Rpt5-Derived Analogs Stimulate Human Proteasome Activity in Cells and Degrade Proteins Forming Toxic Aggregates in Age-Related Diseases.

Aging and age-related diseases are associated with a decline in the capacity of protein turnover. Intrinsically disordered proteins, as well as proteins misfolded and oxidatively damaged, prone to aggregation, are preferentially digested by the ubiquitin-independent proteasome system (UIPS), a major component of which is the 20S proteasome. Therefore, boosting 20S activity constitutes a promising strategy to counteract a decrease in total proteasome activity during aging. One way to enhance the proteolytic removal of unwanted proteins appears to be the use of peptide-based activators of the 20S. In this study, we synthesized a series of peptides and peptidomimetics based on the C-terminus of the Rpt5 subunit of the 19S regulatory particle. Some of them efficiently stimulated human 20S proteasome activity. The attachment of the cell-penetrating peptide TAT allowed them to penetrate the cell membrane and stimulate proteasome activity in HEK293T cells, which was demonstrated using a cell-permeable substrate of the proteasome, TAS3. Furthermore, the best activator enhanced the degradation of aggregation-prone α-synuclein and Tau-441. The obtained compounds may therefore have the potential to compensate for the unbalanced proteostasis found in aging and age-related diseases.

Do long-lasting effects of epidural polarization of afferent fibres depend on persistent sodium current?

Few attempts have so far been made to define the mechanisms underlying the hour-long effects of trans-spinal stimulation combined with epidural polarization. In the present study, we investigated the potential involvement of non-inactivating sodium channels in afferent fibres. To this end, riluzole, a blocker of these channels, was administered locally to the dorsal columns close to the site of the excitation of afferent nerve fibres by epidural stimulation in deeply anaesthetized rats in vivo. Riluzole did not prevent the induction of the polarization-evoked sustained increase in the excitability of dorsal column fibres but tended to weaken it. It likewise weakened but did not abolish the sustained polarization-evoked shortening of the refractory period of these fibres. These results lead to the conclusion that the persistent sodium current may contribute to the sustained post-polarization-evoked effects but is only partly involved in both the induction and the expression of these effects.

Molecular characterization of the PhiKo endolysin from Thermus thermophilus HB27 bacteriophage phiKo and its cryptic lytic peptide RAP-29.

Introduction: In the era of increasing bacterial resistance to antibiotics, new bactericidal substances are sought, and lysins derived from extremophilic organisms have the undoubted advantage of being stable under harsh environmental conditions. The PhiKo endolysin is derived from the phiKo bacteriophage infecting Gram-negative extremophilic bacterium Thermus thermophilus HB27. This enzyme shows similarity to two previously investigated thermostable type-2 amidases, the Ts2631 and Ph2119 from Thermus scotoductus bacteriophages, that revealed high lytic activity not only against thermophiles but also against Gram-negative mesophilic bacteria. Therefore, antibacterial potential of the PhiKo endolysin was investigated in the study presented here. Methods: Enzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3'-Dipropylthiacarbocyanine iodide (DiSC3(5)). Results and discussion: The PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7-7.1 log units and reaching the minimum inhibitory concentration values in the range of 2-31 µM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously.

A Novel Cryptic Clostridial Peptide That Kills Bacteria by a Cell Membrane Permeabilization Mechanism.

