Altered Metabolic Activity Research Articles

Reduction in Corneal Volume with Severity of Keratoconus

Purpose: To compare the corneal volume in keratoconic and normal eyes to improve our understanding of the tissue distribution associated with the disease.Materials and Method: The Oculus Pentacam tomographer (Oculus Inc., Wetzlar, Germany) was used to analyze the corneal volume contained within discs with diameters of 3, 5, 7, and 10 mm in 21 patients with keratoconus and 21 matched healthy control subjects.Results: Corneal volume was significantly decreased in the keratoconus group (keratoconus vs. control group: 3.44 ± 0.39 vs. 4.05 ± 0.29 mm3, 10.34 ± 0.95 vs. 11.79 ± 0.84 mm3, 22.80 ± 1.73 vs. 25.26 ± 1.74 mm3, and 57.17 ± 3.94 vs. 61.90 ± 4.12 mm3 for the 3-, 5-, 7-, and 10-mm diameter discs, respectively; p < 0.001). As the corneal disc diameter analyzed increased, fewer differences were found between the control corneas and keratoconic corneas at different stages of the disease. Within the 3-mm and 5-mm diameter discs, significant differences were detected between the control group, moderate keratoconus, and the severe keratoconus groups (p < 0.05). However, within the 10-mm discs, differences were only detected between the control group and the severe keratoconus group (p = 0.005).Conclusions: Corneal volume was significantly decreased in keratoconus, particularly in the central and paracentral area. The decrease in corneal volume in moderate and severe keratoconus as detected by the Pentacam tomographer, may be explained by loss of corneal tissue. In the early stages of the disease, the altered metabolic activity may cause tissue stretching and, as the disease progresses, this stretching is then accompanied by tissue loss.

Evidence for a Role for the Cytoskeleton in Communication Between the L-Type Calcium Channel and the Mitochondria in Isolated Cardiac Myocytes

Cytoskeletal proteins stabilize cell structure but also regulate subcellular distribution of mitochondria and cardiac L-type Ca2+ channel (LTCC) activity. We have previously demonstrated that mitochondrial function can be regulated by alterations in LTCC activity. This effect was attenuated when the cytoskeleton was disrupted with latrunculin A. To further explore this, we determined whether regulation of mitochondrial function by the LTCC is altered in a murine model of Duchenne Muscular Dystrophy(mdx). Mitochondrial membrane potential (Ψm) and metabolic activity was assessed after activation of the LTCC in cardiac myocytes isolated from C57BL/10ScSn-Dmdmdx/Arc (mdx) and C57BL/10ScSnArc (control) mice. Exposure of control myocytes to 10μM BayK(-) (LTCC agonist) caused a 11.4±1.7% increase in Ψm assessed as alterations in JC-1 compared to myocytes exposed to inactive BayK(+) (n=8, p<0.05). The response was attenuated when myocytes were exposed to LTCC antagonist nisoldipine (n=7). However BayK(-) did not induce any significant alteration in JC-1 signal in myocytes from mdx mice (n=6). In control myocytes BayK(-) caused a 105.4±7.4% increase in metabolic activity assessed using an MTT assay (n=8, p<0.05). The response was attenuated when myocytes were exposed to nisoldipine (n=8) or mitochondrial calcium uniporter inhibitor Ru360 (n=8) but unaltered when exposed to ryanodine receptor antagonist dantrolene (n=4). Exposure of mdx myocytes to BayK(-) did not induce any significant alteration in metabolic activity (n=8). These data confirm that alterations in LTCC activity can modulate mitochondrial function and that the cytoskeleton plays an important role in mediating this response. Since the LTCC is the initiator of contraction it has been proposed that a functional coupling between the LTCC and mitochondria may assist in meeting myocardial energy demand on a beat to beat basis.

