Search

As the world strives towards a low-carbon future, nearly-zero energy buildings (NZEB) have been the goal to reduce carbon emissions. Artificial lighting is estimated to consume as high as 40% of the total energy consumption in a commercial building. By utilising daylighting, which is the practice of allowing natural light into a building, energy consumption by artificial lighting can be reduced. Luminescent solar concentrators (LSCs) can act as a collector and waveguide to transport outdoor light into the building through total internal reflection. Besides, LSCs absorb a part of the solar spectrum and shift them to different wavelengths through up-conversion or down-conversion. Thus, the output spectrum can be manipulated for the desired indoor applications. Circadian rhythm is the periodic variations in behaviour that follows a 24-hour cycle, which is mainly regulated by light response. A regulated circadian rhythm is important for a healthy life, whereas a disturbed circadian rhythm can lead to health issues such as insomnia and mood disorders. There has been a consensus that our circadian rhythm strongly responds to shorter wavelength light, corroborated in studies. Thus, manipulating the output light of LSCs to contain larger proportions of light with shorter wavelengths could enhance circadian regulation. LSC devices have the potential to transport sufficient daylight up to 5m deep into the building, achieving areas beyond the reach of windows. Thus, LSCs can serve as a tool for daylighting purposes, regulating circadian rhythm and providing sufficient light for comfortable indoor visibility.

Luminescent solar concentrators (LSCs) ability to concentrate both direct and diffuse solar irradiation exhibits exciting potential as building-integrated photovoltaics (BIPV) in urban environments. As BIPV elements, LSCs are often imagined as semi-transparent solar windows which can be integrated seamlessly into a building's façade and architectural applications as solar harvesting devices. One application explored in this research is a solar geodesic dome panel for an ongoing community greenhouse development in Derry, N-Ireland. A 4V and 2 m diameter geodesic dome were modelled in Revit, and an Insight Solar Analysis model optimised the LSC-geodesic dome and calculated the solar potential. The triangular LSC panel of 875 cm2 was modelled using raytracing software to obtain efficiency parameters. Subsequently, fabricated using a luminescent acrylic 6T66 waveguide, edge-mounted silicon solar cells and tested outdoors for 29 h. A power conversion efficiency of 0.60% compared to theoretical power conversion efficiency of 1.49% was measured. In the optimum location of the dome, the LSC panel would produce 444.22 Wh and, overall, 74.2 kWh in a year. While this power generation is essential, semi-transparent LSC-geodesic dome panel transmission can downshift solar radiation in the photosynthetically active radiation range, better suited for plant growth and the greenhouse effect.

This paper details the design process, fabrication, optimisation and early-stage performance testing of Luminescent and Plasmonic Luminescent Solar Concentrator (LSC & PLSC) devices. A PLSC is a novel approach to solar concentrator technologies that utilizes the principles of luminescence and plasmonics to enhance the devices' solar energy conversion efficiency. This research analyses various mould dimensions, materials and lightguide fabrication methodologies to ensure equivalent LSC/PLSC devices were created in a reproducible method. The optimisation was an iterative process throughout the production and testing stages after which a 100 × 100 × 5 mm PLSC was identified as the optimal for a rooftop installation. To ensure consistency in production as well as assessing the practicality of PLSC installations for building integration, performance testing has been conducted in both indoor and outdoor environments. Additionally, the lifespan of the devices are currently being investigated through ongoing performance evaluations. The incorporation of a reflective backplate has resulted in device efficiency improvements between 14–18% during indoor tests and was consequently included for all devices during outdoor performance analysis. Power conversion efficiencies of 2.3% and 1.7% have been recorded in sub-optimal conditions as well as concentration ratios of 11 and 9 for the PLSC and LSC devices respectively.

Understanding regional-scale food web structure in the Southern Ocean is critical to informing fisheries management and assessments of climate change impacts on Southern Ocean ecosystems and ecosystem services. Historically, a large component of Southern Ocean ecosystem research has focused on Antarctic krill, which provide a short, highly efficient food chain, linking primary producers to higher trophic levels. Over the last 15 years, the presence of alternative energy pathways has been identified and hypotheses on their relative importance in different regions raised. Using the largest circumpolar dietary database ever compiled, we tested these hypotheses using an empirical circumpolar comparison of food webs across the four major regions/sectors of the Southern Ocean (defined as south of 40°S) within the austral summer period. We used network analyses and generalizations of taxonomic food web structure to confirm that while Antarctic krill are dominant as the mid-trophic level for the Atlantic and East Pacific food webs (including the Scotia Arc and Western Antarctic Peninsula), mesopelagic fish and other krill species are dominant contributors to predator diets in the Indian and West Pacific regions (East Antarctica and the Ross Sea). We also highlight how tracking data and habitat modeling for mobile top predators in the Southern Ocean show that these species integrate food webs over large regional scales. Our study provides a quantitative assessment, based on field observations, of the degree of regional differentiation in Southern Ocean food webs and the relative importance of alternative energy pathways between regions., Competing Interests: The authors declare that there are no conflicts of interest., (© 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

This study aims to evaluate the safety, acceptability, and pharmacokinetics (PK) of an increased dose of nelfinavir (NFV) during the third trimester of pregnancy. The study was registered as part of the International Maternal Pediatric Adolescent AIDS Clinical Trials network (IMPAACT-P1026s), an ongoing multicenter prospective cohort study of antiretroviral PK during pregnancy (NCT00042289). NFV intensive PK evaluations were performed at steady state during the third trimester of pregnancy and 2-3 weeks postpartum. Plasma concentrations of NFV and its active metabolite, hydroxyl-tert-butylamide (M8) were measured using high-performance liquid chromatography with ultraviolet detection. A total of 18 women are included in the analysis. NFV area under the concentration-time curve (AUC) with the increased dose during the third trimester was nearly identical to the standard dose postpartum, with a geometric mean ratio for third trimester to postpartum AUC of 0.98 (90%CI 0.71-1.35). Despite the increased dose, M8 AUC was lower during the third trimester compared to postpartum (0.53, IQR [0.38-0.75]), as was the M8/NFV AUC ratio (0.51, IQR [0.42-0.63]). NFV AUC 0-12 was above target in 15 of 18 (83%) of participants during the third trimester compared to 14 of 16 (88%) postpartum. No major safety concerns were noted. Increasing the NFV dose to 1875 mg twice daily during the third trimester achieved similar concentrations postpartum compared to standard dosing (1250 mg twice daily). Increased NFV dose regimens may still have some benefit to human immunodeficiency virus (HIV)-positive pregnant women living in countries where novel protease inhibitors are currently unavailable or in individuals who are intolerant to ritonavir-boosted HIV medications., (© 2018, The American College of Clinical Pharmacology.)

