Assessment of the effect of candidate anti-inflammatory treatments on the interaction between meningococci and inflammatory cellsin vitro in a whole blood model

A wide range of immunomodulating agents are now available which may be of benefit in reducing inflammatory cell activation in meningococcal sepsis. In order to facilitate selection of candidate anti-inflammatory agents for clinical trials, we have used an in vitro whole blood model to evaluate the effects on meningococcal induced neutrophil and monocyte activation, of dexamethasone, prostacyclin, pentoxifylline and a human IgM anti-lipid A monoclonal antibody (HA-1A). Known concentrations of heat and penicillin killed meningococci were added to whole blood and the time course of cellular activation was determined. Using elastase-α ₁-antitrypsin (elastase-α ₁-AT) and TNFα production as markers of neutrophil and monocyte activation respectively, plasma levels of elastase-α ₁-AT and TNFα were found to increase in a dose-dependant manner. Elastase-α ₁-AT was detected early, with most release occurring between 15–30 min whereas TNFα was detected later, between 120–180 min. Dexamethasone, prostacyclin and pentoxifylline caused a dose dependant inhibition of TNFα release but had no effect on elastase release. HA-1A had no effect on either TNFα or elastase release. This model may be useful in determining the sequence of inflammatory cell activation and in selecting candidate anti-inflammatory agents for evaluation in clinical trials.     

Investigation of some impurity profiles by (n,α) reactions

To investigate some impurity profiles by (n,α) reactions for destructive and nondestructive analysis, two experimental facilities on a horizontal channel of the well-moderated research reactor have been developed. In accordance with an α-particle registration geometry two different measurement techniques based on using a surfacebarrier Si(Au) detector and a counting ionization chamber have been used. The determination limit of boron is ofn x 10¹⁶ cm^-3 or (1 /dv 5) x 10^-5 % with a depth resolution of ±0.03 Μm in semiconductors. A solid-state nuclear track detector (SSNTD) has also been used to determine the boron surface and deep distributions in semiconductors, plants, and other materials.     

Self-hydroxylation of taurine/α-ketoglutarate dioxygenase: evidence for more than one oxygen activation mechanism

2-Aminoethanesulfonic acid (taurine)/α-ketoglutarate (αKG) dioxygenase (TauD) is a mononuclear non-heme iron enzyme that catalyzes the hydroxylation of taurine to generate sulfite and aminoacetaldehyde in the presence of O₂, αKG, and Fe(II). Fe(II)TauD complexed with αKG or succinate, the decarboxylated product of αKG, reacts with O₂ in the absence of prime substrate to generate 550- and 720-nm chromophores, respectively, that are interconvertible by the addition or removal of bound bicarbonate and have resonance Raman features characteristic of an Fe(III)–catecholate complex. Mutagenesis studies suggest that both reactions result in the self-hydroxylation of the active-site residue Tyr73, and liquid chromatography nano-spray mass spectrometry/mass spectrometry evidence corroborates this result for the succinate reaction. Furthermore, isotope-labeling resonance Raman studies demonstrate that the oxygen atom incorporated into the tyrosyl residue derives from H₂ ¹⁸O and ¹⁸O₂ for the αKG and succinate reactions, respectively, suggesting distinct mechanistic pathways. Whereas the αKG-dependent hydroxylation likely proceeds via an Fe(IV)=O intermediate that is known to be generated during substrate hydroxylation, we propose Fe(III)–OOH (or Fe(V)=O) as the oxygenating species in the succinate-dependent reaction. These results demonstrate the two oxygenating mechanisms available to enzymes with a 2-His-1-carboxylate triad, depending on whether the electron source donates one or two electrons.     

