JOURNAL OF THE AMERICAN WATER RESOURCES ASSOCIATION, Issue 3 2000
Kyle E. Juracek
ABSTRACT: The stability of the Neosho River channel downstream from John Redmond Dam, in southeast Kansas, was investigated using multiple-date aerial photographs and stream-gage information. Bankfull channel width was used as the primary indicator variable to assess pre- and post-dam channel change. Five sin-mile river reaches and four stream gages were used in the analysis. Results indicated that, aside from some localized channel widening, the overall channel change has been minor with little post-dam change in bankfull channel width. The lack of a pronounced post-dam channel change may be attributed to a substantial reduction in the magnitude of the post-dam annual peak discharges in combination with the resistance to erosion of the bed and bank materials. Also, the channel may have been overwidened by a series of large floods that predated construction of the dam, including one with an estimated 500-year recurrence interval. [source]

Downstream from calcium signalling: mitochondria, vacuoles and pancreatic acinar cell damage

ACTA PHYSIOLOGICA, Issue 1 2009
S. Voronina
Abstract Ca2+ is one of the most ancient and ubiquitous second messengers. Highly polarized pancreatic acinar cells serve as an important cellular model for studies of Ca2+ signalling and homeostasis. Downstream effects of Ca2+ signalling have been and continue to be an important research avenue. The primary functions regulated by Ca2+ in pancreatic acinar cells , exocytotic secretion and fluid secretion , have been defined and extensively characterized in the second part of the last century. The role of cytosolic Ca2+ in cellular pathology and the related question of the interplay between Ca2+ signalling and bioenergetics are important current research lines in our and other laboratories. Recent findings in these interwoven research areas are discussed in the current review. [source]

Network-scale dynamics of grain-size sorting: implications for downstream fining, stream-profile concavity, and drainage basin morphology

EARTH SURFACE PROCESSES AND LANDFORMS, Issue 4 2004
Nicole M. Gasparini
Abstract We explore the link between channel-bed texture and river basin concavity in equilibrium catchments using a numerical landscape evolution model. Theory from homogeneous sediment transport predicts that river basin concavity directly increases with bed sediment size. If the effective grain size on a river bed governs its concavity, then natural phenomena such as grain-size sorting and channel armouring should be linked to concavity. We examine this hypothesis by allowing the bed sediment texture to evolve in a transport-limited regime using a two grain-size mixture of sand and gravel. Downstream ,ning through selective particle erosion is produced in equilibrium. As the channel-bed texture adjusts downstream so does the local slope. Our model predicts that it is not the texture of the original sediment mixture that governs basin concavity. Rather, concavity is linked to the texture of the sorted surface layer. Two different textural regimes are produced in the experiments: a transitional regime where the mobility of sand and gravel changes with channel-bed texture, and a sand-dominated region where the mobility of sand and gravel is constant. The concavity of these regions varies depending on the median gravel- or sand-grain size, erosion rate, and precipitation rate. The results highlight the importance of adjustments in both surface texture and slope in natural rivers in response to changes in ,uvial and sediment inputs throughout a drainage network. This adjustment can only be captured numerically using multiple grain sizes or empirical downstream ,ning rules. Copyright © 2004 John Wiley & Sons, Ltd. [source]

Reach for the Stars: A Strategic Bidding Game

ECONOMICA, Issue 272 2001
Lynne M. Pepall
We examine two,sided competition in a duopoly market for differentiated products. Downstream, the two firms compete in prices. Upstream, they compete in bidding to hire talent input and there is one unique superstar. The outcome depends on the downstream effect of only one firm employing the superstar. When this intensifies downstream competition, both firms are worse off than they would be if no superstar talent were available. When the hiring of the superstar softens downstream competition, both firms benefit, but a ,winner's curse' emerges in which the firm winning the superstar talent earns less profit than its rival. [source]

Foxo1 regulates marginal zone B-cell development

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2010
Jing Chen
Abstract A fundamental component of signaling initiated by the BCR and CD19 is the activation of phosphoinositide 3-kinase. Downstream of phosphoinositide 3-kinase, the protein kinase AKT phosphorylates several substrates, including members of the forkhead box subgroup O (Foxo) transcription factor family. Among the Foxo proteins, Foxo1 has unique functions in bone marrow B-cell development and peripheral B-cell function. Here, we report a previously unrecognized role for Foxo1 in controlling the ratio of mature B-cell subsets in the spleen. Conditional deletion of Foxo1 in B cells resulted in an increased percentage of marginal zone B cells and a decrease in follicular (FO) B cells. In addition, Foxo1 deficiency corrected the absence of marginal zone B cells that occurs in CD19-deficient mice. These findings show that Foxo1 regulates the balance of mature B-cell subsets and is required for the marginal zone B-cell deficiency phenotype of mice lacking CD19. [source]

Activation of Epstein-Barr virus/C3d receptor (gp140, CR2, CD21) on human cell surface triggers pp60src and Akt-GSK3 activities upstream and downstream to PI 3-kinase, respectively

