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In order to achieve a comprehensive understanding of protein aggregation processes, an exploration of solvation dynamics, a key yet intricate component of biological phenomena, is mandatory. In the present study, we used Fourier transform infrared spectroscopy and terahertz spectroscopy complemented by atomic force microscopy and kinetic experiments utilizing thioflavin T fluorescence to elucidate the changes in solvation dynamics during liquid-liquid phase separation and subsequent amyloid fibril formation, the latter representing a transition from liquid to solid phase separation. These processes are pivotal in the pathology of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. We focus on the ACC 1-13 K 24 -ATP protein complex, which undergoes fibril formation followed by droplet generation. Our investigation reveals the importance of hydration as a driving force in these processes, offering new insights into the molecular mechanisms at play., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Insulin aggregation poses a significant problem in pharmacology and medicine as it occurs during prolonged storage of the hormone and in vivo at insulin injection sites. We have recently shown that dominant forces driving the self-assembly of insulin fibrils are likely to arise from intermolecular interactions involving the N-terminal segment of the A-chain (ACC 1-13 ). Here, we study how proline substitutions within the pilot GIVEQ sequence of this fragment affect its propensity to aggregate in both neutral and acidic environments. In a reasonable agreement with in silico prediction based on the Cordax algorithm, proline substitutions at positions 3, 4, and 5 turn out to be very effective in preventing aggregation according to thioflavin T-fluorescence-based kinetic assay, infrared spectroscopy, and atomic force microscopy (AFM). Since the valine and glutamate side chains within this segment are strongly involved in the interactions with the insulin receptor, we have focused on the possible implications of the Q → P substitution for insulin's stability and interactions with the receptor. To this end, comparative molecular dynamics (MD) simulations of the Q5P mutant and wild-type insulin were carried out for both free and receptor-bound (site 1) monomers. The results point to a mild destabilization of the mutant vis à vis the wild-type monomer, as well as partial preservation of key contacts in the complex between Q5P insulin and the receptor. We discuss the implications of these findings in the context of the design of aggregation-resistant insulin analogues retaining hormonal activity.

Many cellular coassemblies of proteins and polynucleotides facilitate liquid-liquid phase separation (LLPS) and the subsequent self-assembly of disease-associated amyloid fibrils within the liquid droplets. Here, we explore the dynamics of coupled phase and conformational transitions of model adenosine triphosphate (ATP)-binding peptides, ACC 1-13 K n , consisting of the potent amyloidogenic fragment of insulin's A-chain (ACC 1-13 ) merged with oligolysine segments of various lengths (K n , n = 16, 24, 40). The self-assembly of ATP-stabilized amyloid fibrils is preceded by LLPS for peptides with sufficiently long oligolysine segments. The two-component droplets and fibrils are in dynamic equilibria with free ATP and monomeric peptides, which makes them susceptible to ATP-hydrolyzing apyrase and ACC 1-13 K n -digesting proteinase K. Both enzymes are capable of rapid disassembly of amyloid fibrils, producing either monomers of the peptide (apyrase) or free ATP released together with cleaved-off oligolysine segments (proteinase K). In the latter case, the enzyme-sequestered K n segments form subsequent droplets with the co-released ATP, resulting in an unusual fibril-to-droplet transition. In support of the highly dynamic nature of the aggregate-monomer equilibria, addition of superstoichiometric amounts of free peptide to the ACC 1-13 K n -ATP coaggregate causes its disassembly. Our results show that the droplet state is not merely an intermediate phase on the pathway to the amyloid aggregate but may also constitute the final phase of a complex amyloidogenic protein misfolding scenario rich in highly degraded protein fragments incompetent to transition again into fibrils.

Lipidation of the LC3 protein has frequently been employed as a marker of autophagy. However, LC3-lipidation is also triggered by stimuli not related to canonical autophagy. Therefore, characterization of the driving parameters for LC3 lipidation is crucial to understanding the biological roles of LC3. We identified a pseudo-natural product, termed Inducin, that increases LC3 lipidation independently of canonical autophagy, impairs lysosomal function and rapidly recruits Galectin 3 to lysosomes. Inducin treatment promotes Endosomal Sorting Complex Required for Transport (ESCRT)-dependent membrane repair and transcription factor EB (TFEB)-dependent lysosome biogenesis ultimately leading to cell death., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Canonical amyloid fibrils are composed of covalently identical polypeptide chains. Here, we employ kinetic assays, atomic force microscopy, infrared spectroscopy, circular dichroism, and molecular dynamics simulations to study fibrillization patterns of two chimeric peptides, ACC 1-13 E 8 and ACC 1-13 K 8 , in which a potent amyloidogenic stretch derived from the N-terminal segment of the insulin A-chain (ACC 1-13 ) is coupled to octaglutamate or octalysine segments, respectively. While large electric charges prevent aggregation of either peptide at neutral pH, stoichiometric mixing of ACC 1-13 E 8 and ACC 1-13 K 8 triggers rapid self-assembly of two-component fibrils driven by favorable Coulombic interactions. The low-symmetry nonpolar ACC 1-13 pilot sequence is crucial in enforcing the fibrillar structure consisting of parallel β-sheets as the self-assembly of free poly-E and poly-K chains under similar conditions results in amorphous antiparallel β-sheets. Interestingly, ACC 1-13 E 8 forms highly ordered fibrils also when paired with nonpolypeptide polycationic amines such as branched polyethylenimine, instead of ACC 1-13 K 8 . Such synthetic polycations are more effective in triggering the fibrillization of ACC 1-13 E 8 than poly-K (or poly-E in the case of ACC 1-13 K 8 ). The high conformational flexibility of these polyamines makes up for the apparent mismatch in periodicity of charged groups. The results are discussed in the context of mechanisms of heterogeneous disease-related amyloidogenesis.

