RESUMEN

Aminopeptidase A (APA) is a membrane-bound zinc metallopeptidase involved in the production of angiotensin III, one effector peptide of the brain renin-angiotensin system, making brain APA a relevant pharmacological target for the development of novel therapeutic treatments against hypertension and heart failure. The structure-based design of new APA inhibitors is described, based on previously developed thiol-containing inhibitors and APA crystal structure. Chemical synthesis, in vitro assessment against APA activity, pharmacological and pharmacokinetic profiling were performed, ultimately leading to a potent and selective APA inhibitor.

RESUMEN

Non-venomous snakes commonly evolve natural resistance to venom to escape predators. Sinonatrix annularis serum has been shown to inhibit Deinagkistrodon acutus venom-induced hemorrhage and upregulation of serum CK, CK-MB, LDH, AST and ALT levels. Using TMT-labeled proteomics analysis, 168 proteins were found to be altered significantly in the envenomed gastrocnemius muscle and categorized into pathways such as complement and coagulation cascades, leukocyte transendothelial migration, and JAK/STAT signaling. These alterations were mitigated by S. annularis serum. Subsequently, a novel metalloproteinase inhibitor, SaMPI, was isolated from S. annularis serum by two-step chromatography. It showed strong antidotal effects against D. acutus envenomation, including inhibition of subcutaneous bleeding caused by crude venom and DaMP (a metalloproteinase derived from D. acutus) activity in a 1:1 ratio. Histology and immunoblotting analyses demonstrated that SaMPI mitigated myonecrosis, reduced neutrophil infiltration and local inflammatory factor release, and retarded JAK/STAT and MAPK signaling activation. Analysis of the SaMPI gene cloned by 5'-RACE revealed a shared sequence identity of 58-79% with other SVMP inhibitors. These findings demonstrate the protective effects of SaMPI and indicate its potential value as a candidate for viper bite adjuvant therapy.

Asunto(s)

Venenos de Crotálidos , Humanos , Venenos de Crotálidos/toxicidad , Hemorragia , Antídotos , Metaloproteasas

RESUMEN

BACKGROUND: Although sepsis accounts for 1 in 5 deaths globally, few molecular therapies exist for this condition. The development of effective biomarkers and treatments for sepsis requires a more complete understanding of host responses and pathogenic mechanisms at early stages of disease to minimize host-driven pathology. METHODS: An alternative to the current symptom-based approach used to diagnose sepsis is a precise assessment of blood proteomic changes during the onset and progression of Salmonella Typhimurium (ST) murine sepsis. FINDINGS: A distinct pattern of coagulation factor protein abundance was identified in the pre-septic state- prior to overt disease symptoms or bacteremia- that was predictive of the dysregulation of fibrinolytic and anti-coagulant activities and resultant consumptive coagulopathy during ST murine sepsis. Moreover, the changes in protein abundance observed generally have the same directionality (increased or decreased abundance) reported for human sepsis. Significant overlap of ST coagulopathic activities was observed in Gram-negative Escherichia coli- but not in Gram-positive staphylococcal or pneumococcal murine sepsis models. Treatment with matrix metalloprotease inhibitors prevented aberrant inflammatory and coagulopathic activities post-ST infection and increased survival. Antibiotic treatment regimens initiated after specific changes arise in the plasma proteome post-ST infection were predictive of an increase in disease relapse and death after cessation of antibiotic treatment. INTERPRETATION: Altered blood proteomics provides a platform to develop rapid and easy-to-perform tests to predict sepsis for early intervention via biomarker incorporation into existing blood tests prompted by patient presentation with general malaise, and to stratify Gram-negative and Gram-positive infections for appropriate treatment. Antibiotics are less effective in microbial clearance when initiated after the onset of altered blood proteomics as evidenced by increased disease relapse and death after termination of antibiotic therapy. Treatment failure is potentially due to altered bacterial / host-responses and associated increased host-driven pathology, providing insight into why delays in antibiotic administration in human sepsis are associated with increased risk for death. Delayed treatment may thus require prolonged therapy for microbial clearance despite the prevailing notion of antibiotic de-escalation and shortened courses of antibiotics to improve drug stewardship. FUNDING: National Institutes of Health, U.S. Army.