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2003
Monique Barel
Abstract We previously demonstrated that CR2 activation on human B lymphocyte surface specifically triggered tyrosine phosphorylation of the 95-kDa nucleolin, this leading to its binding on SH2 domainsof p85 sub-unit of PI 3-kinase and to activation of this enzyme. The specificity of CR2 pathway was clearly demonstrated as neither CD19 nor BCR could induce tyrosine phosphorylation of nucleolin in normal B lymphocytes. These data led us to investigate herein additional molecular events, which were triggered by CR2 activation, upstream and downstream to PI 3-kinase activation. Upstream, we demonstrated that pp60src, a tyrosine kinase of the src family, was involved in tyrosine phosphorylation of nucleolin, while syk tyrosine kinase was not. We also demonstrated a direct protein-proteininteraction of pp60src with nucleolin in a CR2-dependent and CD19-independent pathway. Downstream, we demonstrated that CR2 activation also triggered Akt and GSK3 enzyme activation, this pathway being under the control of pp60src tyrosine kinase activation. These regulatory functions of activated CR2 were specific as independent of syk tyrosine kinase and of CD19 and BCR activation. Thus, CR2 activation recruits a specific mechanism to activate PI 3-kinase and its subsequent pathways, this mechanism being different to those recruited by CD19 and BCR. [source]

Nitrification in the Schelde estuary: methodological aspects and factors influencing its activity

FEMS MICROBIOLOGY ECOLOGY, Issue 1 2002
Monique J.M. de Bie
Abstract We present a 15-month dataset on nitrification measurements in the Schelde estuary (Belgium and The Netherlands). Nitrification was estimated using the N-serve sensitive dark 14C-bicarbonate incorporation technique. A peak of nitrification activity was observed in the freshwater part of the estuary. Downstream from this peak, nitrification declined, probably because of ammonium limitation. A range of nitrification inhibitors was tested on both a Nitrosomonas europaea culture and estuarine samples. It was found that methyl fluoride and acetylene stimulated dark 14C-bicarbonate incorporation and those inhibitors were therefore considered inappropriate nitrification inhibitors in combination with this technique. The effect of the inhibitor N-serve was studied on the dark incorporation of 13C-bicarbonate into polar lipid derived fatty acids to further identify the dominant chemoautotrophic processes. Inhibition of polar lipid derived fatty acid labelling in the presence of N-serve was complete, suggesting that nitrifying bacteria dominated the chemoautotrophic community. [source]

Human remyelination promoting antibody inhibits apoptotic signaling and differentiation through Lyn kinase in primary rat oligodendrocytes

GLIA, Issue 15 2010
J. Watzlawik
Abstract Purpose: Human remyelination promoting IgM mAbs target oligodendrocytes (OLs) and function in animal models of multiple sclerosis (MS). However, their mechanism of action is unknown. This study seeks to identify the cellular mechanism of action of a recombinant human IgM on OL survival. Methods: Binding of rHIgM22 to the surface of rat OLs was studied by co-localization with various markers. RHIgM22-mediated effects on apoptotic signaling in OLs, differentiation markers, and signaling molecules were detected by Western blotting and immunoprecipitation. Results: RHIgM22 co-localized with integrin ,3 but not other integrin ,-chains in OLs. Downstream of integrin ,3 we identified Src family kinase (SFK) Lyn as a key player of rHIgM22-mediated actions in OLs. Lyn immunoprecipitated in a complex together with integrin ,v,3 and PDGF,R. Lyn expression was 9-fold up-regulated and Lyn activation was 3-fold higher inrHIgM22-treated OL cultures compared with controls. RHIgM22 inhibited apoptotic signaling by greater than 10-fold reduction of caspase-3 and capsase-9 cleavage and reduced by 4-fold expression of differentiation markers MBP and MOG in OLs. SFK inhibitors PP2 and SU6656 inhibited Lyn activity and restored caspase-cleavage in OLs. A human IgM that did not promote remyelination and medium wereused as controls. Conclusions: rHIgM22 prevented apoptotic signaling andinhibited OL differentiation by Lyn implying thatIgM-mediated remyelination is due toprotection of OPC and OLs rather than promotion of OPC differentiation. © 2010 Wiley-Liss, Inc. [source]

Impaired expression and function of toll-like receptor 7 in hepatitis C virus infection in human hepatoma cells,

HEPATOLOGY, Issue 1 2010
Serena Chang
Hepatitis C virus (HCV) interferes with interferon (IFN)-mediated innate immune defenses. Toll-like receptor (TLR) 7 agonists robustly inhibit HCV infection. We hypothesize that HCV infection may interfere with the expression and/or function of TLR7, a sensor of single-stranded RNA. We identified reduced TLR7 RNA and protein levels in hepatoma cells expressing HCV (full-length, BB7-subgenomic, and JFH-1 clone) compared with control HCV-naïve cells. The biological relevance of this finding was confirmed by the observation of decreased TLR7 RNA in livers of HCV-infected patients compared with controls. HCV clearance, by IFN-, treatment or restrictive culture conditions, restored the decreased TLR7 expression. Treatment with RNA polymerase inhibitors revealed a shorter TLR7 half-life in HCV-replicating cells compared with controls. Downstream of TLR7, an increased baseline IRF7 nuclear translocation was observed in HCV-positive cells compared with controls. Stimulation with the TLR7 ligand R837 resulted in significant IRF7 nuclear translocation in control cells. In contrast, HCV-replicating cells showed attenuated TLR7 ligand-induced IRF7 activation. Conclusion: Reduced TLR7 expression, due to RNA instability, directly correlates with HCV replication and alters TLR7-induced IRF7-mediated cell activation. These results suggest a role for TLR7 in HCV-mediated evasion of host immune surveillance. (HEPATOLOGY 2009.) [source]

