A review on the role of angiotensin-converting enzyme 2 in children with coronavirus disease 2019 / 中国当代儿科杂志

With the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) all over the world, there is an increasing number of children with such infection. Angiotensin-converting enzyme 2 (ACE2), one of the binding sites for SARS-CoV-2 infection in humans, can bind to viral spike proteins, allowing transmembrane serine protease (TMPRSS2) to activate S-protein to trigger infection and induce the production of various inflammatory factors such as interleukin-1, interferon-l, and tumor necrosis factor. Compared with adults, children tend to have lower expression levels of ACE2 and TMPRSS2, which are presumed to be associated with milder symptoms and fewer cases in children. The article summarizes the research advances in the role of ACE2 during SARS-CoV-2 infection, in order to help understand the pathogenic mechanism of SARS-CoV-2 and provide a reference for better development of drugs and vaccines to prevent and treat coronavirus disease 2019 in children.

Characterization of digestive enzymes from captive Brazilian flounder Paralichthys orbignyanus / Caracterização de enzimas digestivas do linguado brasileiro de cativeiro Paralichthys orbignyanus

Abstract Knowledge of specific enzyme activity, along with animal habits and digestive capacity is essential in formulating an appropriate diet for any species. In this study, we evaluated and characterized the activity of digestive enzymes present in the liver, intestine, and stomach of Paralichthys orbignyanus. The effects of pH and temperature on enzyme activity were also evaluated via the use of specific substrates. The use of specific substrates and inhibitors showed strong evidence of the presence of trypsin (BApNA= 0.51 ± 0.2 mU mg-1), chimotrypsin (SApNA= 2.62 ± 1.8 mU mg-1), and aminopeptidases (Leu-p-Nan =0.9709 ± 0.83 mU mg-1) in the intestine. Optimum pH for the activity of trypsin, chemotrypsin, leucino aminopeptidase, amilase, and pepsin were 9.5, 9.0, 8.0, 7.5, and 3.5, respectively, while optimum temperatures were 50, 50, 50, 40, and 45 °C, respectively. These results provide additional information regarding the biology of Brazilian flounder and can be used as a basis for further studies regarding fish feeding physiology.

Resumo O conhecimento da atividade enzimática é essencial para formular uma correta dieta específica para espécie, além de estarem correlacionadas com o hábito da alimentação e capacidade digestive. Neste estudo determinamos e caracterizamos a atividade enzimática presente no intestino, estômago e fígado do linguado Paralichthys orbignyanus. Os efeitos da temperatura e pH sobre a atividade enzimática também foram avaliados utilizando substratos específicos. O uso de substratos e inibidores específicos mostrou uma forte evidência da presença da tripsina (BApNA = 0,51 ± 0,2 mU mg-1), quimotripsina (SAPNA = 2,62 ± 1,8 mU mg-1), e as aminopeptidases (Leu-p-Nan = 0,97 ± 0,83 mU mg-1) no intestino. O pH ótimo observado para a atividade de tripsina, quimotripsina, leucino aminopeptidase, amilase e pepsina foi 9,5, 9,0, 8,0, 7,5 e 3,5, respectivamente. A temperatura ótima observada foi 50, 50, 50, 40 e 45 °C, respectivamente. Estes resultados fornecem informações adicionais sobre a biologia do linguado brasileiro e pode ser usado como base para novos estudos sobre fisiologia alimentar.

Immunosuppressive mechanism of Hypoderma lineatum secreted serine esterase, a potential modulatory method used to inhibit transplant rejection