Systems biology of bacterial nitrogen fixation: High-throughput technology and its integrative description with constraint-based modeling

BackgroundBacterial nitrogen fixation is the biological process by which atmospheric nitrogen is uptaken by bacteroids located in plant root nodules and converted into ammonium through the enzymatic activity of nitrogenase. In practice, this biological process serves as a natural form of fertilization and its optimization has significant implications in sustainable agricultural programs. Currently, the advent of high-throughput technology supplies with valuable data that contribute to understanding the metabolic activity during bacterial nitrogen fixation. This undertaking is not trivial, and the development of computational methods useful in accomplishing an integrative, descriptive and predictive framework is a crucial issue to decoding the principles that regulated the metabolic activity of this biological process.ResultsIn this work we present a systems biology description of the metabolic activity in bacterial nitrogen fixation. This was accomplished by an integrative analysis involving high-throughput data and constraint-based modeling to characterize the metabolic activity in Rhizobium etli bacteroids located at the root nodules of Phaseolus vulgaris (bean plant). Proteome and transcriptome technologies led us to identify 415 proteins and 689 up-regulated genes that orchestrate this biological process. Taking into account these data, we: 1) extended the metabolic reconstruction reported for R. etli; 2) simulated the metabolic activity during symbiotic nitrogen fixation; and 3) evaluated the in silico results in terms of bacteria phenotype. Notably, constraint-based modeling simulated nitrogen fixation activity in such a way that 76.83% of the enzymes and 69.48% of the genes were experimentally justified. Finally, to further assess the predictive scope of the computational model, gene deletion analysis was carried out on nine metabolic enzymes. Our model concluded that an altered metabolic activity on these enzymes induced different effects in nitrogen fixation, all of these in qualitative agreement with observations made in R. etli and other Rhizobiaceas.ConclusionsIn this work we present a genome scale study of the metabolic activity in bacterial nitrogen fixation. This approach leads us to construct a computational model that serves as a guide for 1) integrating high-throughput data, 2) describing and predicting metabolic activity, and 3) designing experiments to explore the genotype-phenotype relationship in bacterial nitrogen fixation.

Effects of Gut Microflora on Pharmacokinetics of Hesperidin: A Study on Non-Antibiotic and Pseudo-Germ-Free Rats

Hesperidin is a biologically active flavanone glycoside occurring abundantly in citrus fruits. In the present study, effects of intestinal microflora on pharmacokinetics of hesperidin were investigated using a pseudo-germ-free rat model treated with antibiotics. After administration of hesperidin to rats, hesperetin, hesperetin glucuronides, and metabolites postulated to be eriodictyol, hemoeriodictyol, and their glucuronides were detected in urine while hesperetin glucuronide was predominantly found in plasma. The plasma concentration–time profile of hesperetin was compared between non-antibiotic-exposed and pseudo-germ-free rats administered this compound. The maximal concentration (C max) values of hesperetin in non-antibiotic-exposed and pseudo-germ-free rats were 0.58 and 0.20 μg/ml, respectively, and area under the curve (AUC) values were 6.3 and 2.8 μg-h/ml, respectively. Thus, systemic exposure as evidenced by AUC and C max was significantly higher in normal compared to pseudo-germ-free rats. Fecal β-glucosidase activities of non-antibiotic-exposed and pseudo-germ-free rats were 0.21 and 0.11 nmol/min/mg, while fecal α-rhamnosidase activities were 0.37 and 0.12 nmol/min/mg, respectively. The rate of hesperidin transformation to hesperetin was 6.9 and 2.9 nmol/min/g in fecal samples in non-antibiotic-exposed and pseudo-germ-free rats, respectively. Taken together, these results showed that pharmacokinetic differences between non-antibiotic-exposed and pseudo-germ-free rats may be attributed to differing hesperidin uptake, as well as alterations in metabolic activities of intestinal flora.

