Installing a Green Engine To Drive an Enzyme Cascade: A Light-Powered In Vitro Biosystem for Poly(3-hydroxybutyrate) Synthesis.

Many existing in vitro biosystems harness power from the chemical energy contained in substrates and co-substrates, and light or electric energy provided from abiotic parts, leading to a compromise in atom economy, incompatibility between biological and abiotic parts, and most importantly, incapability to spatiotemporally co-regenerate ATP and NADPH. In this study, we developed a light-powered in vitro biosystem for poly(3-hydroxybutyrate) (PHB) synthesis using natural thylakoid membranes (TMs) to regenerate ATP and NADPH for a five-enzyme cascade. Through effective coupling of cofactor regeneration and mass conversion, 20 mM PHB was yielded from 50 mM sodium acetate with a molar conversion efficiency of carbon of 80.0 % and a light-energy conversion efficiency of 3.04 %, which are much higher than the efficiencies of similar in vitro PHB synthesis biosystems. This suggests the promise of installing TMs as a green engine to drive more enzyme cascades.

Spontaneous Mutants of Streptococcus sanguinis with Defects in the Glucose-Phosphotransferase System Show Enhanced Post-Exponential-Phase Fitness.

Genetic truncations in a gene encoding a putative glucose-phosphotransferase system (PTS) protein (manL, EIIABMan) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. To understand the emergence of these mutants, we engineered deletion mutants of manL and showed that the ManL-deficient strain had improved bacterial viability in the stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H2O2) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small but significant percentage (∼10%) of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. To our surprise, significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e., excess carbohydrates and low pH, are those associated with caries development, we propose that glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H2O2. IMPORTANCE A health-associated dental microbiome provides a potent defense against pathogens and diseases. Streptococcus sanguinis is an abundant member of a health-associated oral flora that antagonizes pathogens by producing hydrogen peroxide. There is a need for a better understanding of the mechanisms that allow bacteria to survive carbohydrate-rich and acidic environments associated with the development of dental caries. We report the isolation and characterization of spontaneous mutants of S. sanguinis with impairment in glucose transport. The resultant reprograming of the central metabolism in these mutants reduced the production of lactic acid and increased pyruvate accumulation; the latter enables these bacteria to better cope with hydrogen peroxide and low pH. The implications of these discoveries in the development of dental caries are discussed.

Optimization of nucleotide sugar supply for polysaccharide formation via thermodynamic buffering.

Plant polysaccharides (cellulose, hemicellulose, pectin, starch) are either direct (i.e. leaf starch) or indirect products of photosynthesis, and they belong to the most abundant organic compounds in nature. Although each of these polymers is made by a specific enzymatic machinery, frequently in different cell locations, details of their synthesis share certain common features. Thus, the production of these polysaccharides is preceded by the formation of nucleotide sugars catalyzed by fully reversible reactions of various enzymes, mostly pyrophosphorylases. These 'buffering' enzymes are, generally, quite active and operate close to equilibrium. The nucleotide sugars are then used as substrates for irreversible reactions of various polysaccharide-synthesizing glycosyltransferases ('engine' enzymes), e.g. plastidial starch synthases, or plasma membrane-bound cellulose synthase and callose synthase, or ER/Golgi-located variety of glycosyltransferases forming hemicellulose and pectin backbones. Alternatively, the irreversible step might also be provided by a carrier transporting a given immediate precursor across a membrane. Here, we argue that local equilibria, established within metabolic pathways and cycles resulting in polysaccharide production, bring stability to the system via the arrangement of a flexible supply of nucleotide sugars. This metabolic system is itself under control of adenylate kinase and nucleoside-diphosphate kinase, which determine the availability of nucleotides (adenylates, uridylates, guanylates and cytidylates) and Mg2+, the latter serving as a feedback signal from the nucleotide metabolome. Under these conditions, the supply of nucleotide sugars to engine enzymes is stable and constant, and the metabolic process becomes optimized in its load and consumption, making the system steady and self-regulated.

Unphosphorylated EIIANtr induces ClpAP-mediated degradation of RpoS in Azotobacter vinelandii.

