Effect of ATRA and ATO on the expression of tissue factor in NB4 acute promyelocytic leukemia cells and regulatory function of the inflammatory cytokines TNF and IL-1β

The characteristic hemorrhages of acute promyelocytic leukemia (APL) are caused in part by the high expression of tissue factor (TF) on leukemic cells, which also produce TNF and IL-1β, proinflammatory cytokines known to increase TF in various cell types. Exposure of NB4 cells, an APL cell line, to all-trans retinoic acid (ATRA) or arsenic trioxide (ATO) rapidly and strongly reduced TF mRNA. Both drugs also reduced TNF mRNA, but later, and moreover increased IL-1β mRNA. The effect on procoagulant activity of cells and microparticles, as measured with calibrated automated thrombography, was delayed and only partial at 24 h. TNF and IL-1β inhibition reduced TF mRNA and activity only partially. Inhibition of the inflammatory signaling intermediate p38 reduced TF mRNA by one third but increased TNF and IL-1β mRNA. NF-κB inhibition reduced, within 1 h, TF and TNF mRNA but did not change IL-1β mRNA, and rapidly and markedly reduced cell survival, with procoagulant properties still being present. In conclusion, although we provide evidence that TNF, IL-1β, and their signaling intermediates have a regulatory function on TF expression by NB4 APL cells, the effect of ATRA and ATO on TF can only partially be accounted for by their impact on these cytokines.     

Evolution of cytokines and inflammatory biomarkers during infliximab induction therapy and the impact of inflammatory burden on primary response in patients with Crohn’s disease

Objective: Primary non-response to infliximab in Crohn’s disease is still incompletely understood. Our aim was to further characterize the role of inflammatory burden during infliximab induction therapy.

Materials and Methods: We studied a well-characterized cohort of 201 anti-TNF naive Crohn’s disease patients treated with infliximab 5mg/kg at week 0, 2, 6 and 14 who had serum samples drawn just before every infusion. All serum samples were analyzed for CRP, albumin, TNF, IFN-γ, IL-6, IL-8, IL-10, infliximab trough concentrations (in-house-developed ELISA) and antibodies to infliximab (HMSA, Prometheus Laboratories Inc., San Diego, CA). Primary non-response was defined as the absence of clinical improvement at week 14.

Results: The incidence of primary non-response to infliximab was 8% (n?=?16). IL-8 concentrations at baseline were higher (p?=?.01) and albumin at week 6 was lower in primary non-responders (p?=?.01) compared to responders. During induction, IFN-γ and IL-6 concentrations decreased significantly at week 2 and week 6 in responders compared to primary non-responders (p?<?.05). Serum TNF increased significantly after each infliximab infusion and this increase from week 0 to week 14 was more pronounced in responders (p?=?.03). Multiple logistic regression identified TNF/CRP ratio at baseline as predictive for primary non-response to infliximab at week 14 (OR 2.8 (95% CI 1.4–5.5; p?=?.003)).

Conclusions: In this intensively sampled cohort of Crohn’s disease patients, we demonstrate that inflammatory burden is more determining for primary non-response than drug exposure or immunogenicity. Our findings furthermore suggest that the contribution of TNF in inflammation might be higher in primary non-response, contradicting the non-TNF-driven concept.     

Induction of macrophage inflammatory protein-1α and vascular endothelial growth factor during inflammatory neovascularization in the mouse cornea