<Emphasis Type="Italic">N</Emphasis>-Methylation of <Emphasis Type="Italic">N</Emphasis><Superscript>α</Superscript>-Acetylated,Fully C<Superscript>α</Superscript>-Ethylated,Linear Peptides

“Mono-N-methyl scan” is a rational approach for the optimization of the peptide biological properties. N-Methylation of the –CONH– functionality is also a useful tool for discriminating solvent exposed from intramolecularly H-bonded secondary amide groups in peptides. We are currently extending this reaction to linear peptides based on C^α-tetrasubstituted α-amino acids. Following our study on the synthesis and conformation of the mono-N-methylated peptides from C^α-methylated residues, in this work we investigated the N-methylation reaction on homo-peptides to the pentamer level from the C^α-ethylated residue C^α,α-diethylglycine. Under the classical experimental conditions used, exclusively mono-N-methylation (on the N-terminal, acetylated residue) takes place, as unambiguously shown by mass spectrometry, 2D-NMR, and X-ray diffraction techniques. This backbone modification does not seem to involve any significant change in the peptide conformation in the crystalline state. Dedicated to the memory of Prof. Miroslav T. Leplawy (Technical University of Łodz, Poland), who performed the first synthesis of the extremely sterically demanding C^α,α-diethylglycine peptides.     

Alkaloid profile and antimicrobial activity of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis> L. alkaloid extract

Purpose of the present study was to evaluate alkaloid profile of the aerial parts of Lupinus angustifolius growing in Turkey by capillary gas chromatography-mass spectrometry (GC-MS). Fifteen alkaloids were identified by capillary GC-MS. 13α-Hydroxylupanine (50.78%) and lupanine (23.55%) were determined as the main alkaloids in the aerial parts of L. angustifolius. Ammodendrine, isoangustifoline, tetrahydrorhombifoline, angustifoline, α-isolupanine, 5,6-dehydrolupanine, 11,12-dehydrolupanine, 13α-acetoxylupanine, 13α-isovaleroyloxylupanine, 13α-valeroyloxylupanine, 13α-tigloyloxylupanine, 13α-cis-cinnamoyloxylupanine and 13α-cis-cinnamoyloxy-17-oxolupanine were identified as the minor alkaloids of the plant. Furthermore, antibacterial and antifungal activities of L. angustifolius alkaloid extract were tested against standard strains of the following bacteria; Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus as well as the fungi; Candida albicans and C. krusei. The alkaloid extract showed significant activity on B. subtilis, S. aureus and P. aeruginosa while it was weakly active on E. coli. On the other hand, the extract possessed moderate activity against C. albicans and C. krusei.     

α2A-Adrenoceptor-specific Stimulation of [35S]GTPγS Binding to Membrane Preparations of Rat Frontal Cortex

Functional activation of α_2A adrenergic receptors in the crude membranes from rat frontal cortex was studied by a [³⁵S]-guanosine 5′-O-(γ-thiotriphosphate) ([³⁵S]GTPγS) binding assay. α_2A agonists UK14304 and guanfacine decreased the ability of GDP to compete with [³⁵S]GTPγS binding to the membranes and 0.1 mM GDP was found to be optimal for the following functional experiments. However, even after careful optimization of experimental conditions the specificity of ligands for rat α₂ adrenoceptors were not sufficient, as agonists as well as antagonists became activators of other signal transduction systems before achieving their maximal effect in the α_2A-adrenergic system. Only using compromising concentration of agonist (up to 1 μM UK14304) and antagonist (up to 1 μM RS79948) to inhibit agonist’s effect, allowed us to filtrate out α_2A specific effect for characterization of signal transduction in rat frontal cortex membranes for the comparison efficacies of this system for different animals from behavioral experiments.     

Gypenoside XLIX isolated from Gynostemma pentaphyllum inhibits nuclear factor-kappaB activation via a PPAR-alpha-dependent pathway

