Presence Of Myoglobin Research Articles

A biomimetic assay for antioxidant reactivity, based on liposomes and myoglobin

Antioxidant assays are typically based on non-physiologically relevant reagents. We describe here a quantitative assay based on the inhibition of the liposome autooxidation in the presence of myoglobin (ILA-Mb), an oxidative process with direct biomedical relevance. Additional advantages of the assay include the use of standard and readily available reagents (lecithin and myoglobin) and the applicability to lipophilic antioxidants. The ILA-Mb assay is based on previously reported qualitative or semi-quantitative ones that employed cytochrome c instead of myoglobin. A number of antioxidants are tested, and their IC50 parameters are discussed and interpreted to involve direct interaction with both myoglobin and the liposomes.

Structure/function relationship of Atlantic bonito (Sarda sarda Bloch) myoglobin and comparison with Atlantic and Tinker mackerel myoglobins

Atlantic bonito meat has economic potential as an alternative to mackerel consumption. Thus, considering the presence of myoglobin (Mb) in red fish muscles, we report the characterisation of Atlantic bonito Mb compared to Atlantic and Tinker mackerel Mbs since this haemoprotein is implicated in lipid oxidation and fish meat preservation. A plethora of biochemical approaches were employed to purified Mb from Atlantic bonito and determine the autoxidation rate constant (0.189 ± 0.009 h−1), melting temperature (Tm = 72.84 ± 1.02 °C) and pseudoperoxidase activity in different conditions (pH and several cations). Atlantic and Tinker mackerel Mbs showed a lower Tm (∼66.85 °C), while oxyMb autoxidation rate constant was higher for Atlantic mackerel (∼1.08-fold) and lower for Tinker mackerel (∼1.35-fold) compared to Atlantic bonito. This Mb had a Michaelis-Menten constant (Km) of 38.63 ± 1.89 μM, ∼2.49-fold and 2.27-fold lower than Atlantic and Tinker mackerel Mbs, respectively. Atlantic bonito Mb primary structure has 146 amino acid residues with the N-terminal acetylated and 25 amino acid substitutions with respect to Atlantic and Tinker mackerel Mbs. In silico analysis revealed that 7 out of 25 substitutions are close to the haem-pocket, while 18 out of 25 are far from this region. All substitutions, except H20, L70 and L81 are exposed on the protein globular surface. Overall, the results of this research provide new information for future studies that will be useful to the fish industry for preservation of frozen or canned Atlantic bonito meat considering the presence of Mb as a reactive haemoprotein.

Influence of myoglobin on the antibacterial activity of carvacrol and the binding mechanism between the two compounds.

Myoglobin (MB), a pigmentation protein, can adversely affect the antibacterial activity of carvacrol (CAR) and weaken its bacteriostasis effect. This study aimed to clarify the influence of MB on the antibacterial activity of CAR and ascertain the mechanism involved in the observed influence, especially the interaction between the two compounds. Microbiological analysis indicated that the presence of MB significantly suppressed the antibacterial activity of CAR against Listeria monocytogenes. Ultraviolet-visible spectrometry and fluorescence spectroscopic analysis confirmed the interaction between CAR and MB. The stoichiometric number was determined as ~0.7 via double logarithmic Stern-Volmer equation analysis, while thermodynamic analysis showed that the conjugation of the two compounds occurred as an exothermal reaction (ΔH° = -32.3 ± 11.4 kJ mol-1 and ΔS° = -75 J mol-1 K-1 ). Circular dichroism, Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy showed hydrogen bonding in the carvacrol-myoglobin complex (CAR-MB). Molecular docking analysis confirmed that amino acid residues, including GLY80 and HIS82, were most likely to form hydrogen bonds with CAR, while hydrogen bonds represented the main driving force for CAR-MB formation. CAR antibacterial activity was significantly inhibited by the presence of MB in the environment due to the notable reduction in the effective concentration of CAR caused by CAR-MB formation. © 2023 Society of Chemical Industry.

