Neutral pH. (neutral + ph)

Distribution by Scientific Domains
Distribution within Chemistry

Selected Abstracts

Unusual stability of human neuroglobin at low pH , molecular mechanisms and biological significance

FEBS JOURNAL, Issue 23 2009
Paola Picotti
Neuroglobin (Ngb) is a recently discovered globin that is predominantly expressed in the brain, retina and other nerve tissues of human and other vertebrates. Ngb has been shown to act as a neuroprotective factor, promoting neuronal survival in conditions of hypoxic,ischemic insult, such as those occurring during stroke. In this work, the conformational and functional stability of Ngb at acidic pH was analyzed, and the results were compared to those obtained with Mb. It was shown by spectroscopic and biochemical (limited proteolysis) techniques that, at pH 2.0, apoNgb is a folded and rigid protein, retaining most of the structural features that the protein displays at neutral pH. Conversely, apoMb, under the same experimental conditions of acidic pH, is essentially a random coil polypeptide. Urea-mediated denaturation studies revealed that the stability displayed by apoNgb at pH 2.0 is very similar to that of Mb at pH 7.0. Ngb also shows enhanced functional stability as compared with Mb, being capable of heme binding over a more acidic pH range than Mb. Furthermore, Ngb reversibly binds oxygen at acidic pH, with an affinity that increases as the pH is decreased. It is proposed that the acid-stable fold of Ngb depends on the particular amino acid composition of the protein polypeptide chain. The functional stability at low pH displayed by Ngb was instead shown to be related to hexacoordination of the heme group. The biological implications of the unusual acid resistance of the folding and function of Ngb are discussed. [source]

Structural model for an AxxxG-mediated dimer of surfactant-associated protein C

FEBS JOURNAL, Issue 11 2004
Visvaldas Kairys
The pulmonary surfactant prevents alveolar collapse and is required for normal pulmonary function. One of the important components of the surfactant besides phospholipids is surfactant-associated protein C (SP-C). SP-C shows complex oligomerization behavior and a transition to ,-amyloid-like fibril structures, which are not yet fully understood. Besides this nonspecific oligomerization, MS and chemical cross-linking data combined with CD spectra provide evidence of a specific, mainly ,-helical, dimer at low to neutral pH. Furthermore, resistance to CNBr cleavage and dual NMR resonances of porcine and human recombinant SP-C with Met32 replaced by isoleucine point to a dimerization site located at the C-terminus of the hydrophobic ,-helix of SP-C, where a strictly conserved heptapeptide sequence is found. Computational docking of two SP-C helices, described here, reveals a dimer with a helix,helix interface that strikingly resembles that of glycophorin A and is mediated by an AxxxG motif similar to the experimentally determined GxxxG pattern of glycophorin A. It is highly likely that mature SP-C adopts such a dimeric structure in the lamellar bilayer systems found in the surfactant. Dimerization has been shown in previous studies to have a role in sorting and trafficking of SP-C and may also be important to the surfactant function of this protein. [source]

Kinetic study of sn -glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1

FEBS JOURNAL, Issue 3 2002
Jin-Suk Han
A gene having high sequence homology (45,49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94,96 C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H- dependent dihydroxyacetone phosphate reduction and NAD+ -dependent,glycerol-1-phosphate (Gro1P) oxidation. NADP+ -dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)+ acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi,bi mechanism. [source]

Cobalamin-Dependent and Cobalamin-Independent Methionine Synthases: Are There Two Solutions to the Same Chemical Problem?

