Molecular identification, characterization, and expression analysis of a serine protease inhibitor gene from cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae) / Identificação molecular, caracterização e análise de expressão de um gene inibidor de serina protease da lagarta Helicoverpa armigera (Lepidoptera: noctuidae) na cultura de algodão

Abstract Serine protease inhibitors (serpins), a superfamily of protease inhibitors, are known to be involved in several physiological processes, such as development, metamorphosis, and innate immunity. In our study, a full-length serpin cDNA, designated Haserpin1, was isolated from the cotton bollworm Helicoverpa armigera. The cDNA sequence of Haserpin1 is 1176 nt long, with an open reading frame encoding 391 amino acids; there is one exon and no intron. The predicted molecular weight of Haserpin1 is 43.53 kDa, with an isoelectric point of 4.98. InterProScan was employed for Haserpin1 functional characterization, which revealed that Haserpin1 contains highly conserved signature motifs, including a reactive center loop (RCL) with a hinge region (E341-N350), the serpin signature, (F367-F375) and a predicted P1-P1′ cleavage site (L357-S358), which are useful for identifying serpins. Transcripts of Haserpin1 were constitutively expressed in the fat body, suggesting that it is the major site for serpin synthesis. During the developmental stages, a fluctuation in the expression level of Haserpin1 was observed, with low expression detected at the 5th-instar larval stage. In contrast, relatively high expression was detected at the prepupal stage, suggesting that Haserpin1 might play a critical role at the H. armigera wandering stage. Although the detailed function of this serpin (Haserpin1) needs to be elucidated, our study provides a perspective for the functional investigation of serine protease inhibitor genes.

Resumo Sabe-se que os inibidores de serina protease (serpinas), uma superfamília de inibidores de protease, estão envolvidos em vários processos fisiológicos, como desenvolvimento, metamorfose e imunidade inata. Neste estudo, um cDNA de serpina de comprimento total, denominado Haserpin1, foi isolado da lagarta Helicoverpa armigera na cultura de algodão. A sequência de ADNc de Haserpin1 tem 1.176 nt de comprimento, com uma grelha de leitura aberta que codifica 391 aminoácidos; existe um éxon, mas nenhum íntron. O peso molecular previsto de Haserpin1 é de 43,53 kDa, com um ponto isoelétrico de 4,98. O InterProScan foi empregado para a caracterização funcional do Haserpin1, que revelou que o Haserpin1 contém motivos de assinatura altamente conservados, incluindo um loop central reativo (RCL) com uma região de dobradiça (E341-N350), a assinatura da serpina (F367-F375) e um local de clivagem previsto de P1-P1' (L357-S358), que são úteis para identificar serpinas. As transcrições de Haserpin1 foram expressas constitutivamente no corpo gordo, sugerindo que é o principal local para a síntese de serpinas. Durante os estágios de desenvolvimento, observou-se uma flutuação no nível de expressão de Haserpin1, com baixa expressão detectada no estágio larval do 5º ínstar. Por outro lado, detectou-se uma expressão relativamente alta no estágio pré-pupal, sugerindo que o Haserpin1 pode desempenhar um papel crítico no estágio errante de H. armigera. Embora a função detalhada dessa serpina (Haserpin1) precise ser elucidada, este estudo fornece uma perspectiva para a investigação funcional dos genes inibidores da serina protease.

Serine protease inhibitor, serpin, is a biomarker following Schistosoma mansoni re-infection in mice

@#Schistosomiasis is a chronic parasitic disease affecting mostly low income and resourcelimited countries. Despite the distribution of the curative medicine, praziquantel (PZQ), the frequency of re-infection is commonly reported, thus, making a difficulty to discriminate treatment failure after re-infection. Therefore, assessing Schistosoma mansoni re-infection after praziquantel administration is crucial to prove the treatment efficacy and to break the transmission of infection in endemic areas. The evolution of highly sensitive and specific diagnostic markers, reliable to detect the re-infection and to evaluate the treatment efficacy, is required to control schistosomiasis. In this study, the potential role of serpin recombinant antigen of S. mansoni as a biomarker of re-infection and chemotherapeutic efficacy has been assessed. Therefore, 20 mice were experimentally challenged and re-challenged with 50 S. mansoni cercariae and divided into 4 equal groups; the first included infected mice (control positive), the second group was twice infected with S. mansoni and left untreated, the third included mice twice infected then treated with praziquantel following the last challenge, and the forth one remained uninfected and untreated (control negative). The current findings demonstrated that high levels of IgG and IgG1 bound to serpin were detected following the re-infection and rapidly declined post treatment. In summary, S. mansoni recombinant serpin could be used as a promising marker to discriminate S. mansoni re-infection and evaluated the efficacy of treatment. The translation of such a potential tool in endemic areas will provide a significant support for the elimination and control programs against schistosomiasis.

