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Conformational studies of a benzodiazepine-like peptide in SDS micelles by circular dichroism, 1 H NMR and molecular dynamics simulation
Abstract
Summary The conformation of a benzodiazepine-like decapeptide corresponding to the YLGYLEQLLR fragment of a casein has been examined
in a sodium dodecyl sulfate micellar medium using circular dichroism, two-dimensional1H NMR spectroscopy and restrained molecular dynamics simulation. The decapeptide adopts an amphipathic 310-helicoid structure in which the E6...R10 ionic bridge stabilizes the C-terminus.
... However, it has been shown that this quieting effect was related not only to suckling but also to milk itself [7]. The milk α S1 -casein hydrolysate and the bioactive de- EJN 534 capeptide [α S1 -CN(f91–100)], a fragment of this hydrolysate that has been spatially modeled [8, 9] , have already shown an anxiolytic-like profile in the conditioned defensive burying test and in the elevated plus-maze in rats [10] , two models used to study anxiolytic agents in rodents. The main objective of the present study was to investigate whether oral intake of this α S1 -casein hydrolysate by healthy human subjects would modify some physiological variables during imposed experimental stress tasks: Stroop test (ST), a mental conflict situation [11] and cold pressor test (CPT), a physical stress situation [12]. ...
mental physical stress, cortisol, Lactium, clinical trial
... A milk caseinate hydrolysate (CH) and a bioactive decapeptide (a s1 -casein-[f91-f100], a-casozepine), a fragment of this hydrolysate that has been spatially modeled (Lecouvey et al., 1997), have already shown an anxiolytic-like profile in the conditioned defensive burying test and in the elevated plus maze in rats, 2 well-known models used to study anxiolytic agents in rodents (Miclo et al., 2001). Moreover, the effect of caseinate hydrolysate on hemodynamic responses was evaluated in healthy human volunteers facing successive mental and physical stress situations (Messaoudi et al., 2004) and in the management of anxious disorders such as social phobia in cats (Beata et al., 2007). ...
The purpose of this randomized, double-blind, placebo-controlled trial was to evaluate the efficacy of a diet containing caseinate hydrolysate (CH) on signs of stress in 2 groups of dogs (defined as Anxious and Nonanxious), using physiological (serum cortisol and lysozyme, N:L ratios and heart rate) and behavioral parameters.From an initial group of 40 female Beagle dogs, ranging in age from 10 months to 4 years (mean = 1.47 years; SD = 0.53) belonging to a dog colony, 32 were selected for this study according to their level of anxiety. A group of 16 Anxious dogs and a group of 16 Nonanxious dogs were identified.A baseline period, aimed to obtain reference values of investigated parameters, preceded the experimental phase. Both groups (Anxious and Nonanxious) were divided into a treatment group, which received the diet containing CH, and a control group which received a placebo diet (PD). Anxious CH and PD groups were balanced for anxiety level. Each dog was evaluated 3 times a day at 4 weeks intervals (T1-T2-T3). Each evaluation lasted 2 days and involved a Reactivity Evaluation Form, a blood sampling, heart rate recording, and a 10-minute behavioral video recording. Results from Reactivity Evaluation Form scores showed that although at T1 Anxious dogs had significantly higher scores (Mann–Whitney test, P < 0.001) compared with Nonanxious dogs, no difference was found between Anxious dogs fed with CH diet and Nonanxious fed with PD or CH diet at T3. Behavioral observations evidenced some signs of improvement in Anxious dog fed with CH diet. Cortisol level significantly decreased in Anxious dogs fed with CH diet (Friedman test, P < 0.05). Individual differences in physiological measures of stress responses may have contributed to the large variability, making interpretation of these measures difficult. These results suggest that CH may be used as a functional ingredient alleviating stress in dogs.
