Expanding the repertoire of DNA-mediated signaling in DNA repair

DNA damage is extremely detrimental to the cell and must be repaired to protect the genome. DNA is capable of conducting charge through the overlapping π-orbitals of stacked bases; this phenomenon is extremely sensitive to the integrity of the π-stack, as perturbations attenuate DNA charge transport (CT). Based on the E. coli base excision repair (BER) proteins EndoIII and MutY, it has recently been proposed that redox-active proteins containing metal clusters can utilize DNA CT to signal one another to locate sites of DNA damage.

To expand our repertoire of proteins that utilize DNA-mediated signaling, we measured the DNA-bound redox potential of the nucleotide excision repair (NER) helicase XPD from Sulfolobus acidocaldarius. A midpoint potential of 82 mV versus NHE was observed, resembling that of the previously reported BER proteins. The redox signal increases in intensity with ATP hydrolysis in only the WT protein and mutants that maintain ATPase activity and not for ATPase-deficient mutants. The signal increase correlates directly with ATP activity, suggesting that DNA-mediated signaling may play a general role in protein signaling. Several mutations in human XPD that lead to XP-related diseases have been identified; using SaXPD, we explored how these mutations, which are conserved in the thermophile, affect protein electrochemistry.

To further understand the electrochemical signaling of XPD, we studied the yeast S. cerevisiae Rad3 protein. ScRad3 mutants were incubated on a DNA-modified electrode and exhibited a similar redox potential to SaXPD. We developed a haploid strain of S. cerevisiae that allowed for easy manipulation of Rad3. In a survival assay, the ATPase- and helicase-deficient mutants show little survival, while the two disease-related mutants exhibit survival similar to WT. When both a WT and G47R (ATPase/helicase deficient) strain were challenged with different DNA damaging agents, both exhibited comparable survival in the presence of hydroxyurea, while with methyl methanesulfonate and camptothecin, the G47R strain exhibits a significant change in growth, suggesting that Rad3 is involved in repairing damage beyond traditional NER substrates. Together, these data expand our understanding of redox-active proteins at the interface of DNA repair.

DNA-mediated charge transport signaling within the cell

DNA possesses the curious ability to conduct charge longitudinally through the π-stacked base pairs that reside within the interior of the double helix. The rate of charge transport (CT) through DNA has a shallow distance dependence. DNA CT can occur over at least 34 nm, a very long molecular distance. Lastly, DNA CT is exquisitely sensitive to disruptions, such as DNA damage, that affect the dynamics of base-pair stacking. Many DNA repair and DNA-processing enzymes are being found to contain 4Fe-4S clusters. These co-factors have been found in glycosylases, helicases, helicase-nucleases, and even enzymes such as DNA polymerase, RNA polymerase, and primase across the phylogeny. The role of these clusters in these enzymes has remained elusive. Generally, iron-sulfur clusters serve redox roles in nature since, formally, the cluster can exist in multiple oxidation states that can be accessed within a biological context. Taken together, these facts were used as a foundation for the hypothesis that DNA-binding proteins with 4Fe-4S clusters utilize DNA-mediated CT as a means to signal one another to scan the genome as a first step in locating the subtle damage that occurs within a sea of undamaged bases within cells.

