Plant Specialized Metabolites in Hazelnut (Corylus avellana) Kernel and Byproducts: An Update on Chemistry, Biological Activity, and Analytical Aspects.

Corylus avellana (hazelnut) is one of the most popular tree nuts on a worldwide basis. The main products of C. avellana are kernels, a nutritious food, with a high content of healthy lipids, contained in a hard shell. In recent years, along with the ongoing research carried out on hazelnut kernels, a growing interest has been addressed to the hazelnut byproducts including hazelnut skin, hazelnut hard shell, and hazelnut green leafy cover as well as hazelnut tree leaf. These byproducts deriving from the roasting, cracking, shelling/hulling, and harvesting processes have been found as a source of "phytochemicals" with biological activity. The aim of this review is to provide a comprehensive and critical update on the chemistry and biological activity of specialized metabolites occurring in hazelnut kernels and byproducts. Phenolics are the most abundant phytochemicals not only in the kernels, but also in other processing byproducts. Attention has been also devoted to taxane derivatives isolated from C. avellana leaves. An overview on the biological activity, mainly antioxidant, antiproliferative, and antimicrobial along with less common biological effects, has been provided, contributing to highlight C. avellana as a source of bioactive phytochemicals with the potential to exert beneficial effects on human health. Finally, analytical techniques for the quali-quantitative analysis of specialized metabolites occurring in the different parts of C. avellana have been reviewed.

Flavopereirine-An Alkaloid Derived from Geissospermum vellosii-Presents Leishmanicidal Activity In Vitro.

Chemotherapy is limited in the treatment of leishmaniasis due to the toxic effects of drugs, low efficacy of alternative treatments, and resistance of the parasite. This work assesses the in vitro activity of flavopereirine on promastigote cultures of Leishmania amazonensis. In addition, an in silico evaluation of the physicochemical characteristics of this alkaloid is performed. The extract and fractions were characterized by thin-layer chromatography and HPLC-DAD, yielding an alkaloid identified by NMR. The antileishmanial activity and cytotoxicity were assayed by cell viability test (MTT). The theoretical molecular properties were calculated on the Molinspiration website. The fractionation made it possible to isolate a beta-carboline alkaloid (flavopereirine) in the alkaloid fraction. Moreover, it led to obtaining a fraction with greater antileishmanial activity, since flavopereirine is very active. Regarding the exposure time, a greater inhibitory effect of flavopereirine was observed at 24 h and 72 h (IC50 of 0.23 and 0.15 µg/mL, respectively). The extract, fractions, and flavopereirine presented low toxicity, with high selectivity for the alkaloid. Furthermore, flavopereirine showed no violation of Lipinski's rule of five, showing even better results than the known inhibitor of oligopeptidase B, antipain, with three violations. Flavopereirine also interacted with residue Tyr-499 of oligopeptidase B during the molecular dynamics simulations, giving a few insights of a possible favorable mechanism of interaction and a possible inhibitory pathway. Flavopereirine proved to be a promising molecule for its antileishmanial activity.

Characterization of PdCP1, a serine carboxypeptidase from Pseudogymnoascus destructans, the causal agent of White-nose Syndrome.

Pseudogymnoascus destructans is a pathogenic fungus responsible for White-nose Syndrome (WNS), a disease afflicting multiple species of North American bats. Pseudogymnoascus destructans infects susceptible bats during hibernation, invading dermal tissue and causing extensive tissue damage. In contrast, other Pseudogymnoascus species are non-pathogenic and cross-species comparisons may therefore reveal factors that contribute to virulence. In this study, we compared the secretome of P. destructans with that from several closely related Pseudogymnoascus species. A diverse set of hydrolytic enzymes were identified, including a putative serine peptidase, PdCP1, that was unique to the P. destructans secretome. A recombinant form of PdCP1 was purified and substrate preference determined using a multiplexed-substrate profiling method based on enzymatic degradation of a synthetic peptide library and analysis by mass spectrometry. Most peptide substrates were sequentially truncated from the carboxyl-terminus revealing that this enzyme is a bona fide carboxypeptidase. Peptides with arginine located close to the carboxyl-terminus were rapidly cleaved, and a fluorescent substrate containing arginine was therefore used to characterize PdCP1 activity and to screen a selection of peptidase inhibitors. Antipain and leupeptin were found to be the most potent inhibitors of PdCP1 activity.

