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D J Kyle

Publications and source records attributed to D J Kyle.

At least 37 records · Page 2Linked to original sources
PubMed1996

Accuracy and reliability of the scaling-relaxation method for loop closure: an evaluation based on extensive and multiple copy conformational samplings.

The accuracy and reliability of the recently proposed scaling-relaxation method for loop closure were examined by using extensive conformational sampling. For each of the eight heptapeptides chosen to represent a variety of protein conformations, 1,000-2,000 conformations were sampled. Each segment contained 14 rotatable backbone dihedral angles. The average root mean square deviations (RMSDs) between the predicted and the native conformations were 0.7 angstrom for the backbone and 1.2 angstroms for the side chain atoms. These predictions were substantially more accurate than the previous predictions (1.1 angstroms for the backbone and 2.2 angstroms for the side chain atoms) of the same eight protein segments based on limited conformational sampling (100 conformations for each segment). Large prediction errors mostly occurred at polar and surface side chains that are unlikely to have any meaningful conformation. Moreover, the reliability of seven of the eight predictions was demonstrated with their energy-RMSD and stability-RMSD correlations of the low-energy conformations, where the conformational stability was estimated by using the multiple copy simultaneous sampling method.

Amino Acid Sequence↗
PubMed1996

Preclinical evaluation of single-cell oils that are highly enriched with arachidonic acid and docosahexaenoic acid.

Arachidonic acid (ARA) and docosahexaenoic acid (DHA) are important in human brain and retina development, and there is growing evidence showing the importance of these fatty acids in infant nutrition. Triglyceride oils, highly enriched in ARA (ARASCO) and DHA (DHASCO), were evaluated using very high dose acute (20 g/kg) and 4-wk subchronic gavage feedings in weanling Sprague-Dawley rats. The combination of these oils, Formulaid, was also tested in the 4-wk subchronic study, ARASCO, DHASCO and Formulaid were found to have a no-observable-adverse-effect level of more than 2.5 g/ kg/day, 1.25 g/kg/day and 3.75 g/kg/day, respectively. This represents a 50-fold safety margin over the intended use of Formulaid in infant formula. Survival, clinical signs, body weight gain, food consumption, haematology, clinical chemistry and histopathological evaluations failed to show any significant differences in animals administered ARASCO, DHASCO or Formulaid compared with that in control animals administered equal amounts of high oleic sunflower oil. The bioavailability of ARASCO, DHASCO and Formulaid was verified by increases in DHA and ARA levels in heart and liver tissues in these animals. Because these oils are enriched in only a single bioactive fatty acid, and they have been shown to be safe, they may offer a new source of these fatty acids in speciality foods such as infant formula.

Administration, Oral↗
PubMed1996

Urinary and plasma purine derivatives in fed and fasted llamas (Lama glama and L. guanacoe).

The changes in urinary and plasma purine derivatives in response to fasting and level of feeding in llamas were examines. In one experiment, four llamas were gradually deprived of feed within 3 days and then fasted for 6 days. Daily urinary excretion of purine derivatives decreased with feed intake and leveled on the last 3 days of fasting at 177 +/- 26 mumol/kg W0.75. Allantoin and uric acid comprised 71% and 15% of total purine derivatives, respectively, in both fed and fasted states, but hypoxanthine plus xanthine increased from 9% to 36%. Plasma concentration of allantoin declined with feed intake reduction, but those of uric acid (217 mumol/l) and hypoxanthine plus xanthine (27 mumol/l) remained relatively unchanged. Concentration of uric acid was higher than that of allantoin, probably due to a high reabsorption of uric acid in renal tubules, which was measured as over 90%. In a second experiment, the four llamas were fed at 860 and 1740 g dry matter/d in a crossover design. Urinary total purine derivatives excretion responded to feed intake (10.4 vs 14.4 mmol/d), although the observed differences did not reach significance. Compared with some ruminant species, it appears that the llama resembles sheep regarding the magnitude of urinary purine derivatives excretion but is unique in maintaining a high concentration of uric acid in plasma, which could be part of the llama's adaptation to their environment.

