Purification of recombinant human growth hormone from CHO cell culture supernatant by Gradiflow preparative electrophoresis technology

Purification of recombinant human growth hormone (rhGH) from Chinese hamster ovary (CHO) cell culture supernatant by Gradiflow large-scale electrophoresis is described. Production of rhGH in CHO cells is an alternative to production in Escherichia coli, with the advantage that rhGH is secreted into protein-free production media, facilitating a more simple purification and avoiding resolubilization of inclusion bodies and protein refolding. As an alternative to conventional chromatography, rhGH was purified in a one-step procedure using Gradiflow technology. Clarified culture supernatant containing rhGH was passed through a Gradiflow BF200 and separations were performed over 60 min using three different buffers of varying pH. Using a 50 mM Tris/Hepes buffer at pH 7.5 together with a 50 kDa separation membrane, rhGH was purified to approximately 98% purity with a yield of 90%. This study demonstrates the ability of Gradiflow preparative electrophoresis technology to purify rhGH from mammalian cell culture supernatant in a one-step process with high purity and yield. As the Gradiflow is directly scalable, this study also illustrates the potential for the inclusion of the Gradiflow into bioprocesses for the production of clinical grade rhGH and other therapeutic proteins. (C) 2003 Elsevier Science (USA). All rights reserved.

The effect of nutrient enrichment on growth, photosynthesis and hydraulic conductance of dwarf mangroves in Panama

1. Dwarf stands of the mangrove Rhizophora mangle L. are extensive in the Caribbean. We fertilized dwarf trees in Almirante Bay, Bocas del Toro Province, north-eastern Panama with nitrogen (N) and phosphorus (P) to determine (1) if growth limitations are due to nutrient deficiency; and (2) what morphological and/or physiological factors underlie nutrient limitations to growth. 2. Shoot growth was 10-fold when fertilized with P and twofold with N fertilization, indicating that stunted growth of these mangroves is partially due to nutrient deficiency. 3. Growth enhancements caused by N or P enrichment could not be attributed to increases in photosynthesis on a leaf area basis, although photosynthetic nutrient-use efficiency was improved. The most dramatic effect was on stem hydraulic conductance, which was increased sixfold by P and 2.5-fold with N enrichment. Fertilization with P enhanced leaf and stem P concentrations and reduced C : N ratio, but did not alter leaf damage by herbivores. 4. Our findings indicate that addition of N and P significantly alter tree growth and internal nutrient dynamics of mangroves at Bocas del Toro, but also that the magnitude, pattern and mechanisms of change will be differentially affected by each nutrient.

Persistence of recipient lymphocytes in NOD mice after irradiation and bone marrow transplantation

The non-obese diabetic (NOD) mouse is a unique and invaluable model of autoimmune disease, in particular type I diabetes. Bone marrow transplantation as a therapy for type I diabetes has been explored in NOD mice. NOD mice require higher doses of conditioning irradiation for successful allogeneic bone marrow transplantation, suggesting that NOD hematopoietic cells are radioresistant compared to those of other mouse strains. However, studies of hematopoietic reconstitution in NOD mice are hampered by the lack of mice bearing a suitable cell-surface marker that would allow transferred cells or their progeny to be distinguished. In order to monitor hematopoietic reconstitution in NOD mice we generated congenic NOD mice that carry the alternative allelic form of the pan-leukocyte alloantigen CD45. Following irradiation and congenic bone marrow transplantation, we found that the myeloid lineage was rapidly reconstituted by cells of donor origin but substantial numbers of recipient T lymphocytes persisted even after supra-lethal irradiation. This indicates that radiation resistance in the NOD hematopoietic compartment is a property primarily of mature T lymphocytes. (C) 2004 Elsevier Ltd. All rights reserved.

