Principal Substrate Research Articles

Substrate Quality Effects on Decomposition of Three Livestock Manure Composts with Similar Stability Degree in an Acid Loamy Soil

Decomposition of compost applied to soils is affected basically by its biological stability; but, many other chemical properties of the compost may also influence compost organic-C mineralization. This study was conducted to investigate the principal substrate quality factors of composts that determine C mineralization of compost with similar stability degree (SD). Three composts samples with similar SD but different chemical properties such as pH, C/N, <TEX>$K_2SO_4$</TEX>-extractable C, and molar ratio of <TEX>$NH_4^+$</TEX> to <TEX>$NO_3^-$</TEX> were mixed with an acid loamy soil and <TEX>$CO_2$</TEX> emission was monitored during the laboratory incubation for 100 days. Temporal pattern of cumulative compost organic-C mineralization expressed as % of total organic C (<TEX>$C_{%\;TOC}$</TEX>) followed double exponential first order kinetics model and the <TEX>$C_{%\;TOC}$</TEX> ranged from 4.8 to 11.8% at the end of incubation. The pattern of C%TOC among the composts was not coincident with the SD pattern (40.1 to 58.6%) of the composts; e.g. compost with the lowest SD resulted in the least <TEX>$C_{%\;TOC}$</TEX> and vice versa. This result indicates that SD of compost can not serve as a concrete predictor of compost mineralization as SD is subject not only to maturity of compost but also to characteristics of co-composting materials such as rice hull (low SD) and sawdust (high SD). Meanwhile, such pattern of <TEX>$C_{%\;TOC}$</TEX> collaborated with pH, C/N, <TEX>$K_2SO_4$</TEX>-extractable C, and molar ratio of <TEX>$NH_4^+$</TEX> to <TEX>$NO_3^-$</TEX> of the composts that are regarded as chemical indices of the progress of composting. Therefore, for better prediction of compost mineralization in soils, it is necessary to consider both SD and other chemical indices (pH, C/N, and molar ratio of <TEX>$NH_4^+$</TEX> to <TEX>$NO_3^-$</TEX>).

Lactate Fuels the Neonatal Brain

Lactate Fuels the Neonatal Brain

Transcription profiling of the isoflavone phenylpropanoid pathway in soybean in response to Bradyrhizobium japonicum inoculation

Isoflavones are legume-specific secondary metabolites that function as defence compounds, signal molecules and regulators of gene expression during both pathogen attack and beneficial plant-microbe interactions. They are synthesised by a branch of the core phenylpropanoid pathway, using several isoenzymes within each enzymatic step. Gene-specific quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to quantify expression of isoflavone synthesis genes in soybean (Glycine max L). Genes encoding chalcone synthase 7 (CHS7), chalcone synthase 8 (CHS8) and isoflavone synthase 1 (IFS1) displayed high basal expression levels in roots compared with hypocotyls, suggesting they could be the gene family members encoding the isoenzyme that contributes the most to the principal substrate flux towards specific isoflavone synthesis in roots. The genes encoding phenylalanine ammonia lyase 1 (PAL1) and IFS1 showed induction in root tissue after inoculation with Bradyrhizobium japonicum (Kirchner) Jordan, suggesting a control point. The absence of a functional nodulation regulator, GmNARK (G. max nodulation autoregulation receptor kinase), in the soybean mutant nts1007 resulted in significantly increased basal expression of PAL1 compared with levels induced by B. japonicum, suggesting that GmNARK is a negative regulator for isoflavone phenylpropanoid pathway genes during nodulation and that distinct genes, as opposed to the complete pathway, are coordinately regulated by the nodulation status of the mutant.

The Major Phagocyte Peroxidase-Derived Oxidant HOSCN Stimulates Gene-Specific p38 MAPK- Dependent, NF-κb-Mediated Activation of Tissue Factor, VCAM-1 and ICAM-1 Expression In Endothelium.