Background: The pharmacokinetics and pharmacodynamics of lumefantrine, a component of the most widely used treatment for malaria, artemether-lumefantrine, has not been adequately characterized in young children., Methods: Capillary whole-blood lumefantrine concentration and treatment outcomes were determined in 105 Ugandan children, ages 6 months to 2 years, who were treated for 249 episodes of Plasmodium falciparum malaria with artemether-lumefantrine., Results: Population pharmacokinetics for lumefantrine used a 2-compartment open model with first-order absorption. Age had a significant positive correlation with bioavailability in a model that included allometric scaling. Children not receiving trimethoprim-sulfamethoxazole with capillary whole blood concentrations <200 ng/mL had a 3-fold higher hazard of 28-day recurrent parasitemia, compared with those with concentrations >200 ng/mL (P = .0007). However, for children receiving trimethoprim-sulfamethoxazole, the risk of recurrent parasitemia did not differ significantly on the basis of this threshold. Day 3 concentrations were a stronger predictor of 28-day recurrence than day 7 concentrations., Conclusions: We demonstrate that age, in addition to weight, is a determinant of lumefantrine exposure, and in the absence of trimethoprim-sulfamethoxazole, lumefantrine exposure is a determinant of recurrent parasitemia. Exposure levels in children aged 6 months to 2 years was generally lower than levels published for older children and adults. Further refinement of artemether-lumefantrine dosing to improve exposure in infants and very young children may be warranted., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.)

Objective: Our goal was to systematically and quantitatively assess treatment response between Asian patients with hepatitis C virus genotype 6 (HCV-6) and hepatitis C virus genotype 1 (HCV-1) treated for 48 weeks with pegylated interferon and ribavirin., Methods: We performed a literature search in MEDLINE and EMBASE for 'genotype 6' in August 2013. Additional abstracts from major international scientific conferences from 2012 to 2013 were reviewed. Studies included were original articles with ≥10 treatment-naïve Asian HCV-6 patients. Exclusion criteria were coinfections with hepatitis B virus, HIV and/or other liver diseases. Heterogeneity was defined as a Cochrane Q test with a p value of 0.10 and an I(2) statistic of >50%. RESULTS of a random-effects model are reported., Results: A total of 1,046 (503 HCV-6; 543 HCV-1) patients from 12 studies were included in the analysis. The pooled sustained virologic response (SVR) rate was 80.2% (95% CI 74.3-85.0, Q statistic = 20.87, p < 0.035; I(2) = 47.3%) for HCV-6 and 62.5% (95% CI 41.9-79.4, Q statistic = 52.41, p < 0.001; I(2) = 92.37) for HCV-1 patients. HCV-6 patients had a significantly higher SVR rate compared to HCV-1 patients (odds ratio 2.73, 95% CI 1.69-4.41, p < 0.001). Approximately one fourth of patients without early virologic response (EVR) achieved SVR, regardless of genotype (HCV-1, n = 6/23; HCV-6, n = 4/21)., Conclusions: Asian patients with HCV-6 can expect higher SVR rates (∼80%) than HCV-1 patients (∼63%). EVR as a stopping rule is less clear in Asian patients with HCV-6 and HCV-1., (© 2015 S. Karger AG, Basel.)

Background: Maternal-to-fetal transfer of antiretroviral drugs contributes to prevention of vertical transmission of HIV., Objective: This systematic review discusses published studies containing data pertaining to the pharmacokinetics of placental transfer of antiretrovirals in humans, including paired cord and maternal plasma samples collected at the time of delivery as well as ex vivo placental perfusion models., Methods: Articles pertaining to placental transfer of antiretrovirals were identified from PubMed, from references of included articles, and from US Department of Health and Human Services Panel on Treatment of HIV-infected Pregnant Women and Prevention of Perinatal Transmission guidelines. Articles from non-human animal models or that had no original maternal-to-fetal transfer data were excluded. PRISMA guidelines were followed., Results: A total of 103 published studies were identified. Data across studies appeared relatively consistent for the nucleoside reverse transcriptase inhibitors (NRTIs) and the non-nucleotide reverse transcriptase inhibitors (NNRTIs), with cord to maternal ratios approaching 1 for many of these agents. The protease inhibitors atazanavir and lopinavir exhibited consistent maternal-to-fetal transfer across studies, although the transfer may be influenced by variations in drug-binding proteins. The protease inhibitors indinavir, nelfinavir, and saquinavir exhibited unreliable placental transport, with cord blood concentrations that were frequently undetectable. Limited data, primarily from case reports, indicate that darunavir and raltegravir provide detectable placental transfer., Conclusion: These findings appear consistent with current guidelines of using two NRTIs plus an NNRTI, atazanavir/ritonavir, or lopinavir/ritonavir to maximize placental transfer as well as to optimally suppress maternal viral load. Darunavir/ritonavir and raltegravir may reasonably serve as second-line agents.

Background: Hepatitis C virus genotype 6 (HCV-6) is common in patients from Southeast Asia and the surrounding regions. Optimal treatment duration for HCV-6 is unknown given the inconclusive evidence from studies with varying methodologies and small sample sizes., Methods: A literature search for 'genotype 6' in MEDLINE and EMBASE in October 2013 produced 161 and 251 articles, respectively. Additional abstracts were identified from four major international GI/liver conferences in 2012/2013. Inclusion criteria were original studies with ≥10 HCV-6 treatment-naïve patients treated with pegylated interferon + ribavirin (PEG IFN+RBV). Exclusion criteria were coinfections with HBV, HIV, other HCV genotypes, and/or other liver diseases. Primary outcome was pooled sustained virologic response (SVR). Heterogeneity was defined by Cochrane Q test (p value of 0.10) and I (2) statistic (≥50 %)., Results: A total of 13 studies with 641 patients were included. The pooled SVR estimate was 77 % (CI 70-83 %) (Q value = 38.4, p value <0.001, I (2) = 68.7 %) overall, 79 % (CI 73-84 %) for the 48-week group and 59 % (CI 46-70 %) for 24-week group, respectively. In studies with direct comparison of the two groups, SVR was superior in patients treated for 48 versus 24 weeks, OR 1.9 (CI 1.08-3.2, p = 0.026). In studies with direct comparison of patients with rapid virologic response (RVR), there was no difference in SVR between 48 versus 24 weeks, OR 1.74 (CI 0.65-4.64, p = 0.27)., Conclusion: Hepatitis C virus genotype 6 patients should be treated for 48 weeks, and those who achieve RVR may receive the shorter 24-week treatment duration. The high SVR (~80 %) with 48 weeks of PEG IFN+RBV therapy may be a cost-effective option for HCV-6 patients from resource-poor regions.