Background: Although immunosuppressive therapies have made organ transplantation a common medical procedure worldwide, chronic toxicity has a major issue for long-term treatment. One method to improve therapies and methods is the application of immunomodulatory agents from parasites such as Hypoderma lineatum. Hypodermin A (HA) is a serine esterase secreted by the larvae of Hypoderma lineatum, several studies demonstrated its immunosuppressive mechanism in vitro, and recently we discovered that HA inhibits the expression of interferon (IFN)-γ and interleukin (IL)-2 and activates IL-10 expression. Therefore, we hypothesized that it might be a potential agent used to block allograft rejections. However, most studies of the immunosuppressive mechanisms associated with HA were undertaken at the cellular level. In order to augment these studies, we evaluated the immunosuppressive effects of HA in vivo using an HA transgenic mouse model. Result: Our results revealed similar findings to those reported by in vitro studies, specifically that HA induced prostaglandin E2 expression, downregulated IFN-γ and IL-2 expression, and promoted IL-10 secretion via E-type prostanoid receptor 4. Additionally, we observed that HA overexpression inhibited lipopolysaccharide-induced TLR4 activation. These findings provide insight into a new potential agent capable of blocking graft rejection. Conclusion: Our founding suggested that HA-related treatment could be a promising option to improve the viability of grafts in human.

Proteolytic activity of recombinant DegP from Chromohalobacter salexigens BKL5

Background: DegP is a serine protease that specifically cleaves and refolds unfolding proteins in the periplasmic space of the cells. To date, there is no information regarding DegP from halophilic bacteria. Chromohalobacter salexigens BKL5 is a moderately halophilic bacterium that has the ability to grow in a media containing more than 15% salt. Therefore, the objectives of this work were to clone and overexpress DegP-encoding gene from C. salexigens BKL5 and characterize its biochemical properties. Results: DegP-encoding gene was overexpressed in Escherichia coli BL21(DE3) CodonPlus in an active form. SDS-PAGE analysis showed that the molecular weight of the recombinant DegP was 45 kDa. Size-exclusion chromatography analysis suggested that recombinant DegP was present in two multimeric states, hexameric and dodecameric, with molecular weights of 297.9 and 579.12 kDa, respectively. Both conformations were enzymatically active when casein was used as substrate for enzymatic assay. Circular dichroism analysis showed that recombinant DegP was composed of 0.21­0.29 helical content, which was comparable to the helical content in the crystal structure of E. coli DegP. The basic/acidic residue ratio of recombinant DegP was 0.56, which was slightly higher than that of DegP from extreme halophiles (average, 0.45) but significantly lower than that of DegP from nonhalophiles (average, 0.94). Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when ß-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.

Características de la estructura molecular de las proteínas E del virus del Zika y E1 del virus de la rubéola y posibles implicaciones en el neurotropismo y en las alteraciones del sistema nervioso / E-ZIKV and E1-RV proteins molecular structure and their potential implications in neurotropism and nervous system disorders

Resumen Introducción. El virus del Zika (ZIKV) es un flavivirus con envoltura, transmitido a los seres humanos principalmente por el vector Aedes aegypti. La infección por ZIKV se ha asociado con un gran neurotropismo y con efectos neuropáticos, como el síndrome de Guillain-Barré en el adulto y la microcefalia fetal y posnatal, así como con un síndrome de infección congénita similar al producido por el virus de la rubéola (RV). Objetivo. Comparar las estructuras moleculares de la proteína de envoltura E del virus del Zika (E-ZIKV) y de la E1 del virus de la rubéola (E1-RV), y plantear posibles implicaciones en el neurotropismo y en las alteraciones del sistema nervioso asociadas con el ZIKV. Materiales y métodos. La secuencia de aminoácidos de la proteína E-ZIKV (PDB: 5iZ7) se alineó con la de la glucopreteína E1 del virus de la rubéola (PDB: 4ADG). Los elementos de la estructura secundaria se determinaron usando los programas Vector NTI Advance®, DSSP y POSA, así como herramientas de gestión de datos (AlignX®). Uno de los criterios principales de comparación y alineación fue la asignación de residuos estructuralmente equivalentes, con más de 70 % de identidad. Resultados. La organización estructural de la proteína E-ZIKV (PDB: 5iZ7) fue similar a la de E1-RV (PDB: 4ADG) (70 a 80 % de identidad), y se observó una correspondencia con la estructura definida para las glucoproteínas de fusión de membrana de clase II de los virus con envoltura. E-ZIKV y E1-RV exhibieron elementos estructurales de fusión muy conservados en la región distal del dominio II, asociados con la unión a los receptores celulares de entrada del virus de la rubéola (glucoproteína de mielina del oligodendrocito, Myelin Oligodendrocyte Glycoprotein, MOG), y con los receptores celulares Axl del ZIKV y de otros flavivirus. Conclusión. La comparación de las proteínas E-ZIKV y E1-RV es un paso necesario hacia la definición de otros factores moleculares determinantes del neurotropismo y la patogenia del ZIKV, el cual puede contribuir a generar estrategias de diagnóstico, prevención y tratamiento de las complicaciones neurológicas inducidas por el ZIKV.