Heavy metal concentrations during storm events in a rehabilitated industrialized catchment

HYDROLOGICAL PROCESSES, Issue 10 2003
W. H. Blake
Abstract Water quality data collected on a fortnightly or monthly basis are inadequate for assessment and modelling of many water quality problems as storm event samples are underrepresented or missed. This paper examines the stormflow dynamics of heavy metals (Pb, Cu, Cd and Zn) in the Nant-y-Fendrod stream, South Wales, which has been affected by 250 years of metal smelting, followed by 35 years of landscape rehabilitation measures. For storm events of contrasting (very dry and very wet) antecedent conditions in May 2000 and February 2001, respectively, temporal changes in streamwater heavy metal concentrations above and below an in-line flood detention lake are analysed. At the upstream site, peaks in total metal concentration were recorded on the rising limb for Pb (0·150 mg l,1) and Cu (0·038 mg l,1) but on the falling limb for Zn (1·660 mg l,1) and Cd (0·006 mg l,1) in the summer 2000 storm event, yielding clockwise and anticlockwise hysteretic loops respectively. In contrast, metal concentrations, although high throughout the winter storm event, were diluted somewhat during the storm peak itself. The Pb and Cu appear to be supplied by quickflow processes and transported in close association with fine sediment, whereas Zn and Cd are delivered to the channel and lake by slower subsurface seepage in dissolved form. In the winter 2001 event, antecedent soil moisture and shallow groundwater levels were anomalously high and seepage sources of dissolved metals dominated. Downstream of the lake, Pb and Cu levels and suspended sediment were high in the summer storm, but low in the winter storm, suggesting retention with deposition of fine sediment in the lake during the latter. In the winter storm, Zn and Cd levels were higher downstream than upstream of the lake, perhaps because of additional seepage inputs from the surrounding slopes, which failed to have an impact during summer. An understanding of the complex interplay of antecedent soil moisture and the dynamics of subsurface seepage pathways in relation to the three-dimensional distribution of sources is important in modelling heavy metal fluxes and levels in contaminated urban catchments. Copyright © 2003 John Wiley & Sons, Ltd. [source]

Stem cell generation and choice of fate: role of cytokines and cellular microenvironment

JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2000
S.N. Constantinescu
Hematopoietic stem cells (HSC) have provided a model for the isolation, enrichment and transplantation of stem cells. Gene targeting studies in mice have shown that expression of the thrombopoietin receptor (TpoR) is linked to the accumulation of HSCs capable to generate long-term blood repopulation when injected into irradiated mice. The powerful increase in vivo in HSC numbers by retrovirally transduced HOX4B, a homeotic gene, along with the role of the TpoR, suggested that stem cell fate, renewal, differentiation and number can be controlled. The discovery of the precise region of the mouse embryo where HSCs originate and the isolation of supporting stromal cell lines open the possibility of identifying the precise signals required for HSC choice of fate. The completion of human genome sequencing coupled with advances in gene expression profiling using DNA microarrays will enable the identification of key genes deciding the fate of stem cells. Downstream from HSCs, multipotent hematopoietic progenitor cells appear to co-express a multiplicity of genes characteristic of different blood lineages. Genomic approaches will permit the identification of the select group of genes consolidated by the commitment of these multipotent progenitors towards one or the other of the blood lineages. Studies with neural stem cells pointed to the unexpected plastic nature of these cells. Isolation of stem cells from multiple tissues may suggest that, providing the appropriate environment/signal, tissues could be regenerated in the laboratory and used for transplantation. A spectacular example of influence of the environment on cell fate was revealed decades ago by using mouse embryonic stem cells (ES). Injected into blastocysts, ES cells contribute to the formation of all adult tissues. Injected into adult mice, ES cells become cancer cells. After multiple passages as ascites, when injected back into the blastocyst environment, ES- derived cancer cells behaved again as ES cells. More recently, the successful cloning of mammals and reprogramming of transferred nuclei by factors in the cytoplasm of oocytes turned back the clock by showing that differentiated nuclei can be "re-booted" to generate again the stem cells for different tissues. [source]

Fish assemblage changes relative to environmental factors and time in the Warta River, Poland, and its oxbow lakes