Structural change, balance-of-payments constraint, and economic growth: evidence from the multisectoral Thirlwall`s law

This paper contributes to the literature on balance-of-payments constrained growth by investigating how structural change identified with changes in the sectoral composition of exports and imports affects the external constraint We test both the original and a multisectoral version of Thirlwall`s law for a sample of Latin American and Asian countries The original Thirlwall s law is found to hold for all sample countries except South Korea, whereas the multisectoral analogue holds for all of them As the sectoral composition of exports and imports is found to matter for growth we analyze the evolution of each country`s weighted trade income elasticities

Import substitution and economic growth

Despite Latin America`s dismal performance between the 1950s and 1980s, the region experienced strong capital deepening. We suggest that these facts can be explained as a consequence of the restrictive trade regime adopted at that time. Our framework is based on a dynamic Heckscher-Ohlin model, with scale economies in the capital-intensive sector. Initially, the economy is open and produces only the labor-intensive good. The trade regime is modeled as a move to a closed economy. The model produces results consistent with the Latin American experience. Specifically, a Sufficiently small Country experiences no long-run income growth, but an increase in capital. (C) 2009 Elsevier B.V. All rights reserved.

Petroleum royalties and regional development in Brazil: The economic growth of recipient towns

In 1997, Brazil approved law n(cr) 9478, establishing new rules for sharing petroleum royalties with Brazilian municipalities. The goal of this paper is to evaluate whether royalties distributed under the new law have contributed for the development of benefited municipalities. For that the difference-indifferences estimator (diff-in-diff) is used, which compares the evolution of the economic product into the municipality affected by the new law with the unaffected ones, by exploring the new legislation as an exogenous change. The data refer to the municipal gross domestic product (GDP) growth rate before and after the event. Results are surprising, showing that royalty receivers grew less than municipalities that did not receive such resources. The difference is small but statistically significant. In general, an increase of one real in royalties per capita reduces the growth rate of the municipal product in 0.002 percentile points. (C) 2009 Elsevier Ltd. All rights reserved.

Salinity tolerance in Schinopsis quebracho colorado: Seed germination, growth, ion relations and metabolic responses

Soil salinity is a major abiotic stress influencing plant productivity worldwide. Schinopsis quebracho colorado is one of the most important woody species in the Gran Chaco, an and and salt-prone subtropical biome of South America. To gain a better understanding of the physiological mechanisms that allow plant establishment under salt conditions, germination and seedling growth of S. quebracho colorado were examined under treatment with a range of NaCl solutions (germination: 0-300 mmol l(-1) NaCl; seedling growth: 0-200 mmol l(-1) NaCl). The aim was to test the hypothesis that S. quebracho colorado is a glycophite that shows different salt tolerance responses with development stage. Proline content, total soluble carbohydrates and Na+, K+ and Cl- concentrations in leaves and roots of seedlings, and the chlorophyll concentration and relative water content of leaves were measured. Germination was not affected by 100 mmol l(-1) NaCl, but decreased at a concentration of 200 mmol l(-1). At 300 mmol l(-1) NaCl, germination did not occur. Seedling growth decreased drastically with increasing salinity. An increase in NaCl from 0 to 100 mmol l(-1) also significantly reduced the leaf relative water content by 22% and increased the proline concentration by 60% in roots. In contrast, total soluble carbohydrates were not significantly affected by salinity. Seedlings showed a sodium exclusion capacity, and there was an inverse correlation between Cl- concentration and the total chlorophyll concentration. S. quebracho colorado was more tolerant to salinity during germination than in the seedling phase. The results suggest that this increased tolerance during germination might, in part, be the result of lower sensitivity to high tissue Na+ concentrations. The significant increment of proline in the roots suggests the positive role of this amino acid as a compatible solute in balancing the accumulation of Na+ and Cl- as a result of salinity. These results help clarify the physiological mechanisms that allow establishment of S. quebracho colorado on salt-affected soils in arid and semi-arid Gran Chaco. (c) 2008 Elsevier Ltd. All rights reserved.

