P-I metalloproteinases and L-amino acid oxidases from Bothrops species inhibit angiogenesis

Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia. In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. Methods: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. Results: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially β chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. Conclusion: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo.(AU)

Extracellular matrix metalloproteinase expression in endometrial tissue after arterial embolization of myomas

OBJECTIVES: Arterial embolization of myomas (AEM) is controversial because of the changes that occur in the extracellular matrix (ECM) of the endometrium and its effect on gestational success in infertile patients desiring reproductive capability. Therefore, we performed this study on the expression of genes in the ECM of the endometrium, such as those coding metalloproteinases (MMP), before and 6 months after embolization of the uterine arteries. METHODS: Seven women with leiomyomas were evaluated, and MMP3 and MMP10 levels were measured. The women underwent pelvic nuclear magnetic resonance (NMR), examination, and endometrial biopsy between the 20th and 24th day of the menstrual cycle, and pre- and post-AEM (after 6 months). For data analysis, the Cq comparative method, also known as the 2-ΔΔCT method, was used to calculate the relative quantities of MMP gene expression among the samples collected. RESULTS: There was a significant decrease by 9.52 times in the expression of MMP3 (p=0.007), and a non-significant change in the expression of MMP10 (p=0.22) in post-AEM-treated women than pre-AEM-treated women. CONCLUSIONS: The results suggest that ECM continues to undergo tissue remodeling 6 months after AEM, at least with regard to MMP3 expression, suggesting that AEM affects the ECM for at least 6 months after the procedure.

Tissue content of metalloproteinase-9 and collagen in the colon with and without fecal stream after intervention with infliximab in rats subjected to Hartmann's surgery

ABSTRACT Purpose Quantify the tissue content of metalloproteinase-9 (MMP-9) and collagen in colic mucosa with and without intestinal transit after infliximab administration in rats subjected to Hartmann's surgery. Methods Twenty-two rats underwent colon diversion by Hartmann's surgery. Animals were maintained with intestinal bypass for 12 weeks to induce development of diversion colitis (DC). Afterwards, animals were divided into three groups: first group received subcutaneous application of saline solution (SS) 0.9%, while the remaining two groups received infliximab subcutaneously at doses of 5 or 10 mg·kg-1·week-1 for five consecutive weeks. After the intervention, animals were sacrificed, removing the segments with and without intestinal transit. Diversion colitis was diagnosed by histological study, and its intensity was determined by a validated inflammatory scale. Tissue expression of MMP-9 was assessed byimmunohistochemistry, while total collagen was assessed by histochemistry. Tissue content of both was measuredby computerized morphometry. Results Colon segments without intestinal transit had a higher degree of inflammation, which improved in animals treated with infliximab. Collagen content was always lower in those without intestinal transit. There was an increase in the collagen content in the colon without transit in animals treated with infliximab, primarily at a dose of 10 mg·kg-1·week-1. There was an increase in the content of MMP-9 in the colon without fecal transit, and a reduction was observed in animals treated with infliximab, regardless of the dose used. Conclusions Application of infliximab reduces inflammation, increases the total collagen content and decreases the content of MMP-9 in the colon without intestinal transit.

In-depth transcriptome reveals the potential biotechnological application of Bothrops jararaca venom gland

Lack of complete genomic data of Bothrops jararaca impedes molecular biology research focusing on biotechnological applications of venom gland components. Identification of full-length coding regions of genes is crucial for the correct molecular cloning design. Methods: RNA was extracted from the venom gland of one adult female specimen of Bothrops jararaca. Deep sequencing of the mRNA library was performed using Illumina NextSeq 500 platform. De novo assembly of B. jararaca transcriptome was done using Trinity. Annotation was performed using Blast2GO. All predicted proteins after clustering step were blasted against non-redundant protein database of NCBI using BLASTP. Metabolic pathways present in the transcriptome were annotated using the KAAS-KEGG Automatic Annotation Server. Toxins were identified in the B. jararaca predicted proteome using BLASTP against all protein sequences obtained from Animal Toxin Annotation Project from Uniprot KB/Swiss-Pro database. Figures and data visualization were performed using ggplot2 package in R language environment. Results: We described the in-depth transcriptome analysis of B. jararaca venom gland, in which 76,765 de novo assembled isoforms, 96,044 transcribed genes and 41,196 unique proteins were identified. The most abundant transcript was the zinc metalloproteinase-disintegrin-like jararhagin. Moreover, we identified 78 distinct functional classes of proteins, including toxins, inhibitors and tumor suppressors. Other venom proteins identified were the hemolytic lethal factors stonustoxin and verrucotoxin. Conclusion: It is believed that the application of deep sequencing to the analysis of snake venom transcriptomes may represent invaluable insight on their biotechnological potential focusing on candidate molecules.(AU)

