A simple and highly accurate numerical differentiation method for sensitivity analysis of large-scale metabolic reaction systems

Numerical differentiation is known to be one of the most difficult numerical calculation methods to obtain reliable calculated values at all times. A simple numerical differentiation method using a combination of finite-difference formulas, derived by approximation of Taylor-series equations, is investigated in order to efficiently perform the sensitivity analysis of large-scale metabolic reaction systems. A result of the application to four basic mathematical functions reveals that the use of the eight-point differentiation formula with a non-dimensionalized stepsize close to 0.01 mostly provides more than 14 digits of accuracy in double precision for the numerical derivatives. Moreover, a result of the application to the modified TCA cycle model indicates that the numerical differentiation method gives the calculated values of steady-state metabolite concentrations within a range of round-off error and also makes it possible to transform the Michaelis-Menten equations into the S-system equations having the kinetic orders whose accuracies are mostly more than 14 significant digits. Because of the simple structure of the numerical differentiation formula and its promising high accuracy, it is evident that the present numerical differentiation method is useful for the analysis of large-scale metabolic reaction systems according to the systematic procedure of BST.     

Proteome analysis of soybean roots under waterlogging stress at an early vegetative stage

To gain better insight into how soybean roots respond to waterlogging stress, we carried out proteomic profiling combined with physiological analysis at two time points for soybean seedlings in their early vegetative stage. Seedlings at the V2 stage were subjected to 3 and 7 days of waterlogging treatments. Waterlogging stress resulted in a gradual increase of lipid peroxidation and in vivo H₂O₂ level in roots. Total proteins were extracted from root samples and separated by two-dimensional gel electrophoresis (2-DE). A total of 24 reproducibly resolved, differentially expressed protein spots visualized by Coomassie brilliant blue (CBB) staining were identified by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis. Of these, 14 proteins were upregulated; 5 proteins were decreased; and 5 were newly induced in waterlogged roots. The identified proteins include well-known classical anaerobically induced proteins as well as novel waterlogging-responsive proteins that were not known previously as being waterlogging responsive. The novel proteins are involved in several processes, i.e. signal transduction, programmed cell death, RNA processing, redox homeostasis and metabolisms of energy. An increase in abundance of several typical anaerobically induced proteins, such as glycolysis and fermentation pathway enzymes, suggests that plants meet energy requirement via the fermentation pathway due to lack of oxygen. Additionally, the impact of waterlogging on the several programmed cell death- and signal transduction-related proteins suggest that they have a role to play during stress. RNA gel blot analysis for three programmed cell death-related genes also revealed a differential mRNA level but did not correlate well with the protein level. These results demonstrate that the soybean plant can cope with waterlogging through the management of carbohydrate consumption and by regulating programmed cell death. The identification of novel proteins such as a translation initiation factor, apyrase, auxin-amidohydrolase and coproporphyrinogen oxidase in response to waterlogging stress may provide new insight into the molecular basis of the waterlogging-stress response of soybean.     

HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase

Background

Deficiency of 3-hydroxy-isobutyryl-CoA hydrolase (HIBCH) caused by HIBCH mutations is a rare cerebral organic aciduria caused by disturbance of valine catabolism. Multiple mitochondrial respiratory chain (RC) enzyme deficiencies can arise from a number of mechanisms, including defective maintenance or expression of mitochondrial DNA. Impaired biosynthesis of iron-sulphur clusters and lipoic acid can lead to pyruvate dehydrogenase complex (PDHc) deficiency in addition to multiple RC deficiencies, known as the multiple mitochondrial dysfunctions syndrome.

Methods

Two brothers born to distantly related Pakistani parents presenting in early infancy with a progressive neurodegenerative disorder, associated with basal ganglia changes on brain magnetic resonance imaging, were investigated for suspected Leigh-like mitochondrial disease. The index case had deficiencies of multiple RC enzymes and PDHc in skeletal muscle and fibroblasts respectively, but these were normal in his younger brother. The observation of persistently elevated hydroxy-C4-carnitine levels in the younger brother led to suspicion of HIBCH deficiency, which was investigated by biochemical assay in cultured skin fibroblasts and molecular genetic analysis.

