Investigation of blood flow regulation and oxygen saturation of the retinal vessels in primary open-angle glaucoma.

PURPOSE: To examine the supply of oxygen to the retina in primary open-angle glaucoma (POAG). METHODS: Forty-one patients with primary open-angle glaucoma (mean age 64.1 ± 12.9 years) and 40 healthy subjects (63.6 ± 14.1 years) were included. Fundus images, centered at the optic disc, were taken using the Retinal Vessel Analyzer (RVA). The vessel diameters were calculated as central retinal artery (CRAE) and vein equivalent (CRVE) from diameter measurements in the peripapillary vessels. The oxygen saturation of the arteries and veins was investigated employing a two-wavelengths technique. After the measurement at baseline, the vascular response to flicker light exposure was measured. RESULTS: In glaucoma patients the mean oxygen saturation of the retinal veins at baseline was higher than in the healthy controls (64.36 ± 7.11 vs. 59.78 ± 8.47, p = 0.01), whereas the mean arteriovenous oxygen saturation difference was lower (33.07 ± 5.24 vs. 37.53 ± 6.95, p = 0.002). The arterial oxygen saturation as well as the arterial and venous diameters showed no difference between the groups. The increase of the CRVE during flicker light stimulation (3.72 ± 3.29 % vs. 5.43 ± 4.04, p = 0.039), as well as the change of the venous oxygen saturation (2.08 ± 3.74 % vs. 4.18 ± 3.88 %, p = 0.016) and the arteriovenous saturation difference (-2.1 ± 3.31 % vs. -4.43 ± 3.6 %, p = 0.003) were smaller in POAG patients than in the healthy group. CONCLUSIONS: The reduction in the arteriovenous difference in oxygen saturation in POAG patients might show a decreased oxygen demand of the retina caused by the glaucomatous loss of neuroretinal tissue. The lower extent of the flicker light-induced change of the diameter of retinal veins and the venous oxygen saturation could indicate an impairment of blood flow regulation.

Structural analysis of FeO(1 1 1)/Ag(0 0 1): undulation of hexagonal oxide monolayers due to square lattice metal substrates.

Iron oxide monolayers are grown on Ag(0 0 1) via reactive molecular beam epitaxy (metal deposition in oxygen atmosphere). The monolayer shows FeO stoichiometry as concluded from x-ray photoemission spectra. Both low energy electron diffraction as well as scanning tunneling microscopy demonstrate that the FeO layer has a quasi-hexagonal (1 1 1) structure although deposited on a surface with square symmetry. Compared to bulk values, the FeO(1 1 1) monolayer is unidirectionally expanded by 3.4% in [Formula: see text] directions while bulk values are maintained in [Formula: see text] directions. In [Formula: see text] directions, this lattice mismatch between FeO(1 1 1) monolayer and Ag(0 0 1) causes a commensurate undulation of the FeO monolayer where 18 atomic rows of the FeO(1 1 1) monolayer match 17 atomic rows of the Ag(0 0 1) substrate. In [Formula: see text] directions, however, the FeO(1 1 1) monolayer has an incommensurate structure.

[Study on the time course of macular pigment density measurement in patients with a macular hole--clinical course and impact of surgery]. / Untersuchungen zum zeitlichen Verlauf der Makulapigmentdichte bei Patienten mit idiopathischem Makulaforamen--klinischer Verlauf und Einfluss der Operation.

