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J Novotny

Publications and source records attributed to J Novotny.

At least 55 records · Page 3Linked to original sources
PubMed1998

The long (Gs(alpha)-L) and short (Gs(alpha)-S) variants of the stimulatory guanine nucleotide-binding protein. Do they behave in an identical way?

The relative proportions and tissue distribution of the long (Gs[alpha]-L) and short (Gs[alpha]-S) variants of the a subunit of the stimulatory G-protein (Gs[alpha]) change under a wide range of metabolic conditions, such as cellular differentiation, ontogenetic development, ageing and various adaptive processes. Although the two variants of Gs(alpha) are generally regarded to be functionally identical, this review summarizes recent experimental support for the non-identical behaviour of these proteins. Similarly, there is no consistent evidence for the functional meaning of these changes as far as regulation of adenylate cyclase activity is concerned. Since it is hard to believe that the complicated scheme of alternative splicing and the energy-consuming synthesis of proteins would be performed for no reason, it is suggested that Gs[alpha] variants might be involved in controlling other effector molecules and processes besides adenylate cyclase and cAMP metabolism. Such an idea is indirectly supported by specific alterations in the Gs[alpha]-L/Gs[alpha]-S ratio under various physiological and pathophysiological conditions.

Animals↗
PubMed1998

Empirical free energy calculations of phage 434 repressor- and cro-DNA complexes support the 'indirect readout' hypothesis of specificity.

Empirical free energy calculations were done for two X-ray DNA-protein complexes. Complex formation energies were estimated for the phage 434 repressor and the 434 cro protein, both in complex with wild-type versions of their respective nucleic acid binding sites. The resulting quantities were subdivided according to the relative contributions of hydrophobicity, electrostatics and entropy (including both conformational changes and those pertinent to physical association of complex components). The non-linearized finite difference Poisson-Boltzmann equation was solved over a range of salt concentrations to determine magnitudes of coulombic and desolvation effects. Though approximate at best, results confirmed (1) the importance of centrally located charged protein residues that do not make direct contact with the DNA, (2) explicit protein backbone interactions with DNA, and (3) the varied parts of the two proteins used to harness the energy of binding. Although a large difference exists between calculated and experimentally measured free energy values, these can be reconciled by assuming that approximately 1.5 e.u. (about 0.4-0.5 kcal at room temperature) of DNA vibrational and/or conformational entropy is lost for every basepair torsion in contact with the protein. Such a conclusion supports an "indirect readout" ("compound recognition") hypothesis of DNA binding specificity, wherein sequence-dependent differences in protein-DNA interaction are due to the topological peculiarities of the interactions rather than intrinsic chemical differences alone.

Bacteriophage lambda↗
PubMed1997

Empirical free energy calculations: a blind test and further improvements to the method.

Empirical Gibbs functions estimate free energies of non-covalent reactions (deltaG) from atomic coordinates of reaction products (e.g. antibody-antigen complexes). The function previously developed by us has four terms that quantify the effects of hydrophobic, electrostatic and entropy changes (conformational, association) upon complexation. The function was used to calculate delta deltaG of ten lysozyme mutants affecting the stability of the HyHEL-10 antibody-lysozyme complex. The mutants were computer-modeled from the X-ray structure of the wild-type, and free energy calculations produced a correlation coefficient of 0.5 with the experimental delta deltaG data (average absolute error +/-3 kcal). The following changes were then introduced into the Gibbs function: (1) the hydrophobic force was made proportional to the molecular surface, as calculated by the GEPOL93 algorithm, with the scaling constant of 70 cal/mol/A2; (2) calculation of the electrostatics of binding was carried out by the finite difference Poisson-Boltzmann algorithm, which employed uniform grid charging, dielectric boundary smoothing and charge anti-aliasing; and (3) side-chain conformational entropy was estimated from the CONGEN sampling of torsional degrees of freedom. In the new calculations, correlation with experimental data improved to 0.6 or 0.8 if a single outlying mutant, K96M, was neglected. Analysis of the errors remaining in our calculations indicated that molecular mechanics-based modeling of the mutants, rather than the form of our amended Gibbs function, was the main factor limiting the accuracy of the free energy estimates.

