Regulation of the homologous two-component systems KvgAS and KvhAS in Klebsiella pneumoniae CG43

In Klebsiella pneumoniae CG43, deletion of the sensor gene kvgS reduced the kvgAS expression in M9 medium with 0.2 mM paraquat, 0.2 mM 2,2-dihydropyridyl, or 300 mM NaCl. This result shows an autoregulatory role of KvgS and a stress-responsive expression of the two-component system (2CS). The kvgS deletion also appeared to decrease the expression of kvhAS, paralogous genes of kvgAS. Additionally, measurements of the promoter activity in kvgA(-) mutant revealed that KvgA is probably an activator for the expression of kvgAS and kvhAS. The subsequent electrophoretic mobility shift assay, indicating a specific binding of the recombinant KvgA to the putative promoters P(kvgAS) and P(kvhAS), also supported an interacting regulation between the 2CSs. In P(kvgAS) and P(kvhAS), the presence of RpoS binding elements suggested an RpoS-dependent regulation. Nevertheless, the rpoS deletion reduced the expression of kvgAS but increased that of kvhAS. Moreover, the kvgA deletion reduced the expression of katG and sodC. The overexpression of KvhA altered the susceptibility to fosfomycin and an increasing activity of UDP-N-acetylglucosamine enolpyruvyl transferase, the target protein of fosfomycin, which suggesting a regulation by KvhA. Taken together, these indicated that the two 2CSs probably belong to different regulatory circuits of the RpoS regulon.     

Comparative assessment of the resistance of the unstirred water layer to solute transport between two different intestinal perfusion systems

The resistance of the unstirred water layer to solute transport was estimated in two different intestinal single-pass perfusion systems for a comparative study, using D-glucose as a model compound. One is a well established perfusion system in anesthetized rats as a standard (system A). The other is the one in unanesthetized rats for comparison (system B). It was demonstrated that in system B as well as in system A the resistance of the unstirred water layer to D-glucose transport should be taken into account and this resistance, accordingly, the effective thickness of the unstirred water layer (delta) which is assumed to be in proportion to its resistance, could be described as a function of the perfusion rate by using a film model. The delta decreased with increasing perfusion rate and was larger in system A than in system B at each perfusion rate; 785 microns in system A versus 319 microns in system B at the perfusion rate of 0.16 ml/min and 337 microns versus 184 micron at that of 2.95 ml/min. Thus in system B the effective thickness, accordingly, the resistance, of the unstirred water layer was reduced to about 50% of that in system A, but the resistance of the unstirred water layer could still account for 85% of the total resistance at the maximum as far as D-glucose absorption was concerned, while 93% in system A. These results suggest that, compared with perfusion experiments in anesthetized rats (system A), the resistance of the unstirred water layer is reduced but cannot be left out of consideration even if perfusion experiments are performed in unanesthetized rats (system B). And the lower resistance of the unstirred water layer in system B was attributed to a turbulent flow in contrary to a laminar flow in system A.     

Escherichia coli K-12 mutants that allow transport of maltose via the beta-galactoside transport system.

We have isolated mutants of Escherichia coli that have an altered beta-galactoside transport system. This altered transport system is able to transport a sugar, maltose, that the wild-type beta-galactoside transport system is unable to transport. The mutation that alters the specificity of the transport system is in the lacY gene, and we refer to the allele as lacYmal. The lacYmal allele was detected originally in strains in which the lac genes were fused to the malF gene. Thus, as a result of gene fusion and isolation of the lacYmal mutation, a new transport system was evolved with regulatory properties and specificity similar to those of the original maltose transport system. Maltose transport via the lacYmal gene product is independent of all of the normal maltose transport system components. The altered transport system shows a higher affinity than the wild-type transport system for two normal substrates of the beta-galactoside transport system, thiomethyl-beta-D-galactoside and o-nitrophenyl-beta-D-galactoside.     

A novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts

Most chloroplast and mitochondrial precursor proteins are targeted specifically to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was specifically targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre-sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre-sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre-sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre-sequence has dual targeting ability, this ability is dependent on the nature of the mature protein.     