BACKGROUND: In a prospective clinical study we examined the course of the optical density of the macular pigment (MPOD) in patients with idiopathic macular holes before and after pars-plana vitrectomy. PATIENTS AND METHODS: Of the 26 patients four had macular holes stage 1, six patients had stage 2 or stage 4 holes and ten people had a macular hole stage 3. The surgical procedure consisted always of a standard 3-port pars plana vitrectomy (PPV) with colouring assisted peeling of the internal limiting membrane. The optical density of macular pigment was determined by the 1-wavelength reflection method before pars plana vitrectomy and after macular hole closure. RESULTS: The macular hole was completely closed in 25 of 26 eyes (96.15 %). This anatomical success is also the basis for a postoperative improvement in the retinal function. In the case of macular holes from stages 3 and 4 the surgery achieved an increase in visual acuity and an increase of the optical density of the the macular pigment. In patients with macular holes stage 2 the visual acuity and the optical density of the macular pigment were reduced after vitrectomy. In the case of stage 1 holes the visual acuity and the volume of macular pigment also were reduced after the operation. DISCUSSION: In the group of stage 4 macular holes there was a significant increase in maximum optical density and volume of macular pigment after successful surgical hole closure, so the functional profit of PPV is very high in these patients. Even in the case of macular holes of stage 3 the PPV effected a functional improvement in the sense of an increase of the optical density of macular pigment. The increase in volume proved to be significant. For macular holes stage 2 the vitrectomy as a therapy option is generally recognised but PPV does not effect positively the height of the optical density of macular pigment. In these patients both the maximum optical density as well as the volume of macular pigment were reduced in the therapeutic course. Whether surgical intervention is indicated in stage 1 is still controversial. Taking into account the development of MPOD the profit from surgery for the patients is very low. This led to a decrease in volume of macular pigment and only a slight increase in maximum optical density after pars plana vitrectomy.

[Time-resolved autofluorescence in retinal vascular occlusions]. / Zeitaufgelöste Autofluoreszenz bei retinalen Gefäßverschlüssen.

BACKGROUND: Cellular metabolism can be evaluated using time-resolved autofluorescence. Because the fluorescence of ocular tissue is an accumulation of the fluorescence of several endogenous fluorophores, it is hard to determine the influence of a single fluorophore. In branch retinal artery occlusion, metabolic changes can be compared with normal tissue. METHOD: Time-resolved autofluorescence was measured in two patients in two spectral channels, K1 (490-560 nm) and K2 (560-700 nm), and was 3-exponentially approximated and compared with representative results of a healthy eye. RESULTS: In K1, lifetime τ1 in the undersupplied tissue was weak, but τ2 was strongly elongated compared with the healthy tissue. In K2, the distribution of τ2 was identical in both tissues. In the healthy eye, there was an equal distribution of all lifetimes in corresponding fundus regions. CONCLUSIONS: The elongation of τ1 in undersupplied tissue is probably caused by a reduced contribution of protein-bound FAD. The elongation of τ2 (about 500 ps) in healthy tissue, compared to about 1.5 ns in undersupplied tissue, is probably caused by protein-bound NADH, which is formed in glycolysis.

[Comparison of parameters of time-resolved autofluorescence between healthy subjects and patients suffering from early AMD]. / Vergleich von Parametern der zeitaufgelösten Autofluoreszenz bei Gesunden und Patienten mit früher AMD.

BACKGROUND: A fluorescence lifetime mapper (FLM) was tested for quantitative estimation of early alterations in age-related macular degeneration (AMD) which are assumed to be in cellular metabolism. METHOD: In FLM time-resolved autofluorescence of the fundus is excited by picosecond (ps) laser impulses at 448 nm and detected in 2 spectral ranges (K1=490-560 nm and K2=560-700 nm) by time-correlated single photon counting. The time-dependent decrease in fluorescence intensity was approximated using 3 decay rates. The calculated lifetimes allow a comparison with endogenous fluorophores of cellular metabolism. RESULTS: Initially mean lifetimes were determined for 8 healthy subjects (K1: tau1=118 ps, tau2=584 ps, tau3=2826 ps, K2: tau1=104 ps, tau2=477 ps, tau3=1623 ps). In 15 AMD patients (AREDS categories I and II) the lifetimes were longer (K1: tau1=166 ps, tau2=986 ps, tau3=3309 ps, K2: tau1=137 ps, tau2=583 ps, tau3=1924 ps). The best separation between healthy subjects and patients with early AMD was possible by parameters 1 and 2 in the short-wave channel. Fluorophore-specific alterations in the macula could be demonstrated in isolated cases with advanced AMD. CONCLUSION: Measurements in the 30 degrees fundus field demonstrated that specific alterations were already present even in early AMD and also outside the macula. These act in the neuronal retina, because parameter tau2 is related to this layer. Increases in the lifetime of parameter tau2 in the short wave channel could at least partially be determined by an increase of protein bound NADH, the content of which increases with reduced cellular respiration.

Regulation of apoptosis and cytokinesis by the anti-apoptotic E2/E3 ubiquitin-ligase BRUCE.

Members of the inhibitor of apoptosis protein (IAP) family are key regulators of apoptosis as they bind and inhibit caspases and other pro-apoptotic factors. Recent findings suggest that these proteins play additional roles, e.g., in cell cycle regulation, angiogenesis, and carcinogenesis. Here, we review the function of BRUCE (BIR repeat-containing ubiquitin-conjugating enzyme), an unusual 528-kDa IAP with ubiquitin ligase activity, and describe its role in apoptosis and cytokinesis. Additionally, we discuss how these seemingly unrelated functions might be linked.

Towards metabolic mapping of the human retina.

Functional alterations are first signs of a starting pathological process. A device that measures parameter for the characterization of the metabolism at the human eye-ground would be a helpful tool for early diagnostics in stages when alterations are yet reversible. Measurements of blood flow and of oxygen saturation are necessary but not sufficient. The new technique of auto-fluorescence lifetime measurement (FLIM) opens in combination with selected excitation and emission ranges the possibility for metabolic mapping. FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Because of its high sensitivity and high temporal resolution, its capability to resolve multi-exponential decay functions, and its easy combination with laser scanner ophthalmoscopy, multi-dimensional time-correlated single photon counting was used for fundus imaging. An optimized set up for in vivo lifetime measurements at the human eye-ground will be explained. In this, the fundus fluorescence is excited at 446 or 468 nm and the time-resolved autofluorescence is detected in two spectral ranges between 510 and 560 nm as well as between 560 and 700 nm simultaneously. Exciting the fundus at 446 nm, several fluorescence maxima of lifetime t1 were detected between 100 and 220 ps in lifetime histograms of 40 degrees fundus images. In contrast, excitation at 468 nm results in a single maximum of lifetime t1 = 190 +/- 16 ps. Several fundus layers contribute to the fluorescence intensity in the short-wave emission range 510-560 nm. In contrast, the fluorescence intensity in the long-wave emission range between 560 and 700 nm is dominated by the fluorescence of lipofuscin in the retinal pigment epithelium. Comparing the lateral distribution of parameters of a tri-exponential model function in lifetime images of the fundus with the layered anatomical fundus structure, the shortest component (t1 = 190 ps) originates from the retinal pigment epithelium and the second lifetime (t2 = 1,000 ps) from the neural retina. The lifetime t3 approximately 5.5 ns might be influenced by the long decay of the fluorescence in the crystalline lens. In vitro analysis of the spectral properties of expected fluorophores under the condition of the living eye lightens the interpretation of in vivo measurements. Taking into account the transmission of the ocular media, the excitation of NADH is unlikely at the fundus.

Analyses of phagocytosis, evoked oxidative burst, and killing of black yeasts by human neutrophils: a tool for estimating their pathogenicity?

The pathogenicity of several dematiaceous yeasts that have, to date, rarely been isolated in humans remains unclear. Because professional phagocytes are prominent in lesions caused by dematiaceous fungi, we address this issue by comparing phagocytosis, evoked oxidative burst and killing by human neutrophils of different black yeasts in vitro. Whereas phagocytosis of all black yeasts tested and evoked oxidative burst yielded comparable results, in contrast, the degree of killing differed significantly after 5 h. Thereby, two groups could be identified; one in which strains are killed at high rates, for example, Hortaea werneckii (81 +/- 11.6%), Exophiala castellanii (96 +/- 8.6%), Phaeoannellomyces elegans (93 +/- 9.7%), Phaeococcomyces exophialae (87 +/- 8.7%), and the other in which strains are killed to a lesser degree, for example, Exophiala dermatitidis (ATCC 34100) (61 +/- 9.5%), E. dermatitidis (CBS 207.35) (66 +/- 7.5%), E. jeanselmei (50 +/- 10.5%), E. mesophila (63 +/- 11.6%), E. bergeri (63 +/- 9.1%), and E. spinifera (57 +/- 9.6%). Non-pigmented yeasts were killed at levels comparable with those at which the white mutant strain of E. dermatitidis (ATCC 44504) was killed (95 +/- 7.5%); the yeast strains tested were Candida albicans (DSM 11943) (95 +/- 4.0% killing) and Saccharomyces cerevisiae (DSM 1333) (95 +/- 10.3%). Comparison of killing rates with the observed pathogenicity of the melanized species suggests that low killing rates might indicate or even predict a high degree of invasiveness. Although previous experiments revealed that melanization conferred killing resistance on E. dermatitidis, the differences in killing rates of other dematious fungi suggest that melanization of the cell wall is in itself insufficient to confer virulence.

Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone.

The OLE pathway of yeast regulates the level of the ER-bound enzyme Delta9-fatty acid desaturase OLE1, thereby controlling membrane fluidity. A central component of this regulon is the transcription factor SPT23, a homolog of mammalian NF-kappaB. SPT23 is synthesized as an inactive, ER membrane-anchored precursor that is activated by regulated ubiquitin/proteasome-dependent processing (RUP). We now show that SPT23 dimerizes prior to processing and that the processed molecule, p90, retains its ubiquitin modification and initially remains tethered to its unprocessed, membrane-bound SPT23 partner. Subsequently, p90 is liberated from its partner for nuclear targeting by the activity of the chaperone-like CDC48(UFD1/NPL4) complex. Remarkably, this enzyme binds preferentially ubiquitinated substrates, suggesting that CDC48(UFD1/NPL4) is qualified to selectively remove ubiquitin conjugates from protein complexes.

Membrane-bound transcription factors: regulated release by RIP or RUP.

Regulated nuclear transport of transcription factors from cytoplasmic pools is a major route by which eukaryotes control gene expression. Exquisite examples are transcription factors that are kept in a dormant state in the cytosol by membrane anchors; such proteins are released from membranes by proteolytic cleavage, which enables these transcription factors to enter the nucleus. Cleavage can be mediated either by regulated intramembrane proteolysis (RIP) catalysed by specific membrane-bound proteases or by regulated ubiquitin/proteasome-dependent processing (RUP). In both cases processing can be controlled by cues that originate at or in the vicinity of the membrane.

SUMO, ubiquitin's mysterious cousin.

Covalent modification of cellular proteins by the ubiquitin-like modifier SUMO regulates various cellular processes, such as nuclear transport, signal transduction, stress response and cell-cycle progression. But, in contrast to ubiquitylation, sumoylation does not tag proteins for degradation, but seems to enhance their stability or modulate their subcellular compartmentalization.

Molecular cloning and characterization of WdPKS1, a gene involved in dihydroxynaphthalene melanin biosynthesis and virulence in Wangiella (Exophiala) dermatitidis.

1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of Wangiella dermatitidis, an agent of phaeohyphomycosis in humans. To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene, WdPKS1, in W. dermatitidis. The resulting wdpks1Delta disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intact WdPKS1 gene was then successfully recovered from two plasmids. The WdPKS1 gene was also isolated independently by complementation of the mel3 mutation in an albino mutant of W. dermatitidis using a cosmid library. Sequence analysis substantiated that WdPKS1 encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a beta-ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all wdpks1Delta strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in a wdpks1Delta strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.

Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing.

Processing of integral membrane proteins in order to liberate active proteins is of exquisite cellular importance. Examples are the processing events that govern sterol regulation, Notch signaling, the unfolded protein response, and APP fragmentation linked to Alzheimer's disease. In these cases, the proteins are thought to be processed by regulated intramembrane proteolysis, involving site-specific, membrane-localized proteases. Here we show that two homologous yeast transcription factors SPT23 and MGA2 are made as dormant ER/nuclear membrane-localized precursors and become activated by a completely different mechanism that involves ubiquitin/proteasome-dependent processing. SPT23 and MGA2 are relatives of mammalian NF-kappaB and control unsaturated fatty acid levels. Intriguingly, proteasome-dependent processing of SPT23 is regulated by fatty acid pools, suggesting that the precursor itself or interacting partners are sensors of membrane composition or fluidity.

Ubiquitin and its kin: how close are the family ties?

Modification of proteins by the covalent attachment of ubiquitin is known to target them for degradation by proteasomes. Several proteins have been discovered recently that are related to ubiquitin or function similarly. Some of these proteins act as modifiers; others bear ubiquitin-like domains embedded in their polypeptide chain but do not form conjugates with cellular proteins. Ubiquitin-like proteins mediate an impressive range of cellular functions, including cell-cycle progression, DNA repair and apoptosis. Recent discoveries endorse the view that, in many cases, the function of the relatives of ubiquitin is linked to the ubiquitin pathway.

Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair.

Two ubiquitin-conjugating enzymes, RAD6 and the heteromeric UBC13-MMS2 complex, have been implicated in post-replicative DNA damage repair in yeast. Here we provide a mechanistic basis for cooperation between the two enzymes. We show that two chromatin-associated RING finger proteins, RAD18 and RAD5, play a central role in mediating physical contacts between the members of the RAD6 pathway. RAD5 recruits the UBC13-MMS2 complex to DNA by means of its RING finger domain. Moreover, RAD5 association with RAD18 brings UBC13-MMS2 into contact with the RAD6-RAD18 complex. Interaction between the two RING finger proteins thus promotes the formation of a heteromeric complex in which the two distinct ubiquitin-conjugating activities of RAD6 and UBC13-MMS2 can be closely coordinated. Surprisingly, UBC13 and MMS2 are largely cytosolic proteins, but DNA damage triggers their redistribution to the nucleus. These findings suggest a mechanism by which the activity of this DNA repair pathway could be regulated.

Conjugation of the ubiquitin-like protein NEDD8 to cullin-2 is linked to von Hippel-Lindau tumor suppressor function.

The von Hippel-Lindau tumor suppressor protein pVHL assembles with cullin-2 (hCUL-2) and elongin B/C forming a protein complex, CBCVHL, that resembles SKP1-CDC53-F-box protein ubiquitin ligases. Here, we show that hCUL-2 is modified by the conserved ubiquitin-like protein NEDD8 and that NEDD8-hCUL-2 conjugates are part of CBCVHL complexes in vivo. Remarkably, the formation of these conjugates is stimulated by the pVHL tumor suppressor. A tumorigenic pVHL variant, however, is essentially deficient in this activity. Thus, ligation of NEDD8 to hCUL-2 is linked to pVHL activity and may be important for pVHL tumor suppressor function.

A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly.

Proteins modified by multiubiquitin chains are the preferred substrates of the proteasome. Ubiquitination involves a ubiquitin-activating enzyme, E1, a ubiquitin-conjugating enzyme, E2, and often a substrate-specific ubiquitin-protein ligase, E3. Here we show that efficient multiubiquitination needed for proteasomal targeting of a model substrate requires an additional conjugation factor, named E4. This protein, previously known as UFD2 in yeast, binds to the ubiquitin moieties of preformed conjugates and catalyzes ubiquitin chain assembly in conjunction with E1, E2, and E3. Intriguingly, E4 defines a novel protein family that includes two human members and the regulatory protein NOSA from Dictyostelium required for fruiting body development. In yeast, E4 activity is linked to cell survival under stress conditions, indicating that eukaryotes utilize E4-dependent proteolysis pathways for multiple cellular functions.