von Willebrand factor binds specifically to sulfated glycolipids

The human plasma glycoprotein Factor VIII/von Willebrand factor (vWF) binds specifically and with high affinity to sulfatides (galactosylceramide-I3-sulfate). vWF does not bind to gangliosides, neutral glycolipids, phospholipids, or cholesterol 3-sulfate. Although the largest oligomers of vWF bind preferentially to sulfatides, vWF monomers and dimers also bind but with reduced affinity. vWF binding is inhibited at high ionic strength or low pH, by some sulfated polysaccharides and by antibodies to vWF. Binding of vWF to sulfatides is probably responsible for its agglutination of aldehyde-fixed erythrocytes and may play a role in vWF-induced platelet adhesion or platelet aggregation.     

Altered processing of integrin receptors during keratinocyte activation

We used monoclonal antibodies against specific integrin subunits to examine the role of integrin receptors in keratinocyte activation. We found that before activation, beta 1 subunits in keratinocytes showed a diffuse distribution, whereas after activation, keratinocytes organized beta 1 receptors into marginal adhesion plaques. In immunoprecipitation experiments with antibodies against beta 1 integrin subunits, we found mostly immature subunits synthesized in keratinocytes freshly harvested from skin. Moreover, integrin receptor complexes immunoprecipitated from these cells by monoclonal antibodies against alpha 2, alpha 3, or alpha 5 subunits contained only immature beta 1 subunits. With keratinocytes cultured 4-7 days, anti-beta 1 antibodies immunoprecipitated mostly mature beta 1 subunits, and integrin complexes immunoprecipitated from cultured cells by anti-alpha subunit antibodies contained mostly mature beta 1 subunits. Antibodies directed against beta 1 subunits also inhibited keratinocyte migration. Based on these results, we suggest that up-regulation of migration by activated keratinocytes depends on changes in processing of pre-beta 1 subunits to mature beta 1 subunits. We also studied the distribution of integrin subunits in skin and on keratinocytes migrating out of skin explants. Whereas beta 1, alpha 2, and alpha 3 subunits were detected in keratinocytes in skin and migrating out of explants, alpha 5 subunits were observed only in migrating cells.     

Enhancement of Survival and Electricity Production in an Engineered Bacterium by Light-Driven Proton Pumping

