Search PubMed⌕ Search

Biomedical subjects

J Frank

Publications and source records attributed to J Frank.

At least 91 records · Page 5Linked to original sources
PubMed2000

Visualization of tRNA movements on the Escherichia coli 70S ribosome during the elongation cycle.

Three-dimensional cryomaps have been reconstructed for tRNA-ribosome complexes in pre- and posttranslocational states at 17-A resolution. The positions of tRNAs in the A and P sites in the pretranslocational complexes and in the P and E sites in the posttranslocational complexes have been determined. Of these, the P-site tRNA position is the same as seen earlier in the initiation-like fMet-tRNA(f)(Met)-ribosome complex, where it was visualized with high accuracy. Now, the positions of the A- and E-site tRNAs are determined with similar accuracy. The positions of the CCA end of the tRNAs at the A site are different before and after peptide bond formation. The relative positions of anticodons of P- and E-site tRNAs in the posttranslocational state are such that a codon-anticodon interaction at the E site appears feasible.

Cryoelectron Microscopy↗
PubMed2000

A ratchet-like inter-subunit reorganization of the ribosome during translocation.

The ribosome is a macromolecular assembly that is responsible for protein biosynthesis following genetic instructions in all organisms. It is composed of two unequal subunits: the smaller subunit binds messenger RNA and the anticodon end of transfer RNAs, and helps to decode the mRNA; and the larger subunit interacts with the amino-acid-carrying end of tRNAs and catalyses the formation of the peptide bonds. After peptide-bond formation, elongation factor G (EF-G) binds to the ribosome, triggering the translocation of peptidyl-tRNA from its aminoacyl site to the peptidyl site, and movement of mRNA by one codon. Here we analyse three-dimensional cryo-electron microscopy maps of the Escherichia coli 70S ribosome in various functional states, and show that both EF-G binding and subsequent GTP hydrolysis lead to ratchet-like rotations of the small 30S subunit relative to the large 50S subunit. Furthermore, our finding indicates a two-step mechanism of translocation: first, relative rotation of the subunits and opening of the mRNA channel following binding of GTP to EF-G; and second, advance of the mRNA/(tRNA)2 complex in the direction of the rotation of the 30S subunit, following GTP hydrolysis.

Biological Transport↗
PubMed2000

Colloidal properties of human transferrin receptor in detergent free solution.

The colloidal properties of transferrin receptor, isolated from human placenta, in detergent free solution has been investigated by light scattering techniques and analytical ultracentrifugation. In detergent free solution at 293.2 K, hTfR forms stable aggregates with an apparent hydrodynamic radius of 17 nm. The molecular mass was determined by ultracentrifugation to lie between (1722+/-87) kDa (sedimentation equilibrium) and (1675+/-46) kDa (sedimentation velocity). This implies that the aggregates are build up from nine hTfR dimers. Based on model calculations, which are in good agreement with the experimental data, we propose a torus-like structure for the aggregates. Upon pH shift from pH 7.5 to 5.0 or removal of the N-linked carbohydrate chains, formation of larger aggregates is induced. These aggregates can be described in terms of porous fractal structures. We propose a simple model, which accounts for that behaviour assuming that the aggregation is mainly due to the reduction of negative surface charge.

Circular Dichroism↗
PubMed2000

Three-dimensional cryo-electron microscopy localization of EF2 in the Saccharomyces cerevisiae 80S ribosome at 17.5 A resolution.

Using a sordarin derivative, an antifungal drug, it was possible to determine the structure of a eukaryotic ribosome small middle dotEF2 complex at 17.5 A resolution by three-dimensional (3D) cryo-electron microscopy. EF2 is directly visible in the 3D map and the overall arrangement of the complex from Saccharomyces cerevisiae corresponds to that previously seen in Escherichia coli. However, pronounced differences were found in two prominent regions. First, in the yeast system the interaction between the elongation factor and the stalk region of the large subunit is much more extensive. Secondly, domain IV of EF2 contains additional mass that appears to interact with the head of the 40S subunit and the region of the main bridge of the 60S subunit. The shape and position of domain IV of EF2 suggest that it might interact directly with P-site-bound tRNA.