Factores que afectan y mejoran la adhesión en dentina, una puesta al día: una revisión de la literatura / Facts that affect and enhance adhesion on dentine, an update: a review of the literature

Objetivo: Analizar la evidencia disponible sobre los componentes de los adhesivos dentinarios, las causas de la falla de la interface dentinaadhesivo, las alternativas para disminuir este fenómeno y aumentar el tiempo de vida de las restauraciones adhesivas. Material y métodos: Se realizó una revisión de la literatura de tipo descriptiva, la búsqueda de artículos se llevó a cabo en diferentes bases de datos, bibliotecas electrónicas, buscadores académicos y búsqueda manual en revistas. Se revisaron 118 artículos, de los cuales se seleccionaron 68. Conclusiones: La adhesión dentinaria sufre degradación hidrolítica y degradación proteolítica; el acondicionado ácido de la dentina promueve la liberación de metaloproteinasas y con ello el inicio de la degradación de la interface dentino-adhesivo, por el momento los adhesivos con MDP brindan la mejor opción ya que presentan los mejores resultados para contrarrestar la degradación, además de encontrarse comercialmente al alcance de los profesionistas. Los sistemas que presentan una simplificación de la técnica en ocasiones pueden ir en detrimento de los resultados. Aún es necesario realizar investigación que conduzca a reducir la falla de la interface adhesiva a largo plazo y obtener restauraciones óptimas, duraderas y libres de microfiltraciones (AU)

Objective: Analyze the available evidence on the components of dentin adhesives, the causes of failure of the dentin-adhesive interface, the alternatives to reduce this phenomenon and increase the lifetime of adhesive restorations. Material and methods: A review of the descriptive literature was made, the search of articles was carried out in different databases, electronic libraries, academic search engines and manual search in journals. 118 articles were reviewed, of which 68 were selected. Conclusions: Dentin adhesion suffers from hydrolytic and proteolytic degradation; the acid conditioning of dentine promotes the release of metalloproteinases and with it the beginning of the degradation of the dentin-adhesive interface, for the moment the adhesives with MDP offer the best option since they present the best results to counteract the degradation besides being commercially available to professionals. Simpli file Systems less can sometimes be detrimental to the results. It is still necessary to carry out investigations that leads to a reduction on the failure of the long-term adhesive interface and obtain optimal, durable and microfiltration-free restorations (AU)

Matrix Metalloproteinase 2: A Possible Role inTooth Development and Eruption / Metaloproteinasa de matriz 2: Un posible papel en el desarrollo dental y la erupción

Abstract 18. Introduction: Tooth development results from a highly coordinated epithelial-mesenchyme interaction in which mesenchyme cells originate the dental papilla and dental follicle, while ectodermal cells originate the enamel organ. Simultaneously, bone tissue is formed around the developing tooth, trapping it in a bony crypt. Tooth eruption requires the resorption of the coronal part of the bony crypt, followed by degradation of the lamina propria, most likely by metalloproteinases (MMPs) activity. Objectives: The aim of this research was to determine MMP-2 expression in the dental germ cells (ameloblasts, odontoblasts, dental papilla and dental follicle) and surrounding tissues (alveolar bone and lamina propria) of rat molars throughout the eruptive process. Material and Methods: A total of 24 rats (4,6,9,11,14 and 16 days old) were used in this study. MMP-2 was detected through immunohistochemistry. A qualitative analysis was performed to investigate the expression of MMP2 in the dental germ cells, lamina propria, and coronal and basal regions of the bony crypt. Results: MMP-2 expression was observed in the dental papilla cells, dental follicle, ameloblasts, odontoblasts and bone cells from the coronal and basal regions of the bony crypt. MMP-2 was also detected in the lamina propria during the mucosal penetration stage of tooth eruption. Conclusion: We conclude that MMP-2 may be important for the extracellular matrix rearrangement necessary for tooth development and secretion of its mineralized tissues. We also conclude that MMP-2 may play a role in the extensive tissue remodeling during the intra-and-extra-osseous phases of the tooth eruption process.