The nitrogen-related phosphotransferase system (PTSNtr ) is composed of the EINtr , NPr and EIIANtr proteins that form a phosphorylation cascade from phosphoenolpyruvate. PTSNtr is a global regulatory system present in most Gram-negative bacteria that controls some pivotal processes such as potassium and phosphate homeostasis, virulence, nitrogen fixation and ABC transport activation. In the soil bacterium Azotobacter vinelandii, unphosphorylated EIIANtr negatively regulates the expression of genes related to the synthesis of the bioplastic polyester poly-ß-hydroxybutyrate (PHB) and cyst-specific lipids alkylresorcinols (ARs). The mechanism by which EIIANtr controls gene expression in A. vinelandii is not known. Here, we show that, in presence of unphosphorylated EIIANtr , the stability of the stationary phase sigma factor RpoS, which is necessary for transcriptional activation of PHB and ARs synthesis related genes, is reduced, and that the inactivation of genes coding for ClpAP protease complex in strains that carry unphosphorylated EIIANtr , restored the levels and in vivo stability of RpoS, as well as the synthesis of PHB and ARs. Taken together, our results reveal a novel mechanism, by which EIIANtr globally controls gene expression in A. vinelandii, where the unphosphorylated EIIANtr induces the degradation of RpoS by the proteolytic complex ClpAP.

Kinase Activity of Fibroblast Growth Factor Receptor 3 Regulates Activity of the Papillomavirus E2 Protein.

The papillomavirus (PV) E2 protein is a DNA binding, protein interaction platform that recruits viral and host factors necessary for transcription and replication. We recently discovered phosphorylation of a tyrosine (Y102) in bovine PV (BPV) E2. To identify the responsible factor, we tested several candidate tyrosine kinases that are highly expressed in keratinocytes for binding to BPV-1 E2. Fibroblast growth factor receptor 3 (FGFR3) coimmunoprecipitated with the BPV-1 E2 protein, as did human papillomavirus 31 (HPV-31) E2, which also colocalized with FGFR3 within the nucleus. A constitutively active mutant form of FGFR3 decreased BPV-1 and HPV-31 transient replication although this result also occurred in a BPV-1 E2 mutant lacking a previously identified phosphorylation site of interest (Y102). Furthermore, FGFR3 depletion in cell lines that maintain HPV-31 episomes increased viral copy number. These results suggest that FGFR3 kinase activity may regulate the PV reproductive program through phosphorylation of the E2 protein although this is unlikely to occur through the Y102 residue of HPV E2.IMPORTANCE The papillomavirus (PV) is a double-stranded DNA tumor virus infecting cervix, mouth, and throat tissues. The viral protein E2 is responsible for the replication of the virus. Understanding the mechanisms of the replicative life cycle of the virus may bring to light direct targets and treatments against viral infection. We recently found that the fibroblast growth factor receptor 3 (FGFR3) interacts with and mediates PV E2 function through phosphorylation of the E2 protein. Our study suggests that the function of the E2 protein may be regulated through a direct FGFR3 target during the maintenance stage of the PV life cycle.

Producing glucose 6-phosphate from cellulosic biomass: structural insights into levoglucosan bioconversion.

The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-ß-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.

Identification and functional analysis of the L-ascorbate-specific enzyme II complex of the phosphotransferase system in Streptococcus mutans.

BACKGROUND: Streptococcus mutans is the primary etiological agent of human dental caries. It can metabolize a wide variety of carbohydrates and produce large amounts of organic acids that cause enamel demineralization. Phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays an important role in carbohydrates uptake of S. mutans. The ptxA and ptxB genes in S. mutans encode putative enzyme IIA and enzyme IIB of the L-ascorbate-specific PTS. The aim of this study was to analyze the function of these proteins and understand the transcriptional regulatory mechanism. RESULTS: ptxA (-), ptxB (-), as well as ptxA (-) , ptxB (-) double-deletion mutants all had more extended lag phase and lower growth yield than wild-type strain UA159 when grown in the medium using L-ascorbate as the sole carbon source. Acid production and acid killing assays showed that the absence of the ptxA and ptxB genes resulted in a reduction in the capacity for acidogenesis, and all three mutant strains did not survive an acid shock. According to biofilm and extracellular polysaccharides (EPS) formation analysis, all the mutant strains formed much less prolific biofilms with small amounts of EPS than wild-type UA159 when using L-ascorbate as the sole carbon source. Moreover, PCR analysis and quantitative real-time PCR revealed that sgaT, ptxA, ptxB, SMU.273, SMU.274 and SMU.275 appear to be parts of the same operon. The transcription levels of these genes were all elevated in the presence of L-ascorbate, and the expression of ptxA gene decreased significantly once ptxB gene was knockout. CONCLUSIONS: The ptxA and ptxB genes are involved in the growth, aciduricity, acidogenesis, and formation of biofilms and EPS of S. mutans when L-ascorbate is the sole carbon source. In addition, the expression of ptxA is regulated by ptxB. ptxA, ptxB, and the upstream gene sgaT, the downstream genes SMU.273, SMU.274 and SMU.275 appear to be parts of the same operon, and L-ascorbate is a potential inducer of the operon.