Abstract Introduction: Zika virus (ZIKV) is an enveloped flavivirus transmitted to humans mainly by Aedes aegypti. ZIKV infection has been associated with high neurotropism and neuropathic effects such as the Guillain-Barré syndrome in adults, and fetal and postnatal microcephaly and the congenital Zika virus syndrome similar to that produced by rubella virus (VR). Objective: To compare Zika virus membrane protein E (E-ZIKV) and rubella virus membrane protein E1 (E1-RV), and to propose possible implications for neurotropism and nervous system disorders associated with ZIKV infections. Materials and methods: The amino acid sequence of E-ZIKV protein (PDB: 5iZ7) was aligned to that of rubella virus glycoprotein E1 (PDB: 4ADG). The secondary structure elements were determined using the programs Vector NTI Advance®, DSSP, and POSA, and integrated data management tools (AlignX®). One of the main comparison and alignment criteria was the allocation of structurally equivalent residues with more than 70% identity. Results: E-ZIKV structural organization (PDB: 5iZ7) was similar to that of E1-RV (PDB: 4ADG) (70%-80% identity), and it was consistent with relevant structural features of viral membrane class II fusion glycoproteins. E-ZIKV and E1-RV exhibited highly conserved fusion structural elements at the distal region of domain II, which has been associated with the RV myelin oligodendrocyte glycoprotein and Axl cell receptors in ZIKV and other flaviviruses. Conclusion: The comparison of E-ZIKV and E1-RV proteins constitutes an essential step towards the definition of ZIKV neurotropism and pathogenesis molecular determinants, and for the adoption of diagnosis, prevention and treatment strategies against neurological complications induced by ZIKV infection.

Anticuerpos policlonales contra la proteína recombinante NS3 del virus del dengue / Polyclonal antibodies against recombinant dengue virus NS3 protein

Resumen Introducción: El dengue es una enfermedad causada por uno de los cuatro serotipos del virus del dengue (DENV) y es endémica en, aproximadamente, 130 países. Su incidencia ha aumentado notablemente en las últimas décadas, así como la frecuencia y la magnitud de los brotes. A pesar de los esfuerzos, no existen tratamientos profilácticos ni terapéuticos contra la enfermedad y, en ese contexto, el estudio de los procesos que gobiernan el ciclo de infección del DENV es esencial para desarrollar vacunas o terapias antivirales. Una de las moléculas del DENV más prometedoras es la proteína no estructural 3 (NS3), la cual es indispensable para la replicación viral y es uno de los principales blancos inmunológicos durante la infección. Objetivo: Producir anticuerpos policlonales para contribuir a los futuros estudios sobre las interacciones entre la proteína NS3 y otras proteínas celulares. Materiales y métodos: Se expresaron dos proteínas recombinantes del dominio helicasa de NS3 del DENV de serotipo 2, las cuales se emplearon para inmunizar ratas y producir anticuerpos policlonales. Resultados: Los anticuerpos producidos fueron útiles en ensayos de Western blot e inmunofluorescencia y se reportó por primera vez un anticuerpo policlonal anti-NS3 que permitió la inmunoprecipitación de la proteína viral y la detecta con Western blot sin necesidad de inducir sobreexpresión de NS3 o de usar extractos de células marcados metabólicamente con radioisótopos. Conclusión: Las proteínas recombinantes expresadas y los anticuerpos producidos constituyen herramientas valiosas para estudiar procesos infecciosos del DENV que involucren a la proteína NS3 y evaluar pruebas dirigidas a interferir las funciones de esta proteína.