Results

Specific spectrophotometric enzyme assay revealed HIBCH activity to be below detectable limits in cultured skin fibroblasts from both brothers. Direct Sanger sequence analysis demonstrated a novel homozygous pathogenic missense mutation c.950G <A; p.Gly317Glu in the HIBCH gene, which segregated with infantile-onset neurodegeneration within the family.

Conclusions

HIBCH deficiency, a disorder of valine catabolism, is a novel cause of the multiple mitochondrial dysfunctions syndrome, and should be considered in the differential diagnosis of patients presenting with multiple RC deficiencies and/or pyruvate dehydrogenase deficiency.

     

Higher Gravidity and Parity Are Associated with Increased Prevalence of Metabolic Syndrome among Rural Bangladeshi Women

Background

Parity increases the risk for coronary heart disease; however, its association with metabolic syndrome among women in low-income countries is still unknown.

Objective

This study investigates the association between parity or gravidity and metabolic syndrome in rural Bangladeshi women.

Methods

A cross-sectional study was conducted in 1,219 women aged 15–75 years from rural Bangladesh. Metabolic syndrome was defined according to the standard NCEP-ATP III criteria. Logistic regression was used to estimate the association between parity and gravidity and metabolic syndrome, with adjustment of potential confounding variables.

Results

Subjects with the highest gravidity (> = 4) had 1.66 times higher odds of having metabolic syndrome compared to those in the lowest gravidity (0-1) (P _trend = 0.02). A similar association was found between parity and metabolic syndrome (P _trend = 0.04), i.e., subjects in the highest parity (> = 4) had 1.65 times higher odds of having metabolic syndrome compared to those in the lowest parity (0-1). This positive association of parity and gravidity with metabolic syndrome was confined to pre-menopausal women (P _trend <0.01). Among the components of metabolic syndrome only high blood pressure showed positive association with parity and gravidity (P _trend = 0.01 and <0.001). Neither Parity nor gravidity was appreciably associated with other components of metabolic syndrome.

Conclusions

Multi parity or gravidity may be a risk factor for metabolic syndrome.     

Staphylococcus aureus protein A mediates invasion across airway epithelial cells through activation of RhoA GTPase signaling and proteolytic activity

Staphyococcus aureus and especially the epidemic methicillin-resistant S. aureus strains cause severe necrotizing pneumonia. The mechanisms whereby these organisms invade across the mucosal epithelial barrier to initiate invasive infection are not well understood. Protein A (SpA), a highly conserved and abundant surface protein of S. aureus, activates TNF receptor 1 and EGF receptor (EGFR) signaling cascades that can perturb the cytoskeleton. We demonstrate that wild-type S. aureus, but not spa mutants, invade across polarized airway epithelial cell monolayers via the paracellular junctions. SpA stimulated a RhoA/ROCK/MLC cascade, resulting in the contraction of the cytoskeleton. SpA(+) but not SpA(-) mutants stimulated activation of EGFR and along with subsequent calpain activity cleaved the membrane-spanning junctional proteins occludin and E-cadherin, facilitating staphylococcal transmigration through the cell-cell junctions. Treatment of polarized human airway epithelial monolayers with inhibitors of ROCK, EGFR, MAPKs, or calpain prevented staphylococcal penetration through the monolayers. In vivo, blocking calpain activity impeded bacterial invasion into the lung parenchyma. Thus, S. aureus exploits multiple receptors available on the airway mucosal surface to facilitate invasion across epithelial barriers.     