Asunto(s)

Bacteriemia , Infecciones Neumocócicas , Sepsis , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Bacteriemia/microbiología , Biomarcadores , Factores de Coagulación Sanguínea/uso terapéutico , Humanos , Ratones , Infecciones Neumocócicas/tratamiento farmacológico , Proteómica , Recurrencia , Sepsis/complicaciones , Sepsis/tratamiento farmacológico

RESUMEN

Bee venom is a mixture of bioactive components that include proteases and protease inhibitors. A metalloprotease inhibitor has been predicted to be a bumblebee-specific toxin in the venom proteome of Bombus terrestris; however, the identification and functional roles of bee venom metalloprotease inhibitors have not been previously determined. In this study, we identified a bumblebee (B. ignitus) venom metalloprotease inhibitor (BiVMPI) that exhibits anti-fibrinolytic activity. BiVMPI contains a trypsin inhibitor-like cysteine-rich domain that exhibits similarity to inducible metalloprotease inhibitor. Using an anti-BiVMPI antibody raised against a recombinant BiVMPI protein produced in baculovirus-infected insect cells, the presence of BiVMPI in the venom gland and secreted venom of B. ignitus worker bees was confirmed. The recombinant BiVMPI protein demonstrated inhibitory activity against a metalloprotease, trypsin, chymotrypsin, protease K, and plasmin, but not subtilisin A, elastase, or thrombin. Additionally, the recombinant BiVMPI bound to plasmin and inhibited the plasmin-mediated degradation of fibrin, demonstrating an anti-fibrinolytic role for BiVMPI as a bee venom metalloprotease inhibitor. Our results provide the first evidence for the identification and anti-fibrinolytic activity of a metalloprotease inhibitor from bee venom.

Asunto(s)

Venenos de Abeja/química , Fibrinógeno/química , Proteínas de Insectos/química , Inhibidores de la Metaloproteinasa de la Matriz/química , Proteínas Recombinantes/química , Animales , Abejas , Fibrinolisina/química , Humanos

RESUMEN

OBJECTIVE: This study investigated the qualitative and semi-quantitative expression of metalloproteinases (MMP) and their tissue inhibitors (TIMP) in trophoblastic tissue during ampullary ectopic pregnancies and correlated that expression with the degree of tubal invasion. STUDY DESIGN: It is a prospective study that included 34 patients diagnosed with ampullary tubal pregnancy who underwent salpingectomy. A histological evaluation of the depth of trophoblastic invasion in the tubes obtained was performed. Subsequently, the expression of the MMP-2, MMP-9, MMP-14, TIMP-1, TIMP-2 and TIMP-3 markers was qualitatively and semi-quantitatively evaluated by indirect immunohistochemistry. In addition, the degree of trophoblastic invasion was correlated with the expression of each marker and with the metalloproteinase/inhibitor ratios. RESULTS: MMP-2 (11.2 %; 3.6-17.9) was the marker with greater expression at the implantation site, both in the qualitative and semi-quantitative assessment, while MMP-9 (2.23 %; 0.2-5.4) and TIMP-3 (2.53 %; 0.1-15.3) were only weakly expressed. CONCLUSION: There was wide variation in expression among the markers and metalloproteinase/inhibitor ratios studied compared to the degrees of invasion.

Asunto(s)

Metaloproteinasas de la Matriz/metabolismo , Embarazo Tubario/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Trofoblastos/metabolismo , Adulto , Biomarcadores/metabolismo , Femenino , Humanos , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Embarazo , Embarazo Tubario/enzimología , Embarazo Tubario/patología , Embarazo Tubario/cirugía , Estudios Prospectivos , Salpingectomía , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Trofoblastos/patología

RESUMEN

BACKGROUND: Pulmonary damage by Pseudomonas aeruginosa during cystic fibrosis lung infection and ventilator-associated pneumonia is mediated both by pathogen virulence factors and host inflammation. Impaired immune function due to tissue damage and inflammation, coupled with pathogen multidrug resistance, complicates the management of these deep-seated infections. Pathological inflammation during infection is driven by interleukin-1ß (IL-1ß), but the molecular processes involved are not fully understood. METHODS: We examined IL-1ß activation in a pulmonary model infection of Pseudomonas aeruginosa and in vitro using genetics, specific inhibitors, recombinant proteins, and targeted reporters of protease activity and IL-1ß bioactivity. FINDINGS: Caspase-family inflammasome proteases canonically regulate maturation of this proinflammatory cytokine, but we report that plasticity in IL-1ß proteolytic activation allows for its direct maturation by the pseudomonal protease LasB. LasB promotes IL-1ß activation, neutrophilic inflammation, and destruction of lung architecture characteristic of severe P. aeruginosa pulmonary infection. INTERPRETATION: Preservation of lung function and effective immune clearance may be enhanced by selectively controlling inflammation. Discovery of this IL-1ß regulatory mechanism provides a distinct target for anti-inflammatory therapeutics, such as matrix metalloprotease inhibitors that inhibit LasB and limit inflammation and pathology during P. aeruginosa pulmonary infections. FUNDING: Full details are provided in the Acknowledgements section.

