Localization of a Mg- or Ca-activated (“basic”) ATPase in skeletal muscle

A chicken pectoralis muscle membrane fraction enriched in a Mg²⁺- or Ca²⁺-activated (‘basic’) ATPase was obtained by sucrose gradient centrifugation. Enzymatic properties of the ‘basic’ ATPase were determined and used to localize its enzymatic activity in situ by ultrastructural cytochemistry. The enzyme was activated by Mg²⁺ or Ca²⁺ but not by Sr²⁺, Ba²⁺, Co²⁺, Ni²⁺ or Pb²⁺. It was present in a membranous fraction with a buoyant density of 1.10-1.12 (24–27.5% (w/w) sucrose). ‘Basic’ ATPase activity had a sedimentation pattern similar to the putative plasma membrane enzymes, 5′-nucleotidase and leucyl β-naphthylamidase, but different from that of sarcoplasmic reticulum Ca²⁺ ATPase. Also unlike sarcoplasmic reticulum Ca²⁺ ATPase, ‘basic’ ATPase was resistant to N-ethylmaleimide and aldehyde fixatives, was active in a medium containing a high Ca²⁺ concentration (3 mM), and was lost when exposed to Triton X-100 or deoxycholate. In cytochemical studies, a low Pb²⁺ concentration was used to capture the enzymatically released phosphate ions. Under conditions which eliminated interfering (Na⁺ + K⁺) ATPase and sarcoplasmic reticulum Ca²⁺ ATPase activities, electron-dense lead precipitates were present at the plasmalemma and T-system membranes. These studies suggest that ‘basic’ ATPase activity is associated with plasmalemma and T-system membranes of skeletal muscle.     

Epistatic interactions in genetic regulation of t-PA and PAI-1 levels in a Ghanaian population

The proteins, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), act in concert to balance thrombus formation and degradation, thereby modulating the development of arterial thrombosis and excessive bleeding. PAI-1 is upregulated by the renin-angiotensin system (RAS), specifically by angiotensin II, the product of angiotensin converting enzyme (ACE) cleavage of angiotensin I, which is produced by the cleavage of angiotensinogen (AGT) by renin (REN). ACE indirectly stimulates the release of t-PA which, in turn, activates the corresponding fibrinolytic system. Single polymorphisms in these pathways have been shown to significantly impact plasma levels of t-PA and PAI-1 differently in Ghanaian males and females. Here we explore the involvement of epistatic interactions between the same polymorphisms in central genes of the RAS and fibrinolytic systems on plasma t-PA and PAI-1 levels within the same population (n = 992). Statistical modeling of pairwise interactions was done using two-way ANOVA between polymorphisms in the ETNK2, RENIN, ACE, PAI-1, t-PA, and AGT genes. The most significant interactions that associated with t-PA levels were between the ETNK2 A6135G and the REN T9435C polymorphisms in females (p = 0.006) and the REN T9435C and the TPA I/D polymorphisms (p = 0.005) in males. The most significant interactions for PAI-1 levels were with REN T9435C and the TPA I/D polymorphisms (p = 0.001) in females, and the association of REN G6567T with the TPA I/D polymorphisms (p = 0.032) in males. Our results provide evidence for multiple genetic effects that may not be detected using single SNP analysis. Because t-PA and PAI-1 have been implicated in cardiovascular disease these results support the idea that the genetic architecture of cardiovascular disease is complex. Therefore, it is necessary to consider the relationship between interacting polymorphisms of pathway specific genes that predict t-PA and PAI-1 levels.     

Can We Identify the Active Ingredients of Behaviour Change Interventions for Coronary Heart Disease Patients? A Systematic Review and Meta-Analysis

BackgroundThe main behaviour change intervention available for coronary heart disease (CHD) patients is cardiac rehabilitation. There is little recognition of what the active ingredients of behavioural interventions for CHD might be. Using a behaviour change technique (BCT) framework to code existing interventions may help to identify this. The objectives of this systematic review are to determine the effectiveness of CHD behaviour change interventions and how this may be explained by BCT content and structure.ConclusionsBehaviour change interventions for CHD patients appear to have a positive impact on a number of outcomes. Using an existing BCT taxonomy to code the interventions helped us to understand which were the most commonly used techniques, providing information and goal setting, but not the active components of these complex interventions.     