Herein we describe a role for 4Fe-4S clusters in DNA-mediated charge transport signaling among EndoIII, MutY, and DinG, which are from distinct repair pathways in E. coli. The DinG helicase is an ATP-dependent helicase that contains a 4Fe-4S cluster. To study the DNA-bound redox properties of DinG, DNA-modified electrochemistry was used to show that the 4Fe-4S cluster of DNA-bound DinG is redox-active at cellular potentials, and shares the 80 mV vs. NHE redox potential of EndoIII and MutY. ATP hydrolysis by DinG increases the DNA-mediated redox signal observed electrochemically, likely reflecting better coupling of the 4Fe-4S cluster to DNA while DinG unwinds DNA, which could have interesting biological implications. Atomic force microscopy experiments demonstrate that DinG and EndoIII cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Knocking out DinG in CC104 cells leads to a decrease in MutY activity that is rescued by EndoIII D138A, but not EndoIII Y82A. DinG, thus, appears to help MutY find its substrate using DNA-mediated CT, but do MutY or EndoIII aid DinG in a similar way? The InvA strain of bacteria was used to observe DinG activity, since DinG activity is required within InvA to maintain normal growth. Silencing the gene encoding EndoIII in InvA results in a significant growth defect that is rescued by the overexpression of RNAseH, a protein that dismantles the substrate of DinG, R-loops. This establishes signaling between DinG and EndoIII. Furthermore, rescue of this growth defect by the expression of EndoIII D138A, the catalytically inactive but CT-proficient mutant of EndoIII, is also observed, but expression of EndoIII Y82A, which is CT-deficient but enzymatically active, does not rescue growth. These results provide strong evidence that DinG and EndoIII utilize DNA-mediated signaling to process DNA damage. This work thus expands the scope of DNA-mediated signaling within the cell, as it indicates that DNA-mediated signaling facilitates the activities of DNA repair enzymes across the genome, even for proteins from distinct repair pathways.

In separate work presented here, it is shown that the UvrC protein from E. coli contains a hitherto undiscovered 4Fe-4S cluster. A broad shoulder at 410 nm, characteristic of 4Fe-4S clusters, is observed in the UV-visible absorbance spectrum of UvrC. Electron paramagnetic resonance spectroscopy of UvrC incubated with sodium dithionite, reveals a spectrum with the signature features of a reduced, [4Fe-4S]+1, cluster. DNA-modified electrodes were used to show that UvrC has the same DNA-bound redox potential, of ~80 mV vs. NHE, as EndoIII, DinG, and MutY. Again, this means that these proteins are capable of performing inter-protein electron transfer reactions. Does UvrC use DNA-mediated signaling to facilitate the repair of its substrates?

UvrC is part of the nucleotide excision repair (NER) pathway in E. coli and is the protein within the pathway that performs the chemistry required to repair bulky DNA lesions, such as cyclopyrimidine dimers, that form as a product of UV irradiation. We tested if UvrC utilizes DNA-mediated signaling to facilitate the efficient repair of UV-induced DNA damage products by helping UvrC locate DNA damage. The UV sensitivity of E. coli cells lacking DinG, a putative signaling partner of UvrC, was examined. Knocking out DinG in E. coli leads to a sensitivity of the cells to UV irradiation. A 5-10 fold reduction in the amount of cells that survive after irradiation with 90 J/m2 of UV light is observed. This is consistent with the hypothesis that UvrC and DinG are signaling partners, but is this signaling due to DNA-mediated CT? Complementing the knockout cells with EndoIII D138A, which can also serve as a DNA CT signaling partner, rescues cells lacking DinG from UV irradiation, while complementing the cells with EndoIII Y82A shows no rescue of viability. These results indicate that there is cross-talk between the NER pathway and DinG via DNA-mediated signaling. Perhaps more importantly, this work also establishes that DinG, EndoIII, MutY, and UvrC comprise a signaling network that seems to be unified by the ability of these proteins to perform long range DNA-mediated CT signaling via their 4Fe-4S clusters.

Self-organized superlattices along the [001] growth direction in In0.52Al0.48As layers grown on nominally (001) InP substrates by molecular beam epitaxy

