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Biomedical subjects

P Walter

Publications and source records attributed to P Walter.

At least 37 records · Page 2Linked to original sources
PubMed2002

Biocompatibility tests on the intraocular vision aid IOVA.

Intraocular miniaturized image transmission systems are developed to restore vision in patients with irreversible destruction of the anterior ocular segment (i.e. chemical burns, explosion trauma, trachoma) and high risk for corneal transplantation, provided that the posterior ocular segment is intact. To ensure safety of such a device biocompatibility tests were conducted. In the present study the effects of light and temperature in various intensities and the toxicity of the implanted materials on neural function in the rabbit retina in-vivo was investigated. All tests give encouraging results concerning the feasibility of intraocular miniaturized image transmission systems.

Animals↗
PubMed2001

Role of SRP RNA in the GTPase cycles of Ffh and FtsY.

The bacterial homologues of the signal recognition particle (SRP) and its receptor, the Ffh*4.5S RNA ribonucleoprotein complex and the FtsY protein, respectively, form a unique complex in which both Ffh and FtsY act as GTPase activating proteins for one another, resulting in the mutual stimulation of GTP hydrolysis by both proteins. Previous work showed that 4.5S RNA enhances the GTPase activity in the presence of both Ffh and FtsY, but it was not clear how this was accomplished. In this work, kinetic and thermodynamic analyses of the GTPase reactions of Ffh and FtsY have provided insights into the role of 4.5S RNA in the GTPase cycles of Ffh and FtsY. We found that 4.5S RNA accelerates the association between Ffh and FtsY 400-fold in their GTP-bound form, analogous to its 200-fold catalytic effect on Ffh*FtsY association previously observed with the GppNHp-bound form [Peluso, P., et al. (2000) Science 288, 1640-1643]. Further, Ffh-FtsY association is rate-limiting for the observed GTPase reaction with subsaturating Ffh and FtsY, thereby accounting for the apparent stimulatory effect of 4.5S RNA on the GTPase activity observed previously. An additional step, GTP hydrolysis from the Ffh*FtsY complex, is also moderately facilitated by 4.5S RNA. These results suggest that 4.5S RNA modulates the conformation of the Ffh*FtsY complex and may, in turn, regulate its GTPase activity during the SRP functional cycle.

Bacterial Proteins↗
PubMed2001

The emergence of different resistance mechanisms toward nucleoside inhibitors is explained by the properties of the wild type HIV-1 reverse transcriptase.

Nucleoside reverse transcriptase inhibitors (NRTIs) represent one of the main drug families used against AIDS. Once incorporated in DNA, they act as chain terminators, due to the lack of a 3'-hydroxyl group. As for the other anti-human immunodeficiency virus type 1 drugs, their efficiency is limited by the emergence of resistant viral strains. Unexpectedly, previous studies indicated that resistance toward NRTIs is achieved via two distinct and generally exclusive mechanisms. Resistance mutations either decrease the efficiency of NRTIs incorporation or increase their excision from the extended primer. To understand the emergence of different resistance mechanisms toward a single inhibitor class, we compared the incorporation and the pyrophosphorolysis of several NRTIs using wild type reverse transcriptase (WT RT). We found that the efficiency of discrimination or excision by pyrophosphorolysis in the presence of nucleotides of a given NRTI is a key determinant in the emergence of one or the other resistance pathway. Indeed, our results suggest that the pathway by which RT become resistant toward a given NRTI can be predicted by studying the inhibition of WT RT, because the resistance mutations do not confer new properties to the mutant enzyme, but rather exacerbate pre-existing properties of the WT enzyme. They also help to understand the low cross-resistance toward d4T observed with the 3'-azido-3'-deoxythymidine (AZT or zidovudine)-resistant RT.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Block of HAC1 mRNA translation by long-range base pairing is released by cytoplasmic splicing upon induction of the unfolded protein response.

Expression of the yeast transcription factor Hac1p, which controls the unfolded protein response, is regulated posttranscriptionally. Hac1p is only produced when an intron at the 3' end of its mRNA is removed by a nonconventional, regulated splicing reaction. We show that a previously unrecognized base-pairing interaction between the intron and the 5' untranslated region is required and sufficient to block mRNA translation. Unspliced HAC1 mRNA is stable, located in the cytosol, and is associated with polyribosomes, yet does not produce protein, indicating that the ribosomes engaged on the mRNA are stalled. We show that the polysomal, cytoplasmic pool of HAC1 mRNA is a substrate for splicing, suggesting that the stalled ribosomes may resume translation after the intron is removed.