Resumen 24. Introducción: el desarrollo del diente resulta de una interacción epitelial-mesénquima altamente coordinada en la cual las células mesénquima originan la papila dental y el folículo dental, mientras que las células ectodérmicas originan el órgano del esmalte. Simultáneamente, el tejido óseo se forma alrededor del diente en desarrollo y lo atrapa en una cripta ósea. La erupción dentaria requiere la resorción de la parte coronal de la cripta ósea, seguida de la degradación de la lámina propia, muy probablemente por la actividad metaloproteinasas (MMPs). Objetivos: el objetivo de esta investigación fue determinar la expresión de MMP-2 en las células germinales dentales (ameloblastos, odontoblastos, papila dentaria y folículo dentario) y tejidos circundantes (hueso alveolar y lámina propia) de molares de rata a lo largo del proceso eruptivo. Material y métodos: en este estudio se utilizó un total de 24 ratas (4,6,9,11,14 y 16 días de edad). MMP-2 se detectó a través de inmunohistoquímica. Un análisis cualitativo fue realizado para investigar la expresión de MMP-2 en las células de germen dentales, el lámina propria, y las regiones coronales y basales de la cripta ósea. Resultados: la expresión de MMP2 fue observada en las células de la papila dental, el folículo dental, el ameloblastos, el odontoblastos y las células del las regiones basales y coronales de la cripta ósea. La expresión de MMP-2 también se detectó en la lámina propia durante la etapa de penetración de la mucosa de la erupción dental. Conclusión: Concluimos que MMP-2 puede ser importante para el cambio extracelular de la matriz necesario para el desarrollo del diente y la secreción de sus tejidos mineralizados. También concluimos que MMP-2 puede desempeñar un papel en la remodelación extensa del tejido durante las fases intra y extraósea del proceso de erupción dental.

tPA Helpers in the Treatment of Acute Ischemic Stroke: Are They Ready for Clinical Use? / 대한뇌졸중학회지

Tissue plasminogen activator (tPA) is the only therapeutic agent approved to treat patients with acute ischemic stroke. The clinical benefits of tPA manifest when the agent is administered within 4.5 hours of stroke onset. However, tPA administration, especially delayed administration, is associated with increased intracranial hemorrhage (ICH), hemorrhagic transformation (HT), and mortality. In the ischemic brain, vascular remodeling factors are upregulated and microvascular structures are destabilized. These factors disrupt the blood brain barrier (BBB). Delayed recanalization of the vessels in the presence of relatively matured infarction appears to damage the BBB, resulting in HT or ICH, also known as reperfusion injury. Moreover, tPA itself activates matrix metalloproteases, further aggravating BBB disruption. Therefore, attenuation of edema, HT, or ICH after tPA treatment is an important therapeutic strategy that may enable clinicians to extend therapeutic time and increase the probability of excellent outcomes. Recently, numerous agents with various mechanisms have been developed to interfere with various steps of ischemia/reperfusion injuries or BBB destabilization. These agents successfully reduce infarct volume and decrease the incidence of ICH and HT after delayed tPA treatment in various animal stroke models. However, only some have entered into clinical trials; the results have been intriguing yet unsatisfactory. In this narrative review, I describe such drugs and discuss the problems and future directions. These “tPA helpers” may be clinically used in the future to increase the efficacy of tPA in patients with acute ischemic stroke.

Conditioned Medium from Tonsil-Derived Mesenchymal Stem Cells Relieves CCl₄-Induced Liver Fibrosis in Mice