Disease-associated progression of protein dysfunction is typically determined by an interplay of transition pathways leading to liquid-liquid phase separation (LLPS) and amyloid fibrils. As LLPS introduces another layer of complexity into fibrillization of metastable proteins, a need for tunable model systems to study these intertwined processes has emerged. Here, we demonstrate the LLPS/fibrillization properties of a family of chimeric peptides, ACC 1-13 K n , in which the highly amyloidogenic fragment of insulin (ACC 1-13 ) is merged with oligolysine segments of various lengths (K n , n = 8, 16, 24, 32, 40). LLPS and fibrillization of ACC 1-13 K n are triggered by ATP through Coulombic interactions with K n fragments. ACC 1-13 K 8 and ACC 1-13 K 16 form fibrils after a short lag phase without any evidence of LLPS. However, in the case of the three longest peptides, ATP triggers instantaneous LLPS followed by the disappearance of droplets occurring in-phase with the formation of amyloid fibrils. The kinetics of the phase transition and the stability of mature co-aggregates are highly sensitive to ionic strength, indicating that electrostatic interactions play a pivotal role in selecting the LLPS-fibrillization transition pathway. Densely packed ionic interactions that characterize ACC 1-13 K n -ATP fibrils render them highly sensitive to hydrostatic pressure due to solvent electrostriction, as demonstrated by infrared spectroscopy. Using atomic force microscopy imaging of rapidly frozen samples, we demonstrate that early fibrils form within single liquid droplets, starting at the droplet/bulk interface through the formation of single bent fibers. A hypothetical molecular scenario underlying the emergence of the LLPS-to-fibrils pathway in the ACC 1-13 K n -ATP system has been put forward.

Aggregation of proteins into amyloid fibrils is driven by interactions between relatively small amyloidogenic segments. The interplay between aggregation-prone and aggregation-resistant fragments within a single polypeptide chain remains obscure. Here, we examine fibrillization behavior of two chimeric peptides, ACC 1 - 13 E 8 and ACC 1 - 13 E 8(L/D) , in which the highly amyloidogenic fragment of insulin (ACC 1 - 13 ) is extended by an octaglutamate segment composed of all-L (E 8 ), or alternating L/D residues (E 8(L/D) ). As separate entities, ACC 1 - 13 readily forms fibrils with the infrared features of parallel β-sheet while E 8 forms antiparallel β-sheets with the distinct infrared characteristics. This contrasts with the profoundly aggregation-resistant E 8(L/D) , although L/D patterns have been hypothesized as compatible with aggregated α-sheets. ACC 1 - 13 E 8 and ACC 1 - 13 E 8(L/D) are found to be equally prone to fibrillization at low pH, or in the presence of Ca 2+ ions. Fibrillar states of both ACC 1 - 13 E 8 and ACC 1 - 13 E 8(L/D) reveal the infrared features of highly ordered parallel β-sheet without evidence of β 2 -aggregates (ACC 1 - 13 E 8 ) or α-sheets (ACC 1 - 13 E 8(L/D) ). Hence, the preferred structural pattern of ACC 1 - 13 overrides the tendency of E 8 to form antiparallel β-sheets and enforces the fibrillar order in E 8(L/D) . We demonstrate how the powerful amyloid stretch determines the overall amyloid structure forcing non-amyloidogenic fragments to participate in its native amyloid pattern., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Computational prediction of molecular structures of amyloid fibrils remains an exceedingly challenging task. In this work, we propose a multi-scale modeling procedure for the structure prediction of amyloid fibrils formed by the association of ACC 1-13 aggregation-prone peptides derived from the N-terminal region of insulin's A-chain. First, a large number of protofilament models composed of five copies of interacting ACC 1-13 peptides were predicted by application of CABS-dock coarse-grained (CG) docking simulations. Next, the models were reconstructed to all-atom (AA) representations and refined during molecular dynamics (MD) simulations in explicit solvent. The top-scored protofilament models, selected using symmetry criteria, were used for the assembly of long fibril structures. Finally, the amyloid fibril models resulting from the AA MD simulations were compared with atomic force microscopy (AFM) imaging experimental data. The obtained results indicate that the proposed multi-scale modeling procedure is capable of predicting protofilaments with high accuracy and may be applied for structure prediction and analysis of other amyloid fibrils.

Tauopathies, including Alzheimer's disease (AD), are manifested by the deposition of well-characterized amyloid aggregates of Tau protein in the brain. However, it is rather unlikely that these aggregates constitute the major form of Tau responsible for neurodegenerative changes. Currently, it is postulated that the intermediates termed as soluble oligomers, assembled on the amyloidogenic pathway, are the most neurotoxic form of Tau. However, Tau oligomers reported so far represent a population of poorly characterized, heterogeneous and unstable assemblies. In this study, to obtain the oligomers, we employed the aggregation-prone K18 fragment of Tau protein with deletion of Lys280 (K18Δ280) linked to a hereditary tauopathy. We have described a new procedure of inducing aggregation of mutated K18 which leads either to the formation of nontoxic amyloid fibrils or neurotoxic globular oligomers, depending on its phosphorylation status. We demonstrate that PKA-phosphorylated K18Δ280 oligomers are toxic to hippocampal neurons, which is manifested by loss of dendritic spines and neurites, and impairment of cell-membrane integrity leading to cell death. We also show that N1, the soluble N-terminal fragment of prion protein (PrP), protects neurons from the oligomers-induced cytotoxicity. Our findings support the hypothesis on the neurotoxicity of Tau oligomers and neuroprotective role of PrP-derived fragments in AD and other tauopathies. These observations could be useful in the development of therapeutic strategies for these diseases., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

ATP acts as a biological hydrotrope preventing protein aggregation. Here, we report a novel chimeric peptide, ACC 1-13 K 8 , with an unusual capacity to bind and incorporate ATP while self-assembling into amyloid fibrils. The amino acid sequence combines a highly amyloidogenic segment of insulin's A-chain (ACC 1-13 ) and octalysine (K 8 ). Fibrillization requires binding 2 ATP molecules per ACC 1-13 K 8 monomer and is not triggered by adenosine di- and monophosphates (ADP, AMP). Infrared and CD spectra and AFM-based morphological analysis reveal tight and orderly entrapment of ATP within superstructural hybrid peptide-ATP fibrils. The incorporation of ATP is an emergent property of ACC 1-13 K 8 not observed for ACC 1-13 and K 8 segments separately. We demonstrate how new functionalities ( e.g. ATP storage) emerge from synergistic coupling of amyloidogenic segments with non-amyloidogenic peptide ligands, and suggest that ATP's role in protein misfolding is more nuanced than previously assumed.

