Nickel resistance in Escherichia coli V38 is dependent on the concentration used for induction

Strain Escherichia coli V38 resistant to 4 mM NiCl₂ was isolated from the city sewage sludge. It showed low nickel accumulation by cells and nickel ion efflux. Cells were pregrown (induced) overnight in the presence of Ni²⁺, then the culture was kept on ice for 20–30 min and transferred to 37°C for further incubation. When the Ni²⁺ concentration during growth was the same as during incubation, there was no noticeable accumulation of Ni²⁺. When the Ni²⁺ concentration during incubation was higher than that used for induction, uptake of ⁶³Ni²⁺ and delayed efflux were seen. The uptake and delay of both efflux and growth were directly proportional to the difference between the concentrations used for induction and incubation. Active nickel ion uptake was seen in cells taken from cultures in the delayed efflux period.     

Wiring of PQQ-dehydrogenases

The performance of pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase (ADH) and two types of PQQ-glucose dehydrogenases in solution and when immobilized on the carbon paste electrodes modified with ferrocene derivatives is investigated. The immobilization of ADH consisting of PQQ and four hemes improves its stability up to 10 times. Both PQQ and heme moieties are involved in the electron transport from substrate to electrode. The ferrocene derivatives improve the electron transport 10-fold. Membrane-bound alcohol dehydrogenase from Gluconobacter sp. 33, intracellular soluble glucose dehydrogenase from Acinetobacter calcoaceticus L.M.D. 79.41 (s-GDH), and the membrane-bound enzyme (m-GDH) from Erwinia sp. 34-1 were purified and investigated. Soluble and membrane-bound PQQ-glucose dehydrogenases display different behavior during the immobilization on the modified carbon electrodes. The immobilization of s-GDH leads to a decrease in both stability and substrate specificity of the enzyme. This suggests that PQQ dissociates from the enzyme active center and operates as a free-diffusing mediator. The rate-limiting step of the process is likely the loading of PQQ onto the apo-enzyme. The immobilization of m-GDH leads to its substantial stabilization and improves the substrate specificity. The nature of m-GDH binding to the electrode surface is presumably similar to the binding to the cell membrane through its anchor-subunit. The enzyme operates as an enzyme and mediator complex.     

Human testis/sperm-specific histone H2B (hTSH2B). Molecular cloning and characterization

Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions. Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B). This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents. Using genomic PCR, two genetic alleles of hTSH2B were found in the human population. The hTSH2B gene is transcribed exclusively in testis, and the corresponding protein is also present in mature sperm. We expressed recombinant hTSH2B and identified this protein with a particular H2B subtype expressed in vivo. The subnuclear distribution of H2B variants in sperm was determined using biochemical fractionation and immunoblotting. The H2B variant associated with telomere-binding activity () was solubilized by Triton X-100 or micrococcal nuclease extraction, whereas hTSH2B was relatively tightly bound in nuclei. Immunofluorescence showed that hTSH2B was concentrated in spots located at the basal nuclear area of a subpopulation (20% of cells) of mature sperm. This fact may be of particular importance, because the hTSH2B "positive" and "negative" sperm cells may undergo significantly different decondensation processes following fertilization.     

SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.     

Selecting neural networks for a committee decision

To improve recognition results, decisions of multiple neural networks can be aggregated into a committee decision. In contrast to the ordinary approach of utilizing all neural networks available to make a committee decision, we propose creating adaptive committees, which are specific for each input data point. A prediction network is used to identify classification neural networks to be fused for making a committee decision about a given input data point. The jth output value of the prediction network expresses the expectation level that the jth classification neural network will make a correct decision about the class label of a given input data point. The proposed technique is tested in three aggregation schemes, namely majority vote, averaging, and aggregation by the median rule and compared with the ordinary neural networks fusion approach. The effectiveness of the approach is demonstrated on two artificial and three real data sets.     

Use of a large multiparent wheat mapping population in genomic dissection of coleoptile and seedling growth

