Biomedical subjects

J Novotny

Publications and source records attributed to J Novotny.

At least 73 records · Page 4Linked to original sources

PubMed1996

Physiological profile of best Czech male and female young triathletes.

To characterize the physiological profile of top young triathletes, 13 top female (mean age = 17.1 +/- 1.4 years, body mass = 58.8 +/- 4.7 kg, body height = 168.4 +/- 2.0 cm and body fat = 10.4 +/- 2.6%) and 23 top male triathletes (age = 17.7 +/- 2.2 years, mass = 66.7 +/- 7.1 kg, height = 176.5 +/- 5.1 cm and fat 8.2 +/- 2.3%) were evaluated by means of an incremental exercise (increment was 1 km.h-1, and exercise starting at 11 km.h-1 in females and 13 km.h-1 in males) on a treadmill with 5% inclination. Mean VO2max was 67.9 +/- 5.9 ml.kg-1 min-1 in boys and 56.1 +/- 2.4 ml.kg-1.min-1 in girls. mean value of maximal running speed was 18.6 +/- 1.2 km.h-1 in mean and 15.4 +/- 0.6 km.h-1 in females and LAmax was 12.5 +/- 2.3 mmol.l-1 in boys and 12.6 +/- 1.2 mmol.l-1 in girls. The selected functional variables at ventilatory threshold (VT) level in boys and girls corresponded to VO2max.kg-1 56.0 +/- 5.4 and 46.6 +/- 2.6 ml.kg-1.min-1, respectively, %VO2max.kg-1 at VT 82.4 +/- 2.1 and 83.1 +/- 1.7%, respectively, speed of running 15.2 +/- 1.4 and 12.7 +/- 0.7 km.h-1, respectively, %Vmax at VT 81.8 +/- 2.6 and 82.3 +/- 1.6%, respectively and the coefficient of energy cost of running c was 3.74 +/- 0.42 and 3.71 +/- 0.39 J.kg-1.m-1, respectively. A comparison of the functional profiles of these triathletes with elite young athletes from the sports of swimming (age 17.5 +/- 2.0 and 17.2 +/- 1.7 years, respectively, VO2max 61.6 +/- 3.6 and 52.1 +/- 3.6 ml.kg-1. min-1, respectively, Vmax 17.5 +/- 0.8 and 15.0 +/- 0.9 km.h-1, respectively, LAmax 11.1 +/- 3.2 and 11.8 +/- 3.3 mmol.l-1, respectively) cycling (17.7 +/- 1.8-17.0 +/- 1.7 years; 65.4 +/- 5.1-55.1 +/- 2.4 ml.kg-1. min-1, 18.2 +/- 0.7-15.2 +/- 0.8 km.h-1; 13.3 +/- 3.5-12.9 +/- 3.7 mmol.l-1) and middle-distance running (17.8 +/- 1.9-17.2 +/- 2.1 years; 66.8 +/- 4.7-57.3 +/- 2.6 ml.kg-1.min-1, 19.1 +/- 0.9-16.1 +/- 0.9 km.h-1; 13.1 +/- 2.6-13.7 +/- 3.0 mmol.l-1) showed the physiological characteristics of triathletes to be similar to those of middle-distance runners. According to our results and according to the data from the literature we can conclude that physiological predispositions for success in international triathlon may be as follows in boys and girls: VO2max.kg-1 higher than 65 and 60 ml.kg-1 min-1, respectively, Vmax (5%) higher than 18 and 16 km.h-1, respectively, LAmax higher than 12 and 11 mmol.l-1, respectively, running speed at "anaerobic threshold" higher than 15.0 and 13 km.h-1, respectively, %VO2max at "anaerobic threshold" level higher than 82.5% in both sexes, and the coefficient of energy cost of running lower than 3.75 and 3.73 J.kg-1.m-1, respectively. As in other sports events of an endurance native, these data are not the sole predictor of racing success. Nevertheless these standards are necessary but not sufficient conditions for success in triathlon. These data play a decisive role in the selection of talent for the triathlon.

Adipose Tissue↗

PubMed1995

The crystal structure of the antibody N10-staphylococcal nuclease complex at 2.9 A resolution.

