Antimicrobial mechanism of antimicrobial peptide and research progress / 中国组织工程研究

BACKGROUND: As a potential candidate that can be extracted from natural sources and used to fight antibiotic-resistant bacteria, antimicrobial peptides have attracted extensive attention of scientists. Familiarity with the antimicrobial mechanism of antimicrobial peptides is conducive to the clinical application of antimicrobial peptides. OBJECTIVE: To review the advance in research of antimicrobial mechanism of antimicrobial peptides. METHODS: The first author conducted a computer-based retrieval of PubMed, Springeriink, Web of Science. ScienceDirect databases for articles regarding the antimicrobial mechanism and research advance published from January 2013 to March 2019. RESULTS AND CONCLUSION: Antimicrobial peptides are a class of special molecules with broad-spectrum antimicrobial activity. In some organisms, antimicrobial peptides arc considered lo bo an important part of innate immune system. The antimicrobial mechanism of antimicrobial peptides can be divided into two main modes: direct killing and immune regulation, and direct killing mechanism can be further divided into membrane targeting and non-membrane targeting. At the same time, based on the extensive application of antimicrobial peptides, it is expected that many resistance strategies have been developed in microbial environments such as staphylococcus, oral bacteria (including streptococcus) and intestinal bacteria (including salmonella). These resistance strategies mainly include passive resistance and induction or adaptive resistance mechanisms. In the future research and application, cationic peptide is an effective choice to solve the increasing multidrug resistance. In addition to the obligation to design new methods to combat the resistance of antimicrobial peptides in bacteria, general preventive measures against the resistance of conventional antibiotics should also be paid attention to.

Preliminary Study of Peptide From Periplaneta americana On Antitumor Immunity in L1210-Bearing Mice / 中国药学杂志

OBJECTIVE: To explore the antitumor effect and immunity change of Periplaneta americana CⅡ3 and related synthetic peptide HFDT1 in mice and to provide a base for clinical application of the materials. METHODS: Sixty BALB/c mouse tumor model with leukemia L1210 were established and divided into five groups. The mice were treated with Periplaneta americana CⅡ3 high dose, low dose, HFDT1, CTX and normal saline for 10 d, respectively. Twelve mice were as normal control group. The tumor inhibition rates and weight of body were observed in different groups. Then the results were observed including thymus and spleen index, the numbers of immune cells in peripheral blood, the ratio of splenic total lymphocytes, splenic B cell(CD45R+) and T cell(CD3+), T cell subset(CD3+ CD4+ and CD3+ CD8+ T cell), and the levels of IgG, IgA and IgM in serum through weight, auto-analysis of blood cell, flow cytometry, sandwich-antibody ELISA from thymus, spleen, peripheral blood in the mice. RESULTS: The results showed that CⅡ3 and HFDT1 inhibited the growth of mouse L1210 tumor and maintained the mouse weights. They increased the mouse thymus indexes and spleen indexes effectively and the numbers of peripheral blood immune cells. They maintained the spleen lymphocyte and regulated the ratio of T and B lymphocytes, CD3+ CD4+ T cell and CD3+ CD8+ T cell. They increased the levels of serum IgG, IgA and IgM. CONCLUSION: Periplaneta americana extract CⅡ3 and related synthetic peptide HFDT1 have a strong inhibition effect to tumor. The antitumor effect of CⅡ3 and HFDT1 may be achieved through to improve immune function. They are hoped to become efficient and low toxicity antitumor drugs. Especially HFDT1, it has an extensive application because it can be synthesized by artificial methods.

Activation of the Mating Pheromone Response Pathway of Lentinula edodes by Synthetic Pheromones

Pheromone (PHB)-receptor (RCB) interaction in the mating pheromone response pathway of Lentinula edodes was investigated using synthetic PHBs. Functionality of the C-terminally carboxymethylated synthetic PHBs was demonstrated by concentration-dependent induction of a mating-related gene (znf2) expression and by pseudoclamp formation in a monokaryotic strain S1-11 of L. edodes. Treatment with synthetic PHBs activated the expression of homeodomain genes (HDs) residing in the A mating type locus, and of A-regulated genes, including znf2, clp1, and priA, as well as genes in the B mating type locus, including pheromone (phb) and receptor (rcb) genes. The synthetic PHBs failed to discriminate self from non-self RCBs. PHBs of the B4 mating type (B4 PHBs) were able to activate the mating pheromone response pathway in both monokaryotic S1-11 and S1-13 strains, whose B mating types were B4 (self) and B12 (non-self), respectively. The same was true for B12 PHBs in the B4 (non-self) and B12 (self) mating types. The synthetic PHBs also promoted the mating of two monokaryotic strains carrying B4-common incompatible mating types (A5B4 × A1B4). However, the dikaryon generated by this process exhibited abnormally high content of hyphal branching and frequent clamp connections and, more importantly, was found to be genetically unstable due to overexpression of mating-related genes such as clp1. Although synthetic PHBs were unable to discriminate self from non-self RCBs, they showed a higher affinity for non-self RCBs, through which the mating pheromone response pathway in non-self cells may be preferentially activated.

