Tyr Gly Research Articles

Peptides N-connected to hydroxycoumarin and cinnamic acid derivatives: synthesis and fluorescence spectroscopic, antioxidant and antimicrobial properties

In this study, we developed an efficient synthesis of tripeptide Tyr–Gly–Ser and a series of conjugations to coumarin, cinnamic and gallic acid and their antioxidant and antimicrobial activities were investigated.

Intramolecular BSSE and dispersion affect the structure of a dipeptide conformer

ABSTRACTB3LYP and MP2 calculations with the commonly-used 6-31+G(d) basis set predict qualitatively different structures for the Tyr–Gly conformer book1, which is the most stable conformer identified in a previous study. The structures differ mainly in the ψtyr Ramachandran angle (138° in the B3LYP structure and 120° in the MP2 structure). The causes for the discrepant structures are attributed to missing dispersion in the B3LYP calculations and large intramolecular BSSE in the MP2 calculations. The correct ψtyr value is estimated to be 130°. The MP2/6-31+G(d) profile identified an additional conformer, not present on the B3LYP surface, with a ψtyr value of 96° and a more folded structure. This minimum is, however, likely an artefact of large intramolecular BSSE values. We recommend the use of basis sets of at least quadruple-zeta quality in density functional theory (DFT), DFTaugmented with an empirical dispersion term (DFT-D) and second-order Møller-Plesset perturbation theory (MP2 ) calculations in cases where intramolecular BSSE is expected to be large.

From preclinical development to clinical application: Kit formulation for radiolabelling the minigastrin analogue CP04 with In-111 for a first-in-human clinical trial

IntroductionA variety of radiolabelled minigastrin analogues targeting the cholecystokinin 2 (CCK2) receptor were developed and compared in a concerted preclinical testing to select the most promising radiotracer for diagnosis and treatment of medullary thyroid carcinoma (MTC). DOTA–DGlu–DGlu–DGlu–DGlu–DGlu–DGlu–Ala–Tyr–Gly–Trp–Met–Asp–Phe–NH2 (CP04) after labelling with 111In displayed excellent characteristics, such as high stability, receptor affinity, specific and persistent tumour uptake and low kidney retention in animal models. Therefore, it was selected for further clinical evaluation within the ERA-NET project GRAN-T-MTC. Here we report on the development of a pharmaceutical freeze-dried formulation of the precursor CP04 for a first multi-centre clinical trial with 111In-CP04 in MTC patients. Materials and methodsThe kit formulation was optimised by adjustment of buffer, additives and radiolabelling conditions. Three clinical grade batches of a final kit formulation with two different amounts of peptide (10 or 50μg) were prepared and radiolabelled with 111In. Quality control and stability assays of both the kits and the resulting radiolabelled compound were performed by HPLC analysis. ResultsUse of ascorbic acid buffer (pH4.5) allowed freeze-drying of the kit formulation with satisfactory pellet-formation. Addition of methionine and gentisic acid as well as careful selection of radiolabelling temperature was required to avoid extensive oxidation of the Met11-residue. Trace metal contamination, in particular Zn, was found to be a major challenge during the pharmaceutical filling process in particular for the 10μg formulation. The final formulations contained 10 or 50μg CP04, 25mg ascorbic acid, 0.5mg gentisic acid and 5mg l-methionine. The radiolabelling performed by incubation of 200–250MBq 111InCl3 at 90°C for 15min resulted in reproducible radiochemical purity (RCP) >94%. Kit-stability was proven for >6months at +5°C and at +25°C. The radiolabelled product was stable for >4h at +25°C. ConclusionA kit formulation to prepare 111In-CP04 for clinical application was developed, showing high stability of the kit as well as high RCP of the final product.