Due to the spontaneous transition of native insulin into therapeutically inactive amyloid, prolonged storage decreases effectiveness of the hormone in treatment of diabetes. Various regions of the amino acid sequence have been implicated in insulin aggregation. Here, we focus on smaller fragments of the highly amyloidogenic H-peptide comprising disulfide-bonded N-terminal sections of insulin's A-chain (13 residues) and B-chain (11 residues). Aggregation patterns of N-terminal fragments of A-chain (ACC 1-13 , ACC 1-11 , ACC 6-13 , ACC 6-11 , all retaining Cys6A-Cys11A disulfide bond) and B-chain (B 1-11(7A) ) are examined at acidic and neutral pH. ACC 1-11 is the smallest fragment found to be amyloidogenic at either pH; removal of the N-terminal GIVEQ section renders this fragment entirely non-amyloidogenic. The self-assembling properties of ACC 1-11 contrast with aggregation-resistant behavior of B 1-11(7A) and its disulfide-linked homodimer, (B 1-11 ) 2 aggregating only at neutral pH. Fibrillar ACC 1-11 is similar to insulin amyloid in terms of morphology and infrared features. Secondary nucleation is likely to account for the detected shortening of insulin aggregation lag phase at neutral pH upon cross-seeding with pre-formed fibrils of ACC 1-11 or (B 1-11 ) 2 . An aggregation-enhancing effect of monomeric ACC 1-11 on co-dissolved native insulin is also observed. Our findings are discussed in the context of mechanisms of insulin aggregation., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Prions, proteins that can convert between structurally and functionally distinct states and serve as non-Mendelian mechanisms of inheritance, were initially discovered and only known in eukaryotes, and consequently considered to likely be a relatively late evolutionary acquisition. However, the recent discovery of prions in bacteria and viruses has intimated a potentially more ancient evolutionary origin. Here, we provide evidence that prion-forming domains exist in the domain archaea, the last domain of life left unexplored with regard to prions. We searched for archaeal candidate prion-forming protein sequences computationally, described their taxonomic distribution and phylogeny, and analyzed their associated functional annotations. Using biophysical in vitro assays, cell-based and microscopic approaches, and dye-binding analyses, we tested select candidate prion-forming domains for prionogenic characteristics. Out of the 16 tested, eight formed amyloids, and six acted as protein-based elements of information transfer driving non-Mendelian patterns of inheritance. We also identified short peptides from our archaeal prion candidates that can form amyloid fibrils independently. Lastly, candidates that tested positively in our assays had significantly higher tyrosine and phenylalanine content than candidates that tested negatively, an observation that may help future archaeal prion predictions. Taken together, our discovery of functional prion-forming domains in archaea provides evidence that multiple archaeal proteins are capable of acting as prions-thus expanding our knowledge of this epigenetic phenomenon to the third and final domain of life and bolstering the possibility that they were present at the time of the last universal common ancestor., (Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2021.)

Disulfide bonds prevent aggregation of globular proteins by stabilizing the native state. However, a disulfide bond within a disordered state may accelerate amyloidogenic nucleation by navigating fluctuating polypeptide chains towards an orderly assembly of β-sheets. Here, the self-assembly behavior of Glu-Cys-(Glu) 4 -Cys-Glu peptide (E 6 C 2 ), in which an intrachain disulfide bond is engineered into an amyloidogenic homopolypeptide motif, is investigated. To this end, the Thioflavin T (ThT) fluorescence kinetic assay is combined with infrared spectroscopy, circular dichroism (CD), atomic force microscopy (AFM) and Raman scattering measurements. Regardless of whether the disulfide bond is intact or reduced, E 6 C 2 monomers remain disordered within a broad range of pH. On the other hand, only reduced E 6 C 2 self-assembles into amyloid fibrils with the unique infrared traits indicative of three-center hydrogen bonds involving main-chain carbonyl as a bifurcating acceptor and main-chain NH and side-chain -COOH groups as hydrogen donors: the bonding pattern observed in so-called β 2 -fibrils. AFM analysis of β 2 -E 6 C 2 reveals tightly packed rectangular superstructures whose presence coincides with strong chiroptical properties. Our findings suggest that formation of chiral amyloid superstructures may be a generic process accessible to various substrates, and that the fully extended conformation of a poly-Glu chain is a condition sine qua non for self-assembly of β 2 -fibrils., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Relatively short amino acid sequences often play a pivotal role in triggering protein aggregation leading to the formation of amyloid fibrils. In the case of insulin, various regions of A- and B-chains have been implicated as the most relevant to the protein's amyloidogenicity. Here, we focus on the highly amyloidogenic H-fragment of insulin comprising the disulfide-bonded N-terminal parts of both chains. Analysis of the aggregation behavior of single-chain peptide derivatives of the H-fragment suggests that the A-chain's part initiates the aggregation process while the disulfide-tethered B-chain reluctantly adapts to amyloid structure. Merging of both A- and B-parts into single-chain continuous peptides (A-B and B-A) results in extreme amyloidogenicity exceeding that of the double-chain H-fragment as reflected by almost instantaneous de novo fibrillization. Amyloid fibrils of A-B and B-A present distinct morphological and infrared traits and do not cross-seed insulin. Our study suggests that the N-terminal part of insulin's A-chain containing the intact Cys6-Cys11 intrachain disulfide bond may constitute insulin's major amyloid stretch which, through its bent conformation, enforces a parallel in-register alignment of β-strands. Comparison of the self-association behavior of H, A-B, and B-A peptides suggests that A-chain's N-terminal amyloid stretch is very versatile and adaptive to various structural contexts.

