Pseudo-peptides derived from isomannide as potential inhibitors of serine proteases

Summary. Hepatitis C, Dengue and West Nile virus are among of the most important flaviviruses that share one important serine protease enzyme. Serine proteases belong to the most studied class of proteolytic enzymes, and are a primary target in the drug development field. In this paper, we describe the synthesis and preliminary molecular modeling studies of a novel class of N-t-Boc amino acid amides derived of isomannide as potential serine proteases inhibitors.     

Threonine is the best substrate for <Emphasis Type="SmallCaps">D</Emphasis>-lactate formation in octopus tentacle

Summary. Carbon sources for D-lactate and enzyme activities related to D-lactate formation were investigated using cell-free homogenates of Octopus vulgaris tentacle tissue. The results are as follows: a) The best precursor for D-lactate formation was threonine and second best precursors were glycine and fructose-1,6-bisphosphate. Threonine and glycine served as precursors only in presence of glutathione. b) Both amino acids were precursors for methylglyoxal from which D-lactate was synthesized. Alanine, cysteine and serine were not precursors. We present a metabolic map for D-lactate formation in octopus in order to explain these experimental results.     

Ethynylglycine synthon from Garner’s aldehyde: a useful precursor for the synthesis of non-natural amino acids

Summary. The ethynylglycine synthon, namely (R)-2,2-dimethyl-3-(tert-butoxycarbonyl)-4-ethynyl-oxazolidine, can be obtained through the synthetic elaboration of naturally occurring serine. This compound has been exploited as a helpful and versatile non-racemic building block to be used for the design and synthesis of biologically important compounds, mainly non-natural α-amino acids. Taking advantage of the terminal acetylene moiety several synthetic applications can be designed. Metalation followed by trapping with electrophiles or Cu/Pd catalysed coupling with aromatic halogenides are shown to deliver useful precursors of ethynylglycine derivatives. Additions of bimetallic reagents like stannyl- or silylcuprates are useful entries for the regio- and stereoselective functionalization of the lateral chain, aimed at the synthesis of modified vinylglycine precursors.An overview of our recent work in the field will be given, and the use of ethynylglycine synthon in the synthesis of non-racemic saturated and unsaturated non-natural amino acids will be briefly reviewed.     

Pseudo-peptides derived from isomannide: inhibitors of serine proteases

In this paper, we describe the synthesis of a novel class of pseudo-peptides derived from isomannide and several oxazolones as potential inhibitors of serine proteases as well as preliminary pharmacological assays for hepatitis C. Hepatitis C, dengue and West Nile fever are among the most important flaviviruses that share one important serine protease enzyme. Serine proteases belong to the most studied class of proteolytic enzymes and are a primary target in the drug development field. Several pseudo-peptides were obtained in good yields from the reaction of isomannide and oxazolones, and their anti-HCV potential using the HCV replicon-based assay was shown.     

Intestinal proteolytic profile changes during larval development of Anticarsia gemmatalis caterpillars

Soybean is one of most consumed and produced grains in the world, and Anticarsia gemmatalis is a pest that causes great damage to this crop due to severe defoliation during its larval phase. Plants have mechanisms that lead to the inhibition of proteases in the intestine of these herbivores, hampering their development. Understanding this complex protease inhibitor is important for pest control. The objective of this study was to evaluate the enzymatic profiles of the intestinal proteases of the soybean caterpillar at different instars. For this, the proteolytic profile of the gut in the third, fourth, and fifth instars were analyzed. Irreversible inhibitors of proteases were separately incubated with A. gemmatalis enzyme extracts at the third, fourth, and fifth instar to assess the contribution of these proteases to total proteolytic activity. The enzymatic extracts were also evaluated with specific substrates to confirm changes in the specific activities of trypsin-like, chymotrypsin-like, and cysteine proteases at different instars. The results showed that the protease profile of A. gemmatalis gut changes throughout its larval development. The activity of cysteine proteases was more intense in the first instar. On the contrary, the serine proteases showed major activities in the late stages of the larval phase. Zymogram analysis and protein identification by liquid chromatography–mass spectrometry indicated serine protease as the main protease class expressed in the fifth instar. These results may shift the focus from the rational development of the protease inhibitor to A. gemmatalis and other Lepidoptera, as the expression of major proteases is not constant.     