Molecular identification, characterization, and expression analysis of a serine protease inhibitor gene from cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract Serine protease inhibitors (serpins), a superfamily of protease inhibitors, are known to be involved in several physiological processes, such as development, metamorphosis, and innate immunity. In our study, a full-length serpin cDNA, designated Haserpin1, was isolated from the cotton bollworm Helicoverpa armigera. The cDNA sequence of Haserpin1 is 1176 nt long, with an open reading frame encoding 391 amino acids; there is one exon and no intron. The predicted molecular weight of Haserpin1 is 43.53 kDa, with an isoelectric point of 4.98. InterProScan was employed for Haserpin1 functional characterization, which revealed that Haserpin1 contains highly conserved signature motifs, including a reactive center loop (RCL) with a hinge region (E341N350), the serpin signature, (F367F375) and a predicted P1P1 cleavage site (L357S358), which are useful for identifying serpins. Transcripts of Haserpin1 were constitutively expressed in the fat body, suggesting that it is the major site for serpin synthesis. During the developmental stages, a fluctuation in the expression level of Haserpin1 was observed, with low expression detected at the 5th-instar larval stage. In contrast, relatively high expression was detected at the prepupal stage, suggesting that Haserpin1 might play a critical role at the H. armigera wandering stage. Although the detailed function of this serpin (Haserpin1) needs to be elucidated, our study provides a perspective for the functional investigation of serine protease inhibitor genes.

Resumo Sabe-se que os inibidores de serina protease (serpinas), uma superfamília de inibidores de protease, estão envolvidos em vários processos fisiológicos, como desenvolvimento, metamorfose e imunidade inata. Neste estudo, um cDNA de serpina de comprimento total, denominado Haserpin1, foi isolado da lagarta Helicoverpa armigera na cultura de algodão. A sequência de ADNc de Haserpin1 tem 1.176 nt de comprimento, com uma grelha de leitura aberta que codifica 391 aminoácidos; existe um éxon, mas nenhum íntron. O peso molecular previsto de Haserpin1 é de 43,53 kDa, com um ponto isoelétrico de 4,98. O InterProScan foi empregado para a caracterização funcional do Haserpin1, que revelou que o Haserpin1 contém motivos de assinatura altamente conservados, incluindo um loop central reativo (RCL) com uma região de dobradiça (E341-N350), a assinatura da serpina (F367-F375) e um local de clivagem previsto de P1-P1' (L357-S358), que são úteis para identificar serpinas. As transcrições de Haserpin1 foram expressas constitutivamente no corpo gordo, sugerindo que é o principal local para a síntese de serpinas. Durante os estágios de desenvolvimento, observou-se uma flutuação no nível de expressão de Haserpin1, com baixa expressão detectada no estágio larval do 5º ínstar. Por outro lado, detectou-se uma expressão relativamente alta no estágio pré-pupal, sugerindo que o Haserpin1 pode desempenhar um papel crítico no estágio errante de H. armigera. Embora a função detalhada dessa serpina (Haserpin1) precise ser elucidada, este estudo fornece uma perspectiva para a investigação funcional dos genes inibidores da serina protease.

A Higher Angiogenin Expression is Associated With a Nonnuclear Maspin Location in Laryngeal Carcinoma

OBJECTIVES: In numerous malignancies, angiogenin (ANG) and Maspin are important proangiogenic and antiangiogenic regulators, respectively. The aim of this study was to identify potential relationships between the biological roles of these two proteins in laryngeal squamous cell carcinoma (LSCC). METHODS: Immunohistochemical staining for ANG and Maspin was performed on specimens from 76 consecutive LSCC patients treated with surgery alone, considering the subcellular pattern of Maspin expression. Univariate and multivariate statistical models were used for prognostic purposes. RESULTS: On univariate analysis, a different level of ANG expression was seen for patients stratified by subcellular Maspin expression pattern: the mean ANG expression was higher in cases with a nonnuclear MASPIN expression than in those with a nuclear pattern (P=0.002). Disease-free survival (DFS; in months) differed significantly when patients were stratified by N stage (P=0.01). Patients whose Maspin expression was nonnuclear (i.e., it was cytoplasmic or there was none) had a significantly higher recurrence rate (P or =5.0% had a significantly shorter DFS than those with an ANG expression or =5.0% was a significant, independent, negative prognostic factor in terms of DFS (P=0.041). CONCLUSION: Our results support the hypothesis that a higher ANG expression is associated with a nonnuclear Maspin expression pattern in patients with LSCC. Further studies are needed to clarify the relationship between the ANG and Maspin pathways, and their potential diagnostic and therapeutic role in LSCC.

