Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

<p>Abstract</p> <p>Background</p> <p>Phosphatidylinositol 4,5-bisphosphate (PIP₂) is required for successful completion of cytokinesis. In addition, both PIP₂and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP₂to yield diacylglycerol (DAG) and inositol trisphosphate (IP₃), which in turn induces calcium (Ca²⁺⁾release from the ER. Several studies suggest PIP₂must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP₂regulates cytokinesis remains to be elucidated.</p> <p>Results</p> <p>Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH₃(edelfosine) on cytokinesis in crane-fly and <it>Drosophila </it>spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH₃produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH₃.</p> <p>Conclusion</p> <p>We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP₂hydrolysis acts via Ca²⁺to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring.</p

Adolescent chronic illness : a qualitative study of psychosocial adjustment

Introduction: The purpose of this study was to investigate the psychosocial issues facing young people living with a chronic medical condition. Materials and Methods: Subjects were young people with a range of medical conditions who were on a waiting list to participate in the Chronic Illness Peer Support programme at the Centre for Adolescent Health, Royal Children\u27s Hospital, Melbourne, Australia. Young people agreed to in-depth interviews which were taped and transcribed. Thematic analysis was undertaken by two researchers working independently. Results: Thirty-five young people were interviewed. Thematic analysis revealed five broad themes: control (in control, under control, out of control); emotional reactions (happiness, frustration, anger, sadness, anxiety); acceptance (of illness, of others, of self); coping strategies, and; a search for meaning. The importance of social connections was emphasised. While illustrating the difficulties of managing a chronic medical condition during adolescence, a generally positive message emerges about these young people. Conclusions: Many young people with chronic illness appear relatively resilient in the face of the adjustment challenges presented by their illness. Interventions that allow a young person to explore meaning, build self-esteem, and acceptance through positive social connections are likely to improve adjustment outcomes in this group.<br /

The holistic phase model of early adult crisis

The objective of the current study was to explore the structural, temporal and experiential manifestations of crisis episodes in early adulthood, using a holistic-systemic theoretical framework. Based on an analysis of 50 interviews with individuals about a crisis episode between the ages of 25 and 35, a holistic model was developed. The model comprises four phases: (1) Locked-in, (2) Separation/Time-out, (3) Exploration and (4) Rebuilding, which in turn have characteristic features at four levels—person-in-environment, identity, motivation and affect-cognition. A crisis starts out with a commitment at work or home that has been made but is no longer desired, and this is followed by an emotionally volatile period of change as that commitment is terminated. The positive trajectory of crisis involves movement through an exploratory period towards active rebuilding of a new commitment, but ‘fast-forward’ and ‘relapse’ loops can interrupt Phases 3 and 4 and make a positive resolution of the episode less likely. The model shows conceptual links with life stage theories of emerging adulthood and early adulthood, and it extends current understandings of the transitional developmental challenges that young adults encounter

A genome-wide association meta-analysis on lipoprotein (a) concentrations adjusted for apolipoprotein (a) isoforms.

High lipoprotein (a) [Lp(a)] concentrations are an independent risk factor for cardiovascular outcomes. Concentrations are strongly influenced by apo(a) kringle IV repeat isoforms. We aimed to identify genetic loci associated with Lp(a) concentrations using data from five genome-wide association studies (n = 13,781). We identified 48 independent SNPs in the &lt;i&gt;LPA&lt;/i&gt; and 1 SNP in the &lt;i&gt;APOE&lt;/i&gt; gene region to be significantly associated with Lp(a) concentrations. We also adjusted for apo(a) isoforms to identify loci affecting Lp(a) levels independently from them, which resulted in 31 SNPs (30 in the &lt;i&gt;LPA&lt;/i&gt; , 1 in the &lt;i&gt;APOE&lt;/i&gt; gene region). Seven SNPs showed a genome-wide significant association with coronary artery disease (CAD) risk. A rare SNP (rs186696265; MAF ∼1%) showed the highest effect on Lp(a) and was also associated with increased risk of CAD (odds ratio = 1.73, &lt;i&gt;P&lt;/i&gt; = 3.35 × 10 &lt;sup&gt;-30&lt;/sup&gt; ). Median Lp(a) values increased from 2.1 to 91.1 mg/dl with increasing number of Lp(a)-increasing alleles. We found the &lt;i&gt;APOE2&lt;/i&gt; -determining allele of rs7412 to be significantly associated with Lp(a) concentrations ( &lt;i&gt;P&lt;/i&gt; = 3.47 × 10 &lt;sup&gt;-10&lt;/sup&gt; ). Each &lt;i&gt;APOE2&lt;/i&gt; allele decreased Lp(a) by 3.34 mg/dl corresponding to ∼15% of the population's mean values. Performing a gene-based test of association, including suspected Lp(a) receptors and regulators, resulted in one significant association of the &lt;i&gt;TLR2&lt;/i&gt; gene with Lp(a) ( &lt;i&gt;P&lt;/i&gt; = 3.4 × 10 &lt;sup&gt;-4&lt;/sup&gt; ). In summary, we identified a large number of independent SNPs in the &lt;i&gt;LPA&lt;/i&gt; gene region, as well as the &lt;i&gt;APOE2&lt;/i&gt; allele, to be significantly associated with Lp(a) concentrations