Cryoelectron Microscopy↗
PubMed2000

Potentiometric detection of carboxylic acids, phosphate esters, and nucleotides in liquid chromatography using anion-selective coated-wire electrodes.

An all-solid-state ion-selective membrane electrode incorporating a lipophilic anion exchanger was used in a flow-through potentiometric detector for the LC determination of organic anions of biological interest. Different metabolic intermediates (mono-, di-. and tricarboxylic acids, sugar phosphates, and nucleotides) were detected sensitively after separation on a pellicular anion-exchange chromatographic column. The electrode was coated by directly casting the electroactive mixture on a glassy carbon support of 3 mm diameter and used in a wall-jet-type flow cell. The analysis conditions were optimized to obtain both efficient separation and sensitive detection. Calibration curves showed a logarithmic dependence on the injected concentration for concentrations higher than 5.0 x 10(-5) M and a linear dependence for injected concentrations below this value. Under isocratic conditions, detection limits of 5.0 x 10(-7) M (25 pmol) were attained when a sodium hydroxide solution was used as an eluent. No suppressor system was needed in this case. The relative standard deviation for consecutive injections was 0.3% (n = 15), and the electrode lifetime was at least 2 months. The utility of potentiometric detection is further demonstrated in a gradient elution separation for single-run analysis of a synthetic mixture of biochemical compounds containing carboxylic acids, phosphate esters, and nucleotides.

Algorithms↗
PubMed2000

Mid-infrared quantum cascade lasers for flow injection analysis

A Fabry-Perot quantum cascade laser (QCL) was used as a powerful light source for mid infrared (MIR) detection in flow injection analysis. The QCL lased at several wavelengths close to each other within a few wavenumbers (990-1010 cm-1), hence fitting well to the broad absorption bands of molecules in liquid phase. As compared with that of a state-of-the-art Fourier transform spectrometer, the signal-to-noise ratio could be improved by a factor of 50. Additionally, by using a QCL as the light source, optical path lengths of more than 100 microns could be used even in aqueous matrixes which reduced the danger of cell clogging. In the example shown here phosphate was determined in Diet Coke samples. The flow injection system used allowed the measurement of the sample at two pH values (5 and 13) at which the analyte was present as H2PO4- and PO4(3-), respectively. As the analytical readout the difference in IR absorption of H2PO4- and PO4(3-) at the laser wavelengths was taken. The FIA-QCL measurements were corroborated by ion chromatography which was used as a reference method.

Journal Article↗
PubMed2000

Solution structure of the E. coli 70S ribosome at 11.5 A resolution.

Over 73,000 projections of the E. coli ribosome bound with formyl-methionyl initiator tRNAf(Met) were used to obtain an 11.5 A cryo-electron microscopy map of the complex. This map allows identification of RNA helices, peripheral proteins, and intersubunit bridges. Comparison of double-stranded RNA regions and positions of proteins identified in both cryo-EM and X-ray maps indicates good overall agreement but points to rearrangements of ribosomal components required for the subunit association. Fitting of known components of the 50S stalk base region into the map defines the architecture of the GTPase-associated center and reveals a major change in the orientation of the alpha-sarcin-ricin loop. Analysis of the bridging connections between the subunits provides insight into the dynamic signaling mechanism between the ribosomal subunits.

Bacterial Proteins↗
PubMed2000

[The quality of the treatment of diabetics in kidney failure in Germany].

BACKGROUND AND OBJECTIVE: In recent years there has been a rapid increase in the number of dialysis-dependent diabetics in Germany. Survival on dialysis is not satisfactory and damage acquired in the preterminal stage of renal failure is thought to play an important role. Late referral to a nephrologist and insufficient quality of medical management are thought to contribute importantly to poor outcome. This hypothesis was evaluated in the present study. PATIENTS AND METHOD: The data of all 173 diabetic patients (16 with type 1, 157 with type 2 diabetes, 90 men, 83 women, mean age 63.3 [31-95] years), who had been referred in 1996 for the first time to five renal units, were retrospectively assessed using a structured protocol. RESULTS: Patients were usually referred in advanced renal failure (median creatinine clearance 29 ml/min, range 1-216) with insufficient control of systolic (170 [120-260] mmHg) and diastolic blood pressure (90 [60-180] mmHg), insufficient antihypertensive therapy (without treatment 32 of 173 patients; median number of classes of antihypertensive agents used 2 [range 1-6]; ACE inhibitors 79 of 173 patients), high HbA1c (7.9 [4.9-15.7]%) and LDL cholesterol (176 [67-307] mg/dl). Immediate dialysis was required in 45 patients. CONCLUSION: The data document insufficient quality of treatment and late incorporation of a nephrologist into the medical team involved in the care of diabetic patients. Changes in the structure of diabetes care are necessary to improve treatment quality.

Adult↗
PubMed2000

Prognostic factors for time receiving workers' compensation benefits in a cohort of patients with low back pain.

STUDY DESIGN: Prospective inception cohort study. OBJECTIVE: To develop a prognostic model that predicts time receiving workers' compensation benefits for low back pain claimants. SUMMARY OF BACKGROUND DATA: As the cost and difficulty of managing low back pain escalate, any predictor of outcome is advantageous. METHODS: To obtain the outcome and predictor variables, patient data from two separate databases were linked: a clinical database and an administrative (Ontario workers' compensation) database. Claimants injured between January 1 and December 31, 1994, were included and observed for 1 year from the date of accident. The outcome variable was cumulative number of calendar days receiving benefits. RESULTS: Multivariable Cox proportional hazards regression (forward stepwise) showed eight significant predictors; five were associated with increased time receiving benefits compared with their reference groups: 1) working in the construction industry, 2) older age, 3) lag time from injury to treatment, 4) pain referred into the leg, and 5) three or more positive Waddell nonorganic signs. Three predictors were associated with reduced time receiving benefits: 1) higher values of questionnaire score, 2) intermittent pain, and 3) a previous episode of back pain. A predictive score was calculated to categorize claimants as at high or low risk for chronicity. When an arbitrary cutoff point was set at the 75th percentile of predictive score, negative predictive value was 94%. CONCLUSION: This research identified eight factors for time receiving workers' compensation benefits among claimants with low back pain. This model discriminates between high- and low-risk claimants. Few low-risk claimants continued to receive benefits for more than 3 months.

Adult↗
PubMed2000

Osteomyocutaneous flap as a preclinical composite tissue allograft: swine model.

Composite tissue allotransplantation (CTA) constitutes one of the last frontiers of microsurgery. Prior to its clinical application, the long-term efficacy of modern immunotherapy must be tested in a pre-clinical CTA model. Based on the concept of osteomyocutaneous forearm flap, we developed a CTA flap model in swine. After identifying the vascular territory of the flaps in six pigs (vascular casting), flaps were transplanted from mismatched donor to recipient pigs (n = 6). Rejection was assessed daily by visual inspection and histopathology of biopsy specimens. Recipient pigs were able to ambulate immediately following surgery. There were no flap failures owing to technical or surgical complications. Rejection occurred over a period of 7 days as manifested by edema, cellular infiltration, epidermalysis, and thrombosis. This pre-clinical flap model is excellent for evaluating the effectiveness of modern immunotherapy because it is anatomically and immunologically relevant and because the minimal morbidity caused to the animal permits long-term studies.

Animals↗
PubMed2000

Recent advances in affinity capillary electrophoresis.

Use of the specificity of (bio)interactions can effectively overcome the selectivity limitation faced in capillary electrophoresis (CE), and the resulting technique usually is referred to as affinity capillary electrophoresis (ACE). Despite the high selectivity of ACE, several important problems still need to be addressed. A major issue in all CE separations, including ACE, is the concentration detection limit. Using UV detection, this is usually in the order of 10(-6) M whereas laser-induced fluorescence (LIF) detection can provide detection limits down to the sub-10(-10) M range. However, a marked disadvantage of LIF is that labeling of the analytes is usually required, which might change the interaction behavior of the solutes under investigation. Additionally, labeling reactions at sub-10(-10) M concentration levels are certainly not trivial and often difficult to perform quantitatively. Alternative and universal detection approaches, particularly mass spectrometric (MS) detection, look very promising but (A) CE-MS techniques are still far from routine application. Important future progress in sensitive detection strategies is likely to increase the use of ACE in the future.

Electrophoresis, Capillary↗
PubMed2000

Effects of dietary phenolic compounds on tocopherol, cholesterol, and fatty acids in rats.

The effects of the phenolic compounds butylated hydroxytoluene (BHT), sesamin (S), curcumin (CU), and ferulic acid (FA) on plasma, liver, and lung concentrations of alpha- and gamma-tocopherols (T), on plasma and liver cholesterol, and on the fatty acid composition of liver lipids were studied in male Sprague-Dawley rats. Test compounds were given to rats ad libitum for 4 wk at 4 g/kg diet, in a diet low but adequate in vitamin E (36 mg/kg of gamma-T and 25 mg/kg of alpha-T) and containing 2 g/kg of cholesterol. BHT significantly reduced feed intake (P < 0.05) and body weight and increased feed conversion ratio; S and BHT caused a significant enlargement of the liver (P < 0.001), whereas CU and FA did not affect any of these parameters. The amount of liver lipids was significantly lowered by BHT (P < 0.01) while the other substances reduced liver lipid concentrations but not significantly. Regarding effects on tocopherol levels, (i) feeding of BHT resulted in a significant elevation (P< 0.001) of alpha-T in plasma, liver, and lung, while gamma-T values remained unchanged; (ii) rats provided with the S diet had substantially higher gamma-T levels (P < 0.001) in plasma, liver, and lung, whereas alpha-T levels were not affected; (iii) administration of CU raised the concentration of alpha-T in the lung (P < 0.01) but did not affect the plasma or liver values of any of the tocopherols; and (iv) FA had no effect on the levels of either homolog in the plasma, liver, or lung. The level of an unknown substance in the liver was significantly reduced by dietary BHT (P < 0.001). BHT was the only compound that tended to increase total cholesterol (TC) in plasma, due to an elevation of cholesterol in the very low density lipoprotein + low density lipoprotein (VLDL + LDL) fraction. S and FA tended to lower plasma total and VLDL + LDL cholesterol concentrations, but the effect for CU was statistically significant (P < 0.05). FA increased plasma high density lipoprotein cholesterol while the other compounds reduced it numerically, but not significantly. BHT, CU, and S reduced cholesterol levels in the liver TC (P < 0.001) and percentages of TC in liver lipids (P < 0.05). With regard to the fatty acid composition of liver lipids, S increased the n-6/n-3 and the 18:3/20:5 polyunsaturated fatty acids (PUFA) ratios, and BHT lowered total monounsaturated fatty acids and increased total PUFA (n-6 + n-3). The effects of CU and FA on fatty acids were not highly significant. These results suggest some in vivo interactions between these phenolic compounds and tocopherols that may increase the bioavailability of vitamin E and decrease cholesterol in rats.

Animals↗
PubMed2000

Domain motions of EF-G bound to the 70S ribosome: insights from a hand-shaking between multi-resolution structures.