The protease inhibitor antipain has a beneficial synergistic effect with trehalose for ram semen cryopreservation.

The objective of this study was to examine the different concentrations of antipain and trehalose combination on post-thawed quality of ram semen cryopreserved in tris extender. Ejaculates were collected from four rams using the artificial vagina, pooled at 37°C and diluted with (A0  Tre0 : antipain 0 µM and trehalose 0 mM (Control); A10  Tre0 ; A50  Tre0 ; A0  Tre30 ; A0  Tre60 ; A10  Tre60 ; A10  Tre30 ; A50  Tre30 and A50  Tre60 ). Diluted semen samples were gradually cooled down from 37 to 5°C in a cold cabinet; then, they were loaded into 0.25 ml straws, frozen and stored in liquid nitrogen. Sperm motility (CASA), viability, membrane functionality and abnormality were evaluated after thawing process. Progressive motility in extender supplemented with A10  Tre0 , A0  Tre30 and A10  Tre60 significantly (p < 0.05) higher as compared to the control (A10  Tre0 ). A10  Tre60 (47.50 ± 0.73) provided the best maintenance of progressive motility in comparison with the control (40.50 ± 0.73). No significant differences were observed between all treated groups in terms of total motility, VAP, VSL, VCL, ALH, BCF, STR and LIN. The percentages of sperm with viable were significantly higher in extenders supplemented with A10  Tre0 , A50  Tre0 , A0  Tre30 and A10  Tre60 , compared to control. Addition of A10  Tre0 , A50  Tre0 and A10  Tre60 to extenders improved the percentages of sperm abnormality, compared to the controls. A10  Tre60 (67.84 ± 1.51) treatment provided the best maintenance of normal morphology compared to the other treatments. The supplementation with A10  Tre0 , A0  Tre60 and A10  Tre60 improved the percentage of sperm membrane functionality when compared to the control (p < 0.05). Comparing these results with those of control diluents, the effects of supplementation were better except for A50  Tre60 group. In conclusion, when combination of antipain (10 µM) and trehalose (30 and 60 mM) was added, they conferred a great cryosurvival capacity with their synergic effects during freeze-thawing process.

Kinetic studies of serine protease inhibitors in simple and rapid 'active barrier' model systems - Diffusion through an inhibitor barrier.

A model based on gelatin for protease activity studies was designed. The model is also extended to study the efficiency of inhibitors in a separate protective layer covering the layer containing the target substrate. A good correlation between protease concentration and the size of erosion wells formed in a plain gelatin layer was observed. Similarly, increased concentration of inhibitors gave a systematic decrease in well area. Kinetic analyses of the two-layer model in a spectrophotometric plate reader with a fixed concentration of substrate in the bottom layer displayed a strict dependence of both inhibitor concentration and thickness of the top "protective" layer. An apparent, but weaker inhibition effect was also observed without inhibitors due to diffusional and erosion delay of enzyme transport to the substrate-containing layer.

Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

In vitro effects of cysteine protease inhibitors on Trichomonas foetus-induced cytopathic changes in porcine intestinal epithelial cells.

