Plant based production of myoglobin - a novel source of the muscle heme-protein.

Myoglobin is a heme-protein in the muscle of vertebrates with important functions in the oxygenation of tissues and as a regulator in nitric oxide signaling. Myoglobin from many species is also an important nutritional source of bioavailable iron. In this study, we have successfully produced human myoglobin in the leaves of Nicotiana benthamiana by transient expression using a viral vector delivered by Agrobacterium tumefaciens. Analyses confirmed that heme was incorporated and the protein was functional, with observed properties consistent with those of native myoglobins. A relatively high degree of purity could be achieved with low cost methods. The results show the high potential of plants as a production platform for heme proteins, a group of proteins of interest for iron nutrition applications and possible future pharmaceutical development.

Theoretical basis of nitrosomyoglobin formation in a dry sausage model by coagulase-negative staphylococci: Behavior and expression of nitric oxide synthase.

Three coagulase-negative staphylococci (CNS) species were investigated for gene expression of nitric oxide synthase (NOS) and the ability of nitrosomyoglobin (NO-Mb) formation in a dry sausage model without nitrite addition. The expression of nos gene was systematically proven from DNA to RNA to protein, and nitric oxide (NO) generation was also directly detected. In the dry sausage model system, the redness (a*-values) of samples inoculated with the three CNS species were higher than those inoculated with Pediococcus pentosaceus and the control (P < 0.05). The results from UV-vis and electron spin resonance spectroscopies revealed that pentacoordinate NO-Mb was formed in the sausages with either CNS or nitrite added. The sausage inoculated with Staphylococcus vitulinus had the highest NO-Mb content among the CNS-treated sausages. Dimer interface residues and phosphorylation sites of NOS in . itulinus differ from the other two CNS species as revealed by amino acid sequences, which may be responsible for the different catalytic activities.

Sucrose enhances colour formation in dry sausages by up-regulating gene expression of nitric oxide synthase in Staphylococcus vitulinus.

The effects of glucose and sucrose on the gene expression of nitric oxide synthase (NOS) in Staphylococcus vitulinus and colour formation in dry sausages were investigated. The results showed that sucrose addition promoted nitric oxide (NO) production in media when compared with glucose. In addition, sucrose could up-regulate nos (encoding NOS) and katA (encoding catalase KatA) gene expression by enhancing oxidative stress levels. In the sausages inoculated with S. vitulinus, a*-values (indicating redness) of the sausages with added sucrose were higher than those of samples with added glucose (P < 0.05) but did not differ from those in the nitrite treatment group (P > 0.05). The UV-vis spectra results showed that nitrosylmyoglobin (NO-Mb) was formed in the sausages with either S. vitulinus or nitrite added. In the S. vitulinus-inoculated sausages, sucrose addition led to a higher NO-Mb content than that after glucose addition, which was attributed to up-regulation of the nos gene. This study provides a potential method to enhance NO yield in S. vitulinus and colour formation in dry sausages without nitrite addition.

Contribution of nitric oxide synthase from coagulase-negative staphylococci to the development of red myoglobin derivatives.

As part of the microbial community of meat or as starter cultures, coagulase-negative staphylococci (CNS) serve several essential technological purposes in meat products, such as color development through the reduction of nitrate to nitrite. As the safety of nitrite as an additive has been questioned, we explored the potential of CNS to develop red myoglobin derivatives such as oxymyoglobin and nitrosomyoglobin. Nitrosoheme was extracted to evaluate NO production. This production could be due to a nitric oxide synthase (NOS) activity. In all CNS strains, a nos gene was identified. The NOS sequences deduced were highly conserved within CNS. A phylogenetic tree based on the NOS sequences revealed that the strains within species were clustered. Ninety-one percent of the strains, whatever the species, were able to form red myoglobin derivatives in aerobic conditions, but a high variability was observed between strains within species. However, NO production was low as nitrosomyoglobin represented 8% to 16% of the red pigments according to the species. Formation of oxymyoglobin, especially under aerobic conditions, was substantial, but varied greatly within species. The mechanism involved in the formation of oxymyoglobin could rely on staphylococcal reductases and remains to be explored.

Acute growth hormone administration increases myoglobin expression and Glut4 translocation in rat cardiac muscle cells.