This work reports detailed characteristics of the antimicrobial peptide Intestinalin (P30), which is derived from the LysC enzyme of Clostridium intestinale strain URNW. The peptide shows a broader antibacterial spectrum than the parental enzyme, showing potent antimicrobial activity against clinical strains of Gram-positive staphylococci and Gram-negative pathogens and causing between 3.04 ± 0.12 log kill for Pseudomonas aeruginosa PAO1 and 7.10 ± 0.05 log kill for multidrug-resistant Acinetobacter baumannii KPD 581 at a 5 µM concentration. Moreover, Intestinalin (P30) prevents biofilm formation and destroys 24-h and 72-h biofilms formed by Acinetobacter baumannii CRAB KPD 205 (reduction levels of 4.28 and 2.62 log CFU/mL, respectively). The activity of Intestinalin is combined with both no cytotoxicity and little hemolytic effect against mammalian cells. The nuclear magnetic resonance and molecular dynamics (MD) data show a high tendency of Intestinalin to interact with the bacterial phospholipid cell membrane. Although positively charged, Intestinalin resides in the membrane and aggregates into small oligomers. Negatively charged phospholipids stabilize peptide oligomers to form water- and ion-permeable pores, disrupting the integrity of bacterial cell membranes. Experimental data showed that Intestinalin interacts with negatively charged lipoteichoic acid (logK based on isothermal titration calorimetry, 7.45 ± 0.44), causes membrane depolarization, and affects membrane integrity by forming large pores, all of which result in loss of bacterial viability. IMPORTANCE Antibiotic resistance is rising rapidly among pathogenic bacteria, becoming a global public health problem that threatens the effectiveness of therapies for many infectious diseases. In this respect, antimicrobial peptides appear to be an interesting alternative to combat bacterial pathogens. Here, we report the characteristics of an antimicrobial peptide (of 30 amino acids) derived from the clostridial LysC enzyme. The peptide showed killing activity against clinical strains of Gram-positive and Gram-negative pathogens. Experimental data and computational modeling showed that this peptide forms transmembrane pores, directly engaging the negatively charged phospholipids of the bacterial cell membrane. Consequently, dissipation of the electrochemical gradient across cell membranes affects many vital processes, such as ATP synthesis, motility, and transport of nutrients. This kind of dysfunction leads to the loss of bacterial viability. Our firm conviction is that the presented study will be a helpful resource in searching for novel antimicrobial peptides that could have the potential to replace conventional antibiotics.

Basic principles of processing of afferent information by spinal interneurons.

Integrative functions of spinal interneurons are well recognized but the relative role of different interneuronal populations in this process continues to be investigated. It therefore appeared useful to review the principles of integration of afferent information by the interneurons analyzed so far as these principles should apply also to those remaining to be analyzed. Considering the results of both functional and morphological studies of spinal interneurons and of the morphology and immunochemistry of afferent fibers that provide input to them, the following five basic principles of processing of afferent information by them will be outlined; 1) afferent information of any origin is forwarded to several neuronal populations, 2) information from any sources of input is distributed unevenly, 3) input from several sources is integrated by individual neurons as well as by their populations, 4) specific combinations of input are integrated by different neuronal populations, and 5) afferent input to spinal interneurons is only one of the features distinguishing their functional populations. As the spinal neuronal organization and properties of neurons and afferent fibers in the so far investigated species (cat, rodents, and primates) have been found to resemble, future studies using molecular techniques in the mouse should allow the new data to integrate with those of the preceding studies and the principles outlined earlier as well as any new ones should apply also in humans.

Long-term modulation of the axonal refractory period.

The main question addressed in this study was whether the refractoriness of nerve fibres can be modulated by their depolarisation and, if so, whether depolarisation of nerve fibres evokes a long-term decrease in the duration of the refractory period as well as the previously demonstrated increase in their excitability. This was investigated on nerve fibres within the dorsal columns, dorsal roots and peripheral nerves in deeply anaesthetised rats in vivo. The results revealed major differences depending on the sites of fibre stimulation and polarisation. Firstly, the relative refractory period was found to be shorter in epidurally stimulated dorsal column fibres than in fibres stimulated at other sites. Secondly, the minimal effective interstimulus intervals reflecting the absolute refractory period were likewise shorter for nerve fibres within the dorsal columns even though action potentials evoked by the second of a pair of stimuli were similarly delayed with respect to the preceding action potentials at all the stimulation sites. Thirdly, the minimal interstimulus intervals were reduced by epidurally applied cathodal direct current polarisation but not at other stimulation sites. Consequently, higher proportions of dorsal column fibres could be excited at higher frequencies, especially following their depolarisation, at interstimulus intervals as short as 0.5-0.7 ms. The results demonstrate that epidural depolarisation results in long-lasting effects not only on the excitability but also on the refractoriness of dorsal column fibres. They also provide further evidence for specific features of afferent fibres traversing the dorsal columns previously linked to properties of their branching regions.