Asunto(s)

Interacciones Huésped-Patógeno , Interleucina-1beta/metabolismo , Pseudomonas aeruginosa/enzimología , Serina Endopeptidasas/metabolismo , Animales , Biomarcadores , Fibrosis Quística/complicaciones , Fibrosis Quística/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática , Inmunohistoquímica , Inflamasomas/metabolismo , Mediadores de Inflamación , Metaloproteasas/antagonistas & inhibidores , Ratones , Ratones Noqueados , Modelos Biológicos , Neumonía Bacteriana/etiología , Neumonía Bacteriana/metabolismo , Neumonía Bacteriana/patología , Unión Proteica , Infecciones por Pseudomonas/etiología , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/patología

RESUMEN

Streptomyces mobaraensis is a key player for the industrial production of the protein cross-linking enzyme microbial transglutaminase (MTG). Extra-cellular activation of MTG by the transglutaminase-activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross-linking are not determined. To address both issues, that is, determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N-terminal peptide by six and three residues respectively. Structural integrity of the mutants was verified by the determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N-terminally shortened variants clearly indicated the highly conserved Leu40-Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40-Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X-ray crystallography. While no structural data could be obtained for the N-terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analysed, which superposes well with SSI-family proteins. ENZYMES: Chymotrypsin, EC3.4.21.1; griselysin (SGMPII, SgmA), EC3.4.24.27; snapalysin (ScNP), EC3.4.24.77; streptogrisin-A (SGPA), EC3.4.21.80; streptogrisin-B (SGPB), EC3.4.21.81; subtilisin BPN', EC3.4.21.62; transglutaminase, EC2.3.2.13; transglutaminase-activating metalloprotease (TAMP), EC3.4.-.-; tri-/tetrapeptidyl aminopeptidase, EC3.4.11.-; trypsin, EC3.4.21.4. DATABASES: The atomic coordinates and structure factors (PDB 6I0I) have been deposited in the Protein Data Bank (http://www.rcsb.org).

Asunto(s)

Proteínas Bacterianas/química , Glutamina/química , Streptomyces/enzimología , Transglutaminasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biotinilación , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Glutamina/metabolismo , Cinética , Modelos Moleculares , Mutación Puntual , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Streptomyces/genética , Especificidad por Sustrato , Transglutaminasas/genética , Transglutaminasas/metabolismo

RESUMEN

The Cry4AaCter tag is a pull-down tag which promotes the formation of inclusion bodies (IBs) that can be resolubilized in an alkaline buffer. Here, we used the Cry4AaCter tag to create a platform for the production of antimicrobial peptides (AMPs) in Escherichia coli featuring a uniform resolubilization process independent of the peptide fused to the pull-down tag. The Cry4AaCter tag conserves the bioactivity of fusion proteins and thus allows the purification of simple AMPs and more complex AMPs stabilized by disulfide bonds. We developed a downstream process (DSP) for the purification of IBs containing the mutated Galleria mellonella insect metalloprotease inhibitor IMPI(I38V), which has a globular structure stabilized by five disulfide bonds. IMPI(I38V) is a potent inhibitor of the M4 metalloproteases used as virulence factors by several human pathogens. We used a single crossflow filtration for the washing and resolubilization of the Cry4AaCter-induced IBs and obtained bioactive IMPI(I38V) after tag removal. We achieved a 68-fold higher protein yield using our IB system compared to an alternative DSP approach in which a GST-fusion strategy was used to produce soluble IMPI(I38V). The Cry4AaCter-based process was transferable to gloverin (another G. mellonella AMP) and the visible marker green fluorescent protein, which accumulated in fluorescent IBs, confirming it is a broadly applicable strategy for the recovery of functional proteins.