Abstract Introduction: Dengue is a disease caused by one of four serotypes of the dengue virus (DENV) and is endemic in approximately 130 countries. The incidence of dengue has increased dramatically in recent decades, as well as the frequency and magnitude of outbreaks. Despite all efforts, there are no prophylactic or therapeutic treatments for the disease. Accordingly, research on the processes governing the DENV infection cycle is essential to develop vaccines or antiviral therapies. One of the most attractive DENV molecules to investigate is nonstructural protein 3 (NS3), which is essential for viral replication and a major immune target for infection. Objective: To produce antibodies to support future studies on NS3 and its cellular interactions with other proteins. Materials and methods: Two recombinant proteins of the helicase domain of DENV NS3 serotype 2 were expressed, and used to immunize mice and produce polyclonal antibodies. Results: The antibodies produced were useful in Western blot and immunofluorescence tests. We report an NS3 antibody that immunoprecipitates the viral protein and detects it in Western blot with no need to over-express it or use cell extracts with metabolic radiolabeling.

Effects of platelet rich plasma on fascial healing in rats with fecal peritonitis

ABSTRACT PURPOSE : To evaluate the effects of platelet rich plasma (PRP) on the healing of fascia wherein peritonitis has been created. METHODS: Twenty eight Wistar Albino rats were divided into four groups. Only a primary fascial repair following laparotomy was performed on Group 1, a primary fascial repair performed and PRP treatment applied following laparotomy on Group 2, and a fecal peritonitis created following laparotomy and a primary fascial repair carried out on Group 3. A fecal peritonitis was created following laparotomy and primary fascial repair and PRP treatment on the fascia was carried out on Group 4. RESULTS: TNF-α was found to be significantly lower in the control group (Group 1). It was detected at the highest level in the group in which fecal peritonitis was created and PRP applied (Group 4). TGF-β was determined as being significantly higher only in Group 4. Histopathologically, the differences between the groups in terms of cell infiltration and collagen deposition were not found to be significant. CONCLUSION: When platelet rich plasma was given histologically and biochemicaly as wound healing parameters cellular infiltration, collagen accumulation, and tissue hydroxyiproline levels were not increased but neovascularization, fibroblast activation and TNF Alfa levels were increased and PRP accelerated wound healing.

Lonomia obliqua Walker (Lepidoptera: Saturniidae): hemostasis implications / Lonomia obliqua Walker (Lepidoptera: Saturniidae): implicações em hemostasia.

Summary In southern Brazil, since 1989, several cases of accidents produced by unwilling contact with the body of poisonous caterpillars of the moth species Lonomia obliqua Walker, 1855 (Lepidoptera: Saturniidae), were described. L. obliqua caterpillars have gregarious behavior and feed on leaves of host trees during the night, staying grouped in the trunk during the day, which favors the occurrence of accidents with the species. This caterpillar has the body covered with bristles that on contact with the skin of individuals, breaks and release their contents, inoculating the venom into the victim. The basic constitution of the venom is protein and its components produce physiological changes in the victim, which include disturbances in hemostasis. Hemorrhagic syndrome associated with consumption coagulopathy, intravascular hemolysis and acute renal failure are some of the possible clinical manifestations related to poisoning by L. obliqua. Specific laboratory tests for diagnosis of poisoning have not been described previously. The diagnosis of poisoning is made based on the patient's medical history, clinical manifestations, erythrocyte levels, and, primarily, parameters that evaluate blood coagulation. Treatment is performed with the use of supportive care and the administration of specific hyperimmune antivenom. Poisoning can be serious and even fatal.

