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Biomedical subjects

J Frank

Publications and source records attributed to J Frank.

At least 109 records · Page 6Linked to original sources
PubMed2000

The ribosome--a macromolecular machine par excellence.

The ribosome is the site in the cell where proteins are synthesized. Cryo-electron microscopy and X-ray crystallography have revealed the ribosome as a particle made of two subunits, each formed as an intricate mesh of RNAs and many proteins. Ligand-binding experiments followed by cryo-electron microscopy have helped to determine some of the key stages of interaction between the ribosome and the main ligand molecules.

Cryoelectron Microscopy↗
PubMed2000

Executive control in set switching: residual switch cost and task-set inhibition.

Executive processes necessary for flexibly switching between different tasks were studied using a set switching paradigm that requires participants to rapidly switch between different tasks across consecutive trials. Switch cost reflects poorer performance for task-switch trials than for consecutive same-task trials. Significant switch cost was observed even with considerable preparation time before a task-switch, an effect known as residual switch cost. This study tested the hypothesis that one process underlying residual switch cost is inhibition of the previous task-set. We used semantic categorization tasks to compare switch cost between alternating task series (ABA) and nonalternating series (ABC) in order to test the generality of a task-set inhibition effect previously observed with perceptual judgment tasks (Mayr & Keele, in press). The results yielded significant switch cost only for alternating tasks, in both response times and errors resulting from performance of the wrong task. Thus, resolving inhibition associated with previously abandoned task-sets may be the main process underlying residual switch costs, suggesting that task-set inhibition is an important executive control process.

Adolescent↗
PubMed2000

FDG positron emission tomography in the diagnosis of peripheral pulmonary focal lesions.

Positron emission tomography (PET) using fluoride-18-marked fluoride deoxyglucose (FDG) represents a metabolically based imaging technique capable of providing information on the potential malignancy of peripheral pulmonary focal lesions. In the present prospective study, we investigated the effectiveness of FDG-PET in determining the dignities of 67 such lesions in 35 patients. Findings of FDG-PET were compared with those of computed tomography (CT), as well as with surgical and histological reports, and the value of FDG-PET as a diagnostic method evaluated. FDG-PET correctly identified 38 lesions as positive for malignancy, 18 correctly as negative, 7 incorrectly as negative, and 4 incorrectly as positive. Based on lesions, this yields a sensitivity of 84.4% and a specificity of 81.8%. All malignant focal lesions with a diameter of over 1.2 cm were correctly identified (sensitivity: 100%). In cases of intense FDG uptake, differentiation between a primary lesion, a metastasis, and an acute inflammation is often not possible.

Adolescent↗
PubMed2000

Trail making test, part B as a measure of executive control: validation using a set-switching paradigm.

Recent controversy surrounds the use of the Trail Making Test as a measure of cognitive flexibility, given that the Trail Making Test, Part B (TMT-B) also differs from Part A (TMT-A) in factors of motor control and perceptual complexity. The present study compared performance in the TMT and a set-switching task in order to test the assumption that cognitive flexibility is captured in TMT-B performance. Set-switching tasks have low motor and perceptual selection demands, and therefore provide a clearer index of executive function. In this study, participants made category judgments for digits, letters, or symbols across a series of trials, and performance for consecutive same-task trials was compared with task-switch trials. Results of the set-switching task indicated significant switch cost, but only for the situation of task alternation (e.g., an ABA series), suggesting that task-set inhibition may play a role in this effect. Alternating-switch cost was significantly correlated with TMT-B performance, especially with the TMT-B to TMT-A ratio (B/A). Cost for alternating switches was especially large for participants with B/A ratio > 3. These results provide direct evidence that the B/A ratio of performance in the TMT provides an index of executive function.

Adult↗
PubMed2000

Identifying male college students' perceived health needs, barriers to seeking help, and recommendations to help men adopt healthier lifestyles.

Seven focus groups at a university campus were formed to identify college men's health concerns, barriers to seeking help, and recommendations to help college men adopt healthier lifestyles. Content analysis was used to identify and organize primary patterns in the focus-group data. Results of the study revealed that the college men were aware that they had important health needs but took little action to address them. The participants identified both physical and emotional health concerns. Alcohol and substance abuse were rated as the most important issues for men. The greatest barrier to seeking services was the men's socialization to be independent and conceal vulnerability. The most frequently mentioned suggestions for helping men adopt healthier lifestyles were offering health classes, providing health information call-in service, and developing a men's center. Implications of the results are discussed.

Adaptation, Psychological↗
PubMed2000

The visualization of oxidant stress in tissues and isolated cells.

Many studies have implicated the role of oxidant stress in a wide range of human diseases and have led to the rapid expansion of research in this area. With many experimental approaches a direct detection of the production of reactive oxygen species (ROS) and free radicals is not possible. Free radicals are very reactive, short-lived and react in a non-specific way, so that ongoing oxidative damage is generally analyzed by measurement of secondary products e.g. H2O2, "oxidized" proteins, peroxidized lipids and their break-down products, "oxidized" DNA or by fluorographic analysis in combination with fluorescent dyes e.g. dichlorofluorescin (DCFH). The histochemical visualization of selected molecular markers for oxidative phenomena can often provide valuable information concerning the distribution of oxidative processes in vivo. A number of biochemical methods are available for the monitoring of almost all oxidant stress-related processes, although their applicability in vivo is limited. This review summarizes the biochemical methods currently available for histochemical detection and indirect visualization of an excess of free radicals and ROS. The cited methods are discussed and the results obtained from their application are critically evaluated.

Animals↗
PubMed2000

An immortalized rat liver stellate cell line (HSC-T6): a new cell model for the study of retinoid metabolism in vitro.

Hepatocytes and hepatic stellate cells play important roles in retinoid storage and metabolism. Hepatocytes process postprandial retinyl esters and are responsible for secretion of retinol bound to retinol-binding protein (RBP) to maintain plasma retinol levels. Stellate cells are the body's major cellular storage sites for retinoid. We have characterized and utilized an immortalized rat stellate cell line, HSC-T6 cells, to facilitate study of the cellular aspects of hepatic retinoid processing. For comparison, we also carried out parallel studies in Hepa-1 hepatocytes. Like activated primary stellate cells, HSC-T6 express myogenic and neural crest cytoskeletal filaments. HSC-T6 cells take up and esterify retinol in a time- and concentration-dependent manner. Supplementation of HSC-T6 culture medium with free fatty acids (up to 300 micrometer) does not affect retinol uptake but does enhance retinol esterification up to 10-fold. RT-PCR analysis indicates that HSC-T6 cells express all 6 retinoid nuclear receptors (RARalpha, -beta, -gamma, and RXRalpha, -beta, -gamma) and like primary stellate cells, HSC-T6 stellate cells express cellular retinol-binding protein, type I (CRBP) but fail to express either retinol-binding protein (RBP) or transthyretin (TTR). Addition of retinol (10(-8)-10(-5) m) or all-trans-retinoic acid (10(-10)-10(-6) m) rapidly up-regulates CRBP expression. Using RAR-specific agonists and antagonists and an RXR-specific agonist, we show that members of the RAR-receptor family modulate HSC-T6 CRBP expression.Thus, HSC-T6 cells display the same retinoid-related phenotype as primary stellate cells in culture and will be a useful tool for study of hepatic retinoid storage and metabolism.

Animals↗
PubMed1999

Direct three-dimensional localization and positive identification of RNA helices within the ribosome by means of genetic tagging and cryo-electron microscopy.

BACKGROUND: Ribosomes are complex macromolecular machines that perform the translation of the genetic message. Cryo-electron microscopic (cryo-EM) maps of the Escherichia coli 70S ribosome are approaching a resolution of 10 A and X-ray maps of the 30S and 50S subunits are now available at 5 A. These maps show a lot of details about the inner architecture of the ribosome and ribosomal RNA helices are clearly visible. However, in the absence of further biological information, even at the higher resolution of the X-ray maps many rRNA helices can be placed only tentatively. Here we show that genetic tagging in combination with cryo-EM can place and orient double-stranded RNA helices with high accuracy. RESULTS: A tRNA sequence inserted into the E. coli 23S ribosomal RNA gene, at one of the points of sequence expansion in eukaryotic ribosomes, is visible in the cryo-EM map as a peripheral 'foot' structure. By tracing its acceptor-stem end, the location of helix 63 in domain IV and helix 98 in domain VI of the 50S subunit could be precisely determined. CONCLUSIONS: Our study demonstrates for the first time that features of a three-dimensional cryo-EM map of an asymmetric macromolecular complex can be interpreted in terms of secondary and primary structure. Using the identified helices as a starting point, it is possible to model and interpret, in molecular terms, a larger portion of the ribosome. Our results might be also useful in interpreting and refining the current X-ray maps.

Base Sequence↗
PubMed1999

Animation of the dynamical events of the elongation cycle based on cryoelectron microscopy of functional complexes of the ribosome.

Using three-dimensional cryoelectron microscopy, the binding positions of tRNA and elongation factors EF-G and EF-Tu (the latter complexed with aminoacyl tRNA and GTP) on the ribosome were determined in previous studies. On the basis of these studies, the dynamical events that take place in the course of the elongation cycle of protein synthesis have been animated. The resulting 3-min movie is accessible on the website of this journal (http://www. idealibrary.com). The following article provides a brief annotation of those frames of the movie for which experimental support is available.

Computer Simulation↗
PubMed1999

Haloarcula marismortui 50S subunit-complementarity of electron microscopy and X-Ray crystallographic information.

The large 50S subunit of the Haloarcula marismortui 70S ribosome was solved to 19 A using cryo-electron microscopy and single particle reconstruction techniques and to 9 A using X-ray crystallography. In the latter case, phases were determined by multiple isomorphous replacement and anomalous scattering from three heavy atom derivatives. The availability of X-ray and electron microscopy (EM) data has made it possible to compare the results of the two experimental methods. In the flexible regions of the 50S subunit, small differences in the mass distribution were detected. These differences can be attributed to the influence of packing in the crystal cell. The rotationally averaged power spectra of X-ray and EM were compared in an overlapping spatial frequency range from 60 to 13 A. The resulting ratio of X-ray to EM power ranges from 1 to 15, reflecting a progressively larger underestimation of the Fourier amplitudes by the electron microscope.

Archaeal Proteins↗
PubMed1999

Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA.

Dynamic changes in secondary structure of the 16S rRNA during the decoding of mRNA are visualized by three-dimensional cryo-electron microscopy of the 70S ribosome. Thermodynamically unstable base pairing of the 912-910 (CUC) nucleotides of the 16S RNA with two adjacent complementary regions at nucleotides 885-887 (GGG) and 888-890 (GAG) was stabilized in either of the two states by point mutations at positions 912 (C912G) and 885 (G885U). A wave of rearrangements can be traced arising from the switch in the three base pairs and involving functionally important regions in both subunits of the ribosome. This significantly affects the topography of the A-site tRNA-binding region on the 30S subunit and thereby explains changes in tRNA affinity for the ribosome and fidelity of decoding mRNA.

Base Sequence↗
PubMed1999

Enhancement of the UVA induction of haem oxygenase-1 expression by beta-carotene in human skin fibroblasts.

beta-Carotene has often been discussed as a means to reduce the risk of skin photodamage. We studied the antioxidative potential of beta-carotene in human skin fibroblasts exposed to ultraviolet A light. Surprisingly, we found a pro-oxidative effect of beta-carotene. Using the induction of haem oxygenase-1 as a marker for oxidative stress, we found a strong enhancement of gene expression by beta-carotene in ultraviolet A-irradiated cells. This effect was clearly suppressed by concomitant addition of vitamin E but only moderately by vitamin C. The results show that beta-carotene has pro-oxidative properties in human skin fibroblasts exposed to ultraviolet-A light.

Antioxidants↗
PubMed1999

Interaction kinetics of tetramethylrhodamine transferrin with human transferrin receptor studied by fluorescence correlation spectroscopy.

We applied fluorescence correlation spectroscopy (FCS) to characterize the interaction dynamics of fluorescence-labeled transferrin with transferrin receptor (hTfR) associates isolated from human placenta. The dissociation constant for the equilibrium binding of TMR-labeled ferri-transferrin to hTfR in detergent free solution was determined to be 7 +/- 3 nM. Binding curves were compatible with equal and independent binding sites present on the hTfR associates. Under pseudo-first-order conditions, with respect to transferrin, complex formation is monophasic. From these curves, association and dissociation rate constants for a reversible bimolecular binding reaction were determined, with (1.1 +/- 0.1) x 10(4) M-1 s-1 for the former and (6 +/- 4) x 10(-)4 s-1 for the latter. In dissociation exchange experiments, biphasic curves and concentration-independent reciprocal relaxation times were determined. From isothermal titration calorimetry experiments, we obtained an enthalpy change of -44.4 kJ/mol associated with the reaction. We thus conclude that the reaction is mainly enthalpy driven.

Calorimetry↗
PubMed1999

Thermodynamics and Structural Studies of the Interaction of Polymyxin B with Deep Rough Mutant Lipopolysaccharides.

Deep rough mutant lipopolysaccharide (ReLPS) dissolved in aqueous solution spontaneously forms supramolecular structures which mainly consist of vesicles. Addition of Polymyxin B (PmB) to these ReLPS vesicles influence the shape of these structures as demonstrated here by electronmicroscopy and dynamic light scattering techniques. The main phase transition of the ReLPS is found at 21.3 +/- 0.1 degrees C for ReLPS from Escherichia coli and at 24.0 +/- 0.5 degrees C for ReLPS from Salmonella minnesota by differential scanning calorimetry (DSC). Using isothermal differential titration calorimetry (ITC), the thermodynamic behavior of the interaction of PmB with ReLPS vesicles has been studied. The stoichiometric ratio for the binding of PmB to ReLPS was found to lie between 0.6 and 1, as determined from ITC and monolayer experiments. No phase transition was observed for ReLPS monolayers saturated with PmB. The results indicate specific interaction of PmB with ReLPS. We propose a two-step mechanism for this interaction, which involves electrostatic attraction between charged parts of the molecules and, in the second step, hydrophobic interactions between the nonpolar parts of both compounds. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

Location of translational initiation factor IF3 on the small ribosomal subunit.

The location of translational initiation factor IF3 bound to the 30S subunit of the Thermus thermophilus ribosome has been determined by cryoelectron microscopy. Both the 30S.IF3 complex and control 30S subunit structures were determined to 27-A resolution. The difference map calculated from the two reconstructions reveals three prominent lobes of positive density. The previously solved crystal structure of IF3 fits very well into two of these lobes, whereas the third lobe probably arises from conformational changes induced in the 30S subunit as a result of IF3 binding. Our placement of IF3 on the 30S subunit allows an understanding in structural terms of the biochemical functions of this initiation factor, namely its ability to dissociate 70S ribosomes into 30S and 50S subunits and the preferential selection of initiator tRNA by IF3 during initiation.

Cell Fractionation↗
PubMed1999

Effect of buffer conditions on the position of tRNA on the 70 S ribosome as visualized by cryoelectron microscopy.

The effect of buffer conditions on the binding position of tRNA on the Escherichia coli 70 S ribosome have been studied by means of three-dimensional (3D) cryoelectron microscopy. Either deacylated tRNAfMet or fMet-tRNAfMet were bound to the 70 S ribosomes, which were programmed with a 46-nucleotide mRNA having AUG codon in the middle, under two different buffer conditions (conventional buffer: containing Tris and higher Mg2+ concentration [10-15 mM]; and polyamine buffer: containing Hepes, lower Mg2+ concentration [6 mM], and polyamines). Difference maps, obtained by subtracting 3D maps of naked control ribosome in the corresponding buffer from the 3D maps of tRNA.ribosome complexes, reveal the distinct locations of tRNA on the ribosome. The position of deacylated tRNAfMet depends on the buffer condition used, whereas that of fMet-tRNAfMet remains the same in both buffer conditions. The acylated tRNA binds in the classical P site, whereas deacylated tRNA binds mostly in an intermediate P/E position under the conventional buffer condition and mostly in the position corresponding to the classical P site, i. e. in the P/P state, under the polyamine buffer conditions.

Acylation↗