Asunto(s)

Antibacterianos/aislamiento & purificación , Escherichia coli/genética , Proteínas de Insectos/aislamiento & purificación , Insectos/genética , Péptidos/aislamiento & purificación , Animales , Escherichia coli/química , Cuerpos de Inclusión/química , Cuerpos de Inclusión/genética , Proteínas de Insectos/genética , Insectos/química , Membranas Artificiales , Péptidos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación

RESUMEN

The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA2). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA2s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), l-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5'-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.

Asunto(s)

Antineoplásicos/aislamiento & purificación , Venenos de Crotálidos/química , Desintegrinas/aislamiento & purificación , Metaloproteasas/aislamiento & purificación , Fosfolipasas A2/aislamiento & purificación , Trimeresurus/fisiología , 5'-Nucleotidasa/química , 5'-Nucleotidasa/aislamiento & purificación , 5'-Nucleotidasa/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fraccionamiento Químico/métodos , Cromatografía Líquida de Alta Presión , Venenos de Crotálidos/aislamiento & purificación , Desintegrinas/química , Desintegrinas/farmacología , Humanos , Concentración 50 Inhibidora , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/farmacología , L-Aminoácido Oxidasa/química , L-Aminoácido Oxidasa/aislamiento & purificación , L-Aminoácido Oxidasa/farmacología , Lectinas Tipo C/química , Lectinas Tipo C/aislamiento & purificación , Espectrometría de Masas , Metaloproteasas/química , Metaloproteasas/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Oligopéptidos/química , Oligopéptidos/aislamiento & purificación , Oligopéptidos/farmacología , Fosfolipasas A2/química , Fosfolipasas A2/farmacología , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/aislamiento & purificación , Hidrolasas Diéster Fosfóricas/farmacología , Serina Proteasas/química , Serina Proteasas/aislamiento & purificación , Serina Proteasas/farmacología

RESUMEN

To control discoloration of dried Japanese common squid (Todarodes pacificus) product, influence of 0-1% organic salts, including sodium gluconate (Na-gluconate), sodium citrate (Na-citrate), sodium phytate (Na-phytate), sodium benzoate (Na-benzoate) and sodium tartrate (Na-tartrate), on Maillard browning in the squid meat during air-drying was investigated. Changes in surface color of the dried squid were mitigated by the addition of organic salts. Organic salts also showed inhibitory effects on autolysis and generation of free amino acids (FAAs), especially arginine, in both the dried squid meat and the model solution. Moreover, Maillard reaction degree of the dried squid was decreased by using organic salts. The addition of 1% Na-citrate or 1% Na-phytate well suppressed the browning of the dried squid (p < 0.05). The results indicated that organic salts having strong chelating ability, including Na-citrate and Na-phytate, can prevent the browning of the dried squid product by inhibiting the generation of FAAs.

Asunto(s)

Decapodiformes/metabolismo , Conservación de Alimentos/métodos , Alimentos Marinos/análisis , Animales , Reacción de Maillard , Carne , Sales (Química)

RESUMEN

Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for numerous industrial applications. Recombinant production requires proteolytic activation of the zymogen. The study provides a convenient procedure for the preparation of the transglutaminase-activating metalloprotease (TAMP) in Escherichia coli. In contrast to wtTAMP, rTAMP exhibited the P domain of convertases as molecular mass of 55.7 kDa suggested. Protein integrity was beneficially influenced by 2-5 mM CaCl2. Study of pH and temperature optima assigned rTAMP to the neutral metalloproteases, more heat-resistant than Dispase but not thermolysin. Zinc had no inhibiting effect but 3.1 µM EDTA completely reduced activity of 5 nM TAMP. MTG, exceeding concentration of rTAMP by three orders of magnitude, was largely activated within few minutes. The kinetic parameters KM (1.31 ±â€¯0.05 mM) and kcat (135 ±â€¯4.3 s-1), monitored by isothermal titration calorimetry (ITC), further highlighted catalytic efficiency (103,053 M-1 s-1) of rTAMP and rapid processing of MTG. ITC even revealed that inhibition of rTAMP by its intrinsic inhibitory protein SSTI was an enthalpy-driven process resulting in Kd of 199 ±â€¯37.9 nM. The production procedure of rTAMP in E. coli closes the gap between production and application of recombinant MTG and may enhance relevance of MTG-mediated reactions in pharmaceutical processes.