Development of a Synthetic Surfactant Using a Surfactant Protein-C Peptide Analog: In Vitro Studies of Surface Physical Properties

PURPOSE: Pulmonary surfactant (PS) replacement has been the gold standard therapy for neonatal respiratory distress syndrome; however, almost all commercial PSs contain animal proteins. We prepared a synthetic PS by using a human surfactant protein (SP) analog and evaluated its in vitro properties. MATERIALS AND METHODS: A peptide sequence (CPVHLKRLLLLLLLLLLLLLLLL) of human SP-C was chosen to develop the peptide analog (SPa-C). The new synthetic SP-C PS (sSP-C PS) was synthesized from SPa-C, dipalmitoyl phosphatidylcholine, phosphatidyl glycerol, and palmitic acid. Physical properties of the sSP-C PS were evaluated by measuring the maximum and minimum surface tensions (STs), surfactant spreading, and adsorption rate. In addition, we recorded an ST-area diagram. The data obtained on sSP-C PS were subsequently compared with those of purified natural bovine surfactant (PNBS), and the commercial product, Surfacten(R). RESULTS: The sSP-C PS and Surfacten(R) were found to have maximum ST values of 32-33 mN/m, whereas that of PNBS was much lower at 19 mN/m. The minimum ST values of all three products were less than 10 mN/m. The values that were measured for the equilibrium ST of rapidly spreading sSP-C PS, Surfacten(R), and PNBS were 27, 27, and 24 mN/m, respectively. The surface adsorptions were found to be the same for all three PSs (20 mN/m). ST-area diagrams of sSP-C PS and Surfacten(R) revealed similar properties. CONCLUSION: In an in vitro experiment, the physical properties exhibited by sSP-C PS were similar to those of Surfacten(R). Further study is required to evaluate the in vivo efficacy.

Study on design and three-dimension structure of a broad-spectrum inhibitory peptide against staphylococcal enterotoxins superantigen / 中国免疫学杂志

Objective:Peptides were designed on the basis of high conservative regions of amino acid sequences and structures of the SEs,three-dimension structure of P72 was constructed.Methods: Bioinformatics analysis softwares such as Vector NTI 10.3, InsightII 2000,Discovery Studio 1.7 were used to analyse and predict the space structure of P72.Results:three-dimensional domains of the peptide P72 from SEA, SEB and SEC were quite similar, Peptide P72 was far away from TCRVβchain and MHC class II molecule.Conclusion:The inhibitory activity of peptide P72 may not due to binding to MHCⅡ and TCRVβchain.The exact mechanism of inhibitory activity of P72 should be explored.

Molecular cloning and antibacterial activity of hepcidin from Chinese rare minnow (Gobiocypris rarus)

Background Hepcidins, a kind of cysteine-rich antimicrobial peptides, play important roles in host immunological processes and iron regulation, which have been identified from several fish species. The rare minnow (Gobiocypris rarus), an endemic cyprinid fish in China, has been used extensively as model animal in laboratory. However, little is known about its hepcidin. Here, we report the cloning and characterization of a hepcidin gene from the liver of Chinese rare minnow. Results The full-length cDNA of rare minnow hepcidin is 662 bp, which contains an ORF of 273 bp encoding a prepropeptide of 90 amino acid residues. The predicted prepropeptide contains three domains: a signal peptide of 24 amino acids, a prodomain of 41 amino acids, and a mature peptide of 25 amino acids. Sequence alignment showed eight conserved cysteine residues in the mature peptide, which formed four disulfide bonds in spatial structure. The deduced structure of mature peptide showed a high degree of homology to the human hepcidin. Phylogenetic analysis showed that it had a close relationship with zebrafish hepcidin, and clustered in a clade with these from Cyprinidae. Synthetic peptide of rare minnow hepcidin could inhibit the growth of Gram positive bacterium Staphylococcus aureus and Gram negative bacteria Escherichia coli and Aeromonas hydrophila. Conclusion These results suggested that rare minnow hepcidin had typical structure of hepcidins and antibacterial activity. It could participate in innate immune response as an antibacterial agent and be used as antibiotic substance.

