High Cell Density Conditions Research Articles

Quorum-sensing regulation in staphylococci-an overview.

Staphylococci are frequent human commensals and some species can cause disease. Staphylococcus aureus in particular is a dangerous human pathogen. In staphylococci, the ability to sense the bacterial cell density, or quorum, and to respond with genetic adaptations is due to one main system, which is called accessory gene regulator (Agr). The extracellular signal of Agr is a post-translationally modified peptide containing a thiolactone structure. Under conditions of high cell density, Agr is responsible for the increased expression of many toxins and degradative exoenzymes, and decreased expression of several colonization factors. This regulation is important for the timing of virulence factor expression during infection and the development of acute disease, while low activity of Agr is associated with chronic staphylococcal infections, such as those involving biofilm formation. Accordingly, drugs inhibiting Agr are being evaluated for their capacity to control acute forms of S. aureus infection.

Human amniotic fluid stem cells: neural differentiation in vitro and in vivo.

The successful integration of stem cells after their implantation into the brain has become a central issue in modern neuroscience. In this study, we test the neural differentiation potential of c-Kit(+)/Oct-4(+) human amniotic fluid stem cells (hAFSCs) in vitro and their survival and integration in vivo. hAFSCs were induced towards neural differentiation and specific markers (GFAP, β-III tubulin, CNPase, MAP2, NeuN, synapsines, S100, PMP22) were detected by immunofluorescence and Western blot analysis. Glial proteins were expressed as early as 2weeks after the initial differentiation stimulus, whereas neuronal markers started to appear from the third week of differentiation under culturing conditions of high cell density. This timeline suggested that glial cells possessed a promoting role in the differentiation of hAFSCs towards a neuronal fate. hAFSCs were then implanted into the lateral ventricle of the brain of 1-day-old rats, since neuronal development occurs up to 1 month after birth in this animal model. Our data showed that hAFSCs survived for up to 6 weeks post-implantation, were integrated into various areas of the central nervous system and migrated away from the graft giving rise to mature neurons and oligodendrocytes. We conclude that hAFSCs are able to differentiate and integrate into nervous tissue during development in vivo.

Long‐term adaptation of Saccharomyces cerevisiae to the burden of recombinant insulin production

High-level production of heterologous proteins is likely to impose a metabolic burden on the host cell and can thus affect various aspects of cellular physiology. A data-driven approach was applied to study the secretory production of a human insulin analog precursor (IAP) in Saccharomyces cerevisiae during prolonged cultivation (80 generations) in glucose-limited aerobic chemostat cultures. Physiological characterization of the recombinant cells involved a comparison with cultures of a congenic reference strain that did not produce IAP, and time-course analysis of both strains aimed at identifying the metabolic adaptation of the cells towards the burden of IAP production. All cultures were examined at high cell density conditions (30 g/L dry weight) to increase the industrial relevance of the results. The burden of heterologous protein production in the recombinant strain was explored by global transcriptome analysis and targeted metabolome analysis, including the analysis of intracellular amino acid pools, glycolytic metabolites, and TCA intermediates. The cellular re-arrangements towards IAP production were categorized in direct responses, for example, enhanced metabolism of amino acids as precursors for the formation of IAP, as well as indirect responses, for example, changes in the central carbon metabolism. As part of the long-term adaptation, a metabolic re-modeling of the IAP-expressing strain was observed, indicating an augmented negative selection pressure on glycolytic overcapacity, and the emergence of mitochondrial dysfunction. The evoked metabolic re-modeling of the cells led to less optimal conditions with respect to the expression and processing of the target protein and thus decreased the cellular expression capacity for the secretory production of IAP during prolonged cultivation.

