A Bcl-2 transgene expressed in hepatocytes does not protect mice from fulminant liver destruction induced by Fas ligand

We compared the biological mechanism of cell death during hepatotoxicity induced by ligation of the Fas receptor in wild-type and liver-specific bcl-2 transgenic mice. Transgenic overexpression of Bcl-2 in mouse hepatocytes can prevent lethal hepatitis induced by agonistic anti-Fas antibodies. In contrast, Fas ligand (FasL)-induced death cannot be overcome in bcl-2 transgenic mice, indicating that anti-Fas antibodies do not reliably mimic the more physiological ligand. Different apoptotic parameters, viz. caspase activation, cytochrome c release and nuclear DNA degradation were analysed. No differences, however, could be observed between wild-type and bcl-2 transgenic mice after injection with a lethal dose of soluble FasL, indicating that apoptosis by FasL-dependent ligation is not modulated by Bcl-2 in vivo. These results demonstrate that the stimulus determines the outcome between type I mitochondria-independent apoptosis, in the case of FasL, or type II mitochondria-dependent and Bcl-2-inhibitable apoptosis, in the case of anti-Fas antibodies.     

Potato Leafroll Virus Binds to the Equatorial Domain of the Aphid Endosymbiotic GroEL Homolog

A GroEL homolog with a molecular mass of 60 kDa, produced by the primary endosymbiotic bacterium (a Buchnera sp.) of Myzus persicae and released into the hemolymph, has previously been shown to be a key protein in the transmission of potato leafroll virus (PLRV). Like other luteoviruses and pea enation mosaic virus, PLRV readily binds to extracellular Buchnera GroEL, and in vivo interference in this interaction coincides with reduced capsid integrity and loss of infectivity. To gain more knowledge of the nature of the association between PLRV and Buchnera GroEL, the groE operon of the primary endosymbiont of M. persicae (MpB groE) and its flanking sequences were characterized and the PLRV-binding domain of Buchnera GroEL was identified by deletion mutant analysis. MpB GroEL has extensive sequence similarity (92%) with Escherichia coli GroEL and other members of the chaperonin-60 family. The genomic organization of the Buchnera groE operon is similar to that of the groE operon of E. coli except that a constitutive promoter sequence could not be identified; only the heat shock promoter was present. By a virus overlay assay of protein blots, it was shown that purified PLRV bound as efficiently to recombinant MpB GroEL (expressed in E. coli) as it did to wild-type MpB GroEL. Mutational analysis of the gene encoding MpB GroEL revealed that the PLRV-binding site was located in the so-called equatorial domain and not in the apical domain which is generally involved in polypeptide binding and folding. Buchnera GroEL mutants lacking the entire equatorial domain or parts of it lost the ability to bind PLRV. The equatorial domain is made up of two regions at the N and C termini that are not contiguous in the amino acid sequence but are in spatial proximity after folding of the GroEL polypeptide. Both the N- and C-terminal regions of the equatorial domain were implicated in virus binding.     

The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

The yeast Ca²⁺ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca²⁺ and Mn²⁺ ions. We show here that addition of Mn²⁺ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca²⁺. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca²⁺-deficient media is overcome by expression of other Ca²⁺ pumps, including a SERCA-type Ca²⁺ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca²⁺ and Mn²⁺ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.     

Genetic sensitivity analysis: Adjusting for genetic confounding in epidemiological associations

Associations between exposures and outcomes reported in epidemiological studies are typically unadjusted for genetic confounding. We propose a two-stage approach for estimating the degree to which such observed associations can be explained by genetic confounding. First, we assess attenuation of exposure effects in regressions controlling for increasingly powerful polygenic scores. Second, we use structural equation models to estimate genetic confounding using heritability estimates derived from both SNP-based and twin-based studies. We examine associations between maternal education and three developmental outcomes – child educational achievement, Body Mass Index, and Attention Deficit Hyperactivity Disorder. Polygenic scores explain between 14.3% and 23.0% of the original associations, while analyses under SNP- and twin-based heritability scenarios indicate that observed associations could be almost entirely explained by genetic confounding. Thus, caution is needed when interpreting associations from non-genetically informed epidemiology studies. Our approach, akin to a genetically informed sensitivity analysis can be applied widely.     

High‐throughput respiration screening of single mitochondrial substrates using permeabilized CHO cells highlights control of mitochondria metabolism

Respiration analysis using isolated mitochondria and electrochemical oxygen sensing has contributed significantly to the knowledge about mitochondrial metabolism, which is involved in energy generation but also in ageing and numerous diseases. Here, we present a high‐throughput respiration screening for functional in situ mitochondrial studies in permeabilized Chinese hamster ovary cells. The determination of oxygen uptake rates allowed a quantitative comparison between different conditions and a distinction of substrates into three groups providing an insight into tricarboxylic acid (TCA) cycle regulation. The mitochondrial metabolization of citrate, isocitrate, glutamine, and glutamate was highly stimulated by ADP supply. In contrast, the metabolization of α‐ketoglutarate, succinate, fumarate, and malate was little controlled by the energy and redox state. Metabolization of pyruvate was very strictly regulated by several independent mechanisms: phosphorylation, feedback inhibition, but also by the availability of CoA. A moderate stimulation of pyruvate metabolization was accomplished by feeding both pyruvate and aspartate simultaneously. The presented high‐throughput respiration screening provides comprehensive information about the effect of single or mixed substrates on mitochondrial metabolic activities, including transport and TCA cycle regulation, and metabolic bottlenecks. This supports the design of efficient mammalian producer strains or feeding strategies, but also the investigation of pathological and toxicological effects related to mitochondrial metabolism.     