The so-called 'H-fragment' of insulin is an extremely amyloidogenic double chain peptide consisting of the N-terminal parts of A-chain and B-chain linked by a disulfide bond between Cys-7A and Cys-7B. Here, we conduct a detailed investigation of the self-association behavior of H-fragment monomers into amyloid-like fibrils using kinetic assays, infrared spectroscopy, circular dichroism (CD), atomic force microscopy (AFM) and molecular dynamics (MD) simulations. Unlike the intact predominantly α-helical insulin, H-fragment remains in a disordered state in aqueous solutions. Its aggregation accelerates with acidification of the environment leading, at pH 1.9, to the formation of thin and structurally homogenous fibrils with the infrared features typical for parallel β-sheet conformation. According to time-lapse AFM morphological analysis both secondary nucleation and fragmentation are involved in later stages of H-fibrils' self-assembly. Based on the low nucleation order (two) obtained from the global fitting of kinetic data, realistic all-atom MD simulations of pairs of interacting H-fragment monomers were subsequently carried out. The molecular self-association scenario emerging from these simulations implicates the intrinsic conformational instability of H-monomer in its tendency to aggregate and form intermolecular β-sheet structure. Our findings provide the new mechanistic context for studies of insulin misfolding and aggregation., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In silico modelling of cascade enzymatic proteolysis is an exceedingly complex and challenging task. Here, we study partial proteolysis of insulin by pepsin: a process leading to the release of a highly amyloidogenic two chain 'H-fragment'. The H-fragment retains several cleavage sites for pepsin. However, under favorable conditions H-monomers rapidly self-assemble into proteolysis-resistant amyloid fibrils whose composition provides snapshots of early and intermediate stages of the proteolysis. In this work, we report a remarkable agreement of experimentally determined and simulation-predicted cleavage sites on different stages of the proteolysis. Prediction of cleavage sites was based on the comprehensive analysis of the docking interactions from direct simulation of coupled folding and binding of insulin (or its cleaved derivatives) to pepsin. The most frequent interactions were found to be between the pepsin's active site, or its direct vicinity, and the experimentally determined insulin cleavage sites, which suggest that the docking interactions govern the proteolytic process., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Conformational transitions involving aggregated proteins or peptides are of paramount biomedical and biotechnological importance. Here, we report an unusual freeze-induced structural reorganization within a β-sheet-rich ionic coaggregate of poly(l-lysine), PLL, and poly(l-glutamic acid), PLGA. Freezing aqueous suspensions of the PLL-PLGA β-aggregate in the presence of low concentrations of salt (NaBr) induces an instantaneous β-sheet-to-disorder transition, as probed by infrared spectroscopy in the amide I' band region. The conformational rearrangement of polypeptide chains appears to be fully synchronized with the global liquid-to-ice phase transition. In contrast to the known freeze-induced transitions, the process described here is fully reversible: the subsequent thawing results in an instantaneous disorder-to-β-sheet "refolding". However, in the absence of traces of soluble salts, the β-sheet framework of the PLL-PLGA aggregate remains resistant to freezing as no transition is observed. We note that the occurrence of the transition depends on the type of salt present in the sample. Our results highlight a hidden dimension of the structural dynamics within β-sheet-rich aggregates. Possible scenarios of freeze-induced salt-bridge rupture and removal of water from nanocanals are discussed.

The presence of disulfide bonds affects the protein stability and therefore tendency to misfold and form amyloid-like fibrils. Insulin's three disulfide bridges stabilize the native state and prevent aggregation. Partial proteolysis of insulin releases highly amyloidogenic and inherently disordered two-chain 'H-fragment' retaining insulin's Cys7A-Cys7B and Cys6A-Cys11A disulfide bonds. The abrupt self-association of H-fragment monomers into fibrils is suppressed in the presence of disulfide-reducing agent. These circumstances make the H-fragment an interesting model to study the impact of disulfide bonds on amyloidogenesis beyond the 'stabilization-of-the-native-state' paradigm. Here, we investigate fibrillization of various synthetic peptides derived from the H-fragment through modifications of Cys7A-Cys7B/Cys6A-Cys11A bonds. In comparison to H-fragment, aggregation of a two-chain 'AB' analog lacking Cys6A-Cys11A bond is decelerated, while the alternative removal of Cys7A-Cys7B bond releases a non-aggregating B-chain and a highly amyloidogenic 'ACC' fragment containing the intrachain Cys6A-Cys11A bond. Our analysis, supported by calculations of configurational entropy, suggests that Cys6A-Cys11A bond is a key factor behind the explosive self-association of H-fragment. The bond restricts the conformational space probed by nucleating monomers which is reflected by an approximately 2.4 kJ·mol -1 K -1 decrease in entropy. The fact that the intact Cys6A-Cys11A bond promotes fibrillization of the H-fragment is remarkable in light of the previously established role of the same disulfide bond in preventing formation of insulin fibrils. Our results imply that a single disulfide bond within a folded protein and its fragment may play entirely different roles in aggregation and that this role may evolve with progressing phases of misfolding., (© 2019 Federation of European Biochemical Societies.)

Bovine serum albumin (BSA) is often employed as a proteinaceous component for synthesis of luminescent protein-stabilized gold nanoclusters (AuNC): intriguing systems with many potential applications. Typically, the formation of BSA-AuNC conjugate occurs under strongly alkaline conditions. Due to the sheer complexity of intertwined chemical and structural transitions taking place upon BSA-AuNC formation, the state of albumin enveloping AuNCs remains poorly characterized. Here, we study the conformational properties of BSA bound to AuNCs using an array of biophysical tools including vibrational spectroscopy, circular dichroism, fluorescence spectroscopy and trypsin digestion. The alkaline conditions of BSA-AuNC self-assembly appear to be primary responsible for the profound irreversible disruption of tertiary contacts, partial unfolding of native α-helices, hydrolysis of disulfide bonds and the protein becoming vulnerable to trypsin digestion. Further unfolding of BSA-AuNC by guanidinium hydrochloride (GdnHCl) is fully reversible equally in terms of albumin's secondary structure and conjugate's luminescent properties. This suggests that binding to AuNCs traps the albumin molecule in a state that is both partly disordered and refractory to irreversible misfolding. Indeed, when BSA-AuNC is subjected to conditions favoring self-association of BSA into amyloid-like fibrils, the buildup of non-native β-sheet conformation is less pronounced than in a control experiment with unmodified BSA. Unexpectedly, BSA-AuNC reveals a tendency to self-assemble into giant twisted superstructures of micrometer lengths detectable with transmission electron microscopy (TEM), a property absent in unmodified BSA. The process is accompanied by ordering of bound AuNCs into elongated streaks and simultaneous decrease in fluorescence intensity. The newly discovered self-association pathway appears to be specifically accessible to protein molecules with a certain restriction on structural dynamics which in the case of BSA-AuNC arises from binding to metal nanoclusters. Our results have been discussed in the context of mechanisms of protein misfolding and applications of BSA-AuNC., Competing Interests: The authors have declared that no competing interests exist.

