Decrease In Binding Capacity Research Articles

Mutation analysis of SUOX in isolated sulfite oxidase deficiency with ectopia lentis as the presenting feature: insights into genotype–phenotype correlation

BackgroundIsolated sulfite oxidase deficiency (ISOD) caused by sulfite oxidase gene (SUOX) mutations is a rare neurometabolic disease associated with ectopia lentis (EL). However, few genotype–phenotype correlations have been established yet.MethodsPotentially pathogenic SUOX mutations were screened from a Chinese cohort of congenital EL using panel-based next-generation sequencing and analyzed with multiple bioinformatics tools. The genotype–phenotype correlations were evaluated via a systematic review of SUOX mutations within our data and from the literature.ResultsA novel paternal missense mutation, c.205G > C (p.A69P), and a recurrent maternal nonsense mutation, c.1200 C > G (p.Y400*), of SUOX were identified in a 4-year-old boy from 312 probands. The biochemical assays manifested elevated urine sulfite and S-sulfocysteine accompanied by decreased homocysteine in the blood. The patient had bilateral EL and normal fundus, yet minimal neurological involvement and normal brain structure. Molecular modeling simulation revealed the p.A69P mutant had an unstable structure but an unchanged affinity for sulfite, while the truncated p.Y400* mutant showed decreased binding capacity. Genotype–phenotype analysis demonstrated patients with biallelic missense mutations had milder symptoms (P = 0.023), later age of onset (P < 0.001), and a higher incidence of regression (P = 0.017) than other genotypes. No correlations were found regarding EL and other neurological symptoms.ConclusionThe data from this study not only enrich the known mutation spectrum of SUOX but also suggest that missense mutations are associated with mild and atypical symptoms.

The identification and function of a Netrin-1 mutation in a pedigree with premature atherosclerosis

Background and aimsNeuroimmune guidance cues have been shown to play a role in atherosclerosis, but their exact role in human pathophysiology is largely unknown. In the current study, we investigated the role of a c.1769G > T variant in Netrin-1 in (premature) atherosclerosis. MethodsTo determine the effect of the genetic variation, purified Netrin-1, either wild type (wtNetrin-1) or the patient observed variation (mutNetrin-1), was used for migration, adhesion, endothelial barrier function and bindings assays. Expression of adhesion molecules and transcription proteins was analyzed by RT-PCR, Western blot or ELISA. To further delineate how mutNetrin-1 mediates its effect on cell migration, lenti-viral knockdown of UNC5B or DCC was used. ResultsBindings assays revealed a decreased binding capacity of mutNetrin-1 to the receptors UNC5B, DCC and β3-integrin and an increased binding capacity to neogenin, heparin and heparan sulfate compared to wtNetrin-1. Exposure of endothelial cells to mutNetrin-1 resulted in enhanced monocyte adhesion and expression of IL-6, CCL2 and ICAM-1 compared to wtNetrin-1. In addition, mutNetrin-1 lacks the inhibitory effect on the NF-κB pathway that is observed for wtNetrin-1. Moreover, the presence of mutNetrin-1 diminished migration of macrophages and smooth muscle cells. Importantly, UNC5B or DCC specific knockdown showed that mutNetrin-1 is unable to act through DCC resulting in enhanced inhibition of migration. ConclusionsOur data demonstrates that mutNetrin-1 fails to exert anti-inflammatory effects on endothelial cells and more strongly blocks macrophage migration compared to wtNetrin-1, suggesting that the carriers of this genetic molecular variant may well be at risk for premature atherosclerosis.

SAT-122 Guanidinylations of albumin decreased binding capacity of hydrophobic metabolites

Since post-translational modifications of proteins may have an impact on the pathogenesis of diseases like atherosclerosis, diabetes mellitus and chronic kidney disease (CKD), post-translational modifications are currently gaining increasing interest. In this study, a comprehensive method for analysis