Role of Hydrogen Bonding and Hydrophobic Interactions on the Stabilization of Myoglobin (Globular Protein)-Primaquine-4-Dicyanomethylene-2,6-Dimethyl-4H-pyran (DDP) Conformers

Molecular docking (MD) approach on the binding interaction of two competing ligands with a well-known globular protein, myoglobin (MB) was carried out. Docking studies of 4-dicyanomethylene- 2,6-dimethyl-4H-pyran (DDP) dye and an anti-malarial drug, primaquine (PRQ) were considered as the guest molecules in the presence of myoglobin as the host molecule. Docking studies of dye-myoglobin conformers reveals that the dye resides predominantly in between the helices C and D, which are stabilized and governed by hydrogen-bonding interactions. Further, the dye is also stabilized through hydrophobic interactions when confined to E and F helices of the protein. However, the drug prefers to reside in all the domains of the polypeptide chain structure of myoglobin resulting in a larger stability when compared to that of dye-protein complex. Interestingly, the displacement of dye from the binding sites of myoglobin was found to be ineffective in the presence of drug. On the contrary, the dye efficiently displaces the drug entirely to a single confined region (between C and D, E and between F and G) of the protein molecule. Docking studies signifies that the hydrogen bonding donor and acceptor sites were involved in the stabilization of complex thereby provides a clear elucidation on the binding nature of two competing guest molecules in the presence of host molecule. Molecular docking technique was used as an efficient and reliable tool to determine the various bimolecular interactions existing between protein-dye and protein- drug complexes.

Preparation of Surface Plasmon Resonance Aptasensor for Human Activated Protein C Sensing.

Nucleic acid aptamers are an emerging class of artificial ligands and have recently gained attention in several areas. Here we report the design of a surface plasmon resonance (SPR) aptasensor for highly sensitive and selective sensing of human activated protein C (APC). First, DNA aptamer (DNA-Apt) specific for APC is complexed with N-methacryloyl-L-cysteine (MAC) monomer. Then, 2-hydroxyethyl methacrylate (HEMA) and cyanamide are mixed with the DNA-Apt/MAC complex. The SPR aptasensor is characterized by atomic force microscopy, ellipsometry, and contact angle measurements. Selectivity of SPR aptasensor is carried out in the presence of myoglobin (Myb), hemoglobin (Hb), and bovine serum albumin (BSA). Limit of detection (LOD) and limit of quantification (LOQ) values are 1.5ngmL-1 and 5.2ngmL-1, respectively. DNA-Apt SPR aptasensor performance for APC detection is also examined in artificial plasma.

Label-Free Electrochemical Sensor Based on Manganese Doped Titanium Dioxide Nanoparticles for Myoglobin Detection: Biomarker for Acute Myocardial Infarction.

A label free electrochemical sensor based on pure titanium oxide and manganese (Mn)-doped titanium oxide (TiO2) nanoparticles are fabricated and characterized for the sensitive detection of myoglobin (Mb) levels to analyze the cardiovascular infarction. Pristine and Mn-doped TiO2 nanoparticles were synthesized via the sol-gel method and characterized in order to understand their structure, morphologies, composition and optical properties. The structural properties revealed that the pure- and doped-TiO2 nanoparticles possess different TiO2 planes. FTIR studies confirm the formation of metal oxide nanoparticles by exhibiting a well-defined peak in the range of 600–650 cm−1. The values of the optical band gap, estimated from UV-Vis spectroscopy, are decreased for the Mn-doped TiO2 nanoparticles. UV-Vis spectra in the presence of myoglobin (Mb) indicated interaction between the TiO2 nanoparticles and myoglobin. The SPE electrodes were then fabricated by printing powder film over the working electrode and tested for label-free electrochemical detection of myoglobin (Mb) in the concentration range of 0–15 nM Mb. The fabricated electrochemical sensor exhibited a high sensitivity of 100.40 μA-cm−2/nM with a lowest detection limit of 0.013 nM (0.22 ng/mL) and a response time of ≤10 ms for sample S3. An interference study with cyt-c and Human Serum Albumin (HSA) of the sensors show the selective response towards Mb in 1:1 mixture.

New Plausible Mechanism for Gastric and Colorectal Carcinogenesis: Free Radical-Mediated Acetaldehyde Generation in a Heme/Myoglobin-Linoleate-Ethanol Mixture.