We predicted that the appearance of macrophages in inflammatory areas is necessary for angiogenic responses in various inflammatory diseases. Using a mouse inflammatory corneal model in which model mouse corneas were cauterized with silver nitrate, we examined the infiltration of macrophages immunohistochemically and the total area of neovascularization quantitively. The expression of macrophage inflammatory protein-1α (MIP-1α) and vascular endothelial growth factor (VEGF) levels were also examined. A day after cauterization, short capillaries began to develop into the corneal stroma, and after 4 or 5 days the neovascularization became maximal and then began to regress. The number of macrophages within the cauterized cornea increased to a maximum at day 3 and began to decrease at day 5. The number of infiltrated macrophages reached maximum at day 3. Both MIP-1α and VEGF protein levels increased markedly immediately after the chemical cauterization, and production of MIP-1α (85.8 pg/4 corneas) and VEGF (206.5 pg/4 corneas) was maximal at 1 day and 0.5 day after cauterization, respectively. MIP-1α and VEGF mRNA levels also increased at 0.5 day after cauterization. In situ hybridization showed that MIP-1α was localized in corneal epithelial cells, and VEGF was localized in corneal epithelial cells and infiltrating inflammatory cells. MIP-1α and VEGF may have an important role in recruiting macrophages and neovascularization. This revised version was published online in June 2006 with corrections to the Cover Date.     

The association of genetic polymorphisms of hypoxia inducible factor-1 alpha and vascular endothelial growth factor with increased risk of chronic obstructive pulmonary disease: A case–control study

Accumulated data over the years have suggested that hypoxia inducible factor-1 alpha (HIF-1α) and its downstream vascular endothelial growth factor (VEGF) gene may be linked with chronic obstructive pulmonary disease (COPD). This study aims to investigate the association of HIF-1α and VEGF genetic polymorphisms and their correlated risks with COPD. COPD patients (case group) and healthy individuals (control group) were recruited. DNA was extracted to detect HIF-1α and VEGF genetic polymorphisms. Basal lung volume and forced expiratory capacity in 1st second (FEV1)/forced vital capacity (FVC) and FEV₁/predicted value (pred)% were calculated. Genotype and allele distributions in HIF-1α and VEGF genes were analyzed. Kaplan–Meier curves and logistic regression model were used for analysis of survival and COPD risk factors. Haplotypes for HIF-1α rs11549465 and rs11549467 were analyzed. FEV₁/FVC and FEV1/pred% in the case group were lower than the control group. Frequencies of HIF-1α rs11549465 CT + TT genotype and T allele, and rs11549467 GA + AA genotype and A allele were higher in the case group than the control group. Patients with rs11549465 CT + TT had higher COPD risk than those with the CC genotype. Patients with rs11549467 GA + AA showed higher COPD risk and lower FEV₁/FVC and FEV₁/pred% than those with the GG genotype. Patients with HIF-1α TA haplotype showed higher COPD risk than those with the CG haplotype. Survival rate of patients with HIF-1α rs11549467 GG genotype was higher than those with the GA + AA genotype. HIF-1α rs11549467 polymorphism may be associated with COPD risk.     

Quantitative analysis of hepatic hypoxia-inducible factor-1α and its abnormal gene expression during the formation of hepatocellular carcinoma

BACKGROUND:Hepatic hypoxia-inducible factor-1(HIF-1) is activated in the progression of hepatocellular carcinoma (HCC).This study aimed to investigate the dynamic alterations of HIF-1αand its gene expression so as to explore the relationship between HIF-1αexpression and hepatocarcinogenesis at the early stage of HCC. METHODS:A hepatoma model was made with 2-fluorenyl- acetamide(2-FAA)in male Sprague-Dawley rats.Morphological changes of rat hepatocytes were assessed pathologically (HE staining).The dynamic e...     

Correction to “Downregulation of FoxM1 inhibits the viability and invasion of gallbladder carcinoma cells,partially dependent on the induction of cellular senescence”

We corrected the mistake of Figure 3, and replaced the incorrect images with the correct ones. The “adenovirus” was a typographical error in writing, and should be revised to “lentivirus”.     

Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3-expressing NOD-SCID IL2Rγ(null) humanized mice

Human hematolymphoid mice have become valuable tools for the study of human hematopoiesis and uniquely human pathogens in vivo. Recent improvements in xenorecipient strains allow for long-term reconstitution with a human immune system. However, certain hematopoietic lineages, for example, the myeloid lineage, are underrepresented, possibly because of the limited cross-reactivity of murine and human cytokines. Therefore, we created a nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-γ-null (NOD-SCID IL2Rγ(null)) mouse strain that expressed human stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3, termed NSG-SGM3. Transplantation of CD34(+) human hematopoietic stem cells into NSG-SGM3 mice led to robust human hematopoietic reconstitution in blood, spleen, bone marrow, and liver. Human myeloid cell frequencies, specifically, myeloid dendritic cells, were elevated in the bone marrow of humanized NSG-SGM3 mice compared with nontransgenic NSG recipients. Most significant, however, was the increase in the CD4(+)FoxP3(+) regulatory T-cell population in all compartments analyzed. These CD4(+)FoxP3(+) regulatory T cells were functional, as evidenced by their ability to suppress T-cell proliferation. In conclusion, humanized NSG-SGM3 mice might serve as a useful model to study human regulatory T-cell development in vivo, but this unexpected lineage skewing also highlights the importance of adequate spatiotemporal expression of human cytokines for future xenorecipient strain development.     

Administration of insulin–like growth factor–1 (IGF–1) improves both structure and function of delta–sarcoglycan deficient cardiac muscle in the hamster

Dilated cardiomyopathies (DCM) are due to progressive dilatation of the cardiac cavities and thinning of the ventricular walls and lead unavoidably to heart failure. They represent a major cause for heart transplantation and, therefore, defining an efficient symptomatic treatment for DCM remains a challenge. We have taken advantage of the hamster strain CHF147 that displays progressive cardiomyopathy leading to heart failure to test whether stimulation of a hypertrophic pathway could delay the process of dilatation.Six month old CHF147 hamsters were treated with IGF–1 so that we could compare the efficacy of systemic administration of human recombinant IGF–1 protein (rh IGF–1) at low dose to that of direct myocardial injections of a plasmid DNA containing IGF–1 cDNA (pCMV–IGF1).IGF-1 treatment did not induce a significant variation of ventricle mass, but preserved left ventricular (LV) wall thickness and delayed dilatation of cardiac cavities when compared to non–treated hamsters. Together with this reduction of dilatation, we also noted a reduction in the amount of interstitial collagen. Furthermore, IGF–1 treatment induced beneficial effects on cardiac function since treated hamsters presented improved cardiac output and stroke volume, decreased end diastolic pressure when compared to nontreated hamsters and also showed a trend towards increased contractility (dP/dt_max).This study provides evidence that IGF–1 treatment induces beneficial structural and functional effects on DCM of CHF147 hamsters, hence making this molecule a promising candidate for future gene therapy of heart failure due to DCM.     

Single-base resolution mapping of H1–nucleosome interactions and 3D organization of the nucleosome

Despite the key role of the linker histone H1 in chromatin structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. In this work we have used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes containing physiologically assembled full-length histone H1 or truncated mutants of this protein. Single-base resolution •OH footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized symmetrically with respect to the nucleosomal dyad. In addition, GH1 interacts with and organizes about one helical turn of DNA in each linker region of the nucleosome. We also find that a seven amino acid residue region (121–127) in the COOH terminus of histone H1 was required for the formation of the stem structure of the linker DNA. A molecular model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within linker DNA.     

Placental growth factor-1 and epithelial haemato–retinal barrier breakdown: potential implication in the pathogenesis of diabetic retinopathy

Aims/hypothesis Disruption of the retinal pigment epithelial (RPE) barrier contributes to sub-retinal fluid and retinal oedema as observed in diabetic retinopathy. High placental growth factor (PLGF) vitreous levels have been found in diabetic patients. This work aimed to elucidate the influence of PLGF-1 on a human RPE cell line (ARPE-19) barrier in vitro and on normal rat eyes in vivo. Methods ARPE-19 permeability was measured using transepithelial resistance and inulin flux under stimulation of PLGF-1, vascular endothelial growth factor (VEGF)-E and VEGF 165. Using RT-PCR, we evaluated the effect of hypoxic conditions or insulin on transepithelial resistance and on PLGF-1 and VEGF receptors. The involvement of mitogen-activated protein kinase (MEK, also known as MAPK)/extracellular signal-regulated kinase (ERK, also known as EPHB2) signalling pathways under PLGF-1 stimulation was evaluated by western blot analysis and specific inhibitors. The effect of PLGF-1 on the external haemato–retinal barrier was evaluated after intravitreous injection of PLGF-1 in the rat eye; evaluation was by semi-thin analysis and zonula occludens-1 immunolocalisation on flat-mounted RPE. Results In vitro, PLGF-1 induced a reversible decrease of transepithelial resistance and enhanced tritiated inulin flux. These effects were specifically abolished by an antisense oligonucleotide directed at VEGF receptor 1. Exposure of ARPE-19 cells to hypoxic conditions or to insulin induced an upregulation of PLGF-1 expression along with increased transcellular permeability. The PLGF-1-induced RPE cell permeability involved the MEK signalling pathway. Injection of PLGF-1 in the rat eye vitreous induced an opening of the RPE tight junctions with subsequent sub-retinal fluid accumulation, retinal oedema and cytoplasm translocation of junction proteins. Conclusions/interpretaion Our results indicate that PLGF-1 may be a potential regulation target for the control of diabetic retinal and macular oedema. This work was presented in part at the Association for Research and Vision in Ophthalmology meeting, Fort Lauderdale, FL, USA, in May 2005.     

A Mitofusin-2–dependent inactivating cleavage of Opa1 links changes in mitochondria cristae and ER contacts in the postprandial liver

Hepatic metabolism requires mitochondria to adapt their bioenergetic and biosynthetic output to accompany the ever-changing anabolic/catabolic state of the liver cell, but the wiring of this process is still largely unknown. Using a postprandial mouse liver model and quantitative cryo-EM analysis, we show that when the hepatic mammalian target of rapamycin complex 1 (mTORC1) signaling pathway disengages, the mitochondria network fragments, cristae density drops by 30%, and mitochondrial respiratory capacity decreases by 20%. Instead, mitochondria–ER contacts (MERCs), which mediate calcium and phospholipid fluxes between these organelles, double in length. These events are associated with the transient expression of two previously unidentified C-terminal fragments (CTFs) of Optic atrophy 1 (Opa1), a mitochondrial GTPase that regulates cristae biogenesis and mitochondria dynamics. Expression of Opa1 CTFs in the intermembrane space has no effect on mitochondria morphology, supporting a model in which they are intermediates of an Opa1 degradation program. Using an in vitro assay, we show that these CTFs indeed originate from the cleavage of Opa1 at two evolutionarily conserved consensus sites that map within critical folds of the GTPase. This processing of Opa1, termed C-cleavage, is mediated by the activity of a cysteine protease whose activity is independent from that of Oma1 and presenilin-associated rhomboid-like (PARL), two known Opa1 regulators. However, C-cleavage requires Mitofusin-2 (Mfn2), a key factor in mitochondria–ER tethering, thereby linking cristae remodeling to MERC assembly. Thus, in vivo, mitochondria adapt to metabolic shifts through the parallel remodeling of the cristae and of the MERCs via a mechanism that degrades Opa1 in an Mfn2-dependent pathway.The last decade expanded our understanding of the importance of mitochondrial shape, position, and interorganellar interactions in the regulation of cell stress. For example, mitochondrial hyperfusion is a stress response that protects against cell death and autophagic degradation, whereas chronic stress triggers mitochondrial fragmentation and cell death. However, the in vivo implications of mitochondrial plasticity under normal physiological conditions are still largely unknown. The liver is a key organ responsible for nutrient sensing and the maintenance of whole-body energy homeostasis. Therefore, we considered the liver as a primary model to examine the changes in mitochondrial plasticity that accompanies physiological transitions in feeding and postprandial metabolism (1–4).The mechanistic target of rapamycin complex 1 (mTORC1) is an evolutionary conserved serine/threonine kinase that plays an important role in regulating metabolism and cell growth in response to anabolic signals (5). Studies indicate that mTORC1, which is activated by growth factors and amino acids, is a key sensor allowing cells and tissues to adapt their metabolism in response to the nutritional state (5). In the liver, it controls the activation of various metabolic processes including lipogenesis (6) and ketogenesis (3). Recent observations indicate that mTORC1 regulates mitochondrial biogenesis and metabolism (7, 8), but the underlying mechanisms remain to be determined.It has been established that the core machinery that governs mitochondrial shape and ultrastructure is essential. Indeed, genetic ablation of its components, which includes the outer mitochondrial membrane (OMM) fusion GTPase Mitofusin-1 (Mfn1) and Mfn2 (9) as well as the inner mitochondrial membrane (IMM) GTPase Optic atrophy 1 (Opa1) (10), is embryonic lethal. Similarly, the loss of the core fission GTPase Drp1 is also lethal (11). Tissue-specific deletions of these genes, including the liver (12, 13), are now emerging and lead to more complex phenotypes (14); however, these models will not inform us on the adaptive mitochondrial response to metabolic changes.Mitochondria cristae shape changes from the “orthodox” state, when oxidative phosphorylation is low, to a more “condensed” form in high respiratory conditions. The mechanisms that regulate these changes have emerged in cultured models (15), but have not been followed within tissues in vivo. The inner membrane GTPase Opa1 has been shown to regulate mitochondrial fusion and cristae architecture (16), where loss of Opa1 in cultured mouse embryonic or adult fibroblast cells compromised the function and assembly of the respiratory chain complexes. Further, recent studies have shown that Opa1 ablation in the liver leads to a loss of cristae (13). However, Opa1 function is complex and can also promote mitochondrial fragmentation (17). Upon mitochondrial dysfunction or depolarization, an inner membrane protease called Oma1 becomes activated, cleaving Opa1 into a short, soluble form that then promotes mitochondrial fragmentation (18, 19). Interestingly, mice lacking Oma1 are not embryonic lethal; rather, they show a metabolic phenotype where they become obese, accompanied by hepatic steatosis when placed on a high fat diet (20). This result is of critical importance because it hints at a major role of Opa1 proteolysis in metabolic adaptation and connects it to the emerging notion that central regulators of mitochondria mitochondria–ER tethering, like Mfn2, might also be central to this process (12). To shed light on this possibility, in this study we have used an in vivo mouse model to link changes in the structure of the mitochondria–ER contacts (MERCs) to that of the cristae. Our findings support a model where mitochondria adapt to the postprandial loss of mTORC1 signaling by activating an Mfn2-dependent degradation program of Opa1. Thus, mitochondria adapt to postprandial metabolic transitions by coupling the machineries that organize cristae architecture and MERC assembly, which were previously thought to operate independently of each other.     

Worldwide transmission and seasonal variation of pandemic influenza A(H1N1)2009 virus activity during the 2009–2010 pandemic

Background

Seasonal influenza activity varies with geography and time of year.

Objective

To describe how pandemic influenza A(H1N1)2009 [A(H1N1)pdm09] activity varied during the 2009–2010 pandemic.

Methods

We analyzed influenza virological data compiled by the World Health Organization from June 2009–August 2010. We calculated weekly proportions of A(H1N1)pdm09‐positive specimens out of all A(H1N1)pdm09‐positive specimens detected during the study period for each country. We compared parameters of pandemic activity (e.g., peak A[H1N1]pdm09 weekly proportion [peak activity], number of weeks between the 5th and 95th percentiles of A(H1N1)pdm09 cumulative weekly proportion [duration of activity]) between countries in temperate and tropical–subtropical regions. We quantified the proportion of A(H1N1)pdm09 out of all influenza A specimens by country and correlated it with countries'' central latitudes.