IgE-Binding Epitope Mapping and Tissue Localization of the Major American Cockroach Allergen Per a 2

PURPOSE: Cockroaches are the second leading allergen in Taiwan. Sensitization to Per a 2, the major American cockroach allergen, correlates with clinical severity among patients with airway allergy, but there is limited information on IgE epitopes and tissue localization of Per a 2. This study aimed to identify Per a 2 linear IgE-binding epitopes and its distribution in the body of a cockroach. METHODS: The cDNA of Per a 2 was used as a template and combined with oligonucleotide primers specific to the target areas with appropriate restriction enzyme sites. Eleven overlapping fragments of Per a 2 covering the whole allergen molecule, except 20 residues of signal peptide, were generated by PCR. Mature Per a 2 and overlapping deletion mutants were affinity-purified and assayed for IgE reactivity by immunoblotting. Three synthetic peptides comprising the B cell epitopes were evaluated by direct binding ELISA. Rabbit anti-Per a 2 antibody was used for immunohistochemistry. RESULTS: Human linear IgE-binding epitopes of Per a 2 were located at the amino acid sequences 57-86, 200-211, and 299-309. There was positive IgE binding to 10 tested Per a 2-allergic sera in 3 synthetic peptides, but none in the controls. Immunostaining revealed that Per a 2 was localized partly in the mouth and midgut of the cockroach, with the most intense staining observed in the hindgut, suggesting that the Per a 2 allergen might be excreted through the feces. CONCLUSIONS: Information on the IgE-binding epitope of Per a 2 may be used for designing more specific diagnostic and therapeutic approaches to cockroach allergy.

Production and evaluation of chicken antibodies against a synthetic peptide from glial growth factor / Producción y evaluación de anticuerpos de gallinas contra un péptido sintético del factor de crecimiento glial

Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFb isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFb1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.

Las Neuregulinas (NRG) son proteínas que pertenecen a la familia de los factores de crecimiento epidermal. Se ha demostrado que estos factores son esenciales para el desarrollo y mantenimiento de la funcionalidad del sistema nervioso. Debido a la dificultad para purificar estas proteínas y la falta de especificidad de los anticuerpos disponibles comercialmente, el objetivo de este trabajo fue producir anticuerpos contra un péptido sintético capaz de detectar e identificar una isoforma de la Neuregulina (GGFb). Para lograr este objetivo, se desarrollaron anticuerpos en gallinas (IgY) contra un péptido sintético diseñado a partir de la secuencia aminoacídica de la región extracelular de GGFb, utilizando programas de predicción de epítopes. Los resultados demuestran que el péptido seleccionado fue immunogénico debido a que estimuló una respuesta inmune específica tipo B en el modelo utilizado. Estos anticuerpos fueron también capaces de reconocer una proteína recombinante e isoformas de GGF presentes en diferentes muestras biológicas. Nuestros resultados demuestran el potencial valor de las inmunoglobulinas Y (IgY) contra péptidos sintéticos como una herramienta de aplicación para la investigación en neurociencia.

Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.

Anti-peptide antibodies: a tool for detecting IL-8 in salmonids

Background: Interleukin 8 is a chemokine that is produced by several types of cells, like macrophages and has chemotactic activity in particular on neutrophils, playing a key role during the inflammatory process. It has been demonstrated at the molecular level that this molecule is present and conserved in several vertebrate groups, pointing its importance. Analysis of the amino acid sequence of IL-8, projected from cDNA of Salmo salar, presents homology with the sequences of mammals, poultry and lamprey, indicating the presence of a homologous molecule in higher fish. However, there is no information at protein level, which allows characterizing the regulatory role of this molecule during the immune response in fish. Results: In this work, we designed and synthesized an epitope peptide of 10 residues with a purity of 95 percent and mass of 1158.7 kDa, which showed a random coil structure. From this peptide it was able to generate a polyclonal mono-specific antibody which was capable of detecting the whole molecule of IL-8 in tissue and cellular model of salmonids. Conclusions: The resulting antibody is a versatile tool for detecting IL-8 by different immune techniques such as ELISA, dot blot, western blotting and immunocytofluorescence. Analysis of IL-8 at proteomic level is a useful method for characterizing immune properties of this molecule in fish.