Asunto(s)

Proteínas Bacterianas , Escherichia coli/metabolismo , Metaloproteasas , Streptomyces/enzimología , Transglutaminasas , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Escherichia coli/genética , Metaloproteasas/genética , Metaloproteasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Transglutaminasas/genética , Transglutaminasas/metabolismo

RESUMEN

The Nose-horned Viper (Vipera ammodytes) is one of the most widespread and venomous snakes in Europe, which causes high frequent snakebite accidents. The first comprehensive venom characterization of the regional endemic Transcaucasian Nose-horned Viper (Vipera ammodytes transcaucasiana) and the Transdanubian Sand Viper (Vipera ammodytes montandoni) is reported employing a combination of intact mass profiling and bottom-up proteomics. The bottom-up analysis of both subspecies identified the major snake protein families of viper venoms. Furthermore, intact mass profiling revealed the presence of two tripeptidic metalloprotease inhibitors and their precursors. While previous reports applied multivariate analysis techniques to clarify the taxonomic status of the subspecies, an accurate classification of Vipera ammodytestranscaucasiana is still part of the ongoing research. The comparative analysis of the viper venoms on the proteome level reveals a close relationship between the Vipera ammodytes subspecies, which could be considered to clarify the classification of the Transcaucasian Nose-horned Viper. However, the slightly different ratio of some venom components could be indicating interspecific variations of the two studied subspecies or intraspecies alternations based on small sample size. Additionally, we performed a bioactivity screening with the crude venoms against several human cancerous and non-cancerous cell lines, which showed interesting results against a human breast adenocarcinoma epithelial cell line. Several fractions of Vipera a. transcaucasiana demonstrated a strong cytotoxic effect on triple negative MDA MB 231 breast cancer cells.

Asunto(s)

Venenos de Víboras/química , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Proteoma , Proteínas de Reptiles/análisis , Proteínas de Reptiles/farmacología , Turquía , Venenos de Víboras/farmacología , Viperidae

RESUMEN

BACKGROUND: The severity and progression of rotator cuff tears have forced research on new treatment pathways such as metalloprotease inhibition, which has shown a reduction in healing time and improvement in the structure of collagen fibers. OBJECTIVE: To evaluate the use of doxycycline as a healing enhancer in rotator cuff tears after surgical treatment. MATERIAL AND METHODS: 20 patients were included; they were divided into two groups, 10 with the use of doxycycline and 10 without it after arthroscopic repair with one-year follow-up. Doxycycline was given orally, 100 mg once a day for one month. Every subject in the test was diagnosed with rotator cuff tear confirmed by MRI with Patte and Goutallier scores below 2. We used the arthroscopic double row technique. Post-op follow-up was 12 months with clinical scales (UCLA, Constant and forward flexion strength). RESULTS: Both groups reported almost complete healing of rotator cuff tears after surgical treatment during the twelve months of follow-up; forward flexion strength was the only score that reported improvement in the doxycycline group during every check-up. DISCUSSION: Doxycycline use after arthroscopic cuff tear repair could improve the clinical outcome, but we do not know how yet; however larger sample and randomized trials should be developed.

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ANTECEDENTES: La severidad y progresión inevitable de la lesión del manguito rotador ha llevado a experimentar con adyuvantes terapéuticos para disminuir el tiempo de recuperación postquirúrgica, así como mejorar la estructura del tendón en recuperación al inhibir la matriz de metaloproteasas. OBJETIVO: Evaluar el uso de la doxiciclina como adyuvante en la cicatrización de lesiones hueso-tendón en la reparación quirúrgica del manguito rotador. MATERIAL Y MÉTODOS: Se reclutaron 20 pacientes con lesión del manguito rotador corroborada por imagen con retracción del supraespinoso grado II (Patte) e infiltración grasa de 50% (Goutallier). Fueron divididos en dos grupos: a 10 se les administró doxiciclina, 100 mg cada 24 horas durante un mes, y el resto fueron un grupo control sin doxiciclina. Ambos fueron tratados quirúrgicamente con técnica de doble hilera vía artroscópica, con seguimiento periódico hasta 12 meses mediante escalas de UCLA, Constant y potencia de flexión anterógrada. RESULTADOS: Se encontró recuperación clínica de la lesión en ambos grupos a los 12 meses, mayor potencia de flexión anterógrada en cada uno de los intervalos de medición para el grupo donde se administró la doxiciclina. Durante la evolución del estudio, se mantuvo constante Constant y UCLA; se encontró mejoría considerable con la potencia de flexión anteró grada como valor independiente. DISCUSIÓN: El uso de doxiciclina podría mejorar de una forma considerable el pronóstico clínico de la reparación artroscópica de mango rotador con el uso de doble hilera, pero aún no sabemos cómo, aunque deberán realizarse estudios adicionales con una muestra mayor.

