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Biomedical subjects

H Gross

Publications and source records attributed to H Gross.

At least 37 records · Page 2Linked to original sources
PubMed1998

Reduction of charging in protein electron cryomicroscopy.

Charging causes a loss of resolution in electron cryomicroscopy with biological specimens prepared without a continuous carbon support film. Thin conductive films were deposited onto catalase crystals prepared across holes using ion-beam sputtering and thermal evaporation and evaluated for the effectiveness of charge reduction. Deposits applied by ion-beam sputtering reduced charging but concurrently resulted in structural damage. Coatings applied by thermal evaporation also reduced charging, and preserved the specimen structure beyond 5 A resolution as judged from electron diffraction patterns and images of glucose-embedded catalase crystals tilted to 45 degrees in the microscope. This study demonstrates for the first time the feasibility of obtaining high-resolution data from unstained, unsupported protein crystals with a conductive surface coating.

Catalase↗
PubMed1997

Prion-inducing domain 2-114 of yeast Sup35 protein transforms in vitro into amyloid-like filaments.

The yeast non-Mendelian genetic factor [PSI], which enhances the efficiency of tRNA-mediated nonsense suppression in Saccharomyces cerevisiae, is thought to be an abnormal cellular isoform of the Sup35 protein. Genetic studies have established that the N-terminal part of the Sup35 protein is sufficient for the genesis as well as the maintenance of [PSI]. Here we demonstrate that the N-terminal polypeptide fragment consisting of residues 2-114 of Sup35p, Sup35pN, spontaneously aggregates to form thin filaments in vitro. The filaments show a beta-sheet-type circular dichroism spectrum, exhibit increased protease resistance, and show amyloid-like optical properties. It is further shown that filament growth in freshly prepared Sup35pN solutions can be induced by seeding with a dilute suspension of preformed filaments. These results suggest that the abnormal cellular isoform of Sup35p is an amyloid-like aggregate and further indicate that seeding might be responsible for the maintenance of the [PSI] element in vivo.

Amyloid↗
PubMed1997

Fluorescence in-situ hybridization (FISH) reveals that in chronic myelogenous leukaemia (CML) following interferon-alpha therapy, normalization of megakaryocyte size is associated with the loss of bcr/abl translocation.

AIMS: In addition to predominant granulocytic proliferation, bone marrow morphology in Philadelphia chromosome positive (Ph1+) CML is characterized by atypical dwarf or microforms of megakaryocytes. However, following therapy with interferon-alpha 2b (IFN), these micromegakaryocytes occur less frequently. The purpose of this study was to elucidate whether the reappearance of normal megakaryocytes may be associated also with a reduction of the bcr/abl-positive cell clone. METHODS AND RESULTS: Fluorescence in-situ hybridization (FISH) technique in combination with immunomorphometry (CD61) was performed on trephine biopsies. A total of 311 CD61-positive megakaryocytes, including precursors and atypical microforms, were evaluated in pre-treatment specimens derived from 11 patients with Ph1+ CML. A specific fusion site marking the bcr/abl translocation was found in 87% of megakaryocytes which showed a size of 169 +/- 35 microns2. In untreated patients, atypical microforms (size 200 microns2) were observed in 66% of the total megakaryocytic population. Following IFN therapy 369 megakaryocytes could be analysed in sequential examinations and were found to display a significant decrease (63%) in positive fusion signals. In addition there was also a significant enhancement in average size (252 +/- 66 microns2) reflecting a reduction in the number of micromegakaryocytes (43%). These findings were particularly conspicuous in three patients with a major to complete cytogenetic remission. CONCLUSIONS: A normalization of megakaryocyte size following IFN therapy in CML is significantly associated with a loss of the bcr/abl translocation site and therefore indicates a (partial) recovery of normal haematopoiesis.

Fusion Proteins, bcr-abl↗
PubMed1997

[Therapeutic problems in tetanus--presented via a case report].

This is the case presentation of a forty-year old female patient, who had incurred a tetanus infection as a result of intravenous drug abuse. Clostridium tetani could be detected repeatedly in abscesses caused by injections. The patient had to be put on continuous relaxation, sedation and artificial respiration for 42 days. Besides the usual intensive care regimen, a high-dose antitoxin therapy was initiated. The areas of abscesses had to be eradicated surgically several times. With the exception of a thrombus of the vena cava superior (without haemodynamic consequences) and a pneumonia, the further course was without any other serious complications. After seven months of hospitalisation the patient could be dismissed at "restitutio ad integrum". The known immunosuppressive effect of a high dosed tetanus antitoxin therapy could be confirmed by the patient's antitoxin titre course. Repeated active immunisation attempts to produce a sufficient endogenous antitoxin titre failed. The existing therapeutic uncertainties regarding the dosage of the tetanus antitoxin therapy, the titre control and the proper antibiotic treatment are described.