Replacing water with dimethyl sulfoxide (DMSO) completely reshapes the free-energy landscapes of solvated proteins. In DMSO, a powerful hydrogen-bond (HB) acceptor, formation of HBs between backbone NH groups and solvent is favored over HBs involving protein's carbonyl groups. This entails a profound structural disruption of globular proteins and proteinaceous aggregates (e.g., amyloid fibrils) upon transfer to DMSO. Here, we investigate an unusual DMSO-induced conformational transition of β 2 -amyloid fibrils from poly-l-glutamic acid (PLGA). The infrared spectra of β 2 -PLGA dissolved in DMSO lack the typical features associated with disordered conformation that are observed when amyloid fibrils from other proteins are dispersed in DMSO. Instead, the frequency and unusual narrowness of the amide I band imply the presence of highly ordered helical structures, which is supported by complementary methods, including vibrational circular dichroism and Raman optical activity. We argue that the conformation most consistent with the spectroscopic data is that of a PLGA chain essentially lacking nonhelical segments such as bends that would provide DMSO acceptors with direct access to the backbone. A structural study of DMSO-dissolved β 2 -PLGA by synchrotron small-angle X-ray scattering reveals the presence of long uninterrupted helices lending direct support to this hypothesis. Our study highlights the dramatic effects that solvation may have on conformational transitions of large polypeptide assemblies.

Amyloid aggregates of Tau protein have been implicated in etiology of many neurodegenerative disorders including Alzheimer's disease (AD). When amyloid growth is induced by seeding with preformed fibrils assembled from the same protein, structural characteristics of the seed are usually imprinted in daughter generations of fibrils. This so-called conformational memory effect may be compromised when the seeding involves proteins with non-identical sequences leading to the emergence of distinct structural variants of fibrils (amyloid 'strains'). Here, we investigate cross-seeding of full-length human Tau (FL Tau) with fibrils assembled from K18 and K18ΔK280 fragments of Tau in the presence of poly-L-glutamate (poly-Glu) as an enhancer of Tau aggregation. To study cross-seeding between Tau polypeptides and the role of the conformational memory effect in induction of Tau amyloid polymorphism, kinetic assays, transmission electron microscopy, infrared spectroscopy and limited proteolysis have been employed. The fastest fibrillization was observed for FL Tau monomers seeded with preformed K18 amyloid yielding daughter fibrils with unique trypsin digestion patterns. Morphological features of daughter FL Tau fibrils induced by K18 and K18ΔK280 seeds were reminiscent of the mother fibrils (i.e. straight paired fibrils and paired helical filaments (PHFs), respectively) but disappeared in the following generations which became similar to unpaired FL Tau amyloid fibrils formed de novo. The structural evolution observed in our study was accompanied by disappearance of the unique proteolysis profile originated from K18. Our findings may have implications for understanding molecular mechanisms of the emergence and stability of Tau amyloid strains., Competing Interests: The authors have declared that no competing interests exist.

Several homopolypeptides including poly-l-glutamic acid (PLGA) form amyloid-like fibrils under favorable physicochemical conditions. We have shown recently that even short uncapped (Glu) n peptides (for n>3) form fibrillar β-aggregates which cross-seed with amyloid fibrils obtained from high molecular weight fractions of PLGA. Here we investigate effects of N-terminal acetylation and C-terminal amidation on the amyloidogenic tendencies of (Glu) n peptides containing 3, 4, and 5 residues. Our results based primarily on time-lapse FT-IR spectroscopy and AFM microscopy indicate that selective modifications of C-termini (and, to a lesser degree, of N-termini) decrease capacity of tetra- and pentapeptides to form fibrils. On the other hand, peptides modified at both ends appear to form fibrils as fast as unmodified analogues. In fact, the double terminal modification enables fibrillation of (Glu) 3 which is not fibrillogenic in the unmodified state. The AFM data suggests that the double capping results in the aggregates becoming more tape-like or acquiring noticeable tendencies to bend. According to seeding and cross-seeding experiments, there is a high degree of promiscuity between modified and unmodified peptides. Possible mechanisms explaining how amyloidogenic propensities of (Glu) n peptides are affected by terminal modifications have been discussed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Due to its symmetric structure and abundance of carboxyl groups, mellitic acid (MA-benzenehexacarboxylic acid) has an uncommon capacity to form highly ordered molecular networks. Dissolved in water, MA dissociates to yield various mellitate anions with pronounced tendencies to form complexes with cations including protonated amines. Deprotonation of MA at physiological pH produces anions with high charge densities (MA5- and MA6-) whose influence on co-dissolved proteins has not been thoroughly studied. As electrostatic attraction between highly symmetric MA6- anions and positively charged low-symmetry globular proteins could lead to interesting self-assembly patterns we have chosen hen egg white lysozyme (HEWL), a basic stably folded globular protein as a cationic partner for mellitate anions to form such hypothetical nanostructures. Indeed, mixing of neutral HEWL and MA solutions does result in precipitation of electrostatic complexes with the stoichiometry dependent on pH. We have studied the self-assembly of HEWL-MA structures using vibrational spectroscopy (infrared absorption and Raman scattering), circular dichroism (CD), atomic force microscopy (AFM). Possible HEWL-MA6- molecular docking scenarios were analyzed using computational tools. Our results indicate that even at equimolar ratios (in respect to HEWL), MA5- and MA6- anions are capable of inducing misfolding and aggregation of the protein upon mild heating which results in non-native intermolecular beta-sheet appearing in the amide I' region of the corresponding infrared spectra. The association process leads to aggregates with compacted morphologies entrapping mellitate anions. The capacity of extremely diluted mellitate anions (i.e. at sub-millimolar concentration range) to trigger aggregation of proteins is discussed in the context of mechanisms of misfolding.