Epidemiological studies have revealed that alcohol, red meat, and cooking oil (or linoleate) are risk factors for both gastric and colon cancers. A survey of the mutation spectra of the p53 tumor suppressor gene in these cancers suggested that the types of mutations and the hot spots are similar to those induced by acetaldehyde (AcAld) in an in vitro p53 mutation analysis system. Accordingly, various combinations of possible factors, components, or model compounds were reacted in an emulsion and tested for the generation of AcAld. Efficient AcAld formation was only observed with combinations of three factors, red meat homogenate (or heme/myoglobin), methyl linoleate, and ethanol, but not by any combination of the two. The generated AcAld levels (ca. 500 μM) far exceeded the minimum mutagenic concentration (40–100 μM) obtained using concentrations of meat homogenate (or heme/Mb), linoleate, and ethanol comparable to those in the stomach after an ordinary meal. A mutagenic level of AcAld (75 μM) was also generated with a physiological concentration of ethanol, heme, and linoleate in the colon. As a mechanism, linoleate hydroperoxide formation and its decomposition in the presence of myoglobin (or heme) to generate the OH radical seem to be involved in the ethanol-to-AcAld conversion.

Think muscle; Think rhabdomyolysis

Rhabdomyolysis is the breakdown of striated muscle cells resulting in leakage of cell matter into the extra-cellular space. It can present with myalgia, muscle weakness and swelling. Episodes of passing of dark tea-coloured urine have also been reported. Raised creatinine kinase is diagnostic of rhabdomyolysis. Raised serum myoglobin levels and presence of myoglobin in the urine (myoglobinuria) help to support the diagnosis. The aetiology for rhabdomyolysis can be both traumatic and non-traumatic. In the case of trauma, individuals with crush injuries trapped in cars or under collapsed buildings , struggling against restraints, immobilized and in the same position for hours due to injuries, comatosed states or positioning during prolonged surgeries and those with high voltage electrical injuries are at increased risk of rhabdomyolysis. Non-traumatic causes of rhabdomyolysis include hyperthermia, metabolic myopathies, drugs and toxins ingestions, electrolyte abnormalities and infections. The mechanism of rhabdomyolysis is often multifactorial. In the case of trauma, direct injury to cell membranes as well as hypoxia from direct compression leading to adenosine triphosphate depletion leads to breakdown of striated muscle cells.. The lysed cells release myoglobin, creatine kinase, urate and phosphate into the interstitium. Direct heme protein-induced toxicity on nephrons can result in acute kidney injury. We report a case of traumatic rhabdomyolysis and share the latest in the literature on the understanding of the subject as well as that of myoglobinuria, with which it is often associated with.

Highly sensitive label-free electrochemiluminescence aptasensor for early detection of myoglobin, a biomarker for myocardial infarction

The authors describe an ultrasensitive electrochemiluminescence (ECL) based aptasensor for myoglobin, a biomarker for the early diagnosis of heart attack (myocardial infarction). A nanocomposite was prepared from reduced graphene oxide, ruthenium(II)-tris(2,2- bipyridyl), samarium oxide and chitosan and placed on the surface of a ccreen-printed carbon electrode (SPCE). The aptamer was immobilized via electrostatic interaction between the negatively charged aptamer and the positively charged nanocomposite which represents a remarkable immobilization platform. In the absence of myoglobin, the ECL signal of the ruthenium complex (generated at 1.2 V and peaking at 530 nm is weak. In the presence of myoglobin, a remarkable increase in ECL is observed due to the formation of the aptamer-myoglobin complex and the corresponding release of the labeled aptamercomplex from the surface. The response of the sensor is linear in the 0.05 to 25 nM myoglobin concentration range, with a 12 pM detection limit (at an S/N ratio of 3). The assay was successfully applied to the determination of myoglobin in spiked human serum and human urine samples where it gave recoveries that ranged from 96 to 105%, and from 95 to 103.7%, respectively."

Rhabdomyolysis in Critically Ill Surgical Patients.