18F-Fallypride binding potential in patients with schizophrenia compared to healthy controls

BackgroundMolecular imaging of dopaminergic parameters has contributed to the dopamine hypothesis of schizophrenia, expanding our understanding of pathophysiology, clinical phenomenology and treatment. Our aim in this study was to compare 18F-fallypride binding potential BPND in a group of patients with schizophrenia-spectrum illness vs. controls, with a particular focus on the cortex and thalamus. MethodsWe acquired 18F-fallypride positron emission tomography images on 33 patients with schizophrenia spectrum disorder (28 with schizophrenia; 5 with schizoaffective disorder) and 18 normal controls. Twenty-four patients were absolutely neuroleptic naïve and nine were previously medicated, although only four had a lifetime neuroleptic exposure of greater than two weeks. Parametric images of 18F-fallypride BPND were calculated to compare binding across subjects. ResultsDecreased BPND was observed in the medial dorsal nucleus of the thalamus, prefrontal cortex, lateral temporal lobe and primary auditory cortex. These findings were most marked in subjects who had never previously received medication. ConclusionsThe regions with decreased BPND tend to match brain regions previously reported to show alterations in metabolic activity and blood flow and areas associated with the symptoms of schizophrenia.

2D-NMR reveals different populations of exposed lysine residues in the apoB-100 protein of electronegative and electropositive fractions of LDL particles

Several potentially atherogenic LDL subfractions present low affinity for the LDL receptor, which result in impaired plasma clearance. Electronegative LDL [LDL(-)] is one of these minor subfractions and the molecular basis for its reduced receptor affinity is not well understood. In the present study, high-resolution 2D-NMR spectroscopy has been employed to characterize the surface-exposed lysine residues of the apolipoprotein (apo)B-100 protein in both LDL(-) and LDL(+) subfractions. LDL(+) showed two populations of lysine residues, similar to those previously described in total LDL. "Normal" Lys have a pk(a) of 10.4 whereas "active" Lys have a pk(a) of 8.8 and have been suggested to be involved in receptor binding. In contrast to LDL(+), the LDL(-) subfraction presented a third type of Lys, named as "intermediate" Lys, with a different microenvironment and higher basicity (pk(a) 10.7). These intermediate Lys cannot be reliably identified by 1D-NMR. Because the abundance of normal Lys is similar in LDL(+) and LDL(-), the intermediate Lys in the apoB-100 molecule of LDL(-) should come from a group of active Lys in LDL(+) particles that have a less basic microenvironment in the LDL(-) particle. These differences between LDL(+) and LDL(-) are indicative of a distinct conformation of apoB-100 that could be related to loss of affinity of LDL(-) for the LDL receptor.

Homozygosity for the EPHX2 K55R polymorphism increases the long-term risk of ischemic stroke in men: a study in Swedes

The soluble epoxide hydrolase (gene name EPHX2) is responsible for metabolism of 8,9 11,12 and 14,15-epoxyeicosatrienoic acids, vasodilator and anti-inflammatory substances. There are several functional polymorphisms in the EPHX2 gene: two of them, the K55R and R287Q, showing an altered metabolic activity in vitro, were associated with coronary heart disease and ischemic stroke in previous studies. The aim of this study was to evaluate the effect of four polymorphisms in the EPHX2 gene on blood pressure levels, hypertension prevalence, and risk of incident cardiovascular events in a large sample of middle-aged Swedes. The incidence of cardiovascular events (coronary events, n = 274; ischemic stroke, n = 197) was monitored over 10 years of follow-up. In the whole population, all polymorphisms had no effect on the studied parameters but a positive interaction between male sex and three SNPs including the K55R was evident: male, but not female, EPHX2 R55R homozygotes had significantly higher crude and adjusted systolic blood pressure and higher hypertension prevalence with respect to K-carriers. Kaplan-Meier curves showed higher incidence of ischemic strokes in male R55R homozygotes with respect to K-carriers (P = 0.015 by log-rank test). After adjustment for major cardiovascular risk factors, the hazard ratio for incident ischemic stroke in male R55R homozygotes remained significantly higher (hazard ratio: 4.8; 95% confidence interval: 1.2-19.9). The functional K55R polymorphism of the EPHX2 gene confers a higher risk of hypertension prevalence and increases the risk of incident ischemic stroke in male homozygotes. Additional studies are needed to confirm these data and to elucidate the interaction between sex and the EPHX2 K55R polymorphism.