Structural Features on the Substrate-Binding Surface of Fungal Lytic Polysaccharide Monooxygenases Determine Their Oxidative Regioselectivity.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave many of nature's most recalcitrant polysaccharides by acting on the C1- and/or C4-carbon of the glycosidic bond. Here, the results of an extensive mutagenesis study on three LPMO representatives, Phanerochaete chrysosporium LPMO9D (C1-oxidizer), Neurospora crassa LPMO9C (C4), and Hypocrea jecorina LPMO9A (C1/C4), are reported. Using a previously published indicator diagram, the authors demonstrate that several structural determinants of LPMOs play an important role in their oxidative regioselectivity. N-glycan removal and alterations of the aromatic residues on the substrate-binding surface are shown to alter C1/C4-oxidation ratios. Removing the carbohydrate binding module (CBM) is found not to alter the regioselectivity of HjLPMO9A, although the effect of mutational changes is shown to increase in a CBM-free context. The accessibility to the solvent-exposed axial position of the copper-site reveales not to be a major regioselectivity indicator, at least not in PcLPMO9D. Interestingly, a HjLPMO9A variant lacking two surface exposed aromatic residues combines decreased binding capacity with a 22% increase in synergetic efficiency. Similarly to recent LPMO10 findings, our results suggest a complex matrix of surface-interactions that enables LPMO9s not only to bind their substrate, but also to accurately direct their oxidative force.

The effect of microwave on the interaction of flavour compounds with G‐actin from grass carp (Catenopharyngodon idella)

In order to investigate the influence of non-thermal effects of microwaves on the flavour of fish and meat products, the G-actin of grass carp in ice baths was exposed to different microwave powers (0, 100, 300 or 500 W); the surface hydrophobicity, sulfhydryl contents, secondary structures and adsorption capacity of G-actin to ketones were determined. As microwave power increased from 0 to 300 W, the surface hydrophobicity, total and reactive sulfhydryls increased; α-helix, β-sheet and random coil fractions turned into β-turn fractions. As microwave power increased from 300 to 500 W, however, hydrophobicity and sulfhydryl contents decreased; β-turn and random coil fractions turned into α-helix and β-sheet fractions. The tendencies of adsorbed capacity of ketones were similar to hydrophobicity and sulfhydryl contents. The increased adsorbing of ketones could be attributed to the unfolding of secondary structures by revealing new binding sites, including thiol groups and hydrophobic binding sites. The decreased binding capacity was related to the refolding and aggregation of protein. The results suggested that microwave powers had obvious effects on the flavour retention and proteins structures in muscle foods. © 2017 Society of Chemical Industry.

MP07-18 IMPACTS OF SMOKING ON THE GLYCOCALYX OF HUMAN SPERMATOZOA

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology I1 Apr 2017MP07-18 IMPACTS OF SMOKING ON THE GLYCOCALYX OF HUMAN SPERMATOZOA Rick Paschold, Susanne Bour, Armin Becker, Christian Stief, and Matthias Trottmann Rick PascholdRick Paschold More articles by this author , Susanne BourSusanne Bour More articles by this author , Armin BeckerArmin Becker More articles by this author , Christian StiefChristian Stief More articles by this author , and Matthias TrottmannMatthias Trottmann More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.284AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES About 10-15% of all infertility patients are diagnosed with idiopathic infertility. Particularly, long term smokers often suffer from a reduction of basic sperm parameters (Ramlau-Hansen, 2007). The effects of smoking on male fertility are still discussed controversially. It was shown, that the essential binding between sperm and oviduct is based on a lectine-carbohydrate interaction (Koelle, 2012). A functional reduction of fertility could occur due to a lack of binding capacity. Therefore, our group characterized the proteins on glycocalyx of human spermatozoa that are capable to bind sugar residues. Further it was evaluated, if smokers show a restricted sugar-binding ability compared to non-smokers. METHODS We separated two study populations (smokers, non-smokers) out of 78 fresh human ejaculate samples. A direct staining with Mitotracker DeepRed, NucBlue (DAPI) and FITC-conjugated sugar residues (sialic acid- (SA), mannose- (MA) and fucose- (FU)) was performed. We used confocal microscopy to examine the fluorescence-marked samples. The fluorescent cells were analysed quantitatively and qualitatively within the study populations.Additionally, we extracted the sperm's proteins from smokers and control group which we applied on SDS-Page (4-12.5 %). Western Blot was used to proof the presence of LMAN2 and CatSper1 proteins on the plasma membrane surface. RESULTS We located proteins at the middle part of the spermatozoa's head that are capable of binding sugar residues.The ratio of fluorescence-labelled cells to the total cell count, which correspond to the capacity of binding sugar residues, was measured. We showed a significant difference between the groups: For smokers, we counted a proportion of 0.07±0.006 (SA), 0.05±0.006 (MA), 0.05±0.005 (FU) compared to 0.15±0.01 (SA), 0.20±0.02 (MA), 0.19±0.02 (FU) for the non-smokers (p<0.05). Fluorescence intensity did not vary significantly between the groups.Protein candidates were found in Western Blot but first experiments did not show a significant difference in the amount between smokers and non-smokers. CONCLUSIONS Our results point out, that smoking could possibly lead to a reduction of sugar binding proteins on the human sperm glycocalyx. This could be a reason for a decreased binding capacity of sperms to the female reproductive tract which could lead to a reduced fertility potential of smokers. Further work is necessary to lighten the exact molecular interaction between spermatozoa and female reproductive tract. Based on these facts it might be possible to examine new diagnostic and therapeutic approach in the future. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e88-e89 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Rick Paschold More articles by this author Susanne Bour More articles by this author Armin Becker More articles by this author Christian Stief More articles by this author Matthias Trottmann More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Guanidinylations of albumin decreased binding capacity of hydrophobic metabolites.

