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.     

Pre-incision infiltration with lidocaine reduces pain and opioid consumption after reduction mammoplasty

BACKGROUND: The main purpose of the reconstruction of the cranium is the protection of the brain. Besides we have to consider important functional and aesthetic necessities in order to achieve satisfactory results. METHODS: Thirty-six clinical cases, operated from November 1991 to June 1996, in which the reconstruction of the cranial vault is carried out by a polymethylmethacrylate acrylic resin are analysed. The causes and locations of the most common bone defects and the main indications for reconstruction are examined. While the repair of the osseous gaps caused by neoplasms is immediate, in the traumatic occurrences, in order to reduce the probability of infectious complications, an average time of 11 months elapsed from the first operation. The surgical technique, with slightest alterations, is the same in all the presented cases, preparing the acrylic resin straight on the operating table. The resin, moulded and adapted to the defect until its complete hardening, presents, thanks to its properties, manifold advantages (and few real disadvantages). RESULTS: The results, in terms of complications, are very satisfactory, with an infectious rate of 2.7%. Besides, in one third of the patients, a considerable clinical improvement after the repair has been observed. CONCLUSIONS: According to personal experience, it is possible to affirm that polymethylmethacrylate, with its remarkable plasticity and stability in time, can always guarantee a satisfactory functional and aesthetic result.     

Potent piperazine hydroxyethylamine HIV protease inhibitors containing novel P3 ligands

The 2-isopropyl thiazolyl group is a highly optimized P3 ligand for C2 symmetry-based HIV protease inhibitors, as exemplified in the drug ritonavir. Here we report that incorporation of this P3 ligand into a piperazine hydroxyethylamine series also yielded novel, highly potent inhibitors. In tissue culture assays, the presence of human serum was less deleterious to the activity of these inhibitors than to that of ritonavir. Furthermore, potent activity against ritonavir resistant HIV was observed.     

Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment

In a pilot study to establish fetal nucleated red blood cell (NRBC) detection in maternal blood, trisomy 13 was diagnosed by FISH analysis at 11 weeks' gestation. The NRBCs were detected after a single-step ficoll density gradient enrichment. In blood samples taken both before and after CVS, 52 and 80 NRBCs, respectively, were found to be positive for fetal haemoglobin. In 47 per cent of these cells, FISH analysis for X and Y chromosomes confirmed the fetal sex. Moreover, 48 per cent of these NRBCs showed three fluorescent signals for a chromosome 13 probe, which confirmed the diagnosis of trisomy 13, previously detected at CVS karyotyping. This is the first report of non-invasive prenatal diagnosis of trisomy 13, i.e., pre-CVS, in the first trimester. The high number of fetal NRBCs detected indicates a connection with aneuploidy, probably due to early impairment of the feto-maternal barrier.     

Development of novel, potent, and selective dopamine reuptake inhibitors through alteration of the piperazine ring of 1-[2-(diphenylmethoxy)ethyl]-and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazines (GBR 12935 and GBR 12909)

The design, synthesis, and biological evaluation of compounds related to the dopamine (DA) uptake inhibitors: 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (1) and 1-[2-[bis-(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (2) (GBR 12395 and GBR 12909, respectively), directed toward the development and identification of new ligands interacting with high potency and selectivity at the dopamine transporter (DAT) is reported. The substitution of the piperazine ring in the GBR structure with other diamine moieties resulted in the retention of the high affinity of new ligands for the DAT. Some of the modified GBR analogs (e.g. 8, 10, (-)-49, or (-)-50) displayed substantially higher selectivity (4736- to 693-fold) for the dopamine (DA) versus the serotonin (5HT) reuptake site than the parent compounds. The bis(p-fluoro) substitution in the (diphenylmethoxy)ethyl fragment slightly increased the affinity of the ligands at the DA reuptake site but reduced their selectivity at this site (e.g. 9 and 8, 11 and 10, or 17 and 16, respectively). Congeners, such as the series of monosubstituted and symmetrically disubstituted piperazines and trans-2,5-dimethylpiperazines, which lack the (diphenylmethoxy)ethyl substituent lost the affinity for the DAT yet exhibited very high potency for binding to the sigma receptors (e.g.28). The chiral pyrrolidine derivatives of 1, (-)-49, and (+)-49, exhibited an enantioselectivity ratio of 181 and 146 for the inhibition of DA reuptake and binding to the DAT, respectively.     