Recirculating bioreactor-separator system for simultaneous biotransformation and recovery of product: Immobilized L-aspartate beta-decarboxylase reactor system

A new combined bioreactor-separator system was designed and its operational feasibility demonstrated in order to develop a bioprocess that enables us to handle simultaneous biotransformation and recovery of product by crystallization. Enzymatic conversion of L-aspartate to L-alanine by L-aspartate beta-decarboxylase from Pseudomonas dacunhae (ATCC 21192) was used as a model system for this study to demonstrate the principles involved in the bioprocess design. Immobilized cells of P. dacunhae containing the enzyme were fluidized in a tapered column type of bioreactor and a filter-crystallizer combination was used as a separator unit in our experimental system.It was found that almost a theoretical yield was achieved, and the process control for both the bioreactor operation and separation was relatively easy. The Production systems, namely, the recirculating bioreactor separator combination system and the conventional batch reactor system, were analyzed and compared based on the results obtained form this study, and it was found that a significant cost reduction, by about 20%, can be achieved when the recirculating bioreactor-separator combination system was employed. Based on these findings, it is anticipated that the conceptual design of the bioreactor-separator combination system evaluated in this study has some potential for industrial application.     

Multiplicity of aspartate transport in thin wastewater biofilms.

This research documents the multiplicity of L-aspartate transport in thin wastewater biofilms. A Line-weaver-Burk analysis of incorporation produced a curvilinear plot (concave down) that suggested active transport by two distinct systems (1 and 2). The inactivation of system 2 with AsO4 or osmotic shock resolved system 1, which was a high-affinity, low-capacity system with an apparent Kt (Michaelis-Menten constant) of 4.3 microM (AsO4) or 4.6 microM (osmotic shock). The inactivation of system 1 with dinitrophenol resolved system 2, which was a low-affinity, high-capacity system with an apparent Kt of 116.7 microM. System 1 was more specific for aspartate than system 2 in the presence of aspartate analogs. Sodium had no discernible effect on the incorporation velocities by either system. These results indicate that system 1 is a membrane-bound proton symport coupled to the proton gradient component of the proton motive force and that system 2 is a binding protein-mediated system coupled to phosphate bond energy. Analyses of diffusional limitations on the derived transport constants indicated that internal resistances were present but that the apparent constants were close to the intrinsic values, especially for system 1. Metabolic inactivation of the biofilm with dinitrophenol and AsO4 did not completely inactivate aspartate incorporation, which indicated that some simple adsorption of the aspartate anion by the biofilm had occurred. These results show that aspartate is transported by wastewater biofilm bacteria via systems with different affinities, specificities, and mechanisms of energy coupling.     

Selection of chemotherapy for patient treatment utilizing a radiometric versus a cloning system

From the 1950s to the 1970s, a number of in vitro systems that measured inhibition of glucose metabolism were used to predict the responsiveness of patients' tumors to chemotherapy. In vitro-in vivo correlations were excellent, with true positive predictions ranging from 68% to 96% and true negative predictions of 95% to 100%. The radiometric system is a new in vitro technique that measures the conversion of 14C-glucose to 14CO2. The system already has been utilized to screen prospective new antineoplastic agents for cytotoxicity. The present study was undertaken to determine if the radiometric system might be used to predict correctly the responsiveness of an individual patient's tumor to single-agent or combination-agent chemotherapy. Fifty-six tumor specimens were divided and tested for drug sensitivity in the radiometric system and a conventional human tumor clonning system. Overall, there was a significant correlation between in vitro and in vivo results for the conventional cloning system (P = 0.03). However, there was no significant relationship between in vitro and in vivo results for the radiometric system. The radiometric system consistently failed to predict the tumor's clinical sensitivity to single agents. A radiometric system is not useful in predicting the responsiveness of a patient's tumor to single agent chemotherapy and is not a replacement for the more biologically attractive human tumor cloning system.     