Summary Nuclear factor (NF)-κB is important in the generation of inflammation. Besides regulating lipid metabolism, peroxisome proliferator-activated receptor (PPAR)-α activators also reduce NF-κB activation to terminate activation of inflammatory pathways. Gynostemma pentaphyllum (GP) has been used to treat various inflammatory diseases and hyperlipidemia. Here, we demonstrate that GP extract and one of its main components, Gypenoside XLIX (Gyp-XLIX) inhibited LPS-induced NF-κB activation in murine macrophages. Furthermore, Gyp-XLIX restored the LPS- and TNF-α-induced decrease in cytosolic I-κBα protein expression and inhibited the translocation of NF-κB(p65) to the nucleus in THP-1 monocyte and HUVEC cells. The inhibition of LPS- and TNF-α-induced NF-κB luciferase activity in macrophages was abolished by MK-886, a selective PPAR-α antagonist. GP extract and Gyp-XLIX (EC₅₀: 10.1 μM) enhanced PPAR-α luciferase activity in HEK293 cells transfected with the tK-PPREx3-Luc reporter plasmid and expression vectors for PPAR-α. Additionally, Gyp-XLIX specifically enhanced PPAR-α mRNA and protein expression in THP-1-derived macrophage cells. The selectivity of Gyp-XLIX for PPAR-α was demonstrated by the activation of only PPAR-α in HEK293 cells transfected with expression vectors for PPAR-α, PPAR-β/δ or PPAR-γ1 plasmids and in THP-1-derived macrophage naturally expressing all three PPAR isoforms. The present study demonstrates that Gyp-XLIX, a naturally occurring gynosaponin, inhibits NF-κB activation via a PPAR-α-dependent pathway.Tom Hsun-Wei Huang and Yuhao Li contributed equally.     

Complete primary structure of a newly characterized galactose-specific lectin from the seeds of <Emphasis Type="Italic">Dolichos lablab</Emphasis>

A new unique lectin (galactose-specific) purified from the seeds of Dolichos lablab, designated as DLL-II is a heterodimer composed of closely related subunits α and β. These were separated by SDS-PAGE and isolated by electroelution. By ESI-MS analysis their molecular masses were found to be 30.746 kDa (α) and 28.815 kDa (β) respectively. Both subunits were glycosylated and displayed similar amino acid composition. Using advanced mass spectrometry in combination with de novo sequencing and database searches for the peptides derived by enzymatic and chemical cleavage of these subunits, the primary sequence was deduced. This revealed DLL-II to be made of two polypeptide chains of 281(α) and 263(β) amino acids respectively. The β subunit differed from the α subunit by the absence of some amino acids at the carboxy terminal end. This structural difference suggests that possibly, the β subunit is derived from the α subunit by posttranslational proteolytic modification at the COOH-terminus. Comparison of the DLL-II sequence to other leguminous seed lectins indicates a high degree of structural conservation. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rho GTPases mediated integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> activation in sphingosine-1-phosphate stimulated chemotaxis of endothelial cells

Integrins, a family of transmembrane heterodimeric polypeptides, mediate various biological responses including cell adhesion and migration. In this report, we show that sphingosine-1-phosphate (S1P) activates integrin α_vβ₃ in endothelial cells (ECs) via the sphingosine-1-phosphate receptor subtype 1 (S1P1)-mediated signaling pathway. S1P treatment results in the activation of integrin α_vβ₃ in the lamellipodia region of ECs, suggesting that integrin α_vβ₃ plays a critical role in the S1P-stimulated chemotactic response of ECs. Indeed, S1P treatment induces the association of focal adhesion kinase (FAK) and cytoskeletal proteins with integrin α_vβ₃, the ligation of α_v and β₃ subunits, as well as enhances endothelial migration on vitronectin-coated substrata. Knockdown endothelial S1P1 receptor, treatments with pertussis toxin or dominant-negative-Rho family GTPases abrogates the S1P-induced integrin α_vβ₃ activation in ECs. Consequently, these treatments markedly inhibit the S1P-induced endothelial migratory response on vitronectin-coated substrata. Collectively, these data indicate that the S1P-mediated signaling via the S1P1/G_i/Rho GTPases pathway activates integrin α_vβ₃, which is indispensable for S1P-stimulated chemotactic response of ECs.     