Allantoin↗
PubMed1996

Computational screening of combinatorial libraries.

We first review existing computational methods with an intrinsic combinatorial feature, then describe a new computational method for screening combinatorial libraries using a recently developed multicopy sampling technique. The new method differs from the existing ones in that it can be used to screen simultaneously an entire library of molecules, instead of the individual molecules in a library. As an example, we have applied the method to study site-directed amino acid substitutions in a protein. After two rounds of library screening, we identified the energetically most stable substitutions along with their optimal conformations from all natural amino acids. In principle, the method is generally applicable to study ligand-host systems.

Amino Acids↗
PubMed1996

Urinary excretion of purine derivatives and tissue xanthine oxidase (EC 1.2.3.2) activity in buffaloes (Bubalis bubalis) with special reference to differences between buffaloes and Bos taurus cattle.

The urinary excretion of purine derivatives (PD) was measured in six buffaloes (Bubalis bubalis) during fasting and in fourteen buffaloes given four restricted levels of roughage (2.5-4.8 kg DM/d). Only allantoin and uric acid, not xanthine and hypoxanthine, were present in the urine, the pattern of excretion being similar to that in cattle. The fasting PD excretion amounted to 0.20 (SD 0.06) mmol/kg metabolic weight (W)0.75 per d, and the rate of PD excretion as a linear function of feed intake was 5.2 mmol/kg digestible organic matter intake. Both values were considerably lower than the values for cattle reported in the literature. Creatinine excretion values were 0.33 (SD 0.06) and 0.44 (SD 0.09) mmol/kg (W)0.75 per d determined in fasting and feeding periods respectively. Fasting N excretion was 257 (SD 49) mg N/kg (W)0.75 per d. Both creatinine and fasting N excretions were also lower than in cattle. The activities of xanthine oxidase (EC 1.2.3.2) in plasma, liver and intestinal mucosa were determined in buffaloes, cattle and sheep. Xanthine oxidase activities in buffaloes were 24.5 (SD 2.7) unit/l plasma and 0.44 (SD 0.02) and 0.31 (SD 0.10) unit/g fresh tissue in liver and intestinal mucosa respectively. These activities were higher than those in cattle and sheep. Xanthine oxidase was practically absent from plasma and intestine of sheep. It is suggested that the differences in PD excretion between buffaloes and cattle were probably due to the smaller proportion of plasma PD that was disposed of in the urine of buffaloes.

Allantoin↗
PubMed1996

Antinociceptive profile of the pseudopeptide B2 bradykinin receptor antagonist NPC 18688 in mice.