Animals↗
PubMed1997

Empirical potentials and functions for protein folding and binding.

Simplified models and empirical potentials are being increasingly used for the analysis of proteins, frequently augmenting or replacing molecular mechanics approaches. Recent folding simulations have employed potentials that, in addition to terms assuring proper polypeptide geometry, include only two noncovalent effects-hydrogen bonding and hydrophobicity, with extremely simple approximations to the latter. The potentials that have been used in the free-energy ranking of protein-ligand complexes have generally been more involved. These potentials have more detailed solvation models and account for both local (hydrophobic and polar) solute-solvent phenomena and long range electrostatic solvation effects. The models of solvation that have been used most frequently are surface area related atomic parameters, knowledge-based models extracted from protein-structure data, and continum electrostatics with an additional area-related parameter. The knowledge-based approaches to solvation, although convenient and accurate enough, are suspect of double counting certain free-energy terms.

Databases, Factual↗
PubMed1997

External audit of photon beams by mailed film dosimetry: feasibility study.

A feasibility study for mailed film dosimetry has been performed. The global reproducibility of the method is better than 2%. It is shown that the normalized sensitometric curve does not depend on photon beam quality in the range from Co-60 gamma-rays to 18 MV x-rays, although the dose per optical density decreases when the energy increases. The fading of the latent image before film processing is only 3% per month and the normalized sensitometric curve is not modified after a period of 51 days between irradiation and processing. Sets of films were mailed to three different institutes for irradiation and returned for processing and evaluation after more than two months in order to verify that mailing of irradiated and unprocessed films does not produce unwanted artefacts. Finally the feasibility of external audits with mailed film dosimetry is illustrated by comparison of beam profiles measured with films and ionization chambers in a polystyrene phantom.

Cobalt Radioisotopes↗
PubMed1997

Bacteremia in cancer patients with solid tumors undergoing chemotherapy versus surgery: risk factors, etiology and outcome in 276 patients.

Etiology, risk factors, outcome and complications of bacteremia in 276 patients with solid tumors were analyzed. A group of 78 patients with solid tumors and surgical therapy only was compared with 172 patients with solid tumors who were treated with chemotherapy only. The most frequently observed risk factors of bacteremia in patients after surgery was urinary catheter insertion, wound as source of bacteremia, age > 60, staphylococci, enterococci and Enterobacteriaceae as etiologic agents. In comparison, viridans streptococci and Pseudomonas aeruginosa as etiologic agents as well as vascular catheters were significantly more frequently found in those treated with chemotherapy only. Patients with bacteremia after surgery only had a lower incidence of septic shock (6.4 vs. 16.9%, P < 0.03) and also lower mortality (5.6 vs. 14.9%, P < 0.04) attributable to shock than patients being treated for solid tumors with chemotherapy only.

Antineoplastic Agents↗
PubMed1997

Do vancomycin serum levels predict failures of vancomycin therapy or nephrotoxicity in cancer patients?