AbstractAbstract 1481Thiocyanate (SCN-) is, unexpectedly, the principal physiologic substrate for eosinophil peroxidase (EPO) and a major (i.e., accounting for 50% of H2O2 consumed) substrate for myeloperoxidase (MPO). The product of these reactions is HOSCN, a weak, exclusively sulfhydryl-reactive oxidant that we have previously shown to be a uniquely potent (up to 100-fold) oxidant transcriptional inducer of human umbilical vein endothelial cell (HUVEC) tissue factor (TF), ICAM-1 and VCAM-1 expression via a mechanism dependent upon NF-κB p65/p50 activation. We hypothesized that oxidative activation of p38 MAPK is a necessary and proximal step in the activation of NF-κB p65/p50 at the TF, VCAM-1 and ICAM-1 promoters. To test this we utilized the p38 MAPK-specific pyridinyl imidazole inhibitor SB 203580 (SB). In HUVEC monolayers exposed 4h to 150 μM HOSCN in M199 medium containing 10% FCS, SB strongly inhibited (>90%, ED50 300 nM) TF activity, decreased VCAM-1 expression assessed by western blot by > 50% but had no discernible effect upon ICAM-1 expression. qRT-PCR analysis confirmed that the effects of SB on these three molecules was transcriptionally mediated. Immunoprecipitation of HOSCN-treated HUVEC whole cell extracts with a polyclonal anti-phospho-p38 MAPK reagent showed rapid (within 1 min) phosphorylation of p38 MAPK that lasted >30 min with consequent kinase activity documented by detection of the downstream target product phospho HSP27. Confocal immunofluorescence confirmed the rapid and durable induction of phospho p38 MAPK from undetectable to abundant, primarily cytoplasmic, but also some nuclear localization. We further hypothesized that the disparate effects of SB on these three molecules reflects differences in the sequences of their NF-κB-binding sites that affect their ability to bind the p65 NF-kB subunit. Chromatin immunoprecipitation (ChIP) analysis of HUVEC treated for 3h with HOSCN using an anti-p65 antibody revealed that SB inhibited by 75% the 10-fold increase in binding of p65 to the authentic endogenous VCAM-1 and NF-κB binding loci but had no discernible effect on p65 binding to the ICAM-1 locus. An identical pattern was seen in HUVEC exposed 30 min to the prototypical inflammatory cytokine TNF. To test the physiologic significance of these findings we assessed the effect of SB upon PMN and eosinophil binding to HOSCN-exposed HUVEC monolayers treated as described above. SB pretreatment of HUVEC blocked by > 80% the 3–8-fold increase in binding of PMN and eosinophils that occurs in HOSCN-exposed monolayers. Blocking antibodies to ICAM-1 and VCAM-1 demonstrate that both of the adhesion molecules contribute to HOSCN-induced PMN and eosinophil adhesion. We conclude that HOSCN generated by PMN and eosinophils attached or subjacent to vascular endothelium has the capacity to rapidly activate endothelial p38 MAPK-dependent activation of p65 binding to the TF and VCAM-1, but not ICAM-1, NF-κB binding loci; and these differences manifest in physiologically relevant transcriptional regulation of protein expression. Therefore, p38 MAPK inhibitors may have anti-thrombotic and anti-inflammatory potential, the latter particularly in states, such as allergic inflammation, that are most dependent on VCAM-1. Disclosures:No relevant conflicts of interest to declare.

Identification of BCAR-1 as a new substrate of Syk tyrosine kinase through a determination of amino acid sequence preferences surrounding the substrate tyrosine residue

Syk, a non-receptor tyrosine kinase, is an essential signaling molecule in B cells and other hematopoietic cells. Recently, its unexpected diverse functions were recognized in the regulation of cellular adhesion, innate immune recognition, vascular development, and carcinogenesis. Despite its pleiotropic role, only a few substrate proteins have been identified. To find new substrate proteins for Syk, we performed a systemic in vitro kinase assay using GST fusion peptides to determine the substrate specificity surrounding the tyrosine residue to be phosphorylated. Substitution of amino acid residues surrounding tyrosine 178 of BLNK, a principal Syk substrate in B cell receptor-mediated signaling, revealed that acidic residues at sites −5 to −1 were necessary for phosphorylation by Syk. Valine at site +1 was also influential in phosphorylation and a substitution of Pro on site +3 to a basic amino acid residue, Lys, resulted in attenuated phosphorylation. On the basis of these results, a general consensus phosphorylation motif for Syk was determined and several new candidate target proteins were identified in protein database searches. Of the candidate proteins, BCAR-1 (breast cancer anti-estrogen resistance 1) was confirmed to be phosphorylated by Syk in an in vitro kinase assay using a full-length protein of BCAR-1. Furthermore, BCAR-1 was tyrosine phosphorylated upon the overexpression of Syk in HEK-293T cells. These results suggest that more Syk substrates can be found using an in vitro kinase approach and show for the first time that BCAR-1 is a physiological substrate of Syk.