Asunto(s)

Antibacterianos , Doxiciclina , Lesiones del Manguito de los Rotadores , Antibacterianos/uso terapéutico , Artroscopía , Doxiciclina/uso terapéutico , Estudios de Seguimiento , Humanos , Imagen por Resonancia Magnética , Manguito de los Rotadores , Lesiones del Manguito de los Rotadores/tratamiento farmacológico , Lesiones del Manguito de los Rotadores/cirugía , Resultado del Tratamiento

RESUMEN

Resumen: Antecedentes: La severidad y progresión inevitable de la lesión del manguito rotador ha llevado a experimentar con adyuvantes terapéuticos para disminuir el tiempo de recuperación postquirúrgica, así como mejorar la estructura del tendón en recuperación al inhibir la matriz de metaloproteasas. Objetivo: Evaluar el uso de la doxiciclina como adyuvante en la cicatrización de lesiones hueso-tendón en la reparación quirúrgica del manguito rotador. Material y métodos: Se reclutaron 20 pacientes con lesión del manguito rotador corroborada por imagen con retracción del supraespinoso grado II (Patte) e infiltración grasa de 50% (Goutallier). Fueron divididos en dos grupos: a 10 se les administró doxiciclina, 100 mg cada 24 horas durante un mes, y el resto fueron un grupo control sin doxiciclina. Ambos fueron tratados quirúrgicamente con técnica de doble hilera vía artroscópica, con seguimiento periódico hasta 12 meses mediante escalas de UCLA, Constant y potencia de flexión anterógrada. Resultados: Se encontró recuperación clínica de la lesión en ambos grupos a los 12 meses, mayor potencia de flexión anterógrada en cada uno de los intervalos de medición para el grupo donde se administró la doxiciclina. Durante la evolución del estudio, se mantuvo constante Constant y UCLA; se encontró mejoría considerable con la potencia de flexión anterógrada como valor independiente. Discusión: El uso de doxiciclina podría mejorar de una forma considerable el pronóstico clínico de la reparación artroscópica de mango rotador con el uso de doble hilera, pero aún no sabemos cómo, aunque deberán realizarse estudios adicionales con una muestra mayor.

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Abstract: Background: The severity and progression of rotator cuff tears have forced research on new treatment pathways such as metalloprotease inhibition, which has shown a reduction in healing time and improvement in the structure of collagen fibers. Objective: To evaluate the use of doxycycline as a healing enhancer in rotator cuff tears after surgical treatment. Material and methods: 20 patients were included; they were divided into two groups, 10 with the use of doxycycline and 10 without it after arthroscopic repair with one-year follow-up. Doxycycline was given orally, 100 mg once a day for one month. Every subject in the test was diagnosed with rotator cuff tear confirmed by MRI with Patte and Goutallier scores below 2. We used the arthroscopic double row technique. Post-op follow-up was 12 months with clinical scales (UCLA, Constant and forward flexion strength). Results: Both groups reported almost complete healing of rotator cuff tears after surgical treatment during the twelve months of follow-up; forward flexion strength was the only score that reported improvement in the doxycycline group during every check-up. Discussion: Doxycycline use after arthroscopic cuff tear repair could improve the clinical outcome, but we do not know how yet; however larger sample and randomized trials should be developed.