Horizontal self-organized superlattice structures consisting of alternating In-rich and Al-rich layers formed naturally during solid-source molecular beam epitaxy (MBE) growth of In0.52Al0.48As on exactly (001) InP substrates, with In and At fluxes unchanged. The growth temperatures were changed from 490 to 510 degrees C, the most commonly used growth temperature for In0.52Al0.48As alloy. No self-organized superlattices (SLs) were observed at the growth temperature 490 degrees C, and self-organized SLs were observed in InAlAs layers at growth temperatures ranging from 498 to 510 degrees C. The results show that the period of the SLs is very highly regular, with the value of similar to 6 nm, and the composition of In or Al varies approximately sinusoidally along the [001] growth direction. The theoretical simulation results confirm that the In composition modulation amplitude is less than 0.02 relative the In composition of the In0.52Al0.48As lattice matched with the InP substrate. The influence of InAs self-organized quantum wires on the spontaneously formed InxAl1-xAs/InyAl1-yAs SLs was also studied and the formation of self-organized InxAl1-xAs/InyAl1-yAs SLs was attributed to the strain-mediated surface segregation process during MBE growth of In0.52Al0.48As alloy. (C) 2005 Published by Elsevier Ltd.

Effects of different modified underlayer surfaces on growth and optical properties of InGaN quantum dots

InGaN/GaN quantum dots were grown on the sapphire (0 0 0 1) substrate in a metalorganic chemical vapor deposition system. The morphologies of QDs deposited on different modified underlayer (GaN) surfaces, including naturally as grown, Ga-mediated, In-mediated, and air-passivated ones, were investigated by atomic force microscopy (AFM). Photo luminescence (PL) method is used to evaluate optical properties. It is shown that InGaN QDs can form directly on the natural GaN layer. However, both the size and distribution show obvious inhomogeneities. Such a heavy fluctuation in size leads to double peaks for QDs with short growth time, and broad peaks for QDs with long growth time in their low-temperature PL spectra. QDs grown on the Ga-mediated GaN underlayer tends to coalesce. Distinct transform takes place from 3D to 2D growth on the In-mediated ones, and thus the formation of QDs is prohibited. Those results clarify Ga and In's surfactant behavior. When the GaN underlayer is passivated in the air, and together with an additional low-temperature-grown seeding layer, however, the island growth mode is enhanced. Subsequently, grown InGaN QDs are characterized by a relatively high density and an improved Gaussian-like distribution in size. Short surface diffusion length at low growth temperature accounts for that result. It is concluded that reduced temperature favors QD's 3D growth and surface passivation can provide another promising way to obtain high-density QDs that especially suits MOCVD system. (c) 2004 Elsevier Ltd. All rights reserved.

In(OH)(3) and In2O3 nanorod bundles and spheres: Microemulsion-mediated hydrothermal synthesis and luminescence properties

Indium hydroxide, In(OH)(3), nano-microstructures with two kinds of morphology, nanorod bundles (around 500 nm in length and 200 nm in diameter) and caddice spherelike agglomerates (around 750 - 1000 nm in diameter), were successfully prepared by the cetyltrimethylammonium bromide (CTAB)/water/cyclohexane/n-pentanol microemulsion-mediated hydrothermal process. Calcination of the In(OH)(3) crystals with different morphologies (nanorod bundles and spheres) at 600 degrees C in air yielded In2O3 crystals with the same morphology. X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and photoluminescence (PL) spectra as well as kinetic decays were used to characterize the samples. The pH values of microemulsion play an important role in the morphological control of the as-formed In(OH)(3) nano-microstructures from the hydrothermal process. The formation mechanisms for the In( OH) 3 nano- microstructures have been proposed on an aggregation mechanism. In2O3 nanorod bundles and spheres show a similar blue emission peaking around 416 and 439 nm under the 383-nm UV excitation, which is mainly attributed to the oxygen vacancies in the In2O3 nano-microstructures.

Axial growth of hexactinellid spicules: Formation of cone-like structural units in the giant basal spicules of the hexactinellid Monorhaphis