Genetic and pharmacologic proteasome augmentation ameliorates Alzheimer's-like pathology in mouse and fly APP overexpression models.

The proteasome has key roles in neuronal proteostasis, including the removal of misfolded and oxidized proteins, presynaptic protein turnover, and synaptic efficacy and plasticity. Proteasome dysfunction is a prominent feature of Alzheimer's disease (AD). We show that prevention of proteasome dysfunction by genetic manipulation delays mortality, cell death, and cognitive deficits in fly and cell culture AD models. We developed a transgenic mouse with neuronal-specific proteasome overexpression that, when crossed with an AD mouse model, showed reduced mortality and cognitive deficits. To establish translational relevance, we developed a set of TAT-based proteasome-activating peptidomimetics that stably penetrated the blood-brain barrier and enhanced 20S/26S proteasome activity. These agonists protected against cell death, cognitive decline, and mortality in cell culture, fly, and mouse AD models. The protective effects of proteasome overexpression appear to be driven, at least in part, by the proteasome's increased turnover of the amyloid precursor protein along with the prevention of overall proteostatic dysfunction.

Peptidomimetics Based on C-Terminus of Blm10 Stimulate Human 20S Proteasome Activity and Promote Degradation of Proteins.

Degradation of misfolded, redundant and oxidatively damaged proteins constitutes one of the cellular processes which are influenced by the 20S proteasome. However, its activity is generally thought to decrease with age which leads to the gradual accumulation of abnormal proteins in cells and their subsequent aggregation. Therefore, increasing proteasomal degradation constitutes a promising strategy to delay the onset of various age-related diseases, including neurodegenerative disorders. In this study we designed and obtained a series of peptidomimetic stimulators of 20S comprising in their sequences the C-terminal fragment of Blm10 activator. Some of the compounds were capable of enhancing the degradation of natively unfolded and oxidatively damaged proteins, such as α-synuclein and enolase, whose applicability as proteasome substrates was evaluated by microscale thermophoresis (MST). Furthermore, they increased the ChT-L activity of the proteasome in HEK293T cell extracts. Our studies indicate that the 20S proteasome-mediated protein substrates hydrolysis may be selectively increased by peptide-based stimulators acting in an allosteric manner. These compounds, after further optimization, may have the potential to counteract proteasome impairment in patients suffering from age-related diseases.

The plasticity of nerve fibers: the prolonged effects of polarization of afferent fibers.

The review surveys various aspects of the plasticity of nerve fibers, in particular the prolonged increase in their excitability evoked by polarization, focusing on a long-lasting increase in the excitability of myelinated afferent fibers traversing the dorsal columns of the spinal cord. We review the evidence that increased axonal excitability 1) follows epidurally applied direct current (DC) as well as relatively short (5 or 10 ms) current pulses and synaptically evoked intrinsic field potentials; 2) critically depends on the polarization of branching regions of afferent fibers at the sites where they bifurcate and give off axon collaterals entering the spinal gray matter in conjunction with actions of extrasynaptic GABAA membrane receptors; and 3) shares the feature of being activity-independent with the short-lasting effects of polarization of peripheral nerve fibers. A comparison between the polarization evoked sustained increase in the excitability of dorsal column fibers and spinal motoneurons (plateau potentials) indicates the possibility that they are mediated by partly similar membrane channels (including noninactivating type L Cav++ 1.3 but not Na+ channels) and partly different mechanisms. We finally consider under which conditions transspinally applied DC (tsDCS) might reproduce the effects of epidural polarization on dorsal column fibers and the possible advantages of increased excitability of afferent fibers for the rehabilitation of motor and sensory functions after spinal cord injuries.NEW & NOTEWORTHY This review supplements previous reviews of properties of nerve fibers by surveying recent experimental evidence for their long-term plasticity. It also extends recent descriptions of spinal effects of DC by reviewing effects of polarization of afferent nerve fibers within the dorsal columns, the mechanisms most likely underlying the long-lasting increase in their excitability and possible clinical implications.