OBJECTIVE: Oxygen (O2) and glucose are important energy sources for the heart. This study sought to investigate the effects of acute growth hormone (GH) administration on the expression of myoglobin (Mb) and Glut4 glucose transporter, two important limiting factors for O2 and glucose utilization for energy production, in cardiac muscle cells of treated rats. METHODS: Male Wistar rats were sacrificed at 30, 45, 90 and 120 min after a single dose of intraperitoneal (ip) rat GH (1.5 mg/kg) or vehicle administration, and total RNA and protein (from whole cell or subcellular fractions) were extracted from cardiomyocytes (left ventricles) of these animals. RESULTS: Acute GH injection led to a significant increase in both Mb mRNA and protein levels, and stimulated Glut4 protein translocation to the plasma membrane of cardiac cells. CONCLUSIONS: These results suggest that GH exerts some of its effects on cardiomyocytes shortly after the first administration inducing the expression of proteins potentially involved in cardiac performance.

Myoglobin expression in prostate cancer is correlated to androgen receptor expression and markers of tumor hypoxia.

Recent studies identified unexpected expression and transcriptional complexity of the hemoprotein myoglobin (MB) in human breast cancer but its role in prostate cancer is still unclear. Expression of MB was immunohistochemically analyzed in three independent cohorts of radical prostatectomy specimens (n = 409, n = 625, and n = 237). MB expression data were correlated with clinicopathological parameters and molecular parameters of androgen and hypoxia signaling. Expression levels of novel tumor-associated MB transcript variants and the VEGF gene as a hypoxia marker were analyzed using qRT-PCR. Fifty-three percent of the prostate cancer cases were MB positive and significantly correlated with androgen receptor (AR) expression (p < 0.001). The positive correlation with CAIX (p < 0.001) and FASN (p = 0.008) as well as the paralleled increased expression of the tumor-associated MB transcript variants and VEGF suggest that hypoxia participates in MB expression regulation. Analogous to breast cancer, MB expression in prostate cancer is associated with steroid hormone signaling and markers of hypoxia. Further studies must elucidate the novel functional roles of MB in human carcinomas, which probably extend beyond its classic intramuscular function in oxygen storage.

Myoglobin overexpression inhibits reperfusion in the ischemic mouse hindlimb through impaired angiogenesis but not arteriogenesis.

Adaptive vascular remodeling in response to arterial occlusion takes the form of capillary growth (angiogenesis) and outward remodeling of pre-existing collateral arteries (arteriogenesis). However, the relative contributions of angiogenesis and arteriogenesis toward the overall reperfusion response are both highly debated and poorly understood. Here, we tested the hypothesis that myoglobin overexpressing transgenic mice (MbTg(+)) exhibit impaired angiogenesis in the setting of normal arteriogenesis in response to femoral artery ligation, and thereby serve as a model for disconnecting these two vascular growth processes. After femoral artery ligation, MbTg(+) mice were characterized by delayed distal limb reperfusion (by laser Doppler perfusion imaging), decreased foot use, and impaired distal limb muscle angiogenesis in both glycolytic and oxidative muscle fiber regions at day 7. Substantial arteriogenesis occurred in the primary collaterals supplying the ischemic limb in both wild-type and MbTg(+) mice; however, there were no significant differences between groups, indicating that myoglobin overexpression does not affect arteriogenesis. Together, these results uniquely demonstrate that functional collateral arteriogenesis alone is not necessarily sufficient for adequate reperfusion after arterial occlusion. Angiogenesis is a key component of an effective reperfusion response, and clinical strategies that target both angiogenesis and arteriogenesis could yield the most efficacious treatments for peripheral arterial disease.

Formation and identification of nitrosylmyoglobin by Staphylococcus xylosus in raw meat batters: a potential solution for nitrite substitution in meat products.

Staphylococcus xylosus and Pediococcus pentosaceus isolated from Chinese dried sausage were assessed for their ability to convert metmyoglobin into nitrosylmyoglobin in Mann-Rogosa-Sharp broth model systems and raw pork meat batters without the addition of nitrite. The results showed that samples in model systems with S. xylosus cultures had an absorption spectra that is typical of nitrosylmyoglobin, an obvious pink colour (judged by visual inspection) and a significantly higher a-value than the control samples or samples inoculated with P. pentosaceus. In raw meat batters, the a-values of the S. xylosus samples were almost the same as those for the meat with nitrite added. The complementary analysis of meat batter samples by photochemical information from UV-vis, electron spin resonance and resonance Raman spectroscopy revealed that the existing status of the myoglobin in meat batters inoculated with S. xylosus was mainly pentacoordinate nitrosylmyoglobin. This study provides a potential solution for nitrite substitute in meat products.

