Acta physiologica Scandinavica, ISSN 0302-2994 vol. 125, suppl. 545, 1985
35 s. : tab., grafy ; 24 cm
Although the fragmentation of the active pharmaceutical ingredient (API) is a phenomenon that is mentioned in many literature sources, no well-suited analytical tools for its investigation are currently known. We used the hot-stage microscopy method, already presented in our previous work, and studied the real fragmentation of the tadalafil particles in model tablets which were prepared under different compaction pressures. The morphology, spectral imaging and evaluation of plastic and elastic energies were also analyzed to support the hot-stage method. The prepared blend of tadalafil and excipients was compacted under a several forces from 5 to 35 kN to reveal the trend of fragmentation. The exact fragmentation of tadalafil with increased compaction pressure was revealed by the hot-stage microscopic method and it was in good agreement with plastic and elastic energies. Conversely, spectral imaging, which is being used for this analysis, was considered to be inaccurate methodology as mainly agglomerates, not individual particles, were measured. The availability of the hot-stage microscopic method equips pharmaceutical scientists with an in vitro assessment technique that will more reliably determine the fragmentation of the API in finished tablets and the behavior of the particles when compacted.
Analysis of glycosylation is challenging due to micro- and macro-heterogeneity of the protein attachment. A combination of LC with MS/MS is one of the most powerful tools for glycopeptide analysis. In this work, we show the effect of various monosaccharide units on the retention time of glycopeptides. Retention behavior of several glycoforms of six peptides obtained from tryptic digest of haptoglobin, hemopexin, and sex hormone-binding globulin was studied on a reversed phase chromatographic column. We observed reduction of the retention time with increasing number of monosaccharide units of glycans attached to the same peptide backbone. Fucosylation of larger glycans provides less significant retention time shift than for smaller ones. Retention times of glycopeptides were expressed as relative retention times. These relative retention times were used for calculation of upper and lower limits of glycopeptide retention time windows under the reversed phase conditions. We then demonstrated on the case of a glycopeptide of haptoglobin that the predicted retention time window boosts confidence of identification and minimizes false-positive identification. Relative retention time, as a qualitative parameter, is expected to improve LC-MS/MS characterization of glycopeptides.
- MeSH
- Chromatography, Reverse-Phase methods MeSH
- Glycopeptides blood chemistry metabolism MeSH
- Glycosylation MeSH
- Humans MeSH
- Nanotechnology methods MeSH
- Peptide Fragments analysis chemistry metabolism MeSH
- Proteomics MeSH
- Sensitivity and Specificity MeSH
- Trypsin metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
This work reveals the growing potential of novel electrochemical methods that are applicable for polysaccharides. It was shown for the first time that the molecules of hyaluronic acid (HA) exhibit electrochemical response using phase-sensitive alternating current (AC) voltammetry in phase-out mode. Adsorption and desorption processes of HA fragments at a charged interface of mercury electrode were observed in buffered HA solutions. Electrostatic and hydrophobic manners of interactions were distinguished for native hyaluronan fragments in a wide electric potential range. The AC voltammetry response depended on the temperature, concentration, and length of HA chains. Results of this work open possibilities for further structural characterization of widely used HA fragments and understanding manners of interactions with charged hydrophobic surfaces that could be useful in the future for understanding HA interactions at biological levels.
- Publication type
- Journal Article MeSH
Progressive cerebral deposition of amyloid beta occurs in Alzheimers disease and during the aging of certain mammals (human, monkey, dog, bear, cow, cat) but not others (rat, mouse). It is possibly due to different amino acid sequences at positions 5, 10 and 13. To address this issue, we performed series of 100 ns long trajectories (each trajectory was run twice with different initial velocity distribution) on amyloid beta (1-42) with the human and rat amino acid sequence in three different environments: water with only counter ions, water with NaCl at a concentration of 0.15 M as a model of intracellular Na(+) concentration at steady state, and water with NaCl at a concentration of 0.30 M as a model of intracellular Na(+) concentration under stimulated conditions. We analyzed secondary structure stability, internal hydrogen bonds, and residual fluctuation. It was observed that the change in ionic strength affects the stability of internal hydrogen bonds. Increasing the ionic strength increases atomic fluctuation in the hydrophobic core of the human amyloid, and decreases the atomic fluctuation in the case of rat amyloid. The secondary structure analyses show a stable α-helix part between residues 10 and 20. However, C-terminus of investigated amyloids is much more flexible showing no stable secondary structure elements. Increasing ionic strength of the solvent leads to decreasing stability of the secondary structural elements. The difference in conformational behavior of the three amino acids at position 5, 10 and 13 for human and rat amyloids significantly changes the conformational behavior of the whole peptide.