The Challenge of Treating Oral Infections Caused by Biofilms

In natural systems, the harsh environments, nutrient/respiration needs, and sheer flow, make a community lifestyle preferable or inevitable for microorganisms. Furthermore, the bacteria take advantage of the communities’ organization to protect them from eradication by conventional antimicrobial therapy. A biofilm community is a microorganism aggregate characterized by cells that are attached to a substratum or interface or to each other; are embedded in a matrix of extracellular polymeric substances (EPS) that they have produced; and exhibit an altered phenotype with respect to growth rate and gene transcription [1]. The oral cavity is an example of a challenging environment for the long-term persistence of bacteria and fungi. Fluctuations in nutrient supply, temperature, pH, and the shear force of saliva flow have selected for a biofilm community adapted to high cell density, species diversity, and dynamic growth conditions [2, 3]. The microorganism production of EPS matrix is also very important for their interaction with the environment [4,5]. The EPS provides a physical barrier that inhibits attachment to macrophages and phagocytes, while the reduced microorganisms’ metabolic activity precludes the use of antibiotics that act on them during growth periods [6-9]. Those are some of the challenges encountered when there is a need to treat or eradicate a disease-causing biofilm. Beyond conventional treatments, which employ a variety of antimicrobial agents, the actual disruption of biofilms currently entails mechanical procedures such as scouring. In order to inactivate the encased microbial cells, antimicrobial molecules must diffuse throughout the biofilm matrix; but the EPS constituting this matrix present a diffusional barrier for these molecules by influencing either their rate of transport to the biofilm interior or by the reaction of the antimicrobial with the matrix material [1]. The viscoelastic properties of the polysaccharides in the EPS are also attributed to the difficulty in disrupting the mature biofilm [10]. Among well-documented

Topical Treatment with Basic Fibroblast Growth Factor Promotes Wound Healing and Barrier Recovery Induced by Skin Abrasion

It has been reported that basic fibroblast growth factor (bFGF) promotes the healing of skin ulceration by inducing fibroblast proliferation, yet the role of bFGF on epidermal barrier function, especially from the perspective of scratch-induced skin abrasion, remains unknown. To this end, we initially developed an epidermal abrasion mouse model induced by scratching with a stainless-steel wire brush, and examined the effects of bFGF on the wound healing induced by skin abrasion. This procedure induced a significant elevation of transepidermal water loss (TEWL) in a scratch-count-dependent manner. This elevated TEWL was significantly decreased following topical application of bFGF to the skin. In addition, bFGF increased the expression of Ki67 in keratinocytes following mechanical scratching. These results suggest that bFGF enhances keratinocyte proliferation, which, in turn, repairs the skin barrier disruption and wounds caused by scratching in mice. Consistently, bFGF stimulated proliferation of normal human epidermal keratinocytes (NHEK). Intriguingly, the effect of bFGF and other growth factors on NHEK proliferation was additive. However, high cell density diminished the effect of bFGF on NHEK proliferation. This particular result can be explained by our observation that FGF receptor mRNA expression in NHEK was low under conditions of high cell density. Our findings suggest that bFGF stimulates keratinocyte proliferation, especially in a lower cell density environment, to repair skin wound in accord with skin barrier recovery.

Effects of mutual shading, pressurization and oxygen partial pressure on the autotrophical cultivation of Scenedesmus obliquus

The appropriate providing of CO2 and the light energy are known to be detrimental to the growth of photosynthetic cells. In the autotrophical cultivation of Scenedesmus obliquus, the available light intensity drops rapidly due to the effects of mutual shading occurred in the conditions of high cell density. The light penetration ratio promptly decreased to less than 1% at a depth of 1cm, while the cell density achieved 3 OD. The mutual shading effects seemed severely limiting the maximum cell concentration attained. With the increase of tank pressure higher than 0.5atm was demonstrated to reversely affect cell growth, which possibly resulted from the inhibition of more dissolved oxygen existing in the medium. The inlet gas with a high oxygen ratio reduced biomass productivity, which was 0.19g/L/day in the oxygen-enriched batch (98% of pure oxygen supplemental in the inlet gas) as compared to 0.44g/L/day in the control. These results indicate that both mutual shading and high oxygen partial pressure impede the growth of Scenedesmus in autotrophical cultivation.

