Coexistence of antibiotic-producing and antibiotic-sensitive bacteria in biofilms is mediated by resistant bacteria

Antibiotic-sensitive bacteria have been found to coexist with antibiotic-producing bacteria in biofilms, but little is known about how the former develop in such an environment. Here we isolated pyocyanin-sensitive bacteria belonging to the genus Brevibacillus from a biofilm derived from soil extract and based on the preestablished biofilm of a pyocyanin producer, Pseudomonas aeruginosa strain P1. In addition, pyocyanin-resistant strains belonging to the genus Raoultella were isolated from the same biofilm. Microbial relationships within biofilms were examined by using three strains, strain P1, Brevibacillus strain S1, and Raoultella strain R1, each of which individually formed a biofilm within 2 days in a flow cell. Strain S1 did not fully develop on the preestablished biofilm of strain P1 during 4 days of cultivation, whereas a mutant of strain P1 which was deficient in pyocyanin production allowed strain S1 to cocolonize within a biofilm. On the other hand, strain R1 developed on the biofilm of strain P1 regardless of pyocyanin production. When mixed 1:1 inocula of strains S1 and R1 were introduced into the strain P1 biofilm, all three species were found in the 4-day biofilm. In the mixed biofilm, strain S1 was surrounded by the layer of strain R1 and seemed to be separated from strain P1 and the outflow solution. However, strain S1 did not survive in a three-species mixed culture under planktonic conditions. These results indicate that the survival of sensitive bacteria in biofilm with a pyocyanin producer is achieved by covering them with a layer of resistant bacteria. We also evaluated the influence of antibiotic production on the producer.     

Determination of glyphosate, glyphosate metabolites, and glufosinate in human serum by gas chromatography-mass spectrometry

This paper describes an assay for the determination of glyphosate (GLYP), glyphosate metabolites [(aminomethyl) phosphonic acid] (AMPA), and glufosinate (GLUF) in human serum. After protein precipitation using acetonitrile and solid-phase extraction, serum samples were derivatized and analyzed by gas chromatography-mass spectrometry (GC-MS). The assay was linear over a concentration range of 3-100.0 microg/ml for GLYP, AMPA, and GLUF. The overall recoveries for the three compounds were >73%. The intra- and inter-day variations were <15%. Precision and accuracy were 6.4-10.6% and 88.2-103.7%, respectively. The validated method was applied to quantify the GLYP and AMPA content in the serum of a GLYP-poisoned patient. In conclusion, the method was successfully applied for the determination of GLYP and its metabolite AMPA in serum obtained from patient of GLYP-poisoning.     

Determination of urinary triclosan by stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry

We have developed an analytical method for the determination of urinary 5-chloro-2-(2,4-dichlorophenoxy)phenol (triclosan), which utilizes stir bar sorptive extraction (SBSE) and thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). Human urine sample is de-conjugated by treatment with beta-glucuronidase and sulfatase. A stir bar coated with polydimethylsiloxane (PDMS) is added to the urine sample in a vial and the sample is stirred for 60 min at room temperature (25 degrees C). Then, the PDMS stir bar is subjected to TD-GC-MS. The detection limit of triclosan is 0.05 ng mL(-1). The method shows linearity over the calibration range (0.1-10 ng mL(-1)) and the correlation coefficient (r) is higher than 0.993 for triclosan standard solution. The average recoveries of triclosan in human urine sample are 102.8-113.1% (RSD: 2.4-6.7%). This simple, sensitive, and selective analytical method may be used in the determination of trace amounts of triclosan in human urine samples.     

Visualizing global properties of large complex networks

For complex biological networks, graphical representations are highly desired for understanding some design principles, but few drawing methods are available that capture topological features of a large and highly heterogeneous network, such as a protein interaction network. Here we propose the circular perspective drawing (CPD) method to visualize global structures of large complex networks. The presented CPD combines the quasi-continuous search (QCS) analogous to the steepest descent method with a random node swapping strategy for an enhanced calculation speed. The CPD depicts a network in an aesthetic manner by showing connection patterns between different parts of the network instead of detailed links between nodes. Global structural features of networks exhibited by CPD provide clues toward a comprehensive understanding of the network organizations. Availability: Software is freely available at http://www.cadlive.jp.     

Protein phosphatase 2Cepsilon is an endoplasmic reticulum integral membrane protein that dephosphorylates the ceramide transport protein CERT to enhance its association with organelle membranes

Protein phosphatase 2Cepsilon (PP2Cepsilon), a mammalian PP2C family member, is expressed in various tissues and is implicated in the negative regulation of stress-activated protein kinase pathways. We show that PP2Cepsilon is an endoplasmic reticulum (ER) transmembrane protein with a transmembrane domain at the amino terminus and the catalytic domain facing the cytoplasm. Yeast two-hybrid screening of a human brain library using PP2Cepsilon as bait resulted in the isolation of a cDNA that encoded vesicle-associated membrane protein-associated protein A (VAPA). VAPA is an ER resident integral membrane protein involved in recruiting lipid-binding proteins such as the ceramide transport protein CERT to the ER membrane. Expression of PP2Cepsilon resulted in dephosphorylation of CERT in a VAPA expression-dependent manner, which was accompanied by redistribution of CERT from the cytoplasm to the Golgi apparatus. The expression of PP2Cepsilon also enhanced the association between CERT and VAPA. In addition, knockdown of PP2Cepsilon expression by short interference RNA attenuated the interaction between CERT and VAPA and the sphingomyelin synthesis. These results suggest that CERT is a physiological substrate of PP2Cepsilon and that dephosphorylation of CERT by PP2Cepsilon may play an important role in the regulation of ceramide trafficking from the ER to the Golgi apparatus.     

