A novel lectin from mushroom Phellodon melaleucus displays hemagglutination activity,and antitumor activity in a B16 melanoma mouse model

A novel lectin(termed PML)was purified from fruiting bodies of the edible mushroom Phellodon melaleucus(division Basidiomycota)by ion exchange,hydrophobic interaction,and gel filtration chromatographies,with overall titer recovery~60%and 20-fold purification.PML displayed hemagglutination activity 13319 units/mg toward rabbit erythrocytes.SDS-PAGE and gel filtration analyses revealed that PML is a homodimeric lectin with a molecular weight of 28.8 kDa.PML hemagglutination activity was not inhibited by various simple sugars or their derivatives,but was enhanced by cations Ca^(2+),Mg^(2+),Zn^(2+),and Cu^(2+).The activity was stable in pH range 6–9 and in the temperature range 20–60°C.Circular dichroism(CD)spectroscopic analysis showed that PML was composed primarily ofβ-sheets with lowα-helix content.In a B16 melanoma mouse model,PML treatment significantly inhibited tumor growth,and increased cytokine IL-10 content.Our findings suggest that PML is a potential anticancer therapeutic agent.

Evaluation of fecal occult blood test with reverse passive hemagglutination for colorectal neoplasm screen *

AIM To evaluate the one sampling and three sampling reverse passive hemagglutination fecal occult blood test (RPHA FOBT) for colorectal neoplasm screening.

Binary Ethylenimine Inactivated Japanese Encephalitis Virus Antigen Reveals Hemagglutination

Severe climate change and global warming may impact significantly on vector-borne disease including Japanese encephalitis (JE) infection in human and animals. Thus, veterinary authority requires large quantity of diagnostic tools to survey vector-borne diseases. New producing method having a relation with JE antigen is needed to substitute conventional sucrose-acetone extraction method using suckling mouse. So, we developed new manufacturing method using polyethylene glycol (PEG) precipitation. Japanese encephalitis virus (JEV) was propagated in roller bottle containing Vero cell and inactivated with two kinds of inactivating reagents. Viability of the supernatant of bulk containing antigen was checked using Vero cell after inactivation. The supernatant did not show hemagglutination (HA) activity with goose erythrocytes. The antigen inactivated by binary ethylenimine (BEI) and concentrated by PEG precipitation method was found to be 2048 HA, but the antigen inactivated by 0.3% formaldehyde solution and concentrated by PEG precipitation method did not show HA titer. The antigen prepared from mice brain using sucrose-acetone extraction method showed 256 HA titer. This BEI inactivation method does not evoke animal welfare problem and can replace the conventional method that required biological hazardous reagents and suckling mice in preparing HA antigen. This new BEI inactivation method was safe in producing HA antigen against JEV in laboratory and can reduce environmental contamination of acetone.

Correlation between Hemagglutination Inhibition Titer and Protection against Infectious Bronchitis Virus Challenge in SPF Layers

[ Objective] To study the correlation between HI titer and protection against IBV challenge in SPF layers. [ Method ] SPF layers were randomly divided into four groups, namely group A1, A2, B1 and B2. The group A1 was immunized with H120 live vaccine. The group A2 was first immunized with H120 live vaccine and later boosted with ND-IB-EDS trivalent inactivated vaccine. The group B1 was used as unimmunized chal- lenge control. The group B2 was kept as unimmunized unchallenged control. The blood samples were taken prior and post-vaccination at intervals and HI tests were conducted. At the laying peak, the group A1, A2 and B1 were challenged with IBV M4t virulent strain. The clinical features and egg production of layers were monitored and recorded. [Result] After 30 d post vaccination with H120 live vaccine, the HI titer reached 4.45 log2; after 30 days boosting with ND-IB-EDS trivalent inactivated vaccine, the HI titer reached to 7.35 log2. Before challenge, HI antibody titer in group A1, A2, B1 and B2 were respectively 4.24 log2, 7.40 Iog2, 2.10 log2 and 2.10 log2. After challenge, chickens in unimmunized challenge control group B1 showed respiratory symptoms, egg production dropped by 30.9%, and they produced more soft-shelled, no-shelled or abnormal eggs. In the group A1, some chickens had light respiratory symptoms and egg production dropped by 11.7%. In the group A2, the egg production of all chickens was as normal as the group B2. [ Conclusion] When the HI titer was over 6 log2, challenge by virulent virus had no impact on egg produc- tion; when the HI titer was 5 log2, 4 log2 and less 3 log2, egg production dropped by 6.0%, 11.3% and 29.6%, respectively. Thus, the HI anti- body level in chickens has close correlation with protection against IBV challenge.

