Hemoglobin induces inflammation after preterm intraventricular hemorrhage by methemoglobin formation.

Cerebral intraventricular hemorrhage (IVH) is a major cause of severe neurodevelopmental impairment in preterm infants. To date, no therapy is available that prevents infants from developing serious neurological disability following IVH. Thus, to develop treatment strategies for IVH, it is essential to characterize the initial sequence of molecular events that leads to brain damage. In this study, we investigated extracellular hemoglobin (Hb) as a causal initiator of inflammation in preterm IVH

Pathophysiology of extracellular haemoglobin : use of animal models to translate molecular mechanisms into clinical significance

The blood's major gas exchange is carried out by haemoglobin, a haeme protein that binds iron and oxygen and can have potentially dangerous side-effects due to redox reactions. Haemoglobin is a very abundant molecule with a concentration of 150 g/l in whole blood, resulting in almost one kg haemoglobin in an adult human body. Normal turnover of red blood cells results in significant haemoglobin release, and pathological conditions that involve haemolysis can lead to massive haemoglobin levels. To control for the potential threat of extracellular haemoglobin, several protective defence systems have evolved. Many pathological conditions, diseases as well as iatrogenic conditions, such as infusion of haemoglobin-based oxygen carriers, cerebral intraventricular haemorrhage, extracorporeal circulation and the pregnancy complication pre-eclampsia, involve abnormal levels of haemolysis and extracellular haemoglobin. Although quite different aetiology, the haemoglobin-induced damage often causes similar clinical sequelae and symptoms. Here, we will give an overview of the pathophysiological mechanisms of extracellular haemoglobin and its metabolites. Furthermore, we will highlight the use of animal models in advancing the understanding of these mechanisms and discuss how to utilize the knowledge in the development of new and better pharmaceutical therapies

The cysteine 34 residue of A1M/α1-microglobulin is essential for protection of irradiated cell cultures and reduction of carbonyl groups.

α1-microglobulin (A1M) is a 26 kDa plasma and a tissue protein belonging to the lipocalin family. The reductase and free radical scavenger A1M has been shown to protect cells and extracellular matrix against oxidative and irradiation-induced damage. The reductase activity was previously shown to depend upon an unpaired cysteinyl side-chain, C34, and three lysyl side-chains, K92, 118, and 130, located around the open end of the lipocalin pocket. The aim of this work was to investigate whether the cell and matrix protection by A1M is a result of its reductase activity by using A1M-variants with site-directed mutations of the C34, K92, K118, and K130 positions. The results show that the C34 side-chain is an absolute requirement for protection of HepG2 cell cultures against alpha-particle irradiation-induced cell death, upregulation of stress response and cell cycle regulation genes. Mutation of C34 also resulted in loss of the reduction capacity toward heme- and hydrogen peroxide-oxidized collagen, and the radical species 2,2´-azino-bis (3-ethyl-benzo-thiazoline-6-sulphonic acid) (ABTS). Furthermore, mutation of C34 significantly suppressed the cell-uptake of A1M. The K92, K118, and K130 side-chains were of minor importance in cell protection and reduction of oxidized collagen but strongly influenced the reduction of the ABTS-radical. It is concluded that antioxidative protection of cells and collagen by A1M is totally dependent on its C34 amino acid residue. A model of the cell protection mechanism of A1M should be based on the redox activity of the free thiolyl group of the C34 side-chain and a regulatory role of the K92, K118, and K130 residues

Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers

Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical alpha- and two identical gamma-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the alpha- and gamma-subunits of HbF were fused in order to stabilize the alpha-subunits and facilitate balanced expression of alpha- and gamma-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes

Pathological Conditions Involving Extracellular Hemoglobin: Molecular Mechanisms, Clinical Significance, and Novel Therapeutic Opportunities for alpha(1)-Microglobulin

