Spore and micro-particle capture on an immunosensor surface in an ultrasound standing wave system

The capture of Bacillus subtilis var. niger spores on an antibody-coated surface can be enhanced when that coated surface acts as an acoustic reflector in a quarter wavelength ultrasonic (3 MHz) standing wave resonator. Immunocapture in such a resonator has been characterised here for both spores and 1 microm diameter biotinylated fluorescent microparticles. A mean spatial acoustic pressure amplitude of 460 kPa and a frequency of 2.82 MHz gave high capture efficiencies. It was shown that capture was critically dependent on reflector thickness. The time dependence of particle deposition on a reflector in a batch system was broadly consistent with a calculated time of 35 s to bring 95% of particles to the coated surface. A suspension flow rate of 0.1 ml/min and a reflector thickness of 1.01 mm gave optimal capture in a 2 min assay. The enhancement of particle detection compared with the control (no ultrasound) situation was x 70. The system detects a total of five particles in 15 fields of view in a 2 min assay when the suspending phase concentration was 10(4) particles/ml. A general expression for the dependence of minimum concentration detectable on; number of fields examined, sample volume flowing through the chamber and assay time shows that, for a practical combination of these variables, the threshold detection concentration can be two orders of magnitude lower.     

Unfolding and refolding of the glutamine-binding protein from Escherichia coli and its complex with glutamine induced by guanidine hydrochloride

The aim of this work was to study the conformational changes of the Escherichia coli glutamine-binding protein (GlnBP) induced by GdnHCl and the effect of the binding of glutamine (Gln) on these processes. To this end, GdnHCl-induced unfolding of GlnBP alone and its GlnBP-Gln complex was studied by protein intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy. The obtained spectroscopic data were interpreted taking into the account the peculiarities of protein three-dimensional structure. In particular, the fact that formation of a complex of GlnBP and Gln, which essentially changes the global structure of protein, affects only insignificantly the microenvironments of tryptophan residues elucidates the similarity of the emission spectra of GlnBP and the GlnBP-Gln complex, and the existence of quenching groups near tyrosine residues and an effective nonradiative Tyr --> Trp and/or Tyr --> Tyr --> Trp energy transfer provide an explanation for the negligibly small contribution of tyrosine to the bulk fluorescence of the native protein and for its increase in protein unfolding. The use of the parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP-Gln unfolding are three-step processes (N --> I(1) --> I(2) --> U), though in the case of the GlnBP-Gln complex these stages essentially overlap. Despite the complex character, GlnBP unfolding is completely reversible. The dramatic shift of the N --> I(1) process to higher GdnHCl concentrations for the GlnBP-Gln complex in comparison with GlnBP was shown.     

Structure- and function-based characterization of a new phosphoglycolate phosphatase from Thermoplasma acidophilum

The protein TA0175 has a large number of sequence homologues, most of which are annotated as unknown and a few as belonging to the haloacid dehalogenase superfamily, but has no known biological function. Using a combination of amino acid sequence analysis, three-dimensional crystal structure information, and kinetic analysis, we have characterized TA0175 as phosphoglycolate phosphatase from Thermoplasma acidophilum. The crystal structure of TA0175 revealed two distinct domains, a larger core domain and a smaller cap domain. The large domain is composed of a centrally located five-stranded parallel beta-sheet with strand order S10, S9, S8, S1, S2 and a small beta-hairpin, strands S3 and S4. This central sheet is flanked by a set of three alpha-helices on one side and two helices on the other. The smaller domain is composed of an open faced beta-sandwich represented by three antiparallel beta-strands, S5, S6, and S7, flanked by two oppositely oriented alpha-helices, H3 and H4. The topology of the large domain is conserved; however, structural variation is observed in the smaller domain among the different functional classes of the haloacid dehalogenase superfamily. Enzymatic assays on TA0175 revealed that this enzyme catalyzed the dephosphorylation of phosphoglycolate in vitro with similar kinetic properties seen for eukaryotic phosphoglycolate phosphatase. Activation by divalent cations, especially Mg2+, and competitive inhibition behavior with Cl- ions are similar between TA0175 and phosphoglycolate phosphatase. The experimental evidence presented for TA0175 is indicative of phosphoglycolate phosphatase.     

UHF-dielectrometry in the assessment of the structural organization of salt solutions and interstitial fluids in normal and cicatricial tissues

The complex dielectric permittivity of salt solutions with positive and negative salvation as well as healthy and cicatricially changed human skin in situ at the frequencies of 42 and 56.6 GHz was measured. The relation between the dielectric characteristics of water and diluted salt solutions and changes in their structural organization conditioned by different temperatures of samples and the type of salvation of electrolytes was studied. The differences in the dielectric characteristics of healthy and cicatricially changed skin are interpreted in terms of the dependence of the structural organization of interstitial fluids on the morphological and functional state of biological tissues.     

General enzymatic screens identify three new nucleotidases in Escherichia coli. Biochemical characterization of SurE, YfbR, and YjjG

To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. These screens identified the presence of nucleotidase activity in three uncharacterized E. coli proteins, SurE, YfbR, and YjjG, that belong to different enzyme superfamilies: SurE-like family, HD domain family (YfbR), and haloacid dehalogenase (HAD)-like superfamily (YjjG). The phosphatase activity of these proteins had a neutral pH optimum (pH 7.0-8.0) and was strictly dependent on the presence of divalent metal cations (SurE: Mn(2+) > Co(2+) > Ni(2+) > Mg(2+); YfbR: Co(2+) > Mn(2+) > Cu(2+); YjjG: Mg(2+) > Mn(2+) > Co(2+)). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate various ribo- and deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates with highest affinity to 3'-AMP. SurE also hydrolyzed polyphosphate (exopolyphosphatase activity) with the preference for short-chain-length substrates (P(20-25)). YfbR was strictly specific to deoxyribonucleoside 5'-monophosphates, whereas YjjG showed narrow specificity to 5'-dTMP, 5'-dUMP, and 5'-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di- and triphosphates: YfbR was equally sensitive to both di- and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli.     