Ribose sugars generate internal glycation cross-links in horse heart myoglobin.

Glycation of horse heart metmyoglobin with d-ribose 5-phosphate (R5P), d-2-deoxyribose 5-phosphate (dR5P), and d-ribose with inorganic phosphate at 37°C generates an altered protein (Myo-X) with increased SDS-PAGE mobility. The novel protein product has been observed only for reactions with the protein myoglobin and it is not evident with other common sugars reacted over a 1 week period. Myo-X is first observed at 1-2 days at 37°C along with a second form that is consistent in mass with that of myoglobin attached to several sugars. MALDI mass spectrometry and other techniques show no evidence of the cleavage of a peptide from the myoglobin chain. Apomyoglobin in reaction with R5P also exhibited this protein form suggesting its occurrence was not heme-related. While significant amounts of O(2)(-) and H(2)O(2) are generated during the R5P glycation reaction, they do not appear to play roles in the formation of the new form. The modification is likely due to an internal cross-link formed during a glycation reaction involving the N-terminus and an internal amine group; most likely the neighboring Lys133. The study shows the unique nature of these common pentose sugars in spontaneous glycation reactions with proteins.

Cytokine responses to carbohydrate ingestion during recovery from exercise-induced muscle injury.

We investigated the effect of carbohydrate ingestion after maximal lengthening contractions of the knee extensors on circulating concentrations of myocellular proteins and cytokines, and cytokine mRNA expression in muscle. Using a cross-over design, 10 healthy males completed 5 sets of 10 lengthening (eccentric) contractions (unilateral leg press) at 120% 1 repetition-maximum. Subjects were randomized to consume a carbohydrate drink (15% weight per volume; 3 g/kg BM) for 3 h after exercise using one leg, or a placebo drink after exercise using the contralateral leg on another day. Blood samples (10 mL) were collected before exercise and after 0, 30, 60, 90, 120, 150, and 180 min of recovery. Muscle biopsies (vastus lateralis) were collected before exercise and after 3 h of recovery. Following carbohydrate ingestion, serum concentrations of glucose (30-90 min and at 150 min) and insulin (30-180 min) increased (P < 0.05) above pre-exercise values. Serum myoglobin concentration increased ( approximately 250%; P < 0.05) after both trials. In contrast, serum cytokine concentrations were unchanged throughout recovery in both trials. Muscle mRNA expression for IL-8 (6.4-fold), MCP-1 (4.7-fold), and IL-6 (7.3-fold) increased substantially after carbohydrate ingestion. TNF-alpha mRNA expression did not change after either trial. Carbohydrate ingestion during early recovery from exercise-induced muscle injury may promote proinflammatory reactions within skeletal muscle.

Quantification of myoglobin deoxygenation and intracellular partial pressure of O2 during muscle contraction during haemoglobin-free medium perfusion.

Although the O(2) gradient regulates O(2) flux from the capillary into the myocyte to meet the energy demands of contracting muscle, intracellular O(2) dynamics during muscle contraction remain unclear. Our hindlimb perfusion model allows the determination of intracellular myoglobin (Mb) saturation ( ) and intracellular oxygen tension of myoglobin ( ) in contracting muscle using near infrared spectroscopy (NIRS). The hindlimb of male Wistar rats was perfused from the abdominal aorta with a well-oxygenated haemoglobin-free Krebs-Henseleit buffer. The deoxygenated Mb ([deoxy-Mb]) signal was monitored by NIRS. Based on the value of [deoxy-Mb], and were calculated, and the time course was evaluated by an exponential function model. Both and started to decrease immediately after the onset of contraction. The steady-state values of and progressively decreased with relative work intensity or muscle oxygen consumption. At the maximal twitch rate, and were 49% and 2.4 mmHg, respectively. Moreover, the rate of release of O(2) from Mb at the onset of contraction increased with muscle oxygen consumption. These results suggest that at the onset of muscle contraction, Mb supplies O(2) during the steep decline in , which expands the O(2) gradient to increase the O(2) flux to meet the increased energy demands.

Expression and functional regulation of myoglobin in epithelial cancers.