Introduction:Rhabdomyolysis is a syndrome of injury of skeletal muscles associated with myoglobinuria, muscle weakness, electrolyte imbalance and often, acute kidney injury as severe complication.The aim:of this study is to detect the incidence of rhabdomyolysis in critically ill patients in the surgical intensive care unit (ICU), and to raise awareness of this medical condition and its treatment among the clinicians.Material and methods:A retrospective review of all surgical and trauma patients admitted to surgical ICU of the University Surgical Clinic “Mother Teresa” in Skopje, Macedonia, from January 1st till December 31st 2015 was performed. Patients medical records were screened for available serum creatine kinase (CK) with levels > 200 U/l, presence of myoglobin in the serum in levels > 80 ng/ml, or if they had a clinical diagnosis of rhabdomyolysis by an attending doctor. Descriptive statistical methods were used to analyze the collected data.Results:Out of totally 1084 patients hospitalized in the ICU, 93 were diagnosed with rhabdomyolysis during the course of one year. 82(88%) patients were trauma patients, while 11(12%) were surgical non trauma patients. 7(7.5%) patients diagnosed with rhabdomyolysis developed acute kidney injury (AKI) that required dialysis. Average values of serum myoglobin levels were 230 ng/ml, with highest values of > 5000 ng/ml. Patients who developed AKI had serum myoglobin levels above 2000 ng/ml. Average values of serum CK levels were 400 U/l, with highest value of 21600 U/l. Patients who developed AKI had serum CK levels above 3000 U/l.Conclusion:Regular monitoring and early detection of elevated serum CK and myoglobin levels in critically ill surgical and trauma patients is recommended in order to recognize and treat rhabdomyolysis in timely manner and thus prevent development of AKI.

Visual detection of myoglobin via G-quadruplex DNAzyme functionalized gold nanoparticles-based colorimetric biosensor

Since myoglobin plays a major role in the diagnosis of acute myocardial infarction (AMI), monitoring of myoglobin in point-of-care is fundamental. Here, a novel colorimetric assay for myoglobin detection was developed based on hemin/G-quadruplex DNAzyme functionalized gold nanoparticles (AuNPs). In the presence of myoglobin, the anti-myoglobin antibody, which was modified on the surface of polystyrene microplate, could first capture the target myoglobin. Then the captured target could further bind to DNA1 probe which contained the aptamer sequence through aptamers/myoglobin interaction. Next, as the DNA2 probe modified AuNPs were introduced, DNA2 probe modified AuNPs could hybridize with the captured DNA1 probe. Subsequently, DNA2 probe which was modified on the AuNPs could fold into a G-quadruplex structure and bind to hemin, and then catalyze the oxidation of colorless ABTS2− to green ABTS+ by H2O2. Consequently, the relationship between the concentration of myoglobin and the absorbance was established. Due to AuNPs amplification, the myoglobin concentration as low as 2.5nM could be detected, which was lower than clinical cutoff for myoglobin in healthy patients. This assay also showed high selectivity for myoglobin and was used for the detection of myoglobin in the human serum samples. This work may provide a simple but effective tool for early diagnosis of AMI in the world, especially in developing countries.

Capture myopathy in a corsican red deer Cervus elaphus corsicanus (Ungulata: Cervidae)

The present study focused on the importance of translocation as stressful event in an endangered cervid species causing fatal consequences during capture operations. Fourteen free-ranging Sardinian red deer (Cervus elaphus corsicanus) have been captured for restocking propose in a protected area of southwestern Sardinia. The cervids were chemically immobilized, transported to a restocking area and released in the wild. Sixteen hours after the release, a pregnant hind was found dead and a complete necropsy was performed. Post-capture blood samples showed increased levels of creatine kinase, lactate dehydrogenase, cortisol and potassium and were highly indicative of stress-linked muscle damage. The macro- and microscopic lesions consisted of muscular and cardiac degeneration, and renal injury. In the kidneys, the presence of myoglobin in intratubular casts, detected by immunohistochemistry assay, suggested an acute renal failure associated with myoglobinuric nephrosis as a consequence of rhabdomyolysis. The pathological findings were indicative of capture myopathy in a Cervus elaphus corsicanus. This condition has never been reported in the endangered Corsican red deer. This report underlines that mortality from capture is a risk that must be considered during restocking programs. Further studies are needed to minimize stress to preserve wildlife from the risk of fatal consequences due to human interactions.

Acute kidney injury and rhabdomyolysis due to multiple wasp stings.

