Ubiquitin Carboxyl-Terminal Hydrolase L1 and Its Role in Parkinson's Disease.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), also known as Parkinson's disease protein 5, is a highly expressed protein in the brain. It plays an important role in the ubiquitin-proteasome system (UPS), where it acts as a deubiquitinase (DUB) enzyme. Being the smallest member of the UCH family of DUBs, it catalyzes the reaction of ubiquitin precursor processing and the cleavage of ubiquitinated protein remnants, thus maintaining the level of ubiquitin monomers in the brain cells. UCHL1 mutants, containing amino acid substitutions, influence catalytic activity and its aggregability. Some of them protect cells and transgenic mice in toxin-induced Parkinson's disease (PD) models. Studies of putative protein partners of UCHL1 revealed about sixty individual proteins located in all major compartments of the cell: nucleus, cytoplasm, endoplasmic reticulum, plasma membrane, mitochondria, and peroxisomes. These include proteins related to the development of PD, such as alpha-synuclein, amyloid-beta precursor protein, ubiquitin-protein ligase parkin, and heat shock proteins. In the context of the catalytic paradigm, the importance of these interactions is not clear. However, there is increasing understanding that UCHL1 exhibits various effects in a catalytically independent manner through protein-protein interactions. Since this protein represents up to 5% of the soluble protein in the brain, PD-related changes in its structure will have profound effects on the proteomes/interactomes in which it is involved. Growing evidence is accumulating that the role of UCHL1 in PD is obviously determined by a balance of canonic catalytic activity and numerous activity-independent protein-protein interactions, which still need better characterization.

Proteasome Interactome and Its Role in the Mechanisms of Brain Plasticity.

Proteasomes are highly conserved multienzyme complexes responsible for proteolytic degradation of the short-lived, regulatory, misfolded, and damaged proteins. They play an important role in the processes of brain plasticity, and decrease in their function is accompanied by the development of neurodegenerative pathology. Studies performed in different laboratories both on cultured mammalian and human cells and on preparations of the rat and rabbit brain cortex revealed a large number of proteasome-associated proteins. Since the identified proteins belong to certain metabolic pathways, multiple enrichment of the proteasome fraction with these proteins indicates their important role in proteasome functioning. Extrapolation of the experimental data, obtained on various biological objects, to the human brain suggests that the proteasome-associated proteins account for at least 28% of the human brain proteome. The proteasome interactome of the brain contains a large number of proteins involved in the assembly of these supramolecular complexes, regulation of their functioning, and intracellular localization, which could be changed under different conditions (for example, during oxidative stress) or in different phases of the cell cycle. In the context of molecular functions of the Gene Ontology (GO) Pathways, the proteins of the proteasome interactome mediate cross-talk between components of more than 30 metabolic pathways annotated in terms of GO. The main result of these interactions is binding of adenine and guanine nucleotides, crucial for realization of the nucleotide-dependent functions of the 26S and 20S proteasomes. Since the development of neurodegenerative pathology is often associated with regioselective decrease in the functional activity of proteasomes, a positive therapeutic effect would be obviously provided by the factors increasing proteasomal activity. In any case, pharmacological regulation of the brain proteasomes seems to be realized through the changes in composition and/or activity of the proteins associated with proteasomes (deubiquitinase, PKA, CaMKIIα, etc.).

Proteomic Profiling of Mouse Brain Pyruvate Kinase Binding Proteins: A Hint for Moonlighting Functions of PKM1?