FAM20A mutations can cause enamel-renal syndrome (ERS).

Enamel-renal syndrome (ERS) is an autosomal recessive disorder characterized by severe enamel hypoplasia, failed tooth eruption, intrapulpal calcifications, enlarged gingiva, and nephrocalcinosis. Recently, mutations in FAM20A were reported to cause amelogenesis imperfecta and gingival fibromatosis syndrome (AIGFS), which closely resembles ERS except for the renal calcifications. We characterized three families with AIGFS and identified, in each case, recessive FAM20A mutations: family 1 (c.992G>A; g.63853G>A; p.Gly331Asp), family 2 (c.720-2A>G; g.62232A>G; p.Gln241_Arg271del), and family 3 (c.406C>T; g.50213C>T; p.Arg136* and c.1432C>T; g.68284C>T; p.Arg478*). Significantly, a kidney ultrasound of the family 2 proband revealed nephrocalcinosis, revising the diagnosis from AIGFS to ERS. By characterizing teeth extracted from the family 3 proband, we demonstrated that FAM20A(-/-) molars lacked true enamel, showed extensive crown and root resorption, hypercementosis, and partial replacement of resorbed mineral with bone or coalesced mineral spheres. Supported by the observation of severe ectopic calcifications in the kidneys of Fam20a null mice, we conclude that FAM20A, which has a kinase homology domain and localizes to the Golgi, is a putative Golgi kinase that plays a significant role in the regulation of biomineralization processes, and that mutations in FAM20A cause both AIGFS and ERS.

Enhanced production of poly-3-hydroxybutyrate by Escherichia coli over-expressing multiple copies of NAD kinase integrated in the host genome.

OBJECTIVES: With the help of attB-attP recombination technique, multiple copies of yfjB gene encoding the NAD kinase of Escherichia coli were inserted into the host chromosome to promote NADPH-dependent poly-3-hydroxybutyrate (PHB) production. RESULTS: The yfjB integration mutant E. coli T2 harbored a similar metabolic profile to that of the wild type control. When PHB biosynthesis operon was introduced, the yfjB integration mutant produced 3 g PHB l(-1) from 18.2 g glucose l(-1), while the wild type consumed 15.7 g glucose l(-1) to afford 2.34 g PHB l(-1) in 48 h of shake-flask cultivation. Transcriptional analysis showed that the transcription levels of genes within the PHB biosynthesis operon were increased by six to eightfold with yfj Bover-expression, which may be the primary reason for the improved PHB production. CONCLUSION: A practical method is demonstrated to construct genetically-stable strains harboring extra copies of NAD kinase to enhance NADPH-dependent reactions.

Does changing the predicted dynamics of a phospholipase C alter activity and membrane binding?

The enzymatic activity of secreted phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes is associated with bacterial virulence. Although the PI-PLC active site has no obvious lid, molecular-dynamics simulations suggest that correlated loop motions may limit access to the active site, and two Pro residues, Pro(245) and Pro(254), are associated with these correlated motions. Whereas the region containing both Pro residues is quite variable among PI-PLCs, it shows high conservation in virulence-associated, secreted PI-PLCs that bind to the surface of cells. These regions of the protein are also associated with phosphatidylcholine binding, which enhances PI-PLC activity. In silico mutagenesis of Pro(245) disrupts correlated motions between the two halves of Bacillus thuringiensis PI-PLC, and Pro(245) variants show significantly reduced enzymatic activity in all assay systems. PC still enhanced activity, but not to the level of wild-type enzyme. Mutagenesis of Pro(254) appears to stiffen the PI-PLC structure, but experimental mutations had minor effects on activity and membrane binding. With the exception of P245Y, reduced activity was not associated with reduced membrane affinity. This combination of simulations and experiments suggests that correlated motions between the two halves of PI-PLC may be more important for enzymatic activity than for vesicle binding.