Na região Sul do Brasil, a partir de 1989, foram descritos inúmeros casos de contato acidental com o corpo de lagartas venenosas da mariposa da espécie Lonomia obliqua Walker, 1855 (Lepidoptera: Saturniidae). As lagartas de L. obliqua possuem comportamento gregário e alimentam- -se de folhas das árvores hospedeiras durante a noite, permanecendo agrupadas no tronco durante o dia, o que favorece a ocorrência de acidentes. Essa lagarta possui o corpo recoberto por cerdas urticantes, que, ao contato com a pele dos indivíduos, se rompem e liberam seu conteúdo, inoculando o veneno na vítima. A constituição básica do veneno é proteica e seus componentes produzem modificações fisiológicas no acidentado, que incluem distúrbios na hemostasia. Síndrome hemorrágica associada a coagulopatia de consumo, hemólise intravascular e insuficiência renal aguda são algumas das manifestações clínicas possíveis relacionadas ao envenenamento por L. obliqua. Ainda não foram descritas avaliações laboratoriais específicas para diagnóstico do envenenamento, que é feito com base na anamnese do paciente, nas manifestações clínicas, nos índices hematimétricos e, principalmente, nos parâmetros que avaliam a coagulação sanguínea. A terapêutica é feita com a utilização de medidas de suporte e com a administração de um soro heterólogo hiperimune específico. Os envenenamentos podem ser graves e até mesmo fatais.

In silico Screening of Chemical Libraries to Develop Inhibitors That Hamper the Interaction of PCSK9 with the LDL Receptor

PURPOSE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) and promotes degradation of the LDLR. Inhibition of PCSK9 either by reducing its expression or by blocking its activity results in the upregulation of the LDLR and subsequently lowers the plasma concentration of LDL-cholesterol. As a modality to inhibit PCSK9 action, we searched the chemical library for small molecules that block the binding of PCSK9 to the LDLR. MATERIALS AND METHODS: We selected 100 chemicals that bind to PCSK9 where the EGF-AB fragment of the LDLR binds via in silico screening of the ChemBridge chemical library, using the computational GOLD algorithm analysis. Effects of chemicals were evaluated using the PCSK9-LDLR binding assay, immunoblot analysis, and the LDL-cholesterol uptake assay in vitro, as well as the fast performance liquid chromatography assay for plasma lipoproteins in vivo. RESULTS: A set of chemicals were found that decreased the binding of PCSK9 to the EGF-AB fragment of the LDLR in a dose-dependent manner. They also increased the amount of the LDLR significantly and subsequently increased the uptake of fluorescence-labeled LDL in HepG2 cells. Additionally, one particular molecule lowered the plasma concentration of total cholesterol and LDL-cholesterol significantly in wild-type mice, while such an effect was not observed in Pcsk9 knockout mice. CONCLUSION: Our findings strongly suggest that in silico screening of small molecules that inhibit the protein-protein interaction between PCSK9 and the LDLR is a potential modality for developing hypercholesterolemia therapeutics.

Forty years of a psychiatric day hospital / Quarenta anos de um hospital de dia de psiquiatria

INTRODUCTION: Day hospitals in psychiatry are a major alternative to inpatient care today, acting as key components of community and social psychiatry. Objective: To study trends in the use of psychiatric day hospitals over the last decades of the 20th century and the first decade of the 21st century, focusing on patient age, sex, and diagnostic group, using data from Centro Hospitalar São João, Porto, Portugal. METHODS: Data corresponding to years 1970 to 2009 were collected from patient files. Patients were classified into seven diagnostic groups considering their primary diagnoses only. RESULTS: Mean age upon admission rose from 32.7±12.1 years in the second half of the 1970s to 43.5±12.2 years in 2005-2009 (p for trend < 0.001). Most patients were female (63.2%), however their proportion decreased from nearly 70% in the 1970s to 60% in the first decade of the 21st century. In males, until the late 1980s, neurotic disorders (E) were the most common diagnosis, accounting for more than one third of admissions. In the subsequent years, this proportion decreased, and the number of admissions for schizophrenia (C) exceeded 50% in 2004- 2009. In females, until the late 1980s, affective disorders (D) and neurotic disorders (E), similarly distributed, accounted for most admissions. From the 1990s on, the proportion of neurotic disorders (E) substantially decreased, and affective disorders (D) came to represent more than 50% of all admissions. CONCLUSIONS: Mean age upon admission rose with time, as did the percentage of female admissions, even though the latter tendency weakened in the last 10 years assessed. There was also an increase in the proportion of patients with schizophrenia. .