1. The purpose of this study was to investigate the topical and systemic anti-hyperalgesic effect of the newly-developed pseudopeptide B2 receptor antagonist, NPC 18688, in different models of nociception in mice. 2. Given systemically 30 min beforehand, NPC 18688 (10-300 nmol kg-1, i.p.) caused no agonist effect, but produced a dose-related and significant inhibition of abdominal constrictions caused by intraperitoneal injection of acetic acid (0.6%), acetylcholine (ACh, 4.5 mg kg-1) or kaolin (50 mg kg-1). The calculated mean ID50s and the percentages of maximal inhibitions (MI) for these effects were: 77, 34 and > 300 nmol kg-1 and 65 +/- 6, 70 +/- 5 and 40 +/- 3%, respectively. The anti-hyperalgesic effect of NPC 18688 (100 nmol kg-1, i.p.) occurred rapidly (30 min) and lasted for at least 150 min. Hoe 140 (3-30 nmol kg-1, i.p.) given 30 min beforehand also inhibited, in a graded manner, acetic acid and ACh-induced writhing, with mean ID50s and MI of 6 and 9 nmol kg-1 and 56 +/- 7 and 62 +/- 6%, respectively. 3. NPC 18688 (10-300 nmol kg-1, i.p.) caused a graded inhibition of both phases of formalin (2.5%)-induced pain, its effects being more potent in relation to the second phase of the formalin test. The calculated mean ID50s and the MI were > 300 and 60 nmol kg-1 and 20 +/- 3 and 60 +/- 5% against the first and second phases of formalin-induced nociception, respectively. NPC 18688 at the same doses also inhibited, in a dose-related manner, formalin-induced paw oedema (MI of 35 +/- 3%). 4. When injected locally in the mouse paw, NPC 18688 (2, 10 and 20 nmol/paw) had no agonist activity. However, when co-injected with formalin NPC 18688 (2-20 nmol/paw), it produced significant inhibition of both phases of formalin response, with MI of 40 +/- 3 and 33 +/- 2%, respectively. NPC 18688 at 10 nmol/paw also significantly inhibited formalin-induced paw oedema (25 +/- 2%). 5. Given intraperitoneally, NPC 18688 (30-300 nmol kg-1) determined a graded inhibition of the nociceptive response caused by intraplantar injection of capsaicin (1.6 micrograms/paw) (40 +/- 2%). However, NPC 18688 (up to 300 nmol kg-1, i.p.), given 30 min beforehand, had no significant analgesic effect when analyzed in the tail flick and in the hot plate pain models, nor did it change the performance of animals in the rota rod test. 6. The action of NPC 18688 was quite selective for the B2 receptor, and like Hoe 140, (1 to 100 nmol kg-1, i.p.) it caused graded inhibition of bradykinin (BK, 3 mol/paw)-induced increase in mouse paw volume, with mean ID50s of 61 and 6 nmol kg-1, respectively. In addition, at 100 nmol kg-1, the dose at which NPC 18688 significantly antagonized BK (3 nmol)-mediated rat paw oedema in naive animals, it had no significant effect on des-Arg9-BK (100 nmol/paw)-induced oedema in paws that had been desensitized to BK. NPC 18688 (210 nmol kg-1), like Hoe 140 (230 nmol kg-1) given s.c. 30 min beforehand, completely abolished BK (28 nmol)-induced hypotension, without affecting the fall of mean arterial blood pressure induced by i.v. injection of ACh (2 nmol kg-1). Finally, NPC 18688 (1 microM) did not affect ACh-mediated contraction in the guinea-pig ileum or toad rectus abdominii in vitro. 7. These results demonstrate that the newly-developed and selective pseudopeptide B2 receptor antagonist, NPC 18688, although less potent than the available second generation of B2 peptide BK receptor antagonists, exhibits topical and long-lasting systemic anti-hyperalgesic properties when analysed in several models of nociception in mice, making it a useful tool for investigating the participation of BK and related kinins in physiological and pathological processes. Finally, this new class of selective pseudopeptide B2 receptor antagonist may constitute a new strategy for developing the third generation of potent and long-lasting orally-active non-peptide BK antagonists, which may be useful for the management of clinical disorders involving BK and relate

Acetylcholine↗
PubMed1996

A pharmacological analysis of receptor subtypes and the mechanisms mediating the biphasic response induced by kinins in the rat stomach fundus in vitro.

Bradykinin (BK), des-Arg9-BK (DABK) and related kinins caused biphasic response (BR) in rat stomach fundus (RSF) precontracted with BaCl2. The B2 receptor antagonist HOE 140 (3-30 nM) produced parallel rightward shifts of the contractile concentration-response curve (CRC) for BK, yielding a pA(2) value of 9.07 +/- 0.27 and slope of 0.99, but caused only discrete rightward shift of the relaxant CRC for BK, leaving the BR CRC to DABK unaffected. The B1 receptor antagonist des-Arg9-NPC 17761 (10 nM to 1 microM) caused graded rightward shifts of the relaxant (but not the contractile) CRC to DABK, yielding a pA2 value of 8.35 +/- 0.05 and slope of 0.59, but had no effect on BK-induced BR. BK- and DABK- (100 nM) induced relaxation were almost suppressed by apamin (1 microM) or by nifedipine (1 nM), but were unaffected by nitric oxide synthase inhibitors, methylene blue, lipo and cyclooxygenase inhibitors, selective receptor antagonist for histamine (H1 and H2), nicotine, platelet activating factor, tachykinins (both NK1 and NK2, calcitonin gene-related peptide, vasoactive intestinal peptide and ganglion blocking agent. BK- and DABK-mediated relaxations were reduced in Ca2+ -free medium plus EGTA, although BK-mediated contraction was more resistant. Escherichia coli endotoxin treatment (10 microgram /rat), 24 hr before, potentiated DABK-induced relaxation, but not contraction, and reduced BK-mediated relaxation (P < .05). It is concluded that RSF express both B1 and B2 receptors. BK-induced contraction involves activation of B2 receptors, although DABK-induced relaxation is mediated by B1 receptors. Both B1 and B2 receptors in RSF are constitutive, but LPS treatment caused induction of B1 and down-regulation of B2 receptors. Finally, kinin-mediated relaxation in RSF are coupled to activation of Ca2+ activated K+ channels, and rely on Ca2+ influx via L-type voltage-sensitive channels.