A genome-wide association meta-analysis on apolipoprotein A-IV concentrations.

Apolipoprotein A-IV (apoA-IV) is a major component of HDL and chylomicron particles and is involved in reverse cholesterol transport. It is an early marker of impaired renal function. We aimed to identify genetic loci associated with apoA-IV concentrations and to investigate relationships with known susceptibility loci for kidney function and lipids. A genome-wide association meta-analysis on apoA-IV concentrations was conducted in five population-based cohorts (n = 13,813) followed by two additional replication studies (n = 2,267) including approximately 10 M SNPs. Three independent SNPs from two genomic regions were significantly associated with apoA-IV concentrations: rs1729407 near APOA4 (P = 6.77 × 10 (-)  (44)), rs5104 in APOA4 (P = 1.79 × 10(-)(24)) and rs4241819 in KLKB1 (P = 5.6 × 10(-)(14)). Additionally, a look-up of the replicated SNPs in downloadable GWAS meta-analysis results was performed on kidney function (defined by eGFR), HDL-cholesterol and triglycerides. From these three SNPs mentioned above, only rs1729407 showed an association with HDL-cholesterol (P = 7.1 × 10 (-)  (07)). Moreover, weighted SNP-scores were built involving known susceptibility loci for the aforementioned traits (53, 70 and 38 SNPs, respectively) and were associated with apoA-IV concentrations. This analysis revealed a significant and an inverse association for kidney function with apoA-IV concentrations (P = 5.5 × 10(-)(05)). Furthermore, an increase of triglyceride-increasing alleles was found to decrease apoA-IV concentrations (P = 0.0078). In summary, we identified two independent SNPs located in or next the APOA4 gene and one SNP in KLKB1 The association of KLKB1 with apoA-IV suggests an involvement of apoA-IV in renal metabolism and/or an interaction within HDL particles. Analyses of SNP-scores indicate potential causal effects of kidney function and by lesser extent triglycerides on apoA-IV concentrations

A comparison of genomic copy number calls by Partek Genomics Suite, Genotyping Console and Birdsuite algorithms to quantitative PCR

<p>Abstract</p> <p>Background</p> <p>Copy number variants are >1 kb genomic amplifications or deletions that can be identified using array platforms. However, arrays produce substantial background noise that contributes to high false discovery rates of variants. We hypothesized that quantitative PCR could finitely determine copy number and assess the validity of calling algorithms.</p> <p>Results</p> <p>Using data from 29 Affymetrix SNP 6.0 arrays, we determined copy numbers using three programs: Partek Genomics Suite, Affymetrix Genotyping Console 2.0 and Birdsuite. We compared array calls at 25 chromosomal regions to those determined by qPCR and found nearly identical calls in regions of copy number 2. Conversely, agreement differed in regions called variant by at least one method. The highest overall agreement in calls, 91%, was between Birdsuite and quantitative PCR. Partek Genomics Suite calls agreed with quantitative PCR 76% of the time while the agreement of Affymetrix Genotyping Console 2.0 with quantitative PCR was 79%.</p> <p>Conclusions</p> <p>In 38 independent samples, 96% of Birdsuite calls agreed with quantitative PCR. Analysis of three copy number calling programs and quantitative PCR showed Birdsuite to have the greatest agreement with quantitative PCR.</p