Background and Objectives: Although many synthetic gonadoliberin analogs have been developed, only a few of them, including buserelin, were introduced into clinical practice. Dalarelin, which differs from buserelin by just one aminoacid in the position 6 (D-Ala), is not widely used so far. Gonadotropin-releasing hormone (GnRH) analogs are used to treat many different illnesses and are available in different forms like solution for injection, nasal spray, microspheres, etc. Unfortunately, none of the above drug formulations can release the hormones for 24 h. We assumed that classical suspension could solve this problem., Methods: Two sets of experiments were performed. In the first one, buserelin and dalarelin were injected into mature female rats in two forms: suspension, in which the analogs are bounded by Zn 2+ ions and solution. The pharmacokinetic parameters and bioavailability of the analogs were calculated, based on their concentration in the plasma measured by high-performance liquid chromatography method (HPLC). In the second experiment, the hormones in two different forms were injected into superovulated immature female rats and then the concentration of Luteinizing hormone (LH), Follicle-stimulating hormone (FSH) and 17β-estradiol in the serum was measured by radioimmunological method., Results: The Extent of Biological Availability (EBA), calculated on the base of AUC 0-∞ , showed that in the form of solution buserelin and dalarelin display, respectively, only 13 and 8 % of biological availability of their suspension counterparts. Comparing both analogs, the EBA of dalarelin was half (53 %) that of buserelin delivered in the form of solution and 83 % when they were delivered in the form of suspension. The injection of buserelin or dalarelin, in the form of solution or suspension, into superovulated female rats increased LH, FSH and estradiol concentration in the serum. However, after injection of the analogs in the form of suspension, the high concentration of LH and FSH in the serum persisted longer., Conclusion: Performed studies indicate that GnRH analogs in the form of suspension have higher bioavailability than their solution counterparts. It influences the effects of their action, especially in relation to LH and FSH.

Typically, elongation of an amyloid fibril entails passing conformational details of the mother seed to daughter generations of fibrils with high fidelity. There are, however, several factors that can potentially prevent such transgenerational structural imprinting from perpetuating, for example heterogeneity of mother seeds or so-called conformational switching. Here, we examine phenotypic persistence of bovine insulin amyloid ([BI]) upon multiple rounds of self-seeding under quiescent conditions. According to infrared spectroscopy, with the following passages of homologous seeding, daughter fibrils gradually depart from the mother seed's spectral characteristics. We note that this transgenerational structural drift in [BI] amyloid leads toward fibrils with infrared, chiroptical, and morphological traits similar to those of the superstructural variant of fibrils which normally forms upon strong agitation of insulin solutions. However, in contrast to agitation-induced insulin amyloid, the superstructural assemblies of daughter fibrils isolated through self-seeding are sonication-resistant. Our results suggest that formation of single amyloid fibrils is not a dead-end of the amyloidogenic self-assembly. Instead, the process appears to continue toward the self-assembly of higher-order structures although on longer time-scales. From this perspective, the fast agitation-induced aggregation of insulin appears to be a shortcut to amyloid superstructures whose formation under quiescent conditions is slow.

Proteases play a well recognized role in the emergence of highly aggregation-prone protein fragments in vivo, whereas in vitro limited proteolysis is often employed to probe different phases of amyloidogenic pathways. Here, we show that addition of moderate amounts of pepsin to acidified bovine insulin at close to physiological temperature results in an abrupt self-assembly of amyloid-like fibrils from partially digested insulin fragments. Biochemical analysis of the pepsin-induced fibrils implicates peptide fragments (named H) consisting of the 13 or 15 N-terminal residues of the A-chain and 11 or 13 N-terminal residues of the B-chain linked by the disulfide bond between Cys-7A-Cys-7B as the main constituents. There are up to eight pepsin-cleavage sites remaining within the double chain peptide, which become protected upon fast fibrillation unless concentration of the enzyme is increased resulting in complete digestion of insulin. Controlled re-association of H-peptides leads to "explosive" fibrillation only under nonreducing conditions implying the key role of the disulfide bond in their amyloidogenicity. Such re-assembled amyloid is similar in terms of morphology and infrared features to typical bovine insulin fibrils, although it lacks the ability to seed the intact protein., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Pyrene, a fluorescent dye, was incorporated into polystyrene particles coated with polypyrrole. The incorporation was achieved by treating the polypyrrole/polystyrene (PPy/PS) beads in a tetrahydrofuran (THF) solution of the pyrene fluorophore followed by rinsing with methanol. The polystyrene cores of the beads swell in THF, allowing penetration of pyrene molecules into the polystyrene structure. The addition of methanol causes contraction of the swollen polystyrene, which encapsulates the dye molecules inside the beads. It is shown that the polypyrrole coating is permeable with respect to both the dye and the solvent, allowing the transport of molecules between the polystyrene cores and the contacting solution. The polypyrrole adlayer can be used as a matrix for the incorporation of magnetic nanoparticles. Embedded particles provide magnetic functionality to the PPy/PS beads. It is demonstrated that the pyrene-loaded beads can be manipulated with an external magnetic field., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Valosin containing protein (VCP) disease (also known as Inclusion Body Myopathy, Paget Disease of Bone and Frontotemporal Dementia [IBMPFD] syndrome) is caused by mutations in the gene encoding VCP classically affecting the muscle, bone and brain. Although the genetic cause has been identified, details regarding the pathogenesis of IBMPFD have not been fully determined. Muscle wasting observed in VCP disease is suggestive of cytokine imbalance. We hypothesized that dysfunctional protein homeostasis caused by VCP mutations leads to cytokine imbalances thereby contributing to the muscle wasting phenotype. Circulating levels of interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF a) and epidermal growth factor (EGF) were measured in plasma of patients with VCP disease or controls. TNF a and EGF were significantly altered in VCP disease as compared to control. TNF a was up-regulated, consistent with a cachexia phenotype and EGF levels were increased. No significant differences were observed in IL-4 and IL-6. Cytokine imbalances may be associated with VCP disease and may play a contributory role in VCP myopathy. Further understanding of how VCP dysfunction leads to aberrant protein homeostasis and subsequent cytokine imbalances may also aid in the understanding of other proteinopathies and in the development of novel treatments., (© 2013 Wiley Periodicals, Inc.)