Characterisation of the bifunctional dihydrofolate synthase–folylpolyglutamate synthase from Plasmodium falciparum; a potential novel target for antimalarial antifolate inhibition

Unusually for a eukaryote, the malaria parasite Plasmodium falciparum expresses dihydrofolate synthase (DHFS) and folylpolyglutamate synthase (FPGS) as a single bifunctional protein. The two activities contribute to the essential pathway of folate biosynthesis and modification. The DHFS activity of recombinant PfDHFS–FPGS exhibited non-standard kinetics at high co-substrate (glutamate and ATP) concentrations, being partially inhibited by increasing concentrations of its principal substrate, dihydropteroate (DHP). Binding of DHP to the catalytic and inhibitory sites exhibited dissociation constants of 0.50μM and 1.25μM, respectively. DHFS activity measured under lower co-substrate concentrations, where data fitted the Michaelis–Menten equation, yielded apparent Km values of 0.88μM for DHP, 22.8μM for ATP and 5.97μM for glutamate. Of the substrates tested in FPGS assays, only tetrahydrofolate (THF) was efficiently converted to polyglutamylated forms, exhibiting standard kinetics with an apparent Km of 0.96μM; dihydrofolate, folate and the folate analogue methotrexate (MTX) were negligibly processed, emphasising the importance of the oxidation state of the pterin moiety. Moreover, MTX inhibited neither DHFS nor FPGS, even at high concentrations. Conversely, two phosphinate analogues of 7,8-dihydrofolate that mimic tetrahedral intermediates formed during DHFS- and FPGS-catalysed glutamylation were powerfully inhibitory. The Ki value of an aryl phosphinate analogue against DHFS was 0.14μM and for an alkyl phosphinate against FPGS 0.091μM, with each inhibitor showing a high degree of specificity. This, combined with the absence of DHFS activity in humans, suggests PfDHFS–FPGS might represent a potential new drug target in the previously validated folate pathway of P. falciparum.

Phospholemman Recruits Peroxiredoxin 6 to the Cardiac Sodium Pump

Phospholemman (FXYD1, PLM), the principal sarcolemmal substrate for protein kinases A and C in the heart, is a regulator of the cardiac sodium pump. We investigated proteins that interact with PLM in adult rat ventricular myocytes using bifunctional crosslinking reagents and co-immunoprecipitation.Digitonin-permeabilized ventricular myocytes were treated with the heterobifunctional crosslinking reagent sulfo-lc-smpt (distance between reactive groups 2nm, reactive towards amino and sulfhydryl amino acid side chains), and cell lysates immunoblotted for PLM. We found sulfo-lc-smpt quantitatively crosslinked PLM to a 20-25kDa protein (electrophoretic mobility of PLM 15kDa, electrophoretic mobility of crosslinked adduct 37kDa). Co-immunoprecipitation experiments indicated that the crosslinked adduct was PLM linked to the anti-oxidant protein peroxiredoxin 6 (prdx6). PLM phosphorylation at serine 68 (protein kinase A activation with 10μM forskolin) or at serines 63, 68 and threonine 69 (protein kinase C activation with 300nM PMA) had no effect on the ability of sulfo-lc-smpt to crosslink PLM to prdx6. Hydrogen peroxide treatment of ventricular myocytes (1-100μM) was also without effect on the binding of prdx6 to PLM.In conclusion, our data suggest a new role for PLM in the heart. As well as being responsible for kinase-mediated regulation of the cardiac sodium pump, it is responsible for recruitment of prdx6 to this ion transporter. Prdx6 catalyzes the reduction of hydrogen peroxide, fatty acid hydroperoxides and phospholipid hydroperoxides using glutathione. Given the well-established sensitivity of the alpha subunit of the sodium pump to cysteine oxidation, recruitment of prdx6 to the sodium pump by PLM may be important to maintain pump activity during periods of oxidative stress.

Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1

FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped α-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase α and β subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na +/K + ion binding site of the Na,K-ATPase α subunit.

Calcineurin Finds a New Partner in the L-Type Ca 2+ Channel

See related articles, pages 51–60 Calcineurin is a Ca2+/calmodulin-sensitive phosphatase that sits near the top of signaling pathways leading to pathological cardiac hypertrophy.1 Pathological stressors activate calcineurin, for which nuclear factor of activated T cells (NFAT) is a principal substrate. Dephosphorylation unmasks a nuclear localization signal on NFAT, which then translocates to the nucleus and, there, serves as part of a transcription factor complex able to initiate gene expression cascades that induce cardiac hypertrophy and subsequent heart failure. Determining how calcineurin is specifically activated in response to hypertrophic stressors by the same Ca2+ signal used for excitation–contraction coupling has been a major challenge. Triggered by Ca2+ influx through CaV1.2 voltage-gated Ca2+ channels at the sarcolemmal membrane, intracellular calcium briefly increases 10-fold with each heart beat to generate myocyte contraction. Almost as quickly, intracellular calcium falls to its baseline level, allowing myocyte relaxation and preparation for the next beat. There are 2 leading proposals for how Ca2+ could activate the calcineurin/NFAT pathway or function as a versatile regulator of multiple other signaling cascades in the face of the large Ca2+ oscillations driving each cycle of contraction and relaxation. One is that modulation of the amplitude and/or frequency of …

Fabry disease urinary globotriaosylceramide/creatinine biomarker evaluation by liquid chromatography–tandem mass spectrometry in healthy infants from birth to 6 months

Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of alpha-galactosidase A, resulting in accumulation of the principal substrate, globotriaosylceramide (Gb 3), in various physiological fluids and tissues in affected patients. The recognition that accumulation of Gb 3 begins in utero, combined with the fact that the diagnosis of the disease is often delayed until after the development of irreversible tissue damage, has generated pressure to develop techniques for the early, pre-symptomatic diagnosis of the disease. Measurements of urinary Gb 3 have been shown to be useful for the diagnosis of Fabry disease in adults. The objective of this work was to measure the Gb 3/creatinine biomarker in urine of healthy infants from birth to 6 months, including the establishment of reference ranges for urinary Gb 3 excretion at various postnatal ages, in male and female infants. We employed liquid chromatography–tandem mass spectrometry (LC–MS/MS) to determine Gb 3/creatinine ratios in urine specimens dried on filter paper and mailed to the laboratory by participating parents. A total of 728 urine specimens were obtained at intervals from birth to 6 months of age from 68 healthy infants (35 male and 33 female). Parental participation was good, with 90% of the expected specimens received by the laboratory. The results of the analyses were grouped by the age of the infants into four periods. We have determined that both postnatal age and sex have an effect on urinary Gb 3 excretion levels which vary considerably in newborns. We conclude that screening for Fabry disease by measurement of urinary Gb 3 excretion is unlikely to be reliable before 30 days of age.