Transport of the Synthetic Opioid Peptide DADLE ([d-Ala2,d-Leu5]–Enkephalin) in Neuronal Cells

The sodium-coupled oligopeptide transporters 1 and 2 (SOPT1 and SOPT2) transport peptides consisting of at least five amino acids and show potential for the delivery of therapeutically relevant peptides/peptidomimetics. Here, we examined the expression of these two transporters in the retinal neuronal cell line RGC-5. These cells showed robust uptake activity for the synthetic pentapeptide DADLE ([D-Ala(2),D-Leu(5)]-Enkephalin). The uptake was Na(+) dependent and saturable (K(t), 6.2 ± 0.6 μM). A variety of oligopeptides inhibited DADLE uptake. The uptake of the competing oligopeptides was directly demonstrated with fluorescein isothiocyanate-labeled Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys in RGC-5 cells and primary mouse retinal ganglion cells. The characteristics of DADLE uptake matched those of SOPT2. We then examined the expression of SOPT1 in these cells with deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH(2)) as the substrate and found that RGC-5 cells also expressed SOPT1. As it is already known that SOPT1 is expressed in the neuronal cell line SK-N-SH, we investigated SOPT2 expression in these cells to determine whether the presence of both oligopeptide transporters is a common feature of neuronal cells. These studies showed that SK-N-SH cells also expressed SOPT2. This constitutes the first report on the functional characterization of SOPT1 and SOPT2 in retinal neuronal cells and on the expression of SOPT2 in nonretinal neuronal cells.

Synthesis and Characterization of the Azido‐Functionalized Ruthenocene Analogue [TpmRu(p‐N3C6H4)Tp]Cl and Its Attachment to Biomolecules by Copper‐Catalyzed Azide–Alkyne Cycloaddition

AbstractThe mixed‐ligand tris(pyrazolyl)borate sandwich compound [TpmRuTp′]Cl (2) was prepared by microwave‐assisted synthesis from Tp′Ru(COD)Cl (1) [Tp = tris(pyrazolyl)borate; Tp′ = p‐bromophenyltris(pyrazolyl)borate; Tpm = tris(pyrazolyl)methane; COD = 1,4‐cyclooctadiene]. Subsequently, 2 was converted to the azide‐functionalized [TpmRu(p‐N3C6H4Tp)] (3), which can be readily coupled to biomolecules by Cu‐catalyzed azide–alkyne cycloaddition (Cu‐AAC) in solution, as exemplified by the covalent attachment to pentyne‐functionalized HC(CH2)2–CO–Val–OtBu (4), and an alkyne derivative of the neuropeptide enkephaline HC(CH2)2–CO–ENK–OH (ENK = enkephaline, Tyr–Gly–Gly–Phe–Leu). The resulting triazole compounds [TpmRu({p‐C2N3H–(CH2)2–CO–Val–OtBu}Tp)]Cl (5) and [TpmRu({p‐C2N3H–(CH2)2–CO–ENK–OH}Tp)]Cl (6), represent the first [3+2] cycloaddition products of azide‐functionalized Tp compounds. They were characterized by NMR spectroscopy and mass spectrometry. Furthermore, 1, 2, and 3 as well as the reaction intermediate [(κ2‐N,N′‐Tpm)RuCl(Tp′)] (2a) were characterized in the solid state by single‐crystal X‐ray diffraction.

Agonist-induced formation of FGFR1 homodimers and signaling differ among members of the FGF family