The three-dimensional structure of the antibody N10 Fab fragment complexed with staphylococcal nuclease (SNase) has been determined to 2.9 A resolution. Eighteen residues from six complementarity-determining regions (CDR) recognize an epitope of five distinct SNase segments with a total of 17 residues. The overall shape of the antibody-antigen interface is U-shaped rather than the more or less rectangular interface seen in other antibody-protein antigen interfaces. Despite the U-shaped interface, the amount of surface buried in the complex, 828 A2 for SNase and 793 A2 for N10, is typical of antibody-protein antigen complexes. Contributing to the shape of the interface is the shortest antibody heavy chain-CDR3 loop reported to date, which probably allows access of bulk solvent in the center of the "U" interface. Another unusual feature of the N10 antibody is the 15 residue antibody light chain-CDR1, a length seen in only three other reported antibodies. Antibody light chain-CDR1 displays a previously unobserved conformation in its distal portion. Finally, although some of the movement observed in the antibody-bound SNase may be due to crystal contacts, it is clear that some side-chain rearrangements are the result of antigen-antibody interaction.

Amino Acid Sequence↗

PubMed1995

Conformational similarity and systematic displacement of complementarity determining region loops in high resolution antibody x-ray structures.

Comparison of seven high resolution x-ray structures shows that the conformations of canonical complementarity determining region (CDR) loops, which are shared by these antibodies, are very similar. However, large spatial displacements (up to 2.7 A) of the essentially identical CDR loops become evident when the antibody beta-sheet frameworks, to which the loops are attached, are least-squares superposed. The loop displacements follow, and amplify, small positional differences in framework/loop splice points. Intradomain structural variability and, to a lesser extent, domain-domain orientation appear to cause the observed loop divergences. The results suggest that the selection of framework regions for loop grafting procedures is more critical than previously thought.

Amino Acid Sequence↗

PubMed1995

Evolutionary conservation of both the hydrophilic and hydrophobic nature of transmembrane residues.

An algorithm (HRG), developed to allow the pairwise comparisons of the aligned residues of several members of large gene families of polytopic integral membrane proteins is described. Using hydrophobicity scales, application of this algorithm allows the number and size of the membrane-spanning domains of bacteriorhodopsin, a polytopic protein whose structure has been partially determined, to be predicted with a high degree of accuracy (sensitivity 94%, specificity 82% for predicting the membrane embedded or extramembranous location of residues). As opposed to previously reported structure-prediction algorithms, delineation of putative transmembrane segments from connecting loops is also more clearly evident with the application of the HRG algorithm, even with proteins from widely divergent species. This indicates strong evolutionary pressure for the conservation of both the hydrophobic and hydrophilic character of residues in membrane-embedded regions of polytopic proteins, such as those of the G-protein-coupled receptor superfamily. These and other structural and functional implications evident from the application of the HRG algorithm are considered.

Algorithms↗

PubMed1995

Characterization of a new four-chain coiled-coil: influence of chain length on stability.

Limited information is available on inherent stabilities of four-chain-coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four-chain coiled-coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four-chain coiled-coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain alpha-helical chains 21, 28, and 35 amino acids in length are 3.1 x 10(-12), 6.7 x 10(-23), and 1.0 x 10(-38) M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 microM, 51 nM, and 280 fM, respectively. Finally, we discuss the rules governing coiled-coil formation in light of the work presented here.

Amino Acid Sequence↗

PubMed1995

Prolonged exposure of hamsters to cold changes the levels of G proteins in brown adipose tissue plasma membranes.

The levels of G proteins in plasma membranes prepared from brown adipose tissue of control and cold-exposed hamsters were determined by quantitative immunoblotting and competitive ELISA. Prolonged (four weeks) exposure of hamsters to cold decreased significantly the total content of the alpha subunits of the stimulatory (Gs alpha) as well as inhibitory (Gi alpha (1,2)) G proteins. Interestingly, the reduction in the Gs alpha content was solely due to a large reduction in the content of the short (45 kDa) isoform of Gs alpha, while the level of the long (52 kDa) isoform of Gs alpha remained unchanged. The level of the beta subunit of G protein was decreased comparably to the reduction in the total content of the alpha subunits. Cold-induced alterations in the G protein network associated with plasma membranes of brown adipose tissue were accompanied by changed characteristics of AlF(4-)-sensitive adenylyl cyclase activity.

