Screening environmental samples for source-specific bacteriophage hosts using a method for the simultaneous pouring of 12 petri plates

A simple apparatus was developed to allow 12 petri plates to be poured simultaneously by hand. It was used when screening bacterial isolates from sewage and dog feces for their ability to detect phages from these sources. This was done to assess the ease with which source-specific phage hosts can be isolated from these sources of fecal pollution. Host bacteria that consistently detected phages from sewage were easily isolated from sewage. These bacterial isolates did not detect phages from dog feces. Host bacteria were not isolated from dog feces even after screening hundreds of colonies from fecal samples from six dogs. Journal of Industrial Microbiology & Biotechnology (2000) 24, 124–126. Received 06 July 1999/ Accepted in revised form 05 November 1999     

Inactivation and sedimentation of mercury from organomercurials by Klebsiella pneumoniae

Abstract A susceptibility of 63 clinical isolates of Klebsiella pneumoniae to inorganic and organic mercuric compounds was determined. 18 of them were found to be resistant to fluorescein mercuric acetate (FMA) and merbromin (MB). Moreover, all the resistant strains inactivate the antibacterial effect of FMA. The changes in the amount of organic mercury at the time of inactivation of the drug and the structures of the end products were examined in detail with the plasmid-bearing strain JK9 and its transconjugants of Escherichia coli .
The results showed that FMA was inactivated by an intracellular enzyme produced inducively and was degraded to fluorescein (sodium salt, uranine), which led to the sedimentation of metallic mercury. The discovery of the genes conferring inducible organic mercury-inactivating enzymes determined by plasmids was the next step and their application in the recovery of metallic mercury from organomercurials is now imminent.     

Some improvements in the quality of NAA procedures and the reliability of the results

After considering the need for quality control in NAA, the concept of quality in NAA procedures themselves is discussed, and some important factors identified. Two approaches to improve quality are then described in more detail. The first concerns the unique ability of NAA using different isotopic reactions and different modes (INAA/RNAA) to provide independent data sets in the same laboratory, thus allowing internal validation or crosschecking. The second discusses the need for chemical yield measurements in RNAA and the advantages of the radioisotopic tracer technique. Some recent advances and further possibilities for this use of tracers are listed.     

Rapid Determination of Conidial Viability for Entomopathogenic Hyphomycetes Using Fluorescence Microscopy Techniques

The viability of conidia from two species of deuteromycetes fungi pathogenic to insects was determined using two fluorochrome stains, fluorescein diacetate (FDA) and propidium iodide (PI). These stains were used either alone or in combination, and results were compared with standard conidial germination tests. FDA fluoresces bright green in viable conidia and PI fluoresces red in non-viable conidia, when viewed using specific fluorescence microscopic techniques. Conidia from two isolates of Paecilomyces fumosoroseus (Wize) Brown and Smith and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated. Conidia were suspended in deionized water and half of each suspension was treated with microwave radiation to kill all the conidia. Conidia were tested for viability in non-microwaved suspensions in a mixture (ca. 1:1) of viable and non-viable conidial suspensions, and in the microwaved suspensions that contained all non-viable conidia. No significant differences were observed for the four isolates tested between germination tests on water and agar and viability tests conducted with FDA alone or FDA in combination with PI. One isolate of B. bassiana that had been damaged in storage was also tested. Differences were observed between the actual germination and the percentage of viability determined using FDA or FDA plus PI. Damaged conidia maintained a measure of viability and fluoresced green, but did not fully germinate.     

Evidence that the Loss of the Voltage-Dependent Mg2+ Block at the N-Methyl-D-Aspartate Receptor Underlies Receptor Activation During Inhibition of Neuronal Metabolism

Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the synaptic vesicle preparation is demonstrated.     