JOURNAL OF FISH BIOLOGY, Issue 2 2004
T. Penczak
Four oxbow lakes and two neighbouring sections of their parent Warta River (Odra River system, Poland) were sampled to investigate differences in fish assemblages between habitats in 1999,2000. Additional comparisons were made with 12 other oxbow lakes in this section of the river that were sampled 30,40 years ago. Downstream of a man-made reservoir, higher species number, diversity and evenness were recorded in oxbow lakes than in the river channel. Upstream of the reservoir, differences in these variables from both habitats were insignificant. Fluvial and stagnant water samples were clearly separated in the multivariate space of a detrended correspondence analysis (DCA). For two oxbow lakes and two neighbouring sections of the Warta River, 12 abiotic and biotic environmental variables were available, and only velocity, water temperature and conductivity were significantly correlated with canonical correspondence analysis (CCA) axes. Fish assemblages of four recently investigated oxbow lakes were clearly separated in the multivariate space of DCA from other neighbouring oxbows sampled 30,40 years ago. Species previously subdominant were becoming rare. Oxbow lakes that are continuously or at least periodically connected with the channel are indispensable for maintaining high biodiversity and a sustainable fishery in the river system. [source]

ASSESSING FLOOD MITIGATION ALTERNATIVES IN SHIJR AREA IN METROPOLITAN TAIPEI,

JOURNAL OF THE AMERICAN WATER RESOURCES ASSOCIATION, Issue 2 2006
Chao-Hsien Liaw
ABSTRACT: The Keelung River Basin in northern Taiwan lies immediately upstream of the Taipei metropolitan area. The Shijr area is in the lower basin and is subject to frequent flooding. This work applies micromanagement and source control, including widely distributed infiltration and detention/ retention runoff retarding measures, in the Wudu watershed above Shijr. A method is also developed that combines a genetic algorithm and a rainfall runoff model to optimize the spatial distribution of runoff retarding facilities. Downstream of Wudu in the Shijr area, five dredging schemes are considered. If 10-year flood flows cannot be confined in the channel, then a levee embankment that corresponds to the respective runoff retarding scheme will be required. The minimum total cost is considered in the rule to select from the regional flood mitigation alternatives. The results of this study reveal that runoff retarding facilities installed in the upper and middle parts of the watershed are most effective in reducing the flood peak. Moreover, as the cost of acquiring land for the levee embankment increases, installing runoff retarding measures in the upper portion of the watershed becomes more economical. [source]

Diabetic neuropathies: components of etiology

JOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 2 2008
David R. Tomlinson
Abstract This review examines the putative role of glucose in the etiology of diabetic neuropathies. Excessive glucose generates several secondary metabolic anomalies , principally oxidative stress (via both the polyol pathway and glucoxidation) and non-enzymic glycation of macromolecules. The latter is also facilitated by glucoxidation. These metabolic deviations trigger cellular responses that are inappropriate to normal function. Principal among these are neurotrophic deficits and phosphorylation of mitogen-activated protein kinases (MAPK). Downstream of these events are aberrant ion channel function and disordered gene expression, leading to changes in cellular phenotype. This leads directly to disordered nerve conduction, a recognised early clinical sign, and indirectly, via as yet undisclosed links, to sensory loss and axonopathy. Recent work also links MAPK activation to the development of neuropathic pain. [source]

Partition of metals in the Vistula River and in effluents from sewage treatment plants in the region of Cracow (Poland)

LAKES & RESERVOIRS: RESEARCH AND MANAGEMENT, Issue 2 2000
C. Guéguen
Abstract The Vistula River suffers from heavy pollution with multiple origins. In the upper reaches, metallic and chlorine pollution originates from the mining and industrial region of Upper Silesia. Downstream from Upper Silesia, urban and industrial sewage adds more metallic and organic contaminants from the large urban agglomeration of Cracow. Although the river status is monitored routinely, little is known about the partition of metals between particulate and dissolved forms. This study focuses on metal partitioning and on the impact of the two main wastewater treatment plants at Cracow on metal concentrations in the Vistula River. The Cd, Co, Cu, Mn, Pb and Zn content was measured in both dissolved and particulate fractions. High metal concentrations in the Vistula River persist, although current levels seem to be lower than those in the past. Metal concentrations in the Vistula River and effluents from the sewage treatment plants at Cracow are similar, indicating a relatively minor contribution from the treated sewage. However, untreated sewage may be a significant source of contaminants. Despite high anthropogenic metal concentrations, the metal partitioning coefficients (Kd) in the Vistula are similar to these found in unpolluted rivers. Within a narrow pH range, Kd values depend on the metal affinity to particles, but there is no evidence of dependence on particle or chloride concentrations. An important fraction of the toxic metals Pb and Cd is associated with particles, which may decrease their immediate availability to the biota of the river. [source]

PY54, a linear plasmid prophage of Yersinia enterocolitica with covalently closed ends

MOLECULAR MICROBIOLOGY, Issue 4 2003
Stefan Hertwig
Summary PY54 is a temperate phage isolated from Yersinia enterocolitica. Lysogenic Yersinia strains harbour the PY54 prophage as a plasmid (pY54). The plasmid has the same size (46 kb) as the PY54 genome isolated from phage particles. By electron microscopy, restriction analysis and DNA sequencing, it was demonstrated that the phage and the plasmid DNAs are linear, circularly permuted molecules. Unusually for phages of Gram-negative bacteria, the phage genome has 3,-protruding ends. The linear plasmid pY54 has covalently closed ends forming telomere-like hairpins. The equivalent DNA sequence of the phage genome is a 42 bp perfect palindrome. Downstream from the palindrome, an open reading frame (ORF) was identified that revealed strong DNA homology to the telN gene of Escherichia coli phage N15 encoding a protelomerase. Similar to PY54, the N15 prophage is a linear plasmid with telomeres. The N15 protelomerase has cleaving/joining activity generating the telomeres by processing a 56 bp palindrome (telomere resolution site tel RL). To study the activity of the PY54 protein, the telN -like gene was cloned and expressed in E. coli. A 77 kDa protein was obtained and partially purified. The protein was found to process recombinant plasmids containing the 42 bp palindrome. Telomere resolution of plasmids under in vivo conditions was also investigated in Yersinia infected with PY54. Processing required a plasmid containing the palindrome as well as adjacent DNA sequences from the phage including an additional inverted repeat. Regions on the phage genome important for plasmid maintenance were defined by the construction of linear and circular miniplasmid derivatives of pY54, of which the smallest miniplasmid comprises a 4.5 kb DNA fragment of the plasmid prophage. [source]