As post-translational modifications of proteins may have an impact on the pathogenesis of diseases such as atherosclerosis, diabetes mellitus and chronic kidney disease (CKD), post-translational modifications are currently gaining increasing interest. In this study, a comprehensive method for analysis of these post-translational modifications is established for the clinical diagnostic routine. Here, we analysed albumin - the most abundant plasma protein in human - isolated from patients with CKD and healthy controls by chromatographic steps and identified by MALDI mass spectrometry. Post-translational modifications of albumin were identified after digestion by analysing mass signal shifts of albumin peptides using pertinent mass databases. Albumin isolated from plasma of patients with CKD but not from healthy control subjects was specifically post-translationally modified by guanidinylation of lysines at positions 249, 468, 548, 565 and 588. After identification of guanidinylations as post-translational modifications of albumin isolated from patients with CKD, these modifications were quantified by mass spectrometry demonstrating a significant increase in the corresponding mass signal intensities in patients with CKD compared to healthy controls. The relative amount of guanidinylation of lysine at position 468 in patients with CKD was determined as 63 ± 32% (N = 3). Subsequently, we characterized the pathophysiological impact of the post-translational guanidinylation on the binding capacity of albumin for representative hydrophobic metabolic waste products. In vitro guanidinylation of albumin from healthy control subjects caused a decreased binding capacity of albumin in a time-dependent manner. Binding of indoxyl sulphate (protein-bound fraction) decreased from 82 ± 1% of not post-translationally modified albumin to 56 ± 1% after in vitro guanidinylation (P < 0.01), whereas the binding of tryptophan decreased from 20 to 4%. These results are in accordance with the binding of indoxyl sulphate to albumin from healthy control subjects and patients with CKD (88 ± 3 vs. 74 ± 10, P < 0.01). Thus, in vitro post-translational guanidinylation of albumin had a direct effect on the binding capacity of hydrophobic metabolites such as indoxyl sulphate and tryptophan. We used a mass spectrometry-based method for the characterization of post-translational modification and demonstrated the pathophysiological impact of a representative post-translational modification of plasma albumin. The data described in this study may help to elucidate the pathophysiological role of protein modifications.

Characterization of little skate (Leucoraja erinacea) recombinant transthyretin: Zinc-dependent 3,3′,5-triiodo-l-thyronine binding

Transthyretin (TTR) diverged from an ancestral 5-hydroxyisourate hydrolase (HIUHase) by gene duplication at some early stage of chordate evolution. To clarify how TTR had participated in the thyroid system as an extracellular thyroid hormone (TH) binding protein, TH binding properties of recombinant little skate Leucoraja erinacea TTR was investigated. At the amino acid level, skate TTR showed 37–46% identities with the other vertebrate TTRs. Because the skate TTR had a unique histidine-rich segment in the N-terminal region, it could be purified by Ni-affinity chromatography. The skate TTR was a 46-kDa homotetramer of 14.5kDa subunits, and had one order of magnitude higher affinity for 3,3′,5-triiodo-l-thyronine (T3) and some halogenated phenols than for l-thyroxine. However, the skate TTR had no HIUHase activity. Ethylenediaminetetraacetic acid (EDTA) treatment inhibited [125I]T3 binding activity whereas the addition of Zn2+ to the EDTA-treated TTR recovered [125I]T3 binding activity in a Zn2+ concentration-dependent manner. Scatchard analysis revealed the presence of two classes of binding site for T3, with dissociation constants of 0.24 and 17nM. However, the high-affinity sites were completely abolished with 1mM EDTA, whereas the remaining low-affinity sites decreased binding capacity. The number of zinc per TTR was quantified to be 4.5–6.3. Our results suggest that skate TTR has tight Zn2+-binding sites, which are essential for T3 binding to at least the high-affinity sites. Zn2+ binding to the N-terminal histidine-rich segment may play an important role in acquisition or reinforcement of TH binding ability during early evolution of TTR.