Microorganisms can use complex photosystems or light-dependent proton pumps to generate membrane potential and/or reduce electron carriers to support growth. The discovery that proteorhodopsin is a light-dependent proton pump that can be expressed readily in recombinant bacteria enables development of new strategies to probe microbial physiology and to engineer microbes with new light-driven properties. Here, we describe functional expression of proteorhodopsin and light-induced changes in membrane potential in the bacterium Shewanella oneidensis strain MR-1. We report that there were significant increases in electrical current generation during illumination of electrochemical chambers containing S. oneidensis expressing proteorhodopsin. We present evidence that an engineered strain is able to consume lactate at an increased rate when it is illuminated, which is consistent with the hypothesis that proteorhodopsin activity enhances lactate uptake by increasing the proton motive force. Our results demonstrate that there is coupling of a light-driven process to electricity generation in a nonphotosynthetic engineered bacterium. Expression of proteorhodopsin also preserved the viability of the bacterium under nutrient-limited conditions, providing evidence that fulfillment of basic energy needs of organisms may explain the widespread distribution of proteorhodopsin in marine environments.Classic experiments in microbial bioenergetics used light-driven reactions from halobacterial bacteriorhodopsin or the photosynthetic reaction center to provide a temporary driving force for understanding transport and chemiosmotic coupling (6, 7, 19, 35). However, light-driven reactions have not been used in metabolic engineering to alter microbial physiology and production of chemicals. The recent discovery of proteorhodopsin (PR) in ocean microorganisms and the ease with which this membrane protein can be functionally expressed by recombinant bacteria have made possible many engineering strategies previously not available (1, 16). In this paper, we describe progress toward the goal of integrating light-driven reactions with biocatalysis.In contrast to the situation for established industrial microorganisms, such as Escherichia coli, our current understanding of less-studied algal and phototrophic bacteria may limit metabolic engineering strategies which require genetic manipulation. Metabolic engineering strategies using photosynthetic bacteria have focused largely on methods to increase hydrogen production, and improvements rely mainly on engineering of nitrogenase and hydrogenase to produce H₂. Algae appear to be suited to large-scale cultivation for lipid production, but so far little has been done to engineer these organisms (36). In principle, platform microbial hosts capable of producing a diverse range of products could be boosted by addition of light-driven processes from phototrophic metabolism.To demonstrate the feasibility of transferring a light-driven process into a nonphotosynthetic bacterium, we chose to study proteorhodopsin (PR) first because it is one of the simplest mechanisms for harnessing the energy from light. The proteorhodopsins are a group of transmembrane proteins that use the light-induced isomerization of retinal, the oxidative cleavage product of the carotenoid β-carotene, either to initiate signaling pathways or to catalyze the transfer of ions across cell membranes (8). PR was discovered by metagenomic analysis of marine samples (1) and is related to the well-studied bacteriorhodopsin of archaea (33) and rhodopsin (34), a eukaryotic light-sensing protein. The membrane potential generated by light-driven proton pumping by PR has been confirmed to drive ATP synthesis in a heterologous system (25). However, bacteria expressing heterologous PR were shown not to benefit from this pumping activity, as no significant increases in growth rates were observed (9). This led to the suggestion that PR may benefit the organism only under starvation conditions. In agreement with this hypothesis, Gomez-Consarnau et al. (10) have reported that the light-dependent growth rates of a marine flavobacterium that has a native PR are increased only when the organism is cultured under energy-limited conditions.Studies of both native and recombinant systems in which rhodopsins are expressed have generated light-dependent membrane potentials. In membrane vesicles isolated from a native host, the light-dependent membrane potential generated by bacteriorhodopsin provides the driving force for ATP synthesis (35) and uptake of leucine and glutamate (20, 22). More recently, studies of recombinant systems have coupled the membrane potential to other transport processes. In one example, the membrane potential-dependent export of specific toxic molecules increased when E. coli cells expressing both an archaeal rhodopsin and a specific efflux pump were exposed to light (17). In another experiment, starved E. coli cells expressing PR increased the swimming motion of their flagella when they were illuminated (44). Based upon measurements of flagellar motion as a function of light intensity and azide concentration, the proton motive force generated by PR was estimated to be −0.2 V, a value similar to the value for aerobic respiration in E. coli (42).As a nonphotosynthetic host for recombinant PR expression, we chose the dissimilatory metal-reducing bacterium Shewanella oneidensis strain MR-1, which is genetically tractable for engineering and is able to use a variety of terminal electron acceptors, including insoluble metal oxides (11, 30). Key to the ability of this bacterium to reduce metal oxides is a multicomponent extracellular respiratory pathway that transports electrons from menaquinol to cytochromes in the outer membrane. This pathway is composed of a cytoplasmic membrane tetraheme protein (CymA), a periplasmic decaheme protein (MtrA), an integral outer membrane protein (MtrB), and a decaheme lipoprotein (MtrC) that is associated with MtrB (14, 37, 40). The ability of S. oneidensis to reduce extracellular metal oxides has made it possible to harvest electrons from this organism by coupling it to an electrode which serves as the electron acceptor (21). The electron flow to the outer surface allows respiration rates to be measured directly by electrochemistry.In the current work, we introduced PR into an electricity-generating bacterium, S. oneidensis strain MR-1, and demonstrated that there was integration of a light-driven process into the metabolism of a previously nonphotosynthetic organism that resulted in a useful output. We show here that PR allows cells to survive for extended periods in stationary phase and that the presence of light results in an increase in electricity generation. A possible physiological model to explain these effects is discussed.     

The carbohydrate of human thrombin: Structural analysis of glycoprotein oligosaccharides by mass spectrometry

The carbohydrate structure of human thrombin has been determined by direct probe mass spectrometry of the oligosaccharides released by trifluoroacetolysis from the asialo glycoprotein. The free oligosaccharides were studied as permethylated and N-trifluoroacetylated oligosaccharide alditols. The structure was confirmed by sequential exoglycosidase digestion of intact thrombin and sugar and methylation analysis of the oligosaccharides by gas-liquid chromatography-mass spectrometry. The results indicate the following structure:

with Fuc present on only about 50% of the oligosaccharides.     