Adenylyl Cyclases↗

PubMed1995

Characterization of elements determining the dimerization properties of RelB and p50.

Members of the Rel/NF-kappa B family of transcription factors share a region of approximately 300 amino acids which mediates dimerization and sequence-specific binding to DNA. Here we report a detailed characterization of the dimerization domain of RelB. The structural core sufficient to form stable Rel/NF-kappa B dimeric complexes consists of about 110 residues. The dimerization and DNA binding properties of more than 50 RelB mutants were analyzed by using p50 and p52 as partners. We present evidence that amino acids of a conserved element in the dimerization domain play a role in the recognition of a kappa B DNA target sequence. The analysis of hybrid molecules with dimerization domains containing different parts of p50 and RelB allowed us to identify some important structural elements determining homo- and heterodimerization properties. Furthermore, we were able to rescue the dimerization-defective mutant RelB-N287D by the introduction of a counteracting mutation intramolecularly (cis), and also intermolecularly (trans) by a mutation in the NF-kappa B dimerization partner p50. Correspondingly, a dimerization defective p50 mutant was effectively rescued by RelB-N287D.

Amino Acid Sequence↗

PubMed1995

Quality assurance network in central Europe. External audit on output calibration for photon beams.

The EROPAQ project for TLD monitoring of photon beams started in June 1994 with the set-up of the TLD system: calibration, reading and evaluation procedures. The acceptance level of +/- 3% was set for the TLD intercomparisons. The policy of the project was to check all beams in 47 participating radiotherapy centres and to recheck all the beams in those centres, where a deviation exceeding +/- 3% occurred in one or more of the beams. Out of 129 beams checked, 100 beams (78%) were found within the +/- 3% limit. Eleven beams show deviations larger than +/- 6%, and immediate corrective action was undertaken. Out of 47 centres checked, 22 did not participate in any external audit in a preceding 5 years. In these centres 68% (34/50) of the total number of gamma and x-ray beams checked but only 59% (20/34) of gamma beams were within the acceptance level, while in the 25 centres, which participated in an external audit before, these figures were 84% (66/79) and 88% (35/40) respectively. The sources of discrepancies were thoroughly investigated, discussed with the participants and the errors corrected. Poor results were in several cases associated with very old design of radiotherapy units and old dosimetry systems, equipped with inadequate ionization chambers. In several centres, an insufficient training of the physicists in clinical dosimetry was observed. Thanks to the corrective action, a great improvement of calibration of the beams was achieved. Standard deviation of the distribution of the results for all x and gamma beams checked decreased from SD = 7.4% at the first check to SD = 2.5% at the second check.

Calibration↗

PubMed1995

Isoproterenol-induced subcellular redistribution of G-protein beta subunits in S49 lymphoma cells demonstrated by a novel competitive ELISA.

A novel competitive ELISA has been developed for the determination of levels of the beta subunit of guanine-nucleotide-binding protein (G-protein) using antipeptide antibodies directed against the amino terminus of the beta subunit. Because beta subunits form highly hydrophobic.heterodimeric complexes with gamma subunits of G-proteins, specific assay conditions were required. Optimal concentrations of antibodies, detergents, Mg2+ as well as ionic strength were determined. In addition, we found that an effective binding of the used antibodies to the beta subunit was ensured only after denaturation of the beta gamma complexes. Subsequently, this ELISA was used for quantitation of the beta subunit in subcellular fractions of S49 lymphoma cells during isoproterenol-mediated desensitization of beta-adrenergic controlled transmembrane signalling system. A 10 min as well as 60 min treatment of the cells with isoproterenol (1 nmol/ml) resulted in a significant shift of G-protein beta subunits (presumably as beta gamma complexes) from the plasma membrane fractions to low-density microsomal fractions. No significant change was detected after the hormone action in the distribution of plasma membrane constitutive enzymes. In conclusion, the developed ELISA helped us to reveal that beta-adrenergic stimulation can induce redistribution of the beta gamma dimer from plasma membranes to low-density microsomes.

