Ion-exchange and potentiometric characterization of Al–cystine and Al–cysteine complexes

The interaction between aluminium and cysteine and cystine was evaluated by means of ion-exchange experiments and potentiometry. Ion-exchange experiments included other ligands with affinity for aluminium and two kinds of resins, either a Na⁺-form or an Al³⁺-form exchanger. The ability of the ligands to keep aluminium in solution in the presence of the Na⁺ exchanger or to withdraw it from the Al³⁺-form resin was evaluated. Aluminium quantification was carried out by either graphite-furnace or flame atomic absorption spectrometry. Aluminium extraction isotherms were linearised using the Scatchard plot, and stability constants were obtained from the curves’ slopes. The experiments showed that the ability of the ligands to withdraw aluminium from the Al³⁺-form resin increased following the order cysteine < oxalate < citrate = cystine < nitrilotriacetic acid < ethylenediaminetetraacetic acid. Potentiometric titrations, carried out in aqueous solution with constant ionic strength and temperature, showed that the predominant species in solution have a metal–ligand proportion of 1:1 for both amino acids. The main species are Al(OH)₃L, with log K of 6.2 for cysteine, and AlL and Al(OH)L, with log K of 10.3 and 1.7, respectively, for cystine. Stability constants obtained from the Scatchard plots showed a linear correlation with the stability constants obtained by potentiometry for cystine and cysteine in this work and those collected from the literature for the other ligands. These results show that cysteine and cystine extract and maintain aluminium in solution, which may explain elevated concentrations of aluminium in parenteral nutrition solutions containing these amino acids.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Metabolomic and elemental analysis of camel and bovine urine by GC–MS and ICP–MS

Recent studies from the author’s laboratory indicated that camel urine possesses antiplatelet activity and anti-cancer activity which is not present in bovine urine. The objective of this study is to compare the volatile and elemental components of bovine and camel urine using GC–MS and ICP–MS analysis. We are interested to know the component that performs these biological activities. The freeze dried urine was dissolved in dichloromethane and then derivatization process followed by using BSTFA for GC–MS analysis. Thirty different compounds were analyzed by the derivatization process in full scan mode. For ICP–MS analysis twenty eight important elements were analyzed in both bovine and camel urine. The results of GC–MS and ICP–MS analysis showed marked difference in the urinary metabolites. GC–MS evaluation of camel urine finds a lot of products of metabolism like benzene propanoic acid derivatives, fatty acid derivatives, amino acid derivatives, sugars, prostaglandins and canavanine. Several research reports reveal the metabolomics studies on camel urine but none of them completely reported the pharmacology related metabolomics. The present data of GC–MS suggest and support the previous studies and activities related to camel urine.     

Cloning and characterization of zebrafish protocadherin–17

Protocadherins constitute the largest subgroup within the cadherin superfamily of cell surface molecules. In this study, we report the molecular cloning and expression analysis of the non-clustered protocadherin-17 (pcdh17) in the embryonic zebrafish nervous system. The zebrafish Pcdh17 protein is highly conserved, exhibiting 73% sequence homology with the human protein. The zebrafish pcdh17 gene consists of four exons spread over 150 kb, and this organization is highly conserved throughout vertebrates. Pcdh17 message is first detectable by 6 h postfertilization in the developing embryo, and the expression is maintained throughout development. Zebrafish embryos express pcdh17 in all of the major subdivisions of the central nervous system, including the telencephalon, diencephalon, mesencephalon, and rhombencephalon. Analysis of the genomic sequence upstream of pcdh17 in several species reveals a pattern of paired CpG islands. While the CpG islands in zebrafish are further upstream than in other teleosts, alignment of the identified sequences reveals a high degree of conservation, suggesting that the sequences may be important for the regulation of pcdh17 expression.     

