Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid

We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid.     

Quantitative determination of ferritin by electroimmunoassay

A method for the quantitative determination of tissue ferritin protein is described. It is based on the electroimmunoassay of Laurell [Laurell, C. B. (1966) Anal. Biochem.15, 45–52] and uses the iron content of ferritin for its identification. It measures as little as 0.1 μg of ferritin protein, requires only a few milligrams of tissue, and is rapidly performed.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I₅₀ = 2 μg/ml) when compared to other naturally occurring glycosaminoglycans. This inhinibition was also appparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E ₂. Heparin was also found to inhibit glucagon-sensitive rat hepatice adenylate cyclase, and the prostaglandin E₁-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfade polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.     

Variation in phosphorus and sulfur content shapes the genetic architecture and phenotypic associations within the wheat grain ionome

Dissection of the genetic basis of wheat ionome is crucial for understanding the physiological and biochemical processes underlying mineral accumulation in seeds, as well as for efficient crop breeding. Most of the elements essential for plants are metals stored in seeds as chelate complexes with phytic acid or sulfur‐containing compounds. We assume that the involvement of phosphorus and sulfur in metal chelation is the reason for strong phenotypic correlations within ionome. Adjustment of element concentrations for the effect of variation in phosphorus and sulfur seed content resulted in drastic change of phenotypic correlations between the elements. The genetic architecture of wheat grain ionome was characterized by quantitative trait loci (QTL) analysis using a cross between durum and wild emmer wheat. QTL analysis of the adjusted traits and two‐trait analysis of the initial traits paired with either P or S considerably improved QTL detection power and accuracy, resulting in the identification of 105 QTLs and 617 QTL effects for 11 elements. Candidate gene search revealed some potential functional associations between QTLs and corresponding genes within their intervals. Thus, we have shown that accounting for variation in P and S is crucial for understanding of the physiological and genetic regulation of mineral composition of wheat grain ionome and can be implemented for other plants.     

Putting the Pieces Together: High-performance LC-MS/MS Provides Network-, Pathway-, and Protein-level Perspectives in Populus