Bacterial production of recombinant human poly(ADP-ribose) glycohydrolase

Poly(ADP-ribosyl)ation, which is mainly involved in DNA repair and replication, is catalyzed mainly by poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Although recombinant human PARP-1 (hPARP-1) is commercially available, there are no reports on the preparation of recombinant human PARG (hPARG). Here, we report the efficient expression and purification of a recombinant hPARG-catalytic domain (hPARG-CD) from Escherichia coli ( E. coli). hPARG-CD was expressed as a fusion protein with a glutathione S-transferase (GST) tag at the N-terminus and a hexahistidine (6His) tag at the C-terminus. Both high cell density and low temperature culture conditions were important for the maximum production of soluble recombinant hPARG-CD. After sequential affinity chromatography using immobilized metal affinity resin and glutathione-Sepharose (GSH-Sephasrose), more than 95% pure recombinant hPARG-CD was obtained with a yield of approximately 2 mg per 1 L of E. coli culture medium. The km and Vmax values of purified recombinant hPARG-CD were 9.0 μM and 35.6 μmol/min/mg protein, respectively. These kinetic values were similar to those of purified endogenous hPARG reported previously. Furthermore, the recombinant hPARG-CD was inhibited by known PARG inhibitors such as adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), eosin Y, and phloxine B. These results show that the recombinant hPARG-CD is useful to search for specific inhibitors and to elucidate the regulatory mechanisms of hPARG.

Online determination of viable biomass up to very high cell densities in Arxula adeninivorans fermentations using an impedance signal

Up to now biomass has been measured online by impedance analysis only at low cell densities in yeast fermentations. As industrial fermentation processes focus, for example, on producing high target concentrations of biocatalysts or pharmaceutical proteins, it is important to investigate cell growth under high cell-density conditions. Therefore, for the first time, biomass has been measured online using impedance analysis in a 50 L high-pressure stirred tank reactor. As model organism the yeast Arxula adeninivorans was cultivated in two chemically defined mineral media at a constant growth rate in fed-batch mode. To ensure aerobic culture conditions over the entire fermentation time, the fermentations were conducted at an elevated headspace overpressure of up to 9.5 bar. The highest oxygen transfer rate value of 0.56 mol L −1 h −1 ever reported for yeast fermentations was measured in these investigations. Unlike previous findings, in this study a linear correlation was found between capacitance and biomass up to concentrations of 174 g L −1 dry cell weight.

Examination of the hdrRM regulon yields insight into the competence system of Streptococcus mutans

Previous studies have identified the hdrRM operon as a novel regulatory system induced by conditions of high cell density. Little is known about the genes under the control of this system, but a variety of important phenotypes are associated with either hdrR overexpression or mutation of hdrM. To characterize the regulatory function of the HdrRM system in Streptococcus mutans we used a microarray approach to compare the transcriptional profiles of an hdrR overexpression strain with an hdrM mutant. Both strains exhibited almost identical profiles, which included all of the known late competence genes as well as a variety of competence-induced bacteriocins. Through a combination of real-time reverse transcription-polymerase chain reaction (PCR), reporter gene analysis and random amplification of complementary DNA ends PCR, we confirmed the role of comX as a central intermediate regulator of numerous genes in the hdrRM regulon. Through these studies, we also identified novel comX-regulated genes required for natural competence. Taken together, our results suggest that the primary function of the HdrRM system is to regulate the late competence genes together with various bacteriocins. This occurs independently of the ComCDE system, even though both systems regulate nearly identical genes. This suggests that S. mutans has multiple parallel input sensory systems that control the same output response: the induction of natural competence and concurrent production of bacteriocins.

Abstract 993: A novel cadherin-dependent Rac/Stat3 pathway in invasive breast cancer