A chitin-like component on sclerotic cells of Fonsecaea pedrosoi inhibits Dectin-1-mediated murine Th17 development by masking β-glucans.

Fonsecaea pedrosoi (F. pedrosoi), a major agent of chromoblastomycosis, has been shown to be recognized primarily by C-type lectin receptors (CLRs) in a murine model of chromoblastomycosis. Specifically, the β-glucan receptor, Dectin-1, mediates Th17 development and consequent recruitment of neutrophils, and is evidenced to have the capacity to bind to saprophytic hyphae of F. pedrosoi in vitro. However, when embedded in tissue, most etiological agents of chromoblastomycosis including F. pedrosoi will transform into the sclerotic cells, which are linked to the greatest survival of melanized fungi in tissue. In this study, using immunocompetent and athymic (nu/nu) murine models infected subcutaneously or intraperitoneally with F. pedrosoi, we demonstrated that T lymphocytes play an active role in the resolution of localized footpad infection, and there existed a significantly decreased expression of Th17-defining transcription factor Rorγt and inefficient recruitment of neutrophils in chronically infected spleen where the inoculated mycelium of F. pedrosoi transformed into the sclerotic cells. We also found that Dectin-1-expressing histocytes and neutrophils participated in the enclosure of transformed sclerotic cells in the infectious foci. Furthermore, we induced the formation of sclerotic cells in vitro, and evidenced a significantly decreased binding capacity of human or murine-derived Dectin-1 to the induced sclerotic cells in comparison with the saprophytic mycelial forms. Our analysis of β-glucans-masking components revealed that it is a chitin-like component, but not the mannose moiety on the sclerotic cells, that interferes with the binding of β-glucans by human or murine Dectin-1. Notably, we demonstrated that although Dectin-1 contributed to the development of IL-17A-producing CD3+CD4+ murine splenocytes upon in vitro-stimulation by saprophytic F. pedrosoi, the masking effect of chitin components partly inhibited Dectin-1-mediated Th17 development upon in vitro-stimulation by induced sclerotic cells. Therefore, these findings extend our understanding of the chronicity of chromoblastomycosis.

Antibiotic stress-induced modulation of the endoribonucleolytic activity of RNase III and RNase G confers resistance to aminoglycoside antibiotics in Escherichia coli

Here, we report a resistance mechanism that is induced through the modulation of 16S ribosomal RNA (rRNA) processing on the exposure of Escherichia coli cells to aminoglycoside antibiotics. We observed decreased expression levels of RNase G associated with increased RNase III activity on rng mRNA in a subgroup of E. coli isolates that transiently acquired resistance to low levels of kanamycin or streptomycin. Analyses of 16S rRNA from the aminoglycoside-resistant E. coli cells, in addition to mutagenesis studies, demonstrated that the accumulation of 16S rRNA precursors containing 3–8 extra nucleotides at the 5’ terminus, which results from incomplete processing by RNase G, is responsible for the observed aminoglycoside resistance. Chemical protection, mass spectrometry analysis and cell-free translation assays revealed that the ribosomes from rng-deleted E. coli have decreased binding capacity for, and diminished sensitivity to, streptomycin and neomycin, compared with wild-type cells. It was observed that the deletion of rng had similar effects in Salmonella enterica serovar Typhimurium strain SL1344. Our findings suggest that modulation of the endoribonucleolytic activity of RNase III and RNase G constitutes a previously uncharacterized regulatory pathway for adaptive resistance in E. coli and related gram-negative bacteria to aminoglycoside antibiotics.