High-performance mass spectrometry (MS)-based proteomics enabled the construction of a detailed proteome atlas for Populus, a woody perennial plant model organism. Optimization of experimental procedures and implementation of current state-of-the-art instrumentation afforded the most detailed look into the predicted proteome space of Populus, offering varying proteome perspectives: (1) network-wide, (2) pathway-specific, and (3) protein-level viewpoints. Together, enhanced protein retrieval through a detergent-based lysis approach and maximized peptide sampling via the dual-pressure linear ion trap mass spectrometer (LTQ Velos), have resulted in the identification of 63,056 tryptic peptides. The technological advancements, specifically spectral-acquisition and sequencing speed, afforded the deepest look into the Populus proteome, with peptide abundances spanning 6 orders of magnitude and mapping to ∼25% of the predicted proteome space. In total, tryptic peptides mapped to 11,689 protein assignments across four organ-types: mature (fully expanded, leaf plastichronic index (LPI) 10–12) leaf, young (juvenile, LPI 4–6) leaf, root, and stem. To resolve protein ambiguity, identified proteins were grouped by sequence similarity (≥ 90%), thereby reducing the protein assignments into 7538 protein groups. In addition, this large-scale data set features the first systems-wide survey of protein expression across different Populus organs. As a demonstration of the precision and comprehensiveness of the semiquantitative analysis, we were able to contrast two stages of leaf development, mature versus young leaf. Statistical comparison through ANOVA analysis revealed 1432 protein groups that exhibited statistically significant (p ≤ 0.01) differences in protein abundance. Experimental validation of the metabolic circuitry expected in mature leaf (characterized by photosynthesis and carbon fixation) compared with young leaf (characterized by rapid growth and moderate photosynthetic activities) strongly testifies to the credibility of the approach. Instead of quantitatively comparing a few proteins, a systems view of all the changes associated with a given cellular perturbation could be made.Mass spectrometry (MS)-based proteomics has experienced tremendous growth in recent years, leading to the establishment of numerous protocols, platforms, and workflows for the characterization of protein expression at the genome level (1). Although these advancements have facilitated comprehensive proteomic investigations of simple bacterial isolates and microbial communities, the application of MS-based proteomics for plants and other higher eukaryotes remains underdeveloped. Recently, large-scale proteomic studies have been directed at characterization of Populus, a woody perennial model organism. With the recent release and subsequent curation of the P. trichocarpa genome (2), these large-scale MS-based proteomic investigations offer the potential to introduce new biological insights into woody perennial plant biology (3, 4, 5). For example, we have recently demonstrated the ability to measure ∼17% of the Populus proteome by coupling multidimensional liquid chromatography (MudPIT)¹ with nano-electrospray tandem mass spectrometry (2D-LC-MS/MS) (6). Relative to the two-dimensional gel-based approaches (7), MudPIT provides enhanced separation and when used in conjunction with MS/MS, surpasses the throughput and number of identifiable proteins detected in complex mixtures (8). Although we have demonstrated the general effectiveness of this approach, the identification and quantitation of the proteins expressed in a plant cell or tissue are still notoriously complicated by a number of factors, including the size and complexity of plant genomes, abundance of protein variants, as well as the dynamic range of protein identification. To overcome these challenges, improvements are needed in sample preparation, MS instrumentation, and data interpretation.The architecture of plant cell walls provides resistance to chemical and biological degradation, thus requiring mechanical and detergent-based lysis for optimal proteome analysis. However, this criterion presents a major challenge for plant proteomic research using electrospray mass spectrometry, as detergent-containing solutions can impede enzymatic digestion and cause significant analyte suppression (9). Therefore, most plant proteomic studies using the “MudPIT” strategy apply mechanical disruption in conjunction with a detergent-free preparation method (10). Typically, strong chaotropic agents such as urea and guanidine hydrochloride are used for the extraction, denaturation, and digestion of proteins. In a recent study, Mann et al. (2009) introduced a filter-aided sample preparation (FASP) method that uses and effectively removes sodium dodecyl sulfate (SDS) before enzymatic digestion and electrospray analysis (11). This study demonstrated enhanced retrieval of peptides from biological materials, yielding a more accurate representation of the proteome. We developed a similar experimental approach for extraction of proteins from plant tissue to obtain a more comprehensive, unbiased proteome characterization well beyond that achievable with currently available methods. Similar to the FASP method, we demonstrate the power of SDS for proteomic sample preparation, not only in its ability to more-thoroughly lyse cells, but also its ability to better solubilize both hydrophilic and hydrophobic proteins. This powerful attribute gives proteolytic enzymes maximum opportunity to generate peptides specific to their cleavage potential so that at least a few representative peptides can be obtained for proteins that would have otherwise been discarded or lost because of insolubility, e.g. membrane-bound proteins. Rather than performing a buffer exchange with urea, depletion of SDS is achieved by precipitating proteins out of solution using trichloroacetic acid.Characterization of protein expression in plants is further complicated by the heterogeneous mixture of various cell types, each with a unique proteome signature and individualized response to environmental chemical or physical signals. This inherent complexity of plant proteomes and the large dynamic range in protein abundance overwhelms current analytical platforms (12). Moreover, biochemical regulatory networks in plants are more elaborate and dynamic than in microbial species; consequently, many biological components are left undiscovered, including modified peptides and low-abundance proteins (13, 14, 15). Recent developments in ion-trap MS instrumentation, namely the dual-pressure linear ion trap mass spectrometer (LTQ Velos), have demonstrated improved ability to comprehensively characterize complex proteomics samples (16). Featuring a newly designed ion source and a two-chamber ion trap mass analyzer, the LTQ Velos achieves greater dynamic range, sensitivity, and speed of spectral acquisition when applied to complex proteomic samples. Cumulatively, the technological advancements afford substantial increases in the detection and identification of both proteins and unique peptides when compared with existing state-of-the-art technologies. Therefore, to satisfy the need for depth of proteome characterization in plants, we apply the newly developed LTQ Velos for mass spectrometry measurements of the Populus proteome.For most terrestrial plants, life begins and ends in the same physical location. For woody perennial plants, this sedentary lifestyle may last thousands of years. One consequence of this lifestyle is that each plant typically experiences dramatic changes in its ambient environment throughout its lifetime and, at any given time, equilibrium between endogenous growth processes and exogenous constraints exerted by the environment must be tightly controlled. To survive under varying environmental conditions, temporal plastic responses evoke patterns of protein expression that progressively influence morphological, anatomical, and functional traits of three principal organs—leaf, root, and stem. Collectively and individually, these organs operate to perceive and respond to periodic and chronic environment conditions. Currently, a comprehensive understanding of the spatial variation in protein expression patterns across the organ types is lacking for woody perennial plants, in which most large-scale proteome analyses with Populus were performed on isolated organs, tissues, organelles, or subcellular structures. For this reason, we combined the state-of-the-art LTQ-Velos platform with the SDS/TCA sample preparation methodology to generate a high-coverage proteome atlas of the principal organ types from Populus.     

