Inhibition of Neuronal Cell Mitochondrial Complex I with Rotenone Increases Lipid β-Oxidation,Supporting Acetyl-Coenzyme A Levels

Rotenone is a naturally occurring mitochondrial complex I inhibitor with a known association with parkinsonian phenotypes in both human populations and rodent models. Despite these findings, a clear mechanistic link between rotenone exposure and neuronal damage remains to be determined. Here, we report alterations to lipid metabolism in SH-SY5Y neuroblastoma cells exposed to rotenone. The absolute levels of acetyl-CoA were found to be maintained despite a significant decrease in glucose-derived acetyl-CoA. Furthermore, palmitoyl-CoA levels were maintained, whereas the levels of many of the medium-chain acyl-CoA species were significantly reduced. Additionally, using isotopologue analysis, we found that β-oxidation of fatty acids with varying chain lengths helped maintain acetyl-CoA levels. Rotenone also induced increased glutamine utilization for lipogenesis, in part through reductive carboxylation, as has been found previously in other cell types. Finally, palmitoylcarnitine levels were increased in response to rotenone, indicating an increase in fatty acid import. Taken together, these findings show that alterations to lipid and glutamine metabolism play an important compensatory role in response to complex I inhibition by rotenone.     

Observational study to characterise 24-hour COPD symptoms and their relationship with patient-reported outcomes: results from the ASSESS study

Background

Few studies have investigated the 24-hour symptom profile in patients with COPD or how symptoms during the 24-hour day are inter-related. This observational study assessed the prevalence, severity and relationship between night-time, early morning and daytime COPD symptoms and explored the relationship between 24-hour symptoms and other patient-reported outcomes.

Methods

The study enrolled patients with stable COPD in clinical practice. Baseline night-time, early morning and daytime symptoms (symptom questionnaire), severity of airflow obstruction (FEV₁), dyspnoea (modified Medical Research Council Dyspnoea Scale), health status (COPD Assessment Test), anxiety and depression levels (Hospital Anxiety and Depression Scale), sleep quality (COPD and Asthma Sleep Impact Scale) and physical activity level (sedentary, moderately active or active) were recorded.

Results

The full analysis set included 727 patients: 65.8% male, mean ± standard deviation age 67.2 ± 8.8 years, % predicted FEV₁ 52.8 ± 20.5%.In each part of the 24-hour day, >60% of patients reported experiencing ≥1 symptom in the week before baseline. Symptoms were more common in the early morning and daytime versus night-time (81.4%, 82.7% and 63.0%, respectively). Symptom severity was comparable for each period assessed. Overall, in the week before baseline, 56.7% of patients had symptoms throughout the whole 24-hour day (3 parts of the day); 79.9% had symptoms in ≥2 parts of the 24-hour day. Symptoms during each part of the day were inter-related, irrespective of disease severity (all p < 0.001).Early morning and daytime symptoms were associated with the severity of airflow obstruction (p < 0.05 for both). Night-time, early morning and daytime symptoms were all associated with worse dyspnoea, health status and sleep quality, and higher anxiety and depression levels (all p < 0.001 versus patients without symptoms in each corresponding period). In each part of the 24-hour day, there was also an association between symptoms and a patient’s physical activity level (p < 0.05 for each period).

Conclusions

More than half of patients experienced COPD symptoms throughout the whole 24-hour day. There was a significant relationship between night-time, early morning and daytime symptoms. In each period, symptoms were associated with worse patient-reported outcomes, suggesting that improving 24-hour symptoms should be an important consideration in the management of COPD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0122-1) contains supplementary material, which is available to authorized users.     