EFFECTS OF ACUPUNCTURE ON THE HIGH HEMAGGLUTINATION STATE, BLOOD-SUGAR-RAISING HORMONE AND IMMUNOCYTE FACTOR LEVELS IN TYPE-II DIABETES PATIENTS

Objective: To investigate the effect of acupuncture on high hemagglutination state, blood sugar raising hormone and immunocyte factor levels in type II diabetes patients. Methods: A total of 120 inpatients and outpatients were randomly divided into acupuncture plus medication group (n=52) and medication group (n=50). In addition, 18 type II diabetes patients formed acupuncture group for comparing their therapeutic effects. Main acupoints used were Pishu (BL 20), Geshu (BL 17), Yishu, Shenshu (BL 23), Zusanli (ST 36), Sanyinjiao (SP 6), etc.. combined with other acupoints according to different sydroms. These acupoints were stimulated by manipulaing the filiform needles with uniform reinforcing and reducing method for 15 min and then stimulated electrically for 15 min with an electroacupuncture therapeutic apparatus. Western medicines used were Glipizide, Dimethyldiguanide Hydrochloride, etc.. The treatment was given once daily, with 10 sessions being a therapeutic course, 2～3 courses altogether. Indexes of external thrombosis length (ETL), platelet agglutination rate (PAgR), fibrinogen (FG), activated partial thromboplastin time (APTT), fasting blood glucose (FBG), prothrombin time(PT), adrenocoticortropic hormone (ACTH), cortisol (CS), growth hormone (GH), glucagon (GL), tumor necrosis factor alpha (TNF α), interleukin 6 (IL 6), insulin (INS) and C peptide (C P) were determined using radioimmunoassay. Results: After 2～3 courses of treatment, both acupuncture group and medication plus acupuncture group could significantly improve high hemagglutination state, lower blood sugar raising hormone level, regulate immunocyte factor level and raise the sensitivity of insulin, which were apparently superior to those of medication group (P<0.05～0.01). Conclusion: Acupuncture therapy can effectively regulate plasma blood sugar raising hormone, immunocyte factor levels, increase the sensitivity of insulin to target cells, resist blood coagulation and improve microcirculation.

The identification of a CD47-blocking“hotspot”and design of a CD47/PD-L1 dual-specific antibody with limited hemagglutination

Dear Editor,By targeting the programmed cell death 1(PD-1)pathway with monoclonal antibodies(mAbs),immune checkpoint therapy(ICT)has achieved unprecedented clinical success in the treatment of multiple tumors.1,2 Cancer cells evade the host immune system via both the tolerance of T cells and functional suppression of innate immune cells.3 CD47 provides a“do not eat me”signal by binding to signal regulatory protein alpha(SIRPα)to prevent innate immune cells from attacking host cells.4 Recently,macrophages were found to restore antitumor reactivity by blocking the interaction between upregulated CD47 on tumor cells and SIRPαon innate immune cells.5 However,it remains unknown whether there are blocking“hotspots”on CD47 for mAb-based anti-CD47 therapy or additional blocking hotspot regions within CD47 for therapeutic mAb development.Although it is overexpressed on tumor cells,CD47 is also expressed in many normal cells,including red blood cells and platelets.6 Some validated CD47-blocking mAbs under clinical investigation induce hemagglutination and anemia.7 Thus,designing an engineered CD47-blocking antibody to exert a therapeutic effect with limited hemagglutination is needed.

Research on the Lectin in Muscle Tissue of Varicorhinus macrolepis

[Objective] The paper aimed at researching lectins in muscles of Varicorhinus macrolepis and providing scientific basis for researching the adaptation mechanism and immune response of V.macrolepis to environment,which were advantageous for the protection and reproduction of V.macrolepis.[Method] V.macrolepis was used as test materials for the hemagglutination test by dialdehyde fixation to prove the existence of lectins in muscle crude homogenate of Varicorhinus macrolepis and study the physical and chemical characters.[Result] Lectins in muscle crude homogenate of V.macrolepis had shown hemagglutination effects on erythrocytes of six types of animals and had the maximum hemagglutination activity against rabbit erythrocytes,which belonged to the S-type lectins with optimal pH ranged from 4 to 8 and optimal temperature at 60 ℃.Results from the saccharide inhibition test had indicated that the sucrose was the only kind of saccharide which had inhibited the hemagglutination,suggesting that sucrose had played an important role in the process of recognition and aggregation of lectins.[Conclusion] It had been speculated that the optimal pH ranges for thermal sensitivity and hemagglutination activity of lectins in different types of aquatic organisms were similar.