BACKGROUND: The liver is an organ with remarkable regenerative capacity; however, once chronic fibrosis occurs, liver failure follows, with high mortality and morbidity rates. Continuous exposure to proinflammatory stimuli exaggerates the pathological process of liver failure; therefore, immune modulation is a potential strategy to treat liver fibrosis. Mesenchymal stem cells (MSCs) with tissue regenerative and immunomodulatory potential may support the development of therapeutics for liver fibrosis. METHODS: Here, we induced hepatic injury in mice by injecting carbon tetrachloride (CCl₄) and investigated the therapeutic potential of conditionedmedium from tonsil-derivedMSCs (T-MSCCM). In parallel, we used recombinant human IL-1Ra,which, as we have previously shown, is secreted exclusively from T-MSCs and resolves the fibrogenic activation of myoblasts. Hepatic inflammation and fibrosis were determined by histological analyses using H&E and Picro-Sirius Red staining. RESULTS: The results demonstrated that T-MSC CM treatment significantly reduced inflammation as well as fibrosis in the CCl₄-injured mouse liver. IL-1Ra injection showed effects similar to T-MSC CM treatment, suggesting that T-MSC CM may exert anti-inflammatory and anti-fibrotic effects via the endogenous production of IL-1Ra. The expression of genes involved in fibrosis was evaluated, and the results showed significant induction of alpha-1 type I collagen, transforming growth factor beta, and tissue inhibitor of metalloproteases 1 upon CCl₄ injection, whereas treatment with T-MSC CM or IL-1Ra downregulated their expression. CONCLUSION: Taken together, these data support the therapeutic potential of T-MSC CM and/or IL-1Ra for the alleviation of liver fibrosis, as well as in treating diseases involving organ fibrosis.

Development of LysargiNase, a mirror trypsin and its application in proteomics / 生物工程学报

Proteomics is a fast-growing discipline that aims at systematic identification, quantification of proteins and their post-translational modifications in cells. Mass spectrometry-based shotgun proteomics technology is currently one of the mainstream methods for proteomics research. With this method, proteins need to be digested to peptides by site-specific proteases before they can be detected with mass spectrometry. Therefore, site-specific proteases played key roles in this process and so far, a variety of specific proteases have been developed and used in proteomics study. Particularly, the identification, characterization and development of proteases that cleave at the N-termini of corresponding amino acid residues, which are just mirrors to those of typical C-termini proteases, provide novel tools for proteomics analysis. In this review, we summarized the proprieties of LysargiNase, a most recently identified mirror trypsin, and its applications in proteomics research to promote its more widespread usage.

Evaluation of Anti-inflammatory and Regenerative Efficiency of Naringin and Naringenin in Degenerated Human Nucleus Pulposus Cells: Biological and Molecular Modeling Studies

STUDY DESIGN: Development of an in vitro model for assessing the anti-inflammatory efficacies of naringin (Nar) and naringenin (NG).PURPOSE: To evaluate the efficacy of natural flavonoids as therapeutic drugs against anti-inflammatory processes in the nucleus pulposus (NP) cells using in-vitro and in-silico methods.OVERVIEW OF LITERATURE: Intervertebral disc (IVD) disease is a common cause of low back pain. Chronic inflammation and degeneration play a significant role in its etiopathology. Thus, a better understanding of anti-inflammatory agents and their role in IVD degeneration and pro-inflammatory cytokines expression is necessary for pain management and regeneration in IVD.METHODS: We performed primary cell culture of NP cells; immunocytochemistry; gene expression studies of cytokines, metalloproteases, extracellular proteins, and apoptotic markers using quantitative polymerase chain reaction and reverse transcription-polymerase chain reaction (RT-PCR); cytotoxicity assay (MTT); and molecular docking studies using AutoDock 4.2 software (Molecular Graphics Laboratory, La Jolla, CA, USA) to confirm the binding mode of proteins and synthesized complexes. We calculated the mean±standard deviation values and performed analysis of variance and t-test using SPSS ver. 17.0 (SPSS, Inc., Chicago, IL, USA).RESULTS: Molecular docking showed that both Nar and NG bind to the selected genes of interest. Semi-quantitative RT-PCR analysis reveals differential gene expression of collagen (COL)9A1, COL9A2, COL9A3, COL11A2, COMT (catechol-O-methyltransferase), and THBS2 (thrombospondin 2); up regulation of ACAN (aggrecan), COL1A1, COL11A1, interleukin (IL)6, IL10, IL18R1, IL18RAP, metalloprotease (MMP)2, MMP3, MMP9, ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5), IGF1R (insulin-like growth factor type 1 receptor), SPARC (secreted protein acidic and cysteine rich), PARK2 (parkin), VDR (vitamin D receptor), and BCL2 (B-cell lymphoma 2); down regulation of IL1A, CASP3 (caspase 3), and nine genes with predetermined concentrations of Nar and NG.CONCLUSIONS: The present study evaluated the anti-inflammatory and regenerative efficiencies of Nar and NG in degenerated human NP cells. Altered gene expressions of cytokines, metalloproteases, extracellular proteins, apoptotic genes were dose responsive. The molecular docking (in silico) studies showed effective binding of these native ligands (Nar and NG) with genes identified as potent inhibitors of inflammation. Thus, these natural flavonoids could serve as anti-inflammatory agents in the treatment of low back pain and sciatica.