Aerobic training increases the expression of adiponectin receptor genes in the peripheral blood mononuclear cells of young men

Little is known about the effect of exercise training on the expression of adiponectin receptor genes in peripheral blood mononuclear cells (PBMCs). In this study, we investigated the effects of aerobic training on the expression of AdipoR1 and AidpoR2 mRNAs in PBMCs, whole body insulin sensitivity, and circulating adiponectins in men. Thirty young men were randomly assigned to either a control (n=15) or an exercise (n=15) group. Subjects assigned to the exercise group underwent a 12-week jogging and/or running programme on a motor-driven treadmill at an intensity of 60%-75% of the age-based maximum heart rate with duration of 40 minutes per session and a frequency of 5 days per week. Two-way mixed ANOVA with repeated measures was used to test any significant time-by-group interaction effects for the measured variables at p=0.05. We found significant time-by-group interaction effects for waist circumference (p=0.001), VO_2max (p<0.001), fasting insulin (p=0.016), homeostasis model assessment for insulin resistance (HOMA-IR) (p=0.010), area under the curve (AUC) for insulin response during the 75-g oral glucose tolerance test (p=0.002), high-molecular weight (HMW) adiponectin (p=0.016), and the PBMC mRNA levels of AdipoR1 (p<0.001) and AdipoR2 (p=0.001). The exercise group had significantly increased mRNA levels of AdipoR1 and AdipoR2 in PBMCs, along with increased whole body insulin sensitivity and HMW adiponectin, decreased waist circumference, and increased VO_2max compared with the control group. In summary, the current findings suggest that exercise training modulates the expression of AdipoR1 and AdipoR2 mRNAs in PBMCs, implying that manipulation of the expression of these genes could be a potential surrogate for lifestyle intervention-mediated improvements of whole body insulin sensitivity and glucose homeostasis.     

Sphingobacterium sp. strain PM2-P1-29 harbours a functional tet(X) gene encoding for the degradation of tetracycline

Aims:  The tet (X) gene has previously been found in obligate anaerobic Bacteroides spp., which is curious because tet (X) encodes for a NADP-dependent monooxygenase that requires oxygen to degrade tetracycline. In this study, we characterized a tetracycline resistant, aerobic, Gram-negative Sphingobacterium sp. strain PM2-P1-29 that harbours a tet (X) gene.
Methods and Results:  Sphingobacterium sp. PM2-P1-29 demonstrated the ability to transform tetracycline compared with killed controls. The presence of the tet (X) gene was verified by PCR and nucleotide sequence analysis. Additional nucleotide sequence analysis of regions flanking the tet (X) gene revealed a mobilizable transposon-like element (Tn 6031 ) that shared organizational features and genes with the previously described Bacteroides conjugative transposon CTnDOT. A circular transposition intermediate of the tet (X) region, characteristic of mobilizable transposons, was detected. However, we could not demonstrate the conjugal transfer of the tet (X) gene using three different recipient strains and numerous experimental conditions.
Conclusions:  This study suggests that Sphingobacterium sp. PM2-P1-29 or a related bacterium may be an ancestral source of the tet (X) gene.
Significance and Impact of the Study:  This study demonstrates the importance of environmental bacteria and lateral gene transfer in the dissemination and proliferation of antibiotic resistance among bacteria.     

Comparison of the specificities of laminin, thrombospondin, and von Willebrand factor for binding to sulfated glycolipids

The adhesive glycoproteins laminin, thrombospondin, and von Willebrand factor bind specifically and with high affinity to sulfated glycolipids. These three glycoproteins differ, however, in their sensitivity to inhibition of binding by sulfated monosaccharides and polysaccharides. Heparin strongly inhibits binding of thrombospondin but only weakly inhibits binding of laminin and von Willebrand factor. Fucoidan strongly inhibits binding of both laminin and thrombospondin but not of von Willebrand factor. Laminin shows significant specificity for inhibition by monosaccharides, whereas thrombospondin does not. Thus, specific spacial orientations of sulfate esters may be primary determinants of binding for the three proteins. Laminin, thrombospondin, and von Willebrand factor also differ in their relative binding affinities for purified sulfated glycosphingolipids. The three proteins strongly prefer terminal-sulfated lipids and bind only weakly to sulfated gangliotriaosyl ceramide with a sulfate ester on the penultimate galactose. Thrombospondin binds with highest affinity to galactosyl sulfatide but only weakly to more complex sulfatides, whereas von Willebrand factor prefers galactosyl sulfatide but binds with moderate affinity to various sulfated glycolipids. Laminin also is less selective than thrombospondin but is less sensitive for detection of low sulfatide concentrations. Galactosyl sulfatide at 1-5 pmol can be detected by staining of lipids separated on high performance TLC with 125I-thrombospondin or 125I-von Willebrand factor. 125I-von Willebrand factor was examined as a reagent for detecting sulfated glycolipids in tissue extracts. Rat kidney lipids contain 5 characterized sulfated glycolipids: galactosyl ceramide I3-sulfate, lactosyl ceramide II3-sulfate, gangliotriaosyl ceramide II3-sulfate, and bis-sulfated gangliotriaosyl and gangliotetraosyl ceramides. von Willebrand factor detects all of these lipids as well as several additional minor sulfated lipids. Complex monosulfated lipids are detected in several human tissues including kidney, erythrocytes, and platelets by this technique.     