A disc-wind model with correct crossing of all magnetohydrodynamic critical surfaces

MONTHLY NOTICES OF THE ROYAL ASTRONOMICAL SOCIETY, Issue 2 2000
N. Vlahakis
The classical Blandford & Payne model for the magneto-centrifugal acceleration and collimation of a disc-wind is revisited and refined. In the original model, the gas is cold and the solution is everywhere subfast magnetosonic. In the present model the plasma has a finite temperature and the self-consistent solution of the MHD equations starts with a subslow magnetosonic speed which subsequently crosses all critical points, at the slow magnetosonic, Alfvén and fast magnetosonic separatrix surfaces. The superfast magnetosonic solution thus satisfies MHD causality. Downstream of the fast magnetosonic critical point the poloidal streamlines overfocus towards the axis and the solution is terminated. The validity of the model to disc winds associated with young stellar objects is briefly discussed. [source]

TLS-CHOP target gene DOL54 expression in liposarcomas and malignant fibrous histiocytomas

PATHOLOGY INTERNATIONAL, Issue 8 2002
Hideharu Domoto
Downstream of the gene for the liposarcoma-associated fusion oncoprotein 54 (DOL54) is a target gene of the myxoid liposarcoma and round cell liposarcoma (M-LPS/RC-LPS) oncogene, TLS/FUS-CHOP. The DOL54 gene product is closely associated with adipogenic differentiation. DOL54 overexpression resulted in tumorigenicity when Chinese Hamster Ovary (CHO) cells were injected subcutaneously into nude mice. The biological significance of DOL54 expression for human malignant soft tissue tumors, however, has not yet been investigated. We examined TLS-CHOP and DOL54 expression in M-LPS/RC-LPS, well-differentiated liposarcoma and malignant fibrous histiocytoma (MFH), a tumor whose cellular origin has not been determined. We observed DOL54 expression in 50% of M-LPS/RC-LPS cases (in which TLS-CHOP was also expressed) and 33% of MFH cases, suggesting that a portion of MFH lesions may either derive from adipocytic precursor cells or have the potential to undergo adipogenic differentiation. In this manner, M-LPS/RC-LPS and MFH lesions may share tumorigenic characteristics, resulting from the unscheduled expression of DOL54. [source]

Connective Tissue Growth Factor Promotes Fibrosis Downstream of TGF, and IL-6 in Chronic Cardiac Allograft Rejection

AMERICAN JOURNAL OF TRANSPLANTATION, Issue 2 2010
A. J. Booth
Cardiac transplantation is an effective treatment for multiple types of heart failure refractive to therapy. Although immunosuppressive therapeutics have increased survival rates within the first year posttransplant, chronic rejection (CR) remains a significant barrier to long-term graft survival. Indicators of CR include patchy interstitial fibrosis, vascular occlusion and progressive loss of graft function. Multiple factors have been implicated in the onset and progression of CR, including TGF,, IL-6 and connective tissue growth factor (CTGF). While associated with CR, the role of CTGF in CR and the factors necessary for CTGF induction in vivo are not understood. To this end, we utilized forced expression and neutralizing antibody approaches. Transduction of allografts with CTGF significantly increased fibrotic tissue development, though not to levels observed with TGF, transduction. Further, intragraft CTGF expression was inhibited by IL-6 neutralization whereas TGF, expression remained unchanged, indicating that IL-6 effects may potentiate TGF,-mediated induction of CTGF. Finally, neutralizing CTGF significantly reduced graft fibrosis without reducing TGF, and IL-6 expression levels. These findings indicate that CTGF functions as a downstream mediator of fibrosis in CR, and that CTGF neutralization may ameliorate fibrosis and hypertrophy associated with CR. [source]

Cell line-specific control of recombinant monoclonal antibody production by CHO cells,

BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2010
Peter M. O'Callaghan
Abstract In this study we compare the cellular control of recombinant human IgG4 monoclonal antibody (Mab) synthesis in different CHO cell lines. Based on comprehensive empirical analyses of mRNA and polypeptide synthetic intermediates we constructed cell line-specific mathematical models of recombinant Mab manufacture in seven GS-CHO cell lines varying in specific production rate (qMab) over 350-fold. This comparative analysis revealed that control of qMab involved both genetic construct and cell line-specific factors. With respect to the former, all cell lines exhibited excess production of light chain (LC) mRNA and polypeptide relative to heavy chain (HC) mediated by more rapid LC transcription and enhanced LC mRNA stability. Downstream of this, cell lines differed markedly in their relative rates of recombinant mRNA translation, Mab assembly and secretion although HC mRNA abundance and the rate of HC translation generally exerted most control over qMab,the latter being directly proportional to qMab. This study shows that (i) cell lines capable of high qMab exceed a threshold functional competency in all synthetic processes, (ii) the majority of cells in parental and transfected cell populations are functionally limited and (iii) cell engineering strategies to increase Mab production should be cell line specific. Biotechnol. Bioeng. 2010;106: 938,951. © 2010 Wiley Periodicals, Inc. [source]

Role of EHEC O157:H7 virulence factors in the activation of intestinal epithelial cell NF-,B and MAP kinase pathways and the upregulated expression of interleukin 8

CELLULAR MICROBIOLOGY, Issue 10 2002
M. Cecilia Berin
Summary Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) is a gastrointestinal pathogen that is generally non-invasive for intestinal epithelial cells, yet causes acute intestinal inflammation, diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome. To study signal transduction pathways activated in human intestinal epithelial cells by EHEC, we took advantage of EHEC O157:H7 and isogenic mutants deficient in the major EHEC virulence factors, intimin (eae,) and Shiga toxin (stx,). Infection with wild-type EHEC activated p38 and ERK MAP kinases and the nuclear translocation of the transcription factor NF-,B. Downstream, this was accompanied by increased expression of mRNA and protein for the neutrophil chemoattractant IL-8. Isogenic eae, and stx, mutants also activated p38 and ERK MAP kinases, and NF-,B and stimulated increases in IL-8 protein secretion similar to those of wild-type EHEC. Further, inhibition of either p38, ERK or NF-,B activation abrogated the IL-8 response induced by wild-type EHEC and the mutants. Epithelial cell MAP kinase and NF-,B pathways leading to IL-8 secretion were also activated by isolated EHEC H7 flagellin, which was active when added to either the apical or basolateral surface of polarized human intestinal epithelial cells. We conclude that EHEC interacting with intestinal epithelial cells activates intracellular signalling pathways and an epithelial cell proinflammatory response independent of either Shiga toxin or intimin, two of the major known virulence factors of EHEC. The activation of proinflammatory signals in human colon epithelial cells in response to this non-invasive pathogen appears to depend to a significant extent on H7 flagellin. [source]

Birth defects caused by mutations in human GLI3 and mouse Gli3 genes

CONGENITAL ANOMALIES, Issue 1 2010
Ichiro Naruse
ABSTRACT GLI3 is the gene responsible for Greig cephalopolysyndactyly syndrome (GCPS), Pallister,Hall syndrome (PHS) and Postaxial polydactyly type-A (PAP-A). Genetic polydactyly mice such as Pdn/Pdn (Polydactyly Nagoya), XtH/XtH (Extra toes) and XtJ/XtJ (Extra toes Jackson) are the mouse homolog of GCPS, and Gli3tmlUrtt/Gli3tmlUrt is produced as the mouse homolog of PHS. In the present review, relationships between mutation points of GLI3 and Gli3, and resulting phenotypes in humans and mice are described. It has been confirmed that mutation in the upstream or within the zinc finger domain of the GLI3 gene induces GCPS; that in the post-zinc finger region including the protease cleavage site induces PHS; and that in the downstream of the GLI3 gene induces PAP-A. A mimicking phenomenon was observed in the mouse homolog. Therefore, human GLI3 and mouse Gli3 genes have a common structure, and it is suggested here that mutations in the same functional regions produce similar phenotypes in human and mice. The most important issue might be that GCPS and PHS exhibit an autosomal dominant trait, but mouse homologs, such as Pdn/Pdn, XtH/XtH, XtJ/XtJ and Gli3tmlUrt/Gli3tmlUrt, are autosomal recessive traits in the manifestation of similar phenotypes to human diseases. It is discussed here how the reduced amounts of the GLI3 protein, or truncated mutant GLI3 protein, disrupt development of the limbs, head and face. [source]

Histamine-1 receptor is not required as a downstream effector of orexin-2 receptor in maintenance of basal sleep/wake states

ACTA PHYSIOLOGICA, Issue 3 2010
M. Hondo
Abstract Aim:, The effect of orexin on wakefulness has been suggested to be largely mediated by activation of histaminergic neurones in the tuberomammillary nucleus (TMN) via orexin receptor-2 (OX2R). However, orexin receptors in other regions of the brain might also play important roles in maintenance of wakefulness. To dissect the role of the histaminergic system as a downstream mediator of the orexin system in the regulation of sleep/wake states without compensation by the orexin receptor-1 (OX1R) mediated pathways, we analysed the phenotype of Histamine-1 receptor (H1R) and OX1R double-deficient (H1R,/,;OX1R,/,) mice. These mice lack OX1R-mediated pathways in addition to deficiency of H1R, which is thought to be the most important system in downstream of OX2R. Methods:, We used H1R deficient (H1R,/,) mice, H1R,/,;OX1R,/, mice, OX1R and OX2R double-deficient (OX1R,/,;OX2R,/,) mice, and wild type controls. Rapid eye movement (REM) sleep, non-REM (NREM) sleep and awake states were determined by polygraphic electroencephalographic/electromyographic recording. Results:, No abnormality in sleep/wake states was observed in H1R,/, mice, consistent with previous studies. H1R,/,;OX1R,/, mice also showed a sleep/wake phenotype comparable to that of wild type mice, while OX1R,/,; OX2R,/, mice showed severe fragmentation of sleep/wake states. Conclusion:, Our observations showed that regulation of the sleep/wake states is completely achieved by OX2R-expressing neurones without involving H1R-mediated pathways. The maintenance of basal physiological sleep/wake states is fully achieved without both H1 and OX1 receptors. Downstream pathways of OX2R other than the histaminergic system might play an important role in the maintenance of sleep/wake states. [source]