Decreased binding capacity (B max) of muscarinic acetylcholine receptors in fibroblasts from boys with attention-deficit/hyperactivity disorder (ADHD)

Monoaminergic dysregulation is implicated in attention-deficit/hyperactivity disorder (ADHD), and methylphenidate and amphetamines are the most frequently prescribed pharmacological agents for treating ADHD. However, it has recently been proposed that the core symptoms of the disorder might be due to an imbalance between monoaminergic and cholinergic systems. In this study, we used fibroblast cell homogenates from boys with and without ADHD as an extraneural cell model to examine the cholinergic receptor density, that is, muscarinic acetylcholine receptors (mAChRs). We found that the binding capacity (B max) of [3H] Quinuclidinyl benzilate (3H-QNB) to mAChRs was decreased by almost 50 % in the children with ADHD (mean = 30.6 fmol/mg protein, SD = 25.6) in comparison with controls [mean = 63.1 fmol/mg protein, SD = 20.5, p ≤ 0.01 (Student’s unpaired t test)]. The decreased B max indicates a reduced cholinergic receptor density, which might constitute a biomarker for ADHD. However, these preliminary findings need to be replicated in larger ADHD and comparison cohorts.

Effect of oxidative stress and endotoxin on human serum albumin in brain-dead organ donors

Albumin, among other molecules, binds and detoxifies endotoxin in healthy people. Oxidative stress leads to protein oxidation and thus to the impaired binding properties of albumin. This property, in combination with increased gut permeability, leads to the appearance of endotoxin in the systemic circulation and to impaired organ function. We hypothesize that these processes occur in the serum of brain-dead organ donors. Endotoxin was determined with an adapted Limulus amoebocyte lysate assay. The albumin fractions and binding capacity were determined by high-performance liquid chromatography (HPLC). FlowCytomix (eBioscience, San Diego, Calif) was used to determine the cytokine levels. Carbonylated proteins (CPs) and myeloperoxidase (MPO) were measured by an enzyme-linked immunosorbent assay (ELISA). Eighty-four brain-dead organ donors were enrolled and categorized by the duration of intensive care unit (ICU) stay. The albumin-binding capacity for dansylsarcosine was reduced in brain-dead patients compared with controls. Endotoxin positivity in 16.7% of donors was associated with decreased binding capacity in donors and worse survival of recipients. The CP and MPO levels of organ donors were significantly higher than in healthy controls. The durations of ICU stay increased albumin oxidation. In addition, interleukin-6 (IL-6), IL-8, IL-10, and IL-1β levels were increased in patients, whereas the interferon-γ (IFN-γ) levels were within the normal range. We conclude that oxidative stress and systemic endotoxemia are present in brain-dead organ donors, which might affect recipient survival. High endotoxin levels might be caused by increased gut permeability and decreased binding capacity of albumin influenced not just by higher albumin oxidation.

Oxidative Modification of Fibrinogen Affects Its Binding Activity to Glycoprotein (GP) IIb/IIIa

Proteins are sensitive biomarkers of human disease condition associated with oxidative stress. Alteration of protein structures by oxidants may result in partial or complete loss of protein functions. We have investigated the effect of structural modifications induced by metal ion catalyzed oxidation of fibrinogen on its binding capacity to glycoprotein IIb/IIIa (GpIIb/IIIa) and human platelets. We identified and quantified of binding capacity of native and oxidized fibrinogen to its receptor in vitro by flow cytometer. Dityrosine formation on oxidized fibrinogen were detected spectrophotometrically. Elevated degradation products of fibrinogen after oxidation were revealed in the HPLC analysis. The native and oxidized fibrinogen were analyzed on mass spectrum upon digestion with trypsin. Oxidatively modified fibrinogen showed less binding activity than native fibrinogen to GpIIb/IIIa coated micro beads and human platelets whereas slightly higher binding capacity to ADP induced stimulated platelets. Formation of di-tyrosines in the amino acid side chains of fibrinogen were observed upon oxidation. Decreased binding capacity of oxidized fibrinogen correlated with intensities of dityrosine formation. Oxidized fibrinogen had more ion-mass intensities at higher than native fibrinogen. Important point is decreased of binding capacity of the oxidized fibrinogen to own receptor. The decreased rate of binding, leading to effect in the diseases of clot formation may account for the association between oxidation of fibrinogen and the incidence of effect in human diseases.

Impaired cytoprotective function of muscle in human gallbladders with cholesterol stones.