The purpose of this study was to determine if patients with high vancomycin (VAN) serum levels experience more toxicity than underdosed patients with lower (VAN) levels, and whether low VAN serum levels cause therapeutic failures in patients with gram-positive bacteremia. In 198 cancer patients trough and peak serum levels of VAN were measured. Acute toxicity (Red Man syndrome) appeared in 3 patients (1.5%). Patients previously or currently treated with other nephrotoxic compounds (134 patients) presented the same incidence of nephrotoxicity as those receiving VAN for the first time in monotherapy (64 patients). VAN did not increase the toxicity when patients were dosed simultaneously or previously with aminoglycosides or amphotericin B. Our second observation, when studying serum levels in our 198 patients was that high VAN trough serum levels (trough > 15 microg/mL) were associated with significantly more nephrotoxicity (33.3% vs. 11.1%, P < 0.03) than low levels in the subgroups of either pretreated patients or unpretreated with other nephrotoxic drugs. None of 198 patients who had trough levels below 15 microg/mL had peak levels exceeding 40 microg/mL. This suggests that only serum monitoring of trough levels may predict nephrotoxicity. A case control study was conducted to compare a group of 22 VAN failures with 22 successfully treated patients matched in underlying disease and neutropenia who were treated in the same period, under the same antibiotic policy, at the same cancer center, for gram-positive bacteremia. Persisting, enterococcal, or mixed enterococcal plus staphylococcal bacteremia were the only statistically significant risk factors which predicted therapy failure in cancer patients. Neither peak nor trough VAN serum levels predicted failure or cure of gram-positive bacteremia in cancer patients.

Aminoglycosides↗
PubMed1996

Design of heterotetrameric coiled coils: evidence for increased stabilization by Glu(-)-Lys(+) ion pair interactions.

Electrostatic interactions between charged amino acids often affect heterospecificity in coiled coils as evidenced by the interaction between the oncoproteins, fos and jun. Such interactions have been successfully exploited in the design of heteromeric coiled coils in a number of laboratories. It has been suggested that heterospecificity in these dimeric coiled-coil systems is driven not by specific electrostatic interactions in the heterodimers but rather by electrostatic repulsion acting to destabilize the homodimer state relative to the heterodimer state. We show that it is possible to design ion pair interactions that directly stabilize the heterotetrameric coiled-coil state. Synthetic peptides were used whose sequences are based on the C-terminal tetramerization domain of Lac repressor, as a model system for four-chain coiled coils (Fairman et al., 1995). These Lac-based peptides, containing either glutamic acid (Lac21E) or lysine (Lac21K) at all b and c heptad positions, only weakly self-associate but, when mixed, afford a highly stable heterotetramer. This study represents the first experimental evidence for the importance of the b and c heptad positions to the stability of coiled coils. Finally, pH dependence and NaCl dependence studies show that heterotetramer stability is driven by ion pair interactions between glutamate and lysine; these interactions contribute about 0.6 kcal/mol of stabilizing free energy for each potential glutamate-lysine pair.

Amino Acid Sequence↗
PubMed1996

Inhibition of beta-adrenergic receptor-mediated signals by a synthetic peptide derived from the alpha subunit of the stimulatory G-protein.

The beta-adrenergic stimulation of adenylyl cyclase is mediated through the stimulatory G-protein (Gs). In this study, three synthetic peptides corresponding to different regions (amino acids 3-14, 72-86 and 325-337) of the alpha subunit of Gs (Gs-alpha) have been employed in competition assays in order to examine more closely the molecular basis of receptor-Gs-alpha interaction. The direct coupling between Gs and adenylyl cyclase was not influenced by any of these peptides and only the peptide representing residues 325-337 of Gs-alpha specifically inhibited beta-adrenergic receptor-mediated activation of Gs. Essentially the same results were obtained when testing beta-1- and beta-2-adrenergic receptors, which supports the notion that both these pharmacologically distinct receptor subtypes exploit at least one identical coupling domain within Gs-alpha for its activation.

Adenylyl Cyclases↗
PubMed1996

Regulation of melanogenesis in B16 mouse melanoma cells by protein kinase C.

