Structural and Functional Impact of Parkinson Disease‐Associated Mutations in the E3 Ubiquitin Ligase Parkin

Mutations in the PARKIN/PARK2 gene that result in loss‐of‐function of the encoded, neuroprotective E3 ubiquitin ligase Parkin cause recessive, familial early‐onset Parkinson disease. As an increasing number of rare Parkin sequence variants with unclear pathogenicity are identified, structure–function analyses will be critical to determine their disease relevance. Depending on the specific amino acids affected, several distinct pathomechanisms can result in loss of Parkin function. These include disruption of overall Parkin folding, decreased solubility, and protein aggregation. However pathogenic effects can also result from misregulation of Parkin autoinhibition and of its enzymatic functions. In addition, interference of binding to coenzymes, substrates, and adaptor proteins can affect its catalytic activity too. Herein, we have performed a comprehensive structural and functional analysis of 21 PARK2 missense mutations distributed across the individual protein domains. Using this combined approach, we were able to pinpoint some of the pathogenic mechanisms of individual sequence variants. Similar analyses will be critical in gaining a complete understanding of the complex regulations and enzymatic functions of Parkin. These studies will not only highlight the important residues, but will also help to develop novel therapeutics aimed at activating and preserving an active, neuroprotective form of Parkin.     

Headgroup interactions in mixed phospholipid bilayers

²H NMR methods have been used to study how bilayer-forming phospholipids interact with each other at the membrane surface. Aqueous dispersions of dimyristoyl-sn-phosphatidylcholine (Myr₂-PtdCho), dimyristoyl-sn-phosphatidylethanolamine (Myr₂-PtdEtn), and dimyristoyl-sn-phosphatidyl-3-glycerol, specifically deuterated at different positions in their headgroups, give well-resolved ²H NMR powder spectra. These spectra are characteristic of a lipid bilayer with quadrupole splittings that are sensitive to the amplitude of headgroup motion. In binary mixed bilayers of deuterated lipids with an unlabeled component, all parts of the deuterated headgroup monitor the presence of the second lipid from changes in the measured quadrupole splittings. The headgroups of the charged lipids, dimyristoyl-sn-phosphatidylserine and dimyristoyl-sn-phosphatidyl-3-glycerol, interact to the largest extent with the choline moiety of Myr₂-PtdCho and the ethanolamine moiety of Myr₂-PtdEtn, whereas a somewhat smaller but still marked alteration in headgroup motion was observed in Myr₂-PtdCho/Myr₂-PtdEtn mixtures. The large changes in the deuterium quadrupole splittings for the zwitterionic lipids after addition of a charged lipid suggest that either a strong perturbation in the hydrogen bonding occurs or changes take place in the water structure at the membrane surface, or possibly both.     

Parkinson’s disease-associated DJ-1 modulates innate immunity signaling in Caenorhabditis elegans

DJ-1 is a neuroprotective gene mutated in recessive Parkinson’s disease (PD). In addition to direct protective functions in neurons, DJ-1 regulates neuroinflammatory signaling in primary mouse brain astrocytes. To assess the influence of DJ-1 on innate immunity signaling in vivo, we have generated djr-1 knockout Caenorhabditis elegans. When grown on pathogenic gram-negative bacteria, djr-1 ^−/− worms showed stronger phosphorylation of p38 mitogen-activated protein kinase (PMK-1) and hyper-induction of PMK-1 target genes. Thus, PD-associated DJ-1 contributes to regulation of innate immunity.     

CPR courses and semi-automatic defibrillators--life saving in cardiac arrest?

OBJECTIVES: The aim was to assess the knowledge of life-supporting first-aid in both cardiac arrest survivors and relatives, and their willingness to have a semi-automatic external defibrillator in their homes and use it in an emergency. MATERIAL AND METHODS: Cardiac arrest survivors, their families, friends, neighbours and co-workers were interviewed by medical students using prepared questionnaires. Their knowledge and self-assessment of life-supporting first-aid, their willingness to have a semi-automatic defibrillator in their homes and their willingness to use it in an emergency before and after a course in cardiopulmonary resuscitation (CPR) with a semi-automatic external defibrillator was evaluated. Courses were taught by medical students who had received special training in basic and advanced life support. RESULTS: Both patients and relatives, after a course of 2-3 h, were no longer afraid of making mistakes by providing life-supporting first-aid. The automated external defibrillator (AED) was generally accepted and considered easy to handle. CONCLUSION: We consider equipping high-risk patients and their families with AEDs as a viable method of increasing their survival in case of a recurring cardiac arrest. This, of course, should be corroborated by further studies.     