Adult↗
PubMed1997

Crystalline mitochondrial inclusion bodies isolated from creatine depleted rat soleus muscle.

Rats were fed a 2% guanidino propionic acid diet for up to 18 weeks to induce cellular creatine depletion by inhibition of creatine uptake by this creatine analogue. Ultrastructural analysis of creatine depleted tissues showed that mitochondrial intermembrane inclusion bodies appeared in all skeletal muscles analysed, after 11 weeks of feeding. Heart had relatively few even after 18 weeks of analogue feeding and none were evident in kidney, brain or liver. These structures were strongly immuno-positive for sarcomeric mitochondrial creatine kinase and upon removal from mitochondria, the inclusion bodies were shown to diffract to a resolution of 2.5 nm. Two-dimensional image analysis and three-dimensional reconstruction revealed arrays of creatine kinase octamers with additional components between the octameric structures. The same mitochondria had a 3-fold higher extractable specific creatine kinase activity than controls. Molecular mass gel filtration of inclusion body containing mitochondrial extracts from analogue fed rat solei revealed mitochondrial creatine kinase eluting as an aggregate of an apparent molecular mass > or = 2,000 kDa. Mitochondrial creatine kinase of control soleus mitochondrial extract eluted as an octamer, with a molecular mass of 340 kDa. Respiration measurements of control solei mitochondria displayed creatine mediated stimulation of oxidative phosphorylation that was absent in analogue-fed rat solei mitochondria. The latter also had 19% and 14% slower rates of state 4 and maximal state 3 respiration, respectively, than control mitochondria. These results indicate that mitochondrial creatine kinase co-crystallises with another component within the inter membrane space of select mitochondria in creatine depleted skeletal muscle, and is inactive in situ.

Adenylate Kinase↗
PubMed1996

Surface topographies at subnanometer-resolution reveal asymmetry and sidedness of aquaporin-1.

Aquaporin-1 (AQP1) is an abundant protein in human erythrocyte membranes which functions as a specific and constitutively active water conducting pore. Solubilized and isolated as tetramer, it forms well-ordered two-dimensional (2D) crystals when reconstituted in the presence of lipids. Several high resolution projection maps of AQP1 have been determined, but information on its three-dimensional (3D) mass distribution is sparse. Here, we present surface reliefs at 0.9 nm resolution that were calculated from freeze-dried unidirectionally metal-shadowed AQP1 crystals as well as surface topographs recorded with the atomic force microscope of native crystals in buffer solution. Our results confirm the 3D map of negatively stained AQP1 crystals, which exhibited tetramers with four major protrusions on one side and a large central cavity on the other side of the membrane. Digestion of AQP1 crystals with carboxypeptidase Y, which cleaves off a 5 kDa intracellular C-terminal fragment, led to a reduction of the major protrusions, suggesting that the central cavity of the tetramer faces the outside of the cell. To interpret the results, sequence based structure predictions served as a guide.

Aquaporin 1↗
PubMed1996

Implications for the domain arrangement of axonin-1 derived from the mapping of its NgCAM binding site.

The neuronal cell adhesion molecule axonin-1 is composed of six immunoglobulin and four fibronectin type III domains. Axonin-1 promotes neurite outgrowth, when presented as a substratum for neurons in vitro, via a neuronal receptor that has been identified as the neuron-glia cell adhesion molecule, NgCAM, based on the blocking effect of polyclonal antibodies directed to NgCAM. Here we report the identification of axonin-1 domains involved in NgCAM binding. NgCAM-conjugated microspheres were tested for binding to COS cells expressing domain deletion mutants of axonin-1. In addition, monoclonal antibodies directed to axonin-1 were assessed for their ability to block the axonin-1-NgCAM interaction, and their epitopes were mapped using the domain deletion mutants. The results suggest that the four amino-terminal immunoglobulin domains of axonin-1 form a domain conglomerate which is necessary and sufficient for NgCAM binding. Surprisingly, NgCAM binding to membrane-bound axonin-1 was increased strongly by deletion of the fifth or sixth immunoglobulin domains of axonin-1. Based on these results and on negative staining electron microscopy, we propose a horseshoe-shaped domain arrangement of axonin-1 that obscures the NgCAM binding site. Neurite outgrowth studies with truncated forms of axonin-1 show that axonin-1 is a neurite outgrowth-promoting substratum in the absence of the NgCAM binding site.