Proteome of fraction from Tityus serrulatus venom reveals new enzymes and toxins

Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and characterization of these components. Although a considerable number of studies have been dedicated to the characterization of this complex cocktail, there is still a limitation of knowledge concerning its venom composition. Most of Ts venom studies aim to isolate and characterize their neurotoxins, which are small, basic proteins and are eluted with high buffer concentrations on cation exchange chromatography. The first and largest fraction from carboxymethyl cellulose-52 (CMC-52) chromatography of Ts venom, named fraction I (Fr I), is a mixture of proteins of high and low molecular masses, which do not interact with the cation exchange resin, being therefore a probable source of components still unknown of this venom. Thus, the present study aimed to perform the proteome study of Fraction I from Ts venom, by high resolution mass spectrometry, and its biochemical characterization, by the determination of several enzymatic activities. Methods: Fraction I was obtained by a cation exchange chromatography using 50 mg of crude venom. This fraction was subjected to a biochemical characterization, including determination of L-amino acid oxidase, phospholipase, hyaluronidase, proteases activities and inhibition of angiotensin converting enzyme (ACE) activity. Fraction I was submitted to reduction, alkylation and digestion processes, and the tryptic digested peptides obtained were analyzed in a Q-Exactive Orbitrap mass spectrometer. Data analysis was performed by PEAKS 8.5 software against NCBI database. Results: Fraction I exhibits proteolytic activity and it was able to inhibit ACE activity. Its proteome analysis identified 8 different classes of venom components, among them: neurotoxins (48%), metalloproteinases (21%), hypotensive peptides (11%), cysteine-rich venom protein (9%), antimicrobial peptides (AMP), phospholipases and other enzymes (chymotrypsin and lysozymes) (3%) and phosphodiesterases (2%). Conclusions: The combination of a proteomic and biochemical characterization strategies leads us to identify new components in the T. serrulatus scorpion venom. The proteome of venom´s fraction can provide valuable direction in the obtainment of components in their native forms in order to perform a preliminary characterization and, consequently, to promote advances in biological discoveries in toxinology.(AU)

Metaloproteinases no tecido laminar do casco de equinos submetidos à obstrução intraluminal do cólon menor / Metalloproteinases in hoof laminar tissue of equine submitted to intraluminal small colon obstruction

O objetivo deste estudo foi avaliar a expressão das MMP-2 e MMP-9 no tecido laminar do casco e o perfil leucocitário de equinos submetidos à obstrução intraluminal do cólon menor. Realizaram-se laparotomia e obstrução do cólon menor de oito equinos hígidos, utilizando-se uma bola inserida no lúmem intestinal. A bola foi inflada à pressão de 80mmHg e a obstrução foi mantida por quatro horas. Foram realizadas coletas sanguíneas antes da obstrução (M0), imediatamente após a desobstrução (M4) e a cada 12 horas após M4, até completar 72 horas (M12, M24, M36, M48, M60 e M72). As biópsias de casco foram realizadas em M0, M4 e M72, e as amostras foram submetidas à análise zimográfica. Foi observado aumento nos leucócitos em M12 e M24, decorrente do aumento de neutrófilos segmentados e bastonetes, os quais diminuíram a partir de M36. Segundo a técnica zimográfica, não se observaram alterações nos valores de MMP-2 e -9, possivelmente devido à baixa intensidade das lesões ocasionadas no cólon menor. Com isso, conclui-se que as alterações inflamatórias decorrentes da obstrução do cólon menor não foram suficientes para ocasionar alterações na expressão das MMP-2 e -9 no tecido laminar podal.(AU)