Two enzymes in Escherichia coli, cobalamin-independent methionine synthase (MetE) and cobalamin-dependent methionine synthase (MetH), catalyze the conversion of homocysteine (Hcy) to methionine using N(5)-methyltetrahydrofolate (CH3 -H4folate) as the Me donor. Despite the absence of sequence homology, these enzymes employ very similar catalytic strategies. In each case, the pKa for the SH group of Hcy is lowered by coordination to Zn2+, which increases the concentration of the reactive thiolate at neutral pH. In each case, activation of CH3 -H4folate appears to involve protonation at N(5). CH3 -H4folate remains unprotonated in binary E,CH3 -H4folate complexes, and protonation occurs only in the ternary E,CH3 -H4folate,Hcy complex in MetE, or in the ternary E,CH3 -H4folate,cob(I)alamin complex in MetH. Surprisingly, the similarities are proposed to extend to the structures of these two unrelated enzymes. The structure of a homologue of the Hcy-binding region of MetH, betainehomocysteine methyltransferase, has been determined. A search of the three-dimensional-structure data base by means of the structure-comparison program DALI indicates similarity of the BHMT structure with that of uroporphyrin decarboxylase (UroD), a homologue of the MT2-A and MT2-M proteins from Archaea, which catalyze Me transfers from methylcorrinoids to coenzyme M and share the Zn-binding scaffold of MetE. Here, we present a model for the Zn binding site of MetE, obtained by grafting the Zn ligands of MT2-A onto the structure of UroD. [source]

Binding of Fusarium mycotoxins by fermentative bacteria in vitro

V. Niderkorn
Abstract Aims:,Fusarium toxins can occur in conserved forages impairing farm animal performances and health. On-farm biological decontamination methods could be an alternative to traditional physico-chemical methods. In this work, the ability to remove Fusarium toxins by fermentative bacteria was evaluated in vitro. Methods and Results:, Twenty-nine strains of lactic (LAB) and propionic acid bacteria (PAB) were tested for their ability to remove deoxynivalenol (DON) and fumonisins B1 and B2 (FB1, FB2) from an acid, pH 4, medium. Mycotoxin removal was widespread for LAB, but differences among strains were large. Removal was up to 55% for DON, 82% for FB1 and 100% for FB2. Selected strains were also capable of removing up to 88% zearalenone. The PAB strains were less efficient than the LAB. Binding, not biodegradation appeared to be the mode of action, as no toxin derivatives were observed and removal was not impaired in nonviable bacteria. Binding was not affected by pH, except for fumonisins that decreased to nearly 0% at neutral pH. Conclusions:, Selected fermentative bacteria are able to bind main Fusarium mycotoxins. Significance and Impact of the Study:, The binding ability of selected strains could be used to decrease the bioavailability of toxins in contaminated silages. [source]

Possibilities of polymer-aided dyeing of cotton fabric with reactive dyes at neutral pH

B. J. Agarwal
Abstract Water-soluble polymers have versatile application, viz., water-soluble polyacrylates have been widely used in the reactive dyeing of cellulosic fibers and the related soaping as an important component of the leveling and washing agent. In this article, one such water-soluble polymer, polyacrylic acid has been synthesized, characterized, and applied in conjunction with various types of reactive dyes, namely triazinyl, vinyl sulfone, high exhaustion, and bifunctional reactive dyes, along with crosslinking agents, namely glycerol 1,3-dichlorohydrin and hexamethylene tetramine-hydroquinone, respectively. One of the crosslinking agents (the former one) has been synthesized in the laboratory. Crosslinking agent is necessary to adhere the dye molecule onto the cellulose macromolecule. Different process sequences have been formulated and explored for dyeing purpose. All such dyeings were carried out at neutral pH. The dyed samples were assessed through color strength in terms of K/S values and their fastness properties were assessed by standard methods. All such dyeings were compared with conventional dyed samples. 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010 [source]

Membrane potential and endocytic activity control disintegration of cell,cell adhesion and cell fusion in vinculin-injected MDBK cells