Construction and elementary mode analysis of a metabolic model for Shewanella oneidensis MR-1

A stoichiometric model describing the central metabolism of Shewanella oneidensis MR-1 wild-type and derivative strains was developed and used in elementary mode analysis (EMA). Shewanella oneidensis MR-1 can anaerobically respire a diverse pool of electron acceptors, and may be applied in several biotechnology settings, including bioremediation of toxic metals, electricity generation in microbial fuel cells, and whole-cell biocatalysis. The metabolic model presented here was adapted and verified by comparing the growth phenotypes of 13 single- and 1 double-knockout strains, while considering respiration via aerobic, anaerobic fumarate, and anaerobic metal reduction (Mtr) pathways, and utilizing acetate, n-acetylglucosamine (NAG), or lactate as carbon sources. The gene ppc, which encodes phosphoenolpyruvate carboxylase (Ppc), was determined to be necessary for aerobic growth on NAG and lactate, while not essential for growth on acetate. This suggests that Ppc is the only active anaplerotic enzyme when cultivated on lactate and NAG. The application of regulatory and substrate limitations to EMA has enabled creation of metabolic models that better reflect biological conditions, and significantly reduce the solution space for each condition, facilitating rapid strain optimization. This wild-type model can be easily adapted to include utilization of different carbon sources or secretion of different metabolic products, and allows the prediction of single- and multiple-knockout strains that are expected to operate under defined conditions with increased efficiency when compared to wild type cells.     

Modularity of the Mtr respiratory pathway of Shewanella oneidensis strain MR‐1

Four distinct pathways predicted to facilitate electron flow for respiration of externally located substrates are encoded in the genome of Shewanella oneidensis strain MR‐1. Although the pathways share a suite of similar proteins, the activity of only two of these pathways has been described. Respiration of extracellular substrates requires a mechanism to facilitate electron transfer from the quinone pool in the cytoplasmic membrane to terminal reductase enzymes located on the outer leaflet of the outer membrane. The four pathways share MtrA paralogues, a periplasmic electron carrier cytochrome, and terminal reductases similar to MtrC for reduction of metals, flavins and electrodes or to DmsAB for reduction of dimethyl sulphoxide (DMSO). The promiscuity of respiratory electron transfer reactions catalysed by these pathways has made studying strains lacking single proteins difficult. Here, we present a comprehensive analysis of MtrA and MtrC paralogues in S. oneidensis to define the roles of these proteins in respiration of insoluble iron oxide, soluble iron citrate, flavins and DMSO. We present evidence that some periplasmic electron carrier components and terminal reductases in these pathways can provide partial compensation in the absence of the primary component, a phenomenon described as modularity, and discuss biochemical and evolutionary implications.     

Lesions in gshA (Encoding gamma-L-glutamyl-L-cysteine synthetase) prevent aerobic synthesis of thiamine in Salmonella enterica serovar typhimurium LT2

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella enterica serovar Typhimurium and other bacteria. In addition to genes encoding enzymes in the biosynthetic pathway, mutations in other metabolic loci have been shown to prevent thiamine synthesis. The latter loci identify the integration of the thiamine biosynthetic pathway with other metabolic processes and can be uncovered when thiamine biosynthesis is challenged. Mutations in gshA, encoding gamma-L-glutamyl-L-cysteine synthetase, prevent the synthesis of glutathione, the major free thiol in the cell, and are shown here to result in a thiamine auxotrophy in some of the strains tested, including S. enterica LT2. Phenotypic characterization of the gshA mutants indicated they were similar enough to apbC and apbE mutants to warrant the definition of a class of mutants unified by (i) a requirement for both the hydroxymethyl pyrimidine (HMP) and thiazole (THZ) moiety of thiamine, (ii) the ability of L-tryosine to satisfy the THZ requirement, (iii) suppression of the thiamine requirement by anaerobic growth, and (iv) suppression by a second-site mutation at a single locus. Genetic data indicated that a defective ThiH generates the THZ requirement in these strains, and we suggest this defect is due to a reduced ability to repair a critical [Fe-S] cluster.