Sir, We read the article, “Acute kidney injury and rhabdomyolysis due to multiple wasp stings” by Radhakrishnan et al.,[1] with great interest. We wish to highlight certain issues in this case. Various case reports are available describing the development of systemic manifestations following wasp or bee stings in patients with two or three stings in contrast to at least 50 stings as described by the author.[2] The authors have described pigmentary nephropathy as the cause of acute kidney injury (AKI) following rhabdomyolysis. But, this is only one of the various pathophysiologic mechanisms; the other important mechanisms are direct hemeprotein-induced oxidant injury and renal vasoconstriction with diminished renal circulation.[3] Myoglobin levels were not performed in this case due to nonavailability, but serum myoglobin levels are not needed for the diagnosis or management of rhabdomyolysis. Myoglobin is cleared more rapidly than creatinine phosphokinase (CK) and therefore is less sensitive for detecting rhabdomyolysis, especially when presentation is delayed. Also, presence of myoglobin in urine is not specific for the development of AKI; therefore, it is not necessary for routine testing.[3] We presume that the author must have monitored serum calcium levels in this case because, while managing a case of rhabdomyolysis with AKI, monitoring of serum calcium levels is very important. Calcium levels are often low initially, secondary to precipitation of calcium with phosphate in damaged muscles. Subsequently, in the recovery phase of rhabdomyolysis and AKI, calcium mobilization from damaged muscles may subsequently result in hypercalcemia.[3] Forced alkaline diuresis is a common intervention in rhabdomyolysis, but evidence of clinical benefit is lacking. The largest retrospective study of bicarbonate and mannitol therapy versus no use of this therapy in trauma patients did not find any difference between groups in the incidence of renal failure, need for dialysis or mortality. A current consensus statement suggests that sodium bicarbonate or mannitol administration is not necessary and is not superior to normal saline diuresis in increasing urine pH.[4] Further, a large dose of bicarbonate may worsen the degree of hypocalcemia, especially if hypovolemia is corrected. In severe rhabdomyolysis, as was evident in this case, intermittent hemodialysis (IHD) may be needed to address rebound hyperkalemia and acidosis. However, conventional hemodialysis may be relatively inefficient at removal of circulating myoglobin owing to its large molecular weight. Continuous renal replacement therapy modes, such as continuous venovenous hemofiltration (CVVH) or continuous venovenous hemodialfiltration (CVVHDF) have the advantage of aborting these rebound complications, and convection removes these larger molecular weight solutes better than diffusion. The Chinese case series as quoted by the author have also reported similar results. In this series, despite the lack of difference in mortality rates, the patients who began renal replacement therapy with CVVH or CVVHDF with plasma exchange experienced a better and more rapid recovery of kidney function than those initiated with IHD.[5] Also, although controversial, myoglobin clearance could theoretically be augmented using novel super high-flow hemofilters (molecular weight cut-off 30-60 kDa).

Rapidly progressing fatal reperfusion syndrome caused by acute critical ischemia of the lower limb

The most severe complication of ischemia-reperfusion injury following lower limb arterial surgery is reperfusion syndrome. Therefore, our aim was to describe the extent of muscle damage and the reperfusion syndrome-related remote organ lesions in detail, through a well-documented case of long-lasting infrarenal aorta thrombosis. After urgent revascularization, several clinical signs of multiple organ dysfunction were detectable, including the circulatory, urinary, respiratory, gastrointestinal, and hemostatic systems. Upon histological examination, intraoperative muscle biopsy showed severe muscle damage. Muscle fiber viability was assessed with a special nitroblue tetrazolium staining-based viability test developed by our team; the obtained results indicated significant degree of muscle damage before this was confirmed by conventional histological methods. Thorough postmortem examination confirmed the presence of remote organ damage. The pathological findings included acute tubular necrosis, myocardial and jejunal infarctions, ischemic pancreatitis, and diffuse alveolar damage with hyaline membrane formation in the lungs and focal centrilobular liver necrosis. By using special staining techniques, the presence of myoglobin and lipofuscin deposits was confirmed in the kidney samples. In this paper, we present a patient who developed all major complications following long-lasting arterial occlusion. We also introduce a novel method to assess the degree of ischemic injury, which may be suitable in the near future for the rapid detection of irreversible muscle injury. Therefore, the mortality of the disease might be reduced.