Riitta Palovuori
Cell fusion occurs during fertilization and in the formation of organs such as muscles, placenta, and bones. We have developed an experimental model for epithelial cell fusion which permits analysis of the processes during junction disintegration and formation of polykaryons (Palovuori and Eskelinen [2000] Eur. J. Cell. Biol. 79: 961,974). In the present work, we analyzed the process in detail. Cell fusion was achieved by microinjecting into the cytoplasm of kidney epithelial Madin-Darby bovine kidney (MDBK) cells TAMRA-tagged vinculin, which incorporated into lateral membranes, focal adhesions and nucleus, and, prior fusion, induced internalization of actin, cadherin and plakoglobin to small clusters in cytoplasm. Injected vinculin was still visible at lateral membranes after removal of junctional proteins indicating that it was tightly associated and perturbed the cell,cell contact sites resulting in membrane fragmentation. Injection of active Rac together with vinculin induced accumulation of cadherin to the membranes, but did not affect vinculin,membrane association. However, it hampered cell fusion probably by supporting adherens junctions. In order to stop endocytosis, we lowered intracellular pH of vinculin-injected cells to 5.5 with the aid of nigericin in KCl buffer. In acidified cells, injected vinculin delineated lateral membranes as thick layers, cadherin remained in situ, and cell fusion was completely inhibited. Since this treatment also leads to cell depolarization, we checked the vinculin incorporation in a KCl solution containing nigericin at neutral pH. In these circumstances, both endogenous and injected vinculin delineated lateral membranes as very thin discontinuous layers, but still fusion was hampered most likely due to perturbation in the initial vinculin,membrane association. We suggest that vinculin might function as a sensor of the environment triggering cell fusion during development in circumstances where membrane potential and local and transient pH gradients play a role. 2004 Wiley-Liss, Inc. [source]


ABSTRACT Lipase from Rhizopus japonicus degraded chitosan resulting in soluble chitosan hydrolysates with molecular weight of about 30,50 kDa as shown by size exclusion chromatography. Optimal temperature for the hydrolysis of chitosan was 40C. The chitosan degradation products were fractionated stepwise according to their molecular weights by ultrafiltration with the filtration range of over 0.1 ,m, 0. l ,m to 30 kDa, 30 kDa to 10 kDa, 10 to 3 kDa, and 3 to 0.2 kDa. These fractions exhibited molecular weights of 50, 41, 41, 35, and 30 kDa, respectively. The molecular weights did not coincide with the pore size of filter membranes. Chitosan hydrolysate exhibited almost the same structural composition in IR spectra as chitosan flakes, except the peak of 1550 nm,1 that appeared to be the COO residue shifted from sodium acetate buffer to amine residue of chitosan. All fractions showed high solubility at neutral pH. The chitosan hydrolysates exhibiting molecular weights between 30 and 41 kDa were considered to be most suitable as a food additive or functional agent as demonstrated by sensory evaluation. [source]

Increase of Conjugated Linoleic Acid Content in Milk by Fermentation with Lactic Acid Bacteria

Y.J. Kim
ABSTRACT: The objectives of this study were to identify the factors and procedures responsible for increasing the conjugated linoleic acid (CLA) content in fermented milk. Fourteen lactic acid bacteria were screened for CLA-producing ability using sunflower oil (containing 70% linoleic acid) as a substrate. Among the screened strains, Lactococcus lactis I-01 showed the highest CLA-producing ability. The optimal concentration of sunflower oil for CLA production was 0.1 g/L in whole milk, which accounted for 0.25% of total milk fat. Our results demonstrated that CLA formation in fermented milk could be affected by numerous factors such as bacterial strain, cell number, optimal substrate concentration, and the period of incubation at neutral pH. [source]

Prediction of the association state of insulin using spectral parameters

Vladimir N. Uversky
Abstract Human insulin exists in different association states, from monomer to hexamer, depending on the conditions. In the presence of zinc the "normal" state is a hexamer. The structural properties of 20 variants of human insulin were studied by near-UV circular dichroism, fluorescence spectroscopy, and small-angle X-ray scattering (SAXS). The mutants showed different degrees of association (monomer, dimers, tetramers, and hexamers) at neutral pH. A correlation was shown between the accessibility of tyrosines to acrylamide quenching and the degree of association of the insulin mutants. The near-UV CD spectra of the insulins were affected by protein association and by mutation-induced structural perturbations. However, the shape and intensity of difference CD spectra, obtained by subtraction of the spectra measured in 20% acetic acid (where all insulin species were monomeric) from the corresponding spectra measured at neutral pH, correlate well with the degree of insulin association. In fact, the near-UV CD difference spectra for monomeric, dimeric, tetrameric, and hexameric insulin are very distinctive, both in terms of intensity and shape. The results show that the spectral properties of the insulins reflect their state of association, and can be used to predict their oligomeric state. 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:847,858, 2003 [source]