Proteasome Composition and Activity Changes in Cultured Fibroblasts Derived From Mucopolysaccharidoses Patients and Their Modulation by Genistein.

In this study, we have asked whether proteasome composition and function are affected in cells derived from patients suffering from all types of mucopolysaccharidosis (MPS), an inherited metabolic disease caused by accumulation of undegraded glycosaminoglycans (GAGs). Moreover, we have tested if genistein, a small molecule proposed previously as a potential therapeutic agent in MPS, can modulate proteasomes, which might shed a new light on the molecular mechanisms of action of this isoflavone as a potential drug for macromolecule storage diseases. Significant changes in expression of various proteasome-linked genes have been detected during transcriptomic (RNA-seq) analyses in vast majority of MPS types. These results were corroborated by demonstration of increased proteasomal activities in MPS cells. However, GAGs were not able to stimulate the 26S proteasome in vitro, suggesting that the observed activation in cells is indirect rather than arising from direct GAG-proteasome interactions. Genistein significantly reduced proteasomal activities in fibroblasts derived from patients suffering from all MPS types, while its effects on in vitro 26S proteasome activity were negligible. Unexpectedly, levels of many proteasomal subunits were increased in genistein-treated MPS cells. On the other hand, this ostensible discrepancy between results of experiments designed for estimation of effects of genistein on proteasome activities and abundance of proteasomal subunits can be explained by demonstration that in the presence of this isoflavone, levels of ubiquitinated proteins were decreased. The genistein-mediated reduction of proteasomal activities might have beneficial effects in cells of MPS patients due to potential increasing of residual activities of defective lysosomal enzymes which would otherwise be subjected to efficient ubiquitination and proteasomal degradation as misfolded proteins. These results indicate another activity of genistein (apart from previously demonstrated reduction of GAG synthesis efficiency, stimulation of lysosomal biogenesis, and activation of the autophagy process) which can be beneficial in the use of this small molecule in treatment of MPS.

Laser induced damage in coatings for cryogenic Yb:YAG active mirror amplifiers.

We report results of a study of the laser induced damage threshold (LIDT) behavior of ion beam sputtered HfO2/SiO2 multilayer coatings on Yb:YAG using 1-on-1 and N-on-1 test protocols. The tests were conducted at ambient, vacuum, and cryogenic conditions using 280 ps pulses at λ=1030nm. The 1-on-1 LIDT of antireflection (AR) stacks is found to be only slightly reduced under vacuum and cryogenic conditions, while that of high reflectivity (HR) stacks is insensitive to environmental conditions within the uncertainty of the measurements. Cryogenic N-on-1 tests show the LIDT of the HR coating is almost the same as in the 1-on-1 tests. Conversely, the cryogenic N-on-1 test of the AR coating shows damage at ∼13J/cm2, a fluence lower than the 20.4J/cm2 of 1-on-1 tests. The AR damage behavior is found to be affected by imperfections at the Yb:YAG surface. These findings show that high surface quality is required to increase energy extraction from active mirror laser amplifiers.

Branching points of primary afferent fibers are vital for the modulation of fiber excitability by epidural DC polarization and by GABA in the rat spinal cord.