Myoglobin is a multifunctional heme protein that is thought to be expressed exclusively in myocytes. Its importance in both oxygen transport and free radical scavenging has been extensively characterized. We hypothesized that solid tumors could take advantage of proteins such as myoglobin to cope with hypoxic conditions and to control the metabolism of reactive oxygen and nitrogen species. We therefore sought to establish whether myoglobin might be expressed and functionally regulated in epithelial tumors that are known to face hypoxia and oxidative stress during disease progression. We analyzed the expression of myoglobin in human epithelial cancers at both transcriptional and protein levels; moreover, we investigated the expression levels of myoglobin in cancer cell lines subjected to different conditions, including hypoxia, oxidative stress, and mitogenic stimuli. We provide evidence that human epithelial tumors, including breast, lung, ovary, and colon carcinomas, express high levels of myoglobin from the earliest stages of disease development. In human cancer cells, myoglobin is induced by a variety of signals associated with tumor progression, including mitogenic stimuli, oxidative stress, and hypoxia. This study provides evidence that myoglobin, previously thought to be restricted to myocytes, is expressed at high levels by human carcinoma cells. We suggest that myoglobin expression is part of a cellular program aimed at coping with changed metabolic and environmental conditions associated with neoplastic growth.

Design of recombinant hemoglobins for use in transfusion fluids.

Molecular biology has been applied to the development of hemoglobin-based oxygen carrier (HBOC) proteins that can be expressed in bacteria or yeast. The transformation of the hemoglobin molecule into an HBOC requires a variety of modifications for rendering the acellular molecule of hemoglobin physiologically acceptable when transfused in circulation. Hemoglobins with different oxygen affinities can be obtained by introducing mutations at the heme pocket, the site of oxygen binding, or by introducing surface mutations that stabilize the hemoglobin molecule in the low-oxygen-affinity state. Modification of the size of the heme pocket is also used to hinder nitric oxide depletion and associated vasoconstriction. Introduction of cysteine residues on the hemoglobin surface allows formation of intermolecular bonds and formation of polymeric HBOCs. These polymers of recombinant hemoglobin have the characteristics of molecular size, molecular stability, and oxygen delivery to hypoxic tissue suitable for an HBOC.

Medulloblastoma with myogenic differentiation showing double immunopositivity for synaptophysin and myoglobin.

Reported herein is a case of medulloblastoma with myogenic differentiation in a 3-year-old girl who died 1 year after appearance of clinical signs. Magnetic resonance imaging indicated a mass lesion in the cerebellar vermis. She underwent total resection of the tumor, followed by chemotherapy and radiotherapy in the brain and spinal cord. The resected specimen mainly consisted of densely packed cells with round-to-oval highly chromatic nuclei surrounded by scanty cytoplasm and focally of long spindle-shaped cells with elongated nuclei and eosinophilic cytoplasm showing discernible cross-striations. Immunohistochemistry indicated partial expression of synaptophysin in the former area and focal expression of desmin in the latter area. The diagnosis was medulloblastoma with myogenic differentiation, also known as medullomyoblastoma. Autopsy indicated disseminated proliferation of immature neuroglial cells with highly chromatic nuclei and scanty cytoplasm showing partial expression of synaptophysin, neurofilaments, and GFAP, and focal proliferation of round-to-oval immature cells showing immunoreactivity of myoglobin. The tumor cells had large nuclei, frequent mitoses, apoptoses, nuclear molding, and cell wrapping, indicating moderate anaplasia. Their Ki-67 labeling index was 54%. In addition, some tumor cells had double immunopositivity for synaptophysin or neurofilament and myoglobin, suggesting that the neuroectodermal cells may undergo differentiation into rhabdomyoblasts.

Folding on the assembly line.

Deciphering the mechanism of folding of newly synthesized proteins in the cell is a major challenge because of the large size and multiplicity of molecular components involved and the asynchrony of biosynthesis. Fluorescently labeled ribosome-bound nascent chains of a defined length were prepared and subjected to dynamic fluorescence depolarization spectroscopy measurements. Nanosecond anisotropy decay correlation times of proteins' nascent chains at different stages of polypeptide elongation were determined for the first time. Striking dependence of the chain dynamics on the stages of elongation was observed and revealed chain length dependence of folding on the ribosome.

Chain dynamics of nascent polypeptides emerging from the ribosome.