Expression of maspin in benign and malignant salivary gland tumors: An immunohistochemical study.

Context: Maspin is a novel serine protease inhibitor (serpin) with multifaceted tumor‑suppressive activities. It was originally identified in normal human breast myoepithelial cells and shows variable expression in different types of cancer cells. Maspin displays anti‑metastatic properties in mammary and prostate cancer. Its expression is maintained during ovarian, lung and pancreatic carcinogenesis, indicating that Maspin regulated metastatic potential is tissue specific. Thus, it is possible that Maspin participates in salivary gland tumor biology as well. In this study, expression pattern of maspin in benign and malignant salivary gland tumors is analyzed, to understand the biological behavior of salivary gland tumors with respect to maspin expression. Aims and Objectives: The aim of this study was to demonstrate, record, and correlate the expression pattern of maspin in benign and malignant salivary gland tumors. Settings and Design: A retrospective study of maspin expression in 30 diagnosed cases of benign and malignant salivary gland tumors retrieved from archives of our department. Materials and Methods: Anti‑maspin antibody and horseradish peroxidase detection system. Statistical Analysis: Descriptive statistical analysis and Chi‑square/Fisher Exact test. Results: Intense expression with P < 0.001 is associated with benign tumors, nuclear staining with P < 0.001 is significantly associated with benign tumors and cytoplasmic staining with P = 0.020 is associated with malignant tumors. Conclusion: Intensity of expression is more in benign tumors when compared with malignant tumors. The benign tumors showed both nuclear and cytoplasmic expression. Some malignant tumors did express maspin, but mainly in the cytoplasm.

Anti-Apoptotic Effects of SERPIN B3 and B4 via STAT6 Activation in Macrophages after Infection with Toxoplasma gondii

Toxoplasma gondii penetrates all kinds of nucleated eukaryotic cells but modulates host cells differently for its intracellular survival. In a previous study, we found out that serine protease inhibitors B3 and B4 (SERPIN B3/B4 because of their very high homology) were significantly induced in THP-1-derived macrophages infected with T. gondii through activation of STAT6. In this study, to evaluate the effects of the induced SERPIN B3/B4 on the apoptosis of T. gondii-infected THP-1 cells, we designed and tested various small interfering (si-) RNAs of SERPIN B3 or B4 in staurosporine-induced apoptosis of THP-1 cells. Anti-apoptotic characteristics of THP-1 cells after infection with T. gondii disappeared when SERPIN B3/B4 were knock-downed with gene specific si-RNAs transfected into THP-1 cells as detected by the cleaved caspase 3, poly-ADP ribose polymerase and DNA fragmentation. This anti-apoptotic effect was confirmed in SERPIN B3/B4 overexpressed HeLa cells. We also investigated whether inhibition of STAT6 affects the function of SERPIN B3/B4, and vice versa. Inhibition of SERPIN B3/B4 did not influence STAT6 expression but SERPIN B3/B4 expression was inhibited by STAT6 si-RNA transfection, which confirmed that SERPIN B3/B4 was induced under the control of STAT6 activation. These results suggest that T. gondii induces SERPIN B3/B4 expression via STAT6 activation to inhibit the apoptosis of infected THP-1 cells for longer survival of the intracellular parasites themselves.

Schistosome serine protease inhibitors: parasite defense or homeostasis?

Serpins are a structurally conserved family of macromolecular inhibitors found in numerous biological systems. The completion and annotation of the genomes of Schistosoma mansoni and Schistosoma japonicum has enabled the identification by phylogenetic analysis of two major serpin clades. S. mansoni shows a greater multiplicity of serpin genes, perhaps reflecting adaptation to infection of a human host. Putative targets of schistosome serpins can be predicted from the sequence of the reactive center loop (RCL). Schistosome serpins may play important roles in both post-translational regulation of schistosome-derived proteases, as well as parasite defense mechanisms against the action of host proteases.