Introduction:  Immunosuppression with glucocorticoids is the method of choice in the treatment of active Graves' ophthalmopathy (GO). However, glucocorticoid therapy may have side effects, among others, it affects bone metabolism., Objectives:  The aim of the study was to compare the effect of methylprednisolone pulse therapy (MPPT) with and without alendronate on bone turnover markers in patients with GO with normal and reduced bone mineral density (BMD)., Patients and Methods:  The study included 53 patients with GO and 20 sex- and age‑matched healthy controls. Twenty patients with normal BMD (17 women, 3 men, aged 45 ±1.0 years) received only MPPT (8 g intravenously during 4 weeks). The remaining patients, with reduced BMD, were randomly assigned either to MPPT without alendronate (10 women, 2 men, aged 47 ±1.0 years) or MPPT with alendronate (18 women, 3 men, aged 47 ±1.0 years). BMD of the lumbar spine and femoral neck was assessed using dual energy X‑ray absorptiometry  (DEXA) before treatment. The markers of bone formation (serum osteocalcin, carboxyterminal propeptide of type I collagen [PICP], alkaline phospatase) and the markers of bone resorption (serum carboxyterminal telopeptide of type I collagen [ICTP], cross‑linked C‑terminal telopeptide of type I collagen [CTX], serum calcium [Ca] and potassium [P], as well as urinary excretion of deoxypyridinoline, Ca, and P) were determined before and after treatment., Results:  MPPT caused a decrease in bone formation markers and an increase in some bone resorption markers. MPPT with alendronate decreased bone formation and bone resorption markers., Conclusions:  A negative effect of MPPT on bone turnover is observed both in patients with GO with normal and with reduced BMD. Simultaneous use of MPPT and alendronate in patients with GO and reduced BMD suppresses bone resorption caused by methylprednisolone.

Background: The aim of the study was to assess diurnal melatonin secretion in children with refractory epilepsy (N=74) as compared to children without epileptic seizures (N=37) and to compare melatonin secretion in children with focal and generalized refractory epilepsy., Material/methods: In the study group 4 subgroups were defined: children with focal symptomatic epilepsy, focal cryptogenic epilepsy, generalized symptomatic epilepsy, and generalized cryptogenic epilepsy. Melatonin level was measured every 3 hours using the RIA method., Results: Analysis of diurnal melatonin secretion indicated a lower level of the hormone in patients with refractory epilepsy. The daily rhythm of melatonin secretion in the study group was maintained, with a peak shift of melatonin secretion especially visible in the subgroup with generalized symptomatic refractory epilepsy in the age group between 6 months and 3 years of age., Conclusions: The hypothesis may be formed that a lowered level of melatonin in the study group in relation to the comparison group is the consequence of the natural course of epilepsy or is influenced by antiepileptic drugs.

Introduction: Cardiovascular abnormalities may be the only manifestations of overt hyperthyroidism. In patients with heart failure and atrial fibrillation digoxin can be beneficial in controlling the symptoms and signs, but hyperthyroid patients show an impaired response or even resistance to digoxin treatment. The aim of the study is to establish: 1. Are there any differences in the pharmacokinetics of a single oral dose of digoxin between hypertyroid and euthyroid patients? 2. Does simultaneous administration of digoxin and methimazole affect the pharmacokinetics of a single oral dose of dogoxin? 3. Does methimazole-induced euthyroidism change the pharmacokinetics of a single oral dose of digoxin?, Material and Methods: The subject of the study were 28 patients with hyperthyroidism and 15 healthy persons. We evaluated the pharmacokinetics of a single oral dose of digoxin. Moreover we evaluated pharmacokinetics of a single dose of digoxin after simultaneous administration of digoxin and methimazole in 12 patients and 12 methimazole treated patients werere-assessed once they had become euthyroid., Results: Hyperthyroid patients showed significantly lower serum digoxin concentrations, shorter T1/2 beta and a significantly smaller area under the concentration curve (AUC) that the control group. Administration of methimazole did not affect digoxin pharmacokinetics., Conclusions: In hyperthyroid patients: 1. the pharmacokinetics of a single oral dose of digoxin does differ from that observed in healthy subjects. 2.methimazole do not alter digoxin pharmacokinetics.

Background: The main aim of this paper is to determine scope of changes in concentration of selected pro-inflammatory factors in blood serum in the initial period after the kidney transplantation from the brain death donor and attempt of establishing which of them are connected with delayed function of the organ, initial condition of both the donor and the recipient., Material/methods: The prospective examination carried out in group of 20 patients that underwent kidney transplantation from 10 donors diagnosed brain death. Blood samples were drawn before the transplantation procedure, 4 hours after procedure and in 4(th) day after the procedure. The blood serum analysis included: interleukin-6 (IL-6), tumor necrosis factor-alpha, interleukin-1beta (IL-1b) and C-reactive protein (CRP)., Results: In perioperative period the dynamic changes of all observed markers were noticed, especially in 4 th hour after the transplantation procedure. Generally, apart from TNFalpha, their concentration in blood serum was raising (the most significantly for IL-6) and then in 4(th) day reached the level equal to or lower than before transplantation., Conclusions: The delayed graft function is accompanied by high CRP level in donors before grafting and sustaining rise of IL-1b content in blood serum in 4(th) day after the transplantation procedure. The IL-6 content in this period revealed similar tendency in recipients' pairs that have been given the kidney from one donor, reflecting the condition of an organ that has been transplanted.

Introduction: Early and precise diagnosis is necessary in successful treatment of the patients with Cushing's disease. Transsphenoidal surgery is the first line treatment option in Cushing's disease. In patients in whom magnetic resonance imaging does not visualise pituitary adenoma patient selection for surgical treatment should be based on convincing results of functional tests., Objectives: The diagnostic accuracy of the desmopressin test was compared with that of the CRH test and the overnight high-dose dexamethasone suppression test in the diagnosis of Cushing's disease., Design and Method: We studied 15 patients with pituitary-dependent Cushing's disease (10 with detected microadenoma and 5 with undetected microadenoma using magnetic resonance imaging). The diagnosis was confirmed during pituitary surgery. The control group included 15 subjects without Cushing's syndrome. Patients underwent tests with desmopressin, CRH and 8 mg overnight dexamethasone., Results: A false negative response to CRH was present in 1 of 15 patients, a false negative response t dexamethasone was present in 1 of 15 patients and a false negative response to desmopressin in 2 of 15 patients. In other patients positive responses to CRH, desmopressin and dexamethasone were found. A positive response to desmopressin was present in 4 of 5 patients with undetected pituitary adenoma. In the control group a positive response to CRH and dexamethazone and a negative response to desmopressin was found in all patients., Conclusions: 1. The highest diagnostic accuracy in the diagnosis of some patients with Cushing's disease is achieved by using a desmopressin test with high-dose dexamethasone test and CRH-test. 2. The desmopressin test can be used to precisely identify healthy individuals.