To evaluate potential calming effects of alpha-casozepine on horses, we blindly compared behavior and training efficiency of adult semi-feral ponies treated with either alpha-casozepine or control supplement during transition to domestic management and handling. Six ponies (three matched pairs) aged 2 to 8 years that had been reared and kept since birth under semi-feral social and environmental conditions were given either alpha-casozepine (1000 mg orally once daily for ponies weighing 160 to 205 kg) or control supplement, beginning 5 days before being moved to a domestic facility for a 2-week introduction to stabling, haltering, leading, tethering, social separation, stall confinement, grooming, simulated girthing, lifting feet, health care treatments, and transportation. Objective quantitative behavior measures (latencies to complete tasks, avoidance responses, and nervous defecations) were derived from video-recorded handling sessions. For each of the 14 sessions, ponies were ranked 1 (best) to 6 (poorest) for calm, compliance, and acclimation/skill progress. All human–animal interactions, video analyses, and rankings were done blindly to supplement assignments. For most daily sessions across the 2-week training period, each of the three alpha-casozepine-treated ponies performed better than their matched control counterparts, and they also had the top three sums of daily session ranks, with a mean of 35.2 compared with 62.8 for control ponies (P < .05, dependent t-test). At 6 weeks after the 2-week training period, the alpha-casozepine-treated ponies retained the best three sum of ranks for the seven specific skills re-assessed at that time. These results provide evidence of the benefit of alpha-casozepine supplementation to horses undergoing potentially stressful situations inherent to domestic management.
Anxiety disorders are recognized worldwide though they may be characterized differently by various researchers and authors. Their treatment involves behavior modification recommendations and often drug therapy. The use of anxiolytic drugs with minimal side effects is recommended for pharmacotherapy. Alpha-casozepine (tryptic hydrolysate of alpha s1-casein) is derived from casein, a naturally occurring milk protein, with a GRAS (Generally Recognised As Safe) status. It has an interesting effect on anxiety that has been evaluated in placebo-controlled studies with rats, human beings, cats and dogs.
2-Hydroxymethylthieno[3,2-e∶4,5-e′
]di[1]benzothiophene and
2-(2,4,5,7-tetranitrofluoren-9-ylideneaminooxy)propionic acid in SDS
micelles initially formed a 1∶2 complex with a negative Cotton effect
around 360 nm, which with time or sonication gradually changed to a
1∶1 complex giving a reversed Cotton effect.
The reaction between the free amino terminus of a solid-supported peptide and a glyoxal leads to two families of pseudopeptides, the ketomethylenimino and the ketomethylenamino peptides. The aim of this study is the synthesis of pseudopeptides on solid supports, in order to quickly obtain modified peptides. We report a convenient step-by-step synthesis of ketomethylenimino ψ[CO-CH=N] and ketomethylenamino ψ[CO-CH 2-NH] peptides. The key is the reaction between the free amino terminus of the supported peptide and a glyoxal-modified amino acid, leading to a ketomethylenimino bond, which can be reduced to a ketomethylenamino bond.
Objective:
To examine the effects of alpha (s1)-casein hydrolysate on females with stress-related symptoms.
Design:
Double-blind, randomized, crossover, placebo-controlled trial.
Setting:
The alpha (s1)-casein hydrolysate was manufactured by INGREDIA (Arras, France) and the placebo was manufactured by DIETAROMA (Bourg, France). Study was designed and performed at PROCLAIM (Rennes, France), and the statistical analyses were performed by D Desor (Nancy, France).
Subjects:
A total of 63 female volunteers suffering from at least one disorder that may be related to stress such as anxiety, sleep problems and general fatigue.
Interventions:
A total of 63 volunteers participated in a double-blind, randomized, crossover, placebo-controlled study. Subjects were randomly allocated to receive either tablets containing alpha (s1)-casein hydrolysate or placebo at the dose of 150 mg/day for 30 days. After a 3 weeks washout period, they were crossed over for a new 30-day period of tablets intake. The outcome measure was a questionnaire including 44 items of symptoms that may be related stress in which the severity of each sign was evaluated using a 10-degree scale. These measures were studied repeatedly at the day of 0, 15 and 30 after the start of each interventional period.
Results:
The 30-day treatment by alpha (s1)-casein hydrolysate in females with stress-related symptoms reduced their symptoms, particularly in digestion (P<0.01), cardiovascular (P<0.05), intellectual (P<0.01), emotional (P<0.05) and social problems (P<0.05).
Conclusion:
This study showed that a 30-day ingestion of alpha (s1)-casein hydrolysate decreased the stress-related symptoms in females suggesting that this product may be used as an effective functional ingredient alleviating such symptoms.
Sponsorship:
This study was partially supported by the INGREDIA of France and Neurobiology Research Program from the Korea Ministry of Science and Technology (2004-01757) of Korea.
