Use of the ARPE-19 cell line as a model of RPE polarity: basolateral secretion of FGF5

PURPOSE: To determine the polarity of fibroblast growth factor 5 (FGF5) secretions from retinal pigment epithelium (RPE) cells and to examine the viability and utility of the ARPE-19 cell line as a model for the study of RPE polarity. METHODS: Influenza infection and adenovirus-mediated gene transfer were used to deliver and express genes encoding influenza hemagglutinin (HA), p75-NTR (a neurotrophin receptor), low-density lipoprotein (LDL) receptor (LDLR), and FGF5 in confluent monolayers of ARPE-19 cells. The localization of HA, p75-NTR, and LDLR was determined by confocal microscopy. Domain selective biotinylation assays were used to quantitatively determine the polarities of p75-NTR and LDLR. The secretion of FGF5 into the apical and basal media of ARPE-19 cultures was examined by immunoblot analysis of conditioned media. RESULTS: Hemagglutinin and p75-NTR were found to be localized on the apical surface of infected and transduced ARPE-19 cells. In contrast, LDLR was associated preferentially with the basolateral membrane of ARPE-19 cells. Biotinylation studies indicated that 84% of p75-NTR was present on the apical surface, and 79% of LDLR was basolaterally polarized. Over the course of 6 hours, more than 90% of the total secreted FGF5 protein accumulated in the basolateral media. CONCLUSIONS: ARPE-19 cells exhibit a polarized distribution of cell surface markers when examined by either confocal microscopy or surface-labeling assays. This indicates that the ARPE-19 cell line is a valid model for studies of RPE cell polarity. FGF5, a secreted protein normally produced by RPE cells, is accumulated preferentially in the basal media after only 6 hours, suggesting that it is vectorially secreted from the basolateral surface of ARPE-19 cells.     

Attenuated hematopoietic response to granulocyte-macrophage colony-stimulating factor in patients with acquired pulmonary alveolar proteinosis

The pathogenesis of acquired pulmonary alveolar proteinosis (PAP), a rare lung disease characterized by excessive surfactant accumulation within the alveolar space, remains obscure. Gene-targeted mice lacking the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) or the signal-transducing beta-common chain of the GM-CSF receptor have impaired surfactant clearance and pulmonary pathology resembling human PAP. We therefore investigated the hematopoietic effects of GM-CSF in patients with PAP. The hematologic response of 5 infants with congenital PAP to 5 microgram/kg/d was of normal magnitude. By contrast, despite normal expression of GM-CSF receptor alpha- and beta-common chains on peripheral blood myelomonocytic cells (n = 6) and normal binding affinity of bone marrow mononuclear cells for GM-CSF (n = 3), each of the 12 patients with acquired PAP treated displayed impaired responses to GM-CSF; 5 microgram/kg/d produced only minor eosinophilia, and doses of 7.5 to 20 microgram/kg were required to induce >/=1.5-fold neutrophil increments in the 3 patients who underwent dose-escalation. However, neutrophilic responses to 5 microgram/kg granulocyte colony-stimulating factor (G-CSF) were normal (n = 4). In vitro, the proportion of hematopoietic progenitors responsive to GM-CSF (16.1% +/- 8.9%; P = .042) or interleukin-3 (IL-3; 19.3% +/- 7.7%; P = .063), both of which utilize the beta-common chain of the GM-CSF receptor complex, were reduced among patients with acquired PAP (n = 4) compared with normal bone marrow donor controls (47.2% +/- 25.9% and 40.9% +/- 18.6%, respectively). In the one individual who had complete resolution of lung disease during the period of study, this was temporally associated with correction of this defective in vitro response to GM-CSF and IL-3 on serial assessment. These data establish that patients with acquired PAP have an associated impaired responsiveness to GM-CSF that is potentially pathogenic in the development of their lung disease. Based on these observations, we propose a model of the pathogenesis of acquired PAP that suggests the disease arises as a consequence of an acquired clonal disorder within the hematopoietic progenitor cell compartment.     