Animals↗
PubMed1995

Synthesis of linoleate and alpha-linolenate by chain elongation in the rat.

The objective was to determine whether rats could synthesize longer chain polyunsaturates from hexadecadienoate (16:2n-6) and hexadecatrienoate (16:3n-3). Rats were gavaged with uniformly 13C-labelled hexadecadienoate or hexadecatrienoate, euthanized 24 h later, and total lipids were extracted from liver and carcass. Gas chromatography/combustion/isotope ratio mass spectrometry was used to measure 13C levels in individual liver, carcass, and whole body fatty acids. 13C Enrichment was present in desaturated and chain-elongated polyunsaturates, including linoleate, arachidonate, alpha-linolenate, and docosahexaenoate at 12-13% of the dose of tracer given. 13C Enrichment from hexadecatrienoate was highest in carcass and liver alpha-linolenate, representing 3.5 and 17.9% of the total alpha-linolenate pool, respectively. For linoleate, arachidonate, or docosahexaenoate, the contribution of 13C did not exceed 0.2% of the total body pool. Green leafy vegetables common in the human diet were shown to contain up to 1.2% of total fatty acids as hexadecadienoate and 11.6% as hexadecatrienoate. Hence, humans consuming green vegetables probably synthesize a small proportion of their total body content of linoleate and alpha-linolenate.

Animals↗
PubMed1995

Effects of long-term protein excess or deficiency on whole-body protein turnover in sheep nourished by intragastric infusion of nutrients.

The effect of long-term dietary protein excess and deficit on whole-body protein-N turnover (WBPNT) was examined in lambs nourished by intragastric infusions of nutrients. Ten sheep were given 500 mg N/kg metabolic weight (W0.75) per d from casein for 2 weeks and then either 50 (L), 500 (M) or 1500 (H) mg N/kgW0.75 per d for 6 weeks. Volatile fatty acids were infused at 500 kJ/kgW0.75 per d. Daily WBPNT was measured by continuous intravenous infusion of [1-13C]leucine 3 d before, and on days 2, 21 and 42 after the alteration in protein intake. Whole-body protein-N synthesis (WBPNS) was calculated as the difference between WBPNT and the protein-N losses as urinary NH3 and urea. Whole-body protein-N degradation (WBPND) was then estimated from WBPNS minus protein gain determined from N balance. Fractional rates of WBPNS and WBPND were calculated against fleece-free body N content. WBPNS rates at the L, M and H intakes were respectively 35.1, 41.5 and 63.7 g/d (P < 0.001) on average over the 6 weeks and WBPND rates were 39.5, 41.1 and 56.8 g/d (P < 0.001). The fractional rates of WBPNS were 5.01, 6.37 and 7.73% per d (P < 0.001) while those of WBPND were 5.64, 6.29 and 6.81% per d (P < 0.005) respectively. On days 2, 21 and 42, WBPNS rates at intake H were 54.0, 61.8 and 75.4 g/d (P = 0.03) respectively, and WBPND rates were 43.2, 56.4 and 70.9 g/d (P = 0.03); at intake L the amounts were 38.2, 34.2 and 32.8 g/d for WBPNS (P = 0.003) and for WBPND were 43.4, 38.0 and 36.9 g/d (P = 0.016) respectively. There were no significant (P > 0.05) differences in fractional rates of WBPNS and WBPND with time at either the L or H intake. We concluded that absolute protein turnover was affected both by dietary protein intake and body condition while the fractional rate of turnover was predominantly influenced by intake.