Salmonella typhi and gallbladder cancer:report from an endemic region

BACKGROUND:Evidence exists of a link between chronic infection by Salmonella typhi(S.typhi) and the development of gallbladder cancer(GBC),but several studies from endemic regions contradict its role in the etiopathogenesis of GBC.This study used various tools to assess the prevalence of S.typhi in patients with GBC and gallstone disease(GSD) in this region with a high incidence of GBC.METHODS:S.typhi was detected in tissue and bile by PCR and culture and in serum by the Widal test and indirect hemagglutination assay(IHA).PCR with two pairs of S.typhi specific primers(flagellin gene H1d and SOP E gene) could detect 0.6 ng of S.typhi DNA.Fifty-four patients with GBC(cases) were matched with 54 patients with GSD(controls).RESULTS:Of the 54 cases,24(44.44%) were positive on the Widal test and 12(22.22%) on IHA,compared to 13(24.07%) and 5(9.26%) respectively in the controls.Eighteen(33.33%) cases showed a positive result on PCR(tissue) and 2 on PCR(bile) vs.none in the controls.Bile culture revealed no Salmonella colonies in either cases or controls.Only 3 cases were positive for Salmonella on tissue culture compared to none in the controls.The sensitivity of PCR(tissue) relative to the Widal test,IHA,culture(bile and tissue) and PCR(bile) was 100% vs.66.67%,11.11%,and 11.11%,and the specificity was 83.33% vs.100%,100%,and 100%,respectively.CONCLUSIONS:S.typhi is significantly associated with GBC compared to GSD(33% vs.0%).PCR appears to be the most specific diagnostic tool,the gold standard for S.typhi in tissue samples.

Immunodiagnostic efficacy of detection of Schistosoma japonicum human infections in China:a meta analysis

Objective:To assess the diagnostic efficacy of the currently most widely used indirect hemagglutination assay(IHA) and enzyme-linked immunosorbent assay(ELISA) for detection of Schistosoma japonicum human infections.Methods:A comprehensive search was undertaken from China National Knowledge Infrastructure,Wanfang Database.VIP Database,PubMed. Cochrane Library,Science Citation Index Expanded.Proquest,and the inclusion and exclusion criteria were strictly settled.The funnel plot was used to assess the publication bias.Cochran’s Q test was employed to measure the homogeneity between studies,a summary receiver operating characteristic(SROC) curve was used to compare the diagnostic accuracy between the IHA and ELISA qualitatively by means of the Weighted Least Square method,the Ordinary Least Square method and the Robust regression method,and the diagnostic odds ratio(DOR) was drawn to compare the accuracy quantitatively.Results:Out of 785 publications,19 papers were eventually selected for analysis.Literature finality assessment indicated that minor publication bias existed in studies pertaining IHA test,but no bias was found in literatures regarding ELISA test.The heterogeneity test showed a heterogeneity between studies was present(χ~2 =466.07 and 34.67. both P values【0.0001).The areas under the SROC curves of IHA were all higher than that of ELISA test using the three methods(Weighted Least Square method:0.766 vs.0.695.Ordinary Least Square method:0.826 vs.0.741.Robust regression:0.815 vs.0.715).The TPR* values for IHA and EUSA were 0.710.0.759.0.749.and 0.650.0.686 and 0.666.respectively,and OR values were 5.997.9.937.8.893.and 3.432.4.784 and 3.959.respectively.The DOR of IHA was 9.41(95% CI:4.88-18.18).and 4.78(95%CI:3.21-7.13) for ELISA.Conclusions:All above results revealed that the diagnostic performance of IHA is better than that of ELISA.However,taking into account their unsatisfactory diagnostic value in areas with low infection intensity,a search for a better diagnostic test that can be applied in field situations in China should be given high priority.