Abstract Stat (signal transducer and activator of transcription) 3 is a transcription factor involved in a number of signaling pathways activated by receptor and non-receptor tyrosine kinases. Constitutively active Stat3 alone is sufficient to induce tumorigenesis. We have previously shown that Stat3 can also be activated by engagement of the epithelial adhesion molecule E-cadherin (MCR 7:1310-27, 2009). An additional novel finding in this study was that levels of total and activated Rac1 proteins increase dramatically, thereby causing the Stat3 stimulation. Interestingly, Stat3 expression was independently found to downregulate p53 expression in several epithelial and fibroblast cell lines. These findings suggest a novel Cadherin/Rac1/Stat3-dependent pathway that causes sustained downregulation of p53, resulting in uncontrolled growth and survival. As a first step to test this hypothesis in breast cancer, we are assessing the expression profiles and localization of these molecules in mouse breast epithelial and carcinoma cell lines. The results revealed that at low cell density in 2-D culture conditions, E-cadherins and b-catenin showed strong cytoplasmic and perinuclear localization, respectively. In contrast, at high cell density both markers showed strong localization to the plasma membrane. On the other hand, a breast carcinoma cell line showed cytoplasmic staining of pan-cadherin and nuclear staining of β-catenin under both low and high cell density conditions. Western blotting revealed that levels of pan and activated (pY705) Stat3, and Rac1 were increased at high compared to low cell densities, consistent with a cadherin-dependent activation of this pathway. We are currently extrapolating these findings to a 3-D Matrigel culture model in which mammary epithelial cells (EPH4) form acini-like spheroids with hollow. Under these conditions, total Stat3 levels are sustained for 4-6 days followed by a transient increase in cyclin D1 expression. We are also performing immunohistochemical analyses of these molecular biomarkers on a cohort of human breast cancer tissues (n=59) to test associations of protein expression profiles in human cancer. Preliminary results have shown increased expression of active Stat3 in breast tumours compared to normal mammoplasty tissues. In addition, increased expression of p53 was observed in a subset (17%) of these breast cancers. Detailed ARIOL imaging analysis is currently in progress to evaluate relative expression levels and associations of Stat3 and p53 in breast cancer tissues. Our findings suggest a novel pathway by which Stat3 activation causes a dramatic decrease in p53 levels, resulting in increased proliferation and survival of cancer cells. (Supported by CIHR and CBCRA). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 993.

Directed evolution of the transcription factor XylS for development of improved expression systems.

SummaryThe inducible Pm promoter together with its cognate positive transcription regulator XylS has been shown to be useful for recombinant protein production under high cell density conditions. Here we report directed evolution of XylS resulting in mutant proteins with increased ability to stimulate transcription in Escherichia coli from Pm. A first round of mutagenesis using error‐prone PCR on xylS was used to construct a library consisting of about 430 000 clones, and this library could be efficiently screened with respect to stimulation of expression from Pm due to a positive correlation between the level of expression of the reporter gene, bla (encoding β‐lactamase), and the ampicillin tolerance of the corresponding host cells. Fourteen different amino acid substitutions in XylS were found to separately lead to up to nearly a threefold stimulation of expression under induced conditions, relative to wild type. These mutations were all located in the part corresponding to the N‐terminal half of the protein. Varying combinations of the mutations resulted in further stimulation, and the best results (about 10‐fold stimulation under induced conditions) were obtained by using a random shuffling procedure followed by a new round of screening. The uninduced levels of expression for the same mutants also increased, but only about four times. Through in silico 3D modelling of the N‐terminal domain of XylS, it was observed that the evolved mutant proteins contained substitutions that were positioned in different parts of the predicted structure, including a β‐barrel putatively responsible for effector binding and a coiled coil probably important for dimerization. The total production of the host‐toxic antibody fragment scFv‐phOx expressed from Pm with the evolved XylS mutant protein StEP‐13 was about ninefold higher than with wild‐type XylS, demonstrating that directed evolution of transcription factors can be an important new tool to achieve high‐level recombinant protein production.

Tgfbr2 is required for development of the skull vault

Transforming growth factor beta (TGFbeta) is known to play important roles in multiple developmental processes. One of the main functions is in skeletal development. Our previous studies demonstrated that loss of Tgfbr2 in Prx1Cre-expressing limb mesenchyme results in defects in the long bones and joints of mice. Here we show that loss of Tgfbr2 also results in defects in the development of the skull vault indicating Tgfbr2 has a critical role in intramembranous bone formation as well as endochondral bone formation. Mutant mice did not survive after birth and demonstrated an open skull. The first signs of skull defects were observed at E14.5 day. Prx1Cre(+)/Tgfbr2(f/f) embryos showed significantly reduced cell proliferation in the developing mesenchyme of the skull by E14.5 day without any detectable alteration in apoptosis suggesting that reduced cell proliferation in Prx1Cre(+)/Tgfbr2(f/f) embryos was at least partially responsible for the defects observed. Immunofluorescent staining showed a significant reduction in the expression of Runx2/Cbfa1 and Osterix/Sp7 in Prx1Cre(+)/Tgfbr2(f/f) embryos suggesting that osteoblast differentiation was also altered in Prx1Cre(+)/Tgfbr2(f/f) embryos. To distinguish between the effects of losing Tgfbr2 on mesenchymal proliferation versus osteoblast differentiation, osteoprogenitor cells from the skulls of Tgfbr2(f/f) embryos were cultured under conditions of high cell density and Tgfbr2 was deleted from the cells using Adeno-Cre virus. RT-PCR analysis showed that the mRNA level of Runx2 and Osterix as well as Dlx5 and Msx2 were down-regulated in Tgfbr2-deleted cultures compared to control cultures indicating that Tgfbr2 regulates osteoblast differentiation independent of regulating proliferation. Together, these results suggest that Tgfbr2 is required for normal development of the skull.