Ionic liquids as mobile phase additives for high-performance liquid chromatography separation of phenoxy acid herbicides and phenols

Xialin Hu
Abstract In this present study, 1-butyl-3-methylimidazolium chloride ([C4MIM]Cl), 1-octyl-3-methylimidazolium chloride ([C8MIM]Cl), and 1-decyl-3-methylimidazolium chloride ([C10MIM]Cl) were adopted as mobile phase additives in the high performance liquid chromatography (HPLC) to simultaneously separate phenoxy acid herbicides and phenols at neutral pH. It was found that by using 20,mM of [C4MIM]Cl, baseline separation and good chromatograms for all the acid compounds were obtained on a normal reversed-phase C18 column. The retention time of the target acid compounds shortened with the increase of the alkyl chain length and the concentrations of ionic liquids, probably due to the delocalization of the positive charge on the imidazolium cation, the repulsion between chlorine ions of ionic liquids and the acid compounds, as well as the stereo-hindrance effect. The mechanism with ionic liquids as mobile additives for the separation of acid compounds was discussed. [source]

Crystallization of parasporin-2, a Bacillus thuringiensis crystal protein with selective cytocidal activity against human cells

Toshihiko Akiba
Bacillus thuringiensis is a valuable source of protein toxins that are specifically effective against certain insects and worms but harmless to mammals. In contrast, a protein toxin obtained from B. thuringiensis strain A1547, designated parasporin-2, is not insecticidal but has a strong cytocidal activity against human cells with markedly divergent target specificity. The 37,kDa inactive protein is proteolytically activated to a 30,kDa active form. The active form of the recombinant protein toxin was crystallized in the presence of ethylene glycol and polyethylene glycol 8000 at neutral pH. The crystals belong to the hexagonal space group P61 or P65, with unit-cell parameters a = b = 134.37, c = 121.24,. Diffraction data from a native crystal were collected to 2.75, resolution using a synchrotron-radiation source. [source]

Crystallization and preliminary X-ray diffraction studies of a mosquito-larvicidal toxin from Bacillus thuringiensis subsp. israelensis

Panadda Boonserm
The Cry4B ,-endotoxin from Bacillus thuringiensis subsp. israelensis is specifically toxic to mosquito larvae. For a better understanding of the mechanism of toxicity, chymotrypsin-activated Cry4B toxin (68,kDa) has been purified and crystallized in sodium bromide at neutral pH. The well formed crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 185.82, c = 187.93,, and diffracted X-rays to 1.75, resolution. The asymmetric unit contains one toxin molecule and 74% solvent content, as shown by molecular replacement from a composite model of the homologous Cry3A and Cry1Aa. The purified protein and crystals both possessed mosquitocidal activity. [source]

Degradation of an Fc-fusion recombinant protein by host cell proteases: Identification of a CHO cathepsin D protease

Flavie Robert
Abstract A host-cell-related proteolytic activity was identified in a recombinant Fc-fusion protein production process. This report describes the strategy applied to characterize and isolate the enzyme responsible for this degradation by combining cell culture investigation and dedicated analytical tools. After isolation and sequencing of the clipped fragment generated in post-capture material, enzymatic activity was traced in different culture conditions, allowing identification of viable CHO cells as the source of protease. Inhibitors and pH screenings showed that the enzyme belongs to an aspartic protease family and is preferably active at acidic pH. The protease was isolated by purification on a pepstatin A column and characterized as a protein related to cathepsin D. An additional metallo-protease inhibited by EDTA was identified with an optimum activity at neutral pH. This study is an example of how quality and stability of therapeutic recombinant molecules are strongly influenced by cell culture parameters. Biotechnol. Bioeng. 2009; 104: 1132,1141. 2009 Wiley Periodicals, Inc. [source]