Multiplicity of aspartate transport in thin wastewater biofilms

This research documents the multiplicity of L-aspartate transport in thin wastewater biofilms. A Line-weaver-Burk analysis of incorporation produced a curvilinear plot (concave down) that suggested active transport by two distinct systems (1 and 2). The inactivation of system 2 with AsO4 or osmotic shock resolved system 1, which was a high-affinity, low-capacity system with an apparent Kt (Michaelis-Menten constant) of 4.3 microM (AsO4) or 4.6 microM (osmotic shock). The inactivation of system 1 with dinitrophenol resolved system 2, which was a low-affinity, high-capacity system with an apparent Kt of 116.7 microM. System 1 was more specific for aspartate than system 2 in the presence of aspartate analogs. Sodium had no discernible effect on the incorporation velocities by either system. These results indicate that system 1 is a membrane-bound proton symport coupled to the proton gradient component of the proton motive force and that system 2 is a binding protein-mediated system coupled to phosphate bond energy. Analyses of diffusional limitations on the derived transport constants indicated that internal resistances were present but that the apparent constants were close to the intrinsic values, especially for system 1. Metabolic inactivation of the biofilm with dinitrophenol and AsO4 did not completely inactivate aspartate incorporation, which indicated that some simple adsorption of the aspartate anion by the biofilm had occurred. These results show that aspartate is transported by wastewater biofilm bacteria via systems with different affinities, specificities, and mechanisms of energy coupling.     

Distribution of the Phosphoenolpyruvate:Glucose Phosphotransferase System in Bacteria

A survey of the occurrence of the phosphoenolpyruvate-dependent glucose phosphotransferase system was carried out in a number of bacteria, representing both gram-positive and gram-negative facultative anaerobic and strictly aerobic types. The system was found to be present in representatives of genera that are characteristically facultative anaerobes, but the system was absent in members of those genera that are strictly aerobic. Thus, although the phosphoenolpyruvate phosphotransferase system is an important system for the transport of sugars in bacteria carrying out anaerobic glycolysis, it plays no role in sugar transport by those organisms having a strictly oxidative physiology. A fundamentally different system, probably not involving phosphorylation during transport, is indicated in this latter group.     

An improved culture system for virus isolation and detection

Cell culture plays an important role in virology. It provides a platform for the detection and isolation of viruses as well as for the biochemistry and molecular biology based studies of viruses. In the present work, a new system that could permits multiple (different) cell lines to be simultaneously cultured in one dish was developed. In the system, each cell line was cultured in an isolated zone in the same dish or well and the system is therefore called an isolated co-culture system. The usefulness of this novel approach for virus isolation was demonstrated using a model system based on adenovirus.     

Relationships between a new type IV secretion system and the icm/dot virulence system of Legionella pneumophila

We describe here a Legionella pneumophila type IV secretion system that is distinct from the previously described icm/dot system. This type IV secretion system contains 11 genes (lvh ) homologous to genes of other type IV secretion systems, arranged in a similar manner. The lvh genes were found to be located on a DNA island with a GC content higher than the L. pneumophila chromosome. In contrast to the icm/dot system that was shown to be required for intracellular growth in HL-60-derived human macrophages and Acanthamoeba castellanii, the lvh system was found to be dispensable for intracellular growth in these two hosts. The lvh system was found to be partially required for RSF1010 conjugation, a process that was previously shown to be completely dependent on several icm/dot genes. However, results obtained from analysis of double mutants in the icm/dot genes and the lvh genes revealed that lvh genes can substitute for some components of the icm/dot system for RSF1010 conjugation, but not for intracellular growth. These results indicate that components of the icm/dot system and components of the lvh type IV secretion system are able to interact with one another.     

Glucose transport by mixed ruminal bacteria from a cow.

The glucose transport of mixed ruminal bacteria harvested from a holstein cow fed 5.0 kg of Italian ryegrass and 1.5 kg of flaked corn a day was investigated. The Eadie-Hofstee plot characterized two transport systems: a high-affinity, low-velocity system and a low-affinity, high-velocity system. The former system (K(m) = 16 microM; Vmax = 2.2 nmol/min/mg of protein) is considered dominant under this feeding condition based on the glucose concentration in the rumen (< 1 mM). In light of the facts that the protonophore SF6847 and the lipophilic triphenylmethyl phosphonium ion had no effect on the high-affinity system and an artificially generated proton gradient and electrical potential across the cell membrane did not increase glucose transport, a proton motive force is not be involved in the system. On the other hand, from the facts that chlorhexidine inhibited about 90% of the high-affinity system while iodoacetate showed no significant effect, and a high phosphoenolpyruvate-dependent phosphorylation of glucose was actually shown, the phosphoenolpyruvate-dependent phosphotransferase system is considered the main system in the high-affinity system. Moreover, as shown by the facts that harmaline inhibited about 30% of the high-affinity system and the artificially generated sodium gradient across the cell membrane significantly stimulated glucose transport, this system also includes sodium symport to some degree. The high-affinity system was sensitive to a decrease in pH (< 6.5) and was inhibited by the presence of sucrose, mannose, and fructose.     