The glass sponge Monorhaphis chuni (Porifera: Hexactinellida) forms the largest bio-silica structures on Earth; their giant basal spicules reach sizes of up to 3 m and diameters of 8.5 mm. Previously, it had been shown that the thickness growth proceeds by appositional layering of individual lamellae; however, the mechanism for the longitudinal growth remained unstudied. Now we show, that the surface of the spicules have towards the tip serrated relief structures that are consistent in size and form with the protrusions on the surface of the spicules. These protrusions fit into the collagen net that surrounds the spicules. The widths of the individual lamellae do not show a pronounced size tendency. The apical elongation of the spicule proceeds by piling up cone-like structural units formed from silica. As a support of the assumption that in the extracellular space silicatein(-like) molecules exist that associate with the external surface of the respective spicule immunogold electron microscopic analyses were performed. With the primmorph system from Suberites domuncula we show that silicatein(-like) molecules assemble as string- and net-like arrangements around the spicules. At their tips the silicatein(-like) molecules are initially stacked and at a later stay also organized into net-like structures. Silicatein(-like) molecules have been extracted from the giant basal spicule of Monorhaphis. Applying the SDS-PAGE technique it could be shown that silicatein molecules associate to dimers and trimers. Higher complexes (filaments) are formed from silicatein(-like) molecules, as can be visualized by electron microscopy (SEM). In the presence of ortho-silicate these filaments become covered with 30-60 nm long small rod-like/cuboid particles of silica. From these data we conclude that the apical elongation of the spicules of Monorhaphis proceeds by piling up cone-like silica structural units, whose synthesis is mediated by silicatein(-like) molecules. (C) 2008 Elsevier Inc. All rights reserved.

Possible suppression of exogenous beta-1,3-glucanase gene gluc78 on rice transformation and growth

Three genes encoding for fungal cell wall degrading enzymes (CWDE), ech42, nag7O and gluc78 from the biocontrol fungus Trichoderma atroviride were transformed into rice mediated by Agrobacterium tumefaciens singly and in all possible combinations. A total of more than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. Our data indicated that gluc78 gene had negative effects on transformation frequency and plant growth. Some regenerated plants with gluc78 gene were stunted; spontaneously produced brown specks; could not tassel. The combination with either one of the two other genes (ech42, nag70) present in the same T-DNA region reduced the negative effect of gluc78 on plant growth. These results indicated that expression of several genes in one T-DNA region interfered with each other and expression of exogenous gene in recipient plant was a complex behavior. (c) 2007 Published by Elsevier Ireland Ltd.

Investigation of lactic acid bacteria mediated bioprotection with applications in cereal industry. Case-study: malting process

Antifungal compounds produced by Lactic acid bacteria (LAB) metabolites can be natural and reliable alternative for reducing fungal infections pre- and post-harvest with a multitude of additional advantages for cereal-base products. Toxigenic and spoilage fungi are responsible for numerous diseases and economic losses. This thesis includes an overview of the impact fungi have on aspects of the cereal food chain. The applicability of LAB in plant protection and cereal industry is discussed in detail. Specific case studies include Fusarium head blight, and the impact of fungi in the malting and baking industry. The impact of Fusarium culmorum infected raw barley on the final malt quality was part of the investigation. In vitro infected barley grains were fully characterized. The study showed that the germinative energy of infected barley grains decreased by 45% and grains accumulated 199 μg.kg-1 of deoxynivalenol (DON). Barley grains were subsequently malted and fully characterized. Fungal biomass increased during all stages of malting. Infected malt accumulated 8-times its DON concentration during malting. Infected malt grains revealed extreme structural changes due to proteolytic, (hemi)-cellulolytic and starch degrading activity of the fungi, this led to increased friability and fragmentation. Infected grains also had higher protease and β-glucanase activities, lower amylase activity, a greater proportion of free amino and soluble nitrogen, and a lower β-glucan content. Malt loss was over 27% higher in infected malt when compared to the control. The protein compositional changes and respective enzymatic activity of infected barley and respective malt were characterized using a wide range of methods. F. culmorum infected barley grains showed an increase in proteolytic activity and protein extractability. Several metabolic proteins decreased and increased at different rates during infection and malting, showing a complex F. culmorum infection interdependence. In vitro F. culmorum infected malt was used to produce lager beer to investigate changes caused by the fungi during the brewing processes and their effect on beer quality attributes. It was found, that the wort containing infected malt had a lower pH, a higher FAN, higher β-glucan and a 45% increase in the purging rate, and led to premature yeast flocculation. The beer produced with infected malt (IB) had also a significantly different amino acid profile. IB flavour characterization revealed a higher concentration of esters, fusel alcohols, fatty acids, ketones, and dimethylsulfide, and in particular, acetaldehyde, when compared to the control. IB had a greater proportion of Strecker aldehydes and Maillard products contributing to an increased beer staling character. IB resulted in a 67% darker colour with a trend to better foam stability. It was also found that 78% of the accumulated mycotoxin deoxynivalenol in the malt was transferred into beer. A LAB cell-freesupernatant (cfs), produced in wort-base substrate, was investigated for its ability to inhibit Fusarium growth during malting. Wort was a suitable substrate for LAB exhibiting antifungal activity. Lactobacillus amylovorus DSM19280 inhibited 104 spores.mL-1 for 7 days, after 120 h of fermentation, while Lactobacillus reuteri R29 inhibited 105 spores.mL-1 for 7 days, after 48 h of fermentation. Both LAB cfs had significant different organic acid profiles. Acid-base antifungal compounds were identified and, phenyllactic, hydroxy-phenyllactic, and benzoic acids were present in higher concentrations when compared to the control. A 3 °P wort substrate inoculated With L. reuteri R29 (cfs) was applied in malting and successfully inhibited Fusarium growth by 23%, and mycotoxin DON by 80%. Malt attributes resulted in highly modified grains, lower pH, higher colouration, and higher extract yield. The implementation of selected LAB producing antifungal compounds can be used successfully in the malting process to reduce mould growth and mycotoxin production.