Up-regulation of microsomal prostaglandin E synthase-1 in COX-1 and COX-2 knock-out mouse fibroblast cell lines

In this paper we investigated the possible involvement of prostaglandin E synthases (PGESs) in compensatory mechanism. Our findings showed that microsomal (m)PGES-1 expression was significantly up-regulated in COX knock-out (K/O) cells whereas the expression of cytosolic PGES was not changed indicating that the induction of mPGES-1 may, at least in part, contribute to the substantial increase of PGE₂ production in COX K/O cell lines. The selective up-regulation of mPGES-1 in COX-2 K/O cells suggests that mPGES-1 may be metabolically coupled with COX-1 for PGE₂ formation. Addition of arachidonic acid caused significant induction of mPGES-1 and COX-2 in WT cells, whereas COX-1 and cPGES were not affected. Our earlier and the current studies demonstrate the coregulation of cPLA₂, COX, and mPGES-1, in PGE₂ synthesis pathway, and that these enzymes contribute to the elevation of PGE₂ level when one COX isoform is absent.     

Lipopolysaccharide prevents apoptosis induced by brefeldin A, an endoplasmic reticulum stress agent, in RAW 264.7 cells

The effect of lipopolysaccharide (LPS) on the cell death induced by endoplasmic reticulum (ER) stress agents in RAW 264.7 cells was studied. LPS prevented the cell death by brefeldin A, but not thapsigargin and tunicamycin. CpG DNA as well as LPS prevented brefeldin A-induced cell death whereas tumor necrosis factor-alpha or interferon-gamma did not. Brefeldin A-induced cell death was mediated with apoptotic cell death and it was significantly inhibited by LPS. LPS abolished the activation of ER stress-related caspases, such as caspases 1, 3, and 4. LPS prevented brefeldin A-induced morphological changes in RAW 264.7 cells. Further, LPS prevented brefeldin A-induced Golgi dispersion. Therefore, LPS was suggested to diminish the stress of ER/Golgi complexes induced by brefeldin A and inhibit apoptosis. The preventive action of LPS on brefeldin A-induced apoptosis is discussed.     

Treatment of CoQ10 Deficient Fibroblasts with Ubiquinone,CoQ Analogs,and Vitamin C: Time- and Compound-Dependent Effects

Background

Coenzyme Q₁₀ (CoQ₁₀) and its analogs are used therapeutically by virtue of their functions as electron carriers, antioxidant compounds, or both. However, published studies suggest that different ubiquinone analogs may produce divergent effects on oxidative phosphorylation and oxidative stress.

Methodology/Principal Findings

To test these concepts, we have evaluated the effects of CoQ₁₀, coenzyme Q₂ (CoQ₂), idebenone, and vitamin C on bioenergetics and oxidative stress in human skin fibroblasts with primary CoQ₁₀ deficiency. A final concentration of 5 µM of each compound was chosen to approximate the plasma concentration of CoQ₁₀ of patients treated with oral ubiquinone. CoQ₁₀ supplementation for one week but not for 24 hours doubled ATP levels and ATP/ADP ratio in CoQ₁₀ deficient fibroblasts therein normalizing the bioenergetics status of the cells. Other compounds did not affect cellular bioenergetics. In COQ2 mutant fibroblasts, increased superoxide anion production and oxidative stress-induced cell death were normalized by all supplements.

Conclusions/Significance

These results indicate that: 1) pharmacokinetics of CoQ₁₀ in reaching the mitochondrial respiratory chain is delayed; 2) short-tail ubiquinone analogs cannot replace CoQ₁₀ in the mitochondrial respiratory chain under conditions of CoQ₁₀ deficiency; and 3) oxidative stress and cell death can be counteracted by administration of lipophilic or hydrophilic antioxidants. The results of our in vitro experiments suggest that primary CoQ₁₀ deficiencies should be treated with CoQ₁₀ supplementation but not with short-tail ubiquinone analogs, such as idebenone or CoQ₂. Complementary administration of antioxidants with high bioavailability should be considered if oxidative stress is present.