The aim of this study was to evaluate the blood leukocytes and the MMP-2 and -9 expression in the hoof laminar tissue of horses undergoing intraluminal small colon obstruction. Laparotomy and the small colon obstruction was performed in eight healthy horses, inserting a ball in the intestinal lumen. The ball was inflated to 80 mmHg pressure and the occlusion was maintained for 4 hours. The blood was collectedBlood samples were taken before the obstruction (M0), immediately after intestinal clearance (M4), and every 12 hours until completeuntil 72 hours (M12, M24, M36, M48, M60 and M72). The hoof biopsies were performed at M0, M4, and M72 and the samples were subjected to zymography analysis. There was an increase in leukocytes in M12 and M24, due to the increase in segmented neutrophils and band neutrophils, which decreased as of M36. According to zymography technique not observed changes were not not observed in MMP-2 and -9, possibly due to the low intensity of the small colon lesions. Wherefore, it is concludedIn conclusion, that the inflammatory changes resulting from small colon obstruction were not enough to cause changes in the expression of MMP-2 and -9 in the hoof laminar tissue.(AU)

Proteases are Modulated by Fascin in Oral Cancer Invasion

BACKGROUND: Cancer invasion is a critical factor for survival and prognosis of patients with cancer. Identifying and targeting factors that influence cancer invasion are an important strategy to overcome cancer. In this study, we investigated the role of fascin known to be associated with cancer invasion. METHODS: Fascin depletion was performed with lentiviral short hairpin RNA against fascin mRNA and stable cell line (Fascin(dep)) was established. Matrigel-Transwell invasion and three-dimensional (3D) culture system were used to observe fascin depletion effects. In order to observe the changes of protease secretion by fascin depleted cancer cells, protease antibody array was performed. RESULTS: Fascin was highly expressed in invasive cancer cells. Fascin-depleted cells showed decreased cancer invasion in Matrigel-Transwell invasion and 3D culture system. In addition, inhibition of proteases secreation and decrease of intracellular proteases mRNA expression were observed in fascin deplete cells. CONCLUSIONS: These results indicates that fascin is closely involved in proteases activity and cancer invasion. Therefore, fascin is a strategically important factor for controlling cancer invasion.

Expression of adhesion molecules on CD34+ cells from steady-state bone marrow before and after mobilization and their association with the yield of CD34+ cells

BACKGROUND: Cell adhesion molecules (CAMs) expressed on hematopoietic progenitor cells (HPCs), endothelial cells, and stromal cells play a pivotal role in the mobilization of CD34+ cells. Herein, we conducted a non-randomized peripheral blood stem cell (PBSC) mobilization study aimed to compare the potential differences in the expressions of several CAMs and chemokines on CD34+ cells obtained from bone marrow aspirate before and after HPC mobilization from patients with hematologic malignancies and healthy donors. METHODS: Three-color cytofluorometric analysis was used to compare the expressions of CAMs and chemokines in the bone marrow before and after mobilization. RESULTS: For all studied groups, CAM expression among those with good and poor yields of CD34+ cells was significantly correlated with VCAM-1 (P=0.007), CD44 (P=0.027), and VLA-4 (P=0.014) expressions. VCAM-1 (P=0.001), FLT-3 (P=0.001), CD44 (P=0.011), VLA-4 (P=0.001), and LFA-1 (P=0.001) expressions were higher before HPC mobilization than after HPC mobilization. By contrast, the expression of CXCR4 significantly varied before and after mobilization only among those with successful PBSC mobilization (P=0.002). CONCLUSION: We attempted to identify particular aspects of CAMs involved in CD34+ cell mobilization, which is a highly complex mechanism that involves adhesion molecules and matrix metalloproteases. The mechanism by which CD34+ cell mobilization is activated through proteolytic enzymes is not fully understood. We believe that CXCR4, VLA-4, CD44, and VCAM-1 are the most important molecules implicated in HPC mobilization, particularly because they show a correlation with the yield of CD34+ cells collected via large volume leukapheresis.

Serum matrix metalloproteinase-1 level represents disease activity as opposed to fibrosis in patients with histologically proven nonalcoholic steatohepatitis