Modulation of direct electron transfer of cytochrome c by use of a molecularly imprinted thin film

We describe the preparation of a molecularly imprinted polymer film (MIP) on top of a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold, where the template cytochrome c (cyt c) participates in direct electron transfer (DET) with the underlying electrode. To enable DET, a non-conductive polymer film is electrodeposited from an aqueous solution of scopoletin and cyt c on to the surface of a gold electrode previously modified with MUA. The electroactive surface concentration of cyt c was 0.5 pmol cm(-2). In the absence of the MUA layer, no cyt c DET was observed and the pseudo-peroxidatic activity of the scopoletin-entrapped protein, assessed via oxidation of Ampliflu red in the presence of hydrogen peroxide, was only 30% of that for the MIP on MUA. This result indicates that electrostatic adsorption of cyt c by the MUA-SAM substantially increases the surface concentration of cyt c during the electrodeposition step, and is a prerequisite for the productive orientation required for DET. After template removal by treatment with sulfuric acid, rebinding of cyt c to the MUA-MIP-modified electrode occurred with an affinity constant of 100,000 mol(-1) L, a value three times higher than that determined by use of fluorescence titration for the interaction between scopoletin and cyt c in solution. The DET of cyt c in the presence of myoglobin, lysozyme, and bovine serum albumin (BSA) reveals that the MIP layer suppresses the effect of competing proteins.

Acute renal failure and/or rhabdomyolysis due to multiple bee stings: A retrospective study

Although multiple bee stings are of rare incidence, they can cause many harmful reactions in humans. Massive inoculation of bee venom is reported to cause skin necrosis, shock hypertension, bleeding, hemolysis, thrombocytopenia, pancreatitis, (adult) respiratory distress syndrome, rhabdomyolysis, and acute renal failure (ARF).[1–12] Generally, three major reactions occur after bee inoculation as 1) local swelling and irritation, 2) generalized anaphylactic responses, and 3) serum sickness like symptoms, which include hemolysis, disseminated intravascular coagulation, rhabdomyolysis, and ARF.[13,14] In India, existences of honey combs are general in rural as well as urban areas but there are sparse data available in the literature on bee sting toxicity. To the best of our knowledge, there are no epidemiological studies reported until now on multiple bee sting-induced ARF and/or rhabdomyolysis in the Indian population. Hence, we performed a retrospective study for multiple bee sting-induced ARF and/or rhabdomyolysis. The cases under International Classification of Diseases 2010 (Code T63.4) i.e., “venom of other arthropods” were screened retrospectively from January 2009 to December 2011 after Institutional Ethics Committee (IEC) approval. A total 32 cases were found. The inclusion criteria were the adult cases diagnosed to have multiple bee sting-induced rhabdomyolysis, ARF and/or other complication. Nine cases were selected in the study [Table 1, Figure ​Figure11 and ​and2],2], out of which six were diagnosed with acute kidney injury (AKI) and rhabdomyolysis, two with AKI only, and one with rhabdomyolysis only. The following type of information was collected from the patient records: Age, sex, complaints on admission, diagnosis, medical, medication, social and family history, days between bee stings and hospital admission, number of hemodialysis given, length of stay in the hospital; laboratory results like serum urea, serum creatinine, aspartate (AST), and alanine (ALT) aminotransferases, alkaline phosphatase (ALP), creatine kinase (CK), lactate dehydrogenase (LDH), total leukocyte count (TLC), hemoglobin (Hb), presence of myoglobin in urine, and other relevant data as per the case were obtained. All the patients had no significant abnormalities in serum potassium and sodium. The distribution of the AKI cases as per the risk, injury, failure, loss, end-stage (RIFLE) criteria was 1-‘Risk’, 6-‘Failure’, 1-‘Loss’. Table 1 Details of the patients Figure 1 Serum urea levels of the patients Figure 2 Serum creatinine of the patients The unique points in each case are discussed below. In all the cases, for anaphylaxis standard treatment was given.

Оксид азоту, міоглобін і кисневий режим клітин: дослідження на математичних моделях

The influence ofnanomolar concentrations of NO on oxygen regimen of muscle cells was investigated. The intracellular distribution of oxygen consumption rates, pO2, NO and myoglobin was calculated by mathematical modeling of diffusion reactions of O2, NO and myoglobin in the muscle cell. The diffusion of O2, and NO from blood vessel surface into cell was taken into account. According to calculations, NO roughly regulates the oxygen regimen of cell, because it increases the tissue pO2 level by inhibition of mitochondria activity in the whole cell space. The presence ofmyoglobin is capable potentially to modulate the influence of NO on oxygen regimen. Myoglobin not only contributes to tissue O2 supply, but it can locally affect the NO concentration. Acting as an NO scavenger under normoxia and as an NO producer under hypoxia, myoglobin together with NO can provide fine adjustment of muscle oxygen regimen via an increase in tissue pO, and elimination a mismatch between oxygen supply and demand.