Identification of alleles towards the selection for improved seedling vigour is a key objective of many wheat breeding programmes. A multiparent advanced generation intercross (MAGIC) population developed from four commercial spring wheat cultivars (cvv. Baxter, Chara, Westonia and Yitpi) and containing ca. 1000 F₂‐derived, F_6:7 RILs was assessed at two contrasting soil temperatures (12 and 20 °C) for shoot length and coleoptile characteristics length and thickness. Narrow‐sense heritabilities were high for coleoptile and shoot length (h² = 0.68–0.70), indicating a strong genetic basis for the differences among progeny. Genotypic variation was large, and distributions of genotype means were approximately Gaussian with evidence for transgressive segregation for all traits. A number of significant QTL were identified for all early growth traits, and these were commonly repeatable across the different soil temperatures. The largest negative effects on coleoptile lengths were associated with Rht‐B1b (?8.2%) and Rht‐D1b (?10.9%) dwarfing genes varying in the population. Reduction in coleoptile length with either gene was particularly large at the warmer soil temperature. Other large QTL for coleoptile length were identified on chromosomes 1A, 2B, 4A, 5A and 6B, but these were relatively smaller than allelic effects at the Rht‐B1 and Rht‐D1 loci. A large coleoptile length effect allele (a = 5.3 mm at 12 °C) was identified on chromosome 1AS despite the relatively shorter coleoptile length of the donor Yitpi. Strong, positive genetic correlations for coleoptile and shoot lengths (r_g = 0.85–0.90) support the co‐location of QTL for these traits and suggest a common physiological basis for both. The multiparent population has enabled the identification of promising shoot and coleoptile QTL despite the potential for the confounding of large effect dwarfing gene alleles present in the commercial parents. The incidence of these alleles in commercial wheat breeding programmes should facilitate their ready implementation in selection of varieties with improved establishment and early growth.     

Comparative study of surface plasmon resonance, electrochemical and electroassisted chemiluminescence methods based immunosensor for the determination of antibodies against human growth hormone

An indirect immunoassay format with human growth hormone (hGH) immobilized on the self-assembled monolayer (SAM) modified surface plasmon resonance (SPR) chip has been shown to detect specific anti-hGH antibodies using the combination of three different physical phenomena in the same channel of the SPR analyzer. For the enhancement of analytical signal and sensitivity of the immunosensor horseradish peroxidase (HRP) labeled secondary antibodies, specifically interacting with the formed immune complexes, were used. The electroassisted chemiluminescence (ECL) protocol offered the limit of detection (LOD) as low as 0.061 nM and this result was very similar to that obtained by SPR, which was 0.051 nM. In the case of anti-hGH detection using pulsed amperometry (PA) with 3,3',5,5'-tetramethylbenzidine (TMB) and H(2)O(2) in the electrochemical system the LOD was the lowest - 0.027 nm. Lower reproducibility of the analytical signal and higher limit of detection was observed using cyclic voltammetry (CV) where LOD was 0.056 nM. PA detection shows 1.89, 2.07 and 2.26 times higher sensitivity if compared with SPR, CV and ECL, respectively. This work demonstrates successful simultaneous exploitation of several techniques to detect the specific anti-hGH antibodies using indirect immunoassay format on the same area of the SPR-chip.     

Amperometric immunosensor for diagnosis of BLV infection

A new amperometric immunosensor for detection of antibodies against bovine leukemia protein (gp51) was designed. The detection of antibody-antigen complex formation was based on application of secondary antibodies labeled with horseradish peroxidase (HRP). Ferrocenecarboxylic acid (FCA) and N,N,N',N'-tetramethylbenzidine (TMB) were selected as suitable mediators for this immunosensor. Optimal conditions for amperometric detection were found. Sensitivity of created system was compared with the results of enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) reaction, and was sufficient for detection of usual anti-gp51 antibody concentration present in the blood serum of BLV-infected cattle.     

A critical role of redox state in determining HL-60 cell granulocytic differentiation and apoptosis via involvement of PKC and NF-κB

The modifications of intracellular redox balance leads to important cellular changes in many cell types. Here, a causal relationship among redox state, granulocytic differentiation induced by all-trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) and apoptosis have been studied in the human acute promyelocytic leukaemia HL-60 cells. The modulation of intracellular reactive oxygen species levels by d, l-buthionine-(S, R) sulfoximide (BSO), and n-acetyl-l-cysteine (NAC) caused inducer- and time-dependent or stage-specific effects on HL-60 cell growth inhibition, differentiation and subsequent apoptosis. The presence of BSO during the commitment stage suppressed RA—but not dbcAMP-mediated differentiation, while NAC inhibited both. BSO alone and in combination with RA or dbcAMP-induced apoptosis, which was prevented by NAC in dbcAMP—but not in RA-treated cells. Using protein kinase C inhibitor, calphostin C, cross-talk effects between the intracellular redox state and PKC signalling was identified by demonstrating inducer-dependent changes in cell differentiation or apoptosis, which were associated with the changes in DNA-NF-κB binding activity. These observations suggest a critical role of redox state in determining HL-60 cell behaviour and provide new insights into the complex effects of redox perturbations on the intracellular signalling network via the involvement of PKC and NF-κB.