OBJECTIVE To investigate the effects of specific cysteine protease (CP) inhibitors on cytopathic changes to porcine intestinal epithelial cells induced by Tritrichomonas foetus isolated from naturally infected cats. SAMPLE T foetus isolates from 4 naturally infected cats and nontransformed porcine intestinal epithelial cells. PROCEDURES T foetus isolates were treated with or without 0.1 to 1.0mM of the CP inhibitors antipain, cystatin, leupeptin, and chymostatin and the vinyl sulfone inhibitors WRR-483 and K11777. In-gel gelatin zymography was performed to evaluate the effects of these inhibitors on CP activity of T foetus isolates. Each treated or untreated isolate was also cocultured with monolayers of porcine intestinal epithelial cells for 24 hours, and cytopathic effects of T foetus were evaluated by light microscopy and crystal violet spectrophotometry. RESULTS Results of in-gel gelatin zymography suggested an ability of WRR-483, K11777, and cystatin to target specific zones of CP activity of the T foetus isolates. These inhibitors had no effect on T foetus growth, and the cytopathic changes to the intestinal epithelium induced by all 4 T foetus isolates were significantly inhibited. CONCLUSIONS AND CLINICAL RELEVANCE This study revealed that certain protease inhibitors were capable of inhibiting regions of CP activity (which has been suggested to cause intestinal cell damage in cats) in T foetus organisms and of ameliorating T foetus-induced cytopathic changes to porcine intestinal epithelium in vitro. Although additional research is needed, these inhibitors might be useful in the treatment of cats with trichomonosis.

Calcium Imaging of Neuronal Activity in Drosophila Can Identify Anticonvulsive Compounds.

Although there are now a number of antiepileptic drugs (AEDs) available, approximately one-third of epilepsy patients respond poorly to drug intervention. The reasons for this are complex, but are probably reflective of the increasing number of identified mutations that predispose individuals to this disease. Thus, there is a clear requirement for the development of novel treatments to address this unmet clinical need. The existence of gene mutations that mimic a seizure-like behaviour in the fruit fly, Drosophila melanogaster, offers the possibility to exploit the powerful genetics of this insect to identify novel cellular targets to facilitate design of more effective AEDs. In this study we use neuronal expression of GCaMP, a potent calcium reporter, to image neuronal activity using a non-invasive and rapid method. Expression in motoneurons in the isolated CNS of third instar larvae shows waves of calcium-activity that pass between segments of the ventral nerve cord. Time between calcium peaks, in the same neurons, between adjacent segments usually show a temporal separation of greater than 200 ms. Exposure to proconvulsants (picrotoxin or 4-aminopyridine) reduces separation to below 200 ms showing increased synchrony of activity across adjacent segments. Increased synchrony, characteristic of epilepsy, is similarly observed in genetic seizure mutants: bangsenseless1 (bss1) and paralyticK1270T (paraK1270T). Exposure of bss1 to clinically-used antiepileptic drugs (phenytoin or gabapentin) significantly reduces synchrony. In this study we use the measure of synchronicity to evaluate the effectiveness of known and novel anticonvulsive compounds (antipain, isethionate, etopiside rapamycin and dipyramidole) to reduce seizure-like CNS activity. We further show that such compounds also reduce the Drosophila voltage-gated persistent Na+ current (INaP) in an identified motoneuron (aCC). Our combined assays provide a rapid and reliable method to screen unknown compounds for potential to function as anticonvulsants.

Mimicking the lipid peroxidation inhibitory activity of phospholipid hydroperoxide glutathione peroxidase (GPx4) by using fatty acid conjugates of a water-soluble selenolane.

A series of fatty acid conjugates of trans-3,4-dihydroxy-1-selenolane (DHS) were synthesized by reacting DHS with appropriate acid chlorides. The obtained monoesters were evaluated for their antioxidant capacities by the lipid peroxidation assay using a lecithin/cholesterol liposome as a model system. The observed antioxidant capacities against accumulation of the lipid hydroperoxide (LOOH) increased with increasing the alkyl chain length and became saturated for dodecanoic acid (C12) or higher fatty acid monoesters, for which the capacities were much greater than those of DHS, its tridecanoic acid (C13) diester, and PhSeSePh. On the other hand, the bacteriostatic activity of myristic acid (C14) monoester, evaluated through the colony formation assay using Bacillus subtilis, indicated that it has higher affinity to bacterial cell membranes than parent DHS. Since DHS-fatty acid conjugates would inhibit lipid peroxidation through glutathione peroxidase (GPx)-like 2e- mechanism, higher fatty acid monoesters of DHS can mimic the function of GPx4, which interacts with LOOH to reduce it to harmless alcohol (LOH). Importance of the balance between hydrophilicity and lipophilicity for the design of effective GPx4 mimics was suggested.

