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Biomedical subjects

M Levitt

Publications and source records attributed to M Levitt.

At least 91 records · Page 5Linked to original sources
PubMed1993

Ondansetron compared with dexamethasone and metoclopramide as antiemetics in the chemotherapy of breast cancer with cyclophosphamide, methotrexate, and fluorouracil.

BACKGROUND: Although ondansetron was found to be effective as an antiemetic in numerous clinical trials of highly emetogenic combination-chemotherapy regimens that included cisplatin, its role in milder emetogenic regimens has not been fully defined. To address its use with a widely used but less emetogenic regimen, we performed a double-blind, randomized clinical trial comparing ondansetron with dexamethasone and metoclopramide in patients with breast cancer receiving chemotherapy with cyclophosphamide, methotrexate, and fluorouracil. METHODS: A total of 165 women with breast cancer from 14 Canadian centers who were about to receive this chemotherapy for the first time were randomly assigned to receive either ondansetron (n = 85) or dexamethasone plus metoclopramide (n = 80), a widely used, standard antiemetic regimen. The patients recorded the incidence of nausea, emesis, and other side effects in diaries, and these data were compared in the two groups. RESULTS: The patients who received dexamethasone and metoclopramide had significantly less nausea during the first 24 hours after chemotherapy was begun. Otherwise, there were no statistically significant differences in efficacy between the regimens. The incidence of drowsiness and increased appetite was higher in the group given dexamethasone and metoclopramide. CONCLUSIONS: For women with breast cancer who are being treated with cyclophosphamide, methotrexate, and fluorouracil, the efficacy of dexamethasone and metoclopramide in controlling nausea and vomiting equaled or exceeded that of ondansetron.

Analysis of Variance↗
PubMed1993

Maturational changes in steroidogenesis in the inner and outer zones of the guinea pig adrenal cortex.

Studies were done to determine the effects of age on steroidogenesis in the inner (zona reticularis) and outer (zona fasciculata plus glomerulosa) zones of the guinea pig adrenal cortex. In 35-day-old animals, cortisol production by adrenal outer zone cells was approximately twice as great as that by inner zone cells. With aging, cortisol secretion by inner zone cells decreased to very low levels, but there was no detectable change in the capacity for cortisol production by the outer zone. However, the outer zone comprised a progressively decreasing fraction of the total adrenal mass in older animals. To determine the basis for the decline in cortisol production by inner zone cells with aging, the activities of several steroidogenic enzymes were determined. Microsomal 21-hydroxylase activity was greater in the inner than outer zone but was not significantly affected by age. By contrast, 17 alpha-hydroxylase activity was greater in the outer zone at all ages, and decreased with aging in the inner but not the outer zone. Mitochondrial cholesterol sidechain cleavage and 11 beta-hydroxylase activities were also higher in the outer than inner zone and declined in the inner zone only in older animals. The decrease in inner zone cholesterol sidechain cleavage activity with aging was proportionately greater than the age-dependent changes in other enzyme activities. The results indicate that the effects of aging on steroidogenesis are both zone- and enzyme-specific. The overall decline in cortisol secretion by the guinea pig adrenal cortex with aging is attributable to both a decrease in cortisol production by the cells of the zone reticularis and a disproportionate increase in the mass of the gland comprised by this zone. The decrease in cortisol secretion correlates closely with a decline in cholesterol sidechain cleavage activity in the zona reticularis, and may be causally related.

Adrenal Cortex↗
PubMed1993

Structural similarity of DNA-binding domains of bacteriophage repressors and the globin core.

BACKGROUND: In recent years, the determination of large numbers of protein structures has created a need for automatic and objective methods for the comparison of structures or conformations. Many protein structures show similarities of conformation that are undetectable by comparing their sequences. Comparison of structures can reveal similarities between proteins thought to be unrelated, providing new insight into the interrelationships of sequence, structure and function. RESULTS: Using a new tool that we have developed to perform rapid structural alignment, we present the highlights of an exhaustive comparison of all pairs of protein structures in the Brookhaven protein database. Notably, we find that the DNA-binding domain of the bacteriophage repressor family is almost completely embedded in the larger eight-helix fold of the globin family of proteins. The significant match of specific residues is correlated with functional, structural and evolutionary information. CONCLUSION: Our method can help to identify structurally similar folds rapidly and with high-sensitivity, providing a powerful tool for analyzing the ever-increasing number of protein structures being elucidated.

Journal Article↗
PubMed1993

The expression of deafferentation dysesthesias reduced by dorsal root entry zone lesions in the rat.

Extensive longitudinal lesions of the dorsal root entry zone (DREZ) are effective in relieving some chronic deafferentation pain in humans. A deafferentation syndrome follows C5-T2 dorsal root ganglionectomies in rats. The syndrome consists of biting and scratching the completely and partially denervated limb areas, respectively. This study examines the effect of DREZ lesions on the deafferentation syndrome in the rat. Of 37 rats, 24 underwent C5-T2 ganglionectomies only, five received C4-T3 micromechanical DREZ lesions only, and eight underwent ganglionectomies plus simultaneous DREZ lesions. The animals were observed for 45 days post-operatively. Histological analysis of the spinal cord lesions was performed. All rats with ganglionectomies alone exhibited the deafferentation syndrome; however, no rats with DREZ lesions alone showed this feature. Only 25% of rats with combined ganglionectomies and DREZ lesions exhibited the deafferentation syndrome in the first 30 days, whereas 80% of the animals with ganglionectomies only did so. Although 75% of the animals with combined lesions eventually bit the insensitive forepaw, this behavior was significantly attenuated: the day of onset was delayed and the extent of self-mutilation was reduced. Postmortem histological examination of the DREZ lesions indicated a close association between the completeness of the dorsal horn destruction and the reduction or prevention of self-mutilation. These data support the validity of the animal model and also the hypothesis stating that the deafferentation syndrome results from abnormal spontaneous neural activity in the dorsal horn. Moreover, the variability of the histological findings in these experiments stresses the importance of making contiguous and complete dorsal horn lesions in human DREZ surgery.

Afferent Pathways↗
PubMed1992

Induced peptide conformations in different antibody complexes: molecular modeling of the three-dimensional structure of peptide-antibody complexes using NMR-derived distance restraints.

Intramolecular interactions in bound cholera toxin peptide (CTP3) in three antibody complexes were studied by two-dimensional transferred NOE spectroscopy. These measurements together with previously recorded spectra that show intermolecular interactions in these complexes were used to obtain restraints on interproton distances in two of these complexes (TE32 and TE33). The NMR-derived distance restraints were used to dock the peptide into calculated models for the three-dimensional structure of the antibody combining site. It was found that TE32 and TE33 recognize a loop comprising the sequence VPGSQHID and a beta-turn formed by the sequence VPGS. The third antibody, TE34, recognizes a different epitope within the same peptide and a beta-turn formed by the sequence IDSQ. Neither of these two turns was observed in the free peptide. The formation of a beta-turn in the bound peptide gives a compact conformation that maximizes the contact with the antibody and that has greater conformational freedom than alpha-helix or beta-sheet secondary structure. A total of 15 antibody residues are involved in peptide contacts in the TE33 complex, and 73% of the contact area in the antibody combining site consists of the side chains of aromatic amino acids. A comparison of the NMR-derived models for CTP3 interacting with TE32 and TE33 with the previously derived model for TE34 reveals a relationship between amino acid sequence and combining site structure and function. (a) The three aromatic residues that interact with the peptide in TE32 and TE33 complexes, Tyr 32L, Tyr 32H, and Trp 50H, are invariant in all light chains sharing at least 65% identity with TE33 and TE32 and in all heavy chains sharing at least 75% identity with TE33. Although TE34 differs from TE32 and TE33 in its fine specificity, these aromatic residues are conserved in TE34 and interact with its antigen. Therefore, we conclude that the role of these three aromatic residues is to participate in nonspecific hydrophobic interactions with the antigen. (b) Residues 31, 31c, and 31e of CDR1 of the light chain interact with the antigen in all three antibodies that we have studied. The amino acids in these positions in TE34 differ from those in TE32 and TE33, and they are involved in specific polar interactions with the antigen. (c) CDR3 of the heavy chain varies considerably both in length and in sequence between TE34 and the two other anti-CTP3 antibodies. These changes modify the shape of the combining site and the hydrophobic and polar interactions of CDR3 with the peptide antigen.