Comprehensive mutational analysis of sucrose-metabolizing pathways in Streptococcus mutans reveals novel roles for the sucrose phosphotransferase system permease.

Sucrose is perhaps the most efficient carbohydrate for the promotion of dental caries in humans, and the primary caries pathogen Streptococcus mutans encodes multiple enzymes involved in the metabolism of this disaccharide. Here, we engineered a series of mutants lacking individual or combinations of sucrolytic pathways to understand the control of sucrose catabolism and to determine whether as-yet-undisclosed pathways for sucrose utilization were present in S. mutans. Growth phenotypes indicated that gtfBCD (encoding glucan exopolysaccharide synthases), ftf (encoding the fructan exopolysaccharide synthase), and the scrAB pathway (sugar-phosphotransferase system [PTS] permease and sucrose-6-PO(4) hydrolase) constitute the majority of the sucrose-catabolizing activity; however, mutations in any one of these genes alone did not affect planktonic growth on sucrose. The multiple-sugar metabolism pathway (msm) contributed minimally to growth on sucrose. Notably, a mutant lacking gtfBC, which cannot produce water-insoluble glucan, displayed improved planktonic growth on sucrose. Meanwhile, loss of scrA led to growth stimulation on fructooligosaccharides, due in large part to increased expression of the fruAB (fructanase) operon. Using the LevQRST four-component signal transduction system as a model for carbohydrate-dependent gene expression in strains lacking extracellular sucrases, a PlevD-cat (EIIA(Lev)) reporter was activated by pulsing with sucrose. Interestingly, ScrA was required for activation of levD expression by sucrose through components of the LevQRST complex, but not for activation by the cognate LevQRST sugars fructose or mannose. Sucrose-dependent catabolite repression was also evident in strains containing an intact sucrose PTS. Collectively, these results reveal a novel regulatory circuitry for the control of sucrose catabolism, with a central role for ScrA.

Staphylococcus aureus SasA is responsible for binding to the salivary agglutinin gp340, derived from human saliva.

Staphylococcus aureus is a major human pathogen that can colonize the nasal cavity, skin, intestine, and oral cavity as a commensal bacterium. gp340, also known as DMBT1 (deleted in malignant brain tumors 1), is associated with epithelial differentiation and innate immunity. In the oral cavity, gp340 induces salivary aggregation with several oral bacteria and promotes bacterial adhesion to tissues such as the teeth and mucosa. S. aureus is often isolated from the oral cavity, but the mechanism underlying its persistence in the oral cavity remains unclear. In this study, we investigated the interaction between S. aureus and gp340 and found that S. aureus interacts with saliva- and gp340-coated resin. We then identified the S. aureus factor(s) responsible for binding to gp340. The cell surface protein SasA, which is rich in basic amino acids (BR domain) at the N terminus, was responsible for binding to gp340. Inactivation of the sasA gene resulted in a significant decrease in S. aureus binding to gp340-coated resin. Also, recombinant SasA protein (rSasA) showed binding affinity to gp340, which was inhibited by the addition of N-acetylneuraminic acid. Surface plasmon resonance analysis showed that rSasA significantly bound to the NeuAcα(2-3)Galß(1-4)GlcNAc structure. These results indicate that SasA is responsible for binding to gp340 via the N-acetylneuraminic acid moiety.

TGF-ß1 sensitizes TRPV1 through Cdk5 signaling in odontoblast-like cells.