Retinal metabolic state of the proline-23-histidine rat model of retinitis pigmentosa

We determined the metabolic changes that precede cell death in the dystrophic proline-23-histidine (P23H) line 3 (P23H-3) rat retina compared with the normal Sprague-Dawley (SD) rat retina. Metabolite levels and metabolic enzymes were analyzed early in development and during the early stages of degeneration in the P23H-3 retina. Control and degenerating retinas showed an age-dependent change in metabolite levels and enzymatic activity, particularly around the time when phototransduction was activated. However, lactate dehydrogenase (LDH) activity was significantly higher in P23H-3 than SD retina before the onset of photoreceptor death. The creatine/phosphocreatine system did not contribute to the increase in ATP, because phosphocreatine levels, creatine kinase, and expression of the creatine transporter remained constant. However, Na(+)-K(+)-ATPase and Mg(2+)-Ca(2+)-ATPase activities were increased in the developing P23H-3 retina. Therefore, photoreceptor apoptosis in the P23H-3 retina occurs in an environment of increased LDH, ATPase activity, and higher-than-normal ATP levels. We tested the effect of metabolic challenge to the retina by inhibiting monocarboxylate transport with alpha-cyano-4-hydroxycinnamic acid or systemically administering the phosphodiesterase inhibitor sildenafil. Secondary to monocarboxylate transport inhibition, the P23H-3 retina did not demonstrate alterations in metabolic activity. However, administration of sildenafil significantly reduced LDH activity in the P23H-3 retina and increased the number of terminal deoxynucleotidyl transferase biotin-dUPT nick end-labeled photoreceptor cells. Photoreceptor cells with a rhodopsin mutation display an increase in apoptotic markers secondary to inhibition of a phototransduction enzyme (phosphodiesterase), suggesting increased susceptibility to altered cation entry.

Inhibition of Mitochondrial Respiratory Chain in the Brain of Adult Rats After Acute and Chronic Administration of Methylphenidate

Methylphenidate (MPH) is frequently prescribed for the treatment of attention deficit/hyperactivity disorder. It was previously demonstrated that MPH altered brain metabolic activity. Most cell energy is obtained through oxidative phosphorylation, in the mitochondrial respiratory chain. However, there are still few studies about MPH effects on the brain of adult rats. Thus, in the present study we evaluated the effect of acute or chronic administration of MPH on the activities of mitochondrial respiratory chain complexes I-IV in the brain of adult rats. For acute administration, a single injection of MPH was given to 60-day-old rats. For chronic administration, MPH injections were given to 60-day-old rats once daily for 28 days. Our results showed that complexes I, II, III and IV were inhibited after acute or chronic MPH administration in the hippocampus, prefrontal cortex, striatum and cerebral cortex. On the other hand, cerebellum was not affected.

Modification of endothelial biology by acute and chronic stress hormones

Objective An increasing number of studies have examined the role of emotional stress and coronary heart disease; the underlying pathophysiology is still poorly understood. The present study was designed to evaluate the relationship between acute (epi- and norepinephrine) and chronic stress hormones (dexamethasone, beta-endorphin, corticotropin releasing hormone) and endothelial dysfunction. Methods Human microvascular endothelial cells were incubated with stress hormones for 6 and 24 h. ET-1 release and ADMA were quantified via ELISA, NO release by using cell permeable 4.5-diaminofluorescein diacetate (DAF2-DA), oxidative stress fluometrically by the ROS-sensitive carboxy-H2-DCFDA method, mitochondrial metabolic activity by using the colorimetric assay WST-1, ET-1 receptor type A (ET AR) protein expression by Western blot, and cell proliferation activity was assessed by the colorimetric assay BrdU. Results With respect to analysed acute and chronic stress hormones, ET-1 release was significantly increased. Likewise, protein expression was enhanced after long term incubation (24 h) with norepinephrine and dexamethasone. In contrast, endothelial NO-levels were only influenced by short term stimulation of dexamethasone (upregulation of NO release) and norepinephrine (downregulation of NO release), whereas modified NO concentration mimics altered mitochondrial metabolic activity. Unexpectedly, both oxidative stress and cell proliferation were not modified by stress hormones. Conclusion Results suggest that acute and chronic stress hormones induce a significant ET-1 release whereas NO release remained mainly unchanged. The imbalance of pro- and antiatherosclerotic factors may play a pivotal role in the initiation of stress-related endothelial dysfunction up to myocardial infarction.