Soil is the vital foundation of terrestrial ecosystems storing water, nutrients, and almost three-quarters of the organic carbon stocks of the Earth's biomes. Soil organic carbon (SOC) stocks vary with land-cover and land-use change, with significant losses occurring through disturbance and cultivation. Although urbanisation is a growing contributor to land-use change globally, the effects of urban land-cover types on SOC stocks have not been studied for densely built cities. Additionally, there is a need to resolve the direction and extent to which greenspace management such as tree planting impacts on SOC concentrations. Here, we analyse the effect of land-cover (herbaceous, shrub or tree cover), on SOC stocks in domestic gardens and non-domestic greenspaces across a typical mid-sized U.K. city (Leicester, 73 km(2), 56% greenspace), and map citywide distribution of this ecosystem service. SOC was measured in topsoil and compared to surrounding extra-urban agricultural land. Average SOC storage in the city's greenspace was 9.9 kg m(-2), to 21 cm depth. SOC concentrations under trees and shrubs in domestic gardens were greater than all other land-covers, with total median storage of 13.5 kg m(-2) to 21 cm depth, more than 3 kg m(-2) greater than any other land-cover class in domestic and non-domestic greenspace and 5 kg m(-2) greater than in arable land. Land-cover did not significantly affect SOC concentrations in non-domestic greenspace, but values beneath trees were higher than under both pasture and arable land, whereas concentrations under shrub and herbaceous land-covers were only higher than arable fields. We conclude that although differences in greenspace management affect SOC stocks, trees only marginally increase these stocks in non-domestic greenspaces, but may enhance them in domestic gardens, and greenspace topsoils hold substantial SOC stores that require protection from further expansion of artificial surfaces e.g. patios and driveways., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Use of pharmacotherapy during pregnancy is common and increasing. Physiologic changes during pregnancy may significantly alter the overall systemic drug exposure, necessitating dose changes. A search of PubMed for pharmacokinetic clinical trials showed 494 publications during pregnancy out of 35,921 total pharmacokinetic published studies (1.29%), from the late 1960s through August 31, 2013. Closer examination of pharmacokinetic studies in pregnant women published since 2008 (81 studies) revealed that about a third of the trials were for treatment of acute labor and delivery issues, a third included studies of infectious disease treatment during pregnancy, and the remaining third were for varied ante-partum indications. Approximately, two-thirds of these recent studies were primarily funded by government agencies worldwide, one-quarter were supported by private non-profit foundations or combinations of government and private funding, and slightly <10% were supported by pharmaceutical industry. As highlighted in this review, vast gaps exist in pharmacology information and evidence for appropriate dosing of medications in pregnant women. This lack of knowledge and understanding of drug disposition throughout pregnancy place both the mother and the fetus at risk for avoidable therapeutic misadventures - suboptimal efficacy or excess toxicity - with medication use in pregnancy. Increased efforts to perform and support obstetric dosing and pharmacokinetic studies are greatly needed.

Soil compaction adversely influences most terrestrial ecosystem services on which humans depend. This global problem, affecting over 68 million ha of agricultural land alone, is a major driver of soil erosion, increases flood frequency and reduces groundwater recharge. Agricultural soil compaction has been intensively studied, but there are no systematic studies investigating the extent of compaction in urban ecosystems, despite the repercussions for ecosystem function. Urban areas are the fastest growing land-use type globally, and are often assumed to have highly compacted soils with compromised functionality. Here, we use bulk density (BD) measurements, taken to 14 cm depth at a citywide scale, to compare the extent of surface soil compaction between different urban greenspace classes and agricultural soils. Urban soils had a wider BD range than agricultural soils, but were significantly less compacted, with 12 per cent lower mean BD to 7 cm depth. Urban soil BD was lowest under trees and shrubs and highest under herbaceous vegetation (e.g. lawns). BD values were similar to many semi-natural habitats, particularly those underlying woody vegetation. These results establish that, across a typical UK city, urban soils were in better physical condition than agricultural soils and can contribute to ecosystem service provision.

A key feature of amphiphilic molecules is their ability to undergo self-assembly, a process in which a complex hierarchical structure is established without external intervention. Ternary systems consisting of aqueous mixtures of cationic and anionic surfactants exhibit a rich array of self-assembled microstructures such as spherical and rodlike micelles, unilamellar and multilamellar vesicles, planar bilayers, and bicontinuous structures. In general, multiple complementary techniques are required to explore the phase behavior and morphology of aqueous systems of oppositely charged surfactants. As a novel and effective alternative approach, we use fluorescence spectroscopic measurements to examine the microstructures of aqueous cationic/anionic surfactant systems in the dilute surfactant region. In particular, we demonstrate that the polarity-sensitive fluorophore prodan can be used to demarcate the surfactant microstructures of the ternary system of cetyltrimethylammonium bromide, sodium octyl sulfate, and water. As the fluorescence signature of this probe is dependent on the nature of the surfactant aggregates present, our method is a promising new approach to effectively map complex surfactant phase diagrams.

The rapid migration of intestinal epithelial cells is important to the healing of mucosal ulcers and wounds. This cell migration requires the presence of polyamines and the activation of RhoA. RhoA activity, however, is not sufficient for migration because polyamine depletion inhibited the migration of IEC-6 cells expressing constitutively active RhoA. The current study examines the role of Rac1 and Cdc42 in cell migration and whether their activities are polyamine-dependent. Polyamine depletion with alpha-difluoromethylornithine inhibited the activities of RhoA, Rac1, and Cdc42. This inhibition was prevented by supplying exogenous putrescine in the presence of alpha-difluoromethylornithine. IEC-6 cells transfected with constitutively active Rac1 and Cdc42 migrated more rapidly than vector-transfected cells, whereas cells expressing dominant negative Rac1 and Cdc42 migrated more slowly. Polyamine depletion had no effect on the migration of cells expressing Rac1 and only partially inhibited the migration of those expressing Cdc42. Although polyamine depletion caused the disappearance of actin stress fibers in cells transfected with empty vector, it had no effect on cells expressing Rac1. Constitutively active Rac1 increased RhoA and Cdc42 activity in both normal and polyamine-depleted cells. These results demonstrate that Rac1, RhoA, and Cdc42 are required for optimal epithelial cell migration and that Rac1 activity is sufficient for cell migration in the absence of polyamines due to its ability to activate RhoA and Cdc42 as well as its own effects on the process of cell migration. These data imply that the involvement of polyamines in cell migration occurs either at Rac1 itself or upstream from Rac1.

Background & Aims: Inhibition of RhoA activity and depletion of polyamines inhibits cell migration and causes changes in the actin cytoskeleton. In this article we have examined the effect of polyamine depletion on RhoA and evaluated these effects on cell migration., Methods: Polyamines were depleted in intestinal epithelial cell (IEC)-6 cells by incubating them for 4 days with 5 mmol/L alpha-difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase, the first rate-limiting enzyme in the synthesis of polyamines. IEC-6 cells were then transfected with vectors containing HA tags and constitutively active (HA-V14) or dominant-negative (HA-N19) RhoA with pcDNA3 (vector)., Results: DFMO caused a significant decrease in Rho levels in the cytoplasm and membranes of IEC-6 cells. This decrease was caused by an approximate 50% inhibition of RhoA protein synthesis. Neither the half-life of RhoA nor the level of RhoA messenger RNA (mRNA) was affected. HA-V14-RhoA cells migrated much more rapidly than vector-transfected cells, and HA-N19-RhoA cells exhibited almost no motility. The migration of HA-V14-RhoA cells, however, was inhibited markedly by polyamine depletion. Polyamine depletion did not affect the activity of RhoA in HA-V14-RhoA cells, but inhibited it dramatically in the vector-transfected cells. In the presence of DFMO, the HA-V14-RhoA cells lost stress fibers and gained the appearance of HA-N19-RhoA cells or wild-type cells treated with DFMO., Conclusions: First, polyamines are essential for the activity and synthesis and, therefore, normal levels of RhoA protein. Second, RhoA does not mediate the inhibitory effects of polyamine depletion on cell migration.