Asunto(s)

Humanos , Doxiciclina/uso terapéutico , Lesiones del Manguito de los Rotadores/cirugía , Lesiones del Manguito de los Rotadores/tratamiento farmacológico , Antibacterianos/uso terapéutico , Artroscopía , Imagen por Resonancia Magnética , Estudios de Seguimiento , Resultado del Tratamiento , Manguito de los Rotadores

RESUMEN

Botulinum neurotoxins (BoNTs) are the most toxic substances known to mankind and are the causative agents of the neuroparalytic disease botulism. Their ease of production and extreme toxicity have caused these neurotoxins to be classified as Tier 1 bioterrorist threat agents and have led to a sustained effort to develop countermeasures to treat intoxication in case of a bioterrorist attack. While timely administration of an approved antitoxin is effective in reducing the severity of botulism, reversing intoxication requires different strategies. In the present study, we evaluated ABS 252 and other mercaptoacetamide small molecule active-site inhibitors of BoNT/A light chain using an integrated multi-assay approach. ABS 252 showed inhibitory activity in enzymatic, cell-based and muscle activity assays, and importantly, produced a marked delay in time-to-death in mice. The results suggest that a multi-assay approach is an effective strategy for discovery of potential BoNT therapeutic candidates.

Asunto(s)

Toxinas Botulínicas Tipo A/antagonistas & inhibidores , Metaloproteasas/antagonistas & inhibidores , Neurotoxinas/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Animales , Toxinas Botulínicas Tipo A/química , Células Cultivadas , Cristalografía por Rayos X , Descubrimiento de Drogas/métodos , Masculino , Ratones , Contracción Muscular/efectos de los fármacos , Músculos/efectos de los fármacos , Músculos/fisiología , Neurotoxinas/química , Inhibidores de Proteasas/química , Ratas Sprague-Dawley

RESUMEN

Resistance of snakes and some other animals to snake envenomation has been attributed to soluble factors present in their tissues. Here we report the isolation of a novel metalloprotease inhibitor from Bothrops alternatus snake serum (named BaltMPI) with high purity, using a four-step chromatographic method. BaltMPI has molecular weights of 60.5 and 42.4kDa, as determined by SDS-PAGE and mass spectrometry, respectively, and pI=5.27. The first 60 amino acids from the N-terminal region of BaltMPI, determined by Edman's degradation, showed high homology (97%) with the snake venom metalloprotease inhibitor (SVMPI) BJ46a and other SVMPIs (78-82%). The chromatographic fractions and purified BaltMPI exhibited anti-hemorrhagic activity against Batroxase and BjussuMP-I. BaltMPI was stable over wide ranges of pH (1, 5, 8, and 9) and temperature (-80, -20, 4, 60, and 100°C), and suppressed the fibrinogenolytic, fibrinolytic, and azocaseinolytic activities of Batroxase. BaltMPI specifically inhibited the activity of metalloproteases, without affecting the activity of serine proteases. Together, our results suggest that BaltMPI and other SVMPIs are promising molecules for the treatment of snake envenomation, in particular that caused by Bothrops sp.

Asunto(s)

Bothrops/sangre , Metaloendopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/aislamiento & purificación , Inhibidores de Proteasas/farmacología , Secuencia de Aminoácidos , Animales , Caseínas/metabolismo , Fibrina/metabolismo , Fibrinógeno/metabolismo , Hemorragia/tratamiento farmacológico , Metaloendopeptidasas/metabolismo , Ratones , Inhibidores de Proteasas/sangre , Inhibidores de Proteasas/química , Proteolisis/efectos de los fármacos

RESUMEN

Proteolytic enzymes and their inhibitors are crucial in host-pathogen interaction. Metalloproteases secreted by pathogenic microbes play an important role in destroying not only host tissues but also their immune proteins. Metalloproteinase inhibitors, in contrast, may serve as effective therapeutic agents, which is especially important because of the increasing number of microorganisms resistant to known antibiotics. The role of metalloproteases produced by the bacterium Pseudomonas aeruginosa in the colonization of the host organism is described. Attention has also been paid to the role of inhibitors of these enzymes in defense responses and underlined their potential role in inhibiting the development of infection.

Asunto(s)

Antibacterianos/farmacología , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/metabolismo , Infecciones por Pseudomonas/prevención & control , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/patogenicidad , Antibacterianos/uso terapéutico , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/uso terapéutico , Proteolisis/efectos de los fármacos , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/fisiología

RESUMEN

Infection by parasitic nematodes is widespread in the developing world causing extensive morbidity and mortality. Furthermore, infection of animals is a global problem, with a substantial impact on food production. Here we identify small molecule inhibitors of a nematode-specific metalloprotease, DPY-31, using both known metalloprotease inhibitors and virtual screening. This strategy successfully identified several µM inhibitors of DPY-31 from both the human filarial nematode Brugia malayi, and the parasitic gastrointestinal nematode of sheep Teladorsagia circumcincta. Further studies using both free living and parasitic nematodes show that these inhibitors elicit the severe body morphology defect 'Dumpy' (Dpy; shorter and fatter), a predominantly non-viable phenotype consistent with mutants lacking the DPY-31 gene. Taken together, these results represent a start point in developing DPY-31 inhibition as a totally novel mechanism for treating infection by parasitic nematodes in humans and animals.