Neuroglobin–prion protein interaction: what's the function?

Neuroglobin and cellular prion protein (PrP(C) ) are expressed in the nervous system and co-localized in the retinal ganglion cell layer. Both proteins do not have an unambiguously assigned function, and it was recently reported that PrP(C) aggregates rapidly in the presence of neuroglobin, whereas it does not aggregate in the presence of myoglobin, another globin with different tissue specificity. Electrostatic complementarity between the unstructured PrP(C) N-terminus and neuroglobin has been proposed to mediate this specific interaction. To verifythis hypothesis experimentally, we have used a combined approach of automated docking and molecular dynamics (MD) studies carried out on short stretches of prion protein (PrP) N-terminus to identify the minimal electrostatically interacting aminoacidic sequences with neuroglobin. Subsequently, we have performed the synthesis of these peptides by solid phase methods, and we tested their interaction with neuroglobin by surface plasmon resonance (SPR). Preliminary results confirm unequivocally the specific interaction between synthetic PrP peptides and neuroglobin suggesting a crucial role of PrP(C) positively charged regions in thisprotein-protein association.

Boronic acid lectin affinity chromatography (BLAC). 3. Temperature dependence of glycoprotein isolation and enrichment

In this paper, the effect of temperature is investigated on the performance of glycoprotein enrichment by boronic acid lectin affinity chromatography (BLAC). Wheat germ agglutinin and m-aminophenyl boronic acid containing stationary phases were evaluated individually and in a mixed mode using an automated liquid handling robot with an integrated 96-well plate temperature controller. Glycoaffinity enrichment of the model proteins of ribonuclease B and trypsin inhibitor was investigated in the presence of the non-glycosylated proteins of myoglobin (neutral) and lysozyme (basic) at a wide temperature range of 5-65 degrees C. Our results revealed that glycoaffinity micropartitioning at the temperature of 25 degrees C provided the highest recovery rate for glycoprotein enrichment. We have also found that a large amount of lysozyme was present in the elution fractions of the m-aminophenyl boronic acid containing micropartitioning columns due to ion-exchange mechanism occurring between the positively charged protein and the negatively charged stationary phase at the operation pH. On the other hand, at high temperature (65 degrees C), non-specific interactions with the agarose carrier prevailed, evidenced by the presence of myoglobin in the eluate.

Hydroxyl radical and glutathione interactions alter calcium sensitivity and maximum force of the contractile apparatus in rat skeletal muscle fibres

Studies on intact muscle fibres indicate that reactive oxygen species (ROS) produced during muscle activity, or applied exogenously, can cause decreased force responses primarily by reducing the Ca(2+) sensitivity of the contractile apparatus. Identification of the molecular basis of this effect is complicated by the fact that studies on skinned muscle fibres in general have not observed reduced contractile Ca(2+) sensitivity when applying ROS, predominantly H(2)O(2). Here, using skinned fibres from rat extensor digitorum longus (EDL) and soleus muscle, it is shown that although H(2)O(2) (> or = 100 microm) has little effect by itself, when added in the presence of myoglobin it causes marked reduction in the Ca(2+) sensitivity of the contractile apparatus, probably due to production of hydroxyl radicals (OH(*)). Maximum force production is also reduced, but only with larger or more prolonged treatments. The effects are not prevented by tempol, a potent superoxide scavenger. Dithiotreitol (DTT) produces little reversal of the sensitivity change if applied afterwards, but it does substantially reverse all the changes if applied before the fibre undergoes an activation sequence. When glutathione (GSH, 5 mM) is present, exposure of EDL fibres to H(2)O(2) and myoglobin causes an increase in Ca(2+) sensitivity, with longer treatments causing a subsequent decrease, whereas in soleus fibres it causes only decreases in sensitivity and maximum force. The increased Ca(2+) sensitivity in EDL fibres is evidently due to the summed actions of (i) a potentiating effect of glutathionylation, which can be reversed by DTT and only occurs in fast-twitch fibres, and (ii) a less reversible reduction in sensitivity. Western blotting showed that reductions in Ca(2+) sensitivity were not due to loss of troponin-C. The present findings help provide a mechanistic basis for diverse findings on the effects of ROS in muscle fibres and implicate OH(*) radicals and glutathione as likely mediators of the effects.