The proliferation of the rat intestinal mucosal IEC-6 cell line requires polyamines, whose synthesis is catalyzed by the enzyme ornithine decarboxylase (ODC). ODC inhibition leads to polyamine depletion, as well as inhibition of both cell proliferation and apoptosis by regulating gene expression. The NF-kappa B transcription factor regulates genes involved in apoptotic, immune, and inflammatory responses. In the present study we tested the hypothesis that NF-kappa B is activated following ODC inhibition. We found that the inhibition of ODC by alpha-difluoromethylornithine (DFMO) resulted in a approximately 50% decrease in intracellular putrescine levels within 1 h. NF-kappa B is activated by DFMO through the degradation of the inhibitory protein I kappa B alpha that sequesters NF-kappa B in the cytoplasm. The DFMO-induced NF-kappa B complexes contain the p65 and p50 members of the Rel protein family. DFMO-induced NF-kappa B activation was accompanied by the translocation of p65 from the cytoplasm into the nucleus. DFMO selectively inhibited a gene reporter construct dependent on the kappa B site present in the HLA-B7 gene. In contrast, DFMO had no effect on a gene reporter construct dependent on the kappa B site present in the interleukin-8 gene. Thus, we report that ODC inhibition activates the NF-kappa B transcription factor, which may mediate the altered physiological state of intestinal cells that occurs following polyamine depletion.

Leeuwenhoek first described polyamines in 1677, but active investigation did not begin until the 1970's. When intracellular polyamine levels are reduced by inhibitors, mutation, or transfection, severe reductions occur in cell division, cell differentiation, and cell migration. These effects are not difficult to demonstrate and measure, and all can be prevented if supplemental exogenous polyamines are supplied. However, linking the overall effects to molecular events remains to be accomplished. In this review, we discuss work (mostly from the last 10 years) that relates to cell migration. Specifically, we have discussed the biology and biochemistry of the polyamines, their transport and regulation, the structure of the cytoskeleton and the mechanics of cell movement. We have also considered four specific processes that polyamines participate in that may affect cell migration significantly. These are: 1) the regulation of intracellular Ca++ concentration by voltage-gated K+ channels, 2) the maintenance of normal RhoA levels that, with Rac, regulate the assembly of actin stress fibers, focal adhesions, and contractility, 3) the formation of ATP-Mg++-polyamine trimers that enhance the phosphorylation activity of ATP toward enzymes in specific signaling pathways and, 4) alterations in the structure of RNA that change translation initiation sites and affect the expression of proteins.

Polyamines are essential to the migration of epithelial cells in the intestinal mucosa. Cells depleted of polyamines do not attach as rapidly to the extracellular matrix and do not form the actin stress fibers essential for migration. Because both attachment and stress fiber formation depend on integrin signaling and the formation of focal adhesions, we examined these and related processes in polyamine-depleted IEC-6 cells. There was general decreased tyrosine phosphorylation of focal adhesion kinase (FAK), and, specifically, decreased phosphorylation of Tyr-925, the paxillin binding site. In control cells, FAK phosphorylation was rapid after attachment to the extracellular matrix, while attached cells depleted of polyamines had significantly delayed phosphorylation. FAK activity was also significantly inhibited in polyamine-depleted cells as was the phosphorylation of paxillin. Polyamine-depleted cells failed to spread normally after attachment, and immunocytochemistry showed little colocalization of FAK and actin compared with controls. Focal adhesion complex formation was greatly reduced in the absence of polyamines. These data suggest that defective integrin signaling may, at least in part, account for the decreased rates of attachment, actin stress fiber formation, spreading, and migration observed in polyamine-depleted cells.

The polyamines spermidine and spermine and their precursor, putrescine, are required for the growth and proliferation of eukaryotic cells. This study compares and contrasts growth arrest caused by polyamine depletion in the untransformed IEC-6 cell line with that in the p53-mutated colon cancer Caco-2 cell line. Cells were grown in the presence or absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, the first rate-limiting enzyme in the synthesis of polyamines. Depletion of polyamines inhibited the growth of both cell lines equally and over the same time frame. However, whereas IEC-6 cells were arrested in the G(1) phase of the cell cycle, there was no accumulation of Caco-2 cells in any particular phase. In IEC-6 cells, growth arrest was accompanied by elevated levels of p53 and p21(Waf1/Cip1) (p21). There were no changes in p53 levels in Caco-2 cells. Levels of p21 increased in Caco-2 cells on day 2 without any effect on cell cycle progression. The amount of cyclin-dependent kinase (Cdk)2 protein was unchanged by polyamine depletion in both cell lines. However, the activity of Cdk2 was significantly inhibited by DFMO in IEC-6 cells. These data suggest that in the untransformed IEC-6 cells the regulation of Cdk2 activity and progression through the cell cycle are p53- and p21 dependent. Growth arrest in the p53-mutated Caco-2 line after polyamine depletion occurs by a different, yet unknown, mechanism.

Polyamines are required for the proliferation of the rat intestinal mucosal IEC-6 cell line. Ornithine decarboxylase (ODC) is the enzyme that catalyzes the first step in polyamine synthesis. ODC inhibition not only leads to polyamine depletion but also leads to inhibition of cell proliferation and regulates the expression of the immediate-early genes c-fos, c-myc, and c-jun. Members of the signal transducers and activators of transcription (STAT) transcription factor family bind to the sis-inducible element (SIE) present in the promoters to regulate the expression of a variety of important genes. In the present study, we tested the hypothesis that the STAT3 transcription factor, which is responsible for activation of the acute phase response genes, is activated after inhibition of ODC. We found that inhibition of ODC rapidly induces STAT3 activation as determined by STAT3 tyrosine phosphorylation, translocation of STAT3 from the cytoplasm into the nucleus, and the presence of STAT3 in SIE-dependent DNA-protein complexes. STAT3 activation upon inhibition of ODC was accompanied by the activation of a STAT3-dependent reporter construct. Moreover, prolonged polyamine depletion resulted in downregulation of cellular STAT3 levels.

The polyamines spermidine and spermine and their precursor putrescine are intimately involved in and are required for cell growth and proliferation. This study examines the mechanism by which polyamines modulate cell growth, cell cycle progression, and signal transduction cascades. IEC-6 cells were grown in the presence or absence of DL-alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the first rate-limiting enzyme for polyamine synthesis. Depletion of polyamines inhibited growth and arrested cells in the G1 phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the level of p53 protein and other cell cycle inhibitors, including p21(Waf1/Cip1) and p27(Kip1). Induction of cell cycle inhibitors and p53 did not induce apoptosis in IEC-6 cells, unlike many other cell lines. Although polyamine depletion decreased the expression of extracellular signal-regulated kinase (ERK)-2 protein, a sustained increase in ERK-2 isoform activity was observed. The ERK-1 protein level did not change, but ERK-1 activity was increased in polyamine-depleted cells. In addition, polyamine depletion induced the stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) type of mitogen-activated protein kinase (MAPK). Activation of JNK-1 was the earliest event; within 5 h after DFMO treatment, JNK activity was increased by 150%. The above results indicate that polyamine depletion causes cell cycle arrest and upregulates cell cycle inhibitors and suggest that MAPK and JNK may be involved in the regulation of the activity of these molecules.

Polyamines are involved in the processes of cell migration and proliferation that result in the repair of mucosal lesions. Depletion of polyamines dramatically alters the arrangement of the cytoskeleton, EGF receptor function, the activities of signal transduction proteins, the levels of several protooncogenes, and the expression and cellular content of at least one growth factor involved in these processes.