Bacillus thuringiensis as a surrogate for Bacillus anthracis in aerosol research

Characterization of candidate surrogate spores prior to experimental use is critical to confirm that the surrogate characteristics are as closely similar as possible to those of the pathogenic agent of interest. This review compares the physical properties inherent to spores of Bacillus anthracis (Ba) and Bacillus thuringiensis (Bt) that impact their movement in air and interaction with surfaces, including size, shape, density, surface morphology, structure and hydrophobicity. Also evaluated is the impact of irradiation on the physical properties of both Bacillus species. Many physical features of Bt and Ba have been found to be similar and, while Bt is considered typically non-pathogenic, it is in the B. cereus group, as is Ba. When cultured and sporulated under similar conditions, both microorganisms share a similar cylindrical pellet shape, an aerodynamic diameter of approximately 1 μm (in the respirable size range), have an exosporium with a hairy nap, and have higher relative hydrophobicities than other Bacillus species. While spore size, morphology, and other physical properties can vary among strains of the same species, the variations can be due to growth/sporulation conditions and may, therefore, be controlled. Growth and sporulation conditions are likely among the most important factors that influence the representativeness of one species, or preparation, to another. All Bt spores may, therefore, not be representative of all Ba spores. Irradiated spores do not appear to be a good surrogate to predict the behavior of non-irradiated spores due to structural damage caused by the irradiation. While the use of Bt as a surrogate for Ba in aerosol testing appears to be well supported, this review does not attempt to narrow selection between Bt strains. Comparative studies should be performed to test the hypothesis that viable Ba and Bt spores will behave similarly when suspended in the air (as an aerosol) and to compare the known microscale characteristics versus the macroscale response.     

A benefit-finding intervention for family caregivers of persons with Alzheimer disease: study protocol of a randomized controlled trial

Background

Patients with ST-elevation myocardial infarction (STEMI) not treated with primary or rescue percutaneous coronary intervention (PCI) are at risk for recurrent ischemia, especially when viability in the infarct-area is present. Therefore, an invasive strategy with PCI of the infarct-related coronary artery in patients with viability would reduce the occurrence of a composite end point of death, reinfarction, or unstable angina (UA).

Methods

Patients admitted with an (sub)acute myocardial infarction, who were not treated by primary or rescue PCI, and who were stable during the first 48 hours after the acute event, were screened for the study. Eventually, we randomly assigned 216 patients with viability (demonstrated with low-dose dobutamine echocardiography) to an invasive or a conservative strategy. In the invasive strategy stenting of the infarct-related coronary artery was intended with abciximab as adjunct treatment. Seventy-five (75) patients without viability served as registry group. The primary endpoint was the composite of death from any cause, recurrent myocardial infarction (MI) and unstable angina at one year. As secondary endpoint the need for (repeat) revascularization procedures and anginal status were recorded.

Results

The primary combined endpoint of death, recurrent MI and unstable angina was 7.5% (8/106) in the invasive group and 17.3% (19/110) in the conservative group (Hazard ratio 0.42; 95% confidence interval [CI] 0.18-0.96; p = 0.032). During follow up revascularization-procedures were performed in 6.6% (7/106) in the invasive group and 31.8% (35/110) in the conservative group (Hazard ratio 0.18; 95% CI 0.13-0.43; p < 0.0001). A low rate of recurrent ischemia was found in the non-viable group (5.4%) in comparison to the viable-conservative group (14.5%). (Hazard-ratio 0.35; 95% CI 0.17-1.00; p = 0.051).

Conclusion

We demonstrated that after acute MI (treated with thrombolysis or without reperfusion therapy) patients with viability in the infarct-area benefit from a strategy of early in-hospital stenting of the infarct-related coronary artery. This treatment results in a long-term uneventful clinical course. The study confirmed the low risk of recurrent ischemia in patients without viability.

Trial registration

ClinicalTrials.gov: NCT00149591.     

Adhesion dynamics: mechanisms and measurements

Adhesion to the extracellular matrix (ECM) is a fundamental requirement for survival, differentiation and migration of numerous cell types during both embryonic development and adult homeostasis. Different types of adhesion structures have been classified in different cell types or tissue environments. The best studied of these are focal adhesions which are found on a wide variety of cell types and will be the main focus of this review. Many years of research into the control of adhesion has yielded a wealth of information regarding the complexity of protein composition of these critical points of cell:ECM contact. Moreover, it has emerged that adhesions are not only highly ordered, but also dynamic structures under tight spatial control at the subcellular level to enable localised responses to extracellular cues. However, it is only in the last decade that the relative dynamics of these adhesion proteins have been closely studied. Here we provide an overview of the imaging strategies that have been developed and implemented to study the intricacies and hierarchy of protein turnover within focal adhesions. The caveats of employing these imaging techniques, as well as future directions will also be discussed.     