Identification and Characterization of Peptide Mimics of Blood Group A Antigen

In order to investigate peptide mimics of carbohydrate blood group A antigen, a phage display 12-mer peptide library was screened with a monoclonal antibody against blood group A antigen, NaM87-1F6. The antibody-binding properties of the selected phage peptides were evaluated by phage ELISA and phage capture assay. The peptides were co-expressed as glutathione S-transferase （GST） fusion proteins. RBC agglutination inhibition assay was performed to assess the natural blood group A antigen-mimicking ability of the fusion proteins. The results showed that seven phage clones selected bound to NaM87-1F6 specifically, among which, 6 clones bore the same peptide sequence, EYWYCGMNRTGC and another harbored a different one QIWYERTLPFTF. The two peptides were successfully expressed at the N terminal of GST protein. Both of the fusion proteins inhibited the RBC agglutination mediated by anti-A serum in a concentration-dependent manner. These results suggested that the fusion proteins based on the selected peptides could mimic the blood group A antigen and might be used as anti-A antibody-adsorbing materials when immunoabsorption was applied in ABO incompatible transplantation.

Evaluation of antimicrobial activity of a lectin isolated and purified from <i>Indigofera heterantha</i>

Indigofera heterantha commonly called indigo bush is a member of leguminoseae family found in the Himalayan region of Kashmir. A lectin has been isolated from the seeds of Indigofera heterantha by the purification procedure involving anion exchange chromatography on DEAE-cellulose followed by gel filtration chromatography on Sephadex G 100. Molecular characterization of the lectin was done by gel filtration and SDS-PAGE. Activity of the lectin was checked by hemagglutination assay and the sugar specificity by sugar inhibition tests. The antimicrobial activity of the purified lectin was carried out by Agar disc diffusion using appropriate standards. On the ion exchange column, the bound protein when eluted with 0-0.5 M NaCl gradient emerged as three peaks—peak I, peak II and peak III out of which only peak II showed the hemagglutinating activity. The lectin further resolved into two peaks G1 and G2 on gel filtration, with the lectin activity residing in G1, corresponding to a molecular weight of 70 KDa. The purified lectin named as Indigofera heterantha Lectin (IHL) produced a single band on SDS PAGE (18 KDa), revealing the tetrameric nature of the lectin. It agglutinated human erythrocytes (A, B, AB, and O). Hemagglutination was inhibited by D-galactose, Dmannose and D-arabinose. The lectin is reasonably thermostable showing full activity within a temperature range of 30&degC to 90&degC. pH stability of the lectin falls in the range of 2-9. IHL demonstrated a remarkable antibacterial activity against the pathogenic bacteria Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, and Bacillus subtilis. IHL also inhibited the growth of phytopathogenic fungi Aspergillus niger, Aspergillus oryzae and Fusarium oxysporum.

Antigenicity and Hemaglutination Activity of a Recombinant Hemagglutinin-Neuraminidase of Paramyxovirus Tianjin Strain

Paramyxovirus Tianjin strain, a new genotype of Sendai virus, was isolated from the lungs of common cotton-eared marmoset that died of severe respiratory infection in the marmoset colonies. The 19.28% IgM positive rate in the young children with acute respiratory tract infection suggested a close relationship between Tianjin strain and humans. Hemagglutinin-neuraminidase (HN) is its major transmembrane glycoprotein responsible for viral attachment, penetration and release. To clear the relationship between HN structure and function of paramyxovirus Tianjin strain, rHN1, rHN2 and rHN3 overlapping the ectodomain of HN protein were expressed. Their antigenicity and hemaglutination activity, as well as cross reactivity to standard antisera against influenza virus type A, type B were analyzed. The results indicated expressed rHNs have the natural antigenicity. The segment rHN2 possesses more linear epitopes exposed on the surface of the native HN protein than found in segments rHN3 and rHN1. The hemagglutination activity of segment rHN3 is higher than that of segments rHN2 and rHN1, and partially dependent on the three-dimensional conformation of HN3 protein. Cross-reactivity between rHNs and standard antisera against influenza virus type A, type B suggested that rHNs might not be the best alternative as specific antigens to detect virus in clinical serum specimens.

Antibodies to Newcastle Disease Virus in Egg Yolks of Great Cormorant (<i>Phalacrocorax carbo</i>) at Qinghai Lake

Newcastle disease virus (NDV) is not considered as cause of serious disease in humans. But, recent data make it clear that, under particular circumstances, it is indeed possible for NDV to cause severe human respiratory disease. Newcastle Disease infection has been reported in many bird species. Cormorants that inhabit at the Qinghai-Tibet Plateau are mainly represented