BACKGROUND/AIMS: Nonalcoholic steatohepatitis (NASH) is prevalent in both economically developed and developing countries. Twenty percent of NASH progresses to cirrhosis with/without hepatocellular carcinoma, and there is an urgent need to find biomarkers for early diagnosis and monitoring progression of the disease. Using immunohistochemical and immunoelectron microscopic examination we previously reported that expression of matrix metalloproteinase-1 (MMP-1) increased in monocytes, Kupffer cells and hepatic stellate cells in early stage NASH. The present study investigated whether serum MMP-1 levels reflect disease activity and pharmaceutical effects in NASH patients. METHODS: We measured the serum levels of MMPs, tissue inhibitors of metalloproteinases (TIMPs), and several cytokines/chemokines in patients with histologically proven early and advanced stages of NASH and compared them with those in healthy controls. RESULTS: Serum MMP-1 levels in stage 1 fibrosis, but not in the more advanced fibrosis stages, were significantly higher than in healthy controls (P=0.019). There was no correlation between serum MMP-1 level and fibrosis stage. Serum MMP- 1 levels in NASH patients represented disease activity estimated by serum aminotransferase values during the follow-up period. In contrast, MMP-2, MMP-9 and TIMPs did not change with disease activity. Consistent with the finding that MMP-1 is expressed predominantly in monocytes and Kupffer cells, serum levels of monocyte chemotactic protein-1 and granulocyte-colony stimulating factor were significantly increased in NASH with stage 1 fibrosis. CONCLUSIONS: These results suggest that serum MMP-1 levels represent disease activity and may serve as a potential biomarker for monitoring the progression of NASH.

Purification and enzymatic characterization of a novel metalloprotease from Lachesis muta rhombeata snake venom

Lachesis muta rhombeata (Lmr) is the largest venomous snake in Latin America and its venom contains mainly enzymatic components, such as serine and metalloproteases, L-amino acid oxidase and phospholipases A2. Metalloproteases comprise a large group of zinc-dependent proteases that cleave basement membrane components such as fibronectin, laminin and collagen type IV. These enzymes are responsible for local and systemic changes, including haemorrhage, myonecrosis and inflammation. This study aimed the isolation and enzymatic characterization of the first metalloprotease (Lmr-MP) from Lmr venom (LmrV). Methods and results: Lmr-MP was purified through two chromatographic steps and submitted to enzymatic characterization. It showed proteolytic activity on azocasein with maximum activity at pH 7.0-9.0. It was inhibited by EDTA (a metal chelator that removes zinc, which is essential for enzymatic activity) and no effect was observed with PMSF, iodoacetic acid or pepstatin (inhibitors of serine, cysteine and aspartyl proteases, respectively). Ca2+, Mg2+ and Ba2+ ions increased its activity, while Al3+, Cu2+, Ni2+ and Zn2+ inhibited it. Additionally, ZnCl2 showed a dose dependent inhibition of the enzyme. Lmr-MP activity was also evaluated upon chromogenic substrates for plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) showing the highest activity on S-2302. The activity in different solutions (5 mM or 50 mM ammonium bicarbonate, pH 7.8; 0.1% trifluoroacetic acid + 50% acetonitrile; phosphate buffer saline, pH 7.4; 50 mM sodium acetate, pH 4.0 or ammonium acetate pH 4.5) was also evaluated and the results showed that its activity was abolished at acidic pHs. Its molecular mass (22,858 Da) was determined by MALDI-TOF and about 90% of its primary structure was verified by high-resolution mass spectrometry using HCD and ETD fragmentations and database search against the sequence of closely related species. It is a novel enzyme which shared high identity with other snake venom metalloproteases (svMPs) belonging to the P-I group. Conclusion: The purification procedure achieved a novel pure highly active metalloprotease from LmrV. This new molecule can help to understand the metalloproteases mechanisms of action, the Lachesis envenoming, as well as to open new perspectives for its use as therapeutic tools.(AU)

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion

Animal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads' secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules. Methods: Total RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman's degradation. Results: We were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion. Conclusions: We identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored.(AU)

Produção e caracterização de protease fibrinolítica de Streptomyces parvulus DPUA 1573 / Production and characterization of fibrinolytic protease from Streptomyces parvulus DPUA 1573