5' Untranslated Regions↗
PubMed2001

Yeast cytoplasmic and mitochondrial methionyl-tRNA synthetases: two structural frameworks for identical functions.

The yeast Saccharomyces cerevisiae possesses two methionyl-tRNA synthetases (MetRS), one in the cytoplasm and the other in mitochondria. The cytoplasmic MetRS has a zinc-finger motif of the type Cys-X(2)-Cys-X(9)-Cys-X(2)-Cys in an insertion domain that divides the nucleotide-binding fold into two halves, whereas no such motif is present in the mitochondrial MetRS. Here, we show that tightly bound zinc atom is present in the cytoplasmic MetRS but not in the mitochondrial MetRS. To test whether the presence of a zinc-binding site is required for cytoplasmic functions of MetRS, we constructed a yeast strain in which cytoplasmic MetRS gene was inactivated and the mitochondrial MetRS gene was expressed in the cytoplasm. Provided that methionine-accepting tRNA is overexpressed, this strain was viable, indicating that mitochondrial MetRS was able to aminoacylate tRNA(Met) in the cytoplasm. Site-directed mutagenesis demonstrated that the zinc domain was required for the stability and consequently for the activity of cytoplasmic MetRS. Mitochondrial MetRS, like cytoplasmic MetRS, supported homocysteine editing in vivo in the yeast cytoplasm. Both MetRSs catalyzed homocysteine editing and aminoacylation of coenzyme A in vitro. Thus, identical synthetic and editing functions can be carried out in different structural frameworks of cytoplasmic and mitochondrial MetRSs.

Acylation↗
PubMed2001

Towards ensuring the safety of vitamins and minerals.

Today, there is a clear need for a risk assessment for vitamins and minerals because the total daily intake for nutrients through regular food, fortified products (Functional Food) and supplements may very well reach critical levels for an individual consumer. Several expert panels of the European Union, the UK, Japan and China will publish their reports on this issue in the near future. The Food and Nutrition Board of the Institute of Medicine of the US/Canada has already published a new standard. The board has defined the so-called Tolerable Upper Intake Level (UL) as the highest level of daily intake that is likely to pose no risk of adverse health effects to almost all individuals in the general population. Their determination involves a multi-step risk assessment procedure on the basis of mainly human data. Several daily UL values that have already been published are of special interest, e.g. 1 mg folic acid, 50 microg vitamin D, 1 g vitamin E, 2 g vitamin C, 2.5 g calcium, 350 mg magnesium.

Humans↗
PubMed2001

Synthesis of analogues of the O-beta-D-ribofuranosyl nucleoside moiety of liposidomycins. Part 2: role of the hydroxyl groups upon the inhibition of MraY.

O-beta-D-ribofuranosyl nucleoside I is the minimal structural entity of liposidomycins that maintains enzyme inhibitory activity on MraY. A set of compounds with hydroxyl patterns different from I has been synthesized. The presence of a hydroxyl group in the 3" position is essential for the activity. The 3'-deoxy derivative (IV), however, shows a 5-fold improved potency.

Aminoglycosides↗
PubMed2001

Improvement of macular function by membrane differential filtration in diabetic retinopathy.

BACKGROUND: Alterations of blood rheology are assumed to substantially contribute to the pathogenesis of diabetic retinopathy. Membrane differential filtration (MDF) is an extracorporeal treatment which is able to optimize rheological parameters by eliminating high molecular weight proteins and lipoproteins from the blood. The present study was designed to investigate the effects of repetitive MDF on visual function in diabetic retinopathy. METHODS: 11 patients (11 eyes) with nonproliferative or inactive proliferative diabetic retinopathy underwent three treatment cycles during a mean period of 18 weeks. The best corrected visual acuity served as the main parameter of the study. The measurement of visual acuity, visual field, biochemical and rheological parameters was carried out 24 hours pre and post each treatment cycle and at follow up. The mean follow up time was 9 weeks during the post treatment period. RESULTS: Compared to baseline examination the visual acuity improved stepwise by a mean value of 1.4 lines (p = 0.02) after the last treatment and remained stable at follow up (1.3 lines, p < 0.001). The mean defect of the visual field was reduced by 2.8 dB (p = 0.13) after the treatment period and by 2.5 dB (p = 0.016) at follow up, respectively. CONCLUSIONS: Repetitive treatment with membrane differential filtration is able to improve visual function in patients with diabetic retinopathy. The present study suggests that repetitive membrane differential filtration treatment could be a useful adjunct along with laser treatment to influence the clinical course of diabetic maculopathy.