Evaluation of drug–drug interactions with fesoterodine

To assess drug-drug interactions of fesoterodine with cytochrome P450 (CYP) 3A4 inhibitor (ketoconazole), inducer (rifampicin), and substrates (ethinylestradiol and levonorgestrel). Effects of ketoconazole 200 mg twice daily and rifampicin 600 mg twice daily on fesoterodine 8 mg once daily were investigated in CYP2D6 extensive metabolizers (EMs) and poor metabolizers (PMs) based on 5-hydroxymethyl tolterodine (5-HMT) pharmacokinetics (principal active fesoterodine metabolite and CYP3A4 substrate). Effects of fesoterodine 8 mg versus placebo once daily on ethinylestradiol and levonorgestrel were investigated based on oral contraceptive pharmacokinetics and on pharmacodynamic effects on progesterone, luteinizing hormone, follicle-stimulating hormone, and estradiol plasma levels. Compared with fesoterodine alone, coadministration of fesoterodine with ketoconazole resulted in increases in mean 5-HMT maximum concentration in plasma (C(max); from 3.0 to 6.0 ng/mL in EMs and from 6.4 to 13.4 ng/mL in PMs) and mean area under the plasma concentration time curve (AUC; from 38.2 to 88.3 ng h/mL in EMs and 88.3 to 217.2 ng h/mL in PMs). Coadministration of festerodine with rifampicin resulted in decreases in mean 5-HMT C(max) (from 5.2 to 1.5 ng/mL in EMs and from 6.8 to 1.9 ng/mL in PMs) and mean AUC (from 62.4 to 14.4 ng h/mL in EMs and from 87.8 to 19.6 ng h/mL in PMs). Fesoterodine did not affect oral contraceptive pharmacokinetics or pharmacodynamics or the suppression of ovulation. Fesoterodine dosage should not exceed 4 mg once daily when taken concomitantly with potent CYP3A4 inhibitors. Coadministration of CYP3A4 inducers with fesoterodine may produce subtherapeutic 5-HMT exposures. No dose adjustment is necessary for concomitant use of fesoterodine with oral contraceptives.

Proposed high‐risk screening protocol for Fabry disease in patients with renal and vascular disease

Fabry disease is a complex, multisystemic and clinically heterogeneous disease with prominent urinary excretion of globotriaosylceramide (Gb(3)), the principal substrate of the deficient enzyme, alpha-galactosidase A. Some measure of specific treatment is possible with enzyme replacement therapy, which can be applied safely and effectively to Fabry patients. Incidence estimations of Fabry disease vary widely from 1:55 000 to 1:3000 male births. The true incidence is likely to be higher than originally thought, owing to the existence of milder variants of the disease. The main complications of Fabry disease are a 100-fold increased risk of ischaemic stroke, cardiac disease, a wide variety of arrhythmias, valvular dysfunction and cardiac vascular disease, as well as progressive renal failure usually associated with significant proteinuria. These clinical manifestations are non-specific and are often mistaken for symptoms of other disorders, thus complicating the confirmation of diagnosis. Other clinical features of the disease are often absent (angiokeratoma), subtle (corneal opacities and hypohidrosis), or unaccompanied by specific physical findings (acroparaesthesias) indicating the true nature of the underlying disease. We propose the hypothesis that alpha-galactosidase A deficiency is a modifiable cardiovascular risk factor in the general population. This hypothesis may be tested by a non-invasive high-risk screening protocol for Fabry patients with ischaemic strokes and a variety of cardiac, and renal complications. These patients would benefit from diagnosis, appropriate treatment, follow-up and surveillance. Early detection of Fabry patients would also benefit affected relatives, many of whom do not have a clear diagnosis of their clinical condition.

Gene-Specific Effects of the Histone Deacetylase Inhibitor TSA on Activation of a Pro-Inflammatory Endothelial Cell Phenotype by the Major Phagocyte Peroxidase-Derived Oxidant HOSCN.