Signal transducers and activators of transcription (STATs) are cytoplasmic proteins that bind to activated membrane receptors, undergo ligand-dependent phosphorylation on tyrosine residues, and translocate to the nucleus, where they induce transcription of specific genes in response to a variety of ligands, including cytokines and some growth factors. Using immunocytochemical and biochemical techniques, we investigated the localization and responses of STAT1 and STAT2 to epidermal growth factor (EGF) stimulation in IEC-6 intestinal epithelial cells and HeLa cells. These studies provide the first description of STAT activation and localization in response to EGF in intestinal epithelial cells and some novel findings regarding the activation and localization of STATs in general. These include the following. First, EGF promoted the tyrosine phosphorylation of STAT1 in IEC-6 cells and caused its translocation to the nucleus. Second, in the absence of EGF stimulation both STAT1 and STAT2 were localized to the Golgi apparatus in IEC-6 cells. Third, EGF caused the translocation of STAT2 to the nucleus in both IEC-6 and HeLa cells without inducing the tyrosine phosphorylation of STAT2.

The cause of reduced migration ability in polyamine-deficient cells is not known, but their actin cytoskeleton is clearly abnormal. We depleted polyamines with alpha-difluoromethylornithine (DFMO) in migrating cells with or without stimulation by epidermal growth factor (EGF) and investigated filamentous (F-) actin, monomeric (G-) actin, and thymosin beta4 (Tbeta4), using immunofluorescent confocal microscopy, DNase assay, and immunoblot analysis. DFMO reduced F-actin in the cell interior, increased it in the cell cortex, redistributed G-actin, and increased nuclear staining of Tbeta4. However, DFMO did not affect the amount of Tbeta4 mRNA. EGF caused a rapid increase in the staining of F-actin in control cells, but DFMO prevented this response to EGF. Despite the visible changes shown by immunocytochemistry, statistically significant changes in the amount of either actin isoform or of total actin did not occur. We propose that DFMO reduces migration by interfering with the sequestration of G-actin by Tbeta4 and the association of F-actin with activated EGF receptors.

Polyamines are essential for the repair of gastric and duodenal erosions. Concentrated NaCl (3.4 M) given intragastrically damages the oxyntic gland mucosa and increases the activity of gastric mucosal ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis. The nature of the process of restitution of damaged mucosa is not well known, except that cell migration and the actin cytoskeleton play a prominent role. Microtubules are cytoskeletal components essential for cell migration. The present investigation determines the relationship between polyamines, the distribution of microtubules, and gastric healing in mucosa damaged with hypertonic NaCl solution. Rats were fasted for 22 h and then given 1.0 ml of 3.4 M NaCl intragastrically. Animals were killed 1, 2, 4, 8, and 10 h after 3.4 M NaCl. The oxyntic gland mucosa was removed, and tubulin was visualized by immunofluorescence. Microtubule density was increased around and below the damaged mucosa in the upper one-third of the glandular epithelium at 2 and 4 h and returned to near control levels by 10 h. In rats damaged with 3.4 M NaCl and pretreated intraperitoneally with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC, microtubule content was reduced significantly at all time points after NaCl treatment. Addition of spermidine after pretreatment with DFMO and 3.4 M NaCl significantly prevented the effects of DFMO. Colchicine, a potent microtubule-disrupting drug, significantly delayed normal gastric mucosal healing with no effect on ODC activity. These data show that polyamines influence the distribution of microtubules during damage in vivo and indicate a partial mechanism for the dependency of mucosal healing on polyamines.

Cell growth and migration are essential processes for the differentiation, maintenance, and repair of the intestinal epithelium. Epidermal growth factor (EGF) is an important factor in the reorganization of the cytoskeleton required for both processes. Because we had previously found significant changes in the cytoskeleton during polyamine deficiency, it was of interest to know whether those changes could prevent EGF from stimulating growth and migration. Polyamine biosynthesis in IEC-6 cells was interrupted by treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, the primary rate-limiting enzyme of polyamine biosynthesis. DFMO halted cell proliferation and inhibited cell migration, and neither function could be normally stimulated by EGF. Immunocytochemistry of the transferrin receptor (used as a marker for the endocytic pathway) revealed an abnormal distribution of the EGF receptor (EGFR) 10 min after binding EGF. Polyamine deficiency depleted the cells of interior microfilaments, thickened the actin cortex, and prevented the prompt association of EGF-bound EGFR with actin. EGF-stimulated 170-kDa protein tyrosine phosphorylation and the kinase activity of purified membrane EGFR were reduced by 50%. Immunoprecipated EGFR protein concentration, however, was not reduced by polyamine deficiency. All of these changes could be prevented by supplementation with putrescine. Cytoskeletal disruption, reduced EGFR phosphorylation and kinase activity, aberrant intracellular EGFR distribution, and delayed association with actin filaments suggest a partial explanation for the dependence of epithelial cell growth and migration on polyamines.

The inhibition of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, with alpha-difluoromethylornithine in IEC-6 cells (small intestinal crypt cell line) reduces cell migration by 70%, inhibits protein cross-linking, and affects the cytoskeletal assembly. The current study examines the effects of intracellular polyamine depletion on attachment of IEC-6 cells to different matrices. Polyamine deficiency inhibited cell attachment to plastic, laminin, fibronectin, collagen IV, and Matrigel by different extents. Intracellular putrescine restored attachment to all matrices. The presence of a specific inhibitor of protein cross-linking also inhibited attachment to laminin in a dose-dependent manner. The inhibition of cell attachment to plastic and Matrigel was correlated with the inhibition of cell migration. Immunofluorescence studies showed that polyamines are essential for the correct expression of the integrin subunit alpha 2 but not for the expression of the alpha 1-subunit. This study demonstrates that polyamines are important for cell attachment and expression of the integrin alpha 2 beta 1, a putative receptor for collagen and laminin. The impairment of protein cross-linking and the inhibition of the expression of cell surface receptors that bind extracellular matrix (ECM) proteins may be part of the mechanism by which polyamine deficiency retards cell migration in the small intestine.

In the intestine, several growth factors stimulate migration of epithelial cells, contributing to the maintenance of tissue integrity. The Ras-like GTPase Rho regulates a signal transduction pathway linking growth factor receptors to the formation of actin stress fibers and focal adhesions, presumed to be important for motility. Using an in vitro wound-induced migration assay, we have examined the role of Rho GTPases in the migration of IEC-6 and Caco-2 cells, and provide evidence that the Rho GTPases play an essential role in the initial phase of mucosal wound healing. Treatment of the cells with Clostridium difficile toxins A and B, inhibitors of the Rho family GTPases inhibited migration in a dose-dependent fashion. Microinjection of the inhibitory exchange factor Rho-guanine nucleotide dissociation inhibitor (GDI), or Clostridium botulinum C3 ADP-ribosyl transferase (C3) toxin, a Rho-ADP-ribosylating exoenzyme, potently inhibited migration. Microinjection of RhoT19N, a dominant negative form of RhoA, or in vitro ADP-ribosylated RhoA impaired the ability of cells to migrate. Rho-GDI and C3 exoenzyme also inhibited EGF-induced migration of IEC-6 cells. These results demonstrate that Rho is required for endogenous and EGF-induced migration of small intestinal crypt cells, and that Rho proteins are essential elements of a mechanism by which growth factors induce cell migration to restitute mucosal integrity.