Blood Proteins↗
PubMed2001

Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals.

Cellular survival of endoplasmic reticulum stress requires the unfolded protein response (UPR), a stress response first elucidated genetically in yeast. While we continue to refine our knowledge of the yeast system, especially the breadth and significance of the transcriptional response, conservation of the system's elements has allowed identification of corresponding and additional components of the mammalian UPR. Recent results reveal that the output of the mammalian UPR reaches beyond transcriptional regulation of secretory pathway components to control of general translation, the cell cycle and programmed cell death.

Animals↗
PubMed2001

Basis for regulated RNA cleavage by functional analysis of RNase L and Ire1p.

RNase L and Ire1p are members of a superfamily of regulated endoribonucleases that play essential roles in mediating diverse types of cellular stress responses. 2'-5' oligoadenylates, produced in response to interferon treatment and viral double-stranded RNA, are necessary to activate RNase L. In contrast, unfolded proteins in the endoplasmic reticulum activate Ire1p, a transmembrane serine/threonine kinase and endoribonuclease. To probe their similarities and differences, molecular properties of wild-type and mutant forms of human RNase L and yeast Ire1p were compared. Surprisingly, RNase L and Ire1p showed mutually exclusive RNA substrate specificity and partially overlapping but not identical requirements for phylogenetically conserved amino acid residues in their nuclease domains. A functional model for RNase L was generated based on the comparative analysis with Ire1p that assigns novel roles for ankyrin repeats and kinase-like domains.

Amino Acid Sequence↗
PubMed2001

Crystal structures of coaxially stacked kissing complexes of the HIV-1 RNA dimerization initiation site.

We describe the crystal structures of the RNA dimerization initiation sites (DIS) of HIV-1 subtypes A and B. Both molecules adopt a hairpin conformation, with loop sequences consisting of 272-AGGUGCACA-280 and 272-AAGCGCGCA-280, respectively. This includes a six-base self-complementary stretch (underlined) that allows homodimerization through the formation of a loop-loop, or 'kissing-loop', complex. The DISs for the two sequences have identical conformations, and the two interacting hairpins show a perfect coaxial alignment. The conserved purines, A272 and R273, are stacked in a bulged-out conformation and symmetrically join the upward and downward strands of each hairpin by crossing the helix major groove. There is good agreement between these structures and previous results from chemical probing in solution, as well as with differences in magnesium dependence for dimerization. The overall shape of the kissing-loop complex is very similar to that of the previously determined subtype A DIS duplex form. Unexpectedly, the purine R273 is the only base seen at a different position and is responsible for the difference in topology between the two forms. We propose that the transition from kissing-loop duplex could occur by a recombination mechanism based on symmetrical chain cleavage at R273 of each hairpin and subsequent cross-religation, rather than by base-pair melting and subsequent reannealing.

Base Pairing↗
PubMed2001

Effects of protein tyrosine kinase inhibitor genistein on retinal function in superfused vertebrate retina.

The aim of this study was to evaluate safe concentrations of genistein for a potential intraocular application using the isolated retina technique on bovine retina preparations. Bovine retinas were isolated and perfused with an oxygen pre-equilibrated standard solution. The electroretinogram (ERG) was recorded as a transretinal potential using silver/silver-chloride electrodes. After recording of stable ERG amplitudes, genistein was added to the solution in different concentrations. The percentage of b-wave reduction under the drug was calculated. We also studied the influence of genistein on the a-wave amplitude. After the addition of aspartate, the b-wave amplitude was reduced continuously until unmasked a-wave amplitudes were reached. Genistein was then added to the aspartate containing perfusate. The percentage of a-wave amplitude reduction under the drug was calculated. Concentrations of 3.3 microMol/l and higher were found to reduce the b-wave amplitude. The a-wave amplitude was not changed by the applied concentrations. The ERG only showed toxic effects from genistein beyond concentrations that were found to inhibit endothelial cell growth in vitro. In previous studies, beneficial effects on trabecular meshwork cells were present for genistein concentrations which are distinctly higher than the maximum nontoxic concentration reported here. It was shown that the photoreceptor layer is not affected at the examined concentration range. Therefore, we attribute the toxic effects to postsynaptic interaction of genistein. Intraocular application of genistein in a sufficient concentration seems possible.

Animals↗
PubMed2001

Multifaceted physiological response allows yeast to adapt to the loss of the signal recognition particle-dependent protein-targeting pathway.

Translational control has recently been recognized as an important facet of adaptive responses to various stress conditions. We describe the adaptation response of the yeast Saccharomyces cerevisiae to the loss of one of two mechanisms to target proteins to the secretory pathway. Using inducible mutants that block the signal recognition particle (SRP) pathway, we find that cells demonstrate a physiological response to the loss of the SRP pathway that includes specific changes in global gene expression. Upon inducing the loss of the SRP pathway, SRP-dependent protein translocation is initially blocked, and cell growth is considerably slowed. Concomitantly, gene expression changes include the induction of heat shock genes and the repression of protein synthesis genes. Remarkably, within hours, the efficiency of protein sorting improves while cell growth remains slow in agreement with the persistent repression of protein synthesis genes. Our results suggest that heat shock gene induction serves to protect cells from mislocalized precursor proteins in the cytosol, whereas reduced protein synthesis helps to regain efficiency in protein sorting by reducing the load on the protein translocation apparatus. Thus, we suggest that cells trade speed in cell growth for fidelity in protein sorting to adjust to life without SRP.

Adaptation, Physiological↗
PubMed2001

An efficient method for solving RNA structures: MAD phasing by replacing magnesium with zinc.

The structure of a 46-nucleotide RNA complex has been successfully solved using multi-wavelength anomalous dispersion (MAD) at the zinc K edge. Taking advantage of the eight magnesium-binding sites, it has been shown that for five of them magnesium could be replaced by zinc. This resulted in an excellent 2.0 A MAD electron-density map. Zinc, in common with some other transition metals, is able to replace magnesium in RNA structures, but zinc has the advantage of its K edge being ideally located at 1.284 A. As most RNA molecules contain magnesium-binding sites, it is suggested that this method could be a valuable alternative to the use of bromo derivatives of bases, which is limited to chemically synthesizable and thus rather short RNA sequences.

Crystallography, X-Ray↗
PubMed2001

Electrophysiological changes after 360 degrees retinotomy and macular translocation for subfoveal choroidal neovascularisation in age related macular degeneration.

AIM: To evaluate electrophysiological changes after 360 degrees retinotomy and macular translocation for subfoveal choroidal neovascularisation in patients with age related macular degeneration (AMD). METHODS: A consecutive series of 32 patients suffering from subfoveal choroidal neovascularisation secondary to AMD underwent 360 degrees retinotomy and macular translocation. The ERG served as the main parameter of the study and was recorded 1 day before the translocation surgery and no earlier than 4 weeks after the silicone oil removal. RESULTS: The scotopic ERG amplitudes were significantly reduced after translocation surgery. Depending on the applied flash luminance the mean b-wave amplitude reduction of the scotopic ERG varied between 67% (0.2 cd.s/m2) and 74% (0.03 cd.s/m2). The a-waves and b-waves of the saturating light response decreased significantly by 46% and 59%, respectively. The photopic a-wave and b-wave amplitudes were significantly lower after the translocation surgery resulting in a mean reduction of 27% and 43%, respectively. CONCLUSIONS: Although macular translocation may provide the potential of preserving and even restoring vision in patients with subfoveal choroidal neovascular membranes secondary to AMD the present study indicates that a significant electrophysiological decrease is caused by surgical procedures associated with this technique. Further research is necessary to clarify if certain modifications of the surgical procedure are able to substantially reduce the neuroretinal trauma.

Aged↗
PubMed2001

The signal recognition particle.

The signal recognition particle (SRP) and its membrane-associated receptor (SR) catalyze targeting of nascent secretory and membrane proteins to the protein translocation apparatus of the cell. Components of the SRP pathway and salient features of the molecular mechanism of SRP-dependent protein targeting are conserved in all three kingdoms of life. Recent advances in the structure determination of a number of key components in the eukaryotic and prokaryotic SRP pathway provide new insight into the molecular basis of SRP function, and they set the stage for future work toward an integrated picture that takes into account the dynamic and contextual properties of this remarkable cellular machine.

Alu Elements↗