Analysis and modeling of time-variant amplitude–frequency couplings of and between oscillations of EEG bursts

Low-frequency (0.5-2.5 Hz) and individually defined high-frequency (7-11 or 8-12 Hz; 11-15 or 14-18 Hz) oscillatory components of the electroencephalogram (EEG) burst activity derived from thiopental-induced burst-suppression patterns (BSP) were investigated in seven sedated patients (17-26 years old) with severe head injury. The predominant high-frequency burst oscillations (>7 Hz) were detected for each patient by means of time-variant amplitude spectrum analysis. Thereafter, the instantaneous envelope (IE) and the instantaneous frequency (IF) were computed for these low- and high-frequency bands to quantify amplitude-frequency dependencies (envelope-envelope, envelope-frequency, and frequency-frequency correlations). Time-variant phase-locking, phase synchronization, and quadratic phase couplings are associated with the observed amplitude-frequency characteristics. Additionally, these time-variant analyses were carried out for modeled burst patterns. Coupled Duffing oscillators were adapted to each EEG burst and by means of these models data-based burst simulations were generated. Results are: (1) strong envelope-envelope correlations (IE courses) can be demonstrated; (2) it can be shown that a rise of the IE is associated with an increase of the IF (only for the frequency bands 0.5-2.5 and 7-11 or 8-12 Hz); (3) the rise characteristics of all individually averaged envelope-frequency courses (IE-IF) are strongly correlated; (4) for the 7-11 or 8-12 Hz oscillation these associations are weaker and the variation between the time courses of the patients is higher; (5) for both frequency ranges a quantitative amplitude-frequency dependency can be shown because higher IE peak maxima are accompanied by stronger IF changes; (6) the time range of significant phase-locking within the 7-11 or 8-12 Hz frequency bands and of the strongest quadratic phase couplings (between 0.5-2.5 and 7-11 or 8-12 Hz) is between 0 and 1,000 ms; (7) all phase coupling characteristics of the modeled bursts accord well with the corresponding characteristics of the measured EEG burst data. All amplitude-frequency dependencies and phase locking/coupling properties described here are known from and can be discussed using coupled Duffing oscillators which are characterized by autoresonance properties.     

The Birth and Life of Species–Cultures

Evolution and life phenomena can be understood as results of history, i.e., as outcomes of cohabitation and collective memory of populations of autonomous entities (individuals) across many generations and vast extent of time. Hence, evolution of distinct lineages of life can be considered as isomorphic with that of cultures. I argue here that cultures and culture-like systems – human culture, natural languages, and life forms – always draw from history, memory, experience, internal dynamics, etc., transforming themselves creatively into new patterns, never foreseen before. This is possible thanks to the fact that all forms of life are descendants of life. Ontogeny and speciation in various lineages draw from continuous re-interpretation of conservative genetic/generic “texts”, as well as from changes of the interpretative process itself. The result is continuous appearances of new lineages-cultures and/or communities-cultures, in a semiotic process of re-interpretation and inventing new ways of living. The topic is developed here on the backgrounds of ideas presented by R. A. Rappaport in “Ritual and religion in the making of humanity” and J. Flegr in “Frozen evolution”.     

Analysis of lignin–carbohydrate and lignin–lignin linkages after hydrolase treatment of xylan–lignin,glucomannan–lignin and glucan–lignin complexes from spruce wood

Xylan–lignin (XL), glucomannan–lignin (GML) and glucan–lignin (GL) complexes were isolated from spruce wood, hydrolyzed with xylanase or endoglucanase/β-glucosidase, and analyzed by analytical pyrolysis and 2D-NMR. The enzymatic hydrolysis removed most of the polysaccharide moieties in the complexes, and the lignin content and relative abundance of lignin–carbohydrate linkages increased. Analytical pyrolysis confirmed the action of the enzymatic hydrolysis, with strong decreases of levoglucosane and other carbohydrate-derived products. Unexpectedly it also revealed that the hydrolase treatment alters the pattern of lignin breakdown products, resulting in higher amounts of coniferyl alcohol. From the anomeric carbohydrate signals in the 2D-NMR spectra, phenyl glycoside linkages (undetectable in the original complexes) could be identified in the hydrolyzed GML complex. Lower amounts of glucuronosyl and benzyl ether linkages were also observed after the hydrolysis. From the 2D-NMR spectra of the hydrolyzed complexes, it was concluded that the lignin in GML is less condensed than in XL due to its higher content in β-O-4′ ether substructures (62 % of side chains in GML vs 53 % in XL) accompanied by more coniferyl alcohol end units (16 vs 13 %). In contrast, the XL lignin has more pinoresinols (11 vs 6 %) and dibenzodioxocins (9 vs 2 %) than the GML (and both have ~13 % phenylcoumarans and 1 % spirodienones). Direct 2D-NMR analysis of the hydrolyzed GL complex was not possible due to its low solubility. However, after sample acetylation, an even less condensed lignin than in the GML complex was found (with up to 72 % β-O-4′ substructures and only 1 % pinoresinols). The study provides evidence for the existence of structurally different lignins associated to hemicelluloses (xylan and glucomannan) and cellulose in spruce wood and, at the same time, offers information on some of the chemical linkages between the above polymers.     