The aim of the study was to examine whether the sustained increases in the excitability of afferent fibers traversing the dorsal columns evoked by their polarization depend on the branching points of these fibers. To this end, the effects of epidural polarization were compared in four spinal regions in deeply anesthetized rats; two with the densest collateralization of muscle afferent fibers (above motor nuclei and Clarke's column) and two where the collateralization is more sparse (rostral and caudal to motor nuclei, respectively. The degree of collateralization in different segments was reconstructed in retrogradely labeled afferent fibers in the rat. Nerve volleys evoked in peripheral nerves by electrical stimulation of the dorsal columns within these regions were used as a measure of the excitability of the stimulated fibers. Potent increases in the excitability were evoked by polarization above motor nuclei and Clarke's column, both during constant direct current (DC) polarization (1 µA for 1 min) and for at least 30 min following DC polarization. Smaller excitability increases occurred during the polarization within other regions and were thereafter either absent or rapidly declined after its termination. The postpolarization increases in excitability were counteracted by the GABAA receptor antagonist bicuculline and the α5GABAA extrasynaptic receptor antagonist L655708 and enhanced by the GABAA receptor agonist muscimol and by ionophoretically applied GABA. As extrasynaptic α5GABAA receptors have been found close to Na channels within branching points, these results are consistent with the involvement of branching points in the induction of the sustained postpolarization increases in fiber excitability.NEW & NOTEWORTHY Polarization of sensory fibers traversing dorsal columns of the spinal cord may considerably increase the excitability of these fibers. We show that this involves the effects of current at branching points of afferent fibers and depends on extrasynaptic effects of GABA. These results contribute to our understanding of the mechanism underlying plasticity of activation of nerve fibers and may be used to increase the effectiveness of epidural stimulation in humans and recovery of spinal functions.

New Peptide-Based Pharmacophore Activates 20S Proteasome.

The proteasome is a pivotal element of controlled proteolysis, responsible for the catabolic arm of proteostasis. By inducing apoptosis, small molecule inhibitors of proteasome peptidolytic activities are successfully utilized in treatment of blood cancers. However, the clinical potential of proteasome activation remains relatively unexplored. In this work, we introduce short TAT peptides derived from HIV-1 Tat protein and modified with synthetic turn-stabilizing residues as proteasome agonists. Molecular docking and biochemical studies point to the α1/α2 pocket of the core proteasome α ring as the binding site of TAT peptides. We postulate that the TATs' pharmacophore consists of an N-terminal basic pocket-docking "activation anchor" connected via a ß turn inducer to a C-terminal "specificity clamp" that binds on the proteasome α surface. By allosteric effects-including destabilization of the proteasomal gate-the compounds substantially augment activity of the core proteasome in vitro. Significantly, this activation is preserved in the lysates of cultured cells treated with the compounds. We propose that the proteasome-stimulating TAT pharmacophore provides an attractive lead for future clinical use.

Pro-apoptotic action of protein-carbohydrate fraction isolated from coelomic fluid of the earthworm Dendrobaena veneta against human colon adenocarcinoma cells.

Earthworm coelomic fluid (CF) is known as a rich source of various bioactive compounds with promising anticancer features. However, it has been demonstrated that CF affects functionality of both, cancer and normal cells. This non-selective activity causes a major problem for medical application of CF. In this study, we present the anticancer activity of the active protein-carbohydrate fraction (AF) isolated from thermally treated CF of earthworm Dendrobaena veneta. The in vitro effect of the AF was examined in human colon model including normal human colon epithelium (CCD 841 CoTr) and human colon adenocarcinoma (HT-29 and LS180) cell lines. We investigated the impact of AF on cell viability neutral red and lactate dehydrogenase assays, morphology May-Grünwald-Giemsa staining assay proliferation MTT tetrazolium salt and BrdU incorporation assays as well as cell cycle progression propidium iodide/RNase staining and the activity of human 20S proteasome the hydrolysis of AMC from a Suc-LLVY-AMC peptide substrate. Additionally, the influence of AF on apoptosis was examined in HT-29 cells by Annexin V/PI, Hoechst 33342 staining and active caspase-3 assays. Our investigation demonstrated that AF at the tested concentration range does not affect the viability and morphology of CCD 841 CoTr cells. Simultaneously, AF inhibits human 20S proteasome activity as well as significantly decreases mitochondrial metabolism, disturbs cell cycle and induces apoptosis via activation of procaspase-3 in HT-29 cancer cells. Obtained results demonstrate the antiproliferative and proapoptotic activity of AF that can be useful in developing therapeutic strategies to treat human colon cancer.