A guide to metabolic flux analysis in metabolic engineering: Methods,tools and applications

The field of metabolic engineering is primarily concerned with improving the biological production of value-added chemicals, fuels and pharmaceuticals through the design, construction and optimization of metabolic pathways, redirection of intracellular fluxes, and refinement of cellular properties relevant for industrial bioprocess implementation. Metabolic network models and metabolic fluxes are central concepts in metabolic engineering, as was emphasized in the first paper published in this journal, “Metabolic fluxes and metabolic engineering” (Metabolic Engineering, 1: 1–11, 1999). In the past two decades, a wide range of computational, analytical and experimental approaches have been developed to interrogate the capabilities of biological systems through analysis of metabolic network models using techniques such as flux balance analysis (FBA), and quantify metabolic fluxes using constrained-based modeling approaches such as metabolic flux analysis (MFA) and more advanced experimental techniques based on the use of stable-isotope tracers, i.e. ¹³C-metabolic flux analysis (¹³C-MFA). In this review, we describe the basic principles of metabolic flux analysis, discuss current best practices in flux quantification, highlight potential pitfalls and alternative approaches in the application of these tools, and give a broad overview of pragmatic applications of flux analysis in metabolic engineering practice.     

Expression and functional analysis of flotillins in Dugesia japonica

FLOTILLIN-1 and FLOTILLIN-2 are membrane rafts associated proteins that have been implicated in insulin and growth factor signaling, endocytosis, cell migration, proliferation, differentiation, cytoskeleton remodeling and membrane trafficking. Furthermore, FLOTILLINs also play important roles in the progression of cancer and neurodegenerative diseases. In this study, the roles of flotillins are investigated in planarian Dugesia japonica. The results show that Djflotillin-1 and Djflotillin-2 play a key role in homeostasis maintenance and regeneration process by regulating the proliferation of the neoblast cells, they are not involved in the maintenance and regeneration of the central nervous system in planarians.     

The role of multidrug resistance protein 1 (MRP1) in transport of fluorescent anions across the human erythrocyte membrane

We employed human red blood cells as a model system to check the affinity of MRP1 (Multidrug Resistance-associated Protein 1) towards fluorescein and a set of its carboxyl derivatives: 5/6-carboxyfluorescein (CF), 2,7-bis-(2-carboxyethyl)-5/6-carboxyfluorescein (BCECF) and calcein (CAL). We found significant differences in the characteristics of transport of the dyes tested across the erythrocyte membrane. Fluorescein is transported mainly in a passive way, while active efflux systems at least partially contribute to the transport of the other compounds. Inside-out vesicle studies revealed that active transport of calcein is masked by another, ATP-independent, transport activity. Inhibitor profiles of CF and BCECF transport are typical for substrates of organic anion transporters. BCECF is transported mainly via MRP1, as proven by the use of QCRL3, a monoclonal antibody known to specifically inhibit MRP1-mediated transport. Lack of effect of QCRL3 on CF uptake excludes the possibility of MRP1 being a transporter of this dye. No inhibition of CF accumulation by cGMP, thioguanine and 6-mercaptopurine suggests also that this fluorescent marker is not a substrate for MRP5, another ABC transporter identified in the human erythrocyte membrane.     

Automated large-volume sample stacking procedure to detect labeled peptides at picomolar concentration using capillary electrophoresis and laser-induced fluorescence detection

We have developed an automated large-volume sample stacking (LVSS) procedure to detect fluorescein isothiocyanate-labeled peptides in the picomolar range. The injection duration is 10 min at 50 mbar to fill 62% of the capillary volume to the detection cell. The calculated limit of detection (S/N=3), filling 1% of the capillary volume, is 74 pM for bradykinin and 45 pM for L-enkephalin with samples diluted in water and analyzed in a 50 mM borate buffer, pH 9.2. With the automated LVSS system, the limits of detection are 7 pM for bradykinin, 3 pM for L-enkephalin and 2 pM for substance P. LVSS is shown to be quantitative from 500 to 10 pM.