Biomedical subjects

A Sarai

Publications and source records attributed to A Sarai.

At least 55 records · Page 3Linked to original sources

PubMed1996

Constructing optimal backbone segments for joining fixed DNA base pairs.

A method is presented to link a sequence of space-fixed base pairs by the sugar-phosphate segments of single nucleotides and to evaluate the effects in the backbone caused by this positioning of the bases. The entire computational unit comprises several nucleotides that are energy-minimized, subject to constraints imposed by the sugar-phosphate backbone segments being anchored to space-fixed base pairs. The minimization schemes are based on two stages, a conjugate gradient method followed by a Newton-Raphson algorithm. Because our purpose is to examine the response, or relaxation, of an artificially stressed backbone, it is essential to be able to obtain, as closely as possible, a lowest minimum energy conformation of the backbone segment in conformational space. For this purpose, an algorithm is developed that leads to the generation of an assembly of many local energy minima. From these sets of local minima, one conformation corresponding to the one with the lowest minimum is then selected and designated to represent the backbone segment at its minimum. The effective electrostatic potential of mean force is expressed in terms of adjustable parameters that incorporate solvent screening action in the Coulombic interactions between charged backbone atoms; these parameters are adjusted to obtain the best fit of the nearest-neighbor phosphorous atoms in an x-ray structure.

Algorithms↗

PubMed1996

Interdependence of conformational variables in double-helical DNA.

DNA exhibits conformational polymorphism, with the details depending on the sequence and its environment. To understand the mechanisms of conformational polymorphism and these transitions, we examine the interrelationships among the various conformational variables of DNA. In particular, we examine the stress-strain relation among conformational variables, describing base-pair morphology and their effects on the backbone conformation. For the calculation of base pairs, we use the method previously developed to calculate averages over conformational variables of DNA. Here we apply this method to calculate the Boltzmann averages of conformational variables for fixed values of one particular conformational variable, which reflects the strain in the structure responding to a particular driving stress. This averaging over all but one driving variable smooths the usual rough energy surface to permit observation of the effects of one conformational variable at a time. The stress-strain analyses of conformational variables of base pair slide, twist, and roll, which exhibit characteristic changes during the conformational transition of DNA, have shown that the conformational changes of base pairs are strongly correlated with one another. Furthermore, the stress-strain relations are not symmetrical with respect to these variables, i.e., the response of one coordinate to another is different from the reverse direction. We also examine the effect of conformational changes in base-pair variables on the sugar-backbone conformation by using the minimization method we developed. The conformational changes of base pairs affect the sugar pucker and other dihedral angles of the backbone of DNA, but each variable affects the sugar-backbone differently. In particular, twist is found to have the most influence in affecting the sugar pucker and backbone conformation. These calculated conformational changes in base pairs and backbone segments are consistent with experimental observations and serve to validate the calculation method.

Base Composition↗

PubMed1996

The cavity in the hydrophobic core of Myb DNA-binding domain is reserved for DNA recognition and trans-activation.

The DNA-binding domain of Myb consists of three imperfect repeats, R1, R2 and R3, each containing a helix-turn-helix motif variation. Among these repeats, R2 has distinct characteristics with high thermal instability. The NMR structure analysis found a cavity inside the hydrophobic core of R2 but not in R1 or R3. Here, we show that R2 has slow conformational fluctuations, and that a cavity-filling mutation which stabilizes the R2 structure significantly reduces specific Myb DNA-binding activity and trans-activation. Structural observations of the free and DNA-complexed stages suggest that the implied inherent conformational flexibility of R2, associated with the presence of the cavity, could be important for DNA recognition by Myb.

Amino Acid Sequence↗

PubMed1995

The affinity maturation of anti-4-hydroxy-3-nitrophenylacetyl mouse monoclonal antibody. A calorimetric study of the antigen-antibody interaction.

To understand the mechanism of affinity maturation, we examined the antigen-antibody interactions between 4-hydroxy-3-nitrophenylacetyl (NP) caproic acid and the Fab fragments of three anti-NP antibodies, N1G9, 3B44, and 3B62, by isothermal titration calorimetry. The analyses have revealed that all of these interactions are mainly driven by negative changes in enthalpy. The enthalpy changes decreased linearly with temperature in the range of 25-45 degrees C, producing negative changes in heat capacity. On the basis of the dependence of binding constants on the sodium chloride concentration, we have shown that, during the affinity maturation of the anti-NP antibody, the electrostatic effect does not significantly contribute to the increase in the binding affinity. We have found that, as the logarithm of the binding constants increases during the affinity maturation of the anti-NP antibody, the magnitudes of the corresponding enthalpy, heat capacity, and unitary entropy changes increase almost linearly. On the basis of this correlation, we have concluded that, during the affinity maturation of the anti-NP antibody, a better surface complementarity is attained in the specific complex in order to obtain a higher binding affinity.