BACKGROUND: Odontoblasts are specialized cells that form dentin and they are believed to be sensors for tooth pain. Transforming growth factor-ß1 (TGF-ß1), a pro-inflammatory cytokine expressed early in odontoblasts, plays an important role in the immune response during tooth inflammation and infection. TGF-ß1 is also known to participate in pain signaling by regulating cyclin-dependent kinase 5 (Cdk5) in nociceptive neurons of the trigeminal and dorsal root ganglia. However, the precise role of TGF-ß1 in tooth pain signaling is not well characterized. The aim of our present study was to determine whether or not in odontoblasts Cdk5 is functionally active, if it is regulated by TGF-ß1, and if it affects the downstream pain receptor, transient receptor potential vanilloid-1 (TRPV1). RESULTS: We first determined that Cdk5 and p35 are indeed expressed in an odontoblast-enriched primary preparation from murine teeth. For the subsequent analysis, we used an odontoblast-like cell line (MDPC-23) and found that Cdk5 is functionally active in these cells and its kinase activity is upregulated during cell differentiation. We found that TGF-ß1 treatment potentiated Cdk5 kinase activity in undifferentiated MDPC-23 cells. SB431542, a specific inhibitor of TGF-ß1 receptor 1 (Tgfbr1), when co-administered with TGF-ß1, blocked the induction of Cdk5 activity. TGF-ß1 treatment also activated the ERK1/2 signaling pathway, causing an increase in early growth response-1 (Egr-1), a transcription factor that induces p35 expression. In MDPC-23 cells transfected with TRPV1, Cdk5-mediated phosphorylation of TRPV1 at threonine-407 was significantly increased after TGF-ß1 treatment. In contrast, SB431542 co-treatment blocked TRPV1 phosphorylation. Moreover, TGF-ß1 treatment enhanced both proton- and capsaicin-induced Ca²âº influx in TRPV1-expressing MDPC-23 cells, while co-treatment with either SB431542 or roscovitine blocked this effect. CONCLUSIONS: Cdk5 and p35 are expressed in a murine odontoblast-enriched primary preparation of cells from teeth. Cdk5 is also functionally active in odontoblast-like MDPC-23 cells. TGF-ß1 sensitizes TRPV1 through Cdk5 signaling in MDPC-23 cells, suggesting the direct involvement of odontoblasts and Cdk5 in dental nociceptive pain transduction.

One-step purification and immobilization of thermophilic polyphosphate glucokinase from Thermobifida fusca YX: glucose-6-phosphate generation without ATP.

The discovery of stable and active polyphosphate glucokinase (PPGK, EC 2.7.1.63) would be vital to cascade enzyme biocatalysis that does not require a costly ATP input. An open reading frame Tfu_1811 from Thermobifida fusca YX encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was overexpressed in Escherichia coli. Mg²âº was an indispensible activator. This enzyme exhibited the highest activity in the presence of 4 mM Mg²âº at 55°C and pH 9.0. Under its suboptimal conditions (pH 7.5), the k (cat) and K(m) values of CBM-PPGK on glucose were 96.9 and 39.7 s⁻¹ as well as 0.77 and 0.45 mM at 37°C and 50°C respectively. The thermoinactivation of CBM-PPGK was independent of its mass concentration. Through one-step enzyme purification and immobilization on a high-capacity regenerated amorphous cellulose, immobilized CBM-PPGK had an approximately eightfold half lifetime enhancement (i.e., t(1/2) = 120 min) as compared to free enzyme at 50°C. To our limited knowledge, this enzyme was the first thermostable PPGK reported. Free PPGK and immobilized CBM-PPGK had total turnover number values of 126,000 and 961,000 mol product per mol enzyme, respectively, suggesting their great potential in glucose-6-phosphate generation based on low-cost polyphosphate.

Rapid selection of glucose-utilizing variants of the polyhydroxyalkanoate producer Ralstonia eutropha H16 by incubation with high substrate levels.

AIMS: The application of Ralstonia eutropha H16 for producing polyhydroxyalkanoates as bioplastics is limited by the incapability of the bacterium to utilize glucose as a growth substrate. This study aims in characterizing glucose-utilizing strains that arose after incubation with high glucose levels, in comparison with previously published mutants, generated either by mutagenesis or by metabolic engineering. METHODS AND RESULTS: Cultivations on solid and liquid media showed that the application of high substrate concentrations rapidly induced a glucose-positive phenotype. The time span until the onset of growth and the frequency of glucose-utilizing colonies were correlated to the initial glucose concentration. All mutants exhibited elevated activities of glucose-6-phosphate dehydrogenase. The glucose-positive phenotype was abolished after deleting genes for the N-acetylglucosamine phosphotransferase system. CONCLUSIONS: A procedure is provided for selecting glucose-utilizing R. eutropha H16 in an unprecedented short time period and without any mutagenic treatment. An altered N-acetylglucosamine phosphotransferase system appears to be a common motif in all glucose-utilizing mutants examined so far. SIGNIFICANCE AND IMPACT OF THE STUDY: The correlation of the applied glucose concentration and the appearance of glucose-utilizing mutants poses questions about the randomness or the specificity of adaptive mutations in general. Furthermore, glucose-adapted strains of R. eutropha H16 could be useful for the production of bioplastics.