Fibroblast growth factor receptor 1 (FGFR1) is known to be activated by homodimerization in the presence of both the FGF agonist ligand and heparan sulfate glycosaminoglycan. FGFR1 homodimers in turn trigger a variety of downstream signaling cascades via autophosphorylation of tyrosine residues in the cytoplasmic domain of FGFR1. By means of Bioluminescence Energy Resonance Transfer (BRET) as a sign of FGFR1 homodimerization, we evaluated in HEK293T cells the effects of all known FGF agonist ligands on homodimer formation. A significant correlation between BRET2 signaling and ERK1/2 phosphorylation was observed, leading to a further characterization of the binding and signaling properties of the FGF subfamilies. FGF agonist ligand-FGFR1 binding interactions appear as the main mechanism for the control of FGFR1 homodimerization and MAPK signaling which demonstrated a high correlation. The bioinformatic analysis demonstrates the interface of the two pro-triplets SSS (Ser–Ser–Ser) and YGS (Tyr–Gly–Ser) located in the extracellular and intracellular domain of the FGFR1. These pro-triplets are postulated participate in the FGFR1 homodimerization interface interaction. The findings also reveal that FGF agonist ligands within the same subfamily of the FGF gene family produced similar increases in FGFR1 homodimer formation and MAPK signaling. Thus, the evolutionary relationship within this gene family appears to have a distinct functional relevance.

Salivary peptidomics

In recent years, there has been an increased interest in the study of saliva. This bodily fluid contains a vast number of protein species, the salivary peptidome, of low molecular weight, comprising approximately 40–50% of the total secreted proteins, in addition to peptides generated by proteolysis of proteins of different sources. Owing to the presence of other components, in particular mucins and enzymes, some distinctive requirements and precautions related to sample collection, time of analysis, sample preservation and treatment are necessary for the successful analysis of salivary peptides. More than 2000 peptides compose the salivary peptidome, from which only 400–600 are directly derived from salivary glands, suggesting an important qualitative peptide contribution of other sources, namely of epithelial cells. Proteolysis events are the main supply for the peptidome and considerable efforts have been made to identify the resulting fragments, the cleavage sites and the involved proteases. The salivary proteins more prone to proteolysis are proline-rich proteins (PRPs; acidic PRPs and basic PRPs), statherin, histatins and P-B peptide. Gln–Gly cleavages are largely associated with PRP classes, while Tyr–Gly cleavages are related to histatin 1 and to the P-B peptide. The interest in saliva has been growing for clinical purposes, as it is an alternative sample to other traditional bodily fluids, such as blood or urine, since it involves an easy and noninvasive collection. In fact, apart from its usefulness as a source of information for the prognosis, diagnosis and treatment of oral diseases, such as Sjögren’s syndrome, gum disease, tooth decay or oral cancer, saliva might also be seen as a potential tool to the diagnosis of systemic diseases. Owing to the enormous amount of previously discovered salivary peptide species, in this article, we attempt to harmonize the nomenclature, following International Union of Pure and Applied Chemistry recommendations.

Even-numbered peptides from a papain hydrolysate of silk fibroin

A protease with broad substrate specificity usually produces a complex peptide mixture. However, even-numbered peptides were obtained at high proportion upon papain hydrolysis of fibroin composed of highly repetitive Ala- and Gly-rich blocks. MALDI-TOF and ESI mass spectrometric analysis revealed that the even-numbered peptides were in the forms of di-, tetra-, hexa-, and octa-peptides with repeating units in combination of Ala–Gly, Ser–Gly, Tyr–Gly, and Val–Gly. Application of tandem mass spectrometry identified the sequences of the tetra-peptides to be in the order of Ala–Gly–X–Gly (X = Tyr or Val). Therefore, the substrate specificity of papain and the unique repetitive sequence of fibroin generated the hydrolysate composed of even number of amino acids at a high percentage. In this work, fibroin hydrolysate was investigated as an example of an end product of protein hydrolysis, which provides a clue to understand the fate of peptides in a protein hydrolysate.

Conductance and Concentration Relationship in a Reduced Model of the K Channel

K+ ions move rapidly through potassium channels more or less ignoring Na+. The mechanism of selectivity is thought to depend on the solvation of K+ and its electrostatic interactions with carbonyl dipoles of the channel wall, made of the side chains Thr Val Gly Tyr Gly TVGYG in many types of potassium channels. We calculate the conductance of the tetrameric KcsA prokaryotic K+ channel measured in solutions of different K+ concentration. The 3D model used here consists of two regions of different dielectric constant, one representing the protein and one representing a bath of implicit water.