INTRODUÇÃO: Os hospitais de dia em psiquiatria representam atualmente uma das principais alternativas ao internamento, atuando como componentes chave na psiquiatria comunitária e social. OBJETIVO: Avaliar tendências na utilização de um hospital de dia no período compreendido entre as últimas décadas do século 20 e a primeira década do século 21, com foco em idade, sexo e grupo diagnóstico, usando dados do Centro Hospitalar São João, Porto, Portugal. MÉTODOS: Dados correspondentes aos anos 1970 a 2009 foram coletados dos prontuários clínicos. Os pacientes foram classificados em sete grupos diagnósticos, tendo em conta o diagnóstico principal. Resultados: A idade média na admissão aumentou de 32.7±12.1 anos na segunda metade da década de 1970 para 43.5±12.2 anos em 2005-2009 (p < 0.001). A maioria dos pacientes era do sexo feminino (63.2%), no entanto sua proporção diminuiu de cerca de 70% na década de 1970 para 60% na primeira década do século 21. Nos homens, até o final dos anos 1980, o grupo das perturbações neuróticas (E) era o diagnóstico mais comum, representando mais de um terço das admissões. Durante os anos seguintes, essa proporção diminuiu, e o número de admissões por esquizofrenia (C) alcançou mais de 50% no período de 2004-2009. Nas mulheres, até o final dos anos 1980, as perturbações afetivas (D) e as perturbações neuróticas (E), distribuídas similarmente, respondiam pela maioria das admissões. A partir dos anos 1990, a proporção das perturbações neuróticas (E) diminuiu substancialmente, e as perturbações afetivas (D) passaram a corresponder a mais de 50% do total das admissões. Conclusões: A idade média na admissão ...

Expression of FAPalpha and TGF-beta1 in sudden deaths due to acute myocardial ischemia / 法医学杂志

OBJECTIVE@#To explore the expression of fibroblast activation protein alpha (FAPalpha) and transforming growth factor-beta1 (TGF-beta1) in myocardial cytoplasm for the cases of sudden death due to acute myocardial ischemia.@*METHODS@#The heart tissues of 47 cases were collected. All cases were divided into three groups: control group, acute myocardial infarction group and recurrent myocardial infarction group. FAPalpha and TGF-beta1 expressions were explored in myocardial cytoplasm by immunohistochemistry technology. The staining results were collected by image analysis system and then the positive area ratio and average optical density were detected. The positive signal differences were compared among the groups.@*RESULTS@#Strong FAPalpha and TGF-beta1 expressions were detected in myocardial cytoplasm in both acute and recurrent myocardial infarction groups. The expression of FAPalpha was not detected in myocardial cytoplasm in control group and TGF-beta1 expression showed a weak positive result. FAPalpha and TGF-beta1 expressions showed the statistical difference (P < 0.05) in myocardial infarction (acute and recurrent) groups and control group.@*CONCLUSION@#FAPalpha and TGF-beta1 can be the diagnostic markers for determing acute myocardial infarction.

Isolation, characterisation and kinetic studies on SEVA TB ES-31 antigen, a metallo-serine protease of interest in serodiagnosis.

