Compound C prevents Hypoxia-Inducible Factor-1α protein stabilization by regulating the cellular oxygen availability via interaction with Mitochondrial Complex I

The transcription factor Hypoxia-Inducible Factor-1α is a master regulator of the cellular response to low oxygen concentration. Compound C, an inhibitor of AMP-activated kinase, has been reported to inhibit hypoxia dependent Hypoxia-Inducible Factor-1α activation via a mechanism that is independent of AMP-activated kinase but dependent on its interaction with the mitochondrial electron transport chain. The objective of this study is to characterize the interaction of Compound C with the mitochondrial electron transport chain and to determine the mechanism through which the drug influences the stability of the Hypoxia-Inducible Factor-1α protein

Feasibility of using quadriceps-strengthening exercise to improve pain and sleep in a severely demented elder with osteoarthritis – a case report

BACKGROUND: Osteoarthritis (OA) of the knee, which is prevalent among older adults in nursing homes, causes significant pain and suffering, including disturbance of nocturnal sleep. One nonpharmacologic treatment option is quadriceps-strengthening exercise, however, the feasibility of such a treatment for reducing pain from OA in severely demented elders has not been studied. This report describes our test of the feasibility of such an exercise program, together with its effects on pain and sleep, in a severely demented nursing home resident. CASE PRESENTATION: The subject was an elderly man with severe cognitive impairment (Mini-Mental Status Exam score 4) and knee OA (Kellgren-Lawrence radiographic grade 4). He was enrolled in a 5-week, 10-session standardized progressive-resistance training program to strengthen the quadriceps, and completed all sessions. Pain was assessed with the Western Ontario and MacMaster OA Index (WOMAC) pain subscale, and sleep was assessed by actigraphy. The patient was able to perform the exercises, with a revision to the protocol. However, the WOMAC OA pain subscale proved inadequate for measuring pain in a patient with low cognitive functioning, and therefore the effects on pain were inconclusive. Although his sleep improved after the intervention, the influence of his medications and the amount of daytime sleep on his nighttime sleep need to be considered. CONCLUSIONS: A quadriceps-strengthening exercise program for treating OA of the knee is feasible in severely demented elders, although a better outcome measure is needed for pain

Molecular evolution of HoxA13 and the multiple origins of limbless morphologies in amphibians and reptiles

Developmental processes and their results, morphological characters, are inherited through transmission of genes regulating development. While there is ample evidence that cis-regulatory elements tend to be modular, with sequence segments dedicated to different roles, the situation for proteins is less clear, being particularly complex for transcription factors with multiple functions. Some motifs mediating protein-protein interactions may be exclusive to particular developmental roles, but it is also possible that motifs are mostly shared among different processes. Here we focus on HoxA13, a protein essential for limb development. We asked whether the HoxA13 amino acid sequence evolved similarly in three limbless clades: Gymnophiona, Amphisbaenia and Serpentes. We explored variation in ω (dN/dS) using a maximum-likelihood framework and HoxA13sequences from 47 species. Comparisons of evolutionary models provided low ω global values and no evidence that HoxA13 experienced relaxed selection in limbless clades. Branch-site models failed to detect evidence for positive selection acting on any site along branches of Amphisbaena and Gymnophiona, while three sites were identified in Serpentes. Examination of alignments did not reveal consistent sequence differences between limbed and limbless species. We conclude that HoxA13 has no modules exclusive to limb development, which may be explained by its involvement in multiple developmental processes

Mitochondrial dysfunction resulting from loss of cytochrome c impairs radiation-induced bystander effect

Cytochrome c is a pivotal protein that resides in mitochondria as component of mitochondria respiration and apoptosis initiator. Using murine cells lacking cytochrome c, we showed here that cytochrome c-deficient cells had attenuated reactive oxygen species/nitric oxide and micronuclei induction to radiation-induced bystander signals, indicating cytochrome c is essential for the bystander effect

Belt restraint reduction in nursing homes: design of a quasi-experimental study

<p>Abstract</p> <p>Background</p> <p>The use of physical restraints still is common practice in the nursing home care. Since physical restraints have been shown to be an ineffective and sometimes even hazardous measure, interventions are needed to reduce their usage. Several attempts have been made to reduce the use of physical restraints. Most studies used educational approaches and introduced a nurse specialist as a consultant. However, the success rate of these interventions has been inconsistent. We developed a new multi-component intervention (EXBELT) comprising an educational intervention for nursing home staff in combination with a policy change (belt use is prohibited by the nursing home management), availability of a nurse specialist and nursing home manager as consultants, and availability of alternative interventions. The first aim of this study is to further develop and test the effectiveness of EXBELT on belt restraint reduction in Dutch psychogeriatric nursing homes. However, the reduction of belts should not result in an increase of other restrictive restraints (such as a chair with locked tray table) or psychoactive drug use. The overall aim is an effective and feasible intervention that can be employed on a large scale in Dutch nursing homes.</p> <p>Methods and design</p> <p>Effects of EXBELT will be studied in a quasi-experimental longitudinal study design. Alongside the effect evaluation, a process evaluation will be carried out in order to further develop EXBELT. Data regarding age, gender, use of physical restraints, the number of falls and fall related injuries, psychoactive drug use, and the use of alternative interventions will be collected at baseline and after four and eight months of follow-up. Data regarding the process evaluation will be gathered in a period of eight months between baseline and the last measurement. Furthermore, changing attitudes will become an important addition to the educational part of EXBELT.</p> <p>Discussion</p> <p>A quasi-experimental study is presented to investigate the effects of EXBELT on the use of belts on wards in psychogeriatric nursing homes. The study will be conducted in 26 wards in 13 psychogeriatric nursing homes. We selected the wards in a manner that contamination between control- and intervention group is prevented.</p> <p>Trial registration</p> <p>(NTR2140)</p