Animals↗
PubMed1996

STM of metal embedded and coated DNA and DNA-protein complexes.

Bare and Pt/Ir/C-coated DNA has been analysed using scanning tunnelling microscopy (STM). To achieve reproducible imaging of bare DNA on mica ethanol/air-dried molecules were embedded in Pt/C. By peeling the metal film off the mica, the previously mica-exposed side of the Pt/C-film with the embedded DNA molecules was accessible for STM analysis. By applying this replica/anchoring technique only hollow trenches in the metal film, and not the DNA itself, could be visualized. The gaps averaged 3.1 nm (+/- 0.9 nm) wide and 1 nm (+/- 0.5 nm) deep. Using scanning force microscopy it could be confirmed that the DNA remained in the Pt/C film during the peel-off procedure. For STM, DNA fragments were also coated with 0.7-1 nm Pt/Ir/C. Owing to the high Z-resolution the STM samples were coated at a high elevation angle (65 degrees), thereby minimizing the problem of self-shadowing. Coating by Pt/Ir/C allowed routine imaging and quantitative analysis of both ethanol/air- and freeze-dried DNA under atmospheric conditions. After ethanol/air drying measured values for DNA width and height were 5.1 nm (+/- 1.8 nm) and 0.9 nm (+/- 0.2 nm), respectively. Freeze-dried DNA averaged 4.2 nm (+/- 1.3 nm) wide and 1.1 nm (+/- 0.1 nm) high. A Pt/Ir/C-coating was also applied to visualize DNA-protein interaction using STM.

Aluminum Silicates↗
PubMed1996

Cerebrovascular reserve capacity in patients with occlusive cerebrovascular disease: assessment with dynamic susceptibility contrast-enhanced MR imaging and the acetazolamide stimulation test.

PURPOSE: To quantitatively assess cerebrovascular reserve capacity in patients with occlusive cerebrovascular disease. MATERIALS AND METHODS: In 21 symptomatic patients with occlusive cerebrovascular disease, magnetic resonance (MR) imaging was performed with a 1.5-T system. Before, during, and after a brief bolus injection of gadopentetate dimeglumine into the antecubital vain, a series of 32 rapid T2*-weighted gradient-echo images of two different sections were acquired simultaneously to measure the concentration-time-curves in the brain tissue and in the brain-feeding arteries. Principles of indicator dilution analysis were applied to compute regional cerebral blood flow (rCBF) and volume. Each patient underwent two examinations, the first before and the second after acetazolamide stimulation. RESULTS: In the asymptomatic hemisphere, a mean increase in rCBF value of 47.1% was observed after acetazolamide stimulation. In the affected areas of the symptomatic hemisphere, a statistically significantly reduced response to acetazolamide stimulation was found, indicating a severely compromised cerebrovascular reserve capacity. CONCLUSION: MR imaging with the described techniques provides quantitative information about the cerebrovascular reserve capacity in patients with occlusive cerebrovascular disease.

Acetazolamide↗
PubMed1996

Local cerebral glucose metabolism in patients with long-term behavioral and cognitive deficits following mild traumatic brain injury.

A retrospective study of 20 patients with mild traumatic brain injury (MTBI) examined brain regions of interest by comparing [18F]-2-deoxyglucose PET, neuropsychological test results, and continuing behavioral dysfunction. Abnormal local cerebral metabolic rates (rLCMs) were most prominent in midtemporal, anterior cingulate, precuneus, anterior temporal, frontal white, and corpus callosum brain regions. Abnormal rLCMs were significantly correlated statistically with 1) overall clinical complaints, most specifically with inconsistent attention/concentration and 2) overall neuropsychological test results. The authors conclude that 1) even mild TBI may result in continuing brain behavioral deficits; 2) PET can help elucidate dysfunctional brain circuitry in neurobehavioral disorders; and 3) specific brain areas may correlate with deficits in daily neurobehavioral functioning and neuropsychological test findings.

Adolescent↗
PubMed1996

Mathematical simulation of retinal image contrast after photorefractive keratectomy with a diaphragm mask.