Ammonia↗
PubMed1994

A proposed model of bradykinin bound to the rat B2 receptor and its utility for drug design.

A putative model of bradykinin bound to the rat B2 receptor was generated using a combination of homology modeling (from the known transmembrane structure of bacteriorhodopsin), energy minimization, molecular dynamics, and a two-stage conformational search as a docking simulation. Overall, the proposed bound ligand adopts a twisted "S" shape, wherein a C-terminal beta-turn is buried in the receptor just below the extracellular boundary of the cell membrane and the N-terminus is interacting with negatively charged residues in extracellular loop 3 of the receptor (most notably Asp268 and Asp286). Mutagenesis experiments describing mutations which result in both a loss of bradykinin affinity as well as those which have no effect on bradykinin affinity are in good agreement with the proposed structure. In short, the mutagenesis results and the computational simulations each point to the same region of the receptor as likely to bind bradykinin. A double mutation, predicted as being likely to have a dramatic effect on bradykinin binding affinity, was confirmed experimentally, adding some validation to the proposed complex. Moreover, a new pseudopeptide bradykinin receptor antagonist (D-Arg0-Arg1-[12-aminododecanoyl]2- Ser3-D-Tic4-Oic5-Arg6) was designed on the basis of the model, and found to have good receptor affinity. Speculation regarding other possible sites for mutagenesis are also described.

Alanine↗
PubMed1994

Multiple copy sampling in protein loop modeling: computational efficiency and sensitivity to dihedral angle perturbations.

Multiple copy sampling and the bond scaling-relaxation technique are combined to generate 3-dimensional conformations of protein loop segments. The computational efficiency and sensitivity to initial loop copy dispersion are analyzed. The multicopy loop modeling method requires approximately 20-50% of the computational time required by the single-copy method for the various protein segments tested. An analytical formula is proposed to estimate the computational gain prior to carrying out a multicopy simulation. When 7-residue loops within flexible proteins are modeled, each multicopy simulation can sample a set of loop conformations with initial dispersions up to +/- 15 degrees for backbone and +/- 30 degrees for side-chain rotatable dihedral angles. The dispersions are larger for shorter and smaller for longer and/or surface loops. The degree of convergence of loop copies during a simulation can be used to complement commonly used target functions (such as potential energy) for distinguishing between native and misfolded conformations. Furthermore, this convergence also reflects the conformational flexibility of the modeled protein segment. Application to simultaneously building all 6 hypervariable loops of an antibody is discussed.

Animals↗
PubMed1994

Multiple copy sampling: rigid versus flexible protein.

Effects of protein flexibility on multiple copy conformational sampling were systematically evaluated by studying the side-chain placement of Phe-14 in protein Zif268. The multiple copy sampling is shown to be significantly more efficient when a flexible but harmonically constrained protein is used instead of a rigid protein. A range of constraint force from 1 to 25 kcal/mol.A per atom is determined to be sufficient to prevent the protein from distortion while allowing the protein to fluctuate for enhanced sampling. The protein fluctuations are essential in smoothing the effective energy surface as shown by the opening-closing of a protein hydrophobic pocket during a multiple copy energy minimization, a phenomenon that has been previously observed only in molecular dynamics. These results provide a practical guidance for applying the multiple copy techniques to molecular modeling and computer-aided drug design.

Aprotinin↗
PubMed1994

Structural features of the bradykinin receptor as determined by computer simulations, mutagenesis experiments, and conformationally constrained ligands: establishing the framework for the design of new antagonists.