Reaction of Cytidine Nucleotides with Cyanoacetylene: Support for the Intermediacy of Nucleoside-2,,3,-cyclic Phosphates in the Prebiotic Synthesis of RNA

CHEMBIOCHEM, Issue 6 2006
Michael A. Crowe
Abstract A robust and prebiotically plausible synthesis of RNA is a key requirement of the "RNA World" hypothesis, but, to date, no such synthesis has been demonstrated. Monomer synthesis strategies involving attachment of preformed nucleobases to sugars have failed, and, even if activated 5,-nucleotides could be made, the hydrolysis of these intermediates in water makes their efficient oligomerisation appear unlikely. We recently reported a synthesis of cytidine-2,,3,-cyclic phosphate 1 (C>p) in which the nucleobase was assembled in stages on a sugar-phosphate template. However, 2,,3,-cyclic nucleotides (N>p's) also undergo hydrolysis, in this case giving a mixture of the 2,- and 3,-monophosphates. This hydrolysis has previously been seen as making the, otherwise promising, oligomerisation of N>p's seem as unlikely as that of the 5,-activated nucleotides. We now find that cyanoacetylene, the reagent used for the second stage of nucleobase assembly in the synthesis of C>p, also reverses the effect of the hydrolysis by driving efficient cyclisation of C2,p and C3,p back to C>p. Excess cyanoacetylene also derivatises the nucleobase, but this modification is reversible at neutral pH. These findings significantly strengthen the case for N>p's in a prebiotic synthesis of RNA. [source]

A New Fluorescent Probe for Zinc(II): An 8-Hydroxy-5- N,N -dimethylaminosulfonylquinoline-Pendant 1,4,7,10-Tetraazacyclododecane

Shin Aoki Prof.
Abstract A new fluorescent probe for Zn2+, namely, 8-hydroxy-5- N,N -dimethylaminosulfonylquinolin-2-ylmethyl-pendant cyclen (L8), was designed and synthesized (cyclen=1,4,7,10-tetraazacyclododecane). By potentiometric pH, 1H NMR, and UV spectroscopic titrations, the deprotonation constants pKa1,pKa6 of L8,4,HCl were determined to be <2, <2, <2 (for amino groups of the cyclen and quinoline moieties), 7.190.05 (for 8-OH of the quinoline moiety), 10.100.05, and 11.490.05, respectively, at 25,C with I=0.1 (NaNO3). The results of 1H NMR, potentiometric pH, and UV titrations, as well as single-crystal X-ray diffraction analysis, showed that L8 and Zn2+ form a 1:1 complex [Zn(H,1L8)], in which the 8-OH group of the quinoline ring of L8 is deprotonated and coordinates to Zn2+, in aqueous solution at neutral pH. On addition of one equivalent of Zn2+ and Cd2+, the fluorescence emission of L8 (5 ,M) at 512 nm in aqueous solution at pH 7.4 [10 mM HEPES with I=0.1 (NaNO3)] and 25,C increased by factors of 17 and 43, respectively. We found that the cyclen moiety has the unique property of quenching the fluorescence emission of the quinolinol moiety when not complexed with metal cations, but enhancing emission when complexed with Zn2+ or Cd2+. In addition, the Zn2+,L8 complex [Zn(H,1L8)] is much more thermodynamically and kinetically stable (Kd{Zn(H,1L8)}=[Zn2+]free[L8]free/[Zn(H,1L8)]=8 fM at pH 7.4) than the Zn2+ complexes of our previous Zn2+ fluorophores ([Zn(H,1L2)] and [Zn(L3)]). Furthermore, formation of [Zn(H,1L8)] is much faster than those of [Zn(H,1L2)] and [Zn(L3)]. The staining of early-stage apoptotic cells with L8 is also described. [source]