Epoxy-α-lapachone has in vitro and in vivo anti-leishmania (Leishmania) amazonensis effects and inhibits serine proteinase activity in this parasite.

Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapeutic treatments present many problems, such as several adverse side effects and the development of resistant strains. Natural compounds have been investigated as potential antileishmanial agents, and the effects of epoxy-α-lapachone on L. (L.) amazonensis were analyzed in the present study. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 h of exposure. This compound also had an effect in experimentally infected BALB/c mice, causing reductions in paw lesions 6 weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area, 24.9 ± 2.0 mm(2)), compared to untreated animals (mean lesion area, 30.8 ± 2.6 mm(2)) or animals treated with Glucantime (mean lesion area, 28.3 ± 1.5 mm(2)). In addition, the effects of this compound on the serine proteinase activities of the parasite were evaluated. Serine proteinase-enriched fractions were extracted from both promastigotes and amastigotes and were shown to act on specific serine proteinase substrates and to be sensitive to classic serine proteinase inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and antipain). These fractions were also affected by epoxy-α-lapachone. Furthermore, in silico simulations indicated that epoxy-α-lapachone can bind to oligopeptidase B (OPB) of L. (L.) amazonensis, a serine proteinase, in a manner similar to that of antipain, interacting with an S1 binding site. This evidence suggests that OPB may be a potential target for epoxy-α-lapachone and, as such, may be related to the compound's effects on the parasite.

"The group facilitates everything": meanings patients with type 2 diabetes mellitus assigned to health education groups / "O grupo facilita tudo": significados atribuídos por pacientes portadores de diabetes mellitus tipo 2 a grupos de educação em saúde / "El grupo facilita todo": significados atribuidos por pacientes con diabetes mellitus tipo 2 a grupos de educación para la salud

OBJECTIVE: to interpret the meanings patients with type 2 diabetes mellitus assign to health education groups. METHOD: ethnographic study conducted with Hyperdia groups of a healthcare unit with 26 informants, with type 2 diabetes mellitus, and having participated in the groups for at least three years. Participant observation, social characterization, discussion groups and semi-structured interviews were used to collect data. Data were analyzed through the thematic coding technique. RESULTS: four thematic categories emerged: ease of access to the service and healthcare workers; guidance on diabetes; participation in groups and the experience of diabetes; and sharing knowledge and experiences. The most relevant aspect of this study is the social use the informants in relation to the Hyperdia groups under study. CONCLUSION: the studied groups are agents producing senses and meanings concerning the process of becoming ill and the means of social navigation within the official health system. We expect this study to contribute to the actions of healthcare workers coordinating these groups given the observation of the cultural universe of these individuals seeking professional care in the various public health care services. .

OBJETIVO: interpretar os significados atribuídos por pacientes portadores de diabetes mellitus tipo 2 a grupos de educação em saúde. MÉTODO: estudo etnográfico em cinco grupos Hiperdia de um centro de saúde, com 26 informantes portadores de diabetes mellitus tipo 2 que participavam dos grupos há, no mínimo, três anos. Para coligir as informações, utilizaram-se observação participante, caracterização social, grupos de discussão e entrevistas semiestruturadas. Os dados foram analisados por meio da técnica de codificação temática. RESULTADOS: emergiram quatro categorias temáticas - facilidades de acesso ao serviço e profissionais de saúde, orientações sobre o diabetes, participação nos grupos e experiência com o diabetes e compartilhamento de saberes e experiências. O aspecto mais relevante deste estudo diz respeito aos usos sociais que os informantes conferiam aos grupos Hiperdia pesquisados. CONCLUSÃO: os grupos estudados mostraram-se como instâncias produtoras de sentidos e de significados, concernentes ao processo de adoecimento e aos modos de navegação social no interior do sistema oficial de saúde. Almeja-se que este estudo possa contribuir para as ações dos profissionais de saúde que atuam nesses grupos, tendo em vista a observação do universo cultural dos indivíduos que procuram por cuidado profissional, nos diversos serviços públicos de saúde. .