Cranberry proanthocyanidins inhibit the adherence properties of Candida albicans and cytokine secretion by oral epithelial cells.

BACKGROUND: Oral candidiasis is a common fungal disease mainly caused by Candida albicans. The aim of this study was to investigate the effects of A-type cranberry proanthocyanidins (AC-PACs) on pathogenic properties of C. albicans as well as on the inflammatory response of oral epithelial cells induced by this oral pathogen. METHODS: Microplate dilution assays were performed to determine the effect of AC-PACs on C. albicans growth as well as biofilm formation stained with crystal violet. Adhesion of FITC-labeled C. albicans to oral epithelial cells and to acrylic resin disks was monitored by fluorometry. The effects of AC-PACs on C. albicans-induced cytokine secretion, nuclear factor-kappa B (NF-κB) p65 activation and kinase phosphorylation in oral epithelial cells were determined by immunological assays. RESULTS: Although AC-PACs did not affect growth of C. albicans, it prevented biofilm formation and reduced adherence of C. albicans to oral epithelial cells and saliva-coated acrylic resin discs. In addition, AC-PACs significantly decreased the secretion of IL-8 and IL-6 by oral epithelial cells stimulated with C. albicans. This anti-inflammatory effect was associated with reduced activation of NF-κB p65 and phosphorylation of specific signal intracellular kinases. CONCLUSION: AC-PACs by affecting the adherence properties of C. albicans and attenuating the inflammatory response induced by this pathogen represent potential novel therapeutic agents for the prevention/treatment of oral candidiasis.

Proteomic and transcriptomic elucidation of the mutant ralstonia eutropha G+1 with regard to glucose utilization.

By taking advantage of the available genome sequence of Ralstonia eutropha H16, glucose uptake in the UV-generated glucose-utilizing mutant R. eutropha G(+)1 was investigated by transcriptomic and proteomic analyses. Data revealed clear evidence that glucose is transported by a usually N-acetylglucosamine-specific phosphotransferase system (PTS)-type transport system, which in this mutant is probably overexpressed due to a derepression of the encoding nag operon by an identified insertion mutation in gene H16_A0310 (nagR). Furthermore, a missense mutation in nagE (membrane component EIICB), which yields a substitution of an alanine by threonine in NagE and may additionally increase glucose uptake, was identified. Phosphorylation of glucose is subsequently mediated by NagF (cytosolic PTS component EIIA-HPr-EI) or glucokinase (GlK), respectively. The inability of the defined deletion mutant R. eutropha G(+)1 ΔnagFEC to utilize glucose strongly confirms this finding. In addition, secondary effects of glucose, which is now intracellularly available as a carbon source, on the metabolism of the mutant cells in the stationary growth phase occurred: intracellular glucose degradation is stimulated by the stronger expression of enzymes involved in the 2-keto-3-deoxygluconate 6-phosphate (KDPG) pathway and in subsequent reactions yielding pyruvate. The intermediate phosphoenolpyruvate (PEP) in turn supports further glucose uptake by the Nag PTS. Pyruvate is then decarboxylated by the pyruvate dehydrogenase multienzyme complex to acetyl coenzyme A (acetyl-CoA), which is directed to poly(3-hydroxybutyrate). The polyester is then synthesized to a greater extent, as also indicated by the upregulation of various enzymes of poly-ß-hydroxybutyrate (PHB) metabolism. The larger amounts of NADPH required for PHB synthesis are delivered by significantly increased quantities of proton-translocating NAD(P) transhydrogenases. The current study successfully combined transcriptomic and proteomic investigations to unravel the phenotype of this hitherto-undefined glucose-utilizing mutant.