Inhibitors of polyamine metabolism: Review article

Summary. The identification of increased polyamine concentrations in a variety of diseases from cancer and psoriasis to parasitic infections has led to the hypothesis that manipulation of polyamine metabolism is a realistic target for therapeutic or preventative intervention in the treatment of certain diseases.The early development of polyamine biosynthetic single enzyme inhibitors such as -difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) showed some interesting early promise as anticancer drugs, but ultimately failed in vivo. Despite this, DFMO is currently in use as an effective anti-parasitic agent and has recently also been shown to have further potential as a chemopreventative agent in colorectal cancer.The initial promise in vitro led to the development and testing of other potential inhibitors of the pathway namely the polyamine analogues. The analogues have met with greater success than the single enzyme inhibitors possibly due to their multiple targets. These include down regulation of polyamine biosynthesis through inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase and decreased polyamine uptake. This coupled with increased activity of the catabolic enzymes, polyamine oxidase and spermidine/spermine N¹-acetyltransferase, and increased polyamine export has made the analogues more effective in depleting polyamine pools. Recently, the identification of a new oxidase (PAO-h1/SMO) in polyamine catabolism and evidence of induction of both PAO and PAO-h1/SMO in response to polyamine analogue treatment, suggests the analogues may become an important part of future chemotherapeutic and/or chemopreventative regimens.     

Activity-based identification of secreted serine proteases of the filamentous fungus, <Emphasis Type="Italic">Ophiostoma</Emphasis>

A general activity probe was synthesized and applied to the supernatant of a filamentous fungus, Ophiostoma, culture to identify directly the secreted serine proteases by covalent enzyme labeling. The activity probe contained a chemically reactive group that reacted with, and thus covalently labeled, the serine residues of only active proteases and not heat-inactivated proteases. The activity probe also contained a fluorescent group that allowed for the subsequent visualization of the captured proteases in 1-D gels and their identification by N-terminal sequencing. This use of a chemical probe led to the rapid discovery of subtilisin-like serine protease of Ophiostoma. Two hypothetical proteins were also captured, with one being a probable endopeptidase K type of protease.     

Tooth morphology of Echimyidae (Rodentia,Caviomorpha): homology assessments,fossils, and evolution

Echimyidae constitute the most important radiation of caviomorph rodents in the Neotropical region, represented by 20 extant genera and several extinct species. Both in extant and fossil forms, this diversity is reflected by a significant morphological variation found in crown structures of the cheek teeth. Different hypotheses of primary homology have been proposed for these structures, which, in turn, support diverse dental evolutionary hypotheses. In this contribution we inspect the main structures (cusps and lophids) of the lower deciduous teeth and molars in extinct and extant Echimyidae, and establish their topological correspondences. Comparisons with cusps and lophids of Erethizontidae are emphasized. We explore the testing of alternative primary hypotheses of lophid correspondences in a cladistic context. Following a ‘dynamic’ approach, we select the hypothesis of primary homology, which produced the more parsimonious results, and evaluate the evolutionary transformations of the dental characters analysed. In this context, the phylogenetic relationships of living Myocastor coypus (Molina, 1782) with the extinct Tramyocastor and Paramyocastor are tested. Our results indicate that pentalophodonty is the derived condition for the lower molars in Echimyidae, that trilophodonty evolved independently at least three times during the evolutionary history of these rodents, and that tetralophodonty represents the plesiomorphic condition. This study shows that dental evolution in echimyids can be better understood when occlusal structures are expressed as reliably comparable characters, and when fossils are taken into account. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 164 , 451–480.     