Results

We analyzed data from 80 countries (47 temperate, 33 tropical–subtropical). The median proportion of cases identified during the peak week was higher in temperate (0·12) than in tropical–subtropical (0·09) regions (P < 0·01). The median duration of activity was longer in tropical–subtropical (27 weeks) than in temperate countries (20 weeks) (P < 0·01). In most temperate countries (98%), peak pandemic activity occurred during the fall–winter period. There was a positive correlation between country central latitude and proportion of A(H1N1)pdm09 out of all influenza A specimens (r: 0·76; P < 0·01).

Conclusions

The transmission of A(H1N1)pdm09 exhibited similarities with seasonal influenza transmission in that activity varied between temperate and tropical–subtropical countries and by time of year. Our findings suggest the potential utility of accounting for these factors during future pandemic planning.     

Plasminogen fragment K1–3 inhibits expression of adhesion molecules and experimental HCC recurrence in the liver

Purpose  There is no established adjuvant or neo-adjuvant treatment to curb tumor recurrence of hepatocellular carcinoma (HCC). Recent data showed that angiostatic factors can inhibit tumor cell adhesion to the endothelium and therefore recurrence/metastasis. We tested a potential preventive, pre-operative strategy using plasminogen kringles 1–3 (K1–3) to overcome this hurdle. Materials and methods  Effects of K1–3 on the intercellular cell adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) expression was analyzed in vitro and in vivo on RNA and protein levels. Influence of K1–3 on HCC recurrence in the liver was analyzed in an orthotopic tumor model. Results  K1–3 decreased ICAM expression in Hepa129 tumor cells and VCAM expression in SVEC4-10 endothelial cells in vitro. In vivo, ICAM was reduced in histological tumor sections. Preventive treatment with AdK1–3 inhibited experimental HCC recurrence and tumor growth in the liver. Conclusions  We were able to show that K1–3 inhibits intrahepatic tumor recurrence. This novel aspect elucidates a possible approach to prevent HCC recurrence.     

Tether mutations that restore function and suppress pleiotropic phenotypes of the C. elegans isp-1(qm150) Rieske iron–sulfur protein