A 39-year-old man with occult eutopic corticotropin microadenoma leading to Cushing's syndrome was presented. Magnetic resonance imaging was unable to identify the pituitary microadenoma. In another department bilateral adrenalectomy was justified. In our department to identify or exclude a pituitary source of autonomous corticotropin secretion desmopressin test was performed. Intravenous desmopressin administration enhanced both circulating concentrations ofACTH and cortisol by 217.36% (peak vs. baseline) and 67% respectively. The patient underwent a transsphenoidal adenomectomy. Immunostaining demonstrated ACTH in adenoma cells. After surgery cortisol levels returned to the normal range, moreover no ACTH and cortisol response was elicited by desmopressin administration. This clinical case represents the demonstration of the possibility to unmask an occult eutopic Cushing's syndrome with the desmopressin test.

Endothelin ET-1 plays an important, however not entirely identified role in physiology and pathology of thyroid gland. The aim of the study was to determine if in patients with non-toxic nodular goiter there is relationship between size of goiter and ET-1 concentration in plasma of peripheral venous blood and plasma of venous blood taken from thyroid vein in patients undergoing strumectomy. The study included 30 patients with non-toxic nodular goiter (29 women and 1 man, mean age 48 year) and 15 healthy persons of control group. In all ET-1 concentration in plasma of venous peripheral blood and T3, T4, TSH serum concentration were estimated using radioimmunologic methods, as well as size of thyroid gland were evaluated in ultrasonography. Patients with nodular goiter underwent strumectomy. During the operation, before strumectomy, blood from thyroid vein was sampled to estimate plasma ET-1 concentration. Patients were divided into two groups: with adenomatous goiter (14 patients) and colloid goiter (16 patients). The goiter size was similar in each group. In all of the groups there was no relationship between goiter size and ET-1 concentration in plasma of peripheral and thyroid vein. Lack of correlation between ET-1 concentration in plasma and goiter size does not exclude possible ET-1 role in goitrogenesis, it suggests that ET-1 may play a role in control of thyroid gland trophic regulation and vascularity as paracrine or autocrine factor. Significantly higher concentration of ET-1 in plasma of thyroid vein in comparison to plasma of peripheral venous blood and positive correlation between ET-1 concentration in thyroid and peripheral plasma were found in both studied groups of patients with goiter and this may speak for increased ET-1 production within the goiter.

We investigated leptin concentration in umbilical cord blood of 51 newborns (mean 5.71 +/- 3.28 ng/ml) and in maternal blood (mean 22.11 +/- 10.95 ng/ml). Leptin concentration in 20 preterm infants (mean 4.73 +/- 2.15 ng/ml) was significantly lower (p < 0.05) than in full-term newborns (mean 6.34 +/- 2.08 ng/ml) and tended to increase according to gestational age and birth weight. We suggested leptin concentration had a role in intrauterine development.

Objectives: Pineal gland hormone, melatonin, is a current issue of interest for accumulating data concerning its diverse physiological functions. The disturbances in melatonin secretion are observed in different pathological conditions involving pineal regions, but it is not ascertained if those disturbances present any clinical implications. The aim of this work was to examine whether pineal gland compression changes melatonin secretion., Setting and Design: The experiment was carried out on adult rats, divided into four equal groups: (i) control (no surgery was performed), (ii) sham-operated, (iii) with sham pineal gland compression and (iv) with pineal gland compression performed by cotton piece application., Methods: The profile of melatonin secretion was assessed in blood samples collected five times daily, every second day, starting from 8 to 14 day following surgery., Results: We found that surgery itself significantly increased night melatonin secretion in comparison to controls. By contrast, in pineal-gland compressed rats, melatonin secretion was lower than in control group, suggesting that the influence of pineal compression overcame that induced by operation stress., Conclusions: In conclusion, we presume that pineal gland compression (like in case of some tumors) results in decrease of the concentration of blood melatonin, that may possibly result in decreased protective action of the indoleamine.

Tumors of the pineal region, though not very common, are recently more often diagnosed due to the introduction of computer tomography and magnetic resonance examinations into neurosurgical practice. However many controversies about the treatment of them still exist. Some of them need aggressive treatment. The others are benign and asymptomatic. Thus it seems to be important to improve our diagnosis of mass lesion of the pineal region, especially before taking a decision for surgery. The purpose of this study was to find any significant changes in the circadian pattern of melatonin secretion in patients with pineal region tumors. Blood samples were collected preoperatively from 21 patients with diagnosed pineal region tumors. In 13 patients sampling was performed at 08:00, 14:00, 20:00 and 02:00 h during a 24-hour period but in 8 former ones only at 02:00 h at night. The samples were immediately centrifuged and the serum stored at -20 degrees C until analysis. The patients stayed in the darkened room from 23:00 to 05:00 h. Plasma melatonin was measured by direct and specific radioimmunoassay. The following three groups of results were found: (1) eight patients showed normal melatonin secretion profiles, (2) six cases with lack of the night maximum plasma value, and (3) seven cases with nocturnal melatonin concentration higher than in the healthy population (>100pg/ml). We observed no correlation between melatonin secretion and histological type of tumor. In conclusion, we suggest that changes of melatonin secretion could indicate the pineal region pathology. However further studies with a larger group of patients, especially with tumors originating from the pineal gland (pinealocytoma, pinealoblastoma), are necessary.

The activity of acetylcholinesterase (AChE 3.1.1.7.) was determined in skeletal muscle homogenates in frogs at various time intervals (15--24 days) after denervation. At the same time changes in AChE activity were compared with morphological changes of neuromuscular end plates in these muscles. Muscle denervation caused initially, within 30 hours, a rise in AChE activity by about 30% in relation to control muscles, followed by its fall to the control values after 4--6 days. The activity decreased further reaching lowest values 15 days after denervation, when it amounted to about 65% of the value of control muscles. After that time, when regeneration of nerves set in, the activity of AChe rose again returning to the control values after 24 hours. In the initial period of denervation no correlation was found between AChE activity and morphological changes in end plates. It was found however in the later period when degenerative changes were most pronounced as well as when reinnervation was in progress.