Seminal Plasma and Semen Amyloids Enhance Cytomegalovirus Infection in Cell Culture

Among the modes of transmission available to the cytomegalovirus (CMV) is sexual transmission, primarily via semen. Both male-to-female (M-F) and male-to-male (M-M) sexual transmission significantly contribute toward the spread of CMV infections in the global population. Semen plays an important role in carrying the viral particle that invades the vaginal or rectal mucosa, thereby initiating viral replication. Both semen and seminal plasma (SP) can enhance HIV-1 infection in cell culture, and two amyloid fibrils, semen-derived enhancer of viral infection (SEVI) and amyloids derived from the semenogelins (SEM amyloids), have been identified as seminal factors sufficient to enhance HIV-1 infection (J. Munch et al., Cell 131:1059–1071, 2007; N. R. Roan et al., Cell Host Microbe 10:541–550, 2011; F. Arnold et al., J. Virol. 86:1244–1249, 2012). Whether SP, SEVI, or SEM amyloids can enhance other viral infections has not been extensively examined. In this study, we found that SP, SEVI, and SEM amyloids strongly enhance both human CMV (HCMV) and murine CMV infection in cell culture. SEVI and SEM amyloids increased infection rates by >10-fold, as determined by both flow cytometry and fluorescence microscopy. Viral replication was increased by 50- to 100-fold. Moreover, viral growth curve assays showed that SP, SEVI, and SEM amyloids sped up the kinetics of CMV replication such that the virus reached its replicative peak more quickly. Finally, we discovered that SEM amyloids and SEVI counteracted the effect of anti-gH in protecting against CMV infection. Collectively, the data suggest that semen enhances CMV infection through interactions between semen amyloid fibrils and viral particles, and these interactions may prevent HCMV from being neutralized by anti-gH antibody.     

Deciphering the structural role of histidine 83 for heme binding in hemophore HasA

Heme carrier HasA has a unique type of histidine/tyrosine heme iron ligation in which the iron ion is in a thermally driven two spin states equilibrium. We recently suggested that the H-bonding between Tyr75 and the invariantly conserved residue His83 modulates the strength of the iron-Tyr75 bond. To unravel the role of His83, we characterize the iron ligation and the electronic properties of both wild type and H83A mutant by a variety of spectroscopic techniques. Although His83 in wild type modulates the strength of the Tyr-iron bond, its removal causes detachment of the tyrosine ligand, thus giving rise to a series of pH-dependent equilibria among species with different axial ligation. The five coordinated species detected at physiological pH may represent a possible intermediate of the heme transfer mechanism to the receptor.     

Barley γ3-hordein: Glycosylation at an atypical site,disulfide bridge analysis,and reactivity with IgE from patients allergic to wheat

Post translational modifications of a seed storage protein, barley γ3-hordein, were determined using immunochemical and mass spectrometry methods. IgE reactivity towards this protein was measured using sera from patients diagnosed with allergies to wheat. N-glycosylation was found at an atypical Asn-Leu-Cys site. The observed glycan contains xylose. This indicates that at least some γ3-hordein molecules trafficked through the Golgi apparatus. Disulfide bridges in native γ3-hordein were almost the same as those found in wheat γ46-gliadin, except the bridge involving the cysteine included in the glycosylation site. IgE reacted more strongly towards the recombinant than the natural γ3-hordein protein. IgE binding to γ3-hordein increased when the protein sample was reduced. Glycosylation and disulfide bridges therefore decrease epitope accessibility. Thus the IgE from patients sensitized to wheat cross-react with γ3-hordein due to sequence homology with wheat allergens rather than through shared carbohydrate determinants.     

The integrative role of cryo electron microscopy in molecular and cellular structural biology

After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo‐EM) is now heading off at unprecedented speed towards high‐resolution analysis of biological objects of various sizes. This ‘revolution in resolution’ is happening largely thanks to new developments of new‐generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo‐EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo‐EM in synergy with other methods such as X‐ray crystallography, fluorescence imaging or focussed‐ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi‐scale and multi‐resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.     

PrimPol-deficient cells exhibit a pronounced G2 checkpoint response following UV damage

PrimPol is a recently identified member of the archaeo-eukaryote primase (AEP) family of primase-polymerases. It has been shown that this mitochondrial and nuclear localized enzyme plays roles in the maintenance of both unperturbed replication fork progression and in the bypass of lesions after DNA damage. Here, we utilized an avian (DT40) knockout cell line to further study the consequences of loss of PrimPol (PrimPol^?/?) on the downstream maintenance of cells after UV damage. We report that PrimPol^?/? cells are more sensitive to UV-C irradiation in colony survival assays than Pol η-deficient cells. Although this increased UV sensitivity is not evident in cell viability assays, we show that this discrepancy is due to an enhanced checkpoint arrest after UV-C damage in the absence of PrimPol. PrimPol^?/? arrested cells become stalled in G2, where they are protected from UV-induced cell death. Despite lacking an enzyme required for the bypass and maintenance of replication fork progression in the presence of UV damage, we show that PrimPol^?/? cells actually have an advantage in the presence of a Chk1 inhibitor due to their slow progression through S-phase.