Defective Peripheral Nerve Development Is Linked to Abnormal Architecture and Metabolic Activity of Adipose Tissue in Nscl-2 Mutant Mice

BackgroundIn mammals the interplay between the peripheral nervous system (PNS) and adipose tissue is widely unexplored. We have employed mice, which develop an adult onset of obesity due to the lack the neuronal specific transcription factor Nscl-2 to investigate the interplay between the nervous system and white adipose tissue (WAT).MethodologyChanges in the architecture and innervation of WAT were compared between wildtype, Nscl2−/−, ob/ob and Nscl2−/−//ob/ob mice using morphological methods, immunohistochemistry and flow cytometry. Metabolic alterations in mutant mice and in isolated cells were investigated under basal and stimulated conditions.Principal FindingsWe found that Nscl-2 mutant mice show a massive reduction of innervation of white epididymal and paired subcutaneous inguinal fat tissue including sensory and autonomic nerves as demonstrated by peripherin and neurofilament staining. Reduction of innervation went along with defects in the formation of the microvasculature, accumulation of cells of the macrophage/preadipocyte lineage, a bimodal distribution of the size of fat cells, and metabolic defects of isolated adipocytes. Despite a relative insulin resistance of white adipose tissue and isolated Nscl-2 mutant adipocytes the serum level of insulin in Nscl-2 mutant mice was only slightly increased.ConclusionsWe conclude that the reduction of the innervation and vascularization of WAT in Nscl-2 mutant mice leads to the increase of preadipocyte/macrophage-like cells, a bimodal distribution of the size of adipocytes in WAT and an altered metabolic activity of adipocytes.

Changes in Relative Glucose Metabolic Rate Following Cortisol Administration in Aging Veterans with Posttraumatic Stress Disorder: An FDG-PET Neuroimaging Study

Changes in Relative Glucose Metabolic Rate Following Cortisol Administration in Aging Veterans with Posttraumatic Stress Disorder: An FDG-PET Neuroimaging Study

Age-associated defects in Tcell metabolism (46.10)

Abstract Aging at the cellular and molecular level causes a decline in immune function. We are characterizing the responses of naïve T-cells to activation. Activated T-cells of aged mice initiate proliferation as well as their young counterparts, but have increased apoptotic rates. However, the mechanisms behind this post- activation death are not completely understood. We will show alterations in metabolic activities in T-cells of aged mice through differences in the expression of glucose transporter (Glut 1), glucose uptake and lactate production. Additionally, we will correlate these findings with autophagocytic vesicle formation by analyzing microtubule-associated protein -1 light chain 3 (LC3) as well as comparing relative amounts of actively respiring mitochondria using Mitotracker Red CM-H2XRos, a membrane potential dependant dye, and total mitochondrial mass by staining with the mitochondrial-selective fluorescence label Mitotracker Green. Levels of reactive oxygen species will be then quantified by staining with ROS specific dye DCF-DA and correlated to the ratio of mitochondrial mass to membrane potential.