OBJETIVO: interpretar los significados atribuidos por pacientes con diabetes mellitus tipo 2 a los grupos de educación para la salud. MÉTODO: estudio etnográfico en cinco grupos Hiperdia de un centro de salud, con 26 informantes con diabetes mellitus tipo 2 que participaban de los grupos hace, por lo menos, tres años. Para recolectar las informaciones se utilizaron la observación participante, la caracterización social, los grupos de discusión y las entrevistas semiestructuradas. Los datos fueron analizados por medio de la técnica de codificación temática. RESULTADOS: surgieron cuatro categorías temáticas: facilidades de acceso al servicio y profesionales de la salud; orientaciones sobre la diabetes; participación en los grupos y experiencia con la diabetes; y, compartir conocimientos y experiencias. El aspecto más relevante de este estudio se refiere a los usos sociales que los informantes daban a los grupos Hiperdia investigados. CONCLUSIÓN: los grupos estudiados se mostraron capaces de producir sentidos y significados concernientes al proceso de enfermarse y a los modos de navegación social en el interior del sistema oficial de salud. El objetivo de este estudio es que pueda contribuir para las acciones de los profesionales de la salud que actúan en esos grupos, considerando la observación del universo cultural de los individuos que buscan cuidados profesionales en los diversos servicios públicos de salud. .

Effect of pH and temperature on stability and kinetics of novel extracellular serine alkaline protease (70 kDa).

A novel extracellular serine protease (70 kDa by SDS-PAGE) was purified and characterized. This enzyme retained more than 93% of its initial activity after preincubation for 30 min at 37 °C in the presence of 25% (v/v) tested organic solvents and showed feather degradation activity. The purified enzyme was deactivated at various combinations of pH and temperature to examine the interactive effect of them on enzyme activity. The deactivation process was modeled as first-order kinetics and the deactivation rate constant (k(d)) was found to be minimum at pH 9 and 37 °C. The kinetic analysis of enzyme over a range of pH values indicated two pK values at 6.21 and at 10.92. The lower pK value was likely due to the catalytic histidine in the free enzyme and higher pK value likely reflected deprotonation of the proline moiety of the substrate but ionization of the active site serine is another possibility. Inhibition kinetic showed that enzyme is serine protease because enzyme was competitively inhibited by antipain and aprotinin as these compounds are known to be competitive inhibitors of serine protease. The organic solvent, thermal and pH tolerances of enzyme suggested that it may have potential for use as a biocatalyst in industry.

Longistatin is an unconventional serine protease and induces protective immunity against tick infestation.

Classical serine proteases use the conserved Ser/His/Asp catalytic triad to hydrolyze substrates. Here, we show that longistatin, a salivary gland protein with two EF-hand domains from the vector tick Haemaphysalis longicornis, does not have the conserved catalytic triad, but still functions as a serine protease. Longistatin was synthesized in and secreted from the salivary glands of ticks, and is injected into host tissues during the acquisition of blood-meals. Longistatin hydrolyzed fibrinogen, an essential plasma protein in the coagulation cascade, and activated plasminogen, into its active form plasmin, a serine protease that dissolves fibrin clots. Longistatin efficiently hydrolyzed several serine protease-specific substrates showing its specificity to the amide bond of Arg. Longistatin did not hydrolyze synthetic substrates specific for other groups of proteases. The enzyme was active at a wide range of temperatures and pHs, with the optimum at 37°C and pH 7. Its activity was efficiently inhibited by various serine protease inhibitors such as phenylmethanesulfonyl fluoride (PMSF), aprotinin, antipain, and leupeptin with the estimated IC(50) of 278.57 µM, 0.35 µM, 41.56 µM and 198.86 µM, respectively. In addition, longistatin was also potently inhibited by Zinc (Zn(2+)) in a concentration-dependent manner with an IC(50) value of 275 µM, and the inhibitory effect of Zn(2+) was revived by ethylenediaminetetra acetic acid (EDTA). Immunization studies revealed that longistatin sharply induced high levels of protective IgG antibodies against ticks. Immunization with longistatin reduced repletion of ticks by about 54%, post engorgement body weight by >11% and molting of nymphs by approximately 34%; thus, the vaccination trial was approximately 73% effective against tick infestation. Taken together, our results suggest that longistatin is a new potent atypical serine protease, and may be an interesting candidate for the development of anti-tick vaccines.