Amino Acid Sequence↗
PubMed1992

Accurate modeling of protein conformation by automatic segment matching.

Segment match modeling uses a data base of highly refined known protein X-ray structures to build an unknown target structure from its amino acid sequence and the atomic coordinates of a few of its atoms (generally only the C alpha atoms). The target structure is first broken into a set of short segments. The data base is then searched for matching segments, which are fitted onto the framework of the target structure. Three criteria are used for choosing a matching data base segment: amino acid sequence similarity, conformational similarity (atomic co-ordinates), and compatibility with the target structure (van der Waals' interactions). The new method works surprisingly well: for eight test proteins ranging in size from 46 to 323 residues, the all-atom root-mean-square deviation of the modeled structures is between 0.93 A and 1.73 A (the average is 1.26 A). Deviations of this magnitude are comparable with those found for protein co-ordinates before and after refinement against X-ray data or for co-ordinates of the same protein in different crystal packings. These results are insensitive to errors in the C alpha positions or to missing C alpha atoms: accurate models can be built with C alpha errors of up to 1 A or by using only half the C alpha atoms. The fit to the X-ray structures is improved significantly by building several independent models based on different random choices and then averaging co-ordinates; this novel concept has general implications for other modeling tasks. The segment match modeling method is fully automatic, yields a complete set of atomic co-ordinates without any human intervention and is efficient (14 s/residue on the Silicon Graphics 4D/25 Personal Iris workstation.

Algorithms↗
PubMed1992

A model of the molten globule state from molecular dynamics simulations.

It is generally accepted that a protein's primary sequence determines its three-dimensional structure. It has proved difficult, however, to obtain detailed structural information about the actual protein folding process and intermediate states. We present the results of molecular dynamics simulations of the unfolding of reduced bovine pancreatic trypsin inhibitor. The resulting partially "denatured" state was compact but expanded relative to the native state (11-25%); the expansion was not caused by an influx of water molecules. The structures were mobile, with overall secondary structure contents comparable to those of the native protein. The protein experienced relatively local unfolding, with the largest changes in the structure occurring in the loop regions. A hydrophobic core was maintained although packing of the side chains was compromised. The properties displayed in the simulation are consistent with unfolding to a molten globule state. Our simulations provide an in-depth view of this state and details of water-protein interactions that cannot yet be obtained experimentally.

Animals↗
PubMed1992

A lattice model for protein structure prediction at low resolution.

The prediction of the folded structure of a protein from its sequence has proven to be a very difficult computational problem. We have developed an exceptionally simple representation of a polypeptide chain, with which we can enumerate all possible backbone conformations of small proteins. A protein is represented by a self-avoiding path of connected vertices on a tetrahedral lattice, with several amino acid residues assigned to each lattice vertex. For five small structurally dissimilar proteins, we find that we can separate native-like structures from the vast majority of non-native folds by using only simple structural and energetic criteria. This method demonstrates significant generality and predictive power without requiring foreknowledge of any native structural details.

Amino Acid Sequence↗
PubMed1992

Molecular dynamics simulations of helix denaturation.

An understanding of the structural transitions that an alpha-helix undergoes will help to elucidate such motions in proteins and their role in protein folding. We present the results of molecular dynamics simulations to investigate these transitions in a short polyalanine peptide (13 residues) both in vacuo and in the presence of solvent. The denaturation of this peptide was monitored as a function of temperature (ranging from 5 to 200 degrees C). In vacuo, the helical state predominated at all temperatures, whereas in solution the helix melted with increasing temperature. The peptide was predominantly helical at low temperature in solution, while at intermediate temperatures the peptide spent the bulk of the time fluctuating between different conformations with intermediate amounts of helix, e.g. not completely helical nor entirely non-helical. Many of these conformations consisted of short helical segments with intervening non-helical residues. At high temperature the peptide unfolded and adopted various collapsed unstructured states. The intrahelical hydrogen bonds that break at high temperature were not fully compensated by hydrogen bonds with water molecules in the partially unfolded forms of the peptide. Increases in temperature disrupted both the helical structure and the peptide-water interactions. Water played a major but indirect role in facilitating unfolding, as opposed to specifically competing for the intrapeptide hydrogen bonds. The implications of our results to protein folding are discussed.

Alanine↗
PubMed1992

Differential effects of adrenocorticotropic hormone on steroid hydroxylase activities in the inner and outer zones of the guinea pig adrenal cortex.

We have studied the effects of ACTH treatment on steroid hydroxylase activities in the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Animals received 5 or 10 U of ACTH daily for 6 days and enzyme activities were then assessed in isolated microsomal or mitochondrial preparations. In control animals, microsomal cytochrome P-450 concentrations were greater in the inner than outer zone, but mitochondrial P-450 levels were similar in the two zones. Microsomal 17 alpha-hydroxylase and mitochondrial 11 beta-hydroxylase activities were greater in the outer than inner zone, but microsomal 21-hydroxylase activity was greater in the inner zone. ACTH treatment decreased cytochrome P-450 concentrations in inner but not outer zone microsomes; mitochondrial P-450 levels were unaffected in both zones. ACTH caused a dose-dependent increase in inner zone 17 alpha-hydroxylase activity and decrease in 21-hydroxylase activity without affecting the activity of either enzyme in outer zone microsomes. ACTH also decreased 11 beta-hydroxylase activity in outer but not inner zone mitochondrial preparations. The net effect of ACTH treatment was to diminish the differences in steroid metabolism between the two zones. The results indicate that the effects of ACTH on steroid hydroxylase activities are both zone- and enzyme-dependent, suggesting the existence of multiple and independent regulatory mechanisms.

Adrenal Cortex↗
PubMed1991

Structural and kinetic studies of the Fab fragment of a monoclonal anti-spin label antibody by nuclear magnetic resonance.

Nuclear magnetic resonance has been used to study the structure of the anti-spin label antibody AN02 combining site and kinetic rates for the hapten-antibody reaction. The association reaction for the hapten dinitrophenyl-diglycine (DNP-diGly) is diffusion-limited. The activation enthalpy for association, 5.1 kcal/mol, is close to the activation enthalpy for diffusion in water. Several reliable resonance assignments have been made with the aid of recently reported crystal structure. Structural data deduced from the nuclear magnetic resonance (n.m.r.) spectra compare favorably with the crystal structure in terms of the combining site amino acid composition, distances of tyrosine residues from the unpaired electron of the hapten, and residues in direct contact with the hapten. Evidence is presented that a single binding site region tyrosine residue can assume two distinct conformations on binding of DNP-diGly. The AN02 antibody is an autoantibody. Dimerization of the Fab fragments is blocked by the hapten DNP-diGly. The n.m.r. spectra suggests that some of the amino acid residues involved in the binding of the DNP-hapten are also involved in the Fab dimerization.

Antibodies, Monoclonal↗
PubMed1991

Accurate prediction of the stability and activity effects of site-directed mutagenesis on a protein core.

Theoretical prediction of the structure, stability and activity of proteins, an important unsolved problem in molecular biology, would be of use for guiding site-directed mutagenesis and other protein-engineering techniques. X-ray diffraction studies have provided extensive structural information for many proteins, challenging theorists to develop reliable techniques able to use such knowledge as a base for prediction of mutants' characteristics. Here we report theoretical calculation of stabilization energies for 78 triple-site sequence variants of lambda repressor characterized experimentally by Lim and Sauer. The calculated energies correlate with the mutants' measured activities; active and inactive mutations are discriminated with 92% reliability. They correlate even more directly with the mutants' thermostabilities, correctly identifying two of the mutants to be more stable than the wild type.

Chemical Phenomena↗
PubMed1991

Real-time interactive frequency filtering of molecular dynamics trajectories.

Molecular dynamics simulations of atomic motion in protein and nucleic acid molecules must be done on a femtosecond time-scale. Much of this rapid motion is unimportant for the slower changes that are most relevant to biological function (conformational changes, substrate binding, protein folding). The high-frequency motion makes simulations computationally expensive. More importantly, the high frequencies obscure visualization of the relevant dynamics processes. Sessions, Dauber-Osguthorpe and Osguthorpe presented a method for removing high-frequency motions from atomic co-ordinates of trajectories generated by simulation. While that study used fast Fourier methods and emphasized the use of filtering for analysis of trajectories, this communication describes a new method that makes it much easier to use frequency filtering in programs that display trajectories as a sequence of moving images. Tests of the method on systems extending from pure water to proteins and nucleic acid molecules in vacuo and in solution have demonstrated its general utility. Impressed with the power and simplicity of the new method, we wish to present it in sufficient detail to allow others to implement it themselves.

Fourier Analysis↗
PubMed1991

Relationship between covalent binding to microsomal protein and the destruction of adrenal cytochrome P-450 by spironolactone.

Previous investigations have demonstrated that guinea pig adrenal microsomes catalyze an NADPH-dependent activation of spironolactone (SL) resulting in the degradation of cytochrome(s) P-450 and decreases in steroidogenic enzyme activities. Studies were done to evaluate the relationship between the destruction of cytochrome P-450 and the covalent binding to microsomal protein by SL and by 7 alpha-thiospironolactone (7 alpha-thio-SL), an obligatory intermediate in the activation pathway. NADPH-dependent irreversible binding to guinea pig adrenal microsomal protein was demonstrable with 22-14C- and with 35S-labelled SL or 7 alpha-thio-SL as substrates. In the absence of NADPH, there was relatively little binding. NADPH-dependent covalent binding was not demonstrable with hepatic microsomal preparations. The amount of covalent binding to adrenal microsomes was far greater with 7 alpha-thio-SL than with SL and also greater with 35S-labelled than with 14C-labelled substrates. The latter results suggest the possibility of more than one reactive metabolite. Time-course experiments revealed a good correlation between covalent binding and P-450 destruction by SL and by 7 alpha-thio-SL. In addition, the 17 alpha-hydroxylase inhibitor, SU-10'603, and the 17 alpha-hydroxylase substrate, progesterone, prevented both the degradation of cytochrome P-450 and the NADPH-dependent covalent binding by 7 alpha-thio-SL. Reduced glutathione also decreased covalent binding but did not diminish P-450 destruction. The latter results indicate that some of the covalent binding is unrelated to the degradation of cytochrome P-450. However, all of the data are consistent with the hypothesis that 7 alpha-thio-SL is a suicide inhibitor of adrenal cytochrome P-450 and that covalent binding to protein is involved in the degradation of cytochrome P-450.

Adrenal Glands↗
PubMed1991

Enhanced stability of subtilisin by three point mutations.

This study was undertaken to characterize the effect of three point mutations made on aprA-subtilisin on the stability of the protein to both heat- and detergent-induced denaturation. Asparagine residues at positions 109 and 218 were replaced with serine residues to prevent the possible cyclization between these asparagines and the adjacent glycine residues and hence to increase the long-term stability. The effect of these substitutions on conformational stability was examined by thermal denaturation. At high calcium concentrations, the Ser109-substituted analog showed a 3 degrees C higher transition temperature than that of aprA-subtilisin, while the Ser218 substituted analog had a 4 degrees C higher transition temperature. The analog with both changes had a 7 degrees C higher transition temperature than that of the original aprA-subtilisin, indicating that the contributions of the individual mutations were additive. The analog with both mutations also exhibited increased stability in the presence of sodium dodecyl sulfate (SDS) when compared to aprA-subtilisin. In addition to the above two mutations, the asparagine at position 76, located in the high affinity Ca(2+) binding loop of subtilisin, was changed to aspartic acid. The effect of this mutation on the thermal stability of the protein was examined at different calcium concentrations. The analog with all three mutations exhibited little dependence on calcium concentration below 1 mM levels, while the proteins without the mutation at asparagine-76 displayed a strong dependence of melting temperature on Ca(2+) concentration in this range. At much higher calcium concentrations, the analog with three mutations showed an increase in stability similar to that observed with aprA-subtilisin. The analog with three mutations also exhibited greater stability to SDS-induced denaturation than both aprA-subtilisin and the Ser109- and Ser218-substituted analogs. The activation energy barrier for loss of structure in 1% SDS for the analog with all three mutations was increased over that for aprA-subtilisin by 16 kcal/ml. These results suggest that the mutation of asparagine-76 to aspartic acid increases the affinity of the primary Ca(2+) binding site.

Circular Dichroism↗