Very little is known about the conformation of polypeptides emerging from the ribosome during protein biosynthesis. Here, we explore the dynamics of ribosome-bound nascent polypeptides and proteins in Escherichia coli by dynamic fluorescence depolarization and assess the population of cotranslationally active chaperones trigger factor (TF) and DnaK. E. coli cell-free technology and fluorophore-linked E. coli Met-tRNA f Met enable selective site-specific labeling of nascent proteins at the N-terminal methionine. For the first time, direct spectroscopic evidence captures the generation of independent nascent chain motions for a single-domain protein emerging from the ribosome (apparent rotational correlation time approximately 5 ns), during the intermediate and late stages of polypeptide elongation. Such motions are detected only for a sequence encoding a globular protein and not for a natively unfolded control, suggesting that the independent nascent chain dynamics may be a signature of folding-competent sequences. In summary, we observe multicomponent, severely rotationally restricted, and strongly chain length/sequence-dependent nascent chain dynamics.

The expression of myoglobin and ROR2 protein in Dupuytren's disease.

BACKGROUND: Dupuytren's disease (DD) is a hand disease inherited as an autosomal dominant trait with variable penetrance, especially among populations of northern European ancestry. The etiology and pathophysiology of DD are not clear. The purpose of this study was to examine the gene expression profiles of palmar fascia of DD and healthy patients using microarray analysis to highlight the genes that might contribute to the pathogenesis of DD. MATERIALS AND METHODS: Dupuytren contracture samples were taken from excised mature cords of DD patients during aponeurectomies. Control samples were collected from healthy hand trauma patients. Microarray analysis was performed with the Affymetrix HGU133A genome array (Affymetrix, Santa Clara, CA). Expression changes of selected proteins were confirmed at the protein level with Western and dot blotting or by immunohistochemistry. RESULTS: At least an 8-fold change in gene expression was found with 127 genes, including a 90-fold down-regulation of myoglobin and a 14-fold up-regulation of tyrosine kinase-like orphan receptor 2 (= ROR2) from absent to present during the disease. The changes in myoglobin and ROR2 expression were confirmed at the protein level. CONCLUSIONS: In this study, we showed for the first time the connection of ROR2 in Dupuytren's disease. ROR2 and myoglobin may play an important role in the pathophysiology of this disease.

On-plate digestion of proteins using novel trypsin-immobilized magnetic nanospheres for MALDI-TOF-MS analysis.

In this study, a novel method of on-plate digestion using trypsin-immobilized magnetic nanospheres was developed followed by MALDI-TOF-MS for rapid and effective analysis and identification of proteins. We utilized a facile one-pot method for the direct preparation of amine-functionalized magnetic nanospheres with highly magnetic properties and the amino groups on the outer surface. Through the reaction of the aldehyde groups with amine groups, trypsin was simply and stably immobilized onto the magnetic nanospheres. The obtained trypsin-linked magnetic nanospheres were then applied for on-plate digestion of sample proteins (myoglobin and Cytochrome c). Moreover, after digestion, the trypsin-linked nanospheres could be easily removed from the plate due to their magnetic property, which would avoid causing contamination on the ion source chamber in MS. The effects of the temperature and incubation time on the digestion efficiency were characterized. Within only 5 min, proteins could be efficiently digested with the peptide sequence coverage higher than or equal to that of the traditional in-solution digestion for 12 h. Furthermore, RPLC fractions of rat liver extract were also successfully processed using this novel method. These results suggested that our improved on-plate digestion protocol for MALDI-MS may find further application in automated analysis of large sets of proteins.

Can myoglobin expression in pancreatic beta cells improve insulin secretion under hypoxia? An exploratory study with transgenic porcine islets.

The feasibility of myoglobin (Mb)-facilitated oxygen transport in improving porcine islet survival under hypoxia was investigated. Discrete groups of islets were transfected with replication-defective adenoviral vector Ad5 respiratory syncitial virus (RSV) to induce expression of Mb or green fluorescent protein (GFP). Native islets served as the controls. In vitro studies at 37 degrees C assessed islet insulin secretion efficacy: (i) to a glucose challenge from 30 to 300 mg/dL at fixed pO2; and (ii) at variable oxygen tensions ranging from 5 to 40 mm Hg over 12 h. The transfection was effective in initiating islet expression of Mb or GFP. Low Mb-expression levels equivalent to 2% the Mb concentration in a muscle cell (0.25 ng of Mb per islet) were documented, with no statistical improvement in insulin secretion. A surprising side note is that insulin secretion was impaired in islets expressing GFP. Improved Mb expression is essential to determine the feasibility of enhancing islet survival under hypoxia.