Shedding of gACE from residual body membrane of rat sperm.

Germinal angiotensin I-converting enzyme (gACE) is expressed only in the testis and is uniquely present in developing spermatids and sperm. We previously purified soluble gACE from porcine seminal plasma, and reported that gACE was secreted from residual body on spermatozoa by the other peptidase(s), namely Sheddase. Using Nma/DNP substrate, it was observed that the shedding activity in testicular fluid was stronger than in the sperm membrane and epididymal fluid. The shedding activity was inhibited by AEBSF and antipain, and not by EDTA and E-64. Accordingly, it is thought that Sheddase is an endo-type of serine peptidase.

Phytomonas serpens: cysteine peptidase inhibitors interfere with growth, ultrastructure and host adhesion.

In this study, we report the ultrastructural and growth alterations caused by cysteine peptidase inhibitors on the plant trypanosomatid Phytomonas serpens. We showed that the cysteine peptidase inhibitors at 10 microM were able to arrest cellular growth as well as promote alterations in the cell morphology, including the parasites becoming short and round. Additionally, iodoacetamide induced ultrastructural alterations, such as disintegration of cytoplasmic organelles, swelling of the nucleus and kinetoplast-mitochondrion complex, which culminated in parasite death. Leupeptin and antipain induced the appearance of microvillar extensions and blebs on the cytoplasmic membrane, resembling a shedding process. A 40 kDa cysteine peptidase was detected in hydrophobic and hydrophilic phases of P. serpens cells after Triton X-114 extraction. Additionally, we have shown through immunoblotting that anti-cruzipain polyclonal antibodies recognised two major polypeptides in P. serpens, including a 40 kDa component. Flow cytometry analysis confirmed that this cruzipain-like protein has a location on the cell surface. Ultrastructural immunocytochemical analysis demonstrated the presence of the cruzipain-like protein on the surface and in small membrane fragments released from leupeptin-treated parasites. Furthermore, the involvement of cysteine peptidases of P. serpens in the interaction with explanted salivary glands of the phytophagous insect Oncopeltus fasciatus was also investigated. When P. serpens cells were pre-treated with either cysteine peptidase inhibitors or anti-cruzipain antibody, a significant reduction of the interaction process was observed. Collectively, these results suggest that cysteine peptidases participate in several biological processes in P. serpens including cell growth and interaction with the invertebrate vector.

Enzymatic characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis.

In the present study, we performed enzymatic characterization of Haemaphysalis longicornis serine proteinase (HlSP) with a view to shed light on the mechanisms of blood digestion in the hard ticks. Escherichia coli-expressed recombinant HlSP (rHlSP) was shown to potently hydrolyze the synthetic substrates Bz-(DL)-Arg-pNA, Z-Ala-Ala-Leu-pNA and Suc-Ala-Ala-Ala-pNA and yielded an activity of 31.5, 88.2 and 18.3 mumol/min/mg protein, respectively at an optimum temperature of 25 degrees C. However, the enzyme showed little activity to hydrolyze the substrates Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-MCA and Pyr-Phe-Leu-pNA. The optimum pH for the enzyme was shown to be 4.0 to 5.0. Several inhibitors such as antipain, leupeptin and phenylmethylsulfonyl fluoride (PMSF), specific for serine proteinase were shown to inhibit enzyme activity by 20-82%, while E-64 (specific for cysteine proteinases) and pepstatinA (specific for aspartic proteinases) had shown only little inhibitory effects on it. This is the first report on enzymatic characterization of a functional serine proteinase from the hard ticks.

Involvement of secreted Aspergillus fumigatus proteases in disruption of the actin fiber cytoskeleton and loss of focal adhesion sites in infected A549 lung pneumocytes.