AuNP-CTG based probing system targeting CAG repeat DNA and RNA sequences

We have developed a AuNP-CTG based probing system that is applicable to the detection of many units of CAG repeat sequences which was synthesized by a rolling circle amplification (RCA) system with changes in fluorescence. We also demonstrate that our AuNP-CTG based probing system could transfect without using transfection reagent and detect target CAG repeat sequences in HeLa cells with dramatic changes in fluorescence. This AuNP-CTG based probing system could also be used, in conjunction with the CAG repeat RCA system, to detect target DNA. This system was so sensitive to the target DNA that it could detect even picomolar amounts with amplification of the fluorescence signal. Furthermore, we have used our gold-based CAG probing system for the detection of RNA CAG repeat sequences.     

Enzymic lipid peroxidation in the microsomal fraction of rat brain

Abstract: An enzymic lipid peroxidation system has been demonstrated in the microsomal fraction of rat brain and the requirements and optimal conditions for assay determined. The involvement of NADPH-cytochrome c reductase was demonstrated in vesicles reconstituted with lipids extracted from the brain microsomal fraction. Further characterization of the system made use of substances shown to inhibit the liver microsomal system. α-Tocopherol was shown to be an effective inhibitor of lipid peroxidation in the brain microsomal system, whereas Na₂SO₃ had no effect, which is indicative that free radical transfer occurs only in the hydrophobic regions. Neither superoxide dismutase nor catalase inhibited lipid peroxidation. The implications of an NADPH-cytochrome c reductase-dependent lipid peroxidation system that is not linked to a drug hydroxylation system and appears to differ from the liver microsomal system in a number of other ways are discussed.     

Identification of the LIV-I/LS system as the third phenylalanine transporter in Escherichia coli K-12

In Escherichia coli, the active transport of phenylalanine is considered to be performed by two different systems, AroP and PheP. However, a low level of accumulation of phenylalanine was observed in an aromatic amino acid transporter-deficient E. coli strain (DeltaaroP DeltapheP Deltamtr Deltatna DeltatyrP). The uptake of phenylalanine by this strain was significantly inhibited in the presence of branched-chain amino acids. Genetic analysis and transport studies revealed that the LIV-I/LS system, which is a branched-chain amino acid transporter consisting of two periplasmic binding proteins, the LIV-binding protein (LIV-I system) and LS-binding protein (LS system), and membrane components, LivHMGF, is involved in phenylalanine accumulation in E. coli cells. The K(m) values for phenylalanine in the LIV-I and LS systems were determined to be 19 and 30 micro M, respectively. Competitive inhibition of phenylalanine uptake by isoleucine, leucine, and valine was observed for the LIV-I system and, surprisingly, also for the LS system, which has been assumed to be leucine specific on the basis of the results of binding studies with the purified LS-binding protein. We found that the LS system is capable of transporting isoleucine and valine with affinity comparable to that for leucine and that the LIV-I system is able to transport tyrosine with affinity lower than that seen with other substrates. The physiological importance of the LIV-I/LS system for phenylalanine accumulation was revealed in the growth of phenylalanine-auxotrophic E. coli strains under various conditions.     

Coupled biological and photo-Fenton pretreatment system for the removal of di-(2-ethylhexyl) phthalate (DEHP) from water

The photo-Fenton coupled with a biological system for the removal of di-(2-ethylhexyl) phthalate (DEHP) in wastewater was analyzed. The toxicity of DEHP-containing wastewater was found to be reduced after pretreatment by the photo-Fenton reaction. The effect of different factors, such as DEHP, Fe³⁺ and H₂O₂ concentrations and the reaction time, on degradation efficiency was investigated. The optimal time to stop the pretreatment process and introduce the effluent to the biological system was 60 min. The results show that effluent of DEHP-containing wastewater pretreated by the photo-Fenton method is biodegradable and that mineralization can be completed when the wastewater is subsequently treated in a biological system. The coupled Fenton and biological treatment system for the degradation of DEHP-containing wastewater can be successfully performed in a semi-continuous mode. These results indicate that the coupled photo-biological system is an effective and potential method for the treatment of DEHP-containing wastewater.     

Properties of an inducible uptake system for beta-ketoadipate in Pseudomonas putida.

