Photoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5

The synthesis is described of the photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4-hydroxylase. The photoaffinity label is a good substrate and is capable of light-induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2-oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L-proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length.     

Reduced regional myocardial perfusion reserve is associated with impaired contractile performance in idiopathic dilated cardiomyopathy

Background. In idiopathic dilated cardiomyopathy (IDC) an imbalance between myocardial oxygen consumption and supply has been postulated. Subclinical myocardial ischaemia may contribute to progressive deterioration of left ventricular function. The relation between regional myocardial perfusion reserve (MPR) and contractile performance was investigated. Methods. Patients with newly diagnosed IDC underwent positron emission tomography (PET) scanning using both ¹³N-ammonia as a perfusion tracer (baseline and dypiridamole stress), and ¹⁸F-fluorodeoxyglucose viability tracer and a dobutamine stress MRI. MPR (assessed by PET) as well as wall motion score (WMS, assessed by MRI) were evaluated in a 17-segment model. Results. Twenty-two patients were included (age 49±11 years; 15 males, LVEF 33±10%). With MRI, a total of 305 segments could be analysed. Wall motion abnormalities at rest were present in 127 (35.5%) segments and in 103 (29.9%) during dobutamine stress. Twenty-one segments deteriorated during stress and 43 improved. MPR was significantly higher in those segments that improved, compared with those that did not change or were impaired during stress (1.87±0.04 vs. 1.56± 0.07 p<0.01.) Conclusion. Signs of regional ischaemia were clearly present in IDC patients. Ischaemic regions displayed impaired contractility during stress. This suggests that impaired oxygen supply contributes to cardiac dysfunction in IDC. (Neth Heart J 2009;17:470–4.)     

Production of thermotolerant entomopathogenic Isaria fumosorosea SFP-198 conidia in corn-corn oil mixture

Low thermotolerance of entomopathogenic fungi is a major impediment to long-term storage and effective application of these biopesticides under seasonal high temperatures. The effects of high temperatures on the viability of an entomopathogenic fungus, Isaria fumosorosea SFP-198 (KCTC 0499BP), produced on different substrates amended with various additives were explored. Ground corn was found to be superior in producing the most thermotolerant conidia compared to yellow soybean, red kidney bean, and rice in a polyethylene bag production system. Using ground corn mixed with corn oil as a substrate resulted in only 7% reduction in germination compared to ground corn alone (67% reduction) after exposure of conidia to 50°C for 2 h. Corn oil as an additive for ground corn was followed by inorganic salts (KCl and NaCl), carbohydrates (sucrose and dextrin), a sugar alcohol (sorbitol), and plant oils (soybean oil and cotton seed oil) in ability to improve conidial thermotolerance. Unsaturated fatty acids, such as linoleic acid and oleic acid, the main components of corn oil, served as effective additives for conidial thermotolerance in a dosage-dependant manner, possibly explaining the improvement by corn oil. This finding suggests that the corn-corn oil mixture can be used to produce highly thermotolerant SFP-198 conidia and provides the relation of unsaturated fatty acids as substrates with conidial thermotolerance.     

Determination of iohexol clearance by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)

We have developed a simple, rapid, and accurate HPLC-MS/MS method for the determination of iohexol in serum. The column used was a Zorbax Eclipse XDB-C8 (100 mm x 2.1 mm i.d., 3.5 microm). Mobile phases consisted of water containing 2mM ammonium acetate and 0.1% formic acid (A) and methanol containing 2 mM ammonium acetate and 0.1% formic acid (B). After simple protein precipitation with ZnSO4, serum samples were mixed with I.S. (bromperidol) and centrifuged for 3 min. The obtained extraction recovery at three levels was 94.6-107.4%. Quantitative analysis was performed in the multiple reaction-monitoring mode (m/z 822.0-->804.0 for iohexol, 420.1-->122.7 for I.S.) with the total running time of 3 min for each sample. The assay was linear between 0.5 and 1500 microg/mL (r2 > 0.997). The intra- and inter-assay coefficient of variations were 2.4-6.2% and 5.5-6.5%, respectively. Our method provided sufficient analytical range and specificity for the 210 clinical samples analyzed.     

Why genetic selection to reduce the prevalence of infectious diseases is way more promising than currently believed

Genetic selection for improved disease resistance is an important part of strategies to combat infectious diseases in agriculture. Quantitative genetic analyses of binary disease status, however, indicate low heritability for most diseases, which restricts the rate of genetic reduction in disease prevalence. Moreover, the common liability threshold model suggests that eradication of an infectious disease via genetic selection is impossible because the observed-scale heritability goes to zero when the prevalence approaches zero. From infectious disease epidemiology, however, we know that eradication of infectious diseases is possible, both in theory and practice, because of positive feedback mechanisms leading to the phenomenon known as herd immunity. The common quantitative genetic models, however, ignore these feedback mechanisms. Here, we integrate quantitative genetic analysis of binary disease status with epidemiological models of transmission, aiming to identify the potential response to selection for reducing the prevalence of endemic infectious diseases. The results show that typical heritability values of binary disease status correspond to a very substantial genetic variation in disease susceptibility among individuals. Moreover, our results show that eradication of infectious diseases by genetic selection is possible in principle. These findings strongly disagree with predictions based on common quantitative genetic models, which ignore the positive feedback effects that occur when reducing the transmission of infectious diseases. Those feedback effects are a specific kind of Indirect Genetic Effects; they contribute substantially to the response to selection and the development of herd immunity (i.e., an effective reproduction ratio less than one).     

Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.     

Role of reactive oxygen species-mediated mitochondrial dysregulation in 3-bromopyruvate induced cell death in hepatoma cells

Hexokinase type II (HK II) is the key enzyme for maintaining increased glycolysis in cancer cells where it is overexpressed. 3-bromopyruvate (3-BrPA), an inhibitor of HK II, induces cell death in cancer cells. To elucidate the molecular mechanism of 3-BrPA-induced cell death, we used the hepatoma cell lines SNU449 (low expression of HKII) and Hep3B (high expression of HKII). 3-BrPA induced ATP depletion-dependent necrosis and apoptosis in both cell lines. 3-BrPA increased intracellular reactive oxygen species (ROS) leading to mitochondrial dysregulation. NAC (N-acetyl-l-cysteine), an antioxidant, blocked 3-BrPA-induced ROS production, loss of mitochondrial membrane potential and cell death. 3-BrPA-mediated oxidative stress not only activated poly-ADP-ribose (PAR) but also translocated AIF from the mitochondria to the nucleus. Taken together, 3-BrPA induced ATP depletion-dependent necrosis and apoptosis and mitochondrial dysregulation due to ROS production are involved in 3-BrPA-induced cell death in hepatoma cells.