Serpinas são uma família de inibidores macromoleculares estruturalmente conservados encontrados em inúmeros sistemas biológicos. O término e a anotação dos genomas de Schistosoma mansoni e de Schistosoma japonicum permitiram a identificação por análise filogenética de dois principais clados de serpinas. S. mansoni mostra uma multiplicidade maior de genes de serpinas, talvez refletindo uma adaptação à infecção de um hospedeiro humano. Alvos putativos das serpinas de esquistossomos podem ser preditos a partir da sequência do "loop" do centro reativo. Serpinas de esquistossomos podem ter importantes papeis tanto na regulação pós-traducional de proteases derivadas do esquistossoma, quanto nos mecanismos de defesa contra a ação de proteases do hospedeiro.

Spi2 gene polymorphism is not associated with recurrent airway obstruction and inflammatory airway disease in thoroughbred horses

The aim was to detect the presence of polymorphisms at exons 1, 2, 3 and 4 of the Spi2 gene, and evaluate a possible association between them and recurrent airway obstruction (RAO) or inflammatory airway disease (IAD) in thoroughbred horses, through single-strand conformational-polymorphism (SSCP) screening. Although polymorphism was not detected in exons 1, 2 and 3, three alleles and six genotypes were identified in exon 4. The frequencies of allele A (0.6388) and genotype AA (0.3888) were higher in horses affected by RAO, although no association was found between polymorphism and horses with either RAO or IAD.

Maspin Suppresses Survival of Lung Cancer Cells through Modulation of Akt Pathway / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Maspin is a tumor suppressor protein that has been reported to stimulate the cell death of cancer and inhibit the metastasis of cancer. The present study aimed to explore the survival pathway by which maspin modulates the resistance of human lung cancer cells to chemotherapeutic drugs, and the consequences of maspin gene therapy in an animal model. MATERIALS AND METHODS: NCI-H157 and A549 cells were transfected with either a mock vector (pCMVTaq4C), maspin (pCMV-maspin), siControl or siMaspin. RT-PCR and Western blot analysis were performed to study the expressions of survival proteins in lung cancer. cDNA microarray analysis was carried out to compare the maspin-modulated gene expression between the xenograft tumors derived from the lung cancer cells that were stably transfected with pCMVTaq4C or pCMV-maspin. Maspin gene therapy was performed by intra-tumoral injections of pCMVTaq4C or pCMV-maspin into the pre-established subcutaneous tumors in nude mice. RESULTS: Maspin significantly decreased the survival to doxorubicin and etoposide, whereas did not affect the survival to cisplatin in the NCI-H157 cells. Interestingly, transfection with a maspin plasmid resulted in a significant reduction of the phosphorylation of Akt in the NCI-H157 cells, whereas knockdown of maspin increased the phosphorylation of Akt in the A549 cells. Microarray analysis of the xenograft tumors revealed a specific gene expression profile, demonstrating that maspin is associated with the differential expressions of PTEN and IGF2R. Direct transfer of pCMV-maspin into the tumor significantly retarded the tumor growth in the animal experiments (p=0.0048). CONCLUSION: Lung cancer cells lacking maspin could be resistant to chemotherapeutic drugs such as doxorubicin or etoposide, at least in part by maintaining Akt phosphorylation.

Vaspin-a novel fat-derived serine protease Inhibitor / 中华内分泌代谢杂志

ncreased. But it remains controversial how vaspin is correlated with insulin sensitivity. More studies based on large population are needed to identify the associ-ation between vaspin and insulin sensitivity.

The inhibitory effect of pigment epithelium-derived factor on migration of gliomas / 中华检验医学杂志

Objective To assess the impact of pigment epithelium-derived factor(PEDF) on the migration of the glioma cells.Methods PEDF(100 ng/ml)was added to U87 cells(U87PFDF),and VEGF of 0.25μg/ml was added to U87PEDF+VEGF while U87 cells as control(U87con).The cell migratory assav was used tbr evaluating its inhibitory migration rate of PEDF.Real time RT-PCR was used for evaluating the expression of Laminin-8 gene.Results The number of migratory cells was higher than those with added PEDF,and the inhibitory rate of migratory was 38% even in the presence of VEGF.Real time RT-PCR revealed that the mRNA expression levels of α4,β1,γl were(1.043±0.090),(0.823±0.012).(0.762±0.05) copy/μl,which were higher than those treated by PEDF(0.633±0.004),(0.442±0.005).(0.424±0.002)copy/μl,respectively (P＜0.05).Conclusion PEDF could decrease the migmtory capacity of the glioma cells even in the presence of VEGF because of the regulation of Laminin-8 in part.

Relationship between serpin peptidase inhibitor 3 gene polymorphism and cerebral infarction / 临床神经病学杂志

0.05),but the rate of GG genotype in cerebral infarction group was significantly higher than that in normal control group(P