BACKGROUND: SEVA TB Excretory secretory-31 (ES-31) antigen, a glycoprotein isolated from M. tb H37Ra culture filtrate, was found to be useful in the serodiagnosis of pulmonary tuberculosis (TB), extrapulmonary TB and in HIV-TB coinfection. Further, it has been shown to be a zinc containing serine protease. AIM: To isolate and purify SEVA TB ES-31 antigen from M. tb H37Ra culture filtrate and study of its enzyme properties and peptide sequence. METHODS: ES-31 antigen was purified from culture filtrate of M. tuberculosis H37Ra strain by ammonium sulphate precipitation, SDS-PAGE fractionation and FPLC. Protease activity of ES-31 antigen was studied using azocasein as substrate. ES-31 antigen was further fractionated by two dimensional polyacrylamide gel electrophoresis (2D PAGE) followed by LCMS-T analysis. RESULTS: Mycobacterial metallo-serine protease was purified 3096 fold from M. tb H37Ra culture filtrate protein. Purified enzyme showed optimum activity at pH 7.0 at 37 degrees C. Of the four substrates explored, the enzyme has shown maximum activity with azocasein and had a Km value of 0.01 mM with specific activity of 6250 x 10(-6) U/mg protein. Further, analysis of ES-31 antigen by 2D PAGE showed two protein spots (A and B). CONCLUSION: Kinetic studies on SEVA TB ES-31 protein, an immunogen with metallo serine protease activity are reported for the first time. Purified enzyme had a Km value of 0.01 mM with azocasein as substrate. Further, study on structure and biological role of serine protease will be of interest.

Expression of FAP and alpha-SMA during the incised wound healing in mice skin / 法医学杂志

UNLABELLED@#OBJECTIVE To investigate the time-dependent expression of fibroblast activation protein (FAP) and alpha-smooth muscle actin(alpha-SMA) during the incised wound healing of the skin in mice.@*METHODS@#The expression of FAP and alpha-SMA in incised wound of mice skin was detected by immunohistochemistry and Western blot.@*RESULTS@#By immunohistochemistry, the expression of FAP and alpha-SMA in the normal skin and the skin 1 h after injury maintained at a very low level, but the positive cells expressing FAP and alpha-SMA started to elevate 6 h after injury and reached its peak on 5 d for FAP and on 3 d for alpha-SMA, then gradually decreased to the normal level on 14 d. The expression of FAP and alpha-SMA was observed throughout the wound healing stages 1 d after injuries by Western blot as well with a peak expression occurring on 5 d for FAP and on 3 d for alpha-SMA after injury.@*CONCLUSION@#FAP may be a potentially useful marker for wound age determination and alpha-SMA may be used as an effective indicator for the mid- and late stage incised wound of mice skin. The combination use of FAP and alpha-SMA may be potentially effective indicators for wound age determination.

Serine protease activities in Oxysarcodexia thornax (Walker) (Diptera: Sarcophagidae) first instar larva

We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 µM), a specific inhibitor of chymotrypsin-like serine proteases.

Inhibition of tryptase release from human colon mast cells by histamine receptor antagonists.

The main objective of this study was to investigate the ability of histamine receptor antagonists to modulate tryptase release from human colon mast cells induced by histamine. Enzymatically dispersed cells from human colon were challenged with histamine in the absence or presence of the histamine receptor antagonists, and the tryptase release was determined. It was found that histamine induced tryptase release from colon mast cells was inhibited by up to approximately 61.5% and 24% by the H1 histamine receptor antagonist terfenadine and the H2 histamine receptor antagonist cimetidine, respectively, when histamine and its antagonists were added to cells at the same time. The H3 histamine receptor antagonist clobenpropit had no effect on histamine induced tryptase release from colon mast cells at all concentrations tested. Preincubation of terfenadine, cimetidine or clobenpropit with cells for 20 minutes before challenging with histamine did not enhance the ability of these antihistamines to inhibit histamine induced tryptase release. Apart from terfenadine at 100 microg/ml, the antagonists themselves did not stimulate tryptase release from colon mast cells following both 15 minutes and 35 minutes incubation periods. It was concluded that H1 and H2 histamine receptor antagonists were able to inhibit histamine induced tryptase release from colon mast cells. This not only added some new data to our hypothesis of self-amplification mechanisms of mast cell degranulation, but also suggested that combining these two types of antihistamine drugs could be useful for the treatment of inflammatory bowel disease (IBD).

Epstein-Barr Virus-Associated Peripheral T-Cell Lymphoma involving Spleen in a Renal Transplant Patient

The incidence of posttransplantation lymphoproliferative disorders (PTLDs) has increased in recent years. Although rare, various types of T-cell lymphoma have been reported and their association with Epstein-Barr virus (EBV) has been compared with B-cell PTLDs. We report a case of splenic peripheral T-cell lymphoma occurring in a 47-yr-old male patient 7 yr after renal allograft transplantation. The spleen showed sinusoidal proliferation of focal CD30 positive, large, atypical lymphoid cells. Positivity for CD3 and cytolytic granule-associated proteins was also demonstrated in the tumor cells, while anaplastic large cell lymphoma kinase (ALK) and CD8 were not expressed. Strong nuclear signals for EBV mRNA were noted by EBER1 in situ hybridization. A molecular genetic study demonstrated a rearrangement of the gamma T-cell receptor gene. To our knowledge, this case is unique in terms of a posttransplant T-cell lymphoma that shows focal CD30, cytolytic granule-associated proteins, and EBV positivity.

The changes of tryptase activity in anaphylactic shock guinea-pigs / 法医学杂志

OBJECTIVE@#To investigate the changes of the activity of tryptase of sera, lungs and bronchial tubes in the guinea-pigs which suffered from hetero-serum anaphylactic shock.@*METHODS@#Sera and tissues were collected from anaphylactic shock guinea-pigs, and the enzyme activity was tested colormetrically using special substrate, BAPNA.@*RESULTS@#The activity of tryptase of sera, lungs and bronchial tubes increased significantly in Anaphylactic guinea-pigs compared with control group.@*CONCLUSION@#The changes of tryptase activity are helpful to diagnose anaphylactic shock.

Granzyme B and TIA-1 Expression in Chronic and Acute on Chronic Renal Allograft Rejection

Although active inflammation may be deleterious and indicate immunologic activation in chronically rejected grafts, the underlying mechanism of tissue destruction has been little studied. Twenty-four cases of chronic rejection (CR) with or without acute rejection (AR) were stained with antibodies against CD3, CD8, CD68, granzyme B and TIA-1, and the number of positive cells were counted. Eleven cases of AR served as controls. The number of CD3 and CD8 positive cells increased in the acute on CR group compared to the CR group. About a half of CD3 positive T cells were CD8 positive in both groups, however, the proportion of TIA-1 or granzyme B positive cells was higher in the acute on CR group. The numbers of CD3, CD68, granzyme B and TIA-1 positive cells were higher in the AR group than the acute on CR group, however, no significant difference was found between the two groups. Serum creatinine level and proteinuria at the time of biopsy and the percentages of late onset AR and graft failure rate were higher in the acute on CR group than the CR group. Summarizing, these results suggest that infiltration of activated T cells containing cytotoxic granules plays a role in graft destruction in acute on CR.

Intactness of zona pellucida does not affect the secretion of a trypsin-like protease from mouse blastocyst

Assisted hatching (AH), which is known to improve the hatching potential of mammalian embryos, has been used to increase the pregnancy rate in in vitro fertilization cycles. However, the effect of AH on a trypsin-like protease, which is known to be associated with the hatching process, has not been studied. In this study, we evaluate whether the intactness of zona pellucida affects the secretion of a trypsin-like protease from mouse blastocyst. Four- to 8-cell stage mouse embryos were collected at 66- to 68 hr after hCG injection and divided into 3 groups according to the manipulation of zona pellucida. The groups are no treatment (control), drilling of zona pellucida (ZD) and thinning of zona pellucida (ZT). The activity of a trypsin-like protease, blastocyst development and hatching rate were compared among the three groups at 110 and 135 hr after hCG injection, respectively. The protease activity and blastocyst development were not significantly different among control, ZD and ZT groups at 110 and 135 hr after hCG injection, respectively. However, the hatching rate of ZD and ZT groups was significantly higher than that of control group at each time, respectively (p>0.001). Even in the zona pellucida removed embryos, the protease activity did not differ from the control group. In conclusion, the secretion of a trypsin-like protease from mouse blastocyst does not seem to be affected by the intactness of zona pellucida.