The S1(1A1)-S0(1A1) Electronic Transition of Jet-Cooled o-Difluorobenzene

Retinal pigment epithelium transplantation has been proposed as adjunctive treatment for age-related macular degeneration following surgical excision of choroidal neovascular membranes. The goal of this study was to develop a model to evaluate retinal pigment epithelium transplantation onto human Bruch's membrane in vitro. We investigated the ability of cultured fetal human retinal pigment epithelium to colonize human cadaver Bruch's membrane, determined the incubation time needed to form a monolayer and to exhibit apical microvilli and tight junctions, and assessed the production of basement membrane. Freshly enucleated (less than 48 hours old) human eyes were cut through the pars plana, and the anterior segment, vitreous, and retina were removed. The native retinal pigment epithelium was debrided with a surgical sponge. Bruch's membrane and choroid at the macula were trephined with a 7.0 mm diameter trephine and then incubated with 1/2 ml of Dulbecco's modified Eagle's medium +15% fetal calf serum+basic fibroblast growth factor (1 ng ml-1), and fetal human retinal pigment epithelium at a concentration of 242,000 cells ml-1. Specimens were incubated for 1, 4, 6, 8, 12, or 24 hours. The specimens were fixed in half strength Karnovsky's fixative, processed, and analysed with scanning and transmission electron microscopy. The retinal pigment epithelium covered the debrided macular specimens to different degrees at different incubation times. After 1 hour, the cells started to attach and flatten (median percent coverage: 78%). The extent of Bruch's membrane coverage by fetal retinal pigment epithelium varied greatly between specimens. After 4-6 hours, the cells covered the entire debrided surface in a monolayer (median percent coverage: 97.2% at 4 hours, 99.8% at 6 hours). Tight junctions were observed, and the cells had few apical microvilli. The lateral cell borders were obliquely oriented with respect to Bruch's membrane, and the nuclei were elongated, exhibited prominent nucleoli, and were oriented parallel to Bruch's membrane. After 6-8 hours, cells started to become hexagonal (median percent coverage at 8 hours: 99.97%). Cells attached to the inner collagenous layer tended to be flatter than cells attached to residual native basement membrane. At 12 and 24 hours, expression of hexagonal shape, tight junctions, and apical microvilli were observed more frequently (median percent coverage: 99.87% at 12 and 100% at 24 hours). No newly formed basement membrane was observed at these time points. In separate experiments comparing attachment in the presence and absence of native RPE basement membrane, the presence of native retinal pigment epithelial basement membrane promoted the early attachment of the cells and more rapid expression of normal morphology. This in vitro system provides a reproducible way to study the adherence of retinal pigment epithelium to normal and diseased human Bruch's membrane.     

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NF1 (neurofibromatosis type 1)

Several distinct Ras GTPase activating proteins (GAPs) from mammals, including Ras GAP of 120 kDa (GAP1) and NF1, stimulate the intrinsic GTPase activity of normal Ras, but not oncogenic Ras mutants (Trahey and McCormick, 1987). That is the reason why normal Ras remains predominantly in the inactive GDP-bound form (D-Ras), whereas oncogenic Ras remains constitutively in the active GTP-bound form (T-Ras). NF1 is a tumor suppressor of 2818 amino acids whose disruption or deletion causes brain tumors called neurofibromatosis type 1 by elevating the T-Ras level. T-Ras activates several distinct oncogenic effectors, including Ser/Thr kinase Raf, GAP1, P1-3 kinase, PKC-zeta and Ra1 GDS. Interestingly, the binding of T-Ras to either GAPs or these oncogenic effectors requires the same effector domain I (residues 32-40) of T-Ras molecule. In other words, these GAPs and effectors compete for binding to T-Ras. Using a series of N- and C-terminal deletion mutants of NF1, we identified a 78 amino acid fragment (NF78, residues 1441-1518) as the minimum GAP domain, and a 56 amino acid fragment (NF 56, residues 1441-1496) as the minimum Ras-binding domain. Furthermore, we identified the Raf fragment of 81 amino acids (Raf81, residues, 51-131) as the minimum Ras-binding domain with a high affinity. We found that (i) these NF1 fragments and Raf81 compete for binding to T-Ras, and that (ii) over-expression of these NF1 or Raf fragments strongly suppresses the malignant transformation caused by oncogenic Ras mutants. Thus, these agents offer a unique opportunity to control the proliferation of T-Ras-associated tumors that represent more than 30% of all human carcinomas including neurofibromatosis type 1.     

Modulation of JunD.AP-1 DNA binding activity by AP-1-associated factor 1 (AF-1)

AP-1-associated factor 1 (AF-1), is a novel protein complex that dramatically enhances the assembly of JunD-containing dimers onto AP-1 consensus sites. We describe the partial purification of AF-1 from nuclear extracts of the T-cell line MLA 144 by ionic, hydrophobic and gel filtration chromatography. AF-1 is a DNA-binding protein composed of low molecular mass polypeptides of 7-17 kDa that exists in solution as a 34-kDa complex. JunD interactions with DNA are accelerated in the presence of AF-1 through the formation of a true tri-molecular complex with JunD dimers and DNA that assembles much more rapidly on DNA than JunD alone. DNA binding analysis of AF-1 interaction with JunD.AP-1 and DNA shows that AF-1 increases the DNA binding affinity of JunD for AP-1 sites over 100-fold. DNA cleavage footprint analysis of isolated AF-1.JunD DNA complexes shows that the ternary complex makes nearly twice as many contacts with DNA than JunD dimers alone. AF-1 interacts readily, but differentially with Jun homodimers and Jun.Fos heterodimers. These findings distinguish AF-1 as a significant protein-specific modulator of AP-1.JunD in T-cells.     

MASP1 (MBL-associated serine protease 1)

The intracellular concentration of many steroids and xenobiotics is influenced by the membrane protein P-glycoprotein (Pgp). It has been inferred that the intracellular retention of many drugs that upregulate Pgp or modulate Pgp function might also be affected by Pgp. However, the ability of Pgp to influence the translocation of these drugs needs to be established to understand Pgp's influence upon their pharmacological effect. We utilized two approaches to determine the interaction of several agents with Pgp: (a) an in vitro system, LLC-PK1 cell lines and derivative LLC cell lines stably expressing on the apical membrane either mouse mdr1a or human MDR1 Pgp grown as polarized epithelium in transwell culture to measure translocation of radiolabeled drugs; and (b) an in vivo system, mdr1a nullizygous and wild-type animals, to compare the contribution of Pgp to in vivo distribution of radiolabeled drugs. In combination these complementary approaches identified erythromycin as a drug whose intracellular retention is influenced by Pgp, while the intracellular accumulation and tissue distribution of retinoic acid and benzo(a)pyrene were unaffected by Pgp.