Interaction between non-anionic phospholipids and cytochrome c induced by reactive oxygen species

The oxidative interaction of cytochrome c (Cyt c) with liposomes of Palmitoyl Linoleyl Phosphatidyl Choline (PLPC) initiated by radio-induced free radicals was investigated. Results showed that the peroxidation of PLPC is decreased in the presence of Cyt c, meaning that this latter is the preferential target of hydroxyl radicals. In addition, when Cyt c was incubated with peroxidized PLPC, it was found to be able to decompose hydroperoxides of PLPC into hydroxides. The peroxidase activity of Cyt c proceeded via the opening of the tertiary structure of Cyt c, as suggested by the loss of the sixth coordination bond of the heme-iron. Even if it is known to preferentially interact with cardiolipin, this work shows that Cyt c is also able to interact with hydroperoxide species of non-anionic phospholipids.     

Students’ Perceptions of Peer-Organized Extra-Curricular Research Course during Medical School: A Qualitative Study

Early integration of research education into medical curricula is crucial for evidence-based practice. Yet, many medical students are graduating with no research experience due to the lack of such integration in their medical school programs. The purpose of this study was to explore the impact of a peer-organized, extra-curricular research methodology course on the attitudes of medical students towards research and future academic careers. Twenty one medical students who participated in a peer-organized research course were enrolled in three focus group discussions to explore their experiences, perceptions and attitudes towards research after the course. Discussions were conducted using a semi-structured interview guide, and were transcribed and thematically analyzed for major and minor themes identification. Our findings indicate that students’ perceptions of research changed after the course from being difficult initially to becoming possible. Participants felt that their research skills and critical thinking were enhanced and that they would develop research proposals and abstracts successfully. Students praised the peer-assisted teaching approach as being successful in enhancing the learning environment and filling the curricular gap. In conclusion, peer-organized extra-curricular research courses may be a useful option to promote research interest and skills of medical students when gaps in research education in medical curricula exist.     

Characterization of Hydrophobic Peptides in the Presence of Detergent by Photoionization Mass Spectrometry

The characterization of membrane proteins is still challenging. The major issue is the high hydrophobicity of membrane proteins that necessitates the use of detergents for their extraction and solubilization. The very poor compatibility of mass spectrometry with detergents remains a tremendous obstacle in studies of membrane proteins. Here, we investigated the potential of atmospheric pressure photoionization (APPI) for mass spectrometry study of membrane proteins. This work was focused on the tetraspanin CD9 and the multidrug transporter BmrA. A set of peptides from CD9, exhibiting a broad range of hydropathicity, was investigated using APPI as compared to electrospray ionization (ESI). Mass spectrometry experiments revealed that the most hydrophobic peptides were hardly ionized by ESI whereas all peptides, including the highly hydrophobic one that corresponds to the full sequence of the first transmembrane domain of CD9, were easily ionized by APPI. The native protein BmrA purified in the presence of the non-ionic detergent beta-D-dodecyl maltoside (DDM) was digested in-solution using trypsin. The resulting peptides were investigated by flow injection analysis of the mixture followed by mass spectrometry. Upon ESI, only detergent ions were detected and the ionic signals from the peptides were totally suppressed. In contrast, APPI allowed many peptides distributed along the sequence of the protein to be detected. Furthermore, the parent ion corresponding to the first transmembrane domain of the protein BmrA was detected under APPI conditions. Careful examination of the APPI mass spectrum revealed a-, b-, c- and y- fragment ions generated by in-source fragmentation. Those fragment ions allowed unambiguous structural characterization of the transmembrane domain. In conclusion, APPI–MS appears as a versatile method allowing the ionization and fragmentation of hydrophobic peptides in the presence of detergent.     

Marked difference in cytochrome c oxidation mediated by HO(*) and/or O(2)(*-) free radicals in vitro

Cytochrome c (cyt c) is an electron carrier involved in the mitochondrial respiratory chain and a critical protein in apoptosis. The oxidation of cytochrome c can therefore be relevant biologically. We studied whether cytochrome c underwent the attack of reactive oxygen species (ROS) during ionizing irradiation-induced oxidative stress. ROS were generated via water radiolysis under ionizing radiation (IR) in vitro. Characterization of oxidation was performed by mass spectrometry, after tryptic digestion, and UV-visible spectrophotometry. When both hydroxyl and superoxide free radicals were generated during water radiolysis, only five tryptic peptides of cyt c were reproducibly identified as oxidized according to a relation that was dependent of the dose of ionizing radiation. The same behavior was observed when hydroxyl free radicals were specifically generated (N(2)O-saturated solutions). Specific oxidation of cyt c by superoxide free radicals was performed and has shown that only one oxidized peptide (MIFAGIK+16), corresponding to the oxidation of Met80 into methionine sulfoxide, exhibited a radiation dose-dependent formation. In addition, the enzymatic site of cytochrome c was sensitive to the attack of both superoxide and hydroxyl radicals as observed through the reduction of Fe(3+), the degradation of the protoporphyrin IX and the oxidative disruption of the Met80-Fe(3+) bond. Noteworthy, the latter has been involved in the conversion of cyt c to a peroxidase. Finally, Met80 appears as the most sensitive residue towards hydroxyl but also superoxide free radicals mediated oxidation.     