The current study tests the hypothesis that intracellular polyamines are involved in the regulation of gene expression of transforming growth factor-beta (TGF-beta) during epithelial cell migration after wounding. Administration of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (the first rate-limiting enzyme for polyamine synthesis), depleted cellular polyamines putrescine, spermidine, and spermine in IEC-6 cells. DFMO also significantly reduced basal levels of TGF-beta mRNA in unwounded cells. Gene expression of TGF-beta was dramatically stimulated after wounding of a monolayer of cells not treated with DFMO. TGF-beta mRNA levels significantly increased from 4 to 12 h after wounding, peaking at 6 h at a level eight times the prewounding control. Increased levels of TGF-beta mRNA in IEC-6 cells after wounding were paralleled by an increase in TGF-beta content. Depletion of intracellular polyamines in DFMO-treated cells significantly inhibited increased expression of the TGF-beta gene in response to wounding. Cell migration also significantly decreased in DFMO-treated cells. In the presence of DFMO, exogenous TGF-beta restored cell migration to normal. These results indicate that 1) polyamine depletion induced by DFMO is associated with decreases in the expression of the TGF-beta gene and cell migration in IEC-6 cells and 2) exogenous TGF-beta reverses the inhibitory effect of polyamine depletion on cell migration. These findings suggest that polyamines are required for epithelial cell migration in association with their ability to regulate TGF-beta gene expression.

Intragastric administration of 3.4 M NaCl damages the gastric mucosa and increases the activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. Polyamines are essential for the repair of gastric erosions. Little is known about the restitution of damaged mucosa except that cell migration is essential. Actin is the principal cytoskeletal protein and is essential for migration. This investigation determines the relationship between polyamines, actin, and gastric healing. Rats were fasted for 22 h and given 1.0 ml of 3.4 M NaCl intragastrically and killed 1, 2, 4, 8, and 10 h later. The mucosa was assayed for ODC activity and stained for G- and F-actin. F-actin was concentrated below the damaged mucosa at 1.5, 2, and 4 h. There was no increase in F-actin distribution at any time point, when NaCl-treated animals were given alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC. In addition, DFMO significantly prevented the healing of the mucosal lesions. Spermidine treatment after DFMO + NaCl significantly prevented the effects of DFMO. Cytochalasin D, a potent actin-disrupting drug, significantly delayed normal gastric mucosal healing. The endogenous polyamines increased significantly in NaCl animals. Data indicate that increases in polyamine synthesis after damage influence the distribution of F-actin in vivo, which may play a part in the healing of mucosal erosions.

The current study determines whether nonmuscle myosin II is involved in the process requiring polyamines for the stimulation of cell migration in an in vitro model that mimics the early stages of epithelial restitution. Treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (ODC), for 4 days totally inhibited ODC activity and depleted intracellular polyamines in the IEC-6 cells. Nonmuscle myosin II concentrations in DFMO-treated cells were decreased by 75%, and stress fibers were sparse or absent. The most striking feature of DFMO-treated cells was the appearance of many small punctate foci of myosin II in the cell interior. Migration of DFMO-treated cells was reduced by 80%. In the presence of DFMO, exogenous putrescine not only returned nonmuscle myosin II levels and distribution toward normal but also restored cell migration to control levels. The administration of wortmannin, an inhibitor of myosin light chain kinase, significantly inhibited cell migration over the denuded area in control cells and in those treated with DFMO + polyamines. These results indicate that 1) polyamine depletion by DFMO is associated with decreased concentration and reorganization of nonmuscle myosin II in IEC-6 cells and 2) exogenous spermidine reverses the inhibitory effects of DFMO.

All epithelial cells require retinoic acid for growth, maintenance, and differentiation. Although the epithelial cells that line the gastrointestinal tract are exposed to extreme retinoid concentration fluctuations in luminal fluid, whether proliferation and differentiation in these cells are significantly affected is not known. We have investigated this question using Caco-2 cells as a model because, although they are derived from a colon adenocarcinoma, they differentiate spontaneously in a manner similar to enterocytes in the small intestine. We found that retinoic acid caused maximum inhibition of cell growth and ornithine decarboxylase activity during the proliferative period. Retinoic acid increased brush border enzyme activities only in differentiating cells but stimulated transglutaminase activity in cells at all stages. In untreated proliferating cells, we found an early peak of transglutaminase activity that has not been reported before. Retinoic acid in intestinal cells acts through its nuclear receptor, RAR beta. The nuclear distribution of this receptor has not been demonstrated. In this study, we show that RAR beta responds to increasing concentrations of retinoic acid with a shift to the nuclear membrane in undifferentiated cells and progressive aggregation, diffusion, and loss in differentiated cells. We conclude that retinoic acid can inhibit proliferation and stimulate differentiation in Caco-2 cells depending on concentration and cell stage, and that these effects are accompanied by changes in distribution, as well as by the loss of RAR beta.

Healing of gastrointestinal mucosal lesions occurs through two processes: an early one involving cell migration and a later one in which cell division replaces lost cells. Both processes require the presence of polyamines, but the mechanism of action of these compounds is unknown. In the present study, we examined the ability of analogues of spermidine and spermine to support migration and growth of IEC-6 cells that have been grown in alpha-difluoromethylornithine to inhibit polyamines. All analogues of spermidine with the general formula x-3 (referring to the numbers of carbon atoms on either side of the central nitrogen), where x = 2-12, competed with spermidine for entry into the cells. However, in addition to spermidine (x = 4), only compounds for which x = 2, 3, or 6 supported migration and only those for which x = 2 or 7 supported growth. Spermine analogues 3-x-3, for which x = 3, 6, 9, or 12, competed for entry into the cells, but only compounds for which x = 3 or 6 supported migration and only the compound for which x = 3, in addition to spermine (x = 4), supported growth. In addition, analogues 2-3-2, 3-2-3, and 2-(3)2, a branched compound, supported both migration and growth but entered the cell via a mechanism different than that for spermidine and spermine. These data define some of the specific structural requirements for polyamines to produce their physiological effects.

Cimetidine has been shown to inhibit normal and carcinoma cell growth but the mechanism of the antiproliferative action is incompletely understood. The current study determined the influence of cimetidine on ornithine decarboxylase (ODC) activity, which is the initial rate-limiting enzyme in polyamine biosynthesis, in rat duodenal mucosa and IEC-6 cells (a line of normal rat intestinal crypt cells). Rats were fasted 22 hr before the various treatments and ODC activity was measured in scraped duodenal mucosa. Administration of pentagastrin and epidermal growth factor (EGF) and refeeding fasted rats significantly increased ODC activity in duodenal mucosa. Cimetidine completely inhibited increases in ODC activity in the mucosa stimulated by pentagastrin and EGF, but not by refeeding. Ranitidine and H1-receptor antagonist chlorpheniramine had similar inhibitory effects on ODC activity induced by gastrin. In cultured IEC-6 cells, cimetidine caused a linear and significant inhibition of the stimulation of ODC activity in response to pentagastrin, EGF, 5% dialyzed fetal bovine serum (FBS) and asparagine. ODC messenger RNA (mRNA) levels in IEC-6 cells were significantly increased after exposure to 5% dialyzed FBS and asparagine. Although cimetidine almost completely prevented the induction of ODC activity in IEC-6 cells exposed to serum or asparagine, the increases in ODC mRNA levels were not inhibited by the compound.(ABSTRACT TRUNCATED AT 250 WORDS)