Calcium Carbonate Particle Growth Depending on Coupling among Adjacent Layers in Hybrid LB/LbL Films

There are practical and academic situations that justify the study of calcium carbonate crystallization and especially of systems that are associated with organic matrices and a confined medium. Despite the fact that many different matrices have been studied, the use of well-behaved, thin organic films may provide new knowledge about this system. In this work, we have studied the growth of calcium carbonate particles on well-defined organic matrices that were formed by layer-by-layer (LbL) polyelectrolyte films deposited on phospholipid Langmuir-Blodgett films (LB). We were able to change the surface electrical charge density of the LB films by changing the proportions of a negatively charged lipid, the sodium salt of dimyristoyl-sn-glycero-phosphatidyl acid (DMPA), and a zwitterionic lipid. dimyristoyl-sn-glycero-phosphatidylethanolamine (DMPE). This affects the subsequent polyelectrolyte LbL film deposition, which also changes the the nature of the bonding (electrostatic interaction or hydrogen bonding). This approach allowed for the formation of calcium carbonate particles of different final shapes, roughnesses, and sizes. The masses of deposited lipids, polyelectrolytes, and calcium cabonate were quantified by the quartz crystal microbalance technique. The structures of obtained particles were analyzed by scanning electron microscopy.

The effect of cholesterol on the reconstitution of alkaline phosphatase into liposomes

Tissue-nonspecific alkaline phosphatase (TNAP), present on the surface of chondrocyte- and osteoblast-derived matrix vesicles (MVs), plays key enzymatic functions during endochondral ossification. Many studies have shown that MVs are enriched in TNAP and also in cholesterol compared to the plasma membrane. Here we have studied the influence of cholesterol on the reconstitution of TNAP into dipalmitoylphosphatidylcholine (DPPC)-liposomes, monitoring the changes in lipid critical transition temperature (T(c)) and enthalpy variation (Delta H) using differential scanning calorimetry (DSC). DPPC-liposomes revealed a T(c) of 41.5 degrees C and Delta H of 7.63 Kcal mol(-1). The gradual increase in cholesterol concentration decrease Delta H values, reaching a Delta H of 0.87 Kcal mol(-1) for DPPC: cholesterol system with 36 mol% of cholesterol. An increase in T(c), up to 47 degrees C for the DPPC:cholesterol liposomes (36 mol% of Chol), resulted from the increase in the area per molecule in the gel phase. TNAP (0.02 mg/mL) reconstitution was done with protein:lipid 1:10,000 (molar ratio), resulting in 85% of the added enzyme being incorporated. The presence of cholesterol reduced the incorporation of TNAP to 42% of the added enzyme when a lipid composition of 36 mol% of Chol was used. Furthermore, the presence of TNAP in proteoliposomes resulted in a reduction in Delta H. The gradual proportional increase of cholesterol in liposomes results in broadening of the phase transition peak and eventually eliminates the cooperative gel-to-liquid-crystalline phase transition of phospholipids bilayers. Thus, the formation of microdomains may facilitate the clustering of enzymes and transporters known to be functional in MVs during endochondral ossification. (C) 2010 Elsevier B.V. All rights reserved.

A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells

The pathways involved in the maintenance of human embryonic stem (hES) cells remain largely unknown, although some signaling pathways have been identified in mouse embryonic stem (mES) cells. Fibroblast feeder layers are used to maintain the undifferentiated growth of hES cells and an examination of the conditioned media (CM) of human neonatal fibroblasts (HNFs) could provide insights into the maintenance of hES cells. The neonatal foreskin fibroblast line (HNF02) used in this study was shown to have a normal 2n = 46, XY chromosomal complement and to support the undifferentiated growth of the Embryonic Stem Cell International Pte. Ltd.-hES3 cell line. The CM of HNF02 was examined using two-dimensional liquid chromatography-tandem mass spectrometry (2-D LCMS) and two-dimensional electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (2-DE/MALDI). A total of 102 proteins were identified, 19 by 2-DE/MALDI, 53 by 2-D LCMS and 30 by both techniques. These proteins were classified into 15 functional groups. Proteins identified in the extracellular matrix and differentiation and growth factor functional categories were considered most likely to be involved in the maintenance of hES cell growth, differentiation and pluripotency as these groups contained proteins involved in a variety of events including cell adhesion, cell proliferation and inhibition of cell proliferation, Writ signaling and inhibition of bone morphogenetic proteins.