Animals↗

PubMed1995

Comparative thermodynamic analyses of the Fv, Fab* and Fab fragments of anti-dansyl mouse monoclonal antibody.

In order to investigate the role of the constant domains on the antigen-binding property of the variable domains, we have carried out a comparative thermodynamic study of the anti-dansyl Fv, Fab* and Fab fragments that possess the identical amino acid sequence of the variable domains. The thermodynamic analyses have shown that binding constants, enthalpy changes and entropy changes are similar for the three antigen-binding fragments, whereas the thermal stability of Fab is much higher than that of Fv and Fab*. We have concluded that (i) the variable domains of the three antigen-binding fragments possess identical intrinsic capability for antigen binding and (ii) the two constant domains serve to improve the stability of the variable domains.

Animals↗

PubMed1995

Human A-myb gene encodes a transcriptional activator containing the negative regulatory domains.

The myb gene family has three members, c-myb, A-myb, and B-myb. A-myb mRNA is mainly expressed in testis and peripheral blood leukocytes. A-Myb can activate transcription from the promoter containing Myb-binding sites in all cells examined. In addition to the two domains (a DNA-binding domain and a transcriptional activation domain), two negative regulatory domains have been identified in A-Myb. These results indicate that A-Myb functions as a transcriptional activator mainly in testis and peripheral blood cells, and the regulatory mechanism of A-Myb activity is similar to that of c-Myb.

Cell Cycle Proteins↗

PubMed1995

Determination of the NMR solution structure of a specific DNA complex of the Myb DNA-binding domain.

The solution structure of a specific DNA complex of the minimum DNA-binding domain of the mouse c-Myb protein was determined by distance geometry calculations using a set of 1732 nuclear Overhauser enhancement (NOE) distance restraints. In order to determine the complex structure independent of the initial guess, we have developed two different procedures for the docking calculation using simulated annealing in four-dimensional space (4D-SA). One is a multiple-step procedure, where the protein and the DNA were first constructed independently by 4D-SA using only the individual intramolecular NOE distance restraints. Here, the initial structure of the protein was a random coil and that of the DNA was a typical B-form duplex. Then, as the starting structure for the next docking procedure, the converged protein and DNA structures were placed in random molecular orientations, separated by 50 A. The two molecules were docked by 4D-SA utilizing all the restraints, including the additional 66 intermolecular distance restraints. The second procedure comprised a single step, in which a random-cell protein and a typical B-form DNA duplex were first placed 70 A from each other. Then, using all the intramolecular and intermolecular NOE distance restraints, the complex structure was constructed by 4D-SA. Both procedures yielded the converged complex structures with similar quality and structural divergence, but the multiple-step procedure has much better convergence power than the single-step procedure. A model study of the two procedures was performed to confirm the structural quality, depending upon the number of intermolecular distance restraints, using the X-ray structure of the engrailed homeodomain-DNA complex.

Animals↗

PubMed1995

Comparison of the free and DNA-complexed forms of the DNA-binding domain from c-Myb.

The DNA-binding domain of c-Myb consists of three imperfect tandem repeats (R1, R2 and R3). The three repeats have similar overall architectures, each containing a helix-turn-helix variation motif. The three conserved tryptophans in each repeat participate in forming a hydrophobic core. Comparison of the three repeat structures indicated that cavities are found in the hydrophobic core of R2, which is thermally unstable. On complexation with DNA, the orientations of R2 and R3 are fixed by tight binding and their conformations are slightly changed. No significant changes occur in the chemical shifts of R1 consistent with its loose interaction with DNA.

Amino Acid Sequence↗

PubMed1995

Pentoxifylline impairs macrophage defense against Mycobacterium avium complex.

Pentoxifylline, a tumor necrosis factor-alpha (TNF) inhibitor, is being tested as a treatment adjunct in human immunodeficiency virus (HIV)-infected patients. However, TNF is important in cellular defense. The effect of pentoxifylline on Mycobacterium avium complex (MAC) growth in exogenously infected macrophages was compared with the effect of dexamethasone. Pentoxifylline, in a concentration that decreased MAC-induced TNF by 48.1%, enhanced MAC growth by 1.9- to 19.6-fold and 1.82- to 4.46-fold in macrophages from normal and HIV-infected patients, respectively. It also induced interleukin-6 (IL-6) in infected macrophages. IL-6 induction correlated with the increase in MAC growth (y = 0.89 + 0.266x, P = .025). Dexamethasone in an equivalent TNF-suppressing concentration also increased MAC growth but was less effective. Unlike pentoxifylline, dexamethasone suppressed IL-6 and the suppression correlated inversely with MAC growth (y = 0.248 + 9.942x, P = .003). Thus, TNF and IL-6 are important in macrophage defense against MAC. Pentoxifylline and dexamethasone should be used with caution in AIDS patients.

CD4 Lymphocyte Count↗

PubMed1995

Identification of the location of antigenic sites of swine vesicular disease virus with neutralization-resistant mutants.

Neutralization sites on swine vesicular disease virus (SVDV) have been identified by sequence analysis of neutralization-resistant mutants. Eight neutralizing monoclonal antibodies (MAbs) were produced and neutralization-resistant mutants were selected with the MAbs. Resistance of the mutants to neutralization was shown using the stab-neutralization method, and the results indicated the presence of five neutralization sites on the virus. The location of each site was identified from amino acid changes resulting from nucleotide substitutions in the mutants, and designated site 1 (residues 87 and 88 of VP1), 2a (residue 163 of VP2), 2b (residue 154 of VP2), 3a (residue 272 and 275 of VP1, 60 of VP3) and 3b (residues 70 and 233 of VP2, 73 and 76 of VP3). The locations of the amino acids substitutions at each site formed a cluster on a computer-simulated three-dimensional model of SVDV and were exposed to the surface of the virion.

Amino Acids↗

PubMed1995

Mechanism of the formation of DNA triplex and effect of chemical modifications on its stability as studied by isothermal titration calorimetry.

The thermodynamic properties of DNA triplex formation with various single-stranded oligo-DNAs as well as mismatched sequences were investigated by isothermal titration calorimetry (ITC). For the triplex formation of perfectly matched sequences, enthalpy changes and dissociation constants were measured and their temperature-dependences suggest that conformational change of pyrimidine single-strand and the intermediate state(s) are involved in the triplex formation. Effects of mismatches and chemical modifications on stability and specificity of the triplex formation will also be discussed.

Base Sequence↗

PubMed1995

Structural polymorphism and thermal stability of telomere DNAs (T2G4)n and (T4G4)n.

The ends of eukaryotic chromosomes, termed telomeres, contain a single-stranded 3' overhang composed of tandemly repeated guanine-rich sequences, such as (T2G4)n and (T4G4)n, along one strand. The sequences can form defined folded tetraplex structures. Here we have systematically examined structural polymorphism and thermal stability of a series of oligonucleotide sequences, Tet n: (T2G4)n and Oxy n: (T4G4)n (n = 1, 2, 3, 4), using circular dichroism (CD) spectroscopy. The CD spectra of Tet 1 and Oxy 1 are consistent with those observed in tetraplex structures consisting of four parallel strands (type I conformation); whereas the spectra of Tet 2, Oxy 2, Oxy 3, and Oxy 4 correspond with those observed for tetraplex conformations where the strands are antiparallel (type II conformation). The spectra of Tet 3 and Tet 4 suggests that Tet 3 and Tet 4 can adopt both type I and type II conformations and they are structurally polymorphic. The melting temperatures of Tet n and Oxy n (n = 1, 2, 3, 4) measured by CD melting are consistent with the previously reported values obtained from differential scanning calorimetry (DSC). Furthermore, the CD melting of Tet 4 suggests that the type II conformation of Tet 4 changes into type I conformation between 55 degrees C and 70 degrees C.

Base Sequence↗

PubMed1994

Solution structure of a specific DNA complex of the Myb DNA-binding domain with cooperative recognition helices.

The DNA-binding region of Myb consists of three imperfect tandem repeats (R1, R2, and R3). We have determined the solution structure of a specific DNA complex of the minimum DNA-binding domain (R2R3) by heteronuclear multidimensional NMR. Both R2 and R3 contain three helices, and the third helix in each is found to be a recognition helix. R2 and R3 are closely packed in the major groove, so that the two recognition helices contact each other directly to bind to the specific base sequence, AACNG cooperatively; this is a significant arrangement of recognition helices. The three key base pairs in this sequence are specifically recognized by Asn-183 (R3), Lys-182 (R3), and Lys-128 (R2). In contrast, R1 has no specific interactions with DNA from our NMR study of the DNA complex of the full DNA-binding domain (R1R2R3).

Amino Acid Sequence↗

PubMed1994

Evolutionary conservation of chymotrypsinogen gene: genomic analysis and protein modeling.

Chymotrypsinogen is widely present in various animal pancreases. To study evolutionary relationship of chymotrypsinogen gene in species, we used a cDNA probe of human prechymotrypsinogen to investigate the species distribution of chymotrypsinogen gene, and designed oligodeoxynucleotide primers to investigate the genomic organization in the three domains of active sites. The genomic analyses showed that chymotrypsinogen gene is evolutionary conserved in species. On the basis of the deduced amino acid residues, a three-dimensional model for human chymotrypsinogen was further built by computer graphics. The model showed high similarity to the X-ray crystal structure of bovine chymotrypsinogen A, thus, demonstrated that the three-dimensional structure is more conserved in evolution than protein sequences.