Oxidized low density lipoprotein activates peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARgamma through MAPK-dependent COX-2 expression in macrophages

It has been reported that oxidized low density lipoprotein (Ox-LDL) can activate both peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARgamma. However, the detailed mechanisms of Ox-LDL-induced PPARalpha and PPARgamma activation are not fully understood. In the present study, we investigated the effect of Ox-LDL on PPARalpha and PPARgamma activation in macrophages. Ox-LDL, but not LDL, induced PPARalpha and PPARgamma activation in a dose-dependent manner. Ox-LDL transiently induced cyclooxygenase-2 (COX-2) mRNA and protein expression, and COX-2 specific inhibition by NS-398 or meloxicam or small interference RNA of COX-2 suppressed Ox-LDL-induced PPARalpha and PPARgamma activation. Ox-LDL induced phosphorylation of ERK1/2 and p38 MAPK, and ERK1/2 specific inhibition abrogated Ox-LDL-induced COX-2 expression and PPARalpha and PPARgamma activation, whereas p38 MAPK-specific inhibition had no effect. Ox-LDL decreased the amounts of intracellular long chain fatty acids, such as arachidonic, linoleic, oleic, and docosahexaenoic acids. On the other hand, Ox-LDL increased intracellular 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) level through ERK1/2-dependent overexpression of COX-2. Moreover, 15d-PGJ(2) induced both PPARalpha and PPARgamma activation. Furthermore, COX-2 and 15d-PGJ(2) expression and PPAR activity were increased in atherosclerotic lesions of apoE-deficient mice. Finally, we investigated the involvement of PPARalpha and PPARgamma on Ox-LDL-induced mRNA expression of ATP-binding cassette transporter A1 and monocyte chemoattractant protein-1. Interestingly, specific inhibition of PPARalpha and PPARgamma suppressed Ox-LDL-induced ATP-binding cassette transporter A1 mRNA expression and enhanced Ox-LDL-induced monocyte chemoattractant protein-1 mRNA expression. In conclusion, Ox-LDL-induced increase in 15d-PGJ(2) level through ERK1/2-dependent COX-2 expression is one of the mechanisms of PPARalpha and PPARgamma activation in macrophages. These effects of Ox-LDL may control excess atherosclerotic progression.     

Functional differences of the catalytic and non-catalytic domains in human ADAMTS-4 and ADAMTS-5 in aggrecanolytic activity

ADAMTS-4 (aggrecanase-1) and ADAMTS-5 (aggrecanase-2) are multidomain metalloproteinases belonging to the ADAMTS family. We have previously reported that human ADAMTS-5 has much higher aggrecanolytic activity than human ADAMTS-4. To investigate the different proteolytic activity of the two enzymes, we generated a series of chimeras by exchanging various non-catalytic domains of the two proteinases. We found that the catalytic domain of ADAMTS-5 has higher intrinsic catalytic ability than that of ADAMTS-4. The studies also demonstrated that the non-catalytic domains of ADAMTS-5 are more effective modifiers than those of ADAMTS-4, making both catalytic domains more active against aggrecan, an Escherichia coli-expressed interglobular domain of aggrecan and fibromodulin. Addition of the C-terminal thrombospondin type I motif of ADAMTS-5 to the C terminus of ADAMTS-4 increased the activity of ADAMTS-4 against aggrecan and fibromodulin severalfold. In contrast to previous reports (Kashiwagi, M., Enghild, J. J., Gendron, C., Hughes, C., Caterson, B., Itoh, Y., and Nagase, H. (2004) J. Biol. Chem. 279, 10109-10119 and Gao, G., Plaas, A., Thompson, V. P., Jin, S., Zuo, F., and Sandy, J. D. (2004) J. Biol. Chem. 279, 10042-10051), our detailed investigation of the role of the C-terminal spacer domain of ADAMTS-4 indicated that full-length ADAMTS-4 is approximately 20-times more active against aggrecan than its spacer domain deletion mutant, even at the Glu373-Ala374 site of the interglobular domain. This discrepancy is most likely due to selective inhibition of full-length ADAMTS-4 by heparin, particularly for cleavage at the Glu373-Ala374 bond. However, removal of the spacer domain from ADAMTS-4 greatly enhanced more general proteolytic activity against non-aggrecan substrates, e.g. E. coli-expressed interglobular domain, fibromodulin, and carboxymethylated transferrin.     

Stabilization of water-in-oil emulsion by <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> B-4 and its application to biotransformation

Rhodococcus opacus B-4, which has recently been isolated as an organic solvent-tolerant bacterium, stabilized water-in-oil (w/o) emulsions by inhibition of droplet coalescence when the cells were dispersed in 90% (v/v) organic solvents. Confocal microscopy revealed that many bacterial cells assembled at the interface between oil and water droplets, though free cells were also detectable at the inside of water droplets. Bacterial cells in the w/o emulsion were capable of utilizing both a water-soluble (glucose) and an oil-soluble substrate (oleic acid) as an energy source. Availability of the w/o emulsion as an immobilized cell system in organic solvents was demonstrated using production of indigo from indole and production of o-cresol from toluene as model conversions. When glucose and oleic acid were simultaneously supplied as energy sources, the w/o emulsion culture of R. opacus B-4 produced indigo and o-cresol at levels of 0.217 and 2.12 mg ml⁻¹, respectively, by 12 h.