Structure and distribution of floral trichomes in Lycaste and Sudamerlycaste (Orchidaceae: Maxillariinae s.l.)

Phylogenetic analysis indicates that Lycastinae should be incorporated into a more broadly defined Maxillariinae. This is supported by several anatomical features, including the presence of sunken, glandular trichomes in both Lycastinae and Maxillariinae s.s. Until recently, these were known only from vegetative organs, but have since been reported from flowers of Maxillaria dichroma. One character currently used to distinguish between Lycaste and Sudamerlycaste is the distribution of floral trichomes. In this article, we test the reliability of this character, describe the floral micromorphology of Lycaste and Sudamerlycaste and investigate whether their flowers bear sunken hairs. Their floral micromorphology is compared with that of other genera currently assigned to Maxillariinae s.l. Flowers of Lycaste and Sudamerlycaste bear conical or obpyriform papillae and unbranched and unequally branched multicellular trichomes. Contrary to previous reports that trichomes are confined to the column in Sudamerlycaste, they also occur in the tepal axils. Labellar trichomes, although often present in Lycaste, are lacking in Sudamerlycaste. In Lycaste sections Lycaste and Aromaticae, floral trichomes tend to be unbranched, whereas section Intermediae has both unbranched and branched hairs. Branched hairs are more common in Sudamerlycaste. Some hairs are tracheoidal, pitted and lignified. These mainly occur in section Lycaste and, to a degree, in section Intermediae, but are absent from section Aromaticae and most species of Sudamerlycaste. Branched column hairs, present in Sudamerlycaste, are absent from all sections of Lycaste, and tracheoidal column hairs occur only in Sudamerlycaste. Sunken floral hairs are absent from both genera. Trichome structure and distribution may prove useful in distinguishing between these taxa and in elucidating the intergeneric relationships of Maxillariinae s.l.© 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164 , 409–421.     

Isolation and characterization of Perkinsus marinus proteases using bacitracin–sepharose affinity chromatography

Extracellular proteases were isolated from the cell-free culture supernatant of the oyster-pathogenic protozoan, Perkinsus marinus, by bacitracin–sepharose affinity chromatography. The purified protease fractions contained >75% of the protease activity initially loaded onto the column with very high specific activity that corresponded to 8–11-fold level of protease enrichment. The isolated proteases hydrolysed a variety of protein substrates including oyster plasma. All of the isolated P. marinus proteases belonged to the serine class of proteases. Inhibitor studies involving spectrophotometric assay and gelatin gel electrophoresis showed high levels of inhibition in the presence of the serine protease inhibitors PMSF, benzamidine and chymostatin, whereas inhibitors of cysteine, aspartic, and metalloproteases showed little or no inhibition. Spectrophotometric assays involving serine-specific peptide substrates further revealed that the isolated proteases belong to the class of chymotrypsin-like serine proteases. A 41.7 kDa monomeric, N-glycosylated, serine protease (designated Perkinsin) has been identified as the major P. marinus extracellular protease.     

A Review of: “The Plasma Proteins,F. W. Putnam Volumes I and II, 2nd Edition, 481 and 436 pages respectively,hardbound; Academic Press, 1975; $37.50; $34.50”

A serine protease was purified 6.9-fold from the leaves of Thespesia populnea using ammonium sulfate fractionation followed by CM-cellulose and Sephadex G-100 chromatography. The purified enzyme was named populnein and was characterized. It was made up of a single polypeptide, and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) analysis showed that the enzyme had a molecular mass of 14,518 Da. Inhibition of enzyme activity by phenyl methane sulfonyl fluoride indicates that populnein belongs to the class of serine proteases. The enzyme had appreciable pH and temperature stability. The activity of the enzyme was optimal at pH 8.0 and temperature 40°C. The enzyme was thermostable and retained 85% of its activity at 70°C after 1 hr. The enzyme was also resistant to autodigestion. The stabilization of the membrane of red blood cells exhibited by the protease populnein was found to be higher than for diclofenac. More studies are necessary to investigate the biological activity and applications of serine protease of T. populnea. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.     