The calcium-conducting ion channel transient receptor potential canonical 6 is involved in macrophage inflammatory protein-2-induced migration of mouse neutrophils,

ACTA PHYSIOLOGICA, Issue 1 2009
N. Damann
Abstract Aim:, The role of the calcium-conducting ion channel transient receptor potential canonical 6 (TRPC6) in macrophage inflammatory protein-2 (MIP-2) induced migration of mouse neutrophils was investigated. Methods:, Neutrophil granulocytes isolated from murine bone marrow of wild-type (TRPC6+/+) and TRPC6 knockout (TRPC6,/,) mice were tested for the presence of TRPC6 channel expression using quantitative real-time polymerase chain reactions and immunocytochemistry. The effect of different stimuli (e.g. MIP-2, 1-oleoyl-2-acetyl-sn-glycerol, formyl-methionyl-leucyl-phenylalanin) on migration of isolated neutrophils was tested by two-dimensional (2D) migration assays, phalloidin staining and intracellular calcium imaging. Results:, We found that neutrophil granulocytes express TRPC6 channels. MIP-2 induced fast cell migration of isolated neutrophils in a 2D cell-tracking system. Strikingly, MIP-2 was less potent in neutrophils derived from TRPC6,/, mice. These cells showed less phalloidin-coupled fluorescence and the pattern of cytosolic calcium transients was altered. Conclusions:, We describe in this paper for the first time a role for transient receptor potential (TRP) channels in migration of native lymphocytes as a new paradigm for the universal functional role of TRPs. Our data give strong evidence that TRPC6 operates downstream to CXC-type Gq -protein-coupled chemokine receptors upon stimulation with MIP-2 and is crucial for the arrangement of filamentous actin in migrating neutrophils. This is a novel cell function of TRP channel beyond their well-recognized role as universal cell sensors. [source]

Decoding epithelial signals: critical role for the epidermal growth factor receptor in controlling intestinal transport function

ACTA PHYSIOLOGICA, Issue 1 2009
D. F. McCole
Abstract The intestinal epithelium engages in bidirectional transport of fluid and electrolytes to subserve the physiological processes of nutrient digestion and absorption, as well as the elimination of wastes, without excessive losses of bodily fluids that would lead to dehydration. The overall processes of intestinal ion transport, which in turn drive the secretion or absorption of water, are accordingly carefully regulated. We and others have identified the epidermal growth factor receptor (EGFr) as a critical regulator of mammalian intestinal ion transport. In this article, we focus on our studies that have uncovered the intricate signalling mechanisms downstream of EGFr that regulate both chloride secretion and sodium absorption by colonocytes. Emphasis will be placed on the EGFr-associated regulatory pathways that dictate the precise outcome to receptor activation in response to signals that may seem, on their face, to be quite similar if not identical. The concepts to be discussed underlie the ability of the intestinal epithelium to utilize a limited set of signalling effectors to produce a variety of outcomes suitable for varying physiological and pathophysiological demands. Our findings therefore are relevant not only to basic biological principles, but also may ultimately point to new therapeutic targets in intestinal diseases where ion transport is abnormal. [source]

Maspin controls mammary tumor cell migration through inhibiting Rac1 and Cdc42, but not the RhoA GTPase

CYTOSKELETON, Issue 5 2007
Heidi Y. Shi
Abstract Rac1 and Cdc42 are members of the Rho family of small GTPases that play essential roles in diverse cellular functions, including cell migration. The activities of these Rho family proteins are controlled by growth factor receptor activation and cell-ECM interactions. Here, we show that maspin, a well-documented tumor suppressor gene, also controls cell motility through inhibiting Rac1/Cdc42 activity. Using the GST-PAK and GST-Rho binding protein pull-down assays for GTP-bound Rac1, Cdc42, and RhoA, we showed that treatment of MDA-MB-231 tumor cells with recombinant maspin for a short time period significantly inhibited the activity of Rac1 and Cdc42, but not RhoA. The reactive site loop (RSL) within maspin protein is the functional domain involved in the inhibition. Maspin mutants with the RSL deleted or a point mutation in the RSL region lost their inhibitory activity. We further examined the ability of maspin to inhibit Rac1- and Cdc42-mediated signaling pathways and transcription factors. Treatment of MDA-MB-231 cells with maspin led to the inhibition of JNK kinase activity as assayed by immuno-kinase assays. In addition, the AP-1 transcription activity downstream of JNK kinase pathway was also reduced. Together, we have identified Rac1 and Cdc42 as the downstream targets that mediate the inhibition of mammary tumor cell migration by maspin. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]