Viscoelastic and Thermal Properties of Collagen–Xanthan Gum and Collagen–Maltodextrin Suspensions During Heating and Cooling

The purpose of this work was to investigate the viscoelastic properties of aqueous suspensions of crude collagen powder extracted from bovine hides and nonsubmitted to the hydrolysis reaction that leads to gelatin. The studied variables included the collagen concentration and the addition of xanthan gum or maltodextrin at varied concentrations during heating/cooling of the mixtures. Differential scanning calorimetry thermograms showed that the addition of polysaccharides decreased the endothermic peak areas observed at the denaturation temperature of collagen. The rheological properties of the pure collagen suspensions were highly dependent on concentration: 4% and 6% collagen suspensions presented a great increase in the storage modulus after heating/cooling, whereas for concentrations of 8% and 10% G′ decreased during heating and did not recover its original value after heating/cooling. The frequency sweeps showed that the thermal treatment was responsible by the strengthening of the interactions that formed the polymer network. Addition of 0.1% xanthan gum to collagen suspensions increased the gel strength, especially after heating/cooling of the system, whereas increasing gum concentration to 0.3% resulted in a weaker gel, which could indicate thermodynamic incompatibility between the biopolymers. Mixtures of collagen and maltodextrin resulted in more fluid structures than those obtained with pure collagen at the same collagen concentration and the range of temperatures in which these mixtures behaved as a gel decreased with increasing concentrations of both collagen and maltodextrin, suggesting incompatibilities between the biopolymers.     

Purification and comparative properties of isoenzymes of nicotinamide–adenine dinucleotide phosphate–isocitrate dehydrogenase from rat heart and liver

1. Rat liver and heart major isoenzymes of NADP-isocitrate dehydrogenase have each been purified about 100-fold by a combination of ammonium sulphate fractionation and chromatography on ion-exchange cellulose and their properties compared. 2. The properties were similar in respect of pH, inhibition by Hg(2+) and Michaelis constants for isocitrate and NADP. 3. Some of the properties of the isoenzymes were different. 4. The heart isoenzyme was activated about 210% by 0.8m-ammonium sulphate whereas the liver isoenzyme was unaffected. The heart isoenzyme showed greater sensitivity to inactivation by heat (30 degrees C for 30min), whereas the liver isoenzyme was more sensitive to inactivation by p-chloromercuribenzoate and by Cu(2+). 5. The Michaelis constants with 3-acetylpyridine-adenine dinucleotide phosphate showed a twofold difference between liver and heart isoenzyme. 6. The differential sensitivity to heat and its mainly non-cytoplasmic location may be an explanation of the failure of plasma isocitrate dehydrogenase activity to increase after a myocardial infarction.     

Comparative biochemistry of chondroitin sulphate–proteins of cartilage and notochord

Fragments that consisted mainly of two polysaccharide chains joined by a short polypeptide bridge (doublets) were prepared from chondroitin sulphate-proteins of lamprey, sturgeon, elasmobranch and ox connective tissues after hydrolysis with trypsin and chymotrypsin. Consideration of molecular parameters, compositions and behaviour on gel electrophoresis and density-gradient fractionation leads to a proposed parent structure for chondroitin sulphate-proteins. A single polypeptide chain of about 2000 amino acid residues contains alternating short and long repeating sequences. A short sequence consists of less than 10 amino acid residues with one N-terminal and one C-terminal serine residue, each of which carries a polysaccharide chain linked glycosidically to its hydroxyl group. This structure constitutes the doublet subunit. Some variation is introduced when the doublet subunit carries only a single polysaccharide chain. The long sequence contains about 35 amino acid residues and is subject to cleavage by trypsin and chymotrypsin. The main polypeptide is probably homologous in the vertebrate sub-phylum with strong conservation of structure suggested for the short sequence. However, polymorphism of polypeptide structures cannot be excluded.     

