Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Even so, the lack of differentiation between DNA from viable and dead cells is one of the major challenges for diagnostic DNA-based methods. Certain nucleic acid-binding dyes can selectively enter dead bacteria and subsequently be covalently linked to DNA. Ethidium monoazide (EMA) is a DNA intercalating dye that enters bacteria with damaged membranes. This dye can be covalently linked to DNA by photoactivation. Our goal was to utilize the irreversible binding of photoactivated EMA to DNA to inhibit the PCR of DNA from dead bacteria. Quantitative 5'-nuclease PCR assays were used to measure the effect of EMA. The conclusion from the experiments was that EMA covalently bound to DNA inhibited the 5'-nuclease PCR. The maximum inhibition of PCR on pure DNA cross-linked with EMA gave a signal reduction of approximately -4.5 log units relative to untreated DNA. The viable/dead differentiation with the EMA method was evaluated through comparison with BacLight staining (microscopic examination) and plate counts. The EMA and BacLight methods gave corresponding results for all bacteria and conditions tested. Furthermore, we obtained a high correlation between plate counts and the EMA results for bacteria killed with ethanol, benzalkonium chloride (disinfectant), or exposure to 70 degrees C. However, for bacteria exposed to 100 degrees C, the number of viable cells recovered by plating was lower than the detection limit with the EMA method. In conclusion, the EMA method is promising for DNA-based differentiation between viable and dead bacteria.     

Development of Agrobacterium tumefaciens C58-induced plant tumors and impact on host shoots are controlled by a cascade of jasmonic acid,auxin, cytokinin,ethylene and abscisic acid

The development of Agrobacterium tumefaciens-induced plant tumors primarily depends on the excessive production of auxin and cytokinin by enzymes encoded on T-DNA genes integrated into the plant genome. The aim of the present study was to investigate the involvement of additional phytohormone signals in the vascularization required for rapid tumor proliferation. In stem tumors of Ricinus communis L., free auxin and zeatin riboside concentrations increased within 2 weeks to 15-fold the concentrations in control stem tissue. Auxin and cytokinin immunolocalization revealed the highest concentrations within and around tumor vascular bundles with concentration gradients. The time-course of changes in free auxin concentration in roots was inversely correlated with that in the tumors. The high ethylene emission induced by increased auxin- and cytokinin correlated with a 36-fold accumulation of abscisic acid in tumors. Ethylene emitted from tumors and exogenously applied ethylene caused an increase in abscisic acid concentrations also in the host leaves, with a diminution in leaf water vapor conductance. Jasmonic acid concentration reached a maximum already within the first week of bacterial infection. A wound effect could be excluded. The results demonstrate the concerted interaction of a cascade of transiently induced, non-T-DNA-encoded phytohormones jasmonic acid, ethylene and abscisic acid with T-DNA-encoded auxin and zeatin riboside plus trans-zeatin, all of which are required for successful plant tumor vascularization and development together with inhibition of host plant growth.     

Sequence analysis and characterization of plasmids from Streptococcus thermophilus

The nucleotide sequences of eight plasmids isolated from seven Streptococcus thermophilus strains have been determined. Plasmids pSt04, pER1-1, and pJ34 are related and replicate via a rolling circle mechanism. Plasmid pJ34 encodes for a replication initiation protein (RepA) and a small polypeptide with unknown function. Plasmids pSt04 and pER1-1 carry in addition to repA genes coding for small heat shock proteins (sHsp). Expression of these proteins is induced at elevated temperatures or low pH and increases the thermo- and acid resistance. Plasmids pER1-2 and pSt22-2 show identical sequences with five putative open reading frames (ORFs). The gene products of ORF1 and ORF4 reveal some similarities to transposon encoded proteins of Bacillus subtilis and Tn916. ORF1 of plasmid pSt106 encodes a protein similar to resolvases of different Gram-positive bacteria. Integrity of ORF2 and 3, encoding a putative DNA primase and a replication protein, is essential for replication. ORF1 to 3 of plasmid pSt08, which are organized in a tricistronic operon, encode a RepA protein, an adenosine-specific methyltransferase, and a type II restriction endonuclease. Another type II restriction-modification (R/M) system is encoded on plasmid pSt0 which is highly similar to those encoded on lactococcal plasmid pHW393 and B. subtilis plasmid pXH13. Plasmid-free derivatives of strains St0 and St08 show increased phage sensitivity, indicating that in the wild-type strains the R/M systems are functionally expressed. Recombinant plasmids based on the replicons of plasmids pSt04, pJ34, pSt106, pSt08, and pSt0, are able to replicate in Lactococcus lactis and B. subtilis, respectively, whereas constructs carrying pER1-2 only replicate in S. thermophilus.     

The role of peptide deformylase in protein biosynthesis: a proteomic study

Recently we investigated the influence of classical and emerging antibiotics on the proteome of Bacillus subtilis including in our studies actinonin, a potent novel inhibitor of peptide deformylase. The protein synthesis pattern under actinonin treatment changed so dramatically that a direct comparison to the control pattern was impossible. Dual channel imaging revealed that actinonin treatment caused the majority of newly synthesised proteins to accumulate in spots different from the ones usually observed, indicating a more acidic isoelectric point. Two strategies were used to investigate the nature of the charge shift. In the first place, protein patterns of a conditional peptide deformylase mutant under nonrepressing and repressing conditions were compared. Secondly, several protein pairs excised from two-dimensional (2-D) gels of the peptide deformylase mutant, exponentially growing untreated wild-type and the actinonin treated wild-type were investigated with matrix-assisted laser desorption/ionization and electrospray ionization (ESI) time of flight mass spectrometry (TOF MS) for the existence of N-terminal formylation. Under nonrepressing conditions the mutant protein pattern resembled that of the wild-type. The loss of peptide deformylase activity under repressing conditions led to the same pI shift observed for actinonin treatment in the wild-type. Quadrupole TOF-MS on 11 protein pairs proved that the remaining N-terminal formyl residue was indeed responsible for the charge shift. Eight of these protein pairs were also present on 2-D gels of exponentially growing B. subtilis, where the more acidic, still formylated protein species represented the smaller parts.     