Targeting A20 decreases glioma stem cell survival and tumor growth.

Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioblastoma stem cells (GSCs). GSCs are regulated by molecular pathways distinct from the bulk tumor that may be useful therapeutic targets. We determined that A20 (TNFAIP3), a regulator of cell survival and the NF-kappaB pathway, is overexpressed in GSCs relative to non-stem glioblastoma cells at both the mRNA and protein levels. To determine the functional significance of A20 in GSCs, we targeted A20 expression with lentiviral-mediated delivery of short hairpin RNA (shRNA). Inhibiting A20 expression decreased GSC growth and survival through mechanisms associated with decreased cell-cycle progression and decreased phosphorylation of p65/RelA. Elevated levels of A20 in GSCs contributed to apoptotic resistance: GSCs were less susceptible to TNFalpha-induced cell death than matched non-stem glioma cells, but A20 knockdown sensitized GSCs to TNFalpha-mediated apoptosis. The decreased survival of GSCs upon A20 knockdown contributed to the reduced ability of these cells to self-renew in primary and secondary neurosphere formation assays. The tumorigenic potential of GSCs was decreased with A20 targeting, resulting in increased survival of mice bearing human glioma xenografts. In silico analysis of a glioma patient genomic database indicates that A20 overexpression and amplification is inversely correlated with survival. Together these data indicate that A20 contributes to glioma maintenance through effects on the glioma stem cell subpopulation. Although inactivating mutations in A20 in lymphoma suggest A20 can act as a tumor suppressor, similar point mutations have not been identified through glioma genomic sequencing: in fact, our data suggest A20 may function as a tumor enhancer in glioma through promotion of GSC survival. A20 anticancer therapies should therefore be viewed with caution as effects will likely differ depending on the tumor type.

Prolactin receptor signaling is essential for perinatal brown adipocyte function: a role for insulin-like growth factor-2.