A change of heart: oxidative stress in governing muscle function?

Biophysical Reviews - Redox/cysteine modification of proteins that regulate calcium cycling can affect contraction in striated muscles. Understanding the nature of these modifications would present...     

Mutations in CAPN1 Cause Autosomal-Recessive Hereditary Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is a genetically and clinically heterogeneous disease characterized by spasticity and weakness of the lower limbs with or without additional neurological symptoms. Although more than 70 genes and genetic loci have been implicated in HSP, many families remain genetically undiagnosed, suggesting that other genetic causes of HSP are still to be identified. HSP can be inherited in an autosomal-dominant, autosomal-recessive, or X-linked manner. In the current study, we performed whole-exome sequencing to analyze a total of nine affected individuals in three families with autosomal-recessive HSP. Rare homozygous and compound-heterozygous nonsense, missense, frameshift, and splice-site mutations in CAPN1 were identified in all affected individuals, and sequencing in additional family members confirmed the segregation of these mutations with the disease (spastic paraplegia 76 [SPG76]). CAPN1 encodes calpain 1, a protease that is widely present in the CNS. Calpain 1 is involved in synaptic plasticity, synaptic restructuring, and axon maturation and maintenance. Three models of calpain 1 deficiency were further studied. In Caenorhabditis elegans, loss of calpain 1 function resulted in neuronal and axonal dysfunction and degeneration. Similarly, loss-of-function of the Drosophila melanogaster ortholog calpain B caused locomotor defects and axonal anomalies. Knockdown of calpain 1a, a CAPN1 ortholog in Danio rerio, resulted in abnormal branchiomotor neuron migration and disorganized acetylated-tubulin axonal networks in the brain. The identification of mutations in CAPN1 in HSP expands our understanding of the disease causes and potential mechanisms.     

Safety and Immunogenicity of a Live Attenuated RSV Vaccine in Healthy RSV-Seronegative Children 5 to 24 Months of Age

Despite substantial morbidity associated with respiratory syncytial virus (RSV) infection, there is no licensed vaccine. MEDI-559 is a live attenuated intranasal vaccine candidate being developed for prevention of lower respiratory illness due to RSV in young children. This randomized, placebo-controlled study evaluated safety of MEDI-559 in healthy, RSV-seronegative children. MEDI-559 or placebo was administered on 3 occasions, 2 months apart. Primary safety was based on solicited symptoms (SSs) and adverse events (AEs) collected for 28 days after each dose. Nasal wash samples were collected 3 times after each dose (days 7-10, 12-18, 28-34) and at sick visits. Serum was collected for measuring antibody immune responses to RSV prior to first vaccination and 28 days post final dose. Long-term safety was monitored for 365 days from first dose. SSs were mild and frequent (MEDI-559 84%; placebo 91%); most common SSs were runny/stuffy nose, cough, and irritability/fussiness. AEs occurred in 67% MEDI-559 and 57% placebo recipients: most common AE was upper respiratory tract infection (MEDI-559 35%; placebo 23%). Higher incidence of medically attended lower respiratory illness within 28 days after dosing occurred in the MEDI-559 arm compared to placebo (none associated with vaccine virus shedding). There was no evidence of enhanced RSV disease. Vaccine virus was detected only in MEDI-559 recipients; shedding occurred in 56%subjects, primarily post dose 1. A functional immune response was observed in 59% and 9% MEDI-559 and placebo recipients, respectively, by an RSV microneutralization assay. Vaccine take, assessed by proportion that shed vaccine-type virus or had a seroresponse against RSV, was seen in 95% MEDI-559 subjects. MEDI-559 is therefore biologically active and immunogenic in this seronegative pediatric population. Although the frequency of SSs and AEs was not considered clinically significant, the increase in medically attended lower respiratory illnesses in the vaccine group warrants expanded safety studies.

Trial Registration

ClinicalTrials.gov NCT00767416