Asunto(s)

Proteínas del Helminto/antagonistas & inhibidores , Nematodos/enzimología , Inhibidores de Proteasas/química , Animales , Sitios de Unión , Brugia Malayi/enzimología , Caenorhabditis elegans/enzimología , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas del Helminto/metabolismo , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/metabolismo , Concentración 50 Inhibidora , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/metabolismo , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Ovinos

RESUMEN

Natural antimicrobial peptides represent a primordial mechanism of immunity in both vertebrate and nonvertebrate organisms. Among them, histatins belong to a family of human salivary metal-binding peptides displaying potent antibacterial, antifungal and wound-healing activities. These properties, along with the ability of histatins to inhibit collagenases and cysteine proteases, have attracted much attention for their potential use in the treatment of several oral diseases. This review critically assesses the studies carried out to date in order to provide a comprehensive and systematic vision of the information accumulated so far. In particular, the relationship between metal-binding and peptide activity is extensively analysed. The review provides important clues for developing possible therapeutic applications of histatins and their synthetic peptide analogues by creating a set of necessary resource materials to support investigators and industries interested in exploiting their unique properties.

Asunto(s)

Antibacterianos/farmacología , Antifúngicos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Cobre/metabolismo , Histatinas/farmacología , Zinc/metabolismo , Animales , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/uso terapéutico , Antifúngicos/química , Antifúngicos/metabolismo , Antifúngicos/uso terapéutico , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Inhibidores de Cisteína Proteinasa/uso terapéutico , Histatinas/química , Histatinas/metabolismo , Histatinas/uso terapéutico , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Inhibidores de la Metaloproteinasa de la Matriz/uso terapéutico , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , Isoformas de Proteínas/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

INTRODUCTION: Peripheral biomarkers to diagnose Alzheimer's disease (AD) have not been established. Given parallels between neuron and platelet biology, we hypothesized platelet membrane-associated protein changes may differentiate patients clinically defined with probable AD from noncognitive impaired controls. METHODS: Purified platelets, confirmed by flow cytometry were obtained from individuals before fractionation by ultracentrifugation. Following a comparison of individual membrane fractions by SDS-PAGE for general proteome uniformity, equal protein weight from the membrane fractions for five representative samples from AD and five samples from controls were pooled. AD and control protein pools were further divided into molecular weight regions by one-dimensional SDS-PAGE, prior to digestion in gel. Tryptic peptides were analyzed by reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Ionized peptide intensities were averaged for each identified protein in the two pools, thereby measuring relative protein abundance between the two membrane protein pools. Log2-transformed ratio (AD/control) of protein abundances fit a normal distribution, thereby permitting determination of significantly changed protein abundances in the AD pool. RESULTS: We report a comparative analysis of the membrane-enriched platelet proteome between patients with mild to moderate AD and cognitively normal, healthy subjects. A total of 144 proteins were determined significantly altered in the platelet membrane proteome from patients with probable AD. In particular, secretory (alpha) granule proteins were dramatically reduced in AD. Of these, we confirmed significant reduction of thrombospondin-1 (THBS1) in the AD platelet membrane proteome by immunoblotting. There was a high protein-protein connectivity of proteins in other pathways implicated by proteomic changes to the proteins that define secretory granules. CONCLUSIONS: Depletion of secretory granule proteins is consistent with a preponderance of post-activated platelets in circulation in AD. Significantly changed pathways implicate additional AD-related defects in platelet glycoprotein synthesis, lipid homeostasis, amyloidogenic proteins, and regulators of protease activity, many of which may be useful plasma membrane-expressed markers for AD. This study highlights the utility of LC-MS/MS to quantify human platelet membrane proteins and suggests that platelets may serve as a source of blood-based biomarkers in neurodegenerative disease.