Matrix Stiffening Enhances DNCB-Induced IL-6 Secretion in Keratinocytes Through Activation of ERK and PI3K/Akt Pathway.

Matrix stiffness, a critical physical property of the cellular environment, is implicated in epidermal homeostasis. In particular, matrix stiffening during the pathological progression of skin diseases appears to contribute to cellular responses of keratinocytes. However, it has not yet elucidated the molecular mechanism underlying matrix-stiffness-mediated signaling in coordination with chemical stimuli during inflammation and its effect on proinflammatory cytokine production. In this study, we demonstrated that keratinocytes adapt to matrix stiffening by increasing cell-matrix adhesion via actin cytoskeleton remodeling. Specifically, mechanosensing and signal transduction are coupled with chemical stimuli to regulate cytokine production, and interleukin-6 (IL-6) production is elevated in keratinocytes on stiffer substrates in response to 2,4-dinitrochlorobenzene. We demonstrated that ß1 integrin and focal adhesion kinase (FAK) expression were enhanced with increasing stiffness and activation of ERK and the PI3K/Akt pathway was involved in stiffening-mediated IL-6 production. Collectively, our results reveal the critical role of matrix stiffening in modulating the proinflammatory response of keratinocytes, with important clinical implications for skin diseases accompanied by pathological matrix stiffening.

Molecular requirements for bi-directional movement of phagosomes along microtubules.

Microtubules facilitate the maturation of phagosomes by favoring their interactions with endocytic compartments. Here, we show that phagosomes move within cells along tracks of several microns centrifugally and centripetally in a pH- and microtubule-dependent manner. Phagosome movement was reconstituted in vitro and required energy, cytosol and membrane proteins of this organelle. The activity or presence of these phagosome proteins was regulated as the organelle matured, with "late" phagosomes moving threefold more frequently than "early" ones. The majority of moving phagosomes were minus-end directed; the remainder moved towards microtubule plus-ends and a small subset moved bi-directionally. Minus-end movement showed pharmacological characteristics expected for dyneins, was inhibited by immunodepletion of cytoplasmic dynein and could be restored by addition of cytoplasmic dynein. Plus-end movement displayed pharmacological properties of kinesin, was inhibited partially by immunodepletion of kinesin and fully by addition of an anti-kinesin IgG. Immunodepletion of dynactin, a dynein-activating complex, inhibited only minus-end directed motility. Evidence is provided for a dynactin-associated kinase required for dynein-mediated vesicle transport. Movement in both directions was inhibited by peptide fragments from kinectin (a putative kinesin membrane receptor), derived from the region to which a motility-blocking antibody binds. Polypeptide subunits from these microtubule-based motility factors were detected on phagosomes by immunoblotting or immunoelectron microscopy. This is the first study using a single in vitro system that describes the roles played by kinesin, kinectin, cytoplasmic dynein, and dynactin in the microtubule-mediated movement of a purified membrane organelle.

Overexpression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly(3-hydroxybutyrate) production.

NAD kinase was overexpressed to enhance the accumulation of poly(3-hydroxybutyrate) (PHB) in recombinant Escherichia coli harboring PHB synthesis pathway via an accelerated supply of NADPH, which is one of the most crucial factors influencing PHB production. A high copy number expression plasmid pE76 led to a stronger NAD kinase activity than that brought about by the low copy number plasmid pELRY. Overexpressing NAD kinase in recombinant E. coli was found not to have a negative effect on cell growth in the absence of PHB synthesis. Shake flask experiments demonstrated that excess NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16-35 wt.% compared with the controls; meanwhile, NADP concentration was enhanced threefold to sixfold. Although the two NAD kinase overexpression recombinants exhibited large disparity on NAD kinase activity, their influence on cell growth and PHB accumulation was not proportional. Under the same growth conditions without process optimization, the NAD kinase-overexpressing recombinant produced 14 g/L PHB compared with 7 g/L produced by the control in a 28-h fermentor study. In addition, substrate to PHB yield Y (PHB/glucose) showed an increase from 0.08 g PHB/g glucose for the control to 0.15 g PHB/g glucose for the NAD kinase-overexpressing strain, a 76% increase for the Y (PHB/glucose). These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis.