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.

[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.

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.

Ubiquitin-dependent protein degradation: a cellular perspective.

A major pathway for protein degradation in eukaryotes is ubiquitin dependent. Substrate-specific ubiquitin-conjugating enzymes and accessory factors recognize specific signals on proteolytic substrates and attach ubiquitin to defined lysine residues of substrate proteins. Ubiquitin-protein conjugates are then degraded by the proteasome, a multicatalytic protease complex. This proteolytic pathway is highly selective and tightly regulated. It mediates the elimination of abnormal proteins and controls the half-lifes of certain regulatory proteins. Targets include transcriptional regulators, p53 and cyclins, pointing to a role of the ubiquitin system in the regulation of gene expression and growth control.

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.

A giant ubiquitin-conjugating enzyme related to IAP apoptosis inhibitors.

Ubiquitin-conjugating enzymes (UBC) catalyze the covalent attachment of ubiquitin to target proteins and are distinguished by the presence of a UBC domain required for catalysis. Previously identified members of this enzyme family are small proteins and function primarily in selective proteolysis pathways. Here we describe BRUCE (BIR repeat containing ubiquitin-conjugating enzyme), a giant (528-kD) ubiquitin-conjugating enzyme from mice. BRUCE is membrane associated and localizes to the Golgi compartment and the vesicular system. Remarkably, in addition to being an active ubiquitin-conjugating enzyme, BRUCE bears a baculovirus inhibitor of apoptosis repeat (BIR) motif, which to this date has been exclusively found in apoptosis inhibitors of the IAP-related protein family. The BIR motifs of IAP proteins are indispensable for their anti-cell death activity and are thought to function through protein-protein interaction. This suggests that BRUCE may combine properties of IAP-like proteins and ubiquitin-conjugating enzymes and indicates that the family of IAP-like proteins is structurally and functionally more diverse than previously expected.

The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme.

Mutants in the gene CDC34 of the yeast Saccharomyces cerevisiae are defective in the transition from G1 to the S phase of the cell cycle. This gene was cloned and shown to encode a 295-residue protein that has substantial sequence similarity to the product of the yeast RAD6 gene. The RAD6 gene is required for a variety of cellular functions including DNA repair and was recently shown to encode a ubiquitin-conjugating enzyme. When produced in Escherichia coli, the CDC34 gene product catalyzed the covalent attachment of ubiquitin to histones H2A and H2B in vitro, demonstrating that the CDC34 protein is another distinct member of the family of ubiquitin-conjugating enzymes. The cell cycle function of CDC34 is thus likely to be mediated by the ubiquitin-conjugating activity of its product.

Ubiquitin-conjugating enzymes: novel regulators of eukaryotic cells.

Covalent attachment of ubiquitin to cellular proteins is essential for cell viability and is catalysed by a set of distinct ubiquitin-conjugating enzymes. Individual members of this novel enzyme family mediate strikingly diverse functions, including DNA repair, cell cycle control, selective protein degradation and essential functions of the stress response.

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.

The ubc-2 gene of Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme involved in selective protein degradation.

The ubiquitin-protein conjugation system is involved in a variety of eukaryotic cell functions, including the degradation of abnormal and short-lived proteins, chromatin structure, cell cycle progression, and DNA repair. The ubiquitination of target proteins is catalyzed by a ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) and in some cases also requires auxiliary substrate recognition proteins (E3s). Multiple E2s have been found, and these likely possess specificity for different classes of target proteins. Here we report the cloning and characterization of a gene (ubc-2) encoding a ubiquitin-conjugating enzyme which is involved in the selective degradation of abnormal and short-lived proteins in the nematode Caenorhabditis elegans. The nematode ubc-2 gene encodes a 16.7-kDa protein with striking amino acid sequence similarity to Saccharomyces cerevisiae UBC4 and UBC5 and Drosophila UbcD1. When driven by the UBC4 promoter, ubc-2 can functionally substitute for UBC4 in yeast cells; it rescues the slow-growth phenotype of ubc4 ubc5 mutants at normal temperature and restores their ability to grow at elevated temperatures. Western blots (immunoblots) of ubc4 ubc5 yeast cells transformed with ubc-2 reveal a protein of the expected size, which cross-reacts with anti-Drosophila UbcD1 antibody. C. elegans ubc-2 is constitutively expressed at all life cycle stages and, unlike yeast UBC4 and UBC5, is not induced by heat shock. Both trans and cis splicing are involved in the maturation of the ubc-2 transcript. These data suggest that yeast UBC4 and UBC5, Drosophila UbcD1, and C. elegans ubc-2 define a highly conserved gene family which plays fundamental roles in all eukaryotic cells.

DNA methyltransferase genes of Bacillus subtilis phages: structural relatedness and gene expression.

The DNA methyltransferase (Mtase) genes of temperate Bacillus subtilis phages phi 3T, rho 11 and SP beta were cloned and expressed in Escherichia coli. Each gene specifies a 47-kDa1 protein, which modifies BsuR (GGCC) and Fnu4HI (GCNGC) target sequences. Transcription is controlled by phage promoters located on the cloned fragments. The direction of transcription and the approximate position of the Mtase genes were determined. DNA/DNA hybridization experiments revealed close structural relatedness of the phi 3T, rho 11 and SP beta genes. A significant degree of homology was also found among these genes and the Mtase gene of related phage SPR, which codes for an enzyme with different modification specificity. These results suggest a common ancestor of the different phage Mtase genes. Phage Z, the only BsuR-sensitive member of this phage group, lacks a modification gene, but contains regions homologous to sequences flanking the SPR, phi 3T, rho 11 and SP beta Mtase genes.

Restriction and modification in Bacillus subtilis: nucleotide sequence, functional organization and product of the DNA methyltransferase gene of bacteriophage SPR.

Bacillus subtilis phage SPR codes for a DNA methyltransferase (Mtase) which methylates the 5' cytosine in the sequence GGCC and both cytosines in the sequence CCGG. A 2126-bp fragment of SPR DNA containing the Mtase gene has been sequenced. This fragment has only one significant open reading frame of 1347 bp, which corresponds to the Mtase gene. Within the sequence the Mtase promoter has been defined by S1 mapping. The size of the SPR Mtase predicted from the deduced amino acid composition is 49.9 kDal. This is in agreement with both the Mr of the purified enzyme and with that of the SPR Mtase gene product identified here by minicell technique. Base changes leading to mutants affected in Mtase activity were localized within the Mtase gene.

Yeast ubiquitin-conjugating enzymes involved in selective protein degradation are essential for cell viability.

Ubiquitin-mediated proteolysis is a major pathway for selective protein degradation in eukaryotic cells. This proteolysis pathway involves the processive covalent attachment of ubiquitin to proteolytic substrates and their subsequent degradation by a specific ATP-dependent protease complex. We have cloned the genes and characterized the function of ubiquitin-conjugating enzymes (UBCs) from the yeast Saccharomyces cerevisiae. UBC1, UBC4 and UBC5 enzymes were found to mediate selective degradation of short-lived and abnormal proteins. These enzymes have overlapping functions and constitute a UBC subfamily essential for growth. UBC1 is specifically required at early stages of growth after germination of spores. UBC4 and UBC5 enzymes generate high molecular weight ubiquitin-protein conjugates and comprise a major ubiquitin-conjugation activity in yeast cells. Moreover, these enzymes are central components of the cellular stress response.

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.

[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.