Molecular modeling and information processing techniques were combined to refine the structure of translocase (EF-G) in the ribosome-bound form against data from cryoelectron microscopy (cryo-EM). We devised a novel multi-scale refinement method based on vector quantization and force-field methods that gives excellent agreement between the flexibly docked structure of GDP. EF-G and the cryo-EM density map at 17 A resolution. The refinement reveals a dramatic "induced fit" conformational change on the 70S ribosome, mainly involving EF-G's domains III, IV, and V. The rearrangement of EF-G's structurally preserved regions, mediated and guided by flexible linkers, defines the site of interaction with the GTPase-associated center of the ribosome.

Computer Simulation↗
PubMed2000

Alveolar epithelial barrier. Role in lung fluid balance in clinical lung injury.

Several studies have established that transport of sodium from the air spaces to the lung interstitium is a primary mechanism driving alveolar fluid clearance, although further work is needed to determine the role of chloride in vectorial fluid transport across the alveolar epithelium. Although there are significant differences among species in the basal rates of sodium and fluid transport, the basic mechanism seems to depend on sodium uptake by channels on the apical membrane of alveolar type II cells, followed by extrusion of sodium on the basolateral surface by Na,K-ATPase. This process can be upregulated by several catecholamine-dependent and independent mechanisms. The identification of water channels expressed in lung, together with the high water permeabilities, suggest a potential role for channel-mediated water movement between the air space and capillary compartments, although definitive evidence will depend on the results of transgenic mouse knock-out studies. The application of this new knowledge regarding salt and water transport in alveolar epithelium in relation to pathologic conditions has been successful in clinically relevant experimental studies, as well as in a few clinical studies. The studies of exogenous and endogenous catecholamine regulation of alveolar fluid clearance are a good example of how new insights into the basic mechanisms of alveolar sodium and fluid transport can be translated to clinically relevant experimental studies. Exogenous catecholamines can increase the rate of alveolar fluid clearance in several species, including the human lung, and it is also apparent that release of endogenous catecholamines can upregulate alveolar fluid clearance in animals with septic or hypovolemic shock. It is possible that therapy with beta-adrenergic agonists might be useful to accelerate the resolution of alveolar edema in some patients. In some patients, the extent of injury to the alveolar epithelial barrier may be too severe for beta-adrenergic agonists to enhance the resolution of alveolar edema, although some experimental studies indicate that alveolar fluid clearance can be augmented in the presence of moderately severe lung injury. A longer-term upregulation of alveolar epithelial fluid transport might be achieved by strategies that accelerate the proliferation of alveolar type II cells repopulating the injured epithelium in clinical lung injury. More clinical research is needed to evaluate the strategies that can upregulate alveolar epithelial fluid transport with both short-term therapy (i.e., beta-agonists) and more sustained, longer-term effects of epithelial mitogens such as keratinocyte growth factor. These approaches may be useful in reducing mortality in the acute respiratory distress syndrome.

Animals↗
PubMed2000

Histochemical visualization of oxidant stress.

Free radicals induce oxidative modification in distinct components of the living matter (lipid, proteins, and DNA). For qualitative and quantitative determination of free radical-induced modifications, different, more or less sensitive biochemical methods are available. Because of the high reactivity and short life of free radicals, ongoing oxidative damage is generally analyzed by measurement of secondary products-such as H(2)O(2), oxidized proteins, peroxidized lipids, and their breakdown products, oxidized DNA-or by fluorographic analysis in combination with fluorescent dyes such as dichlorofluorescin (DCFH). In addition, the determination of free radical-related oxidation products is usually carried out in plasma, urine, or, less frequently, in bioptic material. Consequently, biochemical data seldom reflect the effects of free radical insults in situ. The histochemical visualization of selected molecular markers of oxidative damage can often provide more valuable information concerning the in vivo distribution of oxidative processes. This review summarizes the methods currently available for histochemical detection and indirect visualization of free radical-induced alterations in tissues and isolated cells.

Animals↗