Health Care Supply Chain Design: Toward Linking the Development and Delivery of Care Globally,

DECISION SCIENCES, Issue 2 2009
Kingshuk K. Sinha
ABSTRACT This article is motivated by the gap between the growing demand and available supply of high-quality, cost-effective, and timely health care, a problem faced not only by developing and underdeveloped countries but also by developed countries. The significance of this problem is heightened when the economy is in recession. In an attempt to address the problem, in this article, first, we conceptualize care as a bundle of goods, services, and experiences,including diet and exercise, drugs, devices, invasive procedures, new biologics, travel and lodging, and payment and reimbursement. We then adopt a macro, end-to-end, supply chain,centric view of the health care sector to link the development of care with the delivery of care. This macro, supply chain,centric view sheds light on the interdependencies between key industries from the upstream to the downstream of the health care supply chain. We propose a framework, the 3A-framework, that is founded on three constructs,affordability, access, and awareness,to inform the design of supply chain for the health care sector. We present an illustrative example of the framework toward designing the supply chain for implantable device,based care for cardiovascular diseases in developing countries. Specifically, the framework provides a lens for identifying an integrated system of continuous improvement and innovation initiatives relevant to bridging the gap between the demand and supply for high-quality, cost-effective, and timely care. Finally, we delineate directions of future research that are anchored in and follow from the developments documented in the article. [source]

Targeted gene expression by the Gal4-UAS system in zebrafish

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2008
Kazuhide Asakawa
Targeted gene expression by the Gal4-UAS system is a powerful methodology for analyzing function of genes and cells in vivo and has been extensively used in genetic studies in Drosophila. On the other hand, the Gal4-UAS system had not been applied effectively to vertebrate systems for a long time mainly due to the lack of an efficient transgenesis method. Recently, a highly efficient transgenesis method using the medaka fish Tol2 transposable element was developed in zebrafish. Taking advantage of the Tol2 transposon system, we and other groups developed the Gal4 gene trap and enhancer trap methods and established various transgenic fish expressing Gal4 in specific cells. By crossing such Gal4 lines with transgenic fish lines harboring various reporter genes and effector genes downstream of UAS (upstream activating sequence), specific cells can be visualized and manipulated in vivo by targeted gene expression. Thus, the Gal4 gene trap and enhancer trap approaches together with various UAS lines should be important tools for investigating roles of genes and cells in vertebrates. [source]

RNA interference by expressing short hairpin RNA in the Ciona intestinalis embryo

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2008
Aya Nishiyama
We carried out RNA interference by expressing short hairpin RNA (shRNA) in the Ciona intestinalis embryo. For this purpose, we identified a gene encoding U6 small nuclear RNA (snRNA) in the C. intestinalis genome. The 1-kb sequence upstream of the U6 snRNA gene was sufficient for directing transcription of short RNA as revealed by Northern blot hybridization. An shRNA-expressing plasmid vector was constructed, in which shRNA-encoding oligonucleotides are inserted downstream of the U6 promoter. An shRNA that contained a sequence homologous to the C. intestinalis tyrosinase gene (Ci-tyrosinase) suppressed melanization of pigment cells in the brain of morphologically normal tailbud embryos. An shRNA that perfectly matched the translated sequence of enhanced green fluorescent protein (EGFP) (a mutant type of Aequorea victoria green fluorescent protein) suppressed the expression of the coelectroporated EGFP transgene. These results suggest that the expression of shRNA interferes with functions of both endogenous and exogenous genes. The shRNA-expressing plasmid constructed in the present study provides an easy and inexpensive alternative for the functional analysis of genes in ascidian embryos. [source]

Functional retinoid receptors in budding ascidians

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2000
Mika Kamimura
A homolog of retinoid X receptors (RXR), named PmRXR, was cloned from the budding ascidian, Polyandrocarpa misakiensis. Gel-shift assays revealed that PmRXR and a previously identified P. misakiensis retinoic acid receptor (PmRAR) formed a complex to bind vertebrate-type retinoic acid response element (RARE). Transfection assays were carried out using a reporter gene containing a RARE upstream of lacZ. Two chimeric effector genes were constructed by placing PmRXR and PmRAR cDNA fragments (containing the DNA-binding, ligand-binding and ligand-dependent transactivation domains) downstream of the human RXR, and RAR, cDNA (covering the N-terminal coding region), respectively. Each chimeric cDNA was ligated to a notochord-specific enhancer. In case the embryos were transfected with all three transgenes and treated with retinoic acid (RA), the reporter gene was activated in the notochord cells. The result suggests that the PmRXR/PmRAR complex functions as an RA-dependent transcriptional activator. The PmRXR mRNA was detected in a mesenchymal cell type, called glomerulocyte, in the developing Polyandrocarpa bud. As this cell type has been shown to express PmRAR mRNA, it seems possible that the PmRXR/PmRAR complex mediates RA signaling in this cell type to induce the expression of genes involved in the morphogenesis of the developing bud. [source]