Maintenance of temporal synchrony between syrphid flies and floral resources despite differential phenological responses to climate

Variation in species’ responses to abiotic phenological cues under climate change may cause changes in temporal overlap among interacting taxa, with potential demographic consequences. Here, we examine associations between the abiotic environment and plant–pollinator phenological synchrony using a long‐term syrphid fly–flowering phenology dataset (1992–2011). Degree‐days above freezing, precipitation, and timing of snow melt were investigated as predictors of phenology. Syrphids generally emerge after flowering onset and end their activity before the end of flowering. Neither flowering nor syrphid phenology has changed significantly over our 20‐year record, consistent with a lack of directional change in climate variables over the same time frame. Instead we document interannual variability in the abiotic environment and phenology. Timing of snow melt was the best predictor of flowering onset and syrphid emergence. Snow melt and degree‐days were the best predictors of the end of flowering, whereas degree‐days and precipitation best predicted the end of the syrphid period. Flowering advanced at a faster rate than syrphids in response to both advancing snow melt and increasing temperature. Different rates of phenological advancements resulted in more days of temporal overlap between the flower–syrphid community in years of early snow melt because of extended activity periods. Phenological synchrony at the community level is therefore likely to be maintained for some time, even under advancing snow melt conditions that are evident over longer term records at our site. These results show that interacting taxa may respond to different phenological cues and to the same cues at different rates but still maintain phenological synchrony over a range of abiotic conditions. However, our results also indicate that some individual plant species may overlap with the syrphid community for fewer days under continued climate change. This highlights the role of interannual variation in these flower–syrphid interactions and shows that species‐level responses can differ from community‐level responses in nonintuitive ways.     

miR-192 Directly Binds and Regulates Dicer1 Expression in Neuroblastoma

Neuroblastoma (NB) arises from the embryonic neural crest and is the most common extracranial solid tumor in children under 5 years of age. Reduced expression of Dicer1 has recently been shown to be in correlation with poor prognosis in NB patients. This study aimed to investigate the mechanisms that could lead to the down-regulation of Dicer1 in neuroblastoma. We used computational prediction to identify potential miRs down-regulating Dicer1 in neuroblastoma. One of the miRs that were predicted to target Dicer1 was miR-192. We measured the levels of miR-192 in 43 primary tumors using real time PCR. Following the silencing of miR-192, the levels of dicer1 cell viability, cell proliferation and migration capability were analyzed. Multivariate analysis identified miR-192 as an independent prognostic marker for relapse in neuroblastoma patients (p=0.04). We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3'' UTR of Dicer1 on positions 1232-1238 and 2282-2288. An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic. Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression.     

Comparative Anatomy of Chromosomal Domains with Imprinted and Non-Imprinted Allele-Specific DNA Methylation

Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM.     

Risk of venous thromboembolism in women with polycystic ovary syndrome: a population-based matched cohort analysis

Background:

There is an increased risk of venous thromboembolism among women taking oral contraceptives. However, whether there is an additional risk among women with polycystic ovary syndrome (PCOS) is unknown.

Methods:

We developed a population-based cohort from the IMS LifeLink Health Plan Claims Database, which includes managed care organizations in the United States. Women aged 18–46 years taking combined oral contraceptives and who had a claim for PCOS (n = 43 506) were matched, based on a propensity score, to control women (n = 43 506) taking oral contraceptives. Venous thromboembolism was defined using administrative coding and use of anticoagulation. We used Cox proportional hazards models to assess the relative risk (RR) of venous thromboembolism among users of combined oral contraceptives with and without PCOS.