Wild-type strains of Pseudomonas putida form an inducible uptake system that appears to act on beta-ketoadipate under normal physiological conditions. The system is induced by beta-ketoadipate and is represented by catabolites derived from it. Adipate is metabolized very slowly by wild-type P. putida cultures; [14C]adipate was used as an analogue of beta-ketoadipate to measure the transport activity in wild-type cells and in cells that constitutively produced the uptake system. Constitutive cells that contained high levels of the uptake system concentrated adipate to a level up to 200-fold above the concentration in the external medium. The process was energy dependent. The activity of the system with radioactive adipate was inhibited by beta-ketoadipate, by beta-ketoadipate analogues, and by some compounds (e.g., acetate, glucose) that are structurally unrelated to beta-ketoadipate; it is not known if the inhibitory effects are exerted directly by the compounds themselves or indirectly by catabolites derived from the compounds. The discovery of the beta-ketoadipate uptake system is surprising in view of earlier studies that had indicated that beta-ketoadipate does not permeate the membrane of wild-type P. putida cells. Contradictions between the former investigations and the present analysis are due primarily to the relatively high concentrations of substrate used in the earlier experiments. The existence of the beta-ketoadipate uptake system indicates that beta-ketoadipate may exist as a selective nutrient in the natural niche of P. putida and may play a determinative role in the evolution of induction mechanisms that are characteristic of fluorescent pseudomonads.     

Control of biocorrosion using laboratory and field assessments

Biofouling and biocorrosion can be significant problems in oilfield water injection systems, despite extensive use of chemical biocides. This work set out to establish relevant testing and monitoring procedures to optimise microbiological control in these systems.A combination of laboratory based sessile biocide screening trials and onsite monitoring of biofilm bacteria was developed to ensure that an effective biocontrol programme was set up. It is important to use a mixed population of bacteria freshly isolated from biofilms in the system to ensure that any biocide tolerance of the system bacteria is reflected in the laboratory tests. In general, the results are borne out by the site audits of the control regime against sessile bacteria in the system.In an industrial system, biocide resistant populations develop from time to time. In practice it was found that even a small change to the biocide formulation could improve biocontrol, with concomitant reduction in corrosion rates and maintenance costs.     

5-fluorotryptophan resistant mutants affecting the A and L transport systems in the mouse L cell line A9

Tryptophan transport has been examined in A9 and in mutants resistant to 5-fluorotryptophan (5-FT). Evidence indicates that in A9 cells two systems are present for tryptophan transport, which are analogous to the A and L systems found in Ehrlich ascites cells differing, however, in terms of amino acid specificity. Tryptophan uptake via the L system, a high affinity, low capacity system, is Na⁺ independent and occurs by a counter transport mechanism, while uptake via the A system, a low affinity, high capacity system, is Na⁺ dependent. Alanine, arginine, lysine, proline, asparagine, and aspartate (listed in order of decreasing inhibitory effect) inhibit tryptophan uptake via the A system from approximately 80-50% while having no inhibitory effect on the L system. In addition, glutamine which inhibits tryptophan uptake by 80% via the L system only inhibits to the extent of 20% via the A system. Previous kinetic studies of 5FT resistant clone FT^r37 indicated system A was altered while the analysis of the effects of the mutation on system L was inconclusive. However, in these studies Na⁺ independent uptake was not altered in FT^r 37 indicating system L was not affected. Amino acid competition studies confirmed this observation and suggested that a change in the specificity of system A had occurred in FT^r 37. The amino acid competition studies in FT^r 23, indicated that the specificities of both systems differed from A9. The possibility that this change may be due to a single mutational event is discussed.     

Relationship between values of antioxidant enzyme system activity in various tissues of intact rats]

It was studied the relation between activities of ferments an antioxidant system: of superoxide dismutase, catalase and GSH-peroxidase in the homogenates of livers, lungs and cerebrum of intact rats. When activities were brought to identical units of measurement, it was determined that relation of activities can see with a point to view of chemical kinetics laws for consecutively-parallel reactions. It is followed from the result that the activity of catalase livers can be explained by the participation of catalases in other reactions, which were connected with forming a hydrogen peroxide. From the relations between ferments of antioxidant system it was discovered that GSH-peroxidase is the most important antioxidant enzyme for the cerebrum. Data of the relation of activities ferments of antioxidant system are stipulated by the tissues particularities and they are reflected a contribution of every biocatalyst in that system.