Influence of short peptides with aromatic amino acid residues on aggregation properties of serum amyloid A and its fragments.

Serum amyloid A variant 1.1 (SAA1.1) is an acute phase protein. In response to injury, inflammation or infection its production increases highly, which may lead to aggregation of the protein and accumulation of its deposits in various organs. Due to the cellular toxicity of the aggregates, as well as the fact that accumulated deposits are a burden that obstructs proper functioning of the affected tissues, it is vital to find a way to suppress the process of pathological aggregates formation. To make this possible, it is necessary to investigate thoroughly the oligomerization process and recognize factors that may influence its course. Some previous studies showed that aromatic interactions are important to the potential of an inhibitor to suppress the aggregation process. In our research we had proved that a five-residue peptide RSFFS (saa1-5) is an efficient inhibitor of aggregation of the most amyloidogenic fragment of SAA1.1, SAA1-12. In the present work the oligomerization and aggregation propensity of SAA1-12 was compared to that of SAA1-27, in order to determine the contribution of the sequence which extends beyond the most amyloidogenic region but encompasses residues reportedly involved in the stabilization of the SAA native conformation. Thioflavin T fluorescence assay, quantitative chromatographic analysis of the insoluble fraction and transmission electron microscopy allowed for a deeper insight into the SAA aggregation process and the morphology of aggregates. Substitutions of Phe3 and/or Phe4 residues in saa1-5 sequence with tryptophan, tyrosine, homophenylalanine, naphthylalanine and ß,ß-diphenylalanine allowed to study the influence of different aromatic systems on the aggregation of SAA1-12 and SAA1-27, and evaluate these results in relation to hSAA1.1 protein. Our results indicate that compounds with aromatic moieties can affect the course of the aggregation process and change the ratio between the soluble and insoluble aggregates.

Ephaptic interactions between myelinated nerve fibres of rodent peripheral nerves.

We report evidence that ephaptic interactions may occur between intact mammalian myelinated nerve fibres and not only between demyelinated or damaged mammalian nerve fibres or nerve cells as analysed in previous studies. The ephaptic interactions were investigated between nerve fibres traversing the lumbar dorsal roots and between bundles of fibres in the sciatic nerve in anaesthetized rats in vivo. The interactions were estimated by comparing the excitability of nerve fibres originating from one of the hindlimb nerves (peroneal or sural) under control conditions and when the stimulation of these fibres was combined with stimulation of another nerve (tibial). An increase in nerve volleys recorded from group I muscle afferents in the peroneal nerve and of the fastest skin afferents in the sural nerve was used as a measure of the increase in the excitability. The excitability of these fibres was increased during a fraction of a millisecond, coinciding with the period of passage of nerve impulses evoked by the conditioning stimulation of the tibial nerve. The degree of the increase was comparable to the increases in the excitability evoked by 1-2 min lasting fibre polarization. Ephaptic interactions were found to be more potent and with longer lasting after-effects within the dorsal roots than within the sciatic nerve. We postulate that ephaptic interactions may result in the synchronization of information forwarded via neighbouring afferent nerve fibres prior to their entry into the spinal cord and thereby securing the propagation of nerve impulses across branching points within the spinal grey matter.

Interactions Between Baclofen and DC-induced Plasticity of Afferent Fibers within the Spinal Cord.