Adrenergic beta-Agonists↗

PubMed1995

In vivo/ex vivo T cell depletion reduces the morbidity of allogeneic bone marrow transplantation in patients with acute leukaemias in first remission without increasing the risk of treatment failure: comparison with cyclosporin/methotrexate.

We have performed a non-randomised GVHD prophylaxis trial comparing cyclosporin/methotrexate with in vivo/ex vivo T cell depletion with the monoclonal antibodies Campath 1G/1M in patients with acute leukaemias in first complete remission. We observed significantly less acute and chronic GVHD, neutropenic fever and severe mucositis in the T cell depletion group. The incidence of graft rejection and relapses was no higher than in the cyclosporin/methotrexate group. There is a trend in favour of improved disease-free survival in the in vivo/ex vivo T cell depletion group (80% vs. 62%).

Acute Disease↗

PubMed1995

Successful therapy with donor buffy coat transfusions in patients with relapsed chronic myeloid leukemia after bone marrow transplantation is associated with high frequencies of host-reactive interleukin 2-secreting T helper cells.

Six patients treated for relapsed chronic myeloid leukaemia after allogeneic bone marrow transplantation with donor buffy coat transfusions were investigated. In the 5 patients who achieved molecular remission high frequencies of host-reactive interleukin 2-secreting T helper cell precursors (Th-p) were detectable by limiting dilution analysis. In four of the patients the presence of Th-p was associated with a clinical syndrome similar to transfusion GVHD suggesting a T cell response to minor histocompatibility antigens (minor H) expressed by both malignant haemopoiesis and host tissues. In the fifth responding patient no GVHD or bone marrow hypoplasia was observed. The nature of the antigens recognised by these donor Th-p remains unknown. No host-reactive Th-p were detectable in the non-responder and host-reactive cytotoxic T cell precursors (CTL-p) were not consistently detectable in the responding patients.

Adult↗

PubMed1995

Alterations in G-protein-regulated transmembrane signalling induced in murine myocardium by coxsackievirus B3 infection.

OBJECTIVE: Cardiomyopathy is usually associated with marked alterations in myocardial transmembrane signalling. Although acute viral myocarditis may result in chronic cardiomyopathy in some cases, the possible consequences of viral infection on function of the myocardial signal-transducing complex have not been explored. Therefore, the present study was designed to investigate the G-protein-regulated adenylyl cyclase signalling system in murine myocardium during myocarditis induced by coxsackievirus B3 (CVB3) infection. METHODS: We examined the functional characteristics of adenylyl cyclase as well as the function and distribution of beta-adrenoceptors, m-cholinoceptors and G-proteins in myocardial plasma membranes isolated from the hearts of mice with acute (7 days pi) or late phase (21 days pi) myocarditis and the obtained results were compared with the corresponding data determined in age-matched controls. RESULTS: While the basal adenylyl cyclase activity was not significantly altered, the ability of forskolin, sodium fluoride and GTP gamma S to activate adenylyl cyclase was lowered by about 20% in samples from virus-infected animals. The level of Gs alpha in myocardial plasma membranes as well as the functional activity of Gs alpha was not affected by viral infection, but the Gi alpha content was increased by about 20%. The total number of beta-adrenoceptors in myocardial plasma membranes increased by about 12-15% due to higher content of the beta 2-adrenoceptor subtype. Although the agonist-binding parameters of beta-adrenoceptors were not significantly altered, the ability of these receptors to mediate stimulation of adenylate cyclase was markedly diminished (by 56-80%). The total number of m-cholinoceptors in samples derived from virus-infected mice increased considerably (by 29-59%) and a significant proportion of the receptors shifted to a higher affinity status, but their ability to transduce agonist signals was impaired. CONCLUSIONS: These data are the first to demonstrate that several different sites of the myocardial G-protein-regulated adenylyl cyclase signalling complex are significantly altered in acute as well as in late phase of CVB3-induced myocarditis.

Acute Disease↗

PubMed1995

Model building of antibody combining sites.