Thiocyanate (SCN−) is, unexpectedly, the principal physiologic substrate for eosinophil peroxidase (EPO) and a major (i.e., accounting for 50% of H2O2 consumed) substrate for myeloperoxidase (MPO). The product of these reactions is HOSCN, a weak, exclusively sulfhydryl-reactive oxidant that we have previously shown to be a uniquely potent (up to 100-fold) oxidant transcriptional inducer of human umbilical vein endothelial cell (HUVEC) tissue factor (TF), ICAM-1, E-selectin, and VCAM-1 expression via a mechanism dependent upon NF-κB p65/p50 activation. Histone deacetylase inhibitors (HDACi) have recently been found capable of gene-specific transcriptional regulation by acetylating lysine residues on transcription factors, including p65. Because of previous reports of beneficial effects of the HDACi trichostatin (TSA) in vivo in murine models of SLE and asthma inflammation, we hypothesized that TSA might exert antiinflammatory effects by downregulating endothelial expression of proinflammatory mediators induced by physiologic agonists such as HOSCN. We analyzed the effects of HOSCN and TSA on the HUVEC transcriptome by incubating HUVEC monolayers (n=3, single donor-derived preparations) 4 hours in M199 medium containing 10% FCS supplemented with buffer control, 150 μM HOSCN, 100 nM TSA, or HOSCN + TSA prior to total RNA extraction and analysis using Affymetrix® U133 2.0 Plus Microarray Chips. HOSCN significantly (i.e., > 2-fold) upregulated 0.5% of HUVEC genes, but most strikingly stimulated (10–100 fold) the adhesion molecules VCAM-1, ICAM-1, and E-selectin, chemokines IL-8, MCP-1, CXCL-1 and CXCL2 and COX2, all NF-kB – regulated genes. TSA suppressed 0.03% of genes in HOSCN-treated HUVEC, including, notably, VCAM-1 (but not ICAM-1), IL-8 and CCL2, and COX2. Pursuing the discordant effects of TSA upon HOSCN-stimulated VCAM-1 and ICAM-1 mRNA levels, we confirmed by qRT-PCR that HOSCN increases VCAM-1 mRNA 10–16x at 3–4 h and TSA inhibits this by 85%. In contrast, ICAM-1 mRNA increases 7x in response to HOSCN but is stimulated another 4-fold by TSA. We assessed HUVEC expression of VCAM-1 and ICAM-1 protein by western blot after a 4h exposure to 150 μM HOSCN and found them upregulated 10- and 5-fold, respectively. TSA (ED50 30 nM) suppressed HOSCN-mediated VCAM-1 expression by >90% but increased expression of ICAM-1 2–3x. EMSA and anti-p50 and anti-p65 supershift confirmed HOSCN activation of p65/p50 binding to VCAM-1 and ICAM-1 sequence-derived NF-kB motif oligo probes but, paradoxically, TSA inhibited p65/p50 binding to both VCAM-1 and ICAM-1 probes. In direct contrast, chromatin immunoprecipitation using anti-p65 showed that TSA decreased HOSCN-induced p65 binding to the endogenous HUVEC genomic DNA VCAM-1 NF-kB binding site but did not diminish its binding to the ICAM-1 site. In a static adhesion assay human eosinophils bound to HUVEC exposed 4h to 150 μM HOSCN increased to 4x baseline and 1 μM TSA completely blocked this increase whereas a blocking anti VCAM-1 antibody diminished it by 50%. We conclude that the HDACi TSA is a potent and relatively specific inhibitor of several NF-kB-dependent pro-inflammatory genes, but not ICAM-1, in HUVEC activated by the physiologic oxidant agonist HOSCN. We hypothesize that differential TSA regulation of VCAM-1 and ICAM-1 may be attributable to variations in the nucleotide sequence of their NF-kB-binding motifs or, alternatively, differential recruitment of NFkB transcriptional cofactors such as p300/CBP. We propose that HDACi have significant therapeutic potential as anti-inflammatory agents, particularly in those disease states most dependent upon VCAM-1.

Mucosal Glycan Foraging Enhances Fitness and Transmission of a Saccharolytic Human Gut Bacterial Symbiont

The distal human gut is a microbial bioreactor that digests complex carbohydrates. The strategies evolved by gut microbes to sense and process diverse glycans have important implications for the assembly and operation of this ecosystem. The human gut-derived bacterium Bacteroides thetaiotaomicron forages on both host and dietary glycans. Its ability to target these substrates resides in 88 polysaccharide utilization loci (PULs), encompassing 18% of its genome. Whole genome transcriptional profiling and genetic tests were used to define the mechanisms underlying host glycan foraging in vivo and in vitro. PULs that target all major classes of host glycans were identified. However, mucin O-glycans are the principal host substrate foraged in vivo. Simultaneous deletion of five genes encoding ECF-sigma transcription factors, which activate mucin O-glycan utilization, produces defects in bacterial persistence in the gut and in mother-to-offspring transmission. Thus, PUL-mediated glycan catabolism is an important component in gut colonization and may impact microbiota ecology.