Aprotinin uptake in the proximal tubules in the rat kidney. II. Uptake site relative to glomerulus

Glomerular filtration rates in whole kidney and in outer, middle and inner cortical zones have previously been estimated by measuring the amount of iodinated Aprotinin, filtered and taken up in the first two thirds of the proximal convoluted tubules, in part positioned more superficial than the parent glomerulus. Thus, an appreciable amount of the absorbed Aprotinin may be located superficial to its filtration site and lead to an underestimate of glomerular filtration in deep cortical layers. Therefore, in this study we have measured the distance from the glomerulus to the center of proximal convoluted tubular ball and the site of Aprotinin uptake. Measurements were made on photos of Microfil-injected tubules and on camera lucida drawings of tubular transections from autoradiographs of nephrons containing both Microfil and iodinated Aprotinin. Both techniques showed that the center of the tubular ball was localized more superficial in all cortical layers. The average distance, in percent of cortical thickness, from all proximal convoluted tubular transections to the parent glomerulus was 9% in deep and 13% in middle and superficial cortex. Corresponding distances for tubular transections containing Aprotinin were 7 and 12%. Grain density in five reconstructed proximal convoluted tubules showed a continuous and exponential fall of Aprotinin along the uptake segment. The results may be used to estimate single nephron filtration rate from Aprotinin uptake and glomerular density in outer, middle, and inner cortex.     

Delayed leukocytosis after hard strength and endurance exercise: Aspects of regulatory mechanisms

Background  

During infections, polymorphonuclear neutrophilic granulocytes (PMN) are mobilized from their bone marrow stores, travel with blood to the affected tissue, and kill invading microbes there. The signal(s) from the inflammatory site to the marrow are unknown, even though a number of humoral factors that can mobilize PMN, are well known. We have employed a standardized, non-infectious human model to elucidate relevant PMN mobilizers. Well-trained athletes performed a 60-min strenuous strength workout of leg muscles. Blood samples were drawn before, during and just after exercise, and then repeatedly during the following day. Cortisol, GH, ACTH, complement factors, high-sensitive CRP (muCRP), IL-6, G-CSF, IL-8 (CXCL8) and MIP-1β (CCL4) were measured in blood samples. PMN chemotaxins in test plasma was assessed with a micropore membrane technique.     

High-density tissue-like cultivation of JAR choriocarcinoma cells for the in vitro production of human xylosyltransferase

Human xylosyltransferase is the chain-initiating enzyme involved in the biosynthesis of glycosaminoglycans. Large amounts of xylosyltransferase are required to study the biochemical properties of the native enzyme. To achieve this goal a scale-up of animal cell culture systems was inevitable due to the small amounts of the enzyme present in tissues, e.g. only 0.5 microg XT can be obtained from a chick embryo. JAR choriocarcinoma cells cultured with 10% fetal calf serum were found to secrete xylosyltransferase with relatively high activities (1.10 mU l(-1)). To reduce contaminating proteins JAR cells were adapted to serum-free conditions. Xylosyltransferase activities up to 0.22 mU l(-1) were determined in the harvested cell culture supernatant. Scaling-up of JAR cell culture in the hybrid hollow fiber bioreactor Tecnomouse resulted in the production of 15.8 mU or 270 microg XT in 0.5 l of XT-enriched cell culture supernatant using 57 l of serum-free cell culture medium. The XT activity per ml harvest solution was 200-280-fold higher in this cell culture supernatant than in cell culture flasks. In addition, the specific XT activity of the bioreactor product was 6 microU mg(-1) of total protein, which is 2-fold higher than that obtained under static culture conditions. This study clearly demonstrates the successful high-density, tissue-like cultivation of JAR choriocarcinoma cells in a hollow fiber bioreactor resulting in an effective production of native human xylosyltransferase.     

Six-fold speed-up of Smith-Waterman sequence database searches using parallel processing on common microprocessors

MOTIVATION: Sequence database searching is among the most important and challenging tasks in bioinformatics. The ultimate choice of sequence-search algorithm is that of Smith-Waterman. However, because of the computationally demanding nature of this method, heuristic programs or special-purpose hardware alternatives have been developed. Increased speed has been obtained at the cost of reduced sensitivity or very expensive hardware. RESULTS: A fast implementation of the Smith-Waterman sequence-alignment algorithm using Single-Instruction, Multiple-Data (SIMD) technology is presented. This implementation is based on the MultiMedia eXtensions (MMX) and Streaming SIMD Extensions (SSE) technology that is embedded in Intel's latest microprocessors. Similar technology exists also in other modern microprocessors. Six-fold speed-up relative to the fastest previously known Smith-Waterman implementation on the same hardware was achieved by an optimized 8-way parallel processing approach. A speed of more than 150 million cell updates per second was obtained on a single Intel Pentium III 500 MHz microprocessor. This is probably the fastest implementation of this algorithm on a single general-purpose microprocessor described to date.