Evaluation of functional state of crayfish <Emphasis Type="Italic">Pontastacus leptodactylus</Emphasis> in normal and toxic environment by characteristics of their cardiac activity and hemolymph biochemical parameters

The work deals with study of basic characteristics of cardiac activity (heart rate—HR and stress-index—SI) and hemolymph protein parameters in the crayfish Pontastacus leptodactylus. The main criteria of the crayfish selection are developed for formation of the animal reference groups available for subsequent toxicological experiments. The action of a model toxicant, hydroquinone (often present in waste water), at a concentration of 1 g/l on parameters of the crayfish cardiac activity at various exposition time, its effect on circadian cardioactivity and on the hemolymph total protein were studied. At its short-term action, hydroquinone produced a temporary increase of HR and stress-index, but had no marked effect on total protein parameters in hemolymph. At long action (for one day), hydroquinone led to a considerable tachycardia, disturbance of the circadian cardioactivity and to a decrease by 40% of the hemolymph total protein. In 50% of cases the toxicant caused death of the animals either in the course of its short-term action or at period of washout from the toxicant. Mechanisms of the toxic action of hydroquinone at various levels of organization are discussed.     

Structure and Activity of the Metal-independent Fructose-1,6-bisphosphatase YK23 from Saccharomyces cerevisiae

Fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis and photosynthetic CO₂ fixation, catalyzes the hydrolysis of fructose 1,6-bisphosphate (FBP) to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways. All known FBPases are metal-dependent enzymes, which are classified into five different classes based on their amino acid sequences. Eukaryotes are known to contain only the type-I FBPases, whereas all five types exist in various combinations in prokaryotes. Here we demonstrate that the uncharacterized protein YK23 from Saccharomyces cerevisiae efficiently hydrolyzes FBP in a metal-independent reaction. YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily with homologues found in all organisms. The crystal structure of the YK23 apo-form was solved at 1.75-Å resolution and revealed the core domain with the α/β/α-fold covered by two small cap domains. Two liganded structures of this protein show the presence of two phosphate molecules (an inhibitor) or FBP (a substrate) bound to the active site. FBP is bound in its linear, open conformation with the cleavable C1-phosphate positioned deep in the active site. Alanine replacement mutagenesis of YK23 identified six conserved residues absolutely required for activity and suggested that His¹³ and Glu⁹⁹ are the primary catalytic residues. Thus, YK23 represents the first family of metal-independent FBPases and a second FBPase family in eukaryotes.     

Genetic polymorphism of glutathione-S-transferases and inhibition of DNA repair

A complex investigation of different cell defence systems, such as: DNA repair, antioxidant system (SOD), xenobiotic detoxification system (glutathione-S-transferases M1 and T1), radioadaptive response (RAR) in lymphocytes of patients with hereditary disease of connective tissue (Elers-Danlose syndrome) was carried out. The frequency of genotype GSTM1 (0/0) in children with Elers-Danlose syndrome (23%) is lower as compared to the control group (44%). The lymphocytes of children with Elers-Danlose syndrome were characterized by reduced ability to repair gamma-induced damage of DNA. At given size of the samples of examined children no correlative relationships between GST-status of organism and the condition of other cell defence systems were revealed. The data obtained demonstrate the individual peculiarities of the defence systems in repair-deficient cells of the examined children.     

Role of V1- and V2-receptors in mechanism of physiological paradox--an increase of reabsorption of the solute free water and simultaneous rise of diuresis

In experiments on non-anesthetized rats with administration into stomach of water (5 ml/100 g body mass) direct correlation has been found between an increase of diuresis and excretion of solute free water (r = 0.98, p < 0.01), while after injection to these animals of 5 x 10(-11) M arginine-vasotocin - between an increase of diuresis and simultaneous rise reabsorption of solute free water (r = 0.8, p < 0.01). The rise of diuresis after the vasotocin injection is due to inhibition of sodium re- absorption, with the solute excretion fraction increasing from 2.6 +/- 0.2 % to 11.9 +/- 1.2, p < 0.001. A similar physiological paradox - an increase of diuresis with the simultaneous increase of reabsorption of solute free water - has been revealed at night hours in children with tendency for nocturnal enuresis (r = 0.64, p < 0.01). Mechanism responsible for this phenomenon consists in a rise of diuresis due to a decrease of sodium ion reabsorption in the ascending Henle loop limb. A problem is discussed of the homeostatic significance of a decrease of sodium reabsorption combined with an increase of solute-free water reabsorption; it is suggested that this phenomenon is based on a redistribution of reabsorption inside the nephron - a decrease of ion and water reabsorption in the initial parts of the nephron distal segment and an increase of solute free water reabsorption with the antidiuretic hormone-stimulated high osmotic permeability of terminal parts of renal tubules. An intraperitoneal injection of V1-anatagonist (OPC-21268) decreased the natriuretic component of response to arginine-vasotocin, while injection of V2-antagonist (OPC-31260) eliminated the antidiuretic component.