Aspergillus fumigatus is an opportunistic pathogenic fungus that predominantly infects the respiratory system. Penetration of the lung alveolar epithelium is a key step in the infectious process. The cytoskeleton of alveolar epithelial cells forms the cellular basis for the formation of a physical barrier between the cells and their surroundings. This study focused on the distinct effects of A. fumigatus on the actin cytoskeleton of A549 lung pneumocytes. Of the 3 major classes of cytoskeletal fibers--actin microfilaments, microtubules, and intermediate filaments--only the actin cytoskeleton was found to undergo major structural changes in response to infection, including loss of actin stress fibers, formation of actin aggregates, disruption of focal adhesion sites, and cell blebbing. These changes could be specifically blocked in wild-type strains of A. fumigatus by the addition of antipain, a serine and cysteine protease inhibitor, and were not induced by an alkaline serine protease-deficient strain of A. fumigatus. Antipain also reduced, by approximately 50%, fungal-induced A549 cell detachment from the plates and reduction in viability. Our findings suggest that A. fumigatus breaches the alveolar epithelial cell barrier by secreting proteases that act together to disorganize the actin cytoskeleton and destroy cell attachment to the substrate by disrupting focal adhesions.

The effect of class-specific protease inhibitors on the stabilization of B-type natriuretic peptide in human plasma.

BACKGROUND: B-type natriuretic peptide (BNP) is a cardiac hormone that regulates hemodynamic equilibrium. In the circulation, its activity is controlled by proteolytic factors. Accurate measurement of BNP in a patient's plasma may be affected by degradation due to proteolysis. OBJECTIVE: We report on the identification and performance of classes of protease inhibitors that stabilize BNP in plasma. DESIGN AND METHODS: Using the Bayer ADVIA Centaur BNP assay, we measured the effect of arginine, serine and/or specific kallikrein protease inhibitors (PIs) on exogenous spiked or endogenous BNP in patient plasma. RESULTS: Compared to controls without inhibitor, all PIs were capable, to varying degrees, of retarding the rate of proteolytic degradation. The kallikrein-specific inhibitor, D-Phe-Phe-Arg-chloromethylketone (PPACK II) was most effective as a single constituent and was able to eliminate BNP degradation in patient samples for up to 6-10 days when stored at 2-8 degrees C. CONCLUSIONS: The stability of BNP was markedly increased in the presence of kallikrein-specific PPACK II and a broad spectrum of serine PIs. Use of these compounds offers a simple method of extending sample handling and storage of plasma samples containing BNP.

Isolation and characterization of a novel and potent inhibitor of Arg-gingipain from Streptomyces sp. strain FA-70.

Arg-gingipain (Rgp) is a major cysteine proteinase produced by the oral bacterium Porphyromonas gingivalis, which is a major pathogen of advanced periodontal diseases. This enzyme is important for the bacterium both to exhibit its virulence and to survive in periodontal pockets. The development of Rgp inhibitors thus provides new therapeutic approaches to periodontal diseases. In this study, we first isolated and purified a novel and potent inhibitor of Rgp from the culture supernatant of Streptomyces species strain FA-70, now designated as FA-70C1. This compound was found to be an antipain analog composed of phenylalanyl-ureido-citrullinyl-valinyl-cycloarginal (C27H43N9O7). The Ki value was calculated to be 4.5x10(-9) M when benzyloxycarbonyl-phenylalanyl-arginine-4-methly-coumaryl-7-amide was used as a substrate. This compound also inhibited cathepsins B, L, and H, though their Ki values were much higher than that of Rgp. FA-70C1 had little or no inhibitory activity on Lys-gingipain, another cysteine proteinase of P. gingivalis. The Rgp-induced degradation of various human proteins was completely blocked by this inhibitor. Disruption of both the bactericidal activity of polymorphonuclear leukocytes and the viability of human fibroblasts and umbilical vein endothelial cells induced by the culture supernatant of P. gingivalis was suppressed by the inhibitor in a dose-dependent manner. The enhancement of vascular permeability induced by in vivo administration of the culture supernatant of P. gingivalis was strongly inhibited by the inhibitor. Furthermore, the growth of P. gingivalis was suppressed by FA-70C1 in a dose-dependent manner. These results strongly suggest that FA-70C1 is a useful tool to prevent the virulence of P. gingivalis.