Melanogenesis is regulated by a variety of environmental and hormonal factors. In this study, we showed that protein kinase C (PKC) plays a major role in regulating melanogenesis in B16 mouse melanoma cells. Chronic treatment of B16 cells with phorbol dibutyrate resulted in a concentration-dependent loss of density-dependent induction of tyrosinase activity, which correlated positively with a concentration-dependent loss of PKC enzyme activity. In contrast, B16 clones overexpressing PKC alpha had increased tyrosinase activity. Different phorbol derivatives inhibited tyrosinase activity and depleted cellular PKC alpha in a manner that reflected their reported tumor-promoting activity. Western blotting analysis showed that phorbol dibutyrate decreased the amount of the brown locus gene product (TRP-1) by 50% and lowered the amount of the albino locus gene product (tyrosinase) to undetectable levels. None of the phorbol derivatives affected the level of the slaty locus protein (TRP-2). The decrease in tyrosinase and TRP-1 protein levels was found to be due to a decrease in the mRNA encoded by these genes. In addition to inhibiting the density-dependent increase in tyrosinase activity, phorbol dibutyrate inhibited some, but not all, of the 8-bromocyclic AMP-induced increase in tyrosinase activity. This was accompanied by a decrease in the amount of tyrosinase protein induced by 8-bromocyclic AMP. Although 8-bromocyclic AMP did not change the level of TRP-1, it did reverse the decrease in the amount of this protein induced by phorbol dibutyrate. The amount of TRP-2 was not altered by any of these agents. These data suggest that PKC regulates melanogenesis primarily by controlling the constitutive expression of tyrosinase and, to a lesser extent, TRP-1.

Animals↗
PubMed1996

Factors influencing accuracy of computer-built models: a study based on leucine zipper GCN4 structure.

A three-dimensional model of the leucine zipper GCN4 built from its amino acid sequence had been reported previously by us. When the two alternative x-ray structures of the GCN4 dimer became available, the root mean square (r.m.s.) shifts between our model and the structures were determined as approximately 2.7 A on all atoms. These values are similar to the r.m.s. shift of 2.8 A between the two GCN4 structures in the different crystal forms (C2 and P2(1)2(1)2(1)). CONGEN conformational searches were run to better understand the conditions that may determine the preference of different conformers in different environments and to test the sensitivity of our current modeling techniques. With a judicious choice of CONGEN search parameters, the backbone r.m.s. deviation improved to 0.8 A and 2.5 A on all atoms. The side-chain conformations of Val and Leu at the helical interface were well reproduced (1.2 A r.m.s.), and the large side-chain misplacements occurred with only a small number of charged amino acids and a tyrosine. Inclusion of the crystal environment (C2 symmetry), as a passive background, into the side-chain conformational search further improved the accuracy of the model to an r.m.s. deviation of 2.1 A. Conformational searches carried out in the two different crystal environments and employing the AMBER protein/DNA forcefield, as implemented in CONGEN, gave the r.m.s. values of 2.2 A (for the C2 symmetry) and 2.5 A (for the P2(1)2(1)2(1) symmetry). In the C2 symmetry crystal, as much as 40% of the surface of each dimer was involved in crystal contacts with other dimers, and the charged residues on the surface often interacted with immobilized water molecules. Thus, occasional large r.m.s. deviations between the model and the x-ray side chains were due to specific conditions that did not occur in solution.

Amino Acid Sequence↗
PubMed1996

The influence of the IAEA standard holder on dose evaluated from TLD samples.

The EROPAQ quality assurance project started in 1994 with TLD postal dose intercomparisons for photon beams used in 47 radiotherapy centres in the middle and eastern European countries. The photon dose intercomparisons include beam output checks and beam quality checks. Since an acceptance limit of +/- 3% was chosen for the EROPAQ intercomparisons, any systematic error in dose evaluation by the measuring centre should be minimized. The standard IAEA TLD holder is used in the intercomparisons. In this work the magnitude of the holder attenuation is evaluated and holder corrections derived both for output and photon beam quality checks for beam qualities ranging from Co-60 up to 18 MV. The correction of the dose at the depth of reference does not exceed 1% and decreases with increasing beam energy. A correction of the beam quality ratio of the order of 1% and that is independent of the photon energy has been determined by both calculation and measurements. These corrections are consistent with the preliminary data published by the IAEA and EC QA network.

Biophysical Phenomena↗