The aims of the study were to compare effects of baclofen, a GABAB receptor agonist commonly used as an antispastic drug, on direct current (DC) evoked long-lasting changes in the excitability of afferent fibers traversing the dorsal columns and their terminal branches in the spinal cord, and to examine whether baclofen interferes with the development and expression of these changes. The experiments were performed on deeply anesthetized rats by analyzing the effects of DC before, during and following baclofen administration. Muscle and skin afferent fibers within the dorsal columns were stimulated epidurally and changes in their excitability were investigated following epidural polarization by 1.0-1.1 µA subsequent to i.v. administration of baclofen. Epidural polarization increased the excitability of these fibers during post-polarization periods of at least 1 h. The facilitation was as potent as in preparations that were not pretreated with baclofen, indicating that the advantages of combining epidural polarization with epidural stimulation would not be endangered by pharmacological antispastic treatment with baclofen. In contrast, baclofen-reduced effects of intraspinal stimulation combined with intraspinal polarization (0.3 µA) of terminal axonal branches of the afferents within the dorsal horn or in motor nuclei, whether administered ionophoretically or intravenously. Effects of DC on monosynaptically evoked synaptic actions of these fibers (extracellular field potentials) were likewise reduced by baclofen. The study thus provides further evidence for differential effects of DC on afferent fibers in the dorsal columns and the preterminal branches of these fibers and their involvement in spinal plasticity.

RRMdb-an evolutionary-oriented database of RNA recognition motif sequences.

RNA-recognition motif (RRM) is an RNA-interacting protein domain that plays an important role in the processes of RNA metabolism such as the splicing, editing, export, degradation, and regulation of translation. Here, we present the RNA-recognition motif database (RRMdb), which affords rapid identification and annotation of RRM domains in a given protein sequence. The RRMdb database is compiled from ~57 000 collected representative RRM domain sequences, classified into 415 families. Whenever possible, the families are associated with the available literature and structural data. Moreover, the RRM families are organized into a network of sequence similarities that allows for the assessment of the evolutionary relationships between them.

Proline- and Arginine-Rich Peptides as Flexible Allosteric Modulators of Human Proteasome Activity.

Proline- and arginine-rich peptide PR11 is an allosteric inhibitor of 20S proteasome. We modified its sequence inter alia by introducing HbYX, RYX, or RHbX C-terminal extensions (Hb, hydrophobic moiety; R, arginine; Y, tyrosine; X, any residue). Consequently, we were able to improve inhibitory potency or to convert inhibitors into strong activators: the former with an aromatic penultimate Hb residue and the latter with the HbYX motif. The PR peptide activator stimulated 20S proteasome in vitro to efficiently degrade protein substrates, such as α-synuclein and enolase, but also activated proteasome in cultured fibroblasts. The positive and negative PR modulators differently influenced the proteasome conformational dynamics and affected opening of the substrate entry pore. The resolved crystal structure showed PR inhibitor bound far from the active sites, at the proteasome outer face, in the pocket used by natural activators. Our studies indicate the opportunity to tune proteasome activity by allosteric regulators based on PR peptide scaffold.

DC-Evoked Modulation of Excitability of Myelinated Nerve Fibers and Their Terminal Branches; Differences in Sustained Effects of DC.

Direct current (DC) evokes long-lasting changes in neuronal networks both presynaptically and postsynaptically and different mechanisms were proposed to be involved in them. Different mechanisms were also suggested to account for the different dynamics of presynaptic DC actions on myelinated nerve fibers stimulated before they entered the spinal gray matter and on their terminal branches. The aim of the present study was to examine whether these different dynamics might be related to differences in the involvement of K+ channels. To this end, we compared effects of the K+ channel blocker 4-amino-pyridine (4-AP) on DC-evoked changes in the excitability of afferent fibers stimulated within the dorsal columns (epidurally) and within their projection areas in the dorsal horn and motor nuclei (intraspinally). 4-AP was applied systemically in deeply anesthetized rats. DC-evoked increases in the excitability of epidurally stimulated afferent nerve fibers, and increases in field potentials evoked by these fibers, were not affected by 4-AP. In contrast, sustained decreases rather than increases in the excitability of intraspinally stimulated terminal nerve branches were evoked by local application of DC in conjunction with 4-AP. The study leads to the conclusion that 4-AP-sensitive K+ channels contribute to the sustained DC-evoked post-polarization increases in the excitability at the level of terminal branches of nerve fibers but not of the nodes of Ranvier nor within the juxta-paranodal regions where other mechanisms would be involved in inducing the sustained DC-evoked changes.