A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress

Background: The secretory pathway is a critical index of the capacity of cells to incorporate proteins into cellular membranes and secrete proteins into the extracellular space. Importantly it is disrupted in response to stress to the endoplasmic reticulum that can be induced by a variety of factors, including expression of mutant proteins and physiologic stress. Activation of the ER stress response is critical in the etiology of a number of diseases, such as diabetes and neurodegeneration, as well as cancer. We have developed a highly sensitive assay to monitor processing of proteins through the secretory pathway and endoplasmic reticulum (ER) stress in real-time based on the naturally secreted Gaussia luciferase (Gluc). Methodology/Principle Findings: An expression cassette for Gluc was delivered to cells, and its secretion was monitored by measuring luciferase activity in the conditioned medium. Gluc secretion was decreased down to 90% when these cells were treated with drugs that interfere with the secretory pathway at different steps. Fusing Gluc to a fluorescent protein allowed quantitation and visualization of the secretory pathway in real-time. Expression of this reporter protein did not itself elicit an ER stress response in cells; however, Gluc proved very sensitive at sensing this type of stress, which is associated with a temporary decrease in processing of proteins through the secretory pathway. The Gluc secretion assay was over 20,000-fold more sensitive as compared to the secreted alkaline phosphatase (SEAP), a well established assay for monitoring of protein processing and ER stress in mammalian cells. Conclusions/Significance: The Gluc assay provides a fast, quantitative and sensitive technique to monitor the secretory pathway and ER stress and its compatibility with high throughput screening will allow discovery of drugs for treatment of conditions in which the ER stress is generally induced

Synchronizing chromosome segregation by flux-dependent force equalization at kinetochores

The synchronous movement of chromosomes during anaphase ensures their correct inheritance in every cell division. This reflects the uniformity of spindle forces acting on chromosomes and their simultaneous entry into anaphase. Although anaphase onset is controlled by the spindle assembly checkpoint, it remains unknown how spindle forces are uniformly distributed among different chromosomes. In this paper, we show that tension uniformity at metaphase kinetochores and subsequent anaphase synchrony in Drosophila S2 cells are promoted by spindle microtubule flux. These results can be explained by a mechanical model of the spindle where microtubule poleward translocation events associated with flux reflect relaxation of the kinetochore–microtubule interface, which accounts for the redistribution and convergence of kinetochore tensions in a timescale comparable to typical metaphase duration. As predicted by the model, experimental acceleration of mitosis precludes tension equalization and anaphase synchrony. We propose that flux-dependent equalization of kinetochore tensions ensures a timely and uniform maturation of kinetochore–microtubule interfaces necessary for error-free and coordinated segregation of chromosomes in anaphase

Association of mitochondrial DNA copy number with metabolic syndrome and type 2 diabetes in 14 176 individuals

Background Mitochondria play an important role in cellular metabolism, and their dysfunction is postulated to be involved in metabolic disturbances. Mitochondrial DNA is present in multiple copies per cell. The quantification of mitochondrial DNA copy number (mtDNA-CN) might be used to assess mitochondrial dysfunction. Objectives We aimed to investigate the cross-sectional association of mtDNA-CN with type 2 diabetes and the potential mediating role of metabolic syndrome. Methods We examined 4812 patients from the German Chronic Kidney Disease (GCKD) study and 9364 individuals from the Cooperative Health Research in South Tyrol (CHRIS) study. MtDNA-CN was measured in whole blood using a plasmid-normalized qPCR-based assay. Results In both studies, mtDNA-CN showed a significant correlation with most metabolic syndrome parameters: mtDNA-CN decreased with increasing number of metabolic syndrome components. Furthermore, individuals with low mtDNA-CN had significantly higher odds of metabolic syndrome (OR = 1.025; 95% CI = 1.011–1.039, P = 3.19 × 10−4, for each decrease of 10 mtDNA copies) and type 2 diabetes (OR = 1.027; 95% CI = 1.012–1.041; P = 2.84 × 10−4) in a model adjusted for age, sex, smoking and kidney function in the meta-analysis of both studies. Mediation analysis revealed that the association of mtDNA-CN with type 2 diabetes was mainly mediated by waist circumference in the GCKD study (66%) and by several metabolic syndrome parameters, especially body mass index and triglycerides, in the CHRIS study (41%). Conclusions Our data show an inverse association of mtDNA-CN with higher risk of metabolic syndrome and type 2 diabetes. A major part of the total effect of mtDNA-CN on type 2 diabetes is mediated by obesity parameters