Establishment of Stroma-Free Cultures for Human B Lymphopoiesis: Roles of High Cell Density Condition and Mesenchymal Stem Cell-Secreted Factors.

Background: Due to lacking of appropriate culture systems, it has been difficult to analyze mechanisms of human B lymphocytes. However, we recently found that human mesenchymal stem cells (MSC) have high ability to support human B lymphopoiesis. Although many investigators have believed that stromal layers are essential for supporting the differentiation of human B lymphocyte progenitors, this hypothesis is not a case. By manipulating our co-culture system, we have successfully established stroma-free suspension cultures, which produce CD10+ B cells from human umbilical cord blood (CB) CD34+ cells.Methods: Our stroma-free culture of CB CD34+ cells was performed in QBSF® medium with 10% FCS in the presence of 10 ng/ml stem cell factor (SCF) and 5 ng/ml Flt3-ligand (FL) with or without 5 ng/ml IL-7.Results:1.Even when co-cultures were separated from MSC with membrane filters, they could produce CD10+ cells. Moreover, addition of MSC supernatant to the cultures permitted CD34+ cells to emerge CD10+ cells in the absence of MSC. Therefore, the cell-cell contact between MSC and B lymphocyte progenitors was not essential.2.For stroma-free human B cell cultures with MSC supernatant, QBSF® was the most suitable and serum components were essential while depending on their lot. Although addition of thymic stromal lymphopoietin or IL-7 increased the production of CD10+ cells, neutralizing antibodies for them showed no effect. Addition of Hemokinin-1 antagonist diminished, albeit to a limited degree, the production of CD10+ cells. Addition of neutralizing antibody for CXCR4 had no effect. Therefore, MSC supernatant contains some supportive factors except for them.3.When cultures without MSC or their supernatant stared at 1x104 cells/ml, they could successfully produce CD10+ cells. The density of the cultured cells was critical. The production of CD10+ cells was not detected when CD34+ cells were seeded at low density (1x103/ml). Moreover, when the cultured cells were diluted and adjusted at 1x104/ml weekly, the emergence of CD10+ cells was not observed while the production of CD33+ myeloid cells was enhanced. Therefore, surrounding hematopoietic cells seemed to be required to support human B lymphopoiesis.4.Our suspension cultures of 1x104 CD34+ cells in the presence of SCF, FL, and IL-7 without any stromal materials generated approximately 0.5–1x106 CD10+ cells at 4 week.CD33+ cells were first expanded within 2 weeks, and then CD10+ cells appeared. When the cultured cells were transplanted into NOD/SCID/γcnull mice, they reconstituted both myeloid and B lymphoid lineages. Therefore, some cultured cells maintain stem cell character.Conclusions: We have established stroma-free suspension cultures, which effectively produce CD10+ B cells from CB CD34+ cells. High cell density condition can in part substitute for stromal layers in supporting human B lymphopoiesis although the addition of MSC supernatant enhances the production of CD10+ cells. Our suspension culture does not use any stromal cells, which produce many positive or negative regulators for human B lymphopoiesis. This simplicity proposes that this culture system is useful in a variety of fields such as the screening direct effects of drugs influencing on human B lymphocyte development and the evaluation of progenitors in patients with B-cell malignancies as well as the cloning of human B lymphocyte-supportive molecules.