Performance of the M06-L density functional for a folded Tyr–Gly conformer

The M06-L density functional has been assessed for its ability to predict the correct structure of a Tyr–Gly conformer for which MP2/6-31+G(d) predicts a folded ‘closed book’ conformer, whereas a more open conformation is predicted by B3LYP/6-31+G(d). Potential energy profiles were determined by computing the energy for geometries optimised at various fixed values of a distance that controls the degree of foldedness of the structure. The M06-L/6-31G(d) and M06-L/6-31+G(d) methods yield excellent agreement with the reference df-LCCSD(T0)/aug-cc-pVTZ profile. Thus, M06-L manifests itself as a very promising method to investigate the potential energy surface of small peptides containing aromatic residues.

Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs

Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His–Tyr–Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1 E)-2-(5-imidazolyl)ethenyl]-4-( p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His–Tyr–Gly, Trp–Tyr–Gly, Phe–Trp–Gly, Tyr–Trp–Gly, Asn–Tyr–Gly, Phe–Tyr–Gly, and Tyr–Tyr–Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595 nm was found for the chromophore Trp–Tyr–Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn–Tyr–Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.

Insufficient description of dispersion in B3LYP and large basis set superposition errors in MP2 calculations can hide peptide conformers

B3LYP/6-31+G(d) and MP2/6-31+G(d) calculations predict markedly different structures for one Tyr–Gly conformer. Calculation of the energy profile for rotation around the glycine C α–N bond reveals one minimum in the B3LYP profile ( ϕ gly = 180°) and two in the MP2 profile (∼75° and 280°). Large intramolecular BSSE values are responsible for masking the 180°-minimum in the MP2 profile: approximate elimination of BSSE in the MP2 calculations – by (1) correction using BSSE values from complexes of phenol and N-formylglycine, (2) the application of local MP2, or (3) employing large basis sets (aug-cc-pVTZ/QZ) and density fitting – yields an unambiguous triple-well potential.

The structure of the gas-phase tyrosine–glycine dipeptide

The structural preferences of the neutral dipeptide Tyr–Gly have been investigated using a hierarchical selection scheme. This scheme consists of a hierarchy of increasingly more accurate electronic structure methods (single-point HF/3-21G* energy calculation, HF/3-21G* geometry optimization, B3LYP/6-31+G* geometry optimization, MP2/6-31+G* single-point energy calculation, and MP2/6-31+G* geometry optimization). The conformers are sorted according to their single-point or optimized energy, and only the most stable conformers according to one level are taken through to the next level of calculation. The defining structural characteristics in the 20 most stable Tyr–Gly conformers are the presence or absence of a folded arrangement of the peptide backbone (‘book’) and an OH···O hydrogen bond between the C-terminal hydroxyl group and the carbonyl oxygen of tyrosine (‘OHO’). The most stable conformer is of the book/OHO type. MP2 geometry optimization significantly alters the structure of the book-type conformers, increasing their degree of foldedness. Thus, care has to be taken when applying standard density functionals like B3LYP in structural studies of peptides with aromatic side chains.

Rotaviruses interact with α4β7 and α4β1 integrins by binding the same integrin domains as natural ligands