Acute cholecystitis develops in gallbladders (GB) with excessive bile cholesterol (Ch). Increased membrane Ch content affects membrane function and may affect PGE(2) receptors involved in the cytoprotection against acute inflammation. This study was aimed at determining whether the cytoprotective response to PGE(2) is affected by lithogenic bile with Ch. Muscle cells from human GB with cholesterol stones (ChS) or pigment stones (PS) were obtained by enzymatic digestion. PGE(2) levels were measured by radioimmunoassay, and activities of superoxide dismutase (SOD) and catalase were assayed by spectrophotometry. The contraction in response to H(2)O(2) in muscle cells from PS was 14 +/- 0.3%, not different from normal controls, and decreased after the cells were incubated with Ch-rich liposomes (P < 0.05), which increase the Ch content in the plasma membranes. In muscle cells from GB with ChS, H(2)O(2)-induced contraction was only 9.2 +/- 1.3% and increased to 14 +/- 0.2% after Ch-free liposome treatment to remove Ch from the plasma membranes (P < 0.01). H(2)O(2) caused a similar increase in the levels of lipid peroxidation and PGE(2) content in muscle cells from GBs with ChS and PS. However, the activities of SOD and catalase were significantly lower in muscle cells from GBs with ChS compared with those with PS. The binding capacity of PGE(2) receptors was also significantly lower in muscle cells from GBs with ChS compared with those with PS. In conclusion, the cytoprotective response to reactive oxygen species is reduced in muscle cells from GBs with ChS despite a normal increase in the cellular levels of PGE(2). This impaired cytoprotective response may be due to a dysfunction of PGE(2) receptors with decreased binding capacity resulting from excessive Ch levels in the plasma membrane.

Endothelial lipase-modified high-density lipoprotein exhibits diminished ability to mediate SR-BI (scavenger receptor B type I)-dependent free-cholesterol efflux.

Endothelial lipase (EL) is a phospholipase with little triacylglycerol lipase activity. To assess structural and functional properties of EL-HDL (EL-modified high-density lipoprotein), HDL was incubated with conditioned medium from Cos-7 cells infected with adenovirus encoding human EL. After re-isolation of HDL by ultracentrifugation, TLC and HPLC analyses revealed that EL-HDL was markedly depleted in phosphatidylcholine and enriched in lyso-phosphatidylcholine compared with LacZ-HDL (control HDL) incubated with conditioned medium from Cos-7 cells infected with adenovirus encoding beta-galactosidase. The EL-HDL was enriched in non-esterified fatty acids and, as revealed by lipid electrophoresis, was more negatively charged than control HDL. The HDL particle size as well as the total cholesterol, free cholesterol and triacylglycerol content of HDL were not significantly altered after EL modification. The ability of EL-HDL to mediate 3H-cholesterol efflux from SR-BI (scavenger receptor B type I) overexpressing Chinese-hamster ovary cells was impaired and markedly lower compared with LacZ-HDL at HDL concentrations of 100 microg/ml and above. Studies with 125I-labelled HDL showed almost unaltered binding affinity (K(m) values) and a slightly but significantly decreased binding capacity (B(max) values) of EL-HDL to SR-BI, compared with LacZ-HDL. The ATP-binding-cassette transporter A1-dependent cholesterol and phospholipid effluxes were not affected by EL modification. From these results, we concluded that EL modification alters chemical composition and physical properties of HDL, resulting in its decreased binding capacity to SR-BI and a diminished ability to mediate SR-BI-dependent cholesterol efflux.

Insulin-like growth factor-II delays early but enhances late regeneration of skeletal muscle.

This study tested whether administration of insulin-like growth factor-II (IGF-II) enhances muscle regeneration. Rat biceps femoris muscle was damaged with notexin and then IGF-II was administered for up to 7 days. Results show that the proportion of nuclei containing or surrounded by immunoreactivity to MyoD, myogenin, and developmental myosin heavy chain (dMHC) is less in the IGF-II treatment group relative to the control group on days 1 (p=0.057), 2 (p=0.034), and 3 (p=0.047), respectively. This indicates a delay in muscle precursor cell (MPC) proliferation and differentiation with IGF-II administration. This effect was not associated with decreased binding capacity of the type 1 IGF receptor, as determined by receptor autoradiography in day 1 muscle sections (NS), but was associated with inhibition of phagocytic processes. The cross-sectional area of regenerating muscle fibers was significantly greater in the IGF-II treatment group than in the control group by day 7 (p=0.0092). The enhancing effect of IGF-II on late muscle regeneration, when the main process taking place is fiber enlargement, coincides with the period in which IGF-II is normally expressed by regenerating muscle, indicating that greater endogenous production of IGF-II would be associated with improved regeneration.