Genetic characterization of the hdrRM operon: a novel high-cell-density-responsive regulator in Streptococcus mutans

Many species of bacteria can adhere to surfaces and grow as sessile communities. The continued accumulation of bacteria can eventually lead to the extremely high-cell-density environment characteristic of many biofilms or cell colonies. This is the normal habitat of the cariogenic species Streptococcus mutans, which normally resides in the high-cell-density, multispecies community commonly referred to as dental plaque. Previous work has demonstrated that the transcription of two separate bacteriocins can be activated by the high-cell-density conditions created through the centrifugation and incubation of cell pellets. In this study, we identified an uncharacterized two-gene operon that was induced >10-fold by conditions of high cell density. The genes of the operon encode a putative transcription regulator and a membrane protein, which were renamed as hdrR and hdrM, respectively. A transcription fusion to the hdrRM operon confirmed its induction by high cell density. Mutation of hdrM abolished bacteriocin production, greatly increased natural competence, reduced the growth rate, and severely affected biofilm formation. Interestingly, no obvious phenotypes were observed from a non-polar mutation of hdrR or mutations affecting the entire operon. These data suggest that the hdrRM operon may constitute a novel regulatory system responsible for mediating a cellular response to a high-cell-density environment.

Comparative proteomic analysis of high cell density cultivations with two recombinant Bacillus megaterium strains for the production of a heterologous dextransucrase

High cell density cultivations were performed under identical conditions for two Bacillus megaterium strains (MS941 and WH320), both carrying a heterologous dextransucrase (dsrS) gene under the control of the xylA promoter. At characteristic points of the cultivations (end of batch, initial feeding, before and after induction) the proteome was analyzed based on two dimensional gel electrophoresis and mass spectrometric protein identification using the protein database "bmegMEC.v2" recently made available.High expression but no secretion of DsrS was found for the chemical mutant WH320 whereas for MS 941, a defined protease deficient mutant of the same parent strain (DSM319), not even expression of DsrS could be detected. The proteomic analysis resulted in the identification of proteins involved in different cellular pathways such as in central carbon and overflow metabolism, in protein synthesis, protein secretion and degradation, in cell wall metabolism, in cell division and sporulation, in membrane transport and in stress responses.The two strains exhibited considerable variations in expression levels of specific proteins during the different phases of the cultivation process, whereas induction of DsrS production had, in general, little effect. The largely differing behaviour of the two strains with regard to DsrS expression can be attributed, at least in part, to changes observed in the proteome which predominantly concern biosynthetic enzymes and proteins belonging to the membrane translocation system, which were strongly down-regulated at high cell densities in MS941 compared with WH320. At the same time a cell envelope-associated quality control protease and two peptidoglycan-binding proteins related to cell wall turnover were strongly expressed in MS941 but not found in WH320. However, to further explain the very different physiological responses of the two strains to the same cultivation conditions, it is necessary to identify the mutated genes in WH320 in addition to the known lacZ.In view of the results of this proteomic study it seems that at high cell density conditions and hence low growth rates MS941, in contrast to WH320, does not maintain a vegetative growth which is essential for the expression of the foreign dsrS gene by using the xylA promoter. It is conceivable that applications of a promoter which is highly active under nutrient-limited cultivation conditions is necessary, at least for MS941, for the overexpression of recombinant genes in such B. megaterium fed-batch cultivation process. However to obtain a heterologous protein in secreted and properly folded form stills remains a big challenge.

Inactivation of von Hippel-Lindau Gene Induces Constitutive Phosphorylation of MET Protein in Clear Cell Renal Carcinoma

It is well known that inactivation of von Hippel-Lindau (VHL) gene predisposes for human clear cell renal carcinoma (CCRC). However, details about critical roles of VHL inactivation during tumorigenesis are still unknown. MET protein is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), which regulates cell growth, cell morphology, and cell motility. We showed that MET protein overexpressed in CCRC cells was phosphorylated without HGF/SF. This constitutive phosphorylation of MET protein in CCRC cells was inhibited by the rescue of exogenous wild-type VHL gene without a decrease in expression level of MET protein. Interestingly, wild-type VHL gene suppressed the phosphorylation of MET protein only under high cell density conditions. Additionally, MET protein activated by the inactivation of VHL gene modified cell adherence, including N-cadherin and beta-catenin. When activation of MET protein in CCRC cells was inhibited by the MET inhibitor K252a, the growth of CCRC cells in vitro and the tumorigenesis induced by CCRC cells in nude mice were suppressed. From these results, we concluded that inactivation of VHL gene induced constitutive phosphorylation of MET protein and modified intercellular adherence structure to trigger the cell growth released from contact inhibition, finally resulting in tumorigenesis. This is one of the mechanisms of CCRC oncogenesis, and MET protein has potential as a molecular target for novel CCRC therapies.