Neutral endopeptidase neprilysin is copurified with Na,K-ATPase from rabbit outer medulla and hydrolyzes its α-subunit

Preparations of Na,K-ATPase from outer medulla of rabbit kidney purified in accordance with the method of P. L. Jorgensen were shown to contain as admixture a protease that moves with α-subunit (∼100 kDa) as a single protein band during one-dimensional SDS-PAGE. The electro-elution of proteins of this band from polyacrylamide gel results in the appearance of two protein fragments (∼67 and 55 kDa) that are stained with polyclonal antibodies against Na,K-ATPase α-subunit. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis showed that the neutral membrane-bound endopeptidase neprilysin is located in one protein band together with the Na,K-ATPase α-subunit. Addition of thiorphan, a specific inhibitor of neutral endopeptidase, eliminates proteolysis of the α-subunit. The data demonstrate that Na,K-ATPase α-subunit may be a natural target for neprilysin.     

HIF-1α-dependent gene expression program during the nucleic acid-triggered antiviral innate immune responses

Recent studies suggest a novel role of HIF-1α under non-hypoxic conditions, including antibacterial and antiviral innate immune responses. However, the identity of the pathogen-associated molecular pattern which triggers HIF-1α activation during the antiviral response remains to be identified. Here, we demonstrate that cellular administration of double-stranded nucleic acids, the molecular mimics of viral genomes, results in the induction of HIF-1α protein level as well as the increase in HIF-1α target gene expression. Whole-genome DNA microarray analysis revealed that double-stranded nucleic acid treatment triggers induction of a number of hypoxia-inducible genes, and induction of these genes are compromised upon siRNA-mediated HIF-1α knock-down. Interestingly, HIF-1α knock-down also resulted in down-regulation of a number of genes involved in antiviral innate immune responses. Our study demonstrates that HIF-1α activation upon nucleic acid-triggered antiviral innate immune responses plays an important role in regulation of genes involved in not only hypoxic response, but also immune response. These authors contributed equally to this work.     

Amylases of the thermophilic fungusThermomyces lanuginosus: Their purification,properties, action on starch and response to heat

A thermophilic fungus Thermomyces lanuginous strain IISc 91, secreted one form each of α-amylase and glucoamylase during growth. Both enzymes were purified to homogeneity by ion-exchange and gel-filtration chromatography and obtained in mg quantities. α-Amylase was considered to be a dimeric protein of ∼ 42 kDa and contained 5% (by mass) carbohydrate. It was maximally active at pH 5.6 and at 65°C. It had an activation energy of 44 kJ mol^-1. The apparent K_m for soluble starch was 2.5 mg ml^-1. The enzyme produced exceptionally high levels of maltose from raw potato starch. At 50°C, the enzyme was stable for > 7h. At 65°C, α-amylase was nearly 8-times more stable in the presence of calcium. Addition of calcium increaed the melting temperature of α-amylase from 66°C to 73°C. Upon incubation at 94°C, α-amylase was progressively and irreversibly inactivated, and converted into an inactive 72 kDa trimeric species. Glucoamylase was a monomeric glycoprotein of ∼ 45 kDa with a carbohydrate content of 11% (by mass). It effected up to 76% conversion of starch in 24 h producing glucose as the sole product. Its apparent K_m for soluble starch was 0.04 mg ml^-1 and V_max was 660 Mmol glucose min^-1 mg protein^-1. It also hydrolyzed maltose. Its activity on maltooligosaccharides increased with the chain length of the substrates. Glucoamylase was stable at 60°C for over 7h. Its activation energy was 61 kJ mol^-1 Glucoamylase did not show synergistic effect with α-amylase. The properties of α-amylase and glucoamylase of Thermomyces lanuginosus strain IISc 91 suggest their usefulness in the commercial production of maltose and glucose syrups.     