Hemoglobin is the major oxygen-carrying system of the blood, but has many potentially dangerous side effects due to oxidation and reduction reactions of the heme-bound iron and oxygen. Extracellular hemoglobin, resulting from hemolysis or exogenous infusion, is shown to be an important pathogenic factor in a growing number of diseases. This review briefly outlines the oxidative/reductive toxic reactions of hemoglobin and its metabolites. It also describes physiological protection mechanisms that have evolved against extracellular hemoglobin, with a focus on the most recently discovered: the heme- and radical-binding protein α1-microglobulin (A1M). This protein is found in all vertebrates including man and operates by rapidly clearing cytosols and extravascular fluids of heme groups and free radicals released from hemoglobin. Five groups of pathological conditions with high concentrations of extracellular hemoglobin are described: hemolytic anemias and transfusion reactions, the pregnancy complication preeclampsia, cerebral intraventricular hemorrhage of premature infants, chronic inflammatory leg ulcers, and infusion of hemoglobin-based oxygen carriers as blood substitutes. Finally, possible treatments of these conditions are discussed, giving special attention to the described protective effects of A1M

The radical-binding lipocalin A1M binds to a Complex I subunit and protects mitochondrial structure and function.

Aims: During cell death, energy-consuming cell degradation and recycling programs are performed. Maintenance of energy-delivery during cell death is therefore crucial but the mechanisms to keep the mitochondrial functions intact during these processes are poorly understood. We have investigated the hypothesis that the heme- and radical-binding ubiquitous protein A1M (α1-microglobulin) is involved in protection of the mitochondria against oxidative insult during cell death. Results: Using blood cells, keratinocytes and liver cells, we show that A1M binds with high affinity to apoptosis-induced cells and is localized to mitochondria. The mitochondrial Complex I subunit NDUFAB1 was identified as a major molecular target of the A1M-binding. Furthermore, A1M was shown to inhibit the swelling of mitochondria, and to reverse the severely abrogated ATP-production of mitochondria when exposed to heme and ROS. Innovation: Import of the radical- and heme-binding protein A1M from the extracellular compartment confers protection of mitochondrial structure and function during cellular insult. Conclusion: A1M binds to a subunit of Complex I and has a role in assisting the mitochondria to maintain its energy delivery during cell death. A1M may also, at the same time, counteract and eliminate the ROS generated by the mitochondrial respiration to prevent oxidative damage to surrounding healthy tissue

Urban–Rural Differences in Hip Fracture Mortality: A Nationwide NOREPOS Study

Higher hip fracture incidence in urban than in rural areas has been demonstrated, but urban–rural differences in posthip fracture mortality have been less investigated, and the results are disparate. Hence, the aims of the present register‐based cohort study were to examine possible urban–rural differences in short‐ and long‐term mortality in Norwegian hip fracture patients and their potential associations with sociodemographic variables, and to investigate possible urban–rural differences in excess mortality in hip fracture patients compared with the general population. Data were provided from the NOREPOS hip fracture database, the 2001 Population and Housing Census, and the National Registry. The urbanization degree in each municipality was determined by the proportion of inhabitants living in densely populated areas (rural: 2/3). Age‐adjusted mortality rates and standardized mortality ratios were calculated for hip fracture patients living in rural, semirural, and urban municipalities. A flexible parametric model was used to estimate age‐adjusted average and time‐varying HRs by category of urbanization with the rural category as reference. Among 96,693 hip fracture patients, urban residents had higher mortality than their rural‐dwelling counterparts. The HR of mortality in urban compared with rural areas peaked during the first 1 to 2 years postfracture with a maximum HR of 1.20 (95% CI, 1.10 to 1.30) in men and 1.15 (95% CI, 1.08 to 1.21) in women. The differences were significant during approximately 5 years after fracture. Adjusting for sociodemographic variables did not substantially change the results. However, absolute 30‐day mortality was not significantly different between urban and rural residents, suggesting that health‐care quality immediately postfracture does not vary by urbanization. The novel findings of a higher long‐term mortality in urban hip fracture patients might reflect disparities in health status or lifestyle, differences in posthip fracture health care or rehabilitation, or a combination of several factors

Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion

Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1–10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1–10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1–10 in analyses. The pre-maturated GFP 1–10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1–10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1–10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.Published versio

Heme Induces Endoplasmic Reticulum Stress (HIER Stress) in Human Aortic Smooth Muscle Cells

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