Microsomal and cytosolic cytochrome P450 mediated benzo(a)pyrene hydroxylation in Pleurotus pulmonarius

Summary Microsomal and soluble fractions of Pleurotus pulmonarius exhibited a reduced carbon monoxide difference spectrum with P450 maxima at 448nm and 450–452nm respectively. Substrate induced Type I spectra were observed on addition of benzo(a)pyrene to both fractions. Benzo(a)pyrene hydroxylation was measured using the aryl hydrocarbon hydroxylase assay and was observed to be P450 dependent as indicated by carbon monoxide inhibition together with the substrate binding characteristics. The activity of the fractions were observed to give K_m of 200mM and 660mM and V_max of 1.25 nmol/min/nmol P450 and 0.57 nmol/min/nmol P450 for the microsomal and cytosolic fractions respectively.     

Localization and kinetics of protein kinase C-epsilon anchoring in cardiac myocytes

Protein kinase C-epsilon (PKC-epsilon) plays a central role in cardiac cell signaling, but mechanisms of translocation and anchoring upon activation are poorly understood. Conventional PKC isoforms rely on a rapid Ca2+-mediated recruitment to cell membranes, but this mechanism cannot be employed by PKC-epsilon or other PKC isoforms lacking a Ca2+-binding domain. In this study, we used recombinant green fluorescent protein (GFP) fusion constructs and confocal microscopy to examine the localization, kinetics, and reversibility of PKC-epsilon anchoring in permeabilized rat cardiac myocytes. PKC-epsilon-GFP bound with a striated pattern that co-localized with alpha-actinin, a marker of the Z-line of the sarcomere. Binding required activation of PKC and occurred slowly but reversibly with apparent rate constants of k(on) = 4.6 +/- 1.2 x 10(3) M(-1) x s(-1) and k(off) = 1.4 +/- 0.5 x 10(-3) s(-1) (t1/2 = 8 min) as determined by fluorescence recovery after photobleaching and by perfusion experiments. A truncated construct composed of the N-terminal 144-amino-acid variable region of PKC-epsilon (epsilonV1-GFP), but not an analogous N-terminal domain of PKC-delta, mimicked the Z-line decoration and slow binding rate of the full-length enzyme. These findings suggest that the epsilonV1 domain is important in determining PKC-epsilon localization and translocation kinetics in cardiac muscle. Moreover, PKC-epsilon translocation is not a diffusion-controlled binding process but instead may be limited by intramolecular conformational changes within the V1 domain. The k(off) for epsilonV1-GFP was two- to threefold faster than for full-length enzyme, indicating that other domains in PKC-epsilon contribute to anchoring by prolonging the bound state.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.     

Dysferlin forms a dimer mediated by the C2 domains and the transmembrane domain in vitro and in living cells

Dysferlin was previously identified as a key player in muscle membrane repair and its deficiency leads to the development of muscular dystrophy and cardiomyopathy. However, little is known about the oligomerization of this protein in the plasma membrane. Here we report for the first time that dysferlin forms a dimer in vitro and in living adult skeletal muscle fibers isolated from mice. Endogenous dysferlin from rabbit skeletal muscle exists primarily as a ～460 kDa species in detergent-solubilized muscle homogenate, as shown by sucrose gradient fractionation, gel filtration and cross-linking assays. Fluorescent protein (YFP) labeled human dysferlin forms a dimer in vitro, as demonstrated by fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses. Dysferlin also dimerizes in living cells, as probed by fluorescence resonance energy transfer (FRET). Domain mapping FRET experiments showed that dysferlin dimerization is mediated by its transmembrane domain and by multiple C2 domains. However, C2A did not significantly contribute to dimerization; notably, this is the only C2 domain in dysferlin known to engage in a Ca-dependent interaction with cell membranes. Taken together, the data suggest that Ca-insensitive C2 domains mediate high affinity self-association of dysferlin in a parallel homodimer, leaving the Ca-sensitive C2A domain free to interact with membranes.     

Phosphomimetic mutations increase phospholamban oligomerization and alter the structure of its regulatory complex

To investigate the effect of phosphorylation on the interactions of phospholamban (PLB) with itself and its regulatory target, SERCA, we measured FRET from CFP-SERCA or CFP-PLB to YFP-PLB in live AAV-293 cells. Phosphorylation of PLB was mimicked by mutations S16E (PKA site) or S16E/T17E (PKA+CaMKII sites). FRET increased with protein concentration up to a maximum (FRET(max)) that was taken to represent the intrinsic FRET of the bound complex. The concentration dependence of FRET yielded dissociation constants (K(D)) for the PLB-PLB and PLB-SERCA interactions. PLB-PLB FRET data suggest pseudo-phosphorylation of PLB increased oligomerization of PLB but did not alter PLB pentamer quaternary structure. PLB-SERCA FRET experiments showed an apparent decrease in binding of PLB to SERCA and an increase in the apparent PLB-SERCA binding cooperativity. It is likely that these changes are secondary effects of increased oligomerization of PLB; a change in the inherent affinity of monomeric PLB for SERCA was not detected. In addition, PLB-SERCA complex FRET(max) was reduced by phosphomimetic mutations, suggesting the conformation of the regulatory complex is significantly altered by PLB phosphorylation.     

Methylated DNA/RNA in Body Fluids as Biomarkers for Lung Cancer

DNA/RNA methylation plays an important role in lung cancer initiation and progression. Liquid biopsy makes use of cells, nucleotides and proteins released from tumor cells into body fluids to help with cancer diagnosis and prognosis. Methylation of circulating tumor DNA (ctDNA) has gained increasing attention as biomarkers for lung cancer. Here we briefly introduce the biological basis and detection method of ctDNA methylation, and review various applications of methylated DNA in body fluids in lung cancer screening, diagnosis, prognosis, monitoring and treatment prediction. We also discuss the emerging role of RNA methylation as biomarkers for cancer.     

Phospholamban binds with differential affinity to calcium pump conformers

To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca(2+) elevations, but these increases in Ca(2+) produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca(2+) or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites.     

Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase

Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex.