Biomedical subjects

M G Peter

Publications and source records attributed to M G Peter.

At least 19 recordsLinked to original sources

PubMed2003

In-vivo antimalarial activity of some oxygenated xanthones.

A series of oxygenated xanthones was prepared so that the antimalarial activity of each compound could be evaluated in vivo, using 4-day suppressive assays against Plasmodium berghei ANKA in BALB/c mice. When given in a dose of 20 mg/kg.day for 4 days, most of the compounds produced significant chemosuppression of parasitaemia. The most active compound was 1,3,6,8-tetrahydroxyxanthone, which reduced the percentage of erythrocytes infected by 70.5%, followed by norlichexanthone (44.3%) and its isomer, 1,3,8-trihydroxy-6-methylxanthone (37.0%). Whereas di-C-allyl-dihydroxyxanthone showed lower but still notable activity (33.4%), 1,3-dihydroxyxanthone was much less active (15.1%). This appears to be the first demonstration of the antimalarial activity of some hydroxyxanthones in vivo.

Animals↗

PubMed2001

Structural insights into the catalytic mechanism of a family 18 exo-chitinase.

Chitinase B (ChiB) from Serratia marcescens is a family 18 exo-chitinase whose catalytic domain has a TIM-barrel fold with a tunnel-shaped active site. We have solved structures of three ChiB complexes that reveal details of substrate binding, substrate-assisted catalysis, and product displacement. The structure of an inactive ChiB mutant (E144Q) complexed with a pentameric substrate (binding in subsites -2 to +3) shows closure of the "roof" of the active site tunnel. It also shows that the sugar in the -1 position is distorted to a boat conformation, thus providing structural evidence in support of a previously proposed catalytic mechanism. The structures of the active enzyme complexed to allosamidin (an analogue of a proposed reaction intermediate) and of the active enzyme soaked with pentameric substrate show events after cleavage of the glycosidic bond. The latter structure shows reopening of the roof of the active site tunnel and enzyme-assisted product displacement in the +1 and +2 sites, allowing a water molecule to approach the reaction center. Catalysis is accompanied by correlated structural changes in the core of the TIM barrel that involve conserved polar residues whose functions were hitherto unknown. These changes simultaneously contribute to stabilization of the reaction intermediate and alternation of the pKa of the catalytic acid during the catalytic cycle.

Acetylglucosamine↗

PubMed2001

Modeling, mutagenesis, and structural studies on the fully conserved phosphate-binding loop (loop 8) of triosephosphate isomerase: toward a new substrate specificity.

Loop 8 (residues 232-242) in triosephosphate isomerase (TIM) is a highly conserved loop that forms a tight binding pocket for the phosphate moiety of the substrate. Its sequence includes the fully conserved, solvent-exposed Leu238. The tight phosphate-binding pocket explains the high substrate specificity of TIM being limited to the in vivo substrates dihydroxyacetone-phosphate and D-glyceraldehyde-3-phosphate. Here we use the monomeric variant of trypanosomal TIM for exploring the structural consequences of shortening this loop. The mutagenesis, guided by extensive modeling calculations and followed up by crystallographic characterization, is aimed at widening the phosphate-binding pocket and, consequently, changing the substrate specificity. Two new variants were characterized. The crystal structures of these variants indicate that in monomeric forms of TIM, the Leu238 side-chain is nicely buried in a hydrophobic cluster. Monomeric forms of wild-type dimeric TIM are known to exist transiently as folding intermediates; our structural analysis suggests that in this monomeric form, Leu238 of loop 8 also adopts this completely buried conformation, which explains its full conservation across the evolution. The much wider phosphate-binding pocket of the new variant allows for the development of a new TIM variant with a different substrate specificity.

Amino Acid Sequence↗

PubMed2001

Preparation of insect-cuticle-like biomimetic materials.

A model system of tanning of a protein matrix within a fibrous structure, such as most commonly found in insect cuticle, was developed, using the cellulose of paper in place of chitin. The paper was impregnated with a tripeptide, DOPA-Gly-Gly, or a protein (BSA) plus catechol and treated with tyrosinase to oxidize the catechol. The resulting material was waterproof and had very high wet strength. If the material was wetted and dried repeatedly its water retention decreased by a factor of at least 2.

Animals↗

PubMed2000

Apparent cooperativity in multivalent verotoxin-globotriaosyl ceramide binding: kinetic and saturation binding studies with [(125)I]verotoxin.

Verotoxin (VT) binding to the trisaccharide portion of globotriaosyl ceramide (Gb(3)) is believed to be a crucial step in the development of hemolytic uremic syndrome (HUS) commonly known as 'Hamburger disease'. This interaction is the initial step in the binding process and defines the specificity of verotoxin binding to cellular membranes. Although molecular modeling, co-crystallization and co-NMR studies with VT and the trisaccharide moiety of Gb(3) have indicated potential multiple sites for Gb(3) binding, little is known about their direct effects on kinetic and equilibrium binding. Here we describe how the binding of radiolabeled VT ([(125)I]VT1) to Gb(3) in a microtiter well format, is driven by two different association rate constants (k(+1a)=0.0075 and k(+1b)=0.275 min(-1) nM(-1)) with the high affinity site representing 15% of the total specific binding sites. Binding was reversible at room temperature, reached equilibrium after 2-3 h, and non-specific binding was less than 5%. Equilibrium binding studies defined by [(125)I]VT1 saturation binding to 15, 30, 60 and 120 ng Gb(3)/well, showed the presence of a single site with dissociation constants (K(d)s) ranging between 0.5 and 3 nM. However, the maximum density of specific [(125)I]VT1 binding sites (B(max)) did not directly correlate with the Gb(3) concentration per well: the most[(125)I]VT1 binding was observed for 60 ng Gb(3) (B(max)=1.28 nM; compared to 0. 23 nM for 30 ng Gb(3) and 0.65 nM for 120 ng Gb(3)). Furthermore, while Hill coefficients (n(H)) for 15, 30 and 120 ng Gb(3) were close to unity indicating single interactions, for the saturation isotherm for 60 ng Gb(3)/well n(H) was 1.4. Subsequent Scatchard analysis yielded a concave downward curve for [(125)I]VT1 binding to 60 ng Gb(3)/well, suggesting positive co-operativity. We present, for the first time, conclusive binding data confirming the presence of at least two discrete Gb(3) binding sites: these multivalent interactions between verotoxin VT-1 and Gb(3) were described by association reactions driven by two distinct rate constants, as well as by the positive co-operativity governing binding at a restricted receptor concentration. These results imply that the concentration of Gb(3) on the surface of target cells can have a complex, non-linear effect on verotoxin binding and thereby, on sensitivity to cytotoxicity.

Bacterial Toxins↗

PubMed2000

Two isoflavanones from the stem bark of Erythrina sacleuxii.

From the stem bark of Erythrina sacleuxii two new isoflavanones, (R)-5,7-dihydroxy-2',4',5'-trimethoxyisoflavanone (trivial name, (R)-2,3-dihydro-7-demethylrobustigenin) and (R)-5-hydroxy-2',4',5'-trimethoxy-2",2"-dimethylpyrano[5",6":6,7]isoflavanone (trivial name, (R)-saclenone) were isolated. In addition the known compounds shinpterocarpin, 2,3-dehydrokievitone, abyssinone V, abyssinone V-4'-methyl ether, erythrinasinate and 4'-O-methylsigmoidin B were isolated. The structures were determined on the basis of spectroscopic evidence.

Isoflavones↗

PubMed1999

Alpha-galactose based neoglycopeptides. Inhibition of verotoxin binding to globotriosylceramide.

Solution and solid phase strategies for the synthesis of alpha-galactose based neoglycopeptide derivatives 2-13 were developed. Neoglycopeptides generated were tested for the inhibition of verotoxin binding to globotriosylceramide (Gb3) using ELISA. Among all of the compounds tested, only the lipid derivatives of neoglycopeptides, 11, 12 and 13 were found to be inhibitors, IC50 = 2.0 mM (11b and 12c) and 0.2 mM (11c and 13c). All of the inhibitors (11b, 11c, 12c and 13c) have a similar branching of the two alpha-galactosyl units at the N-terminal glycine residue of a short peptide and a lipid moiety attached at the C-terminal site. Both of these factors seem to be crucial for the inhibition. It is interesting to note that the inhibitors have only a portion of the natural trisaccharide ligand. The secondary groups either may contribute in sub-site oriented interactions with the protein receptors or may mimic the internal sugar units of the cell-surface ligand, Gb3.

Anti-Infective Agents↗

PubMed1998

Reactions of a glucosinolate breakdown product (benzyl isothiocyanate) with myoglobin.

The interaction of various amounts of benzyl isothiocyanate (benzyl-ITC) with myoglobin is known to lead to the formation of derivatives. These have been characterised by the determination of solubility, free amino group, tryptophan content and chromatographic as well as electrophoretic behaviour. In the range between 2.5 and 125 mg benzyl-ITC/g protein, all properties of the reaction products correlate with the concentration of benzyl-ITC. However, at 250 mg benzyl-ITC/g myoglobin, a rather unexpected low degree of derivatization, as well as atypical chromatographic and electrophoretic behaviour, is observed. The proposed explanation was that conformational changes in the presence of a high concentration of hydrophobic benzyl-ITC made fewer amino groups accessible to the reagent. To test this hypothesis we have run the reaction under denaturing conditions. The results showed that the reaction of myoglobin with high concentrations of benzyl-ITC in the presence of 8 M urea led to a higher degree of derivatization than in the presence of water only. In addition, the Mr distribution of the reaction products was determined by MALDI-TOF-mass spectrometry and the overall degree of derivatization calculated from the spectra.

Hydrogen-Ion Concentration↗

PubMed1997

Phenol-oxidizing enzymes: mechanisms and applications in biosensors.

Phenolic compounds are widely distributed in nature. Enzymes which catalyze their oxidation are monophenol monooxygenases, such as tyrosinases and laccases, and peroxidases. Their metabolic role includes the decomposition of natural complex aromatic polymers as well as polymerization of the oxidation products and the degradation of xenobiotics. Their catalytic properties and broad availability gained impact on the development of biosenors for both environmentally important pollutants and clinically relevant metabolites. Mechanisms for the phenol-oxidizine enzymes tyrosinases, laccases, and peroxidases are reviewed and some examples for their use in the construction of phenol selective biosenors are given.

Biosensing Techniques↗

PubMed1996

Characterization of the endothelin receptor selective agonist, BQ3020 and antagonists BQ123, FR139317, BQ788, 50235, Ro462005 and bosentan in the heart.

1. In this study we used ligand binding techniques to determine the affinity and selectivity of endothelin receptor agonists and antagonists in human left ventricle which expresses both ETA and ETB receptors, and compared these results with cardiovascular tissues from rat and porcine hearts. 2. The linear tripeptide antagonist, FR139317 competed for [125I]-ET-1 binding to human left ventricle with over 200,000 fold selectivity for the ETA receptor (KD ETA = 1.20 +/- 0.28 nM, KDETB = 287 +/- 93 microM). The ETA-selective non-peptide antagonist, 50235, competed with lower affinity and selectivity (KDETA = 162 +/- 61 nM, KDETB = 171 +/- 42 microM) in this tissue. BQ123 and FR139317 also showed high selectivity (greater than 20,000 fold) and affinity in rat (BQ123: KDETA = 1.18 +/- 0.16 nM, KDETB = 1370 +/- 1150 microM; FR139317: KDETA = 2.28 +/- 0.30 nM, KDETB = 292 +/- 114 microM) and pig heart (BQ123: KDETA = 0.52 +/- 0.05 nM, KDETB = 70.4 +/- 4.0 microM; FR139317: KDETA = 2.17 +/- 0.51 nM, KDETB = 47.1 +/- 5.7 microM) (n > or = 3 individuals +/- s.e.mean). 3. Although BQ3020 competed with over 1000 fold selectivity for the ETB subtype in human heart (KDETB = 1.38 +/- 0.72 nM, KDETA = 2.04 +/- 0.21 microM) the peptide inhibited only the binding of [125I]-ET-1 at concentrations greater than 100 nM in rat and porcine heart. This is in contrast to the data from the ETA-selective antagonists which indicated the presence of ETB sites in these tissues from animal hearts. 4. The peptide antagonist, BQ788, had a low, micromolar affinity (KD = 1.98 +/- 0.13 microM) using human left ventricle and no significant selectivity for the human ETB-subtype in this tissue. 5. The non-peptide ET antagonists, Ro462005 (KD = 50.3 +/- 9.5 microM) and bosentan (Ro470203; KD = 77.9 +/- 7.9 nM) competed monophasically for [125I]-ET-1 binding sites in human left ventricle. 6. The results show that the ETA antagonists, BQ123 and FR139317, are highly selective for ETA receptors in all cardiac tissues tested, whereas BQ788 has a low affinity and no selectivity in this human tissue. Further we showed that there are species differences in the binding of BQ3020 to the ETB receptors in the hearts derived from human, rat and pig.

Adult↗

PubMed1995

Selectivity of [125I]-PD151242 for human, rat and porcine endothelin ETA receptors in the heart.

1. Endothelin-1 binds with high affinity to heart where it acts as a potent positive inotropic agent. Our aim was to characterize the labelled and unlabelled ETA-selective antagonist PD151242 in heart tissues derived from man, rat and pigs by use of radioligand binding techniques. 2. Binding of [125I]-PD151242 to sections of human left ventricle was time-dependent and reached equilibrium after 120 min at 23 degrees C with an association rate constant of 0.0235 min-1 nM-1. The binding was reversible at 23 degrees C with a dissociation rate constant of 0.00144 min-1. 3. Saturation binding assays with [125I]-PD151242 revealed a single population of high affinity ET receptors in human left ventricle (KD = 1.07 +/- 0.08 nM; Bmax = 29.8 +/- 4.2 fmol mg-1 protein), porcine left ventricle (KD = 1.92 +/- 0.27 nM; Bmax = 493 +/- 248 fmol mg-1 protein), and rat heart (KD = 0.64 +/- 0.08 nM; Bmax = 82.34.7 fmol mg-1 protein). 4. Unlabelled PD151242 competed with specific [125I]-ET-1 binding to human left ventricle tissue in a biphasic manner with high affinity binding to the ETA-site (KD = 7.21 +/- 2.83 nM) and lower affinity for the ETB-subtype (KD = 104 +/- 23 microM), indicating a greater than 10000 fold selectivity to the high affinity site. 5. The ETA-selective ligand FR139317 competed for [125I]-PD151242 binding in human left ventricle with nanomolar affinity (KD = 0.37 +/- 0.10 nM), whereas the ETB-selective compound, BQ3020, competed with only micromolar affinity (KD = 1.5 +/- 0.26 microM). 6. The novel ETA-selective radioligand [125I]-PD151242 binds with high affinity to human, rat and porcine heart. In human tissue, binding was shown to be reversible and highly selective for the ETA-subtype making [1251]-PD151242 a useful selective radioligand for further characterization of the ETA-receptor in human tissue.

Adult↗

PubMed1995

Delineation of endothelin receptors in human left ventricular smooth-muscle cells.

In the human heart, we have previously shown the predominance of endothelin (ET) ETA receptors, in addition to the presence of ET-1, ET-2, ET-3, and big ET-1. ET-1 is a potent constrictor of isolated epicardial coronary arteries, and this action is mediated via ETA receptors. To determine the source of ET in the heart, our aims were to obtain cell cultures from human left ventricle, identify the cell type, and characterize ET secretion and receptor expression. We explanted human left ventricular tissue. Positive staining with alpha-actin antibodies confirmed the presence of smooth-muscle cells, whereas negative staining for sarcomeric actin and von Willebrand factor indicated an absence of cardiac myocytes and endothelial cells, respectively. Therefore, the cultures were identified as human left ventricular smooth-muscle cells (HLVSMCs). Because blood vessels were not macroscopically visible in the ventricular tissue, the HLVSMCs most likely originated from intramyocardial resistance vessels. The cells secreted immunoreactive mature ET and big ET-1 (102 +/- 29 and 73 +/- 10 pM/24 h, respectively; mean of three individuals +/- SEM). Saturation binding studies showed that [125I]ET-1 bind with high affinity in this preparation (Kd 0.21 +/- 0.06 nM; Bmax 15 +/- 4 fmol/mg protein; mean of three individuals +/- SEM). A competition binding study using the ETA-selective antagonist FR139317 (10 pM-10 microM) revealed the predominance of ETA receptors (Kd 0.33 +/- 0.10 nM, n = 3). We have shown that smooth-muscle cells isolated from human left ventricle secrete immunoreactive mature ET and big ET-1, and express mainly ETA receptors. These cells may provide a useful model for studying the effects of ET in the regulation of vascular tone and of the blood supply in the myocardium.

Adult↗

PubMed1990

Chitin biosynthesis enhancement by the endochitinase inhibitor allosamidin.

Membrane preparations of Artemia salina synthetize radiolabelled chitin from UDP-[U-14C]GlcNAc at a low rate (Horst, M.N. (1981) J. Biol. Chem. 256, 1412-1419). We now report that, when the specific endochitinase inhibitor allosamidin is present in addition to the established activators trypsin and GlcNAc, incorporation of [U-14C]GlcNAc into chitin is increased up to 58-fold over the basic synthesis rate. Thus, a greatly enhanced apparent chitin synthase activity is observed in membranes from an arthropod species when simultaneous degradation of chitin is inhibited.

Acetylglucosamine↗

PubMed1990

[A high pressure liquid chromatography method for detecting the isoniazid derivative isonicotinoyl-hydrazine sodium glucuronide, free isoniazid and acetylisoniazid and its use in pharmacologic studies].

A specific direct method for determining the isoniazid derivative isonicotinoyl-hydrazine sodium glucuronide (INHG-Na) with the aid of high-pressure liquid chromatography has been developed, which permits the analysis of this derivative of isoniazid in addition to isoniazid itself and acetylisoniazid in the plasma of patients. In initial pharmacokinetic applications of the method, it has been shown that plasma INHG-Na is a stable substance that liberates only negligible concentrations of INH.

Acetylation↗

PubMed1977