Production of immunoglobulin Y (IgY) against synthetic peptide analogs of the immunogenic epitopes of the hepatitis B surface antigen

INTRODUCTION: Several studies have been conducted on the use of Immunoglobulin Y (IgY) technology in the fields of diagnostics and therapeutics. IgY is the avian counterpart of the mammalian immunoglobulin G (IgG) which is exclusively transferred from the hen to the yolk thus conferring passive immunization to the growing embryo. However, despite the advantages it offers over the use of mammalian immunoglobulin, IgY technology has remained underutilized. OBJECTIVE:The objective of this study is to produce an IgY with activity against synthetic peptide analogs of known immunogenic epitopes of the Hepatitis B Surface Antigen (HBsAg) - a molecular marker of Hepatitis B infection. METHODS: Chickens were immunized with synthetic peptide analogs of previously reported immunogenic epitopes of the S and the pre-S1 regions of the Hepatitis B surface antigen (HBsAg). IgY specific for the synthetic peptides was isolated by delipidation and salt precipitation and was further purified by affinity chromatography. Purity and molecular weights of the whole IgY molecule and its subunits were assessed and determined by SDS-PAGE. Anti-peptide activity and specificity were determined by indirect ELISA. The study was approved by the Ethical Review Board (ERB) and Technical Review Board of the Research Implementation and Development Office (RIDO), University of the Philippines Manila. RESULTS AND CONCLUSION: The IgY that was purified in this study had an approximate molecular weight of 165 kilodaltons. The heavy and light chains are 60 and 28 kilodaltons, respectively. The affinity purified IgY demonstrated anti-peptide antibody activity against synthetic peptide analogs of known immunogenic epitopes of the HBsAg. Specific binding against a battery of synthetic peptides also revealed that the affinity purified IgY specifically binds to the known immunogenic epitope of the HBsAg.

The effects of newly formed synthetic peptide on bone regeneration in rat calvarial defects

PURPOSE: Significant interest has emerged in the design of cell scaffolds that incorporate peptide sequences that correspond to known signaling domains in extracellular matrix and bone morphogenetic protein. The purpose of this study was to evaluate the bone regenerative effects of the synthetic peptide in a critical-size rat calvarial defect model. METHODS: Eight millimeter diameter standardized, circular, transosseus defects created on the cranium of forty rats were implanted with synthetic peptide, collagen, or both synthetic peptide and collagen. No material was was implanted the control group. The healing of each group was evaluated histologically and histomorphometrically after 2- and 8-week healing intervals. RESULTS: Surgical implantation of the synthetic peptide and collagen resulted in enhanced local bone formation at both 2 and 8 weeks compared to the control group. When the experimental groups were compared to each other, they showed a similar pattern of bone formation. The defect closure and new bone area were significantly different in synthetic peptide and collagen group at 8 weeks. CONCLUSIONS: Concerning the advantages of biomaterials, synthetic peptide can be an effective biomaterial for damaged periodontal regeneration.

Detection of the Avian Influenza Viruses Nonstructural Protein 1 for Distinction between Vaccinated and Infected Chickens Using Synthetic Peptide-Based ELISA

Avian influenza (AI) virus infects both animal and human. Low pathogenic AI virus infections (some H7 and H9 subtypes) have been reported all over the world and pose a potential threat to the poultry industry. Vaccination is the most effective way to prevent virus infection. However, vaccination makes it difficult to differentiate between vaccinated chickens and infected chickens. In order to differentiate vaccinated chickens from naturally infected chickens, we adopted synthetic peptide-based enzyme-linked immunosorbent assay (ELISA) using the peptide sequences from nonstructural protein 1 (NS1) of H9N2. Five synthetic peptides were designed using Protein Variability Sever (http://imed.med.ucm.es/PVS/) and synthesized. NS1-1 ~ NS1-4 peptides failed to detect serum antibodies from both vaccinated and naturally infected chickens. NS1-5 peptide from the C-terminal NS1 protein detected serum antibody from naturally infected chickens but not vaccinated chickens. These results imply that NS1-5 peptide may be a useful tool to differentiate naturally infected chicken from vaccinated chicken as being used in the synthetic peptide-based ELISA.