Cytomegalovirus retinitis and viral resistance: 3. Culture results. CMV Retinitis and Viral Resistance Study Group

PURPOSE: The Strong Heart Study is a study of cardiovascular disease and its risk factors among American Indian men and women aged 45-74 yr representative of 13 communities from Arizona (AZ), Oklahoma (OK), and North/South Dakota (N/SD). This investigation sought to characterize the amount and type of physical activity and to determine the association between activity and lipids in this population. METHODS: Total physical activity (occupational plus leisure) was assessed with a validated questionnaire. RESULTS: Men and women from OK (21 +/- 19 and 16 +/- 15 h.wk-1; respectively) and N/SD (23 +/- 21 and 17 +/- 17 h.wk-1; respectively) had activity levels that were similar if not lower than the U.S. population with the AZ communities (17 +/- 21 and 10 +/- 14 h.wk-1; respectively) being substantially lower than the other two communities. Total cholesterol (TC), high density lipoprotein cholesterol (HDL-c), and low density lipoprotein cholesterol (LDL-c) levels were lower than the U.S. population. CONCLUSIONS: For most of the population (diabetic men and nondiabetic men and women), activity was significantly associated (P < 0.05) with apolipoprotein (apo) AI after controlling for covariates. With levels of activity as low if not lower than the general U.S. population coupled with high prevalence of obesity and diabetes, efforts to increase physical activity in American Indians are warranted. Hopefully these increases in activity will result in favorable lipid changes as well as decreasing the risk of diabetes which is epidemic in these populations.     

Cyclosporin A in patients receiving renal allografts from cadaver donors. 1978

A psychrotrophic yeast, Rhodotorula glutinis KUJ 2731, isolated from soil, effectively produced an extracellular endo-beta-glucanase (EC 3.2.1.4). The enzyme was monomeric, and the molecular mass was about 40,000 Da. The N-terminal amino acid sequence was H-Ser-Leu-Pro- Lys-Leu-Gly-Gly-Val-Asp-Leu-Ala-Gly-Leu-Asp-Ile-Gly-Lys-Asp-Lys-Asn-. alpha-Helix content was calculated to be about 32.6%. The isoelectric point was 8.57. The activation energy was 20.9 kJ/mol, which was much smaller than that of mesophilic enzymes. The enzyme was active at temperatures from 0 to 70 degrees C, with a highest initial velocity at 50 degrees C similar to other psychrotrophic enzymes. The enzyme was inhibited by Hg2+. The enzyme catalyzed hydrolysis of carboxymethyl cellulose with an apparent K(m) of 1.1% and Vmax of 556 mumol/min/mg. Products from the enzymatic hydrolysis of carboxymethyl cellulose by the enzyme were glucose, cellobiose, and cellotriose. The enzyme also catalyzed the transglycosylation of p-nitrophenyl-beta-cellotrioside to cellotetraose.     

Physical properties of oleochemical carbonates

Carbonates are a class of compounds that have recently found increasing interest in commercial applications owing to their physical properties and relatively straightforward synthesis. In this work, physical and fuel properties of five straight-chain C_17–39 and three branched C_17–33 oleochemical carbonates were investigated. These properties included cetane number (CN), low-temperature properties, (kinematic) viscosity, lubricity, and surface tension. The carbonates studied had CN ranging from 47 to 107 depending on carbon chain length and branching. For the same number of carbons, the CN of carbonates were lower than those of FA alkyl esters owing to interruption of the CH₂ chain by the carbonate moiety. Kinematic viscosities at 40°C ranged from 4.9 to 22.6 mm²/s whereas m.p. ranged from +3 to below −50°C depending on the carbonate structure. High-frequency reciprocating rig testing showed the neat carbonates to have acceptable lubricity that improved as chain length increased. Finally, the carbonate's ability to influence cold-flow properties in biodiesel (methyl soyate) and lubricity in low-lubricity ultra-low sulfur diesel were examined. The carbonates studied did not significantly affect cold flow or lubricity properties at concentrations up to 10,000 ppm (1 wt%). The properties of the carbonates resemble those of fatty alkyl esters with similar trends resulting from compound structure.     

Low-temperature properties of triglyceride-based diesel fuels: Transesterified methyl esters and petroleum middle distillate/ester blends

This work examines low-temperature properties of triglyceride-based alternate fuels for direct-injection compression-ignition engines. Methyl esters from transesterified soybean oil were studied as neat fuels and in blends with petroleum middle distillates (No. 1 or No. 2 diesel fuel). Admixed methyl esters composed of 5–30 vol% tallowate methyl esters in soyate methyl esters were also examined. Pour points, cloud points, and kinematic viscosities were measured; viscosities at cooler temperatures were studied to evaluate effects of sustained exposure. Low-temperature filterability studies were conducted in accordance with two standard methodologies. The North American standard was the low-temperature flow test (LTFT), and its European equivalent was the cold-filter plugging point (CFPP). With respect to cold-flow properties, blending methyl esters with middle distillates is limited to relatively low ester contents before the properties become preclusive. Under most conditions, cold-flow properties were not greatly affected by admixing the methyl esters with up to 30 vol% tallowate (before blending). Least squares analysis showed that both LTFT and CFPP of formulations containing at least 10 vol% methyl esters are linear functions of cloud point. In addition, statistical analysis of the LTFT data showed a strong 1:1 correlation between LTFT and CP. This result may prove crucial in efforts to improve low-temperature flow properties of alternate diesel fuels that contain methyl esters derived from triglycerides.     

