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Biomedical subjects

V Madison

Publications and source records attributed to V Madison.

At least 19 recordsLinked to original sources
PubMed2002

Binding affinities and geometries of various metal ligands in peptide deformylase inhibitors.

Removal of the N-terminal formyl group from newly synthesized proteins by the enzyme peptide deformylase (PDF) is essential for normal growth of bacteria but not higher organisms. Recently, PDF has been explored as a target for novel antibiotics. Screening a collection of natural products for antimicrobial activity identified actinonin and two matlystatin analogs as potent PDF inhibitors. A number of synthetic analogs of these natural products were prepared and their inhibitory potency determined. Previous work has shown that PDF is an iron metalloproteinase also containing a catalytic glutamic acid residue. Ligation of the ferrous cation is an essential feature of potent inhibitors. The structures of actinonin, a matlystatin analog and a synthetic inhibitor complexed with PDF were determined by crystallography. A quantum mechanics/molecular mechanics (QM/MM) method was used to reproduce the geometry of known complexes, to predict the protonation state in the active site and to predict the geometry of additional complexes. The requirement for protonation of the active site glutamate anion is an important factor in understanding the potency of inhibitors with acidic iron-ligating groups such as hydroxamate and carboxylate. Even though potent inhibitors of PDF have been discovered, their bacteriostatic mechanism of action and the rapid development of resistance in vitro may limit their potential as antibacterial drugs.

Amidohydrolases↗
PubMed2001

Expression, purification, characterization and homology modeling of active Akt/PKB, a key enzyme involved in cell survival signaling.

Akt is a serine/threonine kinase that plays a critical role in cell survival signaling and its activation has been linked to tumorigenesis. Up-regulation of Akt as well as its upstream regulator phosphatidylinositol-3 kinase (PI3K) has been found in many tumors and the negative regulator of this pathway PTEN/MMAC is a tumor suppressor. As a target for drug discovery, we have expressed and purified an active Akt1 enzyme from a recombinant baculovirus-infected Sf9 cell culture. Coexpression of Akt1 with the catalytic subunit of PI3K or treatment with okadaic acid during expression was found to generate an active enzyme in the insect cell culture system. We have optimized the kinase activity and developed a simple quantitative kinase assay using biotinylated peptide substrates. Using the purified active enzyme, we have characterized its physical, catalytic and kinetic properties. Since Akt is closely related to protein kinase C (PKC) and protein kinase A, the issue of obtaining selective inhibitors of this enzyme was addressed by comparison of the structures of catalytic domains of Akt and PKC, derived by homology modeling methods. A number of amino acid differences in the ATP binding regions of these kinases were identified, suggesting that selective inhibitors of Akt can be discovered. However, the ATP binding regions are highly conserved in the three isoforms of Akt implying that the discovery of isoform-selective inhibitors would be very challenging.

Amino Acid Sequence↗
PubMed2001

Effect of naturally occurring active site mutations on hepatitis C virus NS3 protease specificity.

A comparison of the DNA sequences from all available genotypes of HCV indicate that the active site residues of the NS3 protease are strictly conserved with the exception of positions 123 and 168, which border the S(4) subsite. In genotype 3, the canonic arginine and aspartic acid have been replaced with threonine and glutamine, respectively. To determine if these differences contribute to an altered specificity, we characterized single-chain NS3 proteases from strains 1a, 1b, and 3a with peptide substrates and product inhibitors on the basis of the natural cleavage junction sequences, in addition to polyprotein substrates derived from the 1a strain. No statistically significant differences in specificity were observed. To demonstrate that the active sites were actually different, we generated and evaluated peptide substrates with unnatural extended side-chains. These studies confirmed that there are measurable differences between the NS3 proteases of genotypes 1 and 3. Specifically, a 5-fold difference in K(i) was observed between the proteases from genotypes 1 and 3 when a D-Glu occupied P(5), and a 30-fold difference was seen when this position contained a D-homoglutamate. The contribution of residues 123 and 168 toward the altered specificity was then evaluated individually by site-directed mutagenesis. These mutants showed that potency differences within this series could be attributed to the residue that occupied position 123 of the protease. Modeling these unnatural substrate/mutant protease interactions, on the basis of cocrystal structures of enzyme-substrate complexes, provides a structural basis for these observations. Proteins 2001;43:82-88.

Binding Sites↗
PubMed1999

Peptidomimetic compounds that inhibit antigen presentation by autoimmune disease-associated class II major histocompatibility molecules.

We have identified a heptapeptide with high affinity to rheumatoid arthritis-associated class II major histocompatibility (MHC) molecules. Using a model of its interaction with the class II binding site, a variety of mimetic substitutions were introduced into the peptide. Several unnatural amino acids and dipeptide mimetics were found to be appropriate substituents and could be combined into compounds with binding affinities comparable to that of the original peptide. Compounds were designed that were several hundred-fold to more than a thousand-fold more potent than the original peptide in inhibiting T-cell responses to processed protein antigens presented by the target MHC molecules. Peptidomimetic compounds of this type could find therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of autoimmune diseases.