The reconstruction of side-chain conformations from protein Cα coordinates

A model of nine proteins including side-chain atoms have been built from the known C^α coordinates and amino acid sequences using a Monte Carlo Protein Building Annealing method. The Cartesian coordinates for the side-chain atoms were established with bond lengths and angles selected randomly from within previously determined ranges. A simulated annealing technique is used to generate some 300 structures with differing side-chain conformations. The atomic coordinates of the backbone atoms are fixed during the simulated annealing process. The coordinates of the side-chain atoms of 300 low energy conformations are averaged to obtain a mean structure that is minimized with the C^α atoms constrained to their position in the x-ray structure using the OPLS/AMBER force field with the GB/SA water model. The rms deviation of the main-chain atoms (without C^β) compared with the corresponding crystal structures is in the range 0.20–0.64 Å. The rms deviation of the side-chain atoms is between 1.72 and 2.71 Å and for all atoms is between 1.19 and 1.99 Å. The method is insensitive to random errors in the C^α positions and the computational requirement is modest. © 1997 John Wiley & Sons, Inc.     

Drebrin contains a cryptic F-actin–bundling activity regulated by Cdk5 phosphorylation

Drebrin is an actin filament (F-actin)–binding protein with crucial roles in neuritogenesis and synaptic plasticity. Drebrin couples dynamic microtubules to F-actin in growth cone filopodia via binding to the microtubule-binding +TIP protein EB3 and organizes F-actin in dendritic spines. Precisely how drebrin interacts with F-actin and how this is regulated is unknown. We used cellular and in vitro assays with a library of drebrin deletion constructs to map F-actin binding sites. We discovered two domains in the N-terminal half of drebrin—a coiled-coil domain and a helical domain—that independently bound to F-actin and cooperatively bundled F-actin. However, this activity was repressed by an intramolecular interaction relieved by Cdk5 phosphorylation of serine 142 located in the coiled-coil domain. Phospho-mimetic and phospho-dead mutants of serine 142 interfered with neuritogenesis and coupling of microtubules to F-actin in growth cone filopodia. These findings show that drebrin contains a cryptic F-actin–bundling activity regulated by phosphorylation and provide a mechanistic model for microtubule–F-actin coupling.     

INTRAVENOUS CHOLANGIOGRAPHY—Some Observations on the Use of Cholografin

Intravenous cholangiography with cholografin is a safe procedure, most useful for the study of patients who have had cholecystectomy and later have symptoms related to the biliary ducts.When jaundice or liver impairment is present, the examination is usually unsuccessful. However, these conditions are not absolute contraindications to the procedure. There may be failure to visualize the biliary ducts even in the presence of a normal liver.Planigraphy is helpful in eliminating confusing superimposed structures and when there is only faint visualization of the common duct.Intravenous cholecystography is only of questionable value as a supplementary examination to oral cholecystography. It may prove useful in certain instances when patients are unable to retain or absorb the oral media or where emergency operation is contemplated.     

Two soluble glycosyltransferases glycosylate less efficiently in vivo than their membrane bound counterparts

Many Golgi glycosyltransferases are type II membrane proteins which are cleaved to produce soluble forms that are released from cells. Cho and Cummings recently reported that a soluble form of alpha1, 3- galactosyltransferase was comparable to its membrane bound counterpart in its ability to galactosylate newly synthesized glycoproteins (Cho,S.K. and Cummings,R.D. (1997) J. Biol. Chem., 272, 13622-13628). To test the generality of their findings, we compared the activities of the full length and soluble forms of two such glycosyltransferases, ss1,4 N-Acetylgalactosaminyltransferase (GM2/GD2/ GA2 synthase; GalNAcT) and beta galactoside alpha2,6 sialyltransferase (alpha2,6-ST; ST6Gal I), for production of their glycoconjugate products in vivo . Unlike the full length form of GalNAcT which produced ganglioside GM2 in transfected cells, soluble GalNAcT did not produce detectable GM2 in vivo even though it possessed in vitro GalNAcT activity comparable to that of full length GalNAcT. When compared with cells expressing full length alpha2,6-ST, cells expressing a soluble form of alpha2,6-ST contained 3-fold higher alpha2,6-ST mRNA levels and secreted 7-fold greater alpha2,6-ST activity as measured in vitro , but in striking contrast contained 2- to 4-fold less of the alpha2,6-linked sialic acid moiety in cellular glycoproteins in vivo . In summary these results suggest that unlike alpha1,3-galactosyltransferase the soluble forms of these two glycosyltransferases are less efficient at glycosylation of membrane proteins and lipids in vivo than their membrane bound counterparts.