Affinity-based proteomic profiling is widely used for the identification of proteins involved in the formation of various interactomes. Since protein-protein interactions (PPIs) reflect the role of particular proteins in the cell, identification of interaction partners for a protein of interest can reveal its function. The latter is especially important for the characterization of multifunctional proteins, which can play different roles in the cell. Pyruvate kinase (PK), a classical glycolytic enzyme catalyzing the last step of glycolysis, exists in four isoforms: PKM1, PKM2, PKL, and PKR. The enzyme isoform expressed in actively dividing cells, PKM2, exhibits many moonlighting (noncanonical) functions. In contrast to PKM2, PKM1, predominantly expressed in adult differentiated tissues, lacks well-documented moonlighting functions. However, certain evidence exists that it can also perform some functions unrelated to glycolysis. In order to evaluate protein partners, bound to PKM1, in this study we have combined affinity-based separation of mouse brain proteins with mass spectrometry identification. The highly purified PKM1 and a 32-mer synthetic peptide (PK peptide), sharing high sequence homology with the interface contact region of all PK isoforms, were used as the affinity ligands. This proteomic profiling resulted in the identification of specific and common proteins bound to both affinity ligands. Quantitative affinity binding to the affinity ligands of selected identified proteins was validated using a surface plasmon resonance (SPR) biosensor. Bioinformatic analysis has shown that the identified proteins, bound to both full-length PKM1 and the PK peptide, form a protein network (interactome). Some of these interactions are relevant for the moonlighting functions of PKM1. The proteomic dataset is available via ProteomeXchange with the identifier PXD041321.

Atypical Ubiquitination and Parkinson's Disease.

Ubiquitination (the covalent attachment of ubiquitin molecules to target proteins) is one of the main post-translational modifications of proteins. Historically, the type of polyubiquitination, which involves K48 lysine residues of the monomeric ubiquitin, was the first studied type of ubiquitination. It usually targets proteins for their subsequent proteasomal degradation. All the other types of ubiquitination, including monoubiquitination; multi-monoubiquitination; and polyubiquitination involving lysine residues K6, K11, K27, K29, K33, and K63 and N-terminal methionine, were defined as atypical ubiquitination (AU). Good evidence now exists that AUs, participating in the regulation of various cellular processes, are crucial for the development of Parkinson's disease (PD). These AUs target various proteins involved in PD pathogenesis. The K6-, K27-, K29-, and K33-linked polyubiquitination of alpha-synuclein, the main component of Lewy bodies, and DJ-1 (another PD-associated protein) is involved in the formation of insoluble aggregates. Multifunctional protein kinase LRRK2 essential for PD is subjected to K63- and K27-linked ubiquitination. Mitophagy mediated by the ubiquitin ligase parkin is accompanied by K63-linked autoubiquitination of parkin itself and monoubiquitination and polyubiquitination of mitochondrial proteins with the formation of both classical K48-linked ubiquitin chains and atypical K6-, K11-, K27-, and K63-linked polyubiquitin chains. The ubiquitin-specific proteases USP30, USP33, USP8, and USP15, removing predominantly K6-, K11-, and K63-linked ubiquitin conjugates, antagonize parkin-mediated mitophagy.

DJ-1 Protein and Its Role in the Development of Parkinson's Disease: Studies on Experimental Models.

DJ-1, also known as Parkinson's disease protein 7, is a multifunctional protein ubiquitously expressed in cells and tissues. Interacting with proteins of various intracellular compartments, DJ-1 plays an important role in maintaining different cellular functions. Mutant DJ-1 forms containing amino acid substitutions (especially L166P), typical of Parkinson's disease, are characterized by impaired dimerization, stability, and folding. DJ-1 exhibits several types of catalytic activity; however, in the enzyme classification it exists as protein deglycase (EC 3.5.1.124). Apparently, in different cell compartments DJ-1 exhibits catalytic and non-catalytic functions, and their ratio still remains unknown. Oxidative stress promotes dissociation of cytoplasmic DJ-1 dimers into monomers, which are translocated to the nucleus, where this protein acts as a coactivator of various signaling pathways, preventing cell death. In mitochondria, DJ-1 is found in the synthasome, where it interacts with the ß ATP synthase subunit. Downregulation of the DJ-1 gene under conditions of experimental PD increases sensitivity of the cells to neurotoxins, and introduction of the recombinant DJ-1 protein attenuates manifestation of this pathology. The thirteen-membered fragment of the DJ-1 amino acid sequence attached to the heptapeptide of the TAT protein penetrating into the cells exhibited neuroprotective properties in various PD models both in cell cultures and after administration to animals. Low molecular weight DJ-1 ligands also demonstrate therapeutic potential, providing neuroprotective effects seen during their incubation with cells and administration to animals.