Animals↗

PubMed1994

DNA fingerprinting involving fluorescence-labeled termini of any enzymatically generated fragments of DNA.

We have developed a new fluorescence-based method for DNA fingerprinting that does not require a fluorescent linker or a synthetic oligonucleotide primer, both of which are normally used for labeling of DNA. Cosmid DNAs are digested with appropriate restriction enzymes and the 3' termini of DNA fragments are labeled with the corresponding, fluorescent dye-conjugated dideoxynucleotide triphosphate terminator (dye-ddNTP) by the Klenow fragment of DNA polymerase I from Escherichia coli, which has 3'-->5' exonuclease and replacement activities as well as its main 5'-->3' polymerase activity. Samples are separated on a DNA-sequencing gel and data are analyzed by application of both the Version 0.3.8a mapper program (Applied Biosystem Inc., Foster City, CA) and our Overlap I program that facilitate rapid analysis of the frequency of overlapping of cosmid DNAs. Using this method we have determined the overlap frequency of DNA fragments of each cosmid clone from the mouse MHC class I gene cluster.

Animals↗

PubMed1994

Evolution of the IL-6/class IB cytokine receptor family in the immune and nervous systems.

It has been suggested that the cytokine receptor has a structure similar to immunoglobulin and this structural similarity has raised the possibility that they have evolved from a common ancestral molecule. In the early 1970s, it was discovered that developing sympathetic neurons could switch from an adrenergic to cholinergic phenotype. The search for a diffusible factor responsible for this eventually led to the identification of leukemia inhibitory factor (LIF). Cholinergic differentiation factor (CDF)/LIF has turned out to belong to the IL-6/class IB cytokine family. In this article we further speculate on a plausible molecular pathway for the IL6/class IB receptor family in the immune and nervous systems. We think that the evolution of the IL-6/class IB receptor family may have occurred in at least two major steps. Firstly, binding subunits of an IL-6 receptor and for a CDF/LIF receptor evolved and secondly, a third binding subunit of a CNTF receptor evolved. Our evolutional consideration predicts that the binding subunits generally determine the specificity of the receptors and it is possible that novel members of the cytokine family and their receptors exist in the nervous system.

Amino Acid Sequence↗

PubMed1994

Use of pentoxifylline therapy for patients with AIDS-related wasting: pilot study.

Severe weight loss is a common manifestation of advanced infection with the human immunodeficiency virus. The level of tumor necrosis factor alpha (TNF-alpha), an inducer of cachexia in laboratory animals, is elevated in the serum of some patients with AIDS. In a pilot study, five patients with unexplained AIDS-related wasting were treated with pentoxifylline, a known suppressor of TNF-alpha production. Three of the five patients had elevated baseline serum levels of TNF-alpha, and these three patients did not have significant weight gain after 4-8 weeks of pentoxifylline therapy despite the reduction of serum TNF-alpha levels. The remaining two patients, who did not have elevated serum levels of TNF-alpha, continued to lose weight and developed extensive bacterial pneumonia within 3 weeks of starting pentoxifylline therapy. Thus, therapy with pentoxifylline did not clearly benefit the patients with AIDS-related wasting in this uncontrolled pilot study; indeed, it might have been harmful for a subgroup of these patients.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Pentoxifylline aggravates impairment in tumor necrosis factor-alpha secretion and increases mycobacterial load in macrophages from AIDS patients with disseminated Mycobacterium avium-intracellulare complex infection.

Pentoxifylline, which inhibits tumor necrosis factor-alpha (TNF alpha), decreases human immunodeficiency virus replication in peripheral blood mononuclear cells. However, TNF alpha is important in cellular defense against M. avium-intracellulare complex (MAC), a common infection in advanced AIDS. The effect of pentoxifylline on mycobacterial colony counts in macrophages with in vivo MAC infection was evaluated, and differences in lipopolysaccharide (LPS)-induced TNF release in infected and uninfected macrophages were determined. Macrophages with in vivo MAC infection released much less TNF alpha in response to LPS (P = .01). The response was partially restored after antimycobacterial therapy. Pentoxifylline, in a concentration that inhibited LPS-induced TNF alpha by 52.4%, increased MAC counts by 2.5- to 50.0-fold. Thus, macrophages from AIDS patients with disseminated MAC infection are deficient in their ability to release TNF alpha, and further inhibition by pentoxifylline may be detrimental.

AIDS-Related Opportunistic Infections↗