BACKGROUND: The lactogenic hormones prolactin (PRL) and placental lactogens (PL) play central roles in reproduction and mammary development. Their actions are mediated via binding to PRL receptor (PRLR), highly expressed in brown adipose tissue (BAT), yet their impact on adipocyte function and metabolism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: PRLR knockout (KO) newborn mice were phenotypically characterized in terms of thermoregulation and their BAT differentiation assayed for gene expression studies. Derived brown preadipocyte cell lines were established to evaluate the molecular mechanisms involved in PRL signaling on BAT function. Here, we report that newborn mice lacking PRLR have hypotrophic BAT depots that express low levels of adipocyte nuclear receptor PPARgamma2, its coactivator PGC-1alpha, uncoupling protein 1 (UCP1) and the beta3 adrenoceptor, reducing mouse viability during cold challenge. Immortalized PRLR KO preadipocytes fail to undergo differentiation into mature adipocytes, a defect reversed by reintroduction of PRLR. That the effects of the lactogens in BAT are at least partly mediated by Insulin-like Growth Factor-2 (IGF-2) is supported by: i) a striking reduction in BAT IGF-2 expression in PRLR KO mice and in PRLR-deficient preadipocytes; ii) induction of cellular IGF-2 expression by PRL through JAK2/STAT5 pathway activation; and iii) reversal of defective differentiation in PRLR KO cells by exogenous IGF-2. CONCLUSIONS: Our findings demonstrate that the lactogens act in concert with IGF-2 to control brown adipocyte differentiation and growth. Given the prominent role of brown adipose tissue during the perinatal period, our results identified prolactin receptor signaling as a major player and a potential therapeutic target in protecting newborn mammals against hypothermia.

Growth hormone mitigates against lethal irradiation and enhances hematologic and immune recovery in mice and nonhuman primates.

Medications that can mitigate against radiation injury are limited. In this study, we investigated the ability of recombinant human growth hormone (rhGH) to mitigate against radiation injury in mice and nonhuman primates. BALB/c mice were irradiated with 7.5 Gy and treated post-irradiation with rhGH intravenously at a once daily dose of 20 microg/dose for 35 days. rhGH protected 17 out of 28 mice (60.7%) from lethal irradiation while only 3 out of 28 mice (10.7%) survived in the saline control group. A shorter course of 5 days of rhGH post-irradiation produced similar results. Compared with the saline control group, treatment with rhGH on irradiated BALB/c mice significantly accelerated overall hematopoietic recovery. Specifically, the recovery of total white cells, CD4 and CD8 T cell subsets, B cells, NK cells and especially platelets post radiation exposure were significantly accelerated in the rhGH-treated mice. Moreover, treatment with rhGH increased the frequency of hematopoietic stem/progenitor cells as measured by flow cytometry and colony forming unit assays in bone marrow harvested at day 14 after irradiation, suggesting the effects of rhGH are at the hematopoietic stem/progenitor level. rhGH mediated the hematopoietic effects primarily through their niches. Similar data with rhGH were also observed following 2 Gy sublethal irradiation of nonhuman primates. Our data demonstrate that rhGH promotes hematopoietic engraftment and immune recovery post the exposure of ionizing radiation and mitigates against the mortality from lethal irradiation even when administered after exposure.

Beta-arrestin-mediated beta1-adrenergic receptor transactivation of the EGFR confers cardioprotection.

Deleterious effects on the heart from chronic stimulation of beta-adrenergic receptors (betaARs), members of the 7 transmembrane receptor family, have classically been shown to result from Gs-dependent adenylyl cyclase activation. Here, we identify a new signaling mechanism using both in vitro and in vivo systems whereby beta-arrestins mediate beta1AR signaling to the EGFR. This beta-arrestin-dependent transactivation of the EGFR, which is independent of G protein activation, requires the G protein-coupled receptor kinases 5 and 6. In mice undergoing chronic sympathetic stimulation, this novel signaling pathway is shown to promote activation of cardioprotective pathways that counteract the effects of catecholamine toxicity. These findings suggest that drugs that act as classical antagonists for G protein signaling, but also stimulate signaling via beta-arrestin-mediated cytoprotective pathways, would represent a novel class of agents that could be developed for multiple members of the 7 transmembrane receptor family.

Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras.