Prevalence of arbovirus antibodies in sera of animals in Sri Lanka

The sera of cattle, goats, dogs and crows from the Colombo area were tested for antibodies against seven arboviruses of the families Togaviridae and Bunyaviridae by a plaque-reduction neutralization microtest, using Vero cells and a stable line of pig kidney (PS) cells. The overall percentages of positive sera among the mammals were: Bhanja, 92.5%; Calovo (Batai), 30.6%; Sindbis, 13.8%; Langat, 4.8%; Tahyna, 3.9%; West Nile, 1.6%. Among the birds, 23.8% had antibodies to Bhanja virus and 9.5% to Sindbis. No antibodies against tick-borne encephalitis virus were found. The results show that at least two members of the Bunyaviridae family (Bhanja and Calovo) are highly endemic in Colombo.     

Post-mortem modifications of the ependyma of the lateral ventricular wall

Summary The ependymal lining of the lateral ventricles of the brain of rats, rabbits, and man was investigated at several times after death. In contrast to control material that was fixed by the aldehyde perfusing method, the following post-mortem (p.m.) changes were found.(1) Cytoplasmic protrusions of ependymal cells appear 15 min p.m. They are present up to several hours after death. (2) The formation of these protrusions causes the tufts of cilia to clump together and later to become integrated within the ependymal cell. This may simulate an unciliated surface. (3) Small porelike holes, which are present 15 min p.m. in the ependymal cell membrane, enlarge and in later stages produce a meshwork of fibers instead of a closed ependymal lining. (4) TEM observation shows that ependymal cells are separated from each other very soon after death by intercellular gaps. Cell junctions between ependymal cells resist separation over a longer p.m. period.In animal or human material that is fixed at any time after death, such modifications have to be considered very critically. In human p.m. autopsy material they are mostly the expression of a p.m. alteration.     

Cardiac arrest in public locations-An independent predictor for better outcome?

OBJECTIVE: Outcome after cardiac arrest is known to be influenced by immediate access to resuscitation. We aimed to analyse the location of arrest in relation to the prognostic value for outcome. DESIGN: Retrospective review from prospective databases (ambulance routine documentation database and emergency department database on patients treated for cardiac arrest). Setting: Vienna (1.7 million inhabitants) ambulance service and tertiary care facility (university clinics). Patients: Two independent cohorts: (1) a population-based cohort of patients who were treated for cardiac arrest by the municipal ambulance service outside the hospital. The endpoint in this group was survival to hospital admission with spontaneous circulation. (2) A cohort of patients who were admitted to the emergency department after successful out of hospital resuscitation. The endpoint in this group was survival to 6 months with good neurological status (best Cerebral Performance Category 1 or 2 within 6 months). MEASUREMENTS: We analysed whether the location of non-traumatic adult out-of-hospital cardiac arrest (public versus private place) was a predictor for good outcome. RESULTS: Patients who had cardiac arrest in a public location were more likely to arrive in hospital alive (39% versus 31%, crude OR 1.4, 95% CI 1.001-1.975, p=0.049) and were more likely to have a good neurological outcome after 6 months (35% versus 25%, crude OR 1.65, adjusted OR 1.59, 95% CI 1.07-2.36, p=0.023), compared to patients who had cardiac arrest in a non-public location. CONCLUSION: Cardiac arrest in a public location is independently associated with a better outcome.     

Gut-derived bone infection in the neonatal rat.

The risk of osteomyelitis is increased in the premature and critically ill neonate. Although potential sites of bacterial entry are present in many of these infants, the source of infection frequently cannot be established. This study was performed to assess the possible role of bacterial translocation from the intestine in the origin of bone infection using models of breast-fed and formula-fed rat pups. Newborn Sprague-Dawley rats suckled either ad libitum by the dam (n = 30), or were fed a rat milk-simulated formula (n = 30). After 3 d, the animals were killed, and the left femur, heart blood, mesenteric lymph nodes, liver, spleen, and terminal ileum were excised. Organs were analyzed for bacteria by standard microbiologic procedures. Bacterial translocation occurred in 23% of breast-fed rats; the bone was not infected in any of these animals. After feeding of formula diet, bacterial counts of the ileum were markedly elevated (p < 0.001), and the composition of the gut flora was disrupted. Bacterial translocation was noted in all formula-fed rats. Bone cultures were positive in 23 of 30 (77%) rats after formula-feeding (p < 0.001 versus breast-feeding). Organisms translocated to the bone included Enterococci, Proteus, Enterobacter, and Escherichia coli. Bacterial species cultured from the bone correlated with the individual colonization pattern of other extraintestinal organs and with the composition of the ileal flora. Members of the gut flora can escape the intestine and colonize the bone in formula-fed rats. The gut should be considered as a potential source for osteomyelitis in the neonate.