A Neuroprotective Dose of Isatin Causes Multilevel Changes Involving the Brain Proteome: Prospects for Further Research.

Isatin (indole-2,3-dione) is an endogenous regulator, exhibiting a wide range of biological and pharmacological activities. At doses of 100 mg/kg and above, isatin is neuroprotective in different experimental models of neurodegeneration. Good evidence exists that its effects are realized via interaction with numerous isatin-binding proteins identified in the brain and peripheral tissues studied. In this study, we investigated the effect of a single dose administration of isatin to mice (100 mg/kg, 24 h) on differentially expressed proteins and a profile of the isatin-binding proteins in brain hemispheres. Isatin administration to mice caused downregulation of 31 proteins. However, these changes cannot be attributed to altered expression of corresponding genes. Although at this time point isatin influenced the expression of more than 850 genes in brain hemispheres (including 433 upregulated and 418 downregulated genes), none of them could account for the changes in the differentially expressed proteins. Comparative proteomic analysis of brain isatin-binding proteins of control and isatin-treated mice revealed representative groups of proteins sensitive to isatin administration. Control-specific proteins (n = 55) represent specific targets that interact directly with isatin. Appearance of brain isatin-binding proteins specific to isatin-treated mice (n = 94) may be attributed to the formation of new clusters of protein-protein interactions and/or novel binding sites induced by a high concentration of this regulator (ligand-induced binding sites). Thus, isatin administration produces multiple effects in the brain, which include changes in gene expression and also profiles of isatin-binding proteins and their interactomes. Further studies are needed for deeper insight into the mechanisms of the multilevel changes in the brain proteome induced by isatin. In the context of the neuroprotective action, these changes may be aimed at interruption of pathological links that begin to form after initiation of pathological processes.

Renalase Secreted by Human Kidney HEK293T Cells Lacks its N-Terminal Peptide: Implications for Putative Mechanisms of Renalase Action.

BACKGROUND/AIMS: Renalase is a recently discovered flavoprotein involved in regulation of blood pressure. Altered renalase levels have been found in blood of patients with end stage renal disease. The antihypertensive effect of circulating renalase is attributed to putative FAD-dependent monoamine oxidase activity demonstrated by some authors. Being synthesized as an intracellular flavoprotein renalase requires the presence of its N-terminal peptide for FAD accommodation. However, conventional routes of export of secretory proteins outside the cell usually include cleavage of their N-terminal peptide. The aim of this study was to investigate whether renalase is secreted by НЕK293T cells as a full length protein (via proposed nonconventional pathway) or its export is accompanied by the loss of its N-terminal peptide. METHODS: We have expressed human recombinant renalase-1 in human kidney НЕK293T cells and analyzed this protein inside the cells and in the extracellular medium for the presence of the N-terminal peptide by using high resolution targeted MS/MS. RESULTS: Intracellular renalase contained clearly detectable N-terminal peptide, which was absent in extracellular renalase. CONCLUSIONS: Lack of the N-terminal peptide, the structural precondition for FAD binding, suggests that extracellular (circulating) renalase acts in a FAD-independent manner and mechanisms of its action are not associated with FAD.

Protein interactomics based on direct molecular fishing on paramagnetic particles: practical realization and further SPR validation.