Lipopolysaccharide—cell interaction and induced cellular activation in whole blood of septic patients

We used biotinylatedLPS (LPSb) and flow cytometry to study LPS—monocyte interaction and LPS-induced cellular activation in whole blood from septic patients (SP). Expression of surface activation markers was evaluated on monocytes (HLA-DR) and T lymphocytes (CD69 and CD95), and intracellular TNF-α on monocytes. Saturating curve and kinetics of LPSb detection on monocytes were similar in SP and healthy volunteers (HV). LPSb bound to monocytes was detected after 5 min of incubation in both groups, with a more pronounced decay in SP. Monocytes from SP had a lower expression of HLA-DR as compared to HV, both constitutive and upon LPS stimulation. The proportion of monocytes producing TNF-α after LPS stimulus was higher in HV than SP (mean ± SD = 25.2 ± 14.2% and 2.2 ± 2.6%, respectively, P &lt; 0.001). LPS-induced CD69 on T CD8+ and CD8— lymphocytes was similar for patients and controls. Expression of CD95 on T lymphocytes was higher in SP as compared to HV on T CD8+ cells (GMFI, mean ± SD = 22.3 ± 14.6 and 8.6 ± 5.0, respectively, P = 0.01) and CD8— cells (GMFI, mean ± SD = 28.3 ± 7.7 and 14 ± 4.3 respectively, P &lt; 0.001). Thus, monocytes and lymphocytes seem to respond differently to LPS in septic patients. Monocyte hyporesponsiveness appears not to be related to a decreased binding capacity of LPS, but rather to an impaired signal transduction.

1H-NMR study of the effect of acetonitrile on the interaction of ibuprofen with human serum albumin

The effect of acetonitrile (ACN) on the low-affinity interaction between human serum albumin (HSA) and ibuprofen (IBP) was studied using 1H-NMR techniques. Both chemical shift and relaxation measurements showed the addition of ACN to the solutions decreased the binding affinity of IBP to HSA and reduced the hydrophobic interaction between them. The self-diffusion coefficients of IBP were measured as a function of the drug concentration at different ACN concentrations. The association constant, K a, for ligand–HSA complexes and the number of binding sites, n, are evaluated by the application of Langmuir isotherm. The results indicated that the value of n was about 38 without ACN, and about 26 with ACN concentration 12% (v/v%). The decreased binding capacity of IBP to HSA in the presence of ACN was mainly attributed to the competition of ACN with IBP to the low-affinity binding sites of HSA molecule.

Oxidative stress impairs nuclear proteins binding to the insulin responsive element in the GLUT4 promoter.

Substantial evidence suggests an important role for the expression of GLUT4 in adipocytes, in the pathogenesis of insulin resistance and Type II (non-insulin-dependent) diabetes mellitus. We investigated whether oxidative stress decreases GLUT4 expression by impairing DNA binding of nuclear proteins to the insulin responsive element in the GLUT4 promoter. 3T3-L1 adipocytes were exposed to micromolar H2O2 concentrations and GLUT4 expression and binding of nuclear proteins to defined DNA sequences were assessed. GLUT4 mRNA was decreased after at least 4 h exposure to H2O2, without a major change in the stability of GLUT4 transcripts. Nuclear protein extracts prepared from oxidized cells showed decreased binding to the insulin responsive element of the GLUT4 promoter but not to other DNA sequences. The direct effect of oxidation on the binding to the insulin response element was shown by the observation that in vitro oxidation of nuclear extracts with H2O2, n-ethylmaleimide or diamide decreased protein-DNA complex formation. This, and decreased binding capacity observed in nuclear extracts from oxidized cells, were partly reversible by subsequent treatment with a reducing agent. Protein binding to a consensus DNA sequence for nuclear factor 1 transcription factors was decreased 16 % by oxidation, whereas no change was observed in the protein content of several isoforms of these proteins. Oxidative stress causes decreased GLUT4 expression, associated with impaired binding of nuclear proteins to the insulin responsive element in the GLUT4 promoter.

Decreased affinity of PBP3 to methicillin in a clinical isolate of Staphylococcus epidermidis with borderline resistance to methicillin and free of the mecA gene.

Analysis of the antibiotic binding capacity of penicillin-binding proteins (PBPs) in a group of clinical isolates of Staphylococcus epidermidis suggests that the increased level of resistance to methicillin (MIC 4.0 microg/ml) in an isolate free of the mecA gene is due to the decreased binding capacity of PBP3.