Amino Acid Sequence↗
PubMed1995

Insertion of a structural domain of interleukin (IL)-1 beta confers agonist activity to the IL-1 receptor antagonist. Implications for IL-1 bioactivity.

We showed previously that replacement of Lys-145 in the IL-1 receptor antagonist (IL-1ra) with Asp resulted in an analog (IL-1ra K145D) with partial agonist activity. To identify additional amino acids that affect IL-1 bioactivity, we created second site mutations in IL-1ra K145D. Substitutions of single amino acids surrounding position 145 were made; none of these substitutions increased the bioactivity of IL-1ra K145D. However, the insertion of the beta-bulge (QGEESN) of IL-1 beta at the corresponding region of IL-1ra K145D resulted in a 3-4-fold augmentation of bioactivity. An additional increase in agonist activity was observed when the beta-bulge was co-expressed with a second substitution (His-54 --> Pro) in IL-1ra K145D. We also show that the bioactivity of both IL-1ra K145D and the triple mutant IL-1ra K145D/H54P/QGEESN is dependent on interaction with the newly cloned IL-1 receptor accessory protein.

Amino Acid Sequence↗
PubMed1995

Site-directed mutagenesis--molecular biology and rational drug design.

Using site-directed mutagenesis, we have determined the location and composition of the binding sites in human IL-1 alpha and IL-1 beta for the Type I IL-1 receptor (IL-1R). The binding site in each ligand is a discontinuous epitope made up of at least seven amino acids whose side chains are exposed on a contiguous region of the protein surface. Although human IL-1 alpha and IL-1 beta have similar affinities and cross-compete for binding to the human Type I IL-1R, the binding site residues are not identical in the two ligands. In addition, the residues in the binding site of each ligand contribute differently to binding of the human versus the mouse IL-1R. The structure of the IL-1 binding site has implications for the rational design of IL-1 antagonists.

Animals↗
PubMed1994

Biochemical analysis of the transducin-phosphodiesterase interaction.

In vertebrate rod cells, the activated alpha-subunit of rod transducin interacts with the gamma (regulatory) subunits of phosphodiesterase to disinhibit the catalytic subunits. A 22-amino acid long region of rod transducin involved in phosphodiesterase activation has recently been identified. We have used peptides from this region of rod transducin and from several other G protein alpha-subunits to study the nature and specificity of the G protein alpha-effector interaction. Although peptides derived from rod transducin, cone transducin and gustducin are similar, only the rod peptide is capable of activating rod phosphodiesterase. Using substituted peptides we have identified five residues on one exposed face of rod transducin as important to phosphodiesterase activation. These results disagree with previous models which propose that loop regions of rod transducin interact with phosphodiesterase gamma.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Characterization of the human IgE Fc-Fc epsilon RI alpha interaction.

A significant amount of progress has been achieved on characterizing the interaction of the IgE Fc molecule with the Fc epsilon RI alpha. However, there is yet no definitive structural information which precisely defines the nature of this interaction. It is clear that this information will only be provided by the resolution of the X-ray crystallographic structures of the IgE Fc molecule, the Fc epsilon RI alpha subunit extracellular domain, and the IgE Fc-Fc epsilon RI alpha complex. It is anticipated that these structures will be determined in the near future, and that they may provide some insight into the development of potential therapeutics effective in the management of IgE-mediated allergic diseases.

Animals↗
PubMed1992

Morphological development and sex of bovine in vitro-fertilized embryos.

Bovine in vitro-fertilized embryos at the blastocyst stage were collected at days 7, 8, and 10 postinsemination and sex was determined via the polymerase chain reaction (PCR) to compare the embryonic development with the sex of the embryos. The percentages of males (sex ratio) after division of the embryos into three developmental groups were 68%, 48%, and 35% in the fast, intermediate, and slow groups, respectively (P = 0.014). The percentages of males on days 7, 8, and 10 were 60%, 40% and 33%, respectively (P = 0.043). The average sex ratio for the whole material was 50%. It is thus concluded that male bovine preimplantation embryos develop faster than female embryos.

Animals↗
PubMed1991

In vitro fertilization in cattle: a review.

In vitro maturation and fertilization of cattle oocytes and subsequent in vitro culture of zygotes and embryos is discussed in the context of recent encouraging data. Mass production of embryos produced in this way in the future will have a great impact on animal production and animal breeding plans, for example the so-called MO-ET (multiple ovulation and embryo transfer) plan which aims at establishing nucleus breeding herds.

Animal Husbandry↗
PubMed1991

The effect of endotoxin-contaminated medium on in vitro fertilization and development of bovine oocytes matured in vitro.

The purpose of this study was to determine whether the presence of bacterial endotoxin in the bovine serum albumin (BSA) used to supplement media utilized for sperm preparation and co-culture of bovine sperm and oocytes affects in vitro penetration and embryonic development of oocytes matured in vitro. The chromogenic limulus amoebocyte lysate (LAL) test was used for quantification of the content of endotoxin. The proportion of penetrated ova was significantly greater (P greater than 0.0005) for the endotoxin-contaminated group (89%) versus the non-contaminated group (61%), but this was probably not due to endotoxin contamination. The presence of endotoxin resulted in a high rate of polyspermy (27% versus 4%, respectively; P greater than 0.0005), while the occurrence of parthenogenetic activation was the same for each group (8%). The proportion of total embryos put into culture that developed to the blastocyst stage by day 8 was similar (30% and 26%) for the contaminated and non-contaminated group, respectively. Fifty-three and 69%, respectively, hatched on day 10. These results suggest that endotoxin induces polyspermy, but has no adverse effect on embryonic development.

Animals↗
PubMed1990

Bundles of amphipathic transmembrane alpha-helices as a structural motif for ion-conducting channel proteins: studies on sodium channels and acetylcholine receptors.

Channel proteins are transmembrane symmetric (or pseudosymmetric) oligomers organized around a central ionic pore. We present here a molecular model of the pore forming structures of two channel proteins with different primary structures and oligomeric size: the voltage-sensitive sodium channel and the nicotinic cholinergic receptor. We report low-energy arrangements of alpha-helical bundles calculated by semiempiricial potential energy functions and optimization routines and further refined using molecular dynamics. The ion-conducting pore is considered to be a symmetric or pseudosymmetric homooligomer of 3-5 amphipathic alpha-helices arranged such that the polar residues line a central hydrophilic pathway and the apolar residues face the hydrophobic bilayer interior. The channel lining exposes either charged (Asp, Glu, Arg, Lys) or polar-neutral (Ser, Thr) residues. A bundle of four parallel helices constrained to C4 symmetry, the helix axis aligned with the symmetry axis, and the helices constrained to idealized dihedral angles, produces a structure with a pore of the size inferred for the sodium channel protein (area approximately 16 A2). Similarly, a pentameric array optimized with constraints to maintain C5 symmetry and backbone torsions characteristic of alpha-helices adopts a structure that appears well suited to form the lining of the nicotinic cholinergic receptor (pore area approximately 46 A2). Thus, bundles of amphipathic alpha-helices satisfy the structural, energetic, and dynamic requirements to be the molecular structural motif underlying the function of ionic channels.

Amino Acid Sequence↗
PubMed1988

M2 delta, a candidate for the structure lining the ionic channel of the nicotinic cholinergic receptor.

A synthetic 23-mer peptide that mimics the sequence of the putative transmembrane M2 segment of the Torpedo californica acetylcholine receptor (AcChoR) delta subunit--Glu-Lys-Met-Ser-Thr-Ala-Ile-Ser-Val-Leu-Leu-Ala-Gln-Ala-Val-Phe-Leu- Leu-Leu-Thr-Ser-Gln-Arg--forms discrete ionic channels in phosphatidylcholine bilayers. In contrast, a synthetic peptide that mimics the sequence of the putative M1 transmembrane segment of the Torpedo AcChoR delta subunit--Leu-Phe-Tyr-Val-Ile-Asn-Phe-Ile-Thr-Pro-Cys-Val-Leu-Ile-Ser-Phe- Leu-Ala-Ser-Leu-Ala-Phe-Tyr--does not form channels. The synthetic M2 delta channel peptide exhibits features that are characteristic of the authentic AcChoR channel, such as single channel conductances, discrimination of cations over anions, and channel lifetimes for open and closed states in the millisecond time range. Energetic considerations suggest that an aggregate of five amphipathic alpha-helices conforms the channel. Thus, the M2 segment may be a component of the AcChoR channel structure.

Amino Acid Sequence↗
PubMed1982

Unusual cis-trans isomerism in N-acetyl, N'-methylamide derivatives of syn- and anti-5-methylproline.

By means of carbon-13 n.m.r. spectroscopy, we have investigated cis-trans isomerism for N-acetyl, N'-methylamide derivatives of syn- and anti-5-methylproline (syn: the methyl group and carboxamide are on the same side of the proline ring, anti: on opposite sides). For Ac-syn-5-MeProNHMe, we observe about 25% cis peptide bond isomers in most solvents as is common for oligopeptides of proline. The percentage of cis isomer is about 20% greater for Ac-anti-5-MeProNHMe in non-polar solvents. These percentages show little temperature dependence. The free energy barrier for trans to cis isomerization is 19.4 kcal/mol in dioxane and 20.0 kcal/mol in dimethylsulfoxide for Ac-anti-5-MeProNHMe, in agreement with values observed for other peptides. However, value observed for Ac-syn-5-MeProNHMe are about 2 kcal/mol lower. For the latter peptide the barrier is 1.7 kcal/mol higher in water than in three organic solvents. These results indicate that steric interactions of the 5-methyl group destabilized the trans peptide isomer in Ac-anti-5-MeProNHMe without affecting the isomerization barrier. In contrast, for Ac-syn-5-MeProNHMe, the cis and trans peptide isomers are destabilized by the same amount so that the barrier is lowered without altering the equilibrium ratio. The higher energy barrier in water indicates binding of solvent to the plant peptide ground state.

Hydroxyproline↗