There is increasing evidence that proteins function in the cell as integrated stable or temporally formed protein complexes, interactomes. Previously, using model systems we demonstrated applicability of direct molecular fishing on paramagnetic particles for protein interactomics (Ershov et al. Proteomics, 2012, 12, 3295). In the present study, we have used a combination of affinity-based molecular fishing and subsequent MS for investigation of human liver proteins involved in interactions with immobilized microsomal cytochrome b5 (CYB5A), and also transthyretin and BSA as alternative affinity ligands (baits). The LC-MS/MS identification of prey proteins fished on these baits revealed three sets of proteins: 98, 120, and 220, respectively. Comparison analysis of these sets revealed only three proteins common for all the baits. In the case of paired analysis, the number of common proteins varied from 2 to 9. The binding capacity of some identified proteins has been validated by a SPR-based biosensor. All the investigated proteins effectively interacted with the immobilized CYB5A (Kd values ranged from 0.07 to 1.1 µM). Results of this study suggest that direct molecular fishing is applicable for analysis of protein-protein interactions (PPI) under normal and pathological conditions, in which altered PPIs are especially important.

The effects of endogenous non-peptide molecule isatin and hydrogen peroxide on proteomic profiling of rat brain amyloid-ß binding proteins: relevance to Alzheimer's disease?

The amyloid-ß peptide is considered as a key player in the development and progression of Alzheimer's disease (AD). Although good evidence exists that amyloid-ß accumulates inside cells, intracellular brain amyloid-binding proteins remain poorly characterized. Proteomic profiling of rat brain homogenates, performed in this study, resulted in identification of 89 individual intracellular amyloid-binding proteins, and approximately 25% of them were proteins that we had previously identified as specifically binding to isatin, an endogenous neuroprotector molecule. A significant proportion of the amyloid-binding proteins (more than 30%) are differentially expressed or altered/oxidatively modified in AD patients. Incubation of brain homogenates with 70 µM hydrogen peroxide significantly influenced the profile of amyloid-ß binding proteins and 0.1 mM isatin decreased the number of identified amyloid-ß binding proteins both in control and hydrogen peroxide treated brain homogenates. The effects of hydrogen peroxide and isatin have been confirmed in optical biosensor experiments with purified glyceraldehyde-3-phosphate dehydrogenase, one of the known crucial amyloid-ß binding proteins (also identified in this study). Data obtained suggest that isatin protects crucial intracellular protein targets against amyloid binding, and possibly favors intracellular degradation of this protein via preventing formation of amyloid-ß oligomers described in the literature for some isatin derivatives.

Construction of the coding sequence of the transcription variant 2 of the human Renalase gene and its expression in the prokaryotic system.

Renalase is a recently discovered protein, involved in regulation of blood pressure in humans and animals. Although several splice variants of human renalase mRNA transcripts have been recognized, only one protein product, hRenalase1, has been found so far. In this study, we have used polymerase chain reaction (PCR)-based amplification of individual exons of the renalase gene and their joining for construction of full-length hRenalase2 coding sequence followed by expression of hRenalase2 as a polyHis recombinant protein in Escherichia coli cells. To date this is the first report on synthesis and purification of hRenalase2. Applicability of this approach was verified by constructing hRenalase1 coding sequence, its sequencing and expression in E. coli cells. hRenalase1 was used for generation of polyclonal antiserum in sheep. Western blot analysis has shown that polyclonal anti-renalase1 antibodies effectively interact with the hRenalase2 protein. The latter suggests that some functions and expression patterns of hRenalase1 documented by antibody-based data may be attributed to the presence of hRenalase2. The realized approach may be also used for construction of coding sequences of various (especially weakly expressible) genes, their transcript variants, etc.

Affinity-based proteomic profiling: problems and achievements.

Affinity chromatography becomes a more and more popular method used in proteomic studies for separation of various groups of proteins (subproteomes). The review highlights the role of affinity chromatography fractionation for proteomic profiling of the most of intensively studied groups of proteins including cyclic nucleotide-binding proteins, protein kinases (kinomes), phosphoproteins, glycoproteins, ubiquitinated proteins. Special attention is paid to the use of affinity chromatography for the characterization of small-molecule protein targets. The latter is especially important for the elucidation of direct protein targets of potential drug substances for evaluation of their possible side-effects or additional pharmacological application.