Consequences of Interaction of Functional,Somatic, Mental and Social Problems in Community-Dwelling Older People

This study explores the combination of four common health problems in older people and whether problems on four domains result in an additional effect on indicators of poor health. For this purpose, a total of 2681 participants (32% male, mean age 82 years) of the Integrated Systematic Care for Older People (ISCOPE) study were screened on the presence of health problems on four domains (functional, somatic, mental, social) with the postal ISCOPE questionnaire. Extensive interview data on health indicators were obtained at baseline and at 12-months follow-up, including disability (Groningen Activities Restriction Scale, GARS), cognitive function (Mini-Mental State Examination, MMSE), depressive symptoms (Geriatric Depression Scale-15, GDS), loneliness (loneliness scale of De Jong Gierveld), and health-related quality of life (EQ-5D). General practitioner (GP) contact time (min/year) was estimated via GP electronic medical records. Of the study population, 9% had no health problems according to the screening, 8% had problems on one domain, 27% on two, 38% on three and 18% on four domains. At baseline, the number of health domains with problems was associated with poorer scores on the GARS, the MMSE, the GDS-15, the loneliness scale, the EQ-5D and with more GP contact time (p <0.001). Problems on all four domains had an additional negative effect on these health indicators (all p_interaction <0.001). At follow-up, an increased number of domains with problems was associated with an increased decline in health indicators (all p<0.001) and with an additional negative effect on GP contact time of the presence of problems on all four domains (p_interaction <0.001). We conclude that combinations of functional, somatic, mental and social problems are associated with poor health indicators in community-dwelling older people. Since problems on four domains have an additional effect on health, individuals with combined functional, somatic, mental and social problems could benefit from integrated care.

Trial registration

Netherlands Trial Register: NTR1946.     

Venous Thrombosis Risk after Cast Immobilization of the Lower Extremity: Derivation and Validation of a Clinical Prediction Score,L-TRiP(cast), in Three Population-Based Case–Control Studies

Background

Guidelines and clinical practice vary considerably with respect to thrombosis prophylaxis during plaster cast immobilization of the lower extremity. Identifying patients at high risk for the development of venous thromboembolism (VTE) would provide a basis for considering individual thromboprophylaxis use and planning treatment studies.The aims of this study were (1) to investigate the predictive value of genetic and environmental risk factors, levels of coagulation factors, and other biomarkers for the occurrence of VTE after cast immobilization of the lower extremity and (2) to develop a clinical prediction tool for the prediction of VTE in plaster cast patients.

Methods and Findings

We used data from a large population-based case–control study (MEGA study, 4,446 cases with VTE, 6,118 controls without) designed to identify risk factors for a first VTE. Cases were recruited from six anticoagulation clinics in the Netherlands between 1999 and 2004; controls were their partners or individuals identified via random digit dialing. Identification of predictor variables to be included in the model was based on reported associations in the literature or on a relative risk (odds ratio) > 1.2 and p ≤ 0.25 in the univariate analysis of all participants. Using multivariate logistic regression, a full prediction model was created. In addition to the full model (all variables), a restricted model (minimum number of predictors with a maximum predictive value) and a clinical model (environmental risk factors only, no blood draw or assays required) were created. To determine the discriminatory power in patients with cast immobilization (n = 230), the area under the curve (AUC) was calculated by means of a receiver operating characteristic. Validation was performed in two other case–control studies of the etiology of VTE: (1) the THE-VTE study, a two-center, population-based case–control study (conducted in Leiden, the Netherlands, and Cambridge, United Kingdom) with 784 cases and 523 controls included between March 2003 and December 2008 and (2) the Milan study, a population-based case–control study with 2,117 cases and 2,088 controls selected between December 1993 and December 2010 at the Thrombosis Center, Fondazione IRCCS Ca’ Granda–Ospedale Maggiore Policlinico, Milan, Italy.The full model consisted of 32 predictors, including three genetic factors and six biomarkers. For this model, an AUC of 0.85 (95% CI 0.77–0.92) was found in individuals with plaster cast immobilization of the lower extremity. The AUC for the restricted model (containing 11 predictors, including two genetic factors and one biomarker) was 0.84 (95% CI 0.77–0.92). The clinical model (consisting of 14 environmental predictors) resulted in an AUC of 0.77 (95% CI 0.66–0.87). The clinical model was converted into a risk score, the L-TRiP(cast) score (Leiden–Thrombosis Risk Prediction for patients with cast immobilization score), which showed an AUC of 0.76 (95% CI 0.66–0.86). Validation in the THE-VTE study data resulted in an AUC of 0.77 (95% CI 0.58–0.96) for the L-TRiP(cast) score. Validation in the Milan study resulted in an AUC of 0.93 (95% CI 0.86–1.00) for the full model, an AUC of 0.92 (95% CI 0.76–0.87) for the restricted model, and an AUC of 0.96 (95% CI 0.92–0.99) for the clinical model. The L-TRiP(cast) score resulted in an AUC of 0.95 (95% CI 0.91–0.99).Major limitations of this study were that information on thromboprophylaxis was not available for patients who had plaster cast immobilization of the lower extremity and that blood was drawn 3 mo after the thrombotic event.

Conclusions

These results show that information on environmental risk factors, coagulation factors, and genetic determinants in patients with plaster casts leads to high accuracy in the prediction of VTE risk. In daily practice, the clinical model may be the preferred model as its factors are most easy to determine, while the model still has good predictive performance. These results may provide guidance for thromboprophylaxis and form the basis for a management study.     

The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy

The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis in vitro and in vivo indicated that the mutation was responsible for a decrease in posttranslational glycosylation of dystroglycan, eventually interfering with its extracellular-matrix receptor function and laminin binding in skeletal muscle and brain. The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology (Ig-like domain followed by a basket-shaped domain reminiscent of the small subunit ribosomal protein S6) very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159–180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1–B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.