Group A rotaviruses are major intestinal pathogens that express potential alpha4beta1 and alpha4beta7 integrin ligand sequences Leu-Asp-Val and Leu-Asp-Ile in their outer capsid protein VP7, and Ile-Asp-Ala in their spike protein VP4. Monkey rotavirus SA11 can use recombinant alpha4beta1 as a cellular receptor. In this study a new potential alpha4beta1, alpha4beta7 and alpha9beta1 integrin ligand sequence, Tyr-Gly-Leu, was identified in VP4. It was shown that several human and monkey rotaviruses bound alpha4beta1 and alpha4beta7, but not alpha9beta1. Binding to alpha4beta1 mediated the infectivity and growth of monkey rotaviruses, and binding to alpha4beta7 mediated their infectivity. A porcine rotavirus interacted with alpha4 integrins at a post-binding stage to facilitate infection. Activation of alpha4beta1 increased rotavirus infectivity. Cellular treatment with peptides containing the alpha4 integrin ligand sequences Tyr-Gly-Leu and Ile-Asp-Ala eliminated virus binding to alpha4 integrins and infectivity. In contrast, rotavirus recognition of alpha4 integrins was unaffected by a peptide containing the sequence Leu-Asp-Val or by a mutation in the VP7 Leu-Asp-Val sequence. VP4 involvement in rotavirus recognition of alpha4beta1 was demonstrated with rotavirus reassortants. Swapping and point mutagenesis of alpha4 surface loops showed that rotaviruses required the same alpha4 residues and domains for binding as the natural alpha4 integrin ligands: mucosal addressin cell adhesion molecule-1, fibronectin and vascular cell adhesion molecule-1. Several rotaviruses are able to use alpha4beta7 and alpha4beta1 for cell binding or entry, through the recognition of the same alpha4-subunit domains as natural alpha4 ligands.

Calorimetric and thermal decomposition kinetic study of Tb(Tyr)(Gly)3Cl3�3H2O

The solid-state ternary complex of terbium chloride with L-tyrosine and glycine, [Tb(Tyr)(Gly)3Cl3·3H2O], was synthesized and characterized. Using a solution-reaction isoperibol calorimeter, the enthalpy of reaction for the following reaction, TbCl3·6H2O(s)+Tyr(s)+3Gly(s)=Tb(Tyr)(Gly)3Cl3·3H2O(s)+3H2O(l), was determined to be (5.1±0.6) kJ mol-1. The standard enthalpy of formation of Tb(Tyr)(Gly)3Cl3-3H2O at T=298.15 K has been derived as -(4267.3±2.3) kJ mol-1. The thermal decomposition kinetics of the complex was studied by non-isothermal thermogravimetry in the temperature range of 325-675 K. Two main mass loss stages existed in the process of the decomposition of the complex, the kinetic parameters for the second stage were analyzed by means of differential and integral methods, respectively. Comparing the results of differential and integral methods, mechanism functions of the thermal decomposition reaction for its second stage were proposed. The kinetic equation can be expressed as: dα/dt=Aexp(-E/RT)(1-α)2. The average values of the apparent activation energy E and pre-exponential factor A were 213.18 kJ mol-1 and 2.51·1020 s-1, respectively.

Bioimmunotherapy of rodent malaria: co-treatment with recombinant mouse granulocyte-macrophage colony-stimulating factor and an enkephalin fragment peptide Tyr?Gly?Gly

We have earlier shown that recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and methionine-enkephalin co-treatment can protect mice from malaria. We now report the bioimmunotherapeutic effect of rmGM-CSF and a synthetic enkephalin fragment peptide Tyr–Gly–Gly (TGG) co-treatment on blood-induced Plasmodium berghei infection in Swiss mice. Mice were completely aparasitimic following co-treatment with rmGM-CSF (10.0 μg/kg) and TGG (2.0 mg/kg × 3 per day, intraperitoneally (i.p.)) starting from day −1 to day +4; however, in monotherapy, neither of these agents showed any detectable bioimmunotherapeutic effect. Curiously, similar co-treatment with rmGM-CSF (10.0 μg/kg) and higher doses of TGG (10.0 mg/kg) did not protect the mice. The combined bioimmunotherapeutic effect of these agents was abrogated by the separate administration each of rabbit neutralizing anti-rmGM-CSF antibody, non-selective opioid receptor antagonist naltrexone (10.0 mg/kg × 6 per day, i.p.), and silica (3.0 mg per mouse, intravenously (i.v.)). The peritoneal and splenic macrophages from the protected mice showed a significant ( P <0.05) increase in their pool-size and the phagocytic activity, ex vivo. Furthermore, the protected mice, as compared to the unprotected ones, showed a significant ( P <0.05) maximum increase in their serum nitrate and nitrite, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) levels in their splenic homogenates, on the day before the beginning of the resolution of parasitaemia. Selective inhibitors of both inducible (aminoguanidine) and all forms ( l -N G -monomethyl arginine) of nitric oxide (NO) synthase, significantly ( P <0.05) augmented the mortality of co-treated mice, suggesting the role of NO in protection. These data show that, in P. berghei -infected mice, co-treatment with rmGM-CSF and conditional doses of TGG can impart protection, apparently through partly NO-dependent and macrophage-mediated mechanism(s).