Thematic review series: Skin Lipids. Pathogenesis of permeability barrier abnormalities in the ichthyoses: inherited disorders of lipid metabolism

Many of the ichthyoses are associated with inherited disorders of lipid metabolism. These disorders have provided unique models to dissect physiologic processes in normal epidermis and the pathophysiology of more common scaling conditions. In most of these disorders, a permeability barrier abnormality "drives" pathophysiology through stimulation of epidermal hyperplasia. Among primary abnormalities of nonpolar lipid metabolism, triglyceride accumulation in neutral lipid storage disease as a result of a lipase mutation provokes a barrier abnormality via lamellar/nonlamellar phase separation within the extracellular matrix of the stratum corneum (SC). Similar mechanisms account for the barrier abnormalities (and subsequent ichthyosis) in inherited disorders of polar lipid metabolism. For example, in recessive X-linked ichthyosis (RXLI), cholesterol sulfate (CSO(4)) accumulation also produces a permeability barrier defect through lamellar/nonlamellar phase separation. However, in RXLI, the desquamation abnormality is in part attributable to the plurifunctional roles of CSO(4) as a regulator of both epidermal differentiation and corneodesmosome degradation. Phase separation also occurs in type II Gaucher disease (GD; from accumulation of glucosylceramides as a result of to beta-glucocerebrosidase deficiency). Finally, failure to assemble both lipids and desquamatory enzymes into nascent epidermal lamellar bodies (LBs) accounts for both the permeability barrier and desquamation abnormalities in Harlequin ichthyosis (HI). The barrier abnormality provokes the clinical phenotype in these disorders not only by stimulating epidermal proliferation, but also by inducing inflammation.

New directions for carbon-based detectors: exploiting the versatility of carbon substrates in electroanalysis

The different approaches that have been taken in the development of electrochemical detectors incorporating carbon as the principal electrode substrate are reviewed. The multitude of forms that carbon can take provides an extremely versatile platform upon which to enhance the detector performance. The different approaches that have been taken in recent years have been critically appraised and the emergence of new composite technologies is highlighted. The role of carbon and the applications to which it has been employed are presented and compared and contrasted within the remit of electroanalytical investigations.

Suplementação enteral e parenteral com glutamina em neonatos pré-termo e com baixo peso ao nascer

A glutamina é o aminoácido livre mais abundante no sangue e no músculo esquelético, bem como é o principal substrato energético para células de elevado turnover, como enterócitos e leucócitos. Adicionalmente, a glutamina representa o principal aminoácido transferido para o feto pela placenta e, juntamente com o glutamato, constituem os aminoácidos mais abundantes no leite materno. Todavia, bebês nascidos prematuramente sofrem interrupção abrupta do fornecimento placentário de glutamina, o que acarreta em dependência exclusiva da síntese endógena ou do fornecimento exógeno deste aminoácido. Aliado a isso, neonatos pré-termo (PT) e com baixo peso ao nascer (BPN), freqüentemente, recebem apenas nutrição parenteral total nas primeiras semanas de vida, a qual não contém glutamina. Cabe ainda destacar que esses bebês possuem pouca massa muscular e, portanto, seus estoques de glutamina são limitados. Uma vez que neonatos PT e com BPN estão sujeitos a intenso crescimento e a inúmeros estresses fisiológicos, é possível que a glutamina seja um nutriente condicionalmente essencial nessa fase da vida, fato que estimulou a realização de estudos com a finalidade de avaliar os possíveis benefícios clínicos da suplementação enteral e parenteral com glutamina em neonatos PT e com BPN.

Two Distinct Pathways for Cyclooxygenase-2 Protein Degradation

Cyclooxygenases (COX-1 and COX-2) are N-glycosylated, endoplasmic reticulum-resident, integral membrane proteins that catalyze the committed step in prostanoid synthesis. COX-1 is constitutively expressed in many types of cells, whereas COX-2 is usually expressed inducibly and transiently. The control of COX-2 protein expression occurs at several levels, and overexpression of COX-2 is associated with pathologies such as colon cancer. Here we have investigated COX-2 protein degradation and demonstrate that it can occur through two independent pathways. One pathway is initiated by post-translational N-glycosylation at Asn-594. The N-glycosyl group is then processed, and the protein is translocated to the cytoplasm, where it undergoes proteasomal degradation. We provide evidence from site-directed mutagenesis that a 27-amino acid instability motif (27-IM) regulates posttranslational N-glycosylation of Asn-594. This motif begins with Glu-586 8 residues upstream of the N-glycosylation site and ends with Lys-612 near the C terminus at Leu-618. Key elements of the 27-IM include a helix involving residues Glu-586 to Ser-596 with Asn-594 near the end of this helix and residues Leu-610 and Leu-611, which are located in an apparently unstructured downstream region of the 27-IM. The last 16 residues of the 27-IM, including Leu-610 and Leu-611, appear to promote N-glycosylation of Asn-594 perhaps by causing this residue to become exposed to appropriate glycosyl transferases. A second pathway for COX-2 protein degradation is initiated by substrate-dependent suicide inactivation. Suicide-inactivated protein is then degraded. The biochemical steps have not been resolved, but substrate-dependent degradation is not inhibited by proteasome inhibitors or inhibitors of lysosomal proteases. The pathway involving the 27-IM occurs at a constant rate, whereas degradation through the substrate-dependent process is coupled to the rate of substrate turnover.

