Mge1, a nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70 function

Despite the growing evidence of the role of oxidative stress in disease, its molecular mechanism of action remains poorly understood. The yeast Saccharomyces cerevisiae provides a valuable model system in which to elucidate the effects of oxidative stress on mitochondria in higher eukaryotes. Dimeric yeast Mge1, the cochaperone of heat shock protein 70 (Hsp70), is essential for exchanging ATP for ADP on Hsp70 and thus for recycling of Hsp70 for mitochondrial protein import and folding. Here we show an oxidative stress–dependent decrease in Mge1 dimer formation accompanied by a concomitant decrease in Mge1–Hsp70 complex formation in vitro. The Mge1-M155L substitution mutant stabilizes both Mge1 dimer and Mge1–Hsp70 complex formation. Most important, the Mge1-M155L mutant rescues the slow-growth phenomenon associated with the wild-type Mge1 strain in the presence of H₂O₂ in vivo, stimulation of the ATPase activity of Hsp70, and the protein import defect during oxidative stress in vitro. Furthermore, cross-linking studies reveal that Mge1–Hsp70 complex formation in mitochondria isolated from wild-type Mge1 cells is more susceptible to reactive oxygen species compared with mitochondria from Mge1-M155L cells. This novel oxidative sensor capability of yeast Mge1 might represent an evolutionarily conserved function, given that human recombinant dimeric Mge1 is also sensitive to H₂O₂.     

Aquisphaera insulae sp. nov., a new member in the family Isosphaeraceae,isolated from the floating island of Loktak lake and emended description of the genus Aquisphaera

Antonie van Leeuwenhoek - Strain JC669T was isolated from a floating island of Loktak lake, Manipur, India and shares the highest 16S rRNA gene sequence identity with Aquisphaera giovannonii OJF2T....     

Purification and characterization of recombinant FAD synthetase from Neurospora crassa

FAD Synthetase (FADS) [EC 2.7.7.2], the second enzyme in flavin cofactor biosynthetic pathway converts FMN to FAD, plays an important role in many redox reactions. Neurospora crassa FADS (NcFADS) was cloned and overexpressed in E. coli cells. Recombinant NcFADS was purified in high yields of ～8 mg per liter of bacterial culture using a single step glutathione sepharose affinity chromatography. SDS-PAGE and MALDI-MS revealed that NcFADS has a molecular mass of ～31 kDa. Enzyme kinetic analysis monitored by reverse phase HPLC demonstrate a specific activity and k_cat of 1356 nmol/min/mg and 0.69sec^?1 respectively. Steady state kinetic analysis of NcFADS exhibited a K_m of NcFADS for FMN is 2.7 μM and for MgATP^?2 is 88.7 μM. Isothermal titration calorimetry experiments showed that the recombinant protein binds to the substrates with apparent K_d of 20.8 μM for FMN and 16.6 μM for MgATP^?2. Biophysical characterization using intrinsic fluorescence suggests that the enzyme is in folded conformation. Far-UV CD data suggest that the backbone of the enzyme is predominantly in a helical conformation. Differential scanning calorimetry data shows that the T_m is 53 °C ± 1. This is the first report on cloning, purification and characterization of FADS from N. crassa. The specific activity of NcFADS is the highest than any of the reported FADS from any other source. The results obtained in this study is expected to pave way for intensive research aimed to understand the molecular basis for the extraordinarily high turnover rate of NcFADS.     

Influence of carbon and nitrogen sources on ochratoxin A production by two species of Penicillium isolated from poultry feeds

Mycotoxins are natural, secondary metabolites produced by fungi on agricultural commodities in the field and during storage under a wide range of climatic conditions. The ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by several species of Aspergillus and Penicillium. In this study, the influence of carbon and nitrogen sources on ochratoxigenic Penicillium species was assessed. The ochratoxigenic Penicillium species were isolated from poultry feed samples of Andhra Pradesh, India. The isolated ochratoxigenic Penicillium species were identified, screened and characterised as OTA producers by high performance thin layer chromatography (HPTLC) and confirmed by high performance liquid chromatography (HPLC). This experiment was carried out using Czapak yeast Autolysate (CYA) medium with different carbon (C) and nitrogen (N) sources at pH 6.5 and incubated at 25 ± 2°C under dark condition. Maximum OTA production was recorded in the presence of D-glucose followed by D-galactose and D-lactose as carbon sources. Similarly, the maximum amount of OTA production was observed in thiourea followed by potassium nitrate as nitrogen source. However, OTA production, final pH of the medium, and mycelial yield and OTA production of both the species of Penicillium varied with C and N present in the medium. The kinetics of the both species of Penicillium was observed for 30 days at an interval of three days. The maximum amount of OTA was detected by 12 and 15 days incubation periods for Penicillium nordicum and Penicillium verrucosum, respectively.     