Transglutaminases (TGAs) catalyze the cross-linking of proteins through formation of gamma-glutaminyl-epsilon-lysine bonds and incorporation of small-molecular-weight amines, including polyamines, into the gamma-glutamine sites of proteins. Tissue TGA has been shown to establish covalent cross-links between cytoskeletal proteins using polyamines as substrates, and protein-polyamine conjugates have been identified in a variety of cells. We have shown previously that polyamines are required for cell migration in IEC-6 cells [S. A. McCormack, M. J. Viar, and L. R. Johnson. Am. J. Physiol. 264 (Gastrointest. Liver Physiol. 27): G367-G374, 1993]. In this study, we explored the relationship between cell migration, polyamines, and tissue TGA activity in two cell lines and found that while both IEC-6 and Caco-2 cells required normal levels of polyamines to migrate across a denuded surface, tissue TGA activity responded differently to polyamine deficiency brought about by treatment with alpha-difluoromethylornithine (DFMO). DFMO is a specific and irreversible inhibitor of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis. In IEC-6 cells, tissue TGA activity decreased significantly with DFMO treatment concurrent with a rise in inactive TGA protein as measured by Western blot analysis. On the other hand, in Caco-2 cells, tissue TGA activity and protein increased significantly with DFMO treatment. In both cell lines, addition of polyamines to the DFMO treatment restored cell migration, tissue TGA activity, and protein to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)

In earlier work we have shown that polyamine-deficient IEC-6 cells lose most of their ability to migrate. In this report we describe the effect of polyamine deficiency on the cytoskeleton of migrating IEC-6 cells. Cells were grown on cover slips for 4 days. One-third of the monolayer was removed, and the remainder was incubated for 6 h. The monolayers were fixed and stained with rhodamine phalloidin for actin filaments and by immunocytochemistry for tropomyosin. In control cells, actin filaments were found as stress fibers traversing the cell, in a thin actin cortex often visible on only one edge of the cell, and in fine fibers extending into the lamellipodia. Tropomyosin was found in the same distribution. A Western blot showed that tropomyosin was present as 35- and 37-kDa isoforms. In polyamine-deficient cells, actin stress fibers were less dense, whereas the actin cortex was greatly increased in density and lamellipodia were less extensive. Tropomyosin distribution was similar and included a 30-kDa isoform not seen previously. In spite of the obvious changes in the distribution of these cytoskeletal proteins, the concentrations of filamentous actin, beta-actin mRNA, and the higher molecular weight tropomyosin isoforms did not change. In all cases the addition of putrescine to polyamine-deficient cells prevented the changes described. We conclude that polyamines are essential for migration in this system because of their effects on the organization of cytoskeletal actin, tropomyosin, and perhaps other proteins as well.

Ornithine decarboxylase (ODC) catalyzes the first rate-limiting step in polyamine biosynthesis, and increased ODC activity is one of the earliest biochemical events associated with the induction of cellular proliferation. The current study examines the regulation of ODC activity in rat duodenal mucosa and IEC-6 cells (a line of normal rat intestinal crypt cells) in response to the trophic hormone, gastrin, and its inhibitor, secretin. Rats were fasted 22 h before the various treatments, and ODC activity was measured in scraped duodenal mucosa. Gastrin significantly increased ODC activity within 3 h to 4.3 times control levels. The effect of gastrin was totally inhibited by 5 micrograms/kg secretin. In doses of 5 or 10 micrograms/kg, secretin had no effect on basal ODC. Epidermal growth factor (EGF) and refeeding fasted rats also significantly increased ODC activity in duodenal mucosa, but the effects of EGF and refeeding were not prevented by secretin. In cultured IEC-6 cells, ODC activity was significantly increased after exposure to gastrin, 5% dialyzed fetal bovine serum (FBS), EGF, and asparagine. Secretin in doses ranging from 10(-10) to 10(-6) M caused a linear and significant inhibition of the stimulation of ODC activity by gastrin. No dose of secretin affected basal ODC activity or enzyme activity stimulated by 5% dialyzed FBS, EGF, or asparagine in IEC-6 cells. The ODC mRNA levels in IEC-6 cells were also increased after exposure to gastrin. Administration of secretin significantly prevented the stimulated expression of the ODC gene in cells treated with gastrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct exposure of small intestinal mucosal cells to luminal polyamines stimulates proliferation. This study tests the hypothesis that the protooncogenes c-fos, c-myc, c-jun, and junB are involved in the mechanism by which polyamines modulate mucosal growth. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS) in the presence of absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the rate-limiting enzyme for polyamine synthesis. Cellular polyamine levels, cell growth, and relative abundance of c-fos, c-myc, c-jun, and junB mRNAs, were measured at 1, 2, 4, 6, 8, and 12 days after initial plating. The intracellular polyamines, spermidine and spermine, and their precursor, putrescine, in DFMO-treated cells decreased significantly at 2 days and remained depleted thereafter. Although DFMO profoundly decreased growth and final cell number, both control and DFMO-treated cells entered a plateau phase by 6 days. In control cells, c-myc and c-jun mRNA levels significantly increased on days 4-6 and then returned to a basal level of expression, which was maintained thereafter. c-fos mRNA in quiescent cells after 24 h serum deprivation was significantly stimulated by 5% dFBS, although a steady-state level of c-fos mRNA was undetectable in control cells. Treatment with DFMO not only prevented increased expression of c-myc and c-jun protooncogenes at 4 days, but also significantly reduced steady-state levels of c-myc and c-jun mRNA between 6 and 12 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies from our laboratory have shown that polyamines are essential for the normal repair of duodenal erosions induced in vivo in a rat stress-ulcer model. In that model, the inhibition of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, with alpha-difluoromethylornithine (DFMO) almost entirely prevented healing. Healing could be restored by oral polyamines. In this paper, we have investigated whether the polyamines are required for the early stages of epithelial restitution using an IEC-6 cell culture model of cell migration. Treatment of the cells with DFMO for 4 days reduced cell migration 80%. Migration could be restored to normal by concomitant treatment with putrescine (PUT), spermidine (SPD), or spermine (SPM), but not by their addition during the migration period (6 h) only. If DFMO treatment was not begun until the migration period, it still reduced cell migration 20%, and this deficit could not be restored by concomitant addition of the polyamines. Intracellular polyamine levels at these times, i.e., 6 h or 4 days, were an important factor in these results. Only PUT was undetectable after 6 h of DFMO. SPD and SPM were still at normal levels at 6 h. SPD was undetectable at 4 days, but SPM was still at 40% of normal. These data give added importance to PUT because its absence reduced cell migration after only 6 h, while SPD and SPM were still present in normal amounts. Perhaps exogenous SPD and SPM restored cell migration when present with DFMO for 4 days treatment primarily because they contributed to intracellular PUT through the acetyltransferases.(ABSTRACT TRUNCATED AT 250 WORDS)