Oxidative stability of soybean oil fatty acid methyl esters by oil stability index (OSI)

Biodiesel, an alternative diesel fuel derived from transesterification of vegetable oils or animal fats, is composed of saturated and unsaturated long-chain FA alkyl esters. During long-term storage, oxidation caused by contact with air (autoxidation) presents a legitimate concern with respect to monitoring and maintaining fuel quality. Extensive oxidative degradation may compromise quality by adversely affecting kinematic viscosity, acid value, or PV. This work examines the oil stability index (OSI) as a parameter for monitoring the oxidative stability of soybean oil FAME (SME). SME samples from five separate sources and with varying storage and handling histories were analyzed for OSI at 60°C using an oxidative stability instrument. Results showed that OSI may be used to measure relative oxidative stability of SME samples as well as to differentiate between samples from different producers. Although addition of α-tocopherol or TBHQ increased OSI, responses to these antioxidants varied with respect to SME sample. Variations in response to added antioxidant were attributed to aging and other effects that may have caused oxidative degradation in samples prior to acquisition for this study. Results showed that OSI was more sensitive than iodine value in detecting the effects of oxidative degradation in its early stages when monitoring SME during storage.     

Dependence of oil stability index of fatty compounds on their structure and concentration and presence of metals

During storage and use, vegetable oil-derived industrial products such as biodiesel and biodegradable lubricants can be subjected to conditions that promote oxidation of their unsaturated components. The materials arising during oxidation and subsequent degradation can seriously impair the quality and performance of such products. Therefore, oxidative stability is a significant issue facing these vegetable oil-derived products, and enhanced understanding of the influence of various components of vegetable oils and storage parameters is necessary. In this work, the oil stability index (OSI) was used for assessing oxidation of monoalkyl esters of FA by varying several parameters. Neat fatty compounds and prepared mixtures thereof were studied for assessing the influence of compound structure and concentration. Small amounts of more highly unsaturated compounds had a disproportionately strong effect on oxidative stability. The recently developed concept of bis-allylic equivalents correlated more closely than the iodine value with the OSI times of mixtures of fatty esters. The OSI times of free acids were shorter than those of the corresponding alkyl esters. The presence of copper, iron, and nickel also reduced oxidative stability, but their effect was less than the presence of more highly unsaturated fatty compounds. Of these metals, copper had the strongest catalytic effect on OSI time. OSI may be an alternative to long-term storage tests for determining the influence of extraneous materials such as metals on oxidative stability.     

The stress cracking of polyamides by metal salts. Part III. Metal thiocyanates

The stress cracking of polyamide (nylon 6) by a number of metal thiocyanates in aqueous and nonaqueous solutions has been investigated. Lithium, zinc and cobalt_II thiocyanates were the most active and their activity was compared with that of the corresponding metal halides. Stress cracking parameters were determined and the mechanism of cracking studied by infrared techniques. The action of metal thiocyanates on nylon 6 is similar to that of the corresponding metal halides. Some metal cobaltothiocyanates were also found to be active stress cracking agents.     

Design and characterization of a multistage,mechanically stirred column absorber

A multistage, mechanically stirred column absorber has been designed and built with a modular construction, based on preliminary experiments with a test column. The column has been characterized as a gas-liquid contactor by its gas holdup, gas and liquid axial dispersion, mixing times, oxygen transfer coefficients and power consumptions, determined as a function of gas velocity, liquid velocity and impeller speed for one and two impellers per stage.Gassed power was correlated with ungassed power, gas rate and impeller speed. The gas phase axial mixing was essentially plug flow and the liquid phase axial mixing varied between 5 and 12 equivalent stages.Oxygen transfer coefficients were correlated with power consumptions and aeration rates by the equation K_La γ (P/V)^a(υ_sg)^b. The oxygen transfer coefficients with single stiffer stages were 25% above those for the double stirrer stages for equal power consumption and gas rates. Except for the low aeration and high power consumption extremes, the column showed superior oxygen transfer performance. in comparison to tubular loop and tank fermenters.