Limited Mitochondrial Permeabilization Is an Early Manifestation of Palmitate-induced Lipotoxicity in Pancreatic β-Cells

Involvement of the mitochondrial permeability transition (MPT) pore in early stages of lipotoxic stress in the pancreatic beta-cell lines MIN6 and INS-1 was the focus of this study. Both long term (indirect) and acute (direct) effects of fatty acid (FA) application on beta-cell susceptibility to Ca(2+)-induced MPT induction were examined using both permeabilized and intact beta-cells. Long term exposure to moderate (i.e. below cytotoxic) levels of the saturated FA palmitate sensitized beta-cell mitochondria to MPT induced by Ca(2+). Long term exposure to palmitate was significantly a more efficient inducer of MPT than the unsaturated FA oleate, although upon acute application both caused similar MPT activation. Application of antioxidants, inhibitors of the ceramide pathway, or modifiers of membrane fluidity did not protect beta-cell mitochondria from FA exposure. However, significant protection was provided by co-application of the unsaturated FA oleate in a phosphatidylinositol 3-kinase-dependent manner. Characterization of MPT pore opening in response to moderate palmitate treatment revealed the opening of a unique form of MPT in beta-cells as it encompassed features of both low and high conductance MPT states. Specifically, this MPT showed solute selectivity, characteristic of a low conductance MPT; however, it affected mitochondrial respiration and membrane potential in a way typical of a high conductance MPT. Activation of the full-size/high conductance form of MPT required application of high levels of FA that reduced growth and initiated apoptosis. These findings suggest that in the beta-cell, MPTs can act as both initiators of cell death and as versatile modulators of cell metabolism, depending on the mode of the MPT pore induced.

Increased glycogen stores due to γ-AMPK overexpression protects against ischemia and reperfusion damage

During ischemia, endogenous glycogen becomes the principal substrate for energy through glycolysis. Cardiac-specific manipulation of AMP-activated protein kinase (AMPK) by over-expression of its regulatory γ-subunit induces glycogen storage. The aim of this study was to examine whether heart glycogen in transgenic mice overexpressing PRKAG2 may protect from ischemia and reperfusion injury. Isolated hearts were mounted on Langendorff apparatus and subjected to 30min ‘no-flow’ or ‘low-flow’ ischemia and 60min reperfusion. Hemodynamic measurements, tetrazolium staining, glycogen and lactate were used to monitor ischemia reperfusion damage. After low-flow ischemia, left ventricular pressure, coronary flow (CF) and the area of viable myocardium were 20–30% higher in PRKAG2 mice compared to controls. The basal levels of glycogen in PRKAG2 were 9.2μg/g, markedly higher than in controls, but after low-flow ischemia they declined concomitantly with increased lactate washout in the coronary effluent. During no-flow ischemia there was neither protection nor consumption of glycogen in PRKAG2 hearts. Cardioprotection was also eliminated when PRKAG2 hearts were depleted of glycogen prior to low-flow ischemia. AMPK α Thr172 phosphorylation did not differ between PRKAG2 hearts and controls either during low-flow ischemia or reperfusion. We conclude that PRKAG2 hearts resist low-flow ischemia injury better than controls. Improved recovery was associated with increased consumption of glycogen, and was unrelated to AMPK activation. These findings demonstrate the potential of heart protection from ischemia and reperfusion injury through metabolic manipulation increasing the level and utilization of myocardial glycogen.