Preparation and Characterization of Collagen–Chitosan–Chondroitin Sulfate Composite Membranes

Collagen (Col)–chitosan (Chi) membrane was modified by a hot dehydrogenation cross-linking method. Carbodiimide was added for further crossing modification. Chondroitin sulfate (CS) was added so that Col–Chi sulfate composite membranes were prepared. The structure of the composite membranes was characterized by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, and its mechanical properties, degradation, and cytotoxicity were characterized. The composite membrane was applied to a full-thickness skin injury in animal experiments performed in rabbits. Strong interactions and good compatibility among Col, Chi, and CS in the composite membrane were present. The good mechanical properties, biocompatibility, digestion resistance, and wound healing promotion of the composite membrane make it a potential wound dressing or skin scaffold for tissue engineering.     

Chemoenzymatic resolution of racemic Wieland–Miescher and Hajos–Parrish ketones

Enantiospecific microbial reduction of bicyclic ketones was described. Racemic Wieland–Miescher (1) and Hajos–Parrish (2) ketones were used as substrates. In a 4-h biotransformation of Hajos–Parrish ketone (2) using the strain of Didymosphaeria igniaria an optically pure ketone (R)-2 was obtained, whereas the (S)-2 ketone underwent reduction to (4aS,5S)-4 alcohol with 100% of enantiomeric excess and with over 60% of diastereoisomeric excess. Jones oxidation of the alcohol obtained in the biotransformation gave an optically pure ketone (S)-2. Enzymatic system of Coryneum betulinum reduced the (R)-2 ketone to (4aR,5S)-4 alcohol with a high enantiomerical purity in a 6-day reaction. Wieland-Miescher (1) ketone was transformed by these microorganisms in an analogous way, but the reaction times were longer.     

Biosynthesis,properties and potential of natural–synthetic hybrids of polyhydroxyalkanoates and polyethylene glycols

Chemical conjugation with poly(ethylene glycols) (PEGs) are established procedures to facilitate solubilisation of hydrophobic compounds. Such techniques for PEGylation have been applied to polyhydroxybutyrate. ‘BioPEGylation’ of such polyhydroxyalkanoates (PHAs) to form natural–synthetic hybrids has been demonstrated through the addition of PEGs to microbial cultivation systems. The strategic addition of certain PEGs not only supports hybrid synthesis but may also provide a technique for control of PHA composition and molecular mass, and by extension, their physico-mechanical properties. PHA composition and molecular mass control by PEGs is dependent upon the polyethers’ molecular mass, loading in the cultivation system, time of introduction and microbial species. Hybrid characterisation studies are in their infancy, but results to date suggest that PHA–PEG hybrids have subtle, but significant, differences in their physiochemical and material properties as a consequence of the PEGylation.     

Spatial organization of Dps and DNA–Dps complexes

DNA co-crystallization with Dps family proteins is a fundamental mechanism, which preserves DNA in bacteria from harsh conditions. Though many aspects of this phenomenon are well characterized, the spatial organization of DNA in DNA–Dps co-crystals is not completely understood, and existing models need further clarification. To advance in this problem we have utilized atomic force microscopy (AFM) as the main structural tool, and small-angle X-scattering (SAXS) to characterize Dps as a key component of the DNA-protein complex. SAXS analysis in the presence of EDTA indicates a significantly larger radius of gyration for Dps than would be expected for the core of the dodecamer, consistent with the N-terminal regions extending out into solution and being accessible for interaction with DNA. In AFM experiments, both Dps protein molecules and DNA–Dps complexes adsorbed on mica or highly oriented pyrolytic graphite (HOPG) surfaces form densely packed hexagonal structures with a characteristic size of about 9 nm. To shed light on the peculiarities of DNA interaction with Dps molecules, we have characterized individual DNA–Dps complexes. Contour length evaluation has confirmed the non-specific character of Dps binding with DNA and revealed that DNA does not wrap Dps molecules in DNA–Dps complexes. Angle analysis has demonstrated that in DNA–Dps complexes a Dps molecule contacts with a DNA segment of ~6 nm in length. Consideration of DNA condensation upon complex formation with small Dps quasi-crystals indicates that DNA may be arranged along the rows of ordered protein molecules on a Dps sheet.     