The number of high quality crystal structures of antibody fragments available in the Brookhaven Protein Data Bank is rapidly increasing. These structures provide a substantial knowledge base and support model building of novel antibody combining sites. We review some basic principles of antibody structure, describe structure-based modelling procedures, and indicate the strengths and weaknesses of the modelling approach. Applications of antibody models are discussed.

Animals↗

PubMed1994

The short and long forms of the alpha subunit of the stimulatory guanine-nucleotide-binding protein are unequally redistributed during (-)-isoproterenol-mediated desensitization of intact S49 lymphoma cells.

We report here that desensitization of the beta-adrenergic receptor-triggered transmembrane signalling in S49 wild-type lymphoma cells, induced by (-)-isoproterenol (1 microM), results in unequal intracellular redistribution of the splicing variants of the alpha subunit of the stimulatory guanine-nucleotide-binding regulatory (Gs alpha) protein (Gs alpha-short and Gs alpha-long) and alters the functional characteristics of the membrane-associated signal transduction complex. We found that two cellular pools of membranes, light-density membranes and plasma membranes prepared by sucrose-density-gradient centrifugation of cell homogenates differed in their content of Gs alpha splicing subforms and, moreover, that prolonged activation of the beta-adrenergic pathway induced intermembrane redistribution of the splicing variants of Gs alpha. Short (10 min) as well as prolonged (1 h) (-)-isoproterenol treatment of the cells shifted Gs alpha-short from light-density membranes to plasma membranes and increased the total amount of light-density membrane-bound Gs alpha-long; in parallel, the maximal (-)-isoproterenol-stimulated or AlF4(-)-stimulated adenylyl cyclase activities measured in the plasma membrane pools prepared from treated cells decreased. The functional characteristics of the membrane-bound Gs alpha pools were examined by a cyc(-)-reconstitutive adenylyl cyclase assay where extracts of the plasma membrane and light-density-membrane pools, respectively, were mixed with plasma membranes derived from the mutant S49 cell line, cyc-, lacking Gs alpha. The maximal cyc(-)-reconstitutive activities of the extracts prepared from light-density membranes of short-term as well as long-term desensitized cells increased compared to control cells. These findings may indicate differences in the functioning of the splicing variants of Gs alpha.

Adenylyl Cyclases↗

PubMed1994

N9 neuraminidase complexes with antibodies NC41 and NC10: empirical free energy calculations capture specificity trends observed with mutant binding data.

X-ray crystallographic coordinates of influenza virus N9 neuraminidase complexed with monoclonal antibodies NC41 and NC10 [Tulip et al. (1992) J. Mol. Biol. 227, 122-148] served as a starting point for calculations aimed at estimating free energy changes (delta G) of complex formation between the two antibodies and the neuraminidase. Using an empirical function incorporating hydrophobic, electrostatic, and conformational entropy effects, we estimated contributions individual neuraminidase residues make to complex formation (delta G(residue)) and compared the calculated values to experimentally measured differences in antibody binding between the wild-type and mutated neuraminidases [Nuss et al. (1993) Proteins 15, 121-132; calculations done without prior knowledge of the experimental data]. A good correspondence was found between the calculated delta G(residue) values and the mutant binding data in that side chains with large calculated delta G contributions (delta G(residue) < -1 kcal/mol) lie at sites of mutation which cause a marked reduction in antibody binding, and side chains for which delta G(residue) > -1 kcal/mol are sites at which a mutation does not have a marked effect on binding. Because most of the delta G(residue) < -1 kcal/mol side chains also make hydrogen bonds/salt bridges with the antibody, the correspondence of the effect of antibody binding with these electrostatic interactions (18 out of 27 for NC41 and, tentatively, 5 out of 7 for NC10) is about as good as that with predicted energetic residues. All the delta G(residue) < -1 kcal/mol neuraminidase side chains cluster around the most protruding surface regions and are thus spread over different epitope segments. Surprisingly, different residues were found to make the most critical contributions to the NC41 and NC10 complex stabilities despite the fact that the NC41 and NC10 antigenic epitopes overlap, having approximately 70% of surface residues in common. It is thus possible, for two different antibodies, to recognize the same protein surface in strikingly different ways. As only a fraction of the neuraminidase residues appear to make large contributions to antibody binding, the results also support the hypothesis of a "functional" epitope in antigen-antibody interactions. Positive trends between both backbone rigidity and residue accessibility in the complexed state, and contributions of these residues to binding, were also observed for the NC41 complex.