Catabolism of polyamines

Summary. Owing to the establishment of cells and transgenic animals which either lack or over-express acetylCoA:spermidine N¹-acetyltransferase a major progress was made in our understanding of the role of polyamine acetylation. Cloning of polyamine oxidases of mammalian cell origin revealed the existence of several enzymes with different substrate and molecular properties. One appears to be identical with the polyamine oxidase that was postulated to catalyse the conversion of spermidine to putrescine within the interconversion cycle. The other oxidases are presumably spermine oxidases, because they prefer free spermine to its acetyl derivatives as substrate. Transgenic mice and cells which lack spermine synthase revealed that spermine is not of vital importance for the mammalian organism, but its transformation into spermidine is a vitally important reaction, since in the absence of active polyamine oxidase, spermine accumulates in blood and causes lethal toxic effects.Numerous metabolites of putrescine, spermidine and spermine, which are presumably the result of diamine oxidase-catalysed oxidative deaminations, are known as normal constituents of organs of vertebrates and of urine. Reasons for the apparent contradiction that spermine is in vitro a poor substrate of diamine oxidase, but is readily transformed into N⁸-(2-carboxyethyl)spermidine in vivo, will need clarification.Several attempts were made to establish diamine oxidase as a regulatory enzyme of polyamine metabolism. However, diamine oxidase has a slow turnover. This, together with the efficacy of the homeostatic regulation of the polyamines via the interconversion reactions and by transport pathways renders a role of diamine oxidase in the regulation of polyamine concentrations unlikely. 4-Aminobutyric acid, the product of putrescine catabolism has been reported to have antiproliferative properties. Since ornithine decarboxylase and diamine oxidase activities are frequently elevated in tumours, it may be hypothesised that diamine oxidase converts excessive putrescine into 4-aminobutyric acid and thus restricts tumour growth and prevents malignant transformation. This function of diamine oxidase is to be considered as part of a general defence function, of which the prevention of histamine and cadaverine accumulation from the gastrointestinal tract is a well-known aspect.     

Transglutaminase 5 is acetylated at the N-terminal end

Summary. Transglutaminases (TGases) are calcium-dependent enzymes that catalyse cross-linking between proteins by acyl transfer reaction; they are involved in many biological processes including coagulation, differentiation, and tissue repair. Transglutaminase 5 was originally cloned from keratinocytes, and a partial biochemical characterisation showed its involvement in skin differentiation, in parallel to TGase 1 and TGase 3. Here, we demonstrate, by electrospray tandem mass spectrometry that TGase 5 is acetylated at the N-terminal end. Moreover, in situ measurement of TGase activity shows that endogenous TGase 5 is active upon treatment with phorbol acetate, and the enzyme co-localises with vimentin intermediate filaments.     

Modularity in attachment organs of African Cichlidogyrus (Platyhelminthes: Monogenea: Ancyrocephalidae) reflects phylogeny rather than host specificity or geographic distribution

Cichlidogyrus spp. (Monogenea, Ancyrocephalidae) are common parasites of cichlid fishes from Africa and the Levant. They display important morphological variation in their attachment apparatus and infect a broad host spectrum throughout a wide geographic range. Thus, they offer an interesting model to investigate to what extent the phenotypic variability of the attachment organ among congeners is related to host specificity, geographic/environmental components, or phylogeny. A geometric morphometric approach was carried out to analyse the shape variation of sclerotized structures of the attachment organ within 66 African species of the genus Cichlidogyrus. The interspecific shape comparison supports the presence of three main morphological configurations, each consisting of a given combination of particular sclerite shapes. Moreover, data emphasize strong coordination and integration (shape co‐variation) among the different sclerites jointly forming the attachment organ. Although attachment apparatuses are usually considered to be the result of adaptive processes and must be adapted to the hosts and local environmental conditions, we found no relationship between these clusters and host specificity or geographical distribution. Nevertheless, groups are partially congruent with those obtained with the molecular phylogeny of a subset of species, suggesting a phylogenetic constraint rather than an adaptation to either hosts or environment. Because of the necessity to form a functional entity, modularity within attachment organ imposes important evolutionary constraint. This provides new insights into the evolvability of attachment organs, as well as into the morphological basis of host specificity and host–parasite co‐evolutionary interaction in helminth parasites. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 694–706.     

Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus

Two novel extracellular serine proteases were purified to homogeneity from the cell-free culture filtrate of an obligate alkalophilic Bacillus sphaericus by a combination of ultrafiltration, ammonium sulfate precipitation and chromatographic methods. The enzymes showed similar substrate specificities, but differed in hydrophobicity and molecular mass. Protease A was a monomeric protease with a relative molecular mass (M _r) of 28.7 kDa, whereas protease B, with a M _r of 68.0 kDa, apparently consisted of smaller subunits. The purified protease A had a specific activity on hemoglobin of 5.1 U/mg protein compared to 40.9 U/mg protein in the case of protease B. Both proteases were most active on SAAPF-pNa, a substrate for chymotrypsin-like serine proteases. However, the K _m values of these two proteases on SAAPF-pNa were higher than that for α-chymotrypsin, indicating a lower affinity of proteases A and B for this substrate compared to chymotrypsin. Unlike other Bacillus serine proteases, neither protease A nor B stained with Coomasie blue R-250, even with loading of a large amount of protein, and they stained poorly with the silver staining method. However, NH₂-terminal amino acid sequencing of protease B revealed a high similarity with subtilisin Carlsberg (67% homology). Almost total inhibition of both proteases by PMSF, but very little/no inhibition by trypsin and chymotrypsin inhibitors (TPCK and TLCK) or thiol reagents (PCMB and iodoacetic acid), further supported the view that the enzyme belonged to the serine protease family. Journal of Industrial Microbiology & Biotechnology (2001) 26, 387–393. Received 05 November 2000/ Accepted in revised form 23 April 2001     

Excitotoxin-induced changes in transglutaminase during differentiation of cerebellar granule cells

Summary. Excitotoxicity induced by NMDA receptor stimulation is able to increase the activity of many enzymes involved in neuronal cell death. Primary cultures of rat cerebellar granule cells were used to elucidate the role of transglutaminase reaction in the excitotoxic cell response, and to evaluate the role of glutamate receptors in cell survival and degeneration. Granule neurons, maintained in vitro for two weeks, were exposed to NMDA at different stages of differentiation. Following NMDA receptor activation, increases in transglutaminase activity were observed in cell cultures. The levels of enzyme activity were higher in cells at 5 days in vitro than in those at 8–9 or 13–14 days in vitro. Moreover, NMDA exposure up-regulated tTG expression in neurons as young as 5 days in vitro. These cultures also exhibited morphological changes with clear apoptotic features. Results obtained demonstrate that susceptibility of granule cells to excitotoxicity depends on the developmental stage of neurons.     

Modeling of substrate and inhibitory complexes of histidine-aspartic protease

A three-dimensional structure of histo-aspartic protease (HAP), a pepsin-like enzyme from the causative agent of malaria Plasmodium falciparum, is suggested on the basis of homologous modeling followed by equilibration by the method of molecular dynamics. The presence of a His residue in the catalytic site instead of an Asp residue, which is characteristic of pepsin-like enzymes, and replacement of some other conserved residues in the active site make it possible for the enzyme to function by the covalent mechanism inherent in serine proteases. The detailed structures of HAP complexes with pepstatin, a noncovalent inhibitor of aspartic proteases, and phenylmethylsulfonyl fluoride, a covalent inhibitor of serine proteases, as well as with a pentapeptide substrate are discussed.     