- MeSH
- Amyloid beta-Peptides chemistry MeSH
- Sodium Chloride chemistry MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Rats MeSH
- Humans MeSH
- Molecular Sequence Data MeSH
- Osmolar Concentration MeSH
- Peptide Fragments chemistry MeSH
- Surface Properties MeSH
- Protein Structure, Secondary MeSH
- Amino Acid Sequence MeSH
- Molecular Dynamics Simulation * MeSH
- Protein Stability MeSH
- Protein Structure, Tertiary MeSH
- Water chemistry MeSH
- Hydrogen Bonding MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
In this paper, the d(GCGAAGC) heptamer and the closely related d(GCGAGC) hexamer are examined via electrochemical (cyclic voltammetry) and spectroscopic (circular dichroism) methods. Dramatic changes in the CD spectroscopic and CV electrochemical properties, induced by the loss of only one single nucleotide (A), are detected. The CD spectra and native polyacrylamide gel electrophoresis (PAGE) confirmed structural changes taking place in the relevant chain-like oligodeoxynucleotide assemblies. Dedicated studies suggest that the heptamer (Hp) possesses a hairpin structure, whereas the hexamer (Hx) appears to be rather a duplex. Both of the structures exhibited completely different adsorption behavior at the hanging mercury drop electrode, and this factor was readily confirmed by means of elimination voltammetry with linear scan (EVLS). We established that the Hp hairpin (~-1300 mV), compared to the Hx duplex (~-1360 mV), is the thermodynamically favored electron acceptor. The adsorption isotherms were constructed based on the voltammetric peak height values, reflecting the reduction of the adenine (A) and cytosine (C) moieties as well as the oxidation of the 7,8-dihydroguanine (7,8-DHG) moieties. Finally, as revealed by the spectroscopic and electrochemical results, Hx forms a bimolecular antiparallel homo-duplex carrying both Watson-Crick base pairs (CG or GC) and mismatched edge-to-edge base pairs (GA or AG).
V této studii jsme hodnotili, zda-li intracerebroventrikulární (ICV) aplikace (25-35)-P-amyloid fragmentu (AP25.35) vyvolá změny v EEG záznamu a změny v kognitivních a exploračních schopnostech potkanů, které by odpovídaly animálnímu modelu Alzheimerovy demence (AD). EEG záznam byl registrován z 12 aktivních elektrod, následně byl podroben spektrální a koherenční analýze, behaviorální parametry byly hodnoceny v testu rozpoznání nového objektu (novel object recognition test, NOR). Všechny experimenty byly provedeny 4 týdny od ICV aplikace Ap25- 35 a 1 týden po implantaci elektrod. Ap25- 35 zvyšoval relativní výkon v pásmech beta, vysoká beta a gama, dále byl zjevný pík v theta pásmu při 6 Hz. V pásmu delta došlo k mírnému snížení relativního výkonu. AP25 35 aplikace dále významně snižovala koherence. V behaviorální studii pak AP25-35 indukoval výrazné změny v chování, zejména snížení až vymizení základních exploračních aktivit, zkrácení lokomoce a vymizení habituace. Z toho důvodu nebylo ani možné u těchto je¬ dinců hodnotit krátkodobou paměť. Naše nálezy v tomto modelu částečně odpovídají nálezům u AD.
In our study we evaluated whether intracerebroventricular (ICV) application of (25-35)- -amyloid fragment (A 25-35 ) induces changes in rat's EEG signal, cognitive and explorative skills that would correspond to animal model of Alzheimer ́s disease (AD). The EEG signals were registered from 12 active electrodes. Signal was then analyzed by spectral and coherence analysis. Behavioral parameters were measured in the novel object recognition test (NOR). All the experiments were performed 4 week after ICV application of A 25-35 and 1 week after implantation of EEG electrodes. A 25-35 increased the relative power of beta, high beta and gamma, induced significant 6Hz peak in theta band. Slight depres- sion of relative power occurred in the delta band. A significant decrease of coherence also occurred after A 25-35 application. A 25-35 induced also significant changes in the behavior, especially diminution of basic explorative activities, reduction of locomotion and loss of habituation. Because of these alterations animals did not fulfill the criteria for analysis of short term memory in NOR. Our results particularly correspond to the findings found in patients with AD.
- Publication type
- Meeting Abstract MeSH
CART (cocaine- and amphetamine-regulated transcript) peptides are neuropeptides abundant in the central nervous system and periphery found to be involved in the regulation of food intake behavior and other physiological processes. Recently, we reported specific binding of (125)I-CART(61-102) to the rat adrenal pheochromocytoma cell line PC12, both intact cells and cell membranes. In this study, several fragments of CART(61-102) corresponding to its structural loops were synthesized and tested for their potency in binding experiments using PC12 intact cells and cell membranes and in feeding test with fasted mice. From all shorter peptides tested, only CART(74-86) and CART(62-86) containing disulfide bridges kept partial binding potency of the original molecule with K(i) in 10(-5) and 10(-4)M range. However, these fragments were not able to inhibit food intake after their central administration up to a dose of 4 nmol/mouse. The results showed that a compact structure containing three disulfide bridges is necessary for preservation of full biological activity of CART peptides.
- MeSH
- PC12 Cells MeSH
- Financing, Organized MeSH
- Rats MeSH
- Molecular Sequence Data MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Peptide Fragments pharmacology MeSH
- Nerve Tissue Proteins pharmacology chemistry MeSH
- Amino Acid Sequence MeSH
- Feeding Behavior drug effects MeSH
- Structure-Activity Relationship MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