Acyl-biotinyl Exchange Chemistry and Mass Spectrometry-Based Analysis of Palmitoylation Sites of In Vitro Palmitoylated Rat Brain Tubulin

Research has shown that the palmitoyl group of α-tubulin mediates the hydrophobic interaction between microtubules and intracellular membranes and that palmitoylated tubulin plays a role in signal transduction. There are 20 cysteine residues per α/β tubulin heterodimer. C376 of α-tubulin was reported to be predominantly palmitoylated and C20, C213 and C305 of α-tubulin were palmitoylated at lower levels. The previous method used for the analysis of the palmitoylation sites on α-tubulin was based on ³H-labeling, enzymolysis, purification and sequencing. This approach, although efficient, is laborious. Mass spectrometry (MS), especially tandem MS, has been shown to be a successful method for identification of various post-translational modifications of proteins. We report here a convenient MS-based method to comprehensively analyze the palmitoylation sites of the α/β tubulin heterodimer. Acyl-biotinyl exchange chemistry and streptavidin agarose affinity purification were applied to enrich palmitoylated peptides from tubulin. After nano-LC-MS/MS analysis, database searching and manual analysis of the spectra revealed that 11 cysteine residues of the α/β tubulin heterodimer were palmitoylated.     

Determination of mercury in human serum and packed blood cells by neutron activation analysis

A method is described for the determination of mercury in human blood serum and packed blood cells employing neutron activation analysis. Great attention was devoted to the collection and manipulation of the samples. The accuracy and precision of the method were tested by analyzing biological reference materials and by comparing the concentrations measured in a number of serum samples to those obtained by another, independent technique (cold vapor atomic absorption spectrometry) in the same samples. The article reports the levels measured in blood serum and packed blood cells samples from 15 adult volunteers, as well as the figures determined in a “second-generation” biological reference material (freeze-dried human serum), prepared and conditioned at the University of Ghent.     

Relationship among TNF-α, sICAM-1, and selenium in presurgical patients with abdominal aortic aneurysms

Epithermal instrumental neutron activation analysis (EINAA) has been used to determine the iodine content of many individual food materials that constitute the typical Libyan diet. The selected samples include different varieties of local and imported foods such as wheat and barley products, rice, bread, legumes such as chick peas and lentil, table salt, and commonly used spices, including thyme and fenugreek. Both conventional and anticoincidence γ-ray spectrometry techniques have been employed. Epithermal INAA in conjunction with anticoincidence counting has been found to provide the most reliable results. For quality control purposes, a number of NIST biological reference materials were analyzed. The range of daily dietary intake has been calculated as 100–180 μg of iodine per day, which is within the recommended range. Bread was identified as a significant source of iodine in the Libyan diet, as it contributed 99 μg/d.     

Cardiac α1-Adrenoceptors that regulate contractile function: Subtypes and subcellular signal transduction mechanismsthat regulate contractile function: Subtypes and subcellular signal transduction mechanisms

Activation of α₁-adrenoceptors as well as endothelin (ET) and angiotensin II (Ang II) receptors in cardiac muscle is coupled to acceleration of the hydrolysis of phosphoinositide (PI), with resultant production of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol. There is an excellent correlation between the extent of acceleration of the PI hydrolysis and the positive inotropic effect (PIE) under most experimental conditions after the administration of α-adrenoceptor agonists, ET and Ang II in the rabbit ventricular muscle. The PIE of the α-adrenoceptor agonists, ET and Ang II is associated with a negative lusitropic effect and an increase in the sensitivity of myofilaments to Ca²⁺ ions. The PIE can be selectively inhibited by inhibitors of protein kinase C (PKC) such as staurosporine, NA 0345 and H-7, with little effect on the PI hydrolysis and the PIE of isoproterenol and Bay k 8644. Surprisingly, an activator of PKC, phorbol 12,13-dibutyrate (PDBu), selectively and more completely inhibited the PIE and acceleration of PI hydrolysis induced by the α-adrenoceptor agonists as well as by ET and Ang II in the rabbit. These receptor agonists consistently cause intracellular alkalinization by activation of Na⁺−H⁺ exchange, while the effects on membrane ion channel activities are divergent. For example, α-adrenoceptor agonists cause monophasic prolongation of the action potential, the time course of which coincides well with that of the PIE, while ET and Ang II produce a biphasic change in action potential duration, i.e., the long-lasting prolongation preceded by a transient abbreviation. α-Adrenoceptor agonists scarcely affect I_Ca, whereas ET elicits a biphasic alteration of the current. In addition, the potassium current, I_Kl, is markedly suppressed by α-adrenoceptor agonists, but this effect is not revealed with Ang II under the same experimental condition. These results indicate that the effects of α₁-adrenoceptors stimulation are partially shared by those of ET and Ang II receptor activation in the heart. Approximately 60% of the total population of α₁-adrenoceptors in the rabbit ventricle are composed of α_1B subtype, which is susceptible to chlorethylclonidine (CEC) and is predominantly responsible for the α₁-mediated PIE and PI hydrolysis. The remaining fraction that belongs to α_1A-adrenoceptors subtype is further subclassified into the WB 4101-sensitive (partly coupled to PI hydrolysis) and the niguldipinesensitive (PI hydrolysis-unrelated) subtypes. Special issue dedicated to Dr. Kinya Kuriyama.     

