that promote long term maintenance of polarity and liver specific functions. However, culturing hepatocytes at high seeding densities in a standard 24‐well plate poses problems in terms of the mass transport of nutrients and oxygen to the cells. In view of this drawback, we have developed a polydimethylsiloxane (PDMS) bioreactor that was able to maintain the long‐term liver specific functions of a hepatocyte sandwich culture at a high seeding density. The bioreactor was fabricated with PDMS, an oxygen permeable material, which allowed direct oxygenation and perfusion to take place simultaneously. The mass transport of oxygen and the level of shear stress acting on the cells were analyzed by computational fluid dynamics (CFD). The combination of both direct oxygenation and perfusion has a synergistic effect on the liver specific function of a high density hepatocyte sandwich culture over a period of 9 days. Biotechnol. Bioeng. 2013; 110: 1663–1673. © 2012 Wiley Periodicals, Inc.     

A new cryomacroscope device (Type III) for visualization of physical events in cryopreservation with applications to vitrification and synthetic ice modulators

The objective of the current study is to develop a new cryomacroscope prototype for the study of vitrification in large-size specimens. The unique contribution in the current study is in developing a cryomacroscope setup as an add-on device to a commercial controlled-rate cooler and in demonstration of physical events in cryoprotective cocktails containing synthetic ice modulators (SIM)—compounds which hinder ice crystal growth. Cryopreservation by vitrification is a highly complex application, where the likelihood of crystallization, fracture formation, degradation of the biomaterial quality, and other physical events are dependent not only upon the instantaneous cryogenic conditions, but more significantly upon the evolution of conditions along the cryogenic protocol. Nevertheless, cryopreservation success is most frequently assessed by evaluating the cryopreserved product at its end states—either at the cryogenic storage temperature or room temperature. The cryomacroscope is the only available device for visualization of large-size specimens along the thermal protocol, in an effort to correlate the quality of the cryopreserved product with physical events. Compared with earlier cryomacroscope prototypes, the new Cryomacroscope-III evaluated here benefits from a higher resolution color camera, improved illumination, digital recording capabilities, and high repeatability in tested thermal conditions via a commercial controlled-rate cooler. A specialized software package was developed in the current study, having two modes of operation: (a) experimentation mode to control the operation of the camera, record camera frames sequentially, log thermal data from sensors, and save case-specific information; and (b) post-processing mode to generate a compact file integrating images, elapsed time, and thermal data for each experiment. The benefits of the Cryomacroscope-III are demonstrated using various tested mixtures of SIMs with the cryoprotective cocktail DP6, which were found effective in preventing ice growth, even at significantly subcritical cooling rates with reference to the pure DP6.     

嗜碱单胞菌的新型羧基转移酶α亚基基因Aa-accA及其抗盐碱性能

[目的]探讨解淀粉嗜碱单胞菌(Alkalimonas amylolytica)N10来源的羧基转移酶α亚基(Acetyl-coenzyme A carboxylase subunit alpha,AccA)基因Aa-accA对细菌及植物细胞耐盐碱性的作用.[方法]通过PCR方法从嗜碱菌N10基因组中扩增基因Aa-accA,并在大肠杆菌(Escherichia coli)K12中表达,通过测定工程菌及对照菌在不同盐浓度[0%,2%,4%,6%(W/V) NaCl]及不同碱性pH(8.0,8.5,9.0,9.5)的LB中生长12 h后的OD600值,以及二者在分别含6%(W/V) NaCl及pH 9的LB中的生长曲线,评价Aa-accA对大肠杆菌耐盐碱性的影响.同时以pPZP111为载体,构建了植物细胞重组表达载体,通过农杆菌介导方法将该基因转入烟草BY-2悬浮细胞表达,利用FDA染色方法测定经盐碱溶液处理后残存的活细胞数量评价该基因对植物细胞耐盐碱性的影响.[结果]PCR扩增得到基因Aa-accA,其ORF含957 bp,编码318个氨基酸的多肽,BLAST比对显示该基因为羧基转移酶α亚基(AccA)家族中的成员,其氨基酸序列与E.coli的AccA具有76%同源性;含有Aa-accA的E.coli K12相较于对照组在不同NaCl浓度及不同碱性pH的LB中表现出了明显的生长优势,特别是在6%(W/V) NaCl及pH 9的LB中培养12 h后,终OD600分别是对照菌的2.6倍和3.5倍;缺失体实验结果显示基因缺失的突变体E.coli K12△accA在6%(W/V) NaCl及pH 9的LB中不能正常生长,而含有Aa-accA基因的重组质粒使得E.coli K12△accA在同样条件下OD600值达到0.5和0.2;转入此基因的烟草BY-2细胞,经盐碱溶液处理后,其存活细胞比例高于野生型.[结论]本研究首次发现了Aa-accA基因与盐碱性的相关性,可提高大肠杆菌及烟草BY-2细胞的耐盐碱能力.     

Burkholderia sp. SCMS54 reduces cadmium toxicity and promotes growth in tomato

Cadmium (Cd) can enter soil through the use of fertilisers, calcareous, pesticides and industrial and/or domestic effluents. Cd can leach into groundwater and be taken up by plants, potentially leading to reductions in plant growth and yield. In soil, plant roots interact with heavy metal (HM)‐tolerant microorganisms that may promote plant growth. Soil microorganisms may also be able to solubilise or mobilise soil metals, thereby acting as bioremediators. A better understanding of the interaction among plants, metals, microorganisms and soil will lead to improved plant tolerance. Two multi‐tolerant bacteria from the Burkholderia genus were isolated from Cd‐contaminated and Cd‐uncontaminated soil of a coffee plantation. In addition to its high tolerance to Cd, the strain SCMS54 produces indole‐acetic acid (IAA), solubilises inorganic phosphate and produces siderophores, demonstrating its potential to contribute to beneficial plant–microorganism interactions. When interacting with tomato plants exposed to Cd, the bacterium led to decreases in plant peroxide and chlorosis levels, promoted relative plant growth and decreased the root absorption of Cd, resulting in increased plant tolerance to this highly toxic HM. The results indicated that the inoculation of tomato plants with Burkholderia sp. SCMS54 promotes better growth in plants cultivated in the presence of Cd. This phenomenon appears to be attributed to a mechanism that decreases Cd concentrations in the roots via a beneficial interaction between the bacteria and the plant roots.     

Environmental acidification triggers oxidative stress and enhances globin expression in zebrafish gills

Animals in many aquatic ecosystems must cope with changing environmental parameters, such as temperature, oxygen availability or pH. We have investigated the molecular responses to acidification in the gills and body of zebrafish (Danio rerio) by means of quantitative real-time PCR. Expression levels of typical stress genes and genes for antioxidant defense were strongly enhanced in gills, and to lesser extents in the body, suggesting that acidification leads to oxidative stress. Surprisingly, the globins were found to be among the most prominent stress–responsive proteins in our study. Myoglobin showed the strongest response of all investigated genes in the gills, as confirmed by Western blotting. These findings agree with the role of globins in oxidative energy metabolism, but may also hint at a specific function in antioxidative defense.