Bioimmunotherapy of rodent malaria: co-treatment with recombinant mouse granulocyte-macrophage colony-stimulating factor and an enkephalin fragment peptide Tyr–Gly–Gly

We have earlier shown that recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and methionine-enkephalin co-treatment can protect mice from malaria. We now report the bioimmunotherapeutic effect of rmGM-CSF and a synthetic enkephalin fragment peptide Tyr–Gly–Gly (TGG) co-treatment on blood-induced Plasmodium berghei infection in Swiss mice. Mice were completely aparasitimic following co-treatment with rmGM-CSF (10.0 μg/kg) and TGG (2.0 mg/kg × 3 per day, intraperitoneally (i.p.)) starting from day −1 to day +4; however, in monotherapy, neither of these agents showed any detectable bioimmunotherapeutic effect. Curiously, similar co-treatment with rmGM-CSF (10.0 μg/kg) and higher doses of TGG (10.0 mg/kg) did not protect the mice. The combined bioimmunotherapeutic effect of these agents was abrogated by the separate administration each of rabbit neutralizing anti-rmGM-CSF antibody, non-selective opioid receptor antagonist naltrexone (10.0 mg/kg × 6 per day, i.p.), and silica (3.0 mg per mouse, intravenously (i.v.)). The peritoneal and splenic macrophages from the protected mice showed a significant ( P<0.05) increase in their pool-size and the phagocytic activity, ex vivo. Furthermore, the protected mice, as compared to the unprotected ones, showed a significant ( P<0.05) maximum increase in their serum nitrate and nitrite, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) levels in their splenic homogenates, on the day before the beginning of the resolution of parasitaemia. Selective inhibitors of both inducible (aminoguanidine) and all forms ( l-N G-monomethyl arginine) of nitric oxide (NO) synthase, significantly ( P<0.05) augmented the mortality of co-treated mice, suggesting the role of NO in protection. These data show that, in P. berghei-infected mice, co-treatment with rmGM-CSF and conditional doses of TGG can impart protection, apparently through partly NO-dependent and macrophage-mediated mechanism(s).

Isolation, identification and localization of a second beetle antidiuretic peptide

We isolated from head extracts of Tenebrio molitor a peptide that inhibits fluid secretion by the Malpighian tubules of this insect. This second antidiuretic factor, ADFb, like the previously published ADFa, works through cyclic GMP as a second messenger. It has primary structure Tyr–Asp–Asp–Gly–Ser–Tyr–Lys–Pro–His–Ile–Tyr–Gly–Phe–OH with an EC 50 of approximately 240 pM in a fluid secretion assay. This peptide is now the second sequenced endogenous insect ADF which inhibits Malpighian tubule fluid secretion. Immunohistochemical techniques show that the peptide is localized in the brain; it appears to be produced mainly in two pairs of bilaterally symmetrical cells in the protocerebrum.