Mitochondria play an important role in numerous diseases as well as normative aging. Severe reduction in mitochondrial function contributes to childhood disorders such as Leigh Syndrome, whereas mild disruption can extend the lifespan of model organisms. The Caenorhabditis elegans isp-1 gene encodes the Rieske iron–sulfur protein subunit of cytochrome c oxidoreductase (complex III of the electron transport chain). The partial loss of function allele, isp-1(qm150), leads to several pleiotropic phenotypes. To better understand the molecular mechanisms of ISP-1 function, we sought to identify genetic suppressors of the delayed development of isp-1(qm150) animals. Here we report a series of intragenic suppressors, all located within a highly conserved six amino acid tether region of ISP-1. These intragenic mutations suppress all of the evaluated isp-1(qm150) phenotypes, including developmental rate, pharyngeal pumping rate, brood size, body movement, activation of the mitochondrial unfolded protein response reporter, CO₂ production, mitochondrial oxidative phosphorylation, and lifespan extension. Furthermore, analogous mutations show a similar effect when engineered into the budding yeast Rieske iron–sulfur protein Rip1, revealing remarkable conservation of the structure–function relationship of these residues across highly divergent species. The focus on a single subunit as causal both in generation and in suppression of diverse pleiotropic phenotypes points to a common underlying molecular mechanism, for which we propose a “spring-loaded” model. These observations provide insights into how gating and control processes influence the function of ISP-1 in mediating pleiotropic phenotypes including developmental rate, movement, sensitivity to stress, and longevity.Mitochondria are sites for adenosine 5′-triphosphate (ATP) production by oxidative phosphorylation, cellular calcium buffering, iron–sulfur cluster biogenesis, reactive oxygen species (ROS) formation, and regulation of apoptosis. Although inherited defects in mitochondrial function are most often associated with severe childhood disorders, a large number of age-related diseases such as heart disease, cancer, diabetes, obesity, and neurodegeneration have also been linked to mitochondrial dysfunction (1, 2).In Caenorhabditis elegans, multiple studies have demonstrated that reduced electron transport chain activity (ETC) can lead to increased lifespan. These include mutations in the coenzyme Q biosynthetic gene clk-1, the pyrophosphokinase gene tpk-1, and the Rieske iron–sulfur protein isp-1 (3–7). Following RNAi knockdown of ETC components, several other proteins have been implicated in lifespan extension, including HIF-1, GCN-2, CEP-1, CEH-23, TAF-4, AHA-1, CEH-18, JUN-1, NHR-27, and NHR-49 (8–12). In addition, it was proposed that the mitochondrial unfolded protein response (mtUPR) directly mediated lifespan extension from ETC inhibition (13); however, more recent work has suggested that induction of the mtUPR is neither necessary nor sufficient to extend lifespan in worms (14). A self-consistent model is emerging suggesting that isp-1(qm150) animals have increased levels of ROS, which induces activation of the intrinsic apoptotic pathway to extend lifespan (15).ISP-1 is an evolutionary conserved, nuclear-encoded iron–sulfur (2Fe-2S) protein that functions within complex III of the electron transport chain (16). The isp-1(qm150) allele, which results in a proline to serine substitution, has been particularly well studied due to its robust positive effect on lifespan (6). In this context, we set out to further explore the biochemical and molecular mechanisms by which the isp-1(qm150) mutation causes pleiotropic phenotypes including delayed development and increased lifespan. Here we report the identification of intragenic suppressors of isp-1(qm150) all located in the highly conserved six amino acid tether (also sometimes referred to as a “hinge”) region of ISP-1. These mutations suppressed all of the phenotypes associated with isp-1(qm150) examined, including a previously unreported sensitivity to hyperoxia. In addition, we show a similar relationship between the isp-1(qm150) mutation and two of the isolated suppressors in the budding yeast Rieske iron–sulfur protein Rip1, demonstrating a striking conservation of the structure–function relationship across widely divergent phyla. Analysis of the extensive literature on physicochemical parameters involving the role of ISP reveals a “spring-loaded” mechanism of action, summarized in the discussion and further supported in the SI Appendix.     

Disease-associated increased HIF-1, αvβ3 integrin,and Flt-1 do not suffice to compensate the damage-inducing loss of blood vessels in inflammatory myopathies

Objective To analyze the microvascular network in skeletal muscle biopsies from patients with dermatomyositis (DM) and systemic sclerosis (SSc) compared to polymyositis (PM) and systemic lupus erythematosus (SLE), and non-inflammatory myopathies, and to clarify whether reparative angiogenesis-related factors are expressed in parallel to blood vessel damage.Methods Immunohistochemical staining of muscle biopsies (10 DM, 10 SSc, 10 PM, 10 SLE, and 10 non-inflammatory myopathies) with antibodies against von Willebrand factor (vWF), hypoxia-inducible factor-1 (HIF-1), 3 integrin subunit, and vascular endothelial growth factor receptor-1 (VEGFR-1). The TechMate staining robot and biotin-streptavidin protocol were used.Results DM and SSc muscles were characterized by endothelial damage and reduction of blood vessel network. Expression of angiogenesis-related factors (HIF-1, 3, VEGFR-1) was also found in the same biopsies. In contrast, in PM and SLE muscles, vascular networks were apparently not affected and angiogenic stimuli were less expressed if at all.Conclusions This work demonstrates that in inflamed muscles hypoxia/ischemia induces increased expression of angiogenic factors, yet their impact is insufficient to repair disease-associated reduction of the capillary network. This leads to questions considering the usefulness of angiogenic factors in the treatment of ischemic inflammatory myopathies in DM and SSc.