The effect of CpG ODN on the immune responses and immune-contraception induced by ZP~(121-140) synthetic peptide / 中国免疫学杂志

Objective:To investigate the effect of CpG ODN on the immune responses and immune-contraception induced by ZP~(121-140) synthetic peptide.Methods: BALB/e mice were given an injection into the left tibialis anterior muscle of ZP~(121-140) synthetic peptide with 20 μg CpG ODN or CFA,then the mice were given other injections at 2,4,6 weeks using the same formulation.The mice' s blood was collected before each vaccination and after the last vaccination every 2 weeks.The specific IgG and IgA in sere and non-specific cytokines IFN-γ,TNF-α,IL-10 in vaginal mucosa were measured by ELISA.The ovarial pathological changes were undertaken using hemattxylin and eosin-stained paraffin sections.Results:The specific IgG in sera and IgA in vaginal mucosa induced by ZP~(121-140) synthetic peptide combined with CpG ODN were no more than those of ZP~(121-140) synthetic peptide combined with CFA.There were significant increases in IFN-γ,and TNF-α when CpG ODN was mixed with ZP121-140 synthetic peptide and the increase of CpG ODN was more significant than that of CFA.Otherwise there was a significant decrease in IL-10 when CpG ODN was mixed with ZP~(121-140) synthetic peptide and the decrease of CpG ODN was more significant than thai of CFA.There was no significant difference in the rate of pregnancy between CpG ODN group and CFA group,but the average number of birth mice in CpG ODN group was less than that in CFA group.No pathological changes were found in the ovaries of experimental mice.Conclusion: The adjuvant effect of CpG ODN is more advantageous than that of CFA in contraception vaccine research.

Preparation and preliminary application of a novel monoclonal antibody against human SIRPα / 第二军医大学学报

To prepare a monoclonal antibody against human SIRPα using synthetic peptide, and to use it for immune test to assess its efficacy. Methods: The synthetic peptide of SIRPα was linked with KLH and the product was used as antigen for immunization of BALB/c mice. The mAb anti-SIRPα was obtained by hybridoma technique. The produced mAb was used for flow cytometry, Western blotting analysis and immunohistochemistry assay. Cytokines secreted by PMA-treated THP-1 cells were tested by antibody arrays after exposure to the obtained mAb, and the levels of TNF-α and IL-6 were assayed by ELISA. Results: The mAb secreting hybridoma clone was successfully obtained and it had a satisfactory efficacy when used for flow cytometry, Western blotting analysis and immunohistochemistry assay. Compared with negative control and isotype control, the prepared mAb can stimulate TNF-α and IL-6 secretion in PMA-treated THP-1 cells. Conclusion: We have successfully prepared the mAb against SIRPo using synthetic peptide.

Synthetic Smac Peptide Enhances Chemo-sensitivity of Bladder Cancer Cells / 华中科技大学学报（医学）（英德文版）

The effects of synthetic Smac peptide (SmacN7) on chemotherapeutic sensitivity of bladder cancer cells were investigated. SmacN7 penetratin peptide was synthesized and delivered into T24 cells. MTT assay was used to evaluate the viability of T24 cells induced by low-dosage of MMC. Flow cytometry was used to analyze the proportions of apoptosis. Western blot was used to detect the expression of XIAP and Caspase-3. The activity of Caspase-3 was measured and the effect of SmacN7 combined with MMC on T24 cell lines was also determined. The results showed that SmacN7 penetratin peptide could successfully interact with endogenous XIAP, increase the proportions of apoptosis of T24 cell lines induced by low-dosage of MMC in a dose-and time-dependent manner. An obvious down-regulation of XIAP expression and up-regulation of Caspase-3 was identified by Western blot. The activity of Caspase-3 in experimental group was significantly increased as compared with that in the control group. As compared with MMC group, the viability of T24 cells in SmacN7 penetratin peptide + MMC group was markedly decreased to 2.22 and 3.61 folds at 24h and 48h respectively. It was concluded that SmacN7 penetratin peptide could act as a cell-permeable IAP inhibitor, inhibit the proliferation, induce apoptosis and enhance the chemo-sensitivity of bladder cancer cells to MMC. These findings indicate that SmacN7 penetratin peptide may be a very promising ageut for bladder cancer treatment when used in combination with chemotherapy.