Characterization and classification of five cysteine proteinases expressed by Paragonimus westermani adult worm

Three new members of the cysteine proteinase gene family of Paragonimus westermani have been isolated and classified. Comparisons of the predicted amino acid sequences of PwCP2 (U69121), PwCP4 (U56958), and PwCP5 (U33215) were performed with those of the previously reported PwCP1 (U69120) and PwCP3 (U56865) sequence. The amino acid alignment showed conservation of the cysteine, histidine, and asparagine residue that form the catalytic triad. With 57 cysteine proteinases including PwCP1–5, we conducted phylogenetic analysis using neighbor joining method (NJ). A resultant unrooted tree revealed that PwCP1–5 were clustered with cruzipain-like or cathepsin L-like cysteine proteinases. More detailed phylogenetic analyses with a reduced alignment set (22 cysteine proteinases) were performed by NJ and maximum parsimony (MP) methods. The results showed coincidently that PwCP1, 2, 3, and 4 belonged to the group of previously reported cruzipain-like cysteine proteinases (bootstrapping values of 97 and 100% in the MP and NJ trees) but PwCP5 to cathepsin L-like cysteine proteinases (the value of 76 and 100% in MP and NJ trees). Within the cruzipain-like clade, PwCP2 and 4 were found to be the most closely related. PwCP 2, 3, and 4 have five of six cruzipain signature sequences known previously, whereas PwCP5 do not have any cruzipain sequences in the corresponding sites. We found that two signature candidate sites (Gly 174, Asn 175—human cathepsin L numbering) for cathepsin L-like group are conserved in PwCP5, which are conserved within cathepsin L-like group and also different from those of cruzipain and other cysteine proteinase groups. PwCP5 has three-residue insertion (hydrophilic residues, Ser–Tyr–Gly) within the position corresponding to S3 subsite of SmCL2. Compared to the two-residue insertion (Tyr–Gly) in SmCL2, the three-residue insertion appeared in PwCP5 may bring bigger difference in substrate specificity between PwCP1–4 (cruzipain) and PwCP5 (cathepsin L-like). Such presumption is quite plausible considering extremely lower amino acid sequence similarity (18.2%) between PwCP1–4 and PwCP5. The present study is worthy of reporting one another case, the third organism after Schistosoma mansoni and Schistosoma japonicum, which has the two kinds of genes encoding both the cruzipain and cathepsin L-like cysteine proteinases. In addition, the fact that most of cysteine proteinases from P. westermani are cruzipain-like type implies strongly that a new powerful drug for paragonimiasis could be designed and developed if we focus on the exploration of anti-agents against P. westermani cruzipain-like cysteine proteinases.

Characterization of human nasal primary culture systems to investigate peptide metabolism

The objectives of this study were to validate and compare the suitability of different primary cell culture systems as models to investigate peptide enzymatic stability following nasal administration. The degradation kinetics of a model peptide, leucine enkephalin (Tyr–Gly–Gly–Phe–Leu, Leu–Enk), was determined in four nasal cell culture systems: immersion, air–liquid interface, sequential monolayer-suspension, floating collagen. The influence of enzyme inhibitors (bestatin, puromycin) and Leu–Enk metabolite analogs (Tyr–Gly, Phe–Leu, Tyr–Gly–Gly, Gly–Phe–Leu) on the Leu–Enk degradation profile was also investigated. The disappearance of Leu–Enk in all the cell culture systems followed first order kinetics. The specific activity in the cell culture systems followed the rank: sequential monolayer-suspension (32.60 μM min −1 mg −1) >air–liquid interface (15.19 μM min −1 mg −1) >immersion (11.49 μM min −1 mg −1) >floating collagen (4.57 μM min −1 mg −1). At equimolar concentration, bestatin had a higher inhibitory effect than puromycin. The rate of hydrolysis of Leu–Enk was reduced significantly by co-incubation with Leu–Enk metabolite analogs. This study showed that immersion, sequential monolayer-suspension and air–liquid interface culture systems may be potentially suitable for further studies on peptide enzymatic stability following nasal administration.