Synthesis and antibacterial activity of catecholate–ciprofloxacin conjugates

The development of an efficient route to obtain artificial siderophore–antibiotic conjugates active against Gram-negative bacteria is crucial. Herein, a practical access to triscatecholate enterobactin analogues linked to the ciprofloxacin along with their antibacterial evaluation are described. Two series of conjugates were obtained with and without a piperazine linker which is known to improve the pharmacokinetics profile of a drug. A monocatecholate–ciprofloxacin conjugate was also synthesized and evaluated. The antibacterial activities against Pseudomonas aeruginosa for some conjugates are related to the iron concentration in the culture medium and seem to depend on the bacterial iron uptake systems.     

Structures and Energetics of Platinum–Cobalt Bimetallic Clusters

Platinum–cobalt bimetallic clusters (N=11–15) were modeled by a many-body Sutton-Chen (SC) potential. The basin-hopping algorithm and Monte Carlo (MC) energy minimization were used to determine the global minima of bimetallic clusters. The structural changes with cluster size were observed. Most of the structures had built on icosahedral packing. Second energy difference analyzes were performed to investigate the relative stability of a cluster with respect to its size and composition. The dependence of energetics and structures on size and composition is discussed.     

Screening and Characterization of Fructosyl-Valine–Utilizing Marine Microorganisms

We describe the isolation of microorganisms utilizing fructosyl-amine (Amadori compound) from the marine environment and of fructosyl-amine oxidase from a marine yeast. Using fructosyl-valine (Fru-Val), a model Amadori compound for glycated hemoglobin, we isolated 12 microbial strains that grow aerobically in a minimal medium supplemented with Fru-Val as the sole nitrogen source. Among these strains, a yeast strain identified as Pichia sp. N1-1, produced a Fru-Val–oxidizing enzyme. The enzyme was purified in its active form, a single-polypeptide water-soluble protein of 54 kDa by gel electrophoresis, producing H₂O₂ with the oxidation of Fru-Val. By its substrate specificity, the enzyme was categorized as a novel fructosyl-amine oxidase. This is the first study on the isolation of microorganisms utilizing fructosyl-amine in the marine environment and of fructosyl-amine oxidase from budding yeast. Received October 21, 1999; accepted September 12, 2000     

Purification and characterization of HIV–human protein complexes

To fully understand how pathogens infect their host and hijack key biological processes, systematic mapping of intra-pathogenic and pathogen–host protein–protein interactions (PPIs) is crucial. Due to the relatively small size of viral genomes (usually around 10–100 proteins), generation of comprehensive host–virus PPI maps using different experimental platforms, including affinity tag purification-mass spectrometry (AP-MS) and yeast two-hybrid (Y2H) approaches, can be achieved. Global maps such as these provide unbiased insight into the molecular mechanisms of viral entry, replication and assembly. However, to date, only two-hybrid methodology has been used in a systematic fashion to characterize viral–host protein–protein interactions, although a deluge of data exists in databases that manually curate from the literature individual host–pathogen PPIs. We will summarize this work and also describe an AP-MS platform that can be used to characterize viral-human protein complexes and discuss its application for the HIV genome.     

Molecular and practical aspects of the enzymatic properties of human serum albumin and of albumin–ligand complexes

Background

Human serum albumin and some of its ligand complexes possess enzymatic properties which are useful both in vivo and in vitro.

Scope of review

This review summarizes present knowledge about molecular aspects, practical applications and potentials of these properties.

Major conclusions

The most pronounced activities of the protein are different types of hydrolysis. Key examples are esterase-like activities involving Tyr411 or Lys199 and the thioesterase activity of Cys34. In the first case, hydrolysis involves water and both products are released, whereas in the latter cases one of the products is set free, and the other stays covalently bound to the protein. However, the modified Cys34 can be converted back to its reduced form by another compound/enzymatic system. Among the other activities are glucuronidase, phosphatase and amidase as well as isomerase and dehydration properties. The protein has great impact on the metabolism of, for example, eicosanoids and xenobiotics. Albumin with a metal ion-containing complex is capable of facilitating reactions involving reactive oxygen and nitrogen species.

General significance

Albumin is useful in detoxification reactions, for activating prodrugs, and for binding and activating drug conjugates. The protein can be used to construct smart nanotubes with enzymatic properties useful for biomedical applications. Binding of organic compounds with a metal ion often results in metalloenzymes or can be used for nanoparticle formation. Because any compound acting as cofactor and/or the protein can be modified, enzymes can be constructed which are not naturally found and therefore can increase, often stereospecifically, the number of catalytic reactions. This article is part of a Special Issue entitled Serum Albumin.