As proteases fibrinolíticas são capazes de degradar coágulos de fibrina formados dentro dos vasos sanguíneos, evitando a trombose intravascular. Em animais, a tromboflebite, que acomete frequentemente os equinos, ocasiona, em seus casos graves, a obstrução jugular e também um edema de laringe, derivando a obstrução das vias aéreas, o que possibilita um edema cerebral, ocorrendo o óbito do animal. Devido ao fato de o tratamento ser de custo elevado, faz-se necessária a investigação de outras fontesde proteases fibrinolíticas com custos menores e com menos efeitos colaterais. Diante disso, este estudo tem como objetivo produzir e caracterizar proteases fibrinolíticas obtidas de Streptomyces parvulus DPUA 1573. Para produção da enzima, foi utilizado um planejamento fatorial 24 avaliando a concentração da farinha de soja (0,5, 1,0 e 1,5%) e da glicose (0, 0,5 e 1,0g/L), temperatura (28, 32 e 37ºC) e agitação (150, 200 e 250rpm) sobre a biomassa e a atividade fibrinolítica. Pode-se verificar que a protease fibrinolítica apresentou atividade máxima (835U/mL) nas condições de concentração de 1,5% de soja, 1g/L de glicose, 28°C e 150rpm com 48 horas de fermentação. A protease fibrinolítica obtida teve temperatura e pH ótimos de 55°C e pH 9,0, respectivamente. A atividade enzimática foi inibida pelo EDTA, pelo íon Fe2+ e pelo SDS, o que indicou a enzima ser uma metaloprotease. A linhagem Streptomyces parvulus DPUA 1573 foi capaz de produzir protease fibrinolítica, possuindo características bioquímicas favoráveis à aplicação na medicina veterinária e possivelmente humana.(AU)

Fibrinolytic proteases are able to degrade fibrin clot formed in the blood vessel, avoiding intravascular thrombosis. In animals, thrombophlebitis often affects horses, and in severe cases causes obstruction of the jugular and laryngeal edema leading to airway obstruction allowing cerebral edema resulting in the death of the animal. Since treatment is costly, the investigation of other sources of fibrinolytic proteases at lower cost and with fewer side effects is needed. Thus, this study aims to produce and characterize fibrinolytic proteases from Streptomyces parvulus DPUA 1573. For enzyme production, a factorial design was performed to evaluate 24 soybean flour concentration (0.5, 1.0 and 1.5%) and glucose (0, 0.5 and 1.0g/L), temperature (28, 32 and 37°C) and agitation (150, 200 and 250rpm) on biomass and fibrinolytic activity. Fibrinolytic protease showed maximum activity (835 U/mL) under these conditions: 1.5% soybean flour, 1g/L glucose, 28°C, and 150rpm 48 hours of fermentation. The optimal temperature was 55°C and optimal pH was 9.0. Fibrinolytic protease activity was inhibited by EDTA, the ion Fe2+, and by SDS, which indicated that the enzyme is a metallo-protease. The strain Streptomyces parvulus DPUA 1573 was able to produce fibrinolytic protease with biochemical characteristics favorable for application in veterinary and human medicine.(AU)

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 − groups, present in BaltDC, form hydrogen bonds with the PO 2 − groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.(AU)

In vitro characterization of jellyfish venom fibrin(ogen)olytic enzymes from Nemopilema nomurai

Background: Because jellyfish are capable of provoking envenomation in humans, they are considered hazardous organisms. Although the effects of their toxins are a matter of concern, information on the venom components, biological activity and pathological mechanisms are still scarce. Therefore, the aim of the present study was to investigate a serine protease component of Nemopilema nomurai jellyfish venom (NnV) and unveil its characteristics. Methods: To determine the relationship between fibrinolytic activity of NnV and the serine protease, fibrin zymography was performed using metalloprotease and serine protease inhibitors. The biochemical characterization of serine proteases of NnV were determined by the amidolytic assay. Fractions with fibrinolytic activity were obtained by DEAE cation exchange column. Results: NnV displayed fibrinolytic activities with molecular masses of approximately 70, 35, 30, and 28 kDa. The fibrinolytic activity of NnV was completely obliterated by phenylmethylsulfonyl fluoride, a prototype serine protease inhibitor. Based on amidolytic assays using chromogenic substrates specific for various kinds of serine proteases, NnV predominantly manifested a chymotrypsin-like feature. Its activity was completely eliminated at low pH (< 6) and high temperatures (> 37 °C). Some metal ions (Co2+, Cu2+, Zn2+ and Ni2+) strongly suppressed its fibrinolytic activity, while others (Ca2+ and Mg2+) failed to do so. Isolation of a serine protease with fibrionolytic activity from NnV revealed that only p3 showed the fibrinolytic activity, which was completely inhibited by PMSF. Conclusion: The present study showed that N. nomurai jellyfish venom has a chymotrypsin-like serine protease with fibrinolytic activity. Such information might be useful for developing clinical management of jellyfish envenomation and pharmacological agents with therapeutic potential for thrombotic diseases in the future.(AU)