The putative effects of a tryptic bovine αs1-casein hydrolysate on anxious disorders in cats was investigated. This product is known as alpha-casozepine and patented under the name of Zylkene (Ingredia, Arras, France). Within veterinary practices, 34 cats were recruited by certified behaviorist surgeons. This 56-day trial against placebo showed the statistically positive effect of this product in the management of anxious disorders such as social phobias in cats. Global score, as well as different items (fear of strangers, contact with familiars, general fears, fear-related aggressions, autonomic disorders), were all significantly improved by the use of this natural decapeptide.
The putative effects of a tryptic bovine αs1-casein hydrolysate on anxious disorders in cats were investigated. This product is known as alpha-casozepine and patented under the name of lactium. 34 Cats were recruited within veterinary practices by certified behaviorist surgeons. This 56 days trial, against placebo, showed the statistical positive effect of this product to manage anxious disorders such as social phobias in cats. Global score but also different items, (fear of strangers, contact with familiars, general fears, fear related aggressions, autonomous disorders) were all significantly improved by the use of this natural decapeptide.
A chloroform/methanol extract of a peptic digest of bovine K-casein had binding activity to opioid receptors of rat brain. The active peptide was isolated by reverse-phase liquid chromatography on ODS and CN columns. The structure of the peptide was Ser-Arg-Tyr-Pro-Ser-Tyr-OCH3, which corresponds to the methyl ester of the 33 - 38th residues of κ-casein. The peptide was esterified during the extraction process, and the structure was confirmed by a chemical synthesis. The IC50 value of the peptide in a radioreceptor assay in the presence of 1 HM [3H]naloxone was 15 μM. The peptide did not have opioid agonistic activities in any of the assay systems tested. In the guinea pig ileum and rabbit vas deferens preparations, however, the peptide antagonized with morphiceptin and dynorphin A-(1-13), respectively. The peptide did not antagonize with [Leu]enkephalin in the mouse vas deferens preparations. Therefore, the peptide was an antagonist selective for the μ- and κ-types of opioid receptors. The peptide was named casoxin.
A novel approach to tailored selective excitation for the measurement of NMR spectra in non-deuterated aqueous solutions (WATERGATE, WATER suppression by GraAdient-Tailored Excitation) is described. The gradient echo sequence, which effectively combines one selective 180 radiofrequency pulse and two field gradient pulses, achieves highly selective and effective water suppression. This technique is ideally suited for the rapid collection of multi-dimensional data since a single-scan acquisition produces a pure phase NMR spectrum with a perfectly flat baseline, at the highest possible sensitivity. Application to the fast measurement of 2D NOE data of a 2.2. mM solution of a double-stranded DNA fragment in 90% H2O at 5 C is presented.
''Excellent and very timely....It will undoubtedly become a standard reference for the application of circular dichroism (CD) to biomolecules.'' --- Quarterly Review of Biology, March 1997 ''[T]estament to the book's utility is the fact that during the course of my review I had to 'rescue' it from the desks of graduate students on an almost daily basis. In summary, this is a great book.'' --- American Scientist ''Well documented chapters provide a very good insight into the problems surrounding the conformation of biomacromolecules...An indispensible source of information.'' --- Nahrung, 42(2), 1998 Renowned experts present the first state-of-the-art description of circular dichroism spectroscopy (CD). Chapters present in-depth discussions of the history of the field, the theory of CD for application to globular proteins, membrane proteins, peptides, nucleic acids and their interactions, carbohydrates, and instrumentation. Discussions also feature new techniques using synchrotron radiation, vibrational Raman optical activity, and vibrational CD. More than 250 illustrations supplement the text.
The pseudopentapeptide cyclo(-Arg(1)-Gly(2)-Asp(3)-D-Phe(4) psi[CH2NH]Val(5)-) (1) was synthesized by a combination of solution and solid-phase methods. The reduced peptide bond was incorporated as a dipeptide building block, a versatile synthetic alternative that avoids the reduction of imine intermediates. NMR spectroscopy was used to investigate the structure of the peptide. Interproton distances from a NOESY spectrum served as restraints in a molecular dynamics simulation. Though the peptide exhibits a typical beta II',gamma conformation with the D-Phe in the i + 1 position of a gamma turn, this solution conformation is different from that of the parent peptide cyclo(-Arg(1)-Gly(2)-Asp(3)-D-Phe(4)-Val(5)-) with the D-Phe in the i + 1 position of beta II'. The influence of the reduced peptide bond is examined by computational methods. The psi[CH2-NH] bond proved to be a strong hydrogen bond donor and thus rigidifies the peptide backbone.