Antibodies, Monoclonal↗

PubMed1994

Activated Gs alpha but not Gi alpha prevents the thermal inactivation of adenylyl cyclase in plasma membranes derived from S49 lymphoma cells.

The thermal inactivation of adenylyl cyclase was studied in plasma membranes isolated from wild-type and the mutant cell strain cyc- of S49 lymphoma. The half-life of adenylyl cyclase activity at 30 degrees C was decreased from 14.2 min to 3.4 min by the presence of detergents. ATP as well as forskolin prevented the adenylyl cyclase inactivation in a dose-response manner independent of the utilized type of cell membranes. Activation of G-proteins by GTP gamma S or by AlF-4 in wild-type membranes but not in cyc- membranes partially prevented adenylyl cyclase inactivation. Adenylyl cyclase activity in cyc- membranes was preserved in the presence of GTP gamma S or AlF-4 from the observed detergent-induced inactivation by complementation of these membranes with an extract from wild-type membranes. ADP-ribosylation of Gi alpha in cyc- membranes did not influence the kinetics of the inactivation process of adenylyl cyclase, whereas ADP-ribosylated Gs alpha protein protected adenylyl cyclase more effectively than non-ribosylated Gs alpha in wild-type plasma membranes when GTP was used as an activator.

Adenosine Triphosphate↗

PubMed1994

Mutation of peptide binding site in transmembrane region of a G protein-coupled receptor accounts for endothelin receptor subtype selectivity.

The molecular basis for endothelin (ET) isopeptide selectivity between ETA and ETB receptors was studied by examining ligand binding to several site-specific mutants of the human ETA receptor. Based on a computer-built three-dimensional model of the ETA receptor, five non-conserved amino acids, clustered around the putative ligand binding site, were targeted for mutation to alanine. Expression of the wild-type and mutant ETA receptors in COS-7 cells revealed that the binding profile of one of the ETA mutants, Tyr129-->Ala, was characteristic of the ETB receptor. In the Tyr129-->Ala ETA receptor mutant the affinity of two ETB-selective agonists, endothelin-3 and sarafotoxin S6c, was increased 10-200-fold, whereas that for two ETA-selective antagonists, BQ-123 and BMS-182874, was reduced 350-2,000-fold. Thus, mutation of a single amino acid in the second transmembrane region of the wild-type ETA receptor results in subtype conversion. In addition, these data represent the first example of peptide interactions with a transmembrane region of a G protein-coupled receptor and indicate that Tyr129, located in the second transmembrane region of the ETA receptor, is a critical component for determination of endothelin receptor subtype-selective ligand binding.

Amino Acid Sequence↗

PubMed1994

Coxsackievirus B3 entry into the host cell interferes with G-protein-mediated transmembrane signalling.

In the present work we used various cell lines in order to study the possible effect of coxsackievirus B3 (CVB3) entry on the adenylyl cyclase transmembrane signalling system. A significant decrease (by about 10-20%) was found in forskolin-augmented as well as in A1F-4- and GTP gamma S-sensitive adenylyl cyclase activity in plasma membranes isolated from HeLa, HEp-2, Vero and green monkey kidney cells shortly (up to 60 min) preincubated with CVB3 (5 PFU/cell). Moreover, the ability of G-proteins derived from plasma membranes of infected cells to reconstitute AC activity in the cyc- mutant of S49 cells was also reduced. Content of G-protein subunits, however, remained unchanged after CVB3 attachment. Functional alterations in the G-protein-mediated adenylyl cyclase signalling system were accompanied by a marked decrease (by about 20-40%) of intracellular cAMP levels in virus-affected cells. These findings demonstrate clearly that CVB3 may affect functioning of the G-protein regulated adenylyl cyclase transmembrane signalling system in virus-sensitive cells as early as during the first period of its contact with the cellular plasma membrane.

Adenylyl Cyclases↗