Stimulation of Gi-coupled receptors leads to the activation of mitogen-activated protein kinases (MAP kinases). In several cell types, this appears to be dependent on the activation of p21ras (Ras). Which G-protein subunit(s) (G alpha or the G beta gamma complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the beta-adrenergic receptor kinase, containing its G beta gamma-binding domain, is a cellular G beta gamma antagonist capable of specifically distinguishing G alpha- and G beta gamma-mediated processes. Using this G beta gamma inhibitor, we studied Ras and MAP kinase activation through endogenous Gi-coupled receptors in Rat-1 fibroblasts and through receptors expressed by transiently transfected COS-7 cells. We report here that both Ras and MAP kinase activation in response to lysophosphatidic acid is markedly attenuated in Rat-1 cells stably transfected with a plasmid encoding this G beta gamma antagonist. Likewise in COS-7 cells transfected with plasmids encoding Gi-coupled receptors (alpha 2-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed G beta gamma antagonist. Ras-MAP kinase activation mediated through a Gq-coupled receptor (alpha 1-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this G beta gamma antagonist. These results identify G beta gamma as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of Gi-coupled receptors.

NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli.

Outer membrane vesicles (OMVs) are ubiquitously secreted from the outer membrane (OM) of Gram-negative bacteria. These heterogeneous structures are composed of OM filled with periplasmic content from the site of budding. By analyzing mutants that have vesicle production phenotypes, we can gain insight into the mechanism of OMV budding in wild-type cells, which has thus far remained elusive. In this study, we present data demonstrating that the hypervesiculation phenotype of the nlpI deletion mutant of Escherichia coli correlates with changes in peptidoglycan (PG) dynamics. Our data indicate that in stationary phase cultures the nlpI mutant exhibits increased PG synthesis that is dependent on spr, consistent with a model in which NlpI controls the activity of the PG endopeptidase Spr. In log phase, the nlpI mutation was suppressed by a dacB mutation, suggesting that NlpI regulates penicillin-binding protein 4 (PBP4) during exponential growth. The data support a model in which NlpI negatively regulates PBP4 activity during log phase, and Spr activity during stationary phase, and that in the absence of NlpI, the cell survives by increasing PG synthesis. Further, the nlpI mutant exhibited a significant decrease in covalent outer membrane (OM-PG) envelope stabilizing cross-links, consistent with its high level of OMV production. Based on these results, we propose that one mechanism wild-type Gram-negative bacteria can use to modulate vesiculation is by altering PG-OM cross-linking via localized modulation of PG degradation and synthesis.

IGF-1 or insulin, and the TSH cyclic AMP cascade separately control dog and human thyroid cell growth and DNA synthesis, and complement each other in inducing mitogenesis.

The regular doubling of cell mass, and therefore of cell protein content, is required for repetitive cell divisions. Preliminary observations have shown that in dog thyrocytes insulin induces protein accumulation but not DNA synthesis, while TSH does not increase protein accumulation but triggers DNA synthesis in the presence of insulin. We show here that EGF and phorbol myristate ester complement insulin action in the same way. HGF is the only factor activating both protein accumulation and DNA synthesis. The effects of insulin on protein accumulation and in permitting the TSH effect are reproduced by IGF-1 and are mediated, at least in part by the IGF-1 receptor. The concentration effect curves are similar for both effects. Similar results are obtained in human thyrocytes. They reflect true cell growth, as shown by increases in RNA content and cell size. Carbachol and fetal calf serum also stimulate protein synthesis and accumulation without triggering DNA synthesis, but they are not permissive for the mitogenic effects of TSH or of the general adenylate cyclase activator, forskolin. Moreover the mitogenic effect of TSH greatly decreased in cells deprived of insulin for 2 days although these cells remain hypertrophic. Hypertrophy may therefore be necessary for cell division, but it is not sufficient to permit it. Three different mechanisms can therefore be distinguished in the mitogenic action of TSH: (1) the increase of cell mass (hypertrophy) induced by insulin or IGF-1; (2) the permissive effect of insulin or IGF-1 on the mitogenic effect of TSH which may involve both the increase of cell mass and the induction of specific proteins such as cyclin D3 and (3) the mitogenic effect of the TSH cyclic AMP cascade proper.