BACKGROUND: Photorefractive keratectomy (PRK) using a dilating diaphragm mask engraves a delicate three-dimensional staircase pattern into a formerly smooth corneal surface. The created steps are later smoothed by tear film and wound healing processes. The present study investigates, in a mathematical simulation, the effects that such staircase patterns and their smoothing may have on retinal image contrast. METHODS: All simulations are based on the Gullstrand eye model and calculate retinal image contrast from point spread function (PSF) analysis of Gullstrand eyes treated by simulated PRK under various conditions. RESULTS: The simulations indicate that PRK can reduce retinal contrast markedly. The most critical factor for such a reduction is the step height of the ablation pattern. With step heights below 0.4 microns, loss of contrast due to the created staircase pattern is always moderate and should be restored during early wound healing. Complete wound healing may smooth out larger step heights. Micromovements during PRK also can lead to partial loss of retinal image contrast. CONCLUSIONS: Simulation of retinal contrast after PRK shows that step heights below 0.4 microns seem to be acceptable. A minimization of the micromovements during PRK can offset some of the reduction of retinal contrast.

Contrast Sensitivity↗
PubMed1996

[Decrease of retinal image contrast after photorefractive keratectomy, improvement within the scope of surface restitution].

Photorefractive keratectomy (PRK) with the ArF excimer lasers in current use usually approximates the intended corneal curvature by a mean of a delicate step-type pattern that is smooth off afterwards by reepithelialization and tear film. The present study was based on a model eye with axial myopia of -6 D but otherwise the optical and geometric properties of the Gullstrand model eye and was designed to investigate to what extent. (1) corneal step patterns can reduce retinal image contrast and (2) smoothing effects can restore such a loss. METHODS. The corneal surface resulting from PRK in the case of a myopia of -6 D (optical zone diameter 6 mm) is calculated for the parameters of the model eye. The retinal image contrasts of bar patterns are calculated by PSF (point spread function) analysis: varying size of pupil, wavelength, bar width, ablation step height and degree of smoothing. RESULTS. Step height influences retinal image contrast crucially. With step heights above 0.4 micron a massive loss of retinal image contrast must be expected, which can, however, be corrected to a useful extent by surface-smoothing effects. CONCLUSION. This study indicates that PRK with excimer lasers should be performed with low fluence and correspondingly low corneal step heights.

Computer Simulation↗
PubMed1996

Microscopic analysis of DNA and DNA-protein assembly by transmission electron microscopy, scanning tunneling microscopy and scanning force microscopy.

To investigate DNA and DNA-protein assembly, nucleic acids were adsorbed to freshly cleaved mica in the presence of magnesium ions. The efficiency of DNA adhesion and the distribution of the molecules on the mica surface were checked by transmission electron microscopy. In addition, various kinds of DNA-protein interactions including DNA wrapping and DNA supercoiling were analyzed using electron microscopy. In parallel, this Mg2+/mica method can be applied (1) to analyze embedded DNA by scanning tunneling microscopy, (2) to visualize freeze-dried, metal coated DNA-protein complexes by tunneling microscopy, and (3) to image DNA or DNA-protein interaction in air or in liquid by scanning force microscopy. An advantage of such a correlative approach is that parallel imaging can reveal complementary information. The benefit of such a combined approach in analysis of protein-induced DNA bending is discussed.

Aluminum Silicates↗
PubMed1993

[The reliability of the measurement of non-invasive oscillometric blood pressure instruments].

METHODS: The performance of five oscillometric blood pressure monitors was tested by means of a simulator. The oscillometric signals of two healthy patients were replayed. These signals were superimposed by sinusoidal artefacts with increasing amplitude, to examine the influence of artefacts on the accuracy of the measurement. Each measurement was repeated 15 times. RESULTS: The artefacts taken for this examination did affect the scattering of each single measurement rather than the mean value of the repeated measurements. The results did get worse with decreasing signal-to-artefact ratio. However, some monitors are able to handle these artefacts better than others. CONCLUSION: It is obvious that the implemented software for the artefact treatment and the determination of blood pressure values differs from manufacturer to manufacturer. Some devices were able to separate relevant signals from artefacts better than others. The most critical point of the oscillometric method is the correct determination of the maximum amplitude of the oscillations, because it directly influences the accuracy of the displayed blood pressure values. The availability of a simulator to test automated sphygmomanometers has shown to be a good tool to examine the performance of these devices.

Artifacts↗