Results:

The incidence of venous thromboembolism among women with PCOS was 23.7/10 000 person-years, while that for matched controls was 10.9/10 000 person-years. Women with PCOS taking combined oral contraceptives had an RR for venous thromboembolism of 2.14 (95% confidence interval [CI] 1.41–3.24) compared with other contraceptive users. The incidence of venous thromboembolism was 6.3/10 000 person-years among women with PCOS not taking oral contraceptives; the incidence was 4.1/10 000 person-years among matched controls. The RR of venous thromboembolism among women with PCOS not taking oral contraceptives was 1.55 (95% CI 1.10–2.19).

Interpretation:

We found a 2-fold increased risk of venous thromboembolism among women with PCOS who were taking combined oral contraceptives and a 1.5-fold increased risk among women with PCOS not taking oral contraceptives. Physicians should consider the increased risk of venous thromboembolism when prescribing contraceptive therapy to women with PCOS.Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of reproductive age. The National Institutes of Health criteria estimates its prevalence in the United States to be between 6% and 10%, while the Rotterdam criteria estimates the prevalence to be as high as 15%.¹ Although its cause is not entirely known, the diagnostic criteria include oligo- or anovulation, clinical and/or biochemical signs of hyperandrogenism, and polycystic ovaries.² Women often present with clinical manifestations of high androgen levels, including facial hair growth (hirsutism), acne vulgaris and hair loss on the scalp. Previous studies reported the prevalence of impaired glucose tolerance to be 31.1%–35.2% and the prevalence of type 2 diabetes to be 7.5%–9.8% among women with PCOS.^3,4 A recent consensus workshop reported that the prevalence of several known risk factors for cardiovascular disease (hypertension, diabetes, abdominal obesity, psychological factors, smoking, altered apoA1/ApoB ratios) are doubled among women with PCOS compared with matched controls.^1,5Combined oral contraceptives are the mainstay treatment for PCOS. However, they are also known to elevate the risk of venous thromboembolism and cardiovascular disease.⁶ To date, contraceptive studies involving women with PCOS have focused mainly on efficacy, evaluating the effect of combined oral contraceptives on the reduction of hirsutism and hyperandrogenism.^7,8 Two studies assessed the metabolic effects of combined oral contraceptives in PCOS, but these studies had small sample sizes and could not evaluate for cardiovascular events.^9,10Although women with PCOS have an increase in both cardiovascular risk factors and subclinical cardiovascular disease,¹¹ recent guidelines have concluded there are no data in the literature assessing the association between the use of oral contraceptives and cardiovascular disease among women with PCOS.² Because combined oral contraceptives are the mainstay treatment, our objective was to determine whether women with PCOS taking combined oral contraceptives have a greater risk of venous thromboembolism compared with other contraceptive users. We also examined whether women with PCOS not taking oral contraceptives had an increased risk of venous thromboembolism compared with the general population.     

Light at Night Co‐distributes with Incident Breast but not Lung Cancer in the Female Population of Israel

Recent studies of shift‐working women have reported that excessive exposure to light at night (LAN) may be a risk factor for breast cancer. However, no studies have yet attempted to examine the co‐distribution of LAN and breast cancer incidence on a population level with the goal to assess the coherence of these earlier findings with population trends. Coherence is one of Hill's “criteria” (actually, viewpoints) for an inference of causality. Nighttime satellite images were used to estimate LAN levels in 147 communities in Israel. Multiple regression analysis was performed to investigate the association between LAN and breast cancer incidence rates and, as a test of the specificity of our method, lung cancer incidence rates in women across localities under the prediction of a link with breast cancer but not lung cancer. After adjusting for several variables available on a population level, such as ethnic makeup, birth rate, population density, and local income level, a strong positive association between LAN intensity and breast cancer rate was revealed (p<0.05), and this association strengthened (p<0.01) when only statistically significant factors were filtered out by stepwise regression analysis. Concurrently, no association was found between LAN intensity and lung cancer rate. These results provide coherence of the previously reported case‐control and cohort studies with the co‐distribution of LAN and breast cancer on a population basis. The analysis yielded an estimated 73% higher breast cancer incidence in the highest LAN exposed communities compared to the lowest LAN exposed communities.