Use of biotinylated ubiquitin for analysis of rat brain mitochondrial proteome and interactome.

Applicability of in vitro biotinylated ubiquitin for evaluation of endogenous ubiquitin conjugation and analysis of ubiquitin-associated protein-protein interactions has been investigated. Incubation of rat brain mitochondria with biotinylated ubiquitin followed by affinity chromatography on avidin-agarose, intensive washing, tryptic digestion of proteins bound to the affinity sorbent and their mass spectrometry analysis resulted in reliable identification of 50 proteins belonging to mitochondrial and extramitochondrial compartments. Since all these proteins were bound to avidin-agarose only after preincubation of the mitochondrial fraction with biotinylated ubiquitin, they could therefore be referred to as specifically bound proteins. A search for specific ubiquitination signature masses revealed several extramitochondrial and intramitochondrial ubiquitinated proteins representing about 20% of total number of proteins bound to avidin-agarose. The interactome analysis suggests that the identified non-ubiquitinated proteins obviously form tight complexes either with ubiquitinated proteins or with their partners and/or mitochondrial membrane components. Results of the present study demonstrate that the use of biotinylated ubiquitin may be considered as the method of choice for in vitro evaluation of endogenous ubiquitin-conjugating machinery in particular subcellular organelles and changes in ubiquitin/organelle associated interactomes. This may be useful for evaluation of changes in interactomes induced by protein ubiquitination under norm and various brain pathologies.

Tryptophan Metabolites as Mediators of Microbiota-Gut-Brain Communication: Focus on Isatin.

Isatin (indole-2,3-dione) is an endogenous regulator, exhibiting various behavioral, biological, and pharmacological activities. Synthesis of isatin includes several crucial stages: cleavage of the tryptophan side chain and subsequent oxidation of the indole nucleus. Although these stages require concerted action of bacterial and host enzymes, there are two pathways of isatin formation: the host and bacterial pathways. Isatin acts as a neuroprotector in different experimental models of neurodegeneration. Its effects are realized via up- and downregulation of isatin-responsive genes and via interaction with numerous isatin-binding proteins identified in the brain. The effect of isatin on protein-protein interactions in the brain may be important for realization of weak inhibition of multiple receptor targets.

Disulfiram Oxy-Derivatives Suppress Protein Retrotranslocation across the ER Membrane to the Cytosol and Initiate Paraptosis-like Cell Death.

Disulfiram (DSF) and its derivatives were here investigated as antineoplastic agents, and their important feature is the ability to influence the UPS. We have recently shown that hydroxocobalamin catalyzes the aerobic oxidation of diethyldithiocarbamate to form disulfiram and its oxy-derivatives (DSFoxy; i.e., sulfones and sulfoxides), which induce cytoplasm vacuolization and paraptosis-like cancer cell death. We used LC-MS/MS and bioinformatics analysis to determine the key points in these processes. DSFoxy was found to induce an increase in the number of ubiquitinated proteins, including oxidized ones, and a decrease in the monomeric ubiquitin. Enhanced ubiquitination was revealed for proteins involved in the response to exogenous stress, regulation of apoptosis, autophagy, DNA damage/repair, transcription and translation, folding and ubiquitination, retrograde transport, the MAPK cascade, and some other functions. The results obtained indicate that DSF oxy-derivatives enhance the oxidation and ubiquitination of many proteins regulating proteostasis (including E3 ligases and deubiquitinases), which leads to inhibition of protein retrotranslocation across the ER membrane into the cytosol and accumulation of misfolded proteins in the ER followed by ER swelling and initiates paraptosis-like cell death. Our results provide new insight into the role of protein ubiquitination/deubiquitination in regulating protein retrotranslocation across the ER membrane into the cytosol and paraptosis-like cell death.