A block copolymer nanotemplate for mechanically tunable polarized emission from a conjugated polymer

A polymer blend system consisting of polystyrene grafted onto poly (p-phenylene ethynylene) (PS-g-PPE) and poly (styrene-block-isoprene-block-styrene) triblock copolymer (SIS) yields highly polarized emission due to the unidirectional alignment of the PPE molecules. During the roll casting, the triblock copolymer microphase separates and creates unidirectionally aligned PS cylindrical microdomains in the rubbery PI matrix. PPE, a fluorescent conjugated polymer, was grafted with polystyrene (PS) side chains that enabled sequestration and alignment of these rigid backbone emitter molecules into the PS microdomains of the SIS triblock copolymer. Deforming the thermoplastic elastomer in a direction perpendicular to the orientation direction of the cylinders causes rotation of the PS cylinders and the PPE emitter molecules and affords tunable polarized emission due to re-orientation of the PPE containing PS cylinders as well as film thinning from Poisson effect.     

Sulfur-Tolerant NOx Storage Traps: An Infrared and Thermodynamic Study of the Reactions of Alkali and Alkaline-Earth Metal Sulfates

The sulfur tolerance of a barium-containing NO_x storage/reduction trap was investigated using infrared analysis. It was confirmed that barium carbonate could be replaced by barium sulfate by reaction with low concentrations of sulfur dioxide (50 ppm) in the presence of large concentrations of carbon dioxide (10%) at temperatures up to 700 °C. These sulfates could at least be partially removed by switching to hydrogen-rich conditions at elevated temperatures. Thermodynamic calculations were used to evaluate the effects of gas composition and temperature on the various reactions of barium sulfate and carbonate under oxidizing and reducing conditions. These calculations clearly showed that if, under a hydrogen-rich atmosphere, carbon dioxide is included as a reactant and barium carbonate as a product then barium sulfate can be removed by reaction with carbon dioxide at a much lower temperature than is possible by decomposition to barium oxide. It was also found that if hydrogen sulfide was included as a product of decomposition of barium sulfate instead of sulfur dioxide then the temperature of reaction could be significantly lowered. Similar calculations were conducted using a selection of other alkaline-earth and alkali metals. In this case calculations were simulated in a gas mixture containing carbon monoxide, hydrogen and carbon dioxide with partial pressures similar to those encountered in real exhausts during switches to rich conditions. The results indicated that there are metals such as lithium and strontium with less stable sulfates than barium, which may also possess sufficient NO_x storage capacity to give sulfur-tolerant NO_x traps.     

Domain structure of the Fib-1 and Fib-2 regions of human fibronectin. Thermodynamic properties of the type I finger module

The stability of the Fib-1 (29 kDa) and Fib-2 (19 kDa) fragments of human fibronectin as well as several different subfragments and isolated type I "finger" modules were studied under various solvent conditions by differential scanning calorimetry and fluorescence spectroscopy. It was established that all fibronectin fingers constitute independently folded domains whose melting temperatures range from 54 to 108 degrees C. The difference between heat capacities of the native and denatured states (delta Cp) is low, about 0.03 cal/K-g, which is consistent with the relatively low percentage of hydrophobic amino acids and the consequent small change in non-polar surface area exposed to the solvent upon denaturation. The free energy of unfolding at 25 degrees, as calculated from the calorimetric data or measured directly by titration with GdmSCN is also small, in the range of 2.4 to 6.7 kcal/mol. The small delta G value and its flat dependence on temperature (determined by delta Cp) translates the small variations in delta G between fingers into large variations in tm. The small value of delta G also indicates that finger modules are structurally rather fragile which may account for their sensitivity to proteolysis; almost any cleavage within either of the two disulfide loops destroys the cooperative structure and abolishes the corresponding melting transition. The fact that some fingers exhibit large decreases in tm upon separation from more stable neighbors with which they interact can also be viewed as a consequence of the low values of delta G and delta Cp.     

Flap prefabrication: effectiveness of different vascular carriers

A new experimental model of a vascular carrier to prefabricate a "secondary" island flap, the popliteal musculovascular pedicle, was developed in the rat. Using quantitative skin-surface fluorometry 30 minutes after sodium fluorescein injection and a flap survival area in the prefabricated 8 x 2.5-cm abdominal composite island flap, we compared the revascularization ability of our muscular carrier to nonrevascularized controls: the skeletonized arteriovenous pedicle and the fasciovascular pedicle. The free composite graft with no vascular carrier exhibited near-total necrosis. The skeletonized vascular pedicle demonstrated 15.2% +/- 7.8% perfusion of normal skin on dye fluorescence index measurements and 50% flap survival. The fasciovascular pedicle exhibited better revascularization, with a dye fluorescence index of 36.2 +/- 15.5 (p < 0.01) and 90% +/- 10% flap survival (p < 0.001). India ink injection study and histological examination of our model provided visual evidence of revascularization from the musculovascular pedicle, along with preservation of the carrier's muscular architecture. The musculovascular pedicle is a reliable carrier for making new, vascularized composite flaps.