Emended description of the genus Rhodothalassium Imhoff et al., 1998 and proposal of Rhodothalassiaceae fam. nov. and Rhodothalassiales ord. nov

Five strains (JA325, JA389, JA473, JA563 and JA582) of Gram stain-negative, vibrioid to spiral shaped, phototrophic purple bacteria were isolated from solar salterns of India. All strains contained bacteriochlorophyll-a and carotenoids of the spirilloxanthin series as photosynthetic pigments. C_18:1ω7c, C_18:1ω7c 11-methyl and C_16:0 were the major fatty acids of all strains. Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), ornithine lipid (OL), an unidentified phospholipid (PL), and an unidentified aminolipid (AL) were the major polar lipids of all the strains. According to 16S rRNA gene sequences, all strains clustered phylogenetically with the only species of the genus Rhodothalassium (99.8–99.3% sequence similarity) but only strains JA325 and JA563 were distinctly related (60 + 1.5% DNA–DNA hybridization [DDH]) to the type strain Rhodothalassium salexigens DSM 2132^T. However, the genotypic data of strains JA325 and JA563 was not supported because of a large number of phenotypic differences compared to the type strain, therefore, it is proposed that all five newly isolated strains were R. salexigens-like strains. In addition, phylogenetically, the Rhodothalassium clade represented a distinct lineage and formed a deep branch with less than 90% 16S rRNA gene sequence similarity to other orders of the Alphaproteobacteria, and characteristic phenotypic properties also distinguished these bacteria from other purple non-sulfur bacteria. Therefore, the novel family Rhodothalassiaceae fam. nov. and the novel order Rhodothalassiales ord. nov. are proposed for the distinct phyletic line represented by the genus Rhodothalassium.     

An Electrocorticographic Brain Interface in an Individual with Tetraplegia

Brain-computer interface (BCI) technology aims to help individuals with disability to control assistive devices and reanimate paralyzed limbs. Our study investigated the feasibility of an electrocorticography (ECoG)-based BCI system in an individual with tetraplegia caused by C4 level spinal cord injury. ECoG signals were recorded with a high-density 32-electrode grid over the hand and arm area of the left sensorimotor cortex. The participant was able to voluntarily activate his sensorimotor cortex using attempted movements, with distinct cortical activity patterns for different segments of the upper limb. Using only brain activity, the participant achieved robust control of 3D cursor movement. The ECoG grid was explanted 28 days post-implantation with no adverse effect. This study demonstrates that ECoG signals recorded from the sensorimotor cortex can be used for real-time device control in paralyzed individuals.     

Associations of Forest Type,Parasitism and Body Condition of Two European Passerines,Fringilla coelebs and Sylvia atricapilla

Human-induced forest modification can alter parasite-host interactions and might change the persistence of host populations. We captured individuals of two widespread European passerines (Fringilla coelebs and Sylvia atricapilla) in southwestern Germany to disentangle the associations of forest types and parasitism by haemosporidian parasites on the body condition of birds. We compared parasite prevalence and parasite intensity, fluctuating asymmetries, leukocyte numbers, and the heterophil to lymphocyte ratio (H/L-ratio) among individuals from beech, mixed-deciduous and spruce forest stands. Based on the biology of bird species, we expected to find fewer infected individuals in beech or mixed-deciduous than in spruce forest stands. We found the highest parasite prevalence and intensity in beech forests for F. coelebs. Although, we found the highest prevalence in spruce forests for S. atricapilla, the highest intensity was detected in beech forests, partially supporting our hypothesis. Other body condition or health status metrics, such as the heterophil to lymphocyte ratio (H/L-ratio), revealed only slight differences between bird populations inhabiting the three different forest types, with the highest values in spruce for F. coelebs and in mixed-deciduous forests for S. atricapilla. A comparison of parasitized versus non-parasitized individuals suggests that parasite infection increased the immune response of a bird, which was detectable as high H/L-ratio. Higher infections with blood parasites for S. atricapilla in spruce forest indicate that this forest type might be a less suitable habitat than beech and mixed-deciduous forests, whereas beech forests seem to be a suboptimal habitat regarding parasitism for F. coelebs.     

Post-translational protein modification by carotenoid cleavage products

Carotenoids are known to generate various aldehydes, known as carotenoid-derived aldehydes (CDAs), which could efficiently react with protein or DNA. In this in vitro model study, interaction between CDA and protein has been studied. Various proteins were incubated with CDA, and protein modification and adduct formation were confirmed by using matrix-assisted laser desorption and ionization time-of-flight, amino acid analysis, and measuring enzyme activity on modification with CDA. Using radiolabeled NaB((3) H)H(4) and Raney nickel as well as sulfhydryl assay (Ellman's reagent), we confirmed that CDA could conjugate with cysteine through a thioether linkage. The carbonyl assay using 2,4-dinitrophenylhydrazine revealed the possible involvement of Schiff's base reaction between CDA and lysine. The adducts formed between β-apo-8-carotenal (BA8C) and N-acetylcysteine and BA8C and N-acetyllysine were confirmed by HPLC and ESI-MS. Our results suggest that CDA could alter protein function by post-translational interaction with cysteine and lysine by thioether linkage and by schiff's based bonds, respectively. Thus, the formation of CDA adducts with proteins could alter functional properties of proteins responsible for maintaining cell homeostasis and thereby cause cellular toxicity. In view of these observations, further studies are required to understand the delicate balance between beneficial and/or harmful effects of carotenoids as a dietary supplement to slow age-related macular degeneration progression.