1. In recent years, two classes of second generation bradykinin receptor antagonists have been reported. Both are of the general sequence D-Arg0-Arg1-Pro2-W3-Gly4-X5-Ser6-Y7-Z8+ ++-Arg9, where W is either Pro or Hyp, and X is an aromatic or aliphatic side chain-containing amino acid. Y and Z are unnatural amino acids, presumed to enforce a beta-turn structure. The de novo design of a non-peptide receptor antagonist (or the optimization of a lead discovered by random screening) will ultimately require knowledge about the receptor topology. In the absence of an experimentally determined structure of the bradykinin-bradykinin receptor complex, we have attempted to gain insights from other sources. 2. We have synthesized conformationally constrained ligands and completed extensive computer modeling on the bradykinin receptor. Moreover, using systematic synthetic modifications, we have explored the relative importances of selected amide bonds and side chains in second generation peptides and have made a series of C alpha- and/or N-methyl substitutions at positions four and five which led to the discovery of two new cyclic peptide antagonists. 3. Computational simulations led to a proposed model of bradykinin bound to its receptor which was found to be in good agreement with mutagenesis results. This model led ultimately to the design and synthesis of D-Arg0-Arg1-(12-aminododecanoyl)2-Ser3-D-Tic4-Oic5+ ++-Arg6. Consideration of this new lead compound, together with the extensive structure-activity relationship (SAR) which has been developed for peptide ligands and the receptor, represents a tangible framework for the design of more potent and longer-lasting antagonists of the bradykinin receptor.

Amino Acid Sequence↗
PubMed1993

Measurement of allantoin in urine and plasma by high-performance liquid chromatography with pre-column derivatization.

A method is reported for determination of allantoin in urine and plasma based on high-performance liquid chromatography (HPLC) and pre-column derivatization. In the derivatization procedure, allantoin is converted to glyoxylic acid which forms a hydrazone with 2,4-dinitrophenylhydrazine. The hydrazone appears as syn and anti isomers at a constant ratio. These derivatives are separated by HPLC using a reversed-phase C18 column from hydrazones of other keto acids possibly present in urine and plasma and then monitored at 360 nm. All components were completely resolved in 15 min. Both the reagents and derivatization products are stable. Recovery of allantoin added to urine and plasma was 95 +/- 3.7% (n = 45) and 100 +/- 7.5% (n = 64), respectively. The lowest allantoin concentration that gave a reproducible integration was 5 mumol/l. The between-assay and within-day coefficients of variation were 2.8 and 0.6%, respectively.

Allantoin↗
PubMed1993

NMR and computational evidence that high-affinity bradykinin receptor antagonists adopt C-terminal beta-turns.

Three tetrapeptides were prepared, each corresponding to the four C-terminal amino acid residues of highly potent, second-generation bradykinin receptor antagonists. The tetrapeptides are (IA) Ser-D-Phe-Oic-Arg, (IIA) Ser-D-Tic-Oic-Arg, and (IIIA) Ser-D-Hype(trans-propyl)-Oic-Arg. Solution conformations for each were determined by incorporating interproton distance restraints, determined by 2D NMR experiments performed in water at neutral pH, into a series of distance geometry/simulated annealing model building calculations. Similarly, systematic conformational analyses were performed for each using molecular mechanics calculations. Both the NMR-derived structures, as well as the calculated structures, are shown to adopt a beta-turn as the primary conformation. Excellent agreement between the predicted structures and the NMR-derived structures is demonstrated. Aside from being the first examples of linear tetrapeptides reported to be ordered in aqueous solvent, the results presented support the hypothesis that high-affinity bradykinin receptor antagonists must adopt C-terminal beta-turn conformations.

Amino Acid Sequence↗
PubMed1993

Disbudding of red deer stag calves to prevent antler growth.

Red deer stag calves aged five to seven months were disbudded with a standard cattle disbudding iron as a means of preventing antler growth and development. Two sizes of iron, one 2.2 cm in diameter and one 1.5 cm in diameter, were compared at disbudding in November or January. Disbudding in November and January with the 2.2 cm iron had success rates of 97 per cent and 92 per cent, respectively. Treatment with the 1.5 cm iron was less effective at both times. There was no significant difference between the liveweights of the treated groups and a non-disbudded control group at turnout in the spring or at slaughter in November when the stags were 16 months of age.

Anesthesia, Local↗