Effects of antenatal glucocorticoids on cerebral substrate metabolism in the preterm ovine fetus

Although the benefits of antenatal glucocorticoids are well known for infants who are born preterm, there is increasing evidence of adverse effects on brain development, which may relate to altered metabolic activity. We have determined the effect of maternal glucocorticoid administration at doses that are used clinically on cerebral substrate metabolism in the preterm ovine fetus. Chronically instrumented pregnant sheep at 0.85 gestation received 2 intramuscular injections of betamethasone at 170 microg/kg maternal weight (n = 13) or saline (n = 10) 24 hours apart together with a continuous infusion of L-[1-(13)C] leucine to the fetus. Fetal cerebral substrate arteriovenous differences (O2, glucose, leucine, leucine enrichment) and blood flow (fluorescent microspheres) were measured at baseline, 24 hours after the first betamethasone/saline injection (late beta/saline 1), and 4 hours after the second betamethasone/saline injection (early beta/saline 2) to obtain substrate deliveries and fractional extractions. Fetal pH, blood gases, and metabolites were little changed in either group over the course of the study, except for glucose values in the betamethasone animals, which increased 1.4- and 1.9-fold, measured late beta 1 and early beta 2, respectively (both P < .01). Cerebral blood flow, although little changed in the control group or at late beta 1, was decreased at early beta 2 by approximately 30% (P < .05). As such, early beta 2 animals showed a decrease in cerebral O2 delivery of approximately 20% (P = .06) and conversely an increase in cerebral glucose delivery of 1.4- and 1.3-fold at late beta 1 (P < .05) and early beta 2 (P = .08), respectively. Fractional extraction values for these substrates were not changed significantly, which resulted in corresponding decreases in estimated O2 uptake and increases in estimated glucose uptake, such that the glucose/oxygen quotient (as an index of glucose oxidative metabolism) measured 1.6 at early beta 2, which was considerably greater than baseline values at 1.1 (P < .05). Fractional extraction values for leucine and leucine enrichment averaged 2%-3%; although somewhat higher in the betamethasone animals, none of the between or within group differences were significant. Fetal cerebral metabolism in the preterm ovine fetus is altered by antenatal glucocorticoid administration, which is comparable with that used in human pregnancy, and includes an acute decrease in cerebral blood flow and a probable increase in anaerobic glucose metabolism. Although likely of short duration in conjunction with peak glucocorticoid levels, these metabolic effects may place the developing brain at added risk for superimposed hypoxic injury.

Chronic administration of methylphenidate activates mitochondrial respiratory chain in brain of young rats

Methylphenidate is frequently prescribed for the treatment of attention deficit/hyperactivity disorder. Psychostimulants can cause long-lasting neurochemical and behavioral adaptations. The exact mechanisms underlying its therapeutic and adverse effects are still not well understood. In this context, it was previously demonstrated that methylphenidate altered brain metabolic activity, evaluated by glucose consumption. Most cell energy is obtained through oxidative phosphorylation, in the mitochondrial respiratory chain. Tissues with high energy demands, such as the brain, contain a large number of mitochondria. In this work, our aim was to measure the activities of mitochondrial respiratory chain complexes II and IV and succinate dehydrogenase in cerebellum, prefrontal cortex, hippocampus, striatum, and cerebral cortex of young rats (starting on 25th post-natal day and finishing on 53rd post-natal day) chronically treated with methylphenidate. Our results showed that mitochondrial respiratory chain enzymes activities were increased by chronic administration of this drug. Succinate dehydrogenase was activated in cerebellum, prefrontal cortex and striatum, but did not change in hippocampus and brain cortex. Complex II activity was increased in cerebellum and prefrontal cortex and was not affected in hippocampus, striatum and brain cortex. Finally, complex IV activity was increased in cerebellum, hippocampus, striatum and brain cortex, and was not affected in prefrontal cortex. These findings suggest that chronic exposure to methylphenidate in young rats increases mitochondrial enzymes involved in brain metabolism. Further research is being carried out in order to better understand the effects of this drug on developing nervous system and the potential consequences in adulthood resulting from early-life drug exposure.