20S and 26S proteasome-binding proteins of the rabbit brain: A proteomic dataset.

Fractions of 26S and 20S proteasomes isolated from the rabbit brain by the method of salt fractionation (salt-induced precipitation) contain intrinsic proteasome proteins responsible for assembly of the core particle and regulatory particle of proteasome and also proteasome-binding proteins. These proteasome-binding proteins include components of the ubiquitin-proteasome system, some ubiquitinated proteins, as well as cytoskeleton components, protective proteins, regulators of gene expression, cell division, and differentiation, and multifunctional proteins (mainly, glycolytic enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase, etc.). The multifunctional proteins also known as "moonlighting proteins" are involved in various (regulatory) processes in the cell and obviously represent important components of the proteasome interactome rather than contaminants of the 26S and 20S proteasome fractions.

Isatin-binding proteins of rat and mouse brain: proteomic identification and optical biosensor validation.

Isatin (indole-2,3-dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Isatin itself and its analogues exhibit a wide range of pharmacological activities but its specific biological targets still are not well characterized. Affinity chromatography of Triton X-100 lysates of soluble and particulate fractions of mouse and rat whole brain homogenates on 5-aminocaproyl-isatin-Sepharose followed by subsequent proteomic analysis resulted in identification of 65 and 64 individual proteins, respectively. Isatin-binding capacity of some of the identified proteins has been validated in an optical biosensor study using a Biacore 3000 optical biosensor, 5-aminocarproyl-isatin, and 5-aminoisatin as the affinity ligands. The K(d) values (of 0.1-20 microM) obtained during the optical biosensor experiments were consistent with the range of K(d) values recently reported for [(3)H]isatin binding to brain sections. Although the number of isatin-binding proteins identified in the mouse and rat brain was similar, only 21 proteins (about one-third) were identical in the two species. This may be one reason for the differences in isatin effects in rats and mice reported in the literature.

Mitochondrial Dysfunction in Parkinson's Disease: Focus on Mitochondrial DNA.

Mitochondria, the energy stations of the cell, are the only extranuclear organelles, containing their own (mitochondrial) DNA (mtDNA) and the protein synthesizing machinery. The location of mtDNA in close proximity to the oxidative phosphorylation system of the inner mitochondrial membrane, the main source of reactive oxygen species (ROS), is an important factor responsible for its much higher mutation rate than nuclear DNA. Being more vulnerable to damage than nuclear DNA, mtDNA accumulates mutations, crucial for the development of mitochondrial dysfunction playing a key role in the pathogenesis of various diseases. Good evidence exists that some mtDNA mutations are associated with increased risk of Parkinson's disease (PD), the movement disorder resulted from the degenerative loss of dopaminergic neurons of substantia nigra. Although their direct impact on mitochondrial function/dysfunction needs further investigation, results of various studies performed using cells isolated from PD patients or their mitochondria (cybrids) suggest their functional importance. Studies involving mtDNA mutator mice also demonstrated the importance of mtDNA deletions, which could also originate from abnormalities induced by mutations in nuclear encoded proteins needed for mtDNA replication (e.g., polymerase γ). However, proteomic studies revealed only a few mitochondrial proteins encoded by mtDNA which were downregulated in various PD models. This suggests nuclear suppression of the mitochondrial defects, which obviously involve cross-talk between nuclear and mitochondrial genomes for maintenance of mitochondrial functioning.

Isatin binding proteins in rat brain: in situ imaging, quantitative characterization of specific [3H]isatin binding, and proteomic profiling.