Molecular characterization of soluble factors from human menstrual effluent that induce epithelial to mesenchymal transitions in mesothelial cells

We have studied menstrual effluent in order to identify soluble menstrual factors that induce epithelial to mesenchymal transitions (EMT) in mesothelial cells. A variety of molecules, such as nitric oxide and its reaction products, proteases (i.e. matrix metalloproteinases, plasmin) and proteins and/or peptides (i.e. growth factors: b-fibroblast growth factor, epidermal growth factor, hepatocyte growth factor, transforming growth factor-β; cytokines: interleukin 1β, tumour necrosis factor-α [TNF-α]) may be involved in this process. We have demonstrated that TNF-α is involved in EMT, whereas the other molecules are not. Biochemical analysis has shown that the inducing menstrual factors are heat-labile molecules, are uncharged at neutral pH, have a molecular weight between 50–70 kDa (or are bound in complexes of that size) and are eluted in the albumin fraction during gel filtration chromatography. Further analysis of this fraction by using proteomics and mass spectrometry has led to the identification of α-enolase and haemoglobin whose inhibition partially prevents EMT. When antibodies against TNF-α, α-enolase and haemoglobin are combined, EMT is almost completely inhibited. Thus, the candidates for soluble menstrual factors that induce mesothelial EMT are TNF-α, α-enolase and haemoglobin.     

Phosphorylation of eIF2 alpha in <Emphasis Type="Italic">Sf9</Emphasis> cells: a stress,survival and suicidal signal

An analysis of the stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor (eIF2α) involved in translation regulation, in the ovarian cells of Spodoptera frugiperda (Sf9) for its role in cell survival and death reveals that it stimulates casapase activation and cell death in the absence of BiP, a chaperone and stress marker of the endoplasmic reticulum (ER). While Phospho-JNK and GADD-153 levels are elevated in non-ER stress-induced eIF2α phosphorylation-mediated cell death, ATF4 levels are elevated both in response to ER and non-ER stress-induced eIF2α phosphorylation. Infection of Sf9 cells by wt and a mutant Δpk2 baculovirus that harbor the anti-apoptotic p35 gene induces BiP expression. However, UV-induced eIF2α phosphorylation and caspase activation are mitigated more efficiently by wt, but not by Δpk2 baculovirus that lacks pk2, an inhibitor of eIF2α kinase. z-VAD-fmk, a caspase inhibitor reduces the late stages, but not the initial stages of non-ER stress-induced eIF2α phosphorylation, thereby suggesting that eIF2α phosphorylation is a cause and consequence of caspase activation. The importance of BiP affecting the delicate balance between eIF2α phosphorylation-mediated cell survival and death is further supported by the findings that tunicamycin-treated cells expressing BiP resist eIF2α phosphorylation-mediated cell death and addition of a purified recombinant mutant phosphomimetic form, but not wt eIF2α, stimulates caspase activation in cell extracts devoid of BiP. These findings therefore suggest that eIF2α phosphorylation is primarily a stress signal and evokes adaptive or apoptotic responses depending on its cellular location, changes in gene expression, coincident signaling activities, and inter-protein interactions.