Hepatocyte growth factor increases mitochondrial mass in glioblastoma cells

Hepatocyte growth factor/scatter factor (HGF) is a multifunctional growth factor that is linked to the initiation and/or progression of numerous malignancies. HGF also alters cancer cell responses to DNA damaging cytotoxic agents. Many cell responses to Met activation require alterations in metabolic activity but how the metabolic machinery responds to Met activation remains poorly defined. Treating human glioblastoma cells with HGF followed by the topoisomerase inhibitor camptothecin was found to increase the activity per cell of the mitochondrial respiratory chain enzyme succinate-tetrazolium reductase (>80% increase, p < 0.05) and the tricarboxylic acid cycle enzyme succinate dehydrogenase (>25% increase, p < 0.05). Treatment with either HGF or camptothecin alone had no effect on enzyme activity. The mitochondrial enzymatic response to HGF was dose- and time-dependent with the maximum increase occurring in cells pre-treated with 30 ng/ml HGF for 48 h prior to camptothecin exposure. This enzymatic response was associated with a concurrent increase in mitochondrial mass of comparable magnitude (∼56%, p < 0.05) as measured by fluorescent mitochondrial staining and flow cytometry. The mitochondrial mass response to HGF was prevented by the MAP-kinase pathway inhibitor PD98059 and was unaffected by the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin. These findings suggest that HGF influences cell responses to chemotherapeutic stress, in part, by altering mitochondrial functions through a MAP-kinase dependent increase in mitochondrial mass.

Alterations of A549 lung cell gene expression in response to biochemical toxins

Health risks associated with the inhalation of potentially toxic materials have been a topic of great public concern. In vitro cellular analyses can provide mechanistic information on the molecular-level responses of lung-derived cell lines to a variety of these hazards. This understanding may be used to develop methods to reduce the damage from such toxins or to detect early stages of their effects. Here we describe an evaluation of the alterations in gene expression of an immortalized lung cell line (A549, human type II epithelia) to a variety of inhalation health hazards including etoposide, gliotoxin, streptolysin O, methyl methansesulfonate (MMS), and Triton X-100. The A549 cells display a dose-response relationship to each toxin with initial responses including alterations in metabolic activity, increases in membrane permeability, and initiation of response genes. In general, membrane-damaging agents (streptolysin O and Triton X-100) induce production of new ion channel proteins, structural proteins, and metabolic enzymes. Gliotoxin impacted the metabolic machinery, but also altered ion channels. Etoposide and MMS caused alterations in the cell cycle, induced DNA repair enzymes, and initiated apoptotic pathways, but MMS also induced immune response cascades. The mechanism of cell response to each toxin is supported by physiological analyses that indicated a fairly slow initiation of cell response to all compounds tested, except for Triton, which caused rapid decline in cell function due to solubilization of the cell membrane. However, Triton does induce production of a number of cell membrane-associated proteins and so its effects at low concentrations are likely translated throughout the cell. Together these results indicate a broader array of cellular responses to each of the test toxins than have previously been reported.

Comparative proteome analysis of Staphylococcus aureus biofilm and planktonic cells and correlation with transcriptome profiling

Pathogenic staphylococci can form biofilms in which they show a higher resistance to antibiotics and the immune defense system than their planktonic counterparts, which suggests that the cells in a biofilm have an altered metabolic activity. Here, 2-D PAGE was used to identify secreted, cell wall-associated and cytoplasmic proteins expressed in Staphylococcus aureus after 8 and 48 h of growth. The proteins were separated at pH ranges of 4-7 or 6-11. The protein patterns revealed significant differences in 427 protein spots; from these, 258 non-redundant proteins were identified using ESI-MS/MS. Biofilm cells expressed higher levels of proteins associated with cell attachment and peptidoglycan synthesis, and in particular fibrinogen-binding proteins. Enzymes involved in pyruvate and formate metabolism were upregulated. Furthermore, biofilm cells expressed more staphylococcal accessory regulator A protein (SarA), which corroborates the positive effect of SarA on the expression of the intercellular adhesion operon ica and biofilm growth. In contrast, proteins, such as proteases and particularly immunodominant antigen A (IsaA) and staphylococcal secretory antigen (SsaA), were found in lower amounts. The RNA expression profiling largely supports the proteomic data. The results were mapped onto KEGG pathways.