Two key enzymes in polyamine biosynthesis are ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). SAMDC decarboxylates S-adenosylmethionine, which then donates aminopropyl groups for spermidine and spermine synthesis. The purpose of our study was to determine whether putrescine, taken up from medium or synthesized endogenously by ODC, alters SAMDC activity. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS). They were deprived of serum for 24 h before experiments. Basal SAMDC activity was increased significantly by > or = 10(-4) M of putrescine. Lower doses had no significant effect. The same doses of putrescine decreased ODC activity to near zero. Asparagine at 10 mM or 5% dFBS not only stimulated ODC activity and the intracellular putrescine levels but also increased significantly SAMDC activity as well. ODC activity peaked at 3 h, and the maximum level of SAMDC occurred 3-4 h after exposure to asparagine or serum. Treatment with DL-alpha-difluoromethylornithine (DFMO), a specific ODC inhibitor, prevented the increases in both cellular putrescine levels and SAMDC activity in asparagine- and serum-treated cells. In the presence of DFMO, exogenous putrescine returned SAMDC activity toward control levels but had no effect on ODC. A very slight increase of SAMDC half-life in IEC-6 cells grown in the presence of putrescine was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell migration is the principal force behind the early restitution of erosions of the mucosa of the gastrointestinal tract. Despite the importance of cell migration to healing, no attempts to study the process in culture have been reported. We have attempted to standardize conditions for migration and test the migration responses of the small intestinal epithelial crypt cell line IEC-6 in some experimental situations already well known in vivo. We found good correspondence between in culture and in vivo on the following points: 1) migration was independent of DNA synthesis; 2) DNA synthesis was not concentrated at the wound edge; and 3) inhibition of actin polymerization stopped migration altogether. In addition, the presence of an extracellular matrix maximized migration. Protein inhibitors with different modes of action inhibited cell migration to different degrees, not always commensurate with their inhibition of protein synthesis. Cell surface proteoglycans were important; hyaluronic acid had an effect, but the secretion of a migration-stimulating substance by wounded cells was equivocal. Significantly, alpha-difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase and polyamine synthesis, almost totally prevented cell migration. Because DFMO also prevents healing of mucosal erosions in vivo, we believe that this model can be used, keeping in mind its spatial limitations, to study the process of cell migration involved in the early restitution of mucosal erosions.

The purpose of this study was to determine whether luminal polyamines stimulate intestinal mucosal growth in vivo. Rats received 2% alpha-difluoromethylornithine (DFMO) added to their drinking water throughout the experiment. The polyamines spermidine and spermine (3 mg each/100 g body wt) were given intragastrically in combined doses once at 9:30 A.M. and again at 5:30 P.M. Duodenal and jejunal mucosal ornithine decarboxylase (ODC) activity in the DFMO-treated rats was inhibited significantly for the duration of the study. DFMO also markedly decreased the rate of [3H]thymidine incorporation into DNA of duodenal and jejunal mucosa. The decrease in [3H]thymidine incorporation was significant 4 days and maximal 6 and 8 days after beginning treatment with DFMO. Decreased ODC activity and DNA synthesis were paralleled by decreases in total mucosal DNA, RNA, and protein content. Administration of the polyamines significantly reversed the effects of DFMO except the inhibition of ODC. In fact, there were no significant differences in mucosal growth parameters between the controls (without DFMO) and those treated with DFMO plus polyamines. Oral administration of spermidine and spermine at a dose of 4.5 mg each/100 g body wt for 6 days to rats not treated with DFMO increased the normal rate of mucosal growth in the duodenum and jejunum as well. Polyamine accumulation in IEC-6 cells was measured to determine whether it was altered by DFMO. IEC-6 cells took up [3H]putrescine and [3H]spermidine from their surrounding environment and the uptake was stimulated by serum. DFMO (5 mM) totally inhibited the increase in ODC activity but had no effect on the cellular uptake of polyamines in the presence of putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)

IEC-6 cells were cultured on permeable filter inserts with separate access to the apical and basolateral sides. [3H]Putrescine uptake favored the apical side and its release (in Earle's balanced salt solution containing 0.1% bovine serum albumin) was six times greater in the apical-to-basolateral than in the basolateral-to-apical direction. Release in DMEM did not show this preference. The uptake of [3H]putrescine was stimulated approximately 1.3 times the basal level by 10 mM asparagine (ASN) or 5% dialyzed fetal bovine serum whether the [3H]putrescine was added at a concentration of 1 or 100 nM. The increased uptake was maintained for up to 6 h. When [3H]putrescine was removed after 4 h of uptake, the cells continued to release it into the medium on both sides for up to 4 h. Stimulated cells released only 50% as much as unstimulated cells. Unlabeled putrescine reduced the uptake of [3H]putrescine with an IC50 of 1.81 x 10(-6) M (r = 0.9476) and 1.02 x 10(-6) M (r = 0.9967) for unstimulated and ASN-stimulated cells, respectively. When the intracellular putrescine was reduced by difluoromethylornithine, the uptake of [3H]-putrescine was not changed, but its release was inhibited. Sodium was not required for [3H]putrescine uptake or release. Although the stimulated cells attained intracellular levels of [3H]putrescine which, if expressed as concentration based on cell volume, were up to 500 times the original extracellular concentration, a true concentration gradient could not be proven because 85% of the [3H]putrescine was probably bound to polyanions as shown by butanol extraction.

The role of Na+ and Na(+)-H+ exchange in the stimulation of ornithine decarboxylase (ODC) activity has been investigated in a human colon adenocarcinoma cell line, LoVo. Asparagine (Asn; 10 mM) or 10% fetal bovine serum (FBS) increased ODC activity from undetectable levels to greater than 500 pmol CO2.mg protein-1.h-1 in 4 h. This increase could be reduced 50% by concentrations of Na(+)-H+ exchange inhibitors that did not reduce protein synthesis. (approximately 0.2 mM for amiloride and 0.05 mM for hexamethyleneamiloride). Asn was able to double the uptake of 22Na+, whether an ionic (choline chloride) or nonionic (D-mannitol) substance was substituted for Na+, and the substitution of these compounds as well as N-methyl-glucamine for Na+ largely prevented the stimulation of ODC by Asn. Another factor influencing ODC activity was extracellular pH (pHo). When pHo was lowered, intracellular pH (pHi) also fell, and ODC activity was reduced. When pHo was raised, pHi also rose, and ODC activity increased. The well-known correlation between increased pHi and Na+ uptake with the stimulation of growth may be due to their influence on ODC activity.

In an attempt to identify the factors which might affect the measurement of water proton relaxation times in cultured cells, we have begun a long-term study of two human breast cancer cell lines, MDA-MB-231 and MDA-MB-435s. We tested growth rates and cell cycle distribution as intrinsic properties of the cells as well as methodological steps which might affect the measurement of T1 and T2. A detailed examination of the growth rates of the two cell lines, easily recognized as slow (231) and fast (435s) in culture, revealed that this attribute is difficult to correlate precisely with T1s or T2s. The reason is that the relaxation times are necessarily measured at one point in time while the growth rates are a summation of ongoing processes occurring over hours. Cell cycle distribution, on the other hand, can be measured simultaneously with the relaxation times by using cells quick-frozen from the same suspension. By this method, cell cycle distribution appears to be reflected through an effect on T1s. For example, cell pellets distributed 72:15:14 in G0G1:S:G2M has longer T1s (p less than 0.01) than those distributed 43:34:23 in G0G1:S:G2M. Regarding methodological factors, trypsin appeared to lower water content and T2s in the 231 cell line. Drift in the cell cycle distribution after sample preparation did not become significant until after 2 hours in the NMR tube. It was important to standardize the force and duration of centrifugation of the cell pellets to minimize the contribution of the suspending medium without affecting cell viability. We conclude that, given careful control of methodological factors, differences in T1 may reflect metabolic differences as demonstrated by T1 differences in cell pellets showing divergent cell cycle distribution.