HIF-Independent Regulation of Thioredoxin Reductase 1 Contributes to the High Levels of Reactive Oxygen Species Induced by Hypoxia

Cellular adaptation to hypoxic conditions mainly involves transcriptional changes in which hypoxia inducible factors (HIFs) play a critical role. Under hypoxic conditions, HIF protein is stabilized due to inhibition of the activity of prolyl hydroxylases (EGLNs). Because the reaction carried out by these enzymes uses oxygen as a co-substrate it is generally accepted that the hypoxic inhibition of EGLNs is due to the reduction in oxygen levels. However, several studies have reported that hypoxic generation of mitochondrial reactive oxygen species (ROS) is required for HIF stabilization. Here, we show that hypoxia downregulates thioredoxin reductase 1 (TR1) mRNA and protein levels. This hypoxic TR1 regulation is HIF independent, as HIF stabilization by EGLNs inhibitors does not affect TR1 expression and HIF deficiency does not block TR1 hypoxic-regulation, and it has an effect on TR1 function, as hypoxic conditions also reduce TR1 activity. We found that, when cultured under hypoxic conditions, TR1 deficient cells showed a larger accumulation of ROS compared to control cells, whereas TR1 over-expression was able to block the hypoxic generation of ROS. Furthermore, the changes in ROS levels observed in TR1 deficient or TR1 over-expressing cells did not affect HIF stabilization or function. These results indicate that hypoxic TR1 down-regulation is important in maintaining high levels of ROS under hypoxic conditions and that HIF stabilization and activity do not require hypoxic generation of ROS

Modular protein-RNA interactions regulating mRNA metabolism: a role for NMR

Here we review the role played by transient interactions between multi-functional proteins and their RNA targets in the regulation of mRNA metabolism, and we describe the important function of NMR spectroscopy in the study of these systems. We place emphasis on a general approach for the study of different features of modular multi-domain recognition that uses well-established NMR techniques and that has provided important advances in the general understanding of post-transcriptional regulation

Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cells

<p>Abstract</p> <p>Background</p> <p>Hypoxia inducible factor-1 (HIF-1) is a transcription factor that functions to maintain cellular homeostasis in response to hypoxia. There is evidence that HIF-1 can also trigger apoptosis, possibly when cellular responses are inadequate to meet energy demands under hypoxic conditions.</p> <p>Methods</p> <p>Cardiac derived H9c2 and renal tubular epithelial HK-2 cells expressing either the wild type oxygen regulated subunit of HIF-1 (pcDNA3-Hif-1α) or a dominant negative version that lacked both DNA binding and transactivation domains (pcDNA3-DN-Hif-1α), were maintained in culture and exposed to hypoxia. An RNA interference approach was also employed to selectively knockdown expression of Hif-1α. Apoptosis was analyzed in both H9c2 and HK-2 cells by Hoechst and TUNEL staining, caspase 3 activity assays and activation of pro-apoptotic Bcl2 family member Bax.</p> <p>Results</p> <p>Overexpression of pcDNA3-DN-Hif-1α led to a significant reduction in hypoxia -induced apoptosis (17 ± 2%, <it>P </it>< 0.01) in H9c2 cells compared to both control-transfected and wild type Hif-1α transfected cells. Moreover, selective ablation of HIF-1α protein expression by RNA interference in H9c2 cells led to 55% reduction of caspase 3 activity and 46% reduction in the number of apoptotic cells as determined by Hoechst 33258 staining, after hypoxia. Finally, upregulation of the pro-apoptotic protein, Bax, was found in H9c2 cells overexpressing full-length pcDNA3-HA-HIF-1α exposed to hypoxia. In HK-2 cells overexpression of wild-type Hif-1α led to a two-fold increase in Hif-1α levels during hypoxia. This resulted in a 3.4-fold increase in apoptotic cells and a concomitant increase in caspase 3 activity during hypoxia when compared to vector transfected control cells. HIF-1α also induced upregulation of Bax in HK-2 cells. In addition, introduction of dominant negative Hif-1α constructs in both H9c2 and HK-2 -cells led to decreased active Bax expression.</p> <p>Conclusion</p> <p>These data demonstrate that HIF-1α is an important component of the apoptotic signaling machinery in the two cell types.</p

Effects of TLR Agonists on the Hypoxia-Regulated Transcription Factor HIF-1α and Dendritic Cell Maturation under Normoxic Conditions

Dendritic cells (DC) are professional antigen presenting cells that represent an important link between innate and adaptive immunity. Danger signals such as toll-like receptor (TLR) agonists induce maturation of DC leading to a T-cell mediated adaptive immune response. In this study, we show that exogenous as well as endogenous inflammatory stimuli for TLR4 and TLR2 induce the expression of HIF-1α in human monocyte-derived DC under normoxic conditions. On the functional level, inhibition of HIF-1α using chetomin (CTM), YC-1 and digoxin lead to no consistent effect on MoDC maturation, or cytokine secretion despite having the common effect of blocking HIF-1α stabilization or activity through different mechanisms. Stabilization of HIF-1α protein by hypoxia or CoCl2 did not result in maturation of human DC. In addition, we could show that TLR stimulation resulted in an increase of HIF-1α controlled VEGF secretion. These results show that stimulation of human MoDC with exogenous as well as endogenous TLR agonists induces the expression of HIF-1α in a time-dependent manner. Hypoxia alone does not induce maturation of DC, but is able to augment maturation after TLR ligation. Current evidence suggests that different target genes may be affected by HIF-1α under normoxic conditions with physiological roles that differ from those induced by hypoxia