A novel anti-plant viral protein from coelomic fluid of the earthworm Eisenia foetida: purification, characterization and its identification as a serine protease

A novel protein showing strong antiviral activities against cucumber mosaic virus (CMV) and tomato mosaic virus (TMV) was purified from the coelomic fluid of the earthworm Eisenia foetida. The protein was characterized as a cold-adapted serine protease. Its molecular weight was estimated to be 27,000 by SDS-PAGE. The enzyme was most active at pH 9.5 and 40–50 °C. The protease activity at 4 °C was 60% of that obtained at the optimal temperature. The activity was suppressed by various serine protease inhibitors. Partial N-terminal amino acid sequence of the enzyme showed homology with serine proteases of earthworms, E. foetida and Lumbricus rubellus previously studied. Our results suggest that the enzyme can be applicable as a potential antiviral factor against CMV, TMV, and other plant viruses.     

Phylogenetics and biogeography of the Balkan ‘sand gobies’ (Teleostei: Gobiidae): vulnerable species in need of taxonomic revision

Within the Atlantic–Mediterranean region, the ‘sand gobies’ are abundant and widespread, and play an important role in marine, brackish, and freshwater ecosystems. They include the smallest European freshwater fish, Economidichthys trichonis, which is threatened by habitat loss and pollution, as are several other sand gobies. Key to good conservation management is an accurate account of the number of evolutionary significant units. Nevertheless, many taxonomic and evolutionary questions remain unresolved within the clade, and molecular studies are lacking, especially in the Balkans. Using partial 12S and 16S mitochondrial ribosomal DNA sequences of 96 specimens of at least eight nominal species (both freshwater and marine populations), we assess species relationships and compare molecular and morphological data. The results obtained do not support the monophyly of Economidichthys, suggesting the perianal organ to be a shared adaptation to hole‐brooding rather than a synapomorphy, and urge for a taxonomic revision of Knipowitschia. The recently described Knipowitschia montenegrina seems to belong to a separate South‐East Adriatic lineage. Knipowitschia milleri, an alleged endemic of the Acheron River, and Knipowitschia cf. panizzae, are shown to be very closely related to other western Greek Knipowitschia populations, and appear conspecific. A distinct Macedonian–Thessalian lineage is formed by Knipowitschia thessala, whereas Knipowitschia caucasica appears as an eastern lineage, with populations in Thrace and the Aegean. The present study combines the phylogeny of a goby radiation with insights on the historical biogeography of the eastern Mediterranean, and identifies evolutionary units meriting conservation attention. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 73–91.     

Using enzymes to remove biofilms of bacterial isolates sampled in the food-industry

The aim of this study was to analyze the cleaning efficiency of polysaccharidases and proteolytic enzymes against biofilms of bacterial species found in food industry processing lines and to study enzyme effects on the composition of extracellular polymeric substances (EPS) and biofilm removal in a Clean-in-Place (CIP) procedure. The screening of 7 proteases and polysaccharidases for removal of biofilms of 16 bacterial species was first evaluated using a microtiter plate assay. The alkaline pH buffer removed more biofilm biomass as well as affecting a larger range of bacterial species. The two serine proteases and α-amylase were the most efficient enzymes. Proteolytic enzymes promoted biofilm removal of a larger range of bacterial species than polysaccharidases. Using three isolates derived from two bacterial species widely found in food processing lines (Pseudomonas fluorescens and the Bacillus cereus group), biofilms were developed on stainless steel slides and enzymatic solutions were used to remove the biofilms using CIP procedure. Serine proteases were more efficient in removing cells of Bacillus biofilms than polysaccharidases. However, polysaccharidases were more efficient in removing P. fluorescens biofilms than serine proteases. Solubilization of enzymes with a buffer containing surfactants, and dispersing and chelating agents enhanced the efficiency of polysaccharidases and proteases respectively in removing biofilms of Bacillus and P. fluorescens. A combination of enzymes targeting several components of EPS, surfactants, dispersing and chelating agents would be an efficient alternative to chemical cleaning agents.