Intein-mediated protein purification of fusion proteins expressed under high-cell density conditions in E. coli

The intein-mediated purification system has the potential to significantly reduce the recovery costs of industrial recombinant proteins. The ability of inteins to catalyze a controllable peptide bond cleavage reaction can be used to separate a recombinant protein from its affinity tag during affinity purification. Inteins have been combined with a chitin-binding domain to serve as a self-cleaving affinity tag, facilitating highly selective capture of the fusion protein on an inexpensive substrate--chitin (IMPACT) system, New England Biolabs, Beverly, MA). This purification system has been used successfully at a lab scale in low cell density cultures, but has not been examined comprehensively under high-cell density conditions in defined medium. In this study, the intein-mediated purification of three commercially relevant proteins expressed under high-cell density conditions in E. coli was studied. Additionally, losses during the purification process were quantified. The data indicate that the intein fusion proteins expressed under high cell density fermentations were stable in vivo after induction for a significant duration, and the intein fusion proteins could undergo thiol or pH and temperature initiated cleavage reaction in vitro. Thus, the intein-mediated protein purification system potentially could be employed for the production of recombinant proteins at the industrial-scale.

Rethinking 'secondary' metabolism: physiological roles for phenazine antibiotics

Microorganisms exist in the environment as multicellular communities that face the challenge of surviving under nutrient-limited conditions. Chemical communication is an essential part of the way in which these populations coordinate their behavior, and there has been an explosion of understanding in recent years regarding how this is accomplished. Much less, however, is understood about the way these communities sustain their metabolism. Bacteria of the genus Pseudomonas are ubiquitous, and are distinguished by their production of colorful secondary metabolites called phenazines. In this article, we suggest that phenazines, which are produced under conditions of high cell density and nutrient limitation, may be important for the persistence of pseudomonads in the environment.

Global expression profiling of Bacillus subtilis cells during industrial‐close fed‐batch fermentations with different nitrogen sources

A detailed gene expression analysis of industrial-close Bacillus subtilis fed-batch fermentation processes with casamino acids as the only nitrogen source and with a reduced casamino acid concentration but supplemented by ammonia was carried out. Although glutamine and arginine are supposed to be the preferred nitrogen sources of B. subtilis, we demonstrate that a combined feeding of ammonia and casamino acids supports cell growth under fed-batch fermentation conditions. The transcriptome and proteome analyses revealed that the additional feeding of ammonia in combination with a reduced amino acid concentration results in a significantly lower expression level of the glnAR or tnrA genes, coding for proteins, which are mainly involved in the nitrogen metabolism of B. subtilis. However, the mRNA levels of the genes of the ilvBHC-leuABD and hom-thrCB operons were significantly increased, indicating a valine, leucine, isoleucine, and threonine limitation under these fermentation conditions. In contrast, during the fermentation with casamino acids as the only nitrogen source, several genes, which play a crucial role in nitrogen metabolism of B. subtilis (e.g., glnAR, nasCDE, nrgAB, and ureABC), were up-regulated, indicating a nitrogen limitation under these conditions. Furthermore, increased expression of genes, which are involved in motility and chemotaxis (e.g., hag, fliT) and in acetoin metabolism (e.g., acoABCL), was determined during the fermentation with the mixed nitrogen source of casamino acids and ammonia, indicating a carbon limitation under these fermentation conditions. Under high cell density and slow growth rate conditions a weak up-regulation of autolysis genes could be observed as well as the induction of a number of genes involved in motility, chemotaxis and general stress response. Results of this study allowed the selection of marker genes, which could be used for the monitoring of B. subtilis fermentation processes. The data suggest for example acoA as a marker gene for glucose limitation or glnA as an indicator for nitrogen limitation.

Layers of Signaling in a Bacterium-Host Association

Quorum sensing, the monitoring of population density by bacteria, is used to coordinately control gene expression and therefore particular behaviors under conditions of high cell density. Such group behaviors provide advantages to organisms under certain conditions, such as during pathogenic colonization when virulence traits are induced by a group of bacteria. In the accompanying paper, Lupp and Ruby describe the requirement for a quorum-sensing system at relatively low cell density during colonization by Vibrio fischeri of its symbiotic host, Euprymna scolopes (18).