Isatin (indole-2,3-dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Its biological targets remain poorly characterized, although [(3)H]isatin binding sites have been demonstrated in various brain structures. In this study, by using a real-time beta-imager, [(3)H]isatin radioligand binding analysis, and proteomic identification of proteins specifically bound to the affinity sorbent 5-aminocaproyl-isatin-Sepharose, we have investigated the distribution of [(3)H]isatin specific binding sites in the rat brain, characterized their K(d) and B(max), and identified some individual brain isatin binding proteins. The binding of [(3)H]isatin to rat brain sections was saturable and characterized by K(d) values (of 0.2-0.3 microM) consistent with physiological concentrations. The highest B(max) was found in the hypothalamus, consistent with a role in stress. In most brain regions, the homologous inhibition of [(3)H]isatin binding by increasing concentrations of cold isatin demonstrated complex behavior suggesting involvement of various binding proteins characterized by different affinity to isatin. Affinity chromatography of Triton X-100 lysates of whole-brain homogenates on 5-aminocaproyl-isatin-Sepharose followed by subsequent proteomic analysis resulted in identification of 25 individual proteins, including glyceraldehyde-3-phosphate dehydrogenase, one of few previously reported isatin binding proteins, and a group of cytoskeleton-related proteins. These binding sites may be related to the known antiproliferative and proapoptotic activities of isatin.

Monoamine oxidase and semicarbazide sensitive amine oxidase activities in normal and inflamed human dental pulp.

BACKGROUND: Human dental pulp contains monoamine oxidase (MAO) and semicarbazide sensitive amine oxidase (SSAO). In other tissues SSAO is involved in oxidative stress and inflammation, but the role of MAO and SSAO in human pulp and changes of their activities in reversible pulpitis still remains poorly understood. MATERIAL/METHODS: We investigated MAO labeling with mechanism-based inhibitor [3H]pargyline activities of MAO A, MAO B, and SSAO in healthy and inflamed human dental pulp. RESULTS: Incubation of human dental pulp homogenates with [3H]pargyline caused MAO labeling. MAO activity assayed with 100 microM [14C]5HT or 10 microM [14C]PEA was sensitive to selective inhibitors of MAO A and MAO B, respectively. MAO activity with 50 microM [14C]PEA was partially inhibited by clorgyline, and total inhibition was achieved only by the combination of clorgyline and semicarbazide, suggesting the presence of SSAO. Inflammation of the dental pulp was accompanied by a significant decrease in MAO labeling, MAO B (but not MAO A) activity and the increase in SSAO activity. CONCLUSIONS: The results of the present study suggest that the increase of dental pulp SSAO activity contributes to the development of inflammation in the dental pulp. The decrease in MAO B activity and lack of significant changes in MAO A activity may be associated with an anti-inflammatory response - inflamed pulp MAO A still effectively deaminates the inflammatory mediator 5HT, whereas inhibition of MAO B could result in some decrease of hydrogen peroxide generation, essential for the tissue damage in inflammation.

The effect of short-term administration of (-)-deprenyl and isatin on the expressions of some genes in the mouse brain cortex.

BACKGROUND: Isatin (indoledione 2,3) is an endogenous indole found in the mammalian brain, peripheral tissues, and body fluids. It exhibits many neurophysiological and neuropharmacological effects. It shares some common molecular targets with (-)-deprenyl, a neuroprotective pharmacological drug. Some isatin effects imply a possible influence of gene expression; however, no isatin-responsive genes have yet been identified. MATERIAL/METHODS: In this study the effects of a three-week administration of isatin (20 mg/kg) or (-)-deprenyl (1 mg/kg) on the expressions of several putative isatin/deprenyl-responsive genes in the mouse cortex were compared using real-time PCR. RESULTS: Both treatments caused similarly significant decreases in superoxide dismutase (SOD) mRNA. Treatment of mice with either drug decreased glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, although only in the deprenyl-treated mice was this significant (p<0.01). No significant changes were found in cortex mRNA content of monoamine oxidase A or monoamine oxidase B. CONCLUSIONS: The results suggest that isatin and (-)-deprenyl have some common target genes and this supports the idea that isatin may be an endogenous partial functional agonist of (-)-deprenyl. Since GAPDH mRNA expression is sensitive to the pharmacological treatments, these results also question the applicability of GAPDH as a reference gene in gene expression studies.