A novel test for the selective identification of anxiolytic and anxiogenic drug effects in the rat is described, using an elevated + -maze consisting of two open arms and two enclosed arms. The use of this test for detecting such drug effects was validated behaviourally, physiologically, and pharmacologically. Rats made significantly fewer entries into the open arms than into the closed arms, and spent significantly less time in open arms. Confinement to the open arms was associated with the observation of significantly more anxiety-related behaviours, and of significantly greater plasma corticosterone concentrations, than confinement to the closed arms. Neither novelty nor illumination was a significant contributor to the behaviour of the rats on the + -maze. A significant increase in the percentage of time spent on the open arms and the number of entries into the open arms was observed only within clinically effective anxiolytics (chlordiazepoxide, diazepam and, less effectively, phenobarbitone). Compounds that cause anxiety in man significantly reduced the percentage of entries into, and time spent on, the open arms (yohimbine, pentylenetetrazole, caffeine, amphetamine). Neither antidepressants nor major tranquilisers had a specific effect. Exposure to a holeboard immediately before placement on the + -maze showed that behaviour on the maze was not clearly correlated either with exploratory head-dipping or spontaneous locomotor activity.
Chloroform-methanol extracts of lyophilized milk, of commercially available dried milk or baby food and of casein digests were tested for opioid activity on the guinea-pig ileum longitudinal muscle-myenteric plexus preparation. Compounds with opioid activity--which proved to be resistant to peptidases--were detected in certain batches of baby food, casein digest, and cow milk in considerably varying amounts.
An improved model for calculating the CD of polypeptides has been developed. Excited state wavefunctions were derived from CNDO/S (complete neglect of differential overlap, spectroscopic) calculations on N-methylacetamide. Four discrete peptide-localized transitions were employed: pi 0 pi* (NV1), pi* + pi* (NV2), n pi*, and n' pi*. Inclusion of the pi + pi transition (lambda 0 = 140 nm) significantly improves the accuracy of the calculated CD spectra in the 180-250-nm region. Spectra were computed for various helical structures, including right-handed alpha-, alpha II-, omega-, pi-, 3(10-), and poly (proline) I-helices, and the left-handed poly (proline) II-helix. Sensitivity to changes in the peptide backbone geometry and chain length are examined. Electronic factors such as ground-state charge distribution, hybridization effects, and basis set deorthogonalization have been investigated. The nonconservative nature of the poly (Pro) I and II CD spectra is reproduced, and the helix band present in earlier exciton calculations on the alpha-helix has been diminished.
The heptadecapeptides bombolitin I and bombolitin III are two members of a series of biologically active peptides postulated to be membrane active. In order to understand the effects of the membrane on the secondary structure of the peptides, we have carried out the conformational characterization of bombolitins I and III in the presence of SDS micelles using circular dichroism, nuclear magnetic resonance, and computer simulations. The characteristic bands in the circular dichroism spectra indicate an alpha-helix content of approximately 60% in bombolitin III and 70% in bombolitin I. The observation of NOE's quite distinctive for such secondary structure strongly supports the CD results. The conformational preferences of the two bombolitins derived from CD and NMR were then energetically refined with molecular dynamics simulations. The results from the spectroscopic examination were utilized as input for the simulations, the CD results for generation of the initial structure, and the NOE's as constraints during the simulations. The results from the different techniques employed are in complete agreement.
Ro 15-1788 (flumazepil) is an imidazodiazepine that is able to antagonise most of the behavioural actions of the benzodiazepines, as well as having some intrinsic effects. Acute administration of Ro 15-1788 (10 mg/kg) decreases social interaction between male rats and elevates exploratory head-dipping. After 5 days of pretreatment there was tolerance to the former effect, although Ro 15-1788 retained its ability to antagonise the effects on social interaction of the beta-carboline, FG 7142. Ro 15-1788 also retained its ability to elevate head-dipping: additionally, the chronically-treated rats had elevated motor activity and rearing scores. The acute effects of lorazepam in the holeboard were unchanged by chronic pretreatment with Ro-15-1788. The plasma and brain concentrations after acute administration of lorazepam were unchanged following chronic administration of Ro 15-1788. After chronic treatment the brain concentrations of Ro 15-1788 were unchanged. It is unlikely that pharmacokinetic factors could underlie the different behavioural changes following chronic treatment.