Characterization of HC58cDNA, a putative cysteine protease from the parasite Haemonchus contortus

Because of the complexity of the cathepsin B-like (CBL) family, an information on the biological and biochemical characteristics of individual CBL genes is lacking. In this study, we investigated the degradative effects of the recombinant HC58 protein isolated from Haemonchus contortus parasites on protein substrates over a broad pH range in vitro. This protein, which hydrolyzed the synthetic peptide substrates Z-FR-AMC and Z-RR-AMC, had characteristics of the cysteine protease class of proteins. In the acidic pH range, the isolated protein actively degraded hemoglobin (Hb), the heavy chain of goat immunoglobulin G, and azocasein. By contrast, it degraded fibrinogen in the alkaline pH range. These activities were strongly inhibited in the presence of the cysteine protease inhibitor E-64. While the protein digested Hb, it did not induce the agglutination of erythrocytes from its natural host. These results suggest that the HC58 protein may play a role in the nutrition of this parasite.

Experiment of promoting chemosensitivity of bladder cancer cell by synthetic Smac peptide / 中国癌症杂志

Background and purpose:Smac/DIABLO was the only apoptosis-related protein that could inhibit IAPs directly and simultaneously.The four amino-residual AVPI(Ala-Val-Pro-lie)in its N-terminal was the very important domain that could stimulate apoptosis.This study investigated the effect of synthetic Smac peptide (SmacN7) on chemotherapy sensitivity of bladder cancer cells.Methods:SmacN7 penetratin peptide was synthesized and delivered into T24 cells.MTT assay was adopted to evaluate the viability of T24 cells induced by low-dosage of MMC. Flow cytometry was applied to analyze the proportion of apoptosis and Western blot was used to detect the expression of XIAP and caspase-3;The activity of caspase-3 was measured and the effect of SmacN7 combined with MMC on T24 cell lines was also determined.Results:SmacN7 penetratin peptide could successfully interact with endogenous XIAP and increase the proportions of apoptosis of T24 cell lines induced by low-dosage of MMC in a dose-and time- dependent manner.An obvious down-regulation of XIAP expression and up-regulation of caspase-3 was identified by Western blot.The activity of caspase-3 in experimental group was significantly increased as compared with that in the control group;Combining the treatment with SmacN7 penetratin peptide,the viability of T24 cells decreased to 55% and 72.7% in 24 hrs and 48 hrs respectively.Conclusion:SmacN7 penetratin peptide could act as a cell-permeable IAP inhibitor,inhibit the proliferation,induce apoptosis and enhance the chemo-sensitivity of bladder cancer cells to MMC. When combined with chemotherapy,it may be a very promising strategy for bladder cancer therapy.

Detection of Differences in Light Chain Isotype (?/?) Expression of Anti-HCV Antibodies after HCV Infection and Its Clinical Significance / 中国医师杂志

Objective To study the cause of protective immunodeficiency of patients with hepatitis C. Methods An antigen capture ELISA in which HCV synthetic peptides SP42, CP10 and CP9 derived from HCV NS4 and core gene region, respectively, were used as solid-phase antigens was used to detect the differences in light chain isotype expression of anti-HCV antibodies. Results Antibodies in 84 sera of HCV-infected patients against HCV SP42, CP10 and CP9 were characterized by a skewed light chain isotype expression. Eighty-two out of 84 sera of HCV infection (97 62%) showed at least one of the three anti-HCV antibodies skewed from the normal ratio of light chain isotype kappa/lambda. The kappa/lambda ratios of anti-HCV antibodies in all patients with hepatitis C were found to be unique and constant during one year follow-up, and 11 of them received two years follow-up. Conclusions Anti-HCV response was stable and clonally restricted in HCV infection. B-cell clonal dominance may be the cause of human protective immunodeficiency after HCV infection.

Establishment of ELISA Method of Detecting Human Cytomegalovirus Using Synthetic Peptide and its Application / 中国医师杂志

Objective To establish the ELISA method by using synthetic peptide for detection of IgM antibodies to human cytomegalovirus(HCMV).Methods According to HCMV PP150 sequence of amino acid and nucleotide,the peptide sequence was decided by computer with some program of software of protein.The ELISA method using synthetic peptide as antigen was developed and was applied to detected anti-HCMV IgM in sera of normal pregnant woman and habitual abortion woman.The HCMV DNA were detected by polymerase chain reaction(PCR).Results The positive rates of anti-HCMV IgM in different populations were as follows:9 17%(11/120) in normal pregnant woman,37 5%(24/64) in habitual abortion woman.The ELISA method have good speciality.The positive rate of HCMV DNA was significantly related to that of anti-HCMV IgM(P