Alterations of metabolic activity in human osteoarthritic osteoblasts by lipid peroxidation end product 4-hydroxynonenal

4-Hydroxynonenal (HNE), a lipid peroxidation end product, is produced abundantly in osteoarthritic (OA) articular tissues, but its role in bone metabolism is ill-defined. In this study, we tested the hypothesis that alterations in OA osteoblast metabolism are attributed, in part, to increased levels of HNE. Our data showed that HNE/protein adduct levels were higher in OA osteoblasts compared to normal and when OA osteoblasts were treated with H2O2. Investigating osteoblast markers, we found that HNE increased osteocalcin and type I collagen synthesis but inhibited alkaline phosphatase activity. We next examined the effects of HNE on the signaling pathways controlling cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6) expression in view of their putative role in OA pathophysiology. HNE dose-dependently decreased basal and tumour necrosis factor-α (TNF-α)-induced IL-6 expression while inducing COX-2 expression and prostaglandin E2 (PGE2) release. In a similar pattern, HNE induces changes in osteoblast markers as well as PGE2 and IL-6 release in normal osteoblasts. Upon examination of signaling pathways involved in PGE2 and IL-6 production, we found that HNE-induced PGE2 release was abrogated by SB202190, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Overexpression of p38 MAPK enhanced HNE-induced PGE2 release. In this connection, HNE markedly increased the phosphorylation of p38 MAPK, JNK2, and transcription factors (CREB-1, ATF-2) with a concomitant increase in the DNA-binding activity of CRE/ATF. Transfection experiments with a human COX-2 promoter construct revealed that the CRE element (-58/-53 bp) was essential for HNE-induced COX-2 promoter activity. However, HNE inhibited the phosphorylation of IκBα and subsequently the DNA-binding activity of nuclear factor-κB. Overexpression of IKKα increased TNF-α-induced IL-6 production. This induction was inhibited when TNF-α was combined with HNE. These findings suggest that HNE may exert multiple effects on human OA osteoblasts by selective activation of signal transduction pathways and alteration of osteoblastic phenotype expression and pro-inflammatory mediator production.

Differential Gene Expression Profiling of Staphylococcus aureus Cultivated under Biofilm and Planktonic Conditions

It is well known that biofilm formation by pathogenic staphylococci on implanted medical devices leads to "chronic polymer-associated infections." Bacteria in these biofilms are more resistant to antibiotics and the immune defense system than their planktonic counterparts, which suggests that the cells in a biofilm have altered metabolic activity. To determine which genes are up-regulated in Staphylococcus aureus biofilm cells, we carried out a comparative transcriptome analysis. Biofilm growth was simulated on dialysis membranes laid on agar plates. Staphylococci were cultivated planktonically in Erlenmeyer flasks with shaking. mRNA was isolated at five time points from cells grown under both conditions and used for hybridization with DNA microarrays. The gene expression patterns of several gene groups differed under the two growth conditions. In biofilm cells, the cell envelope appeared to be a very active compartment since genes encoding binding proteins, proteins involved in the synthesis of murein and glucosaminoglycan polysaccharide intercellular adhesin, and other enzymes involved in cell envelope synthesis and function were significantly up-regulated. In addition, evidence was obtained that formate fermentation, urease activity, the response to oxidative stress, and, as a consequence thereof, acid and ammonium production are up-regulated in a biofilm. These factors might contribute to survival, persistence, and growth in a biofilm environment. Interestingly, toxins and proteases were up-regulated under planktonic growth conditions. Physiological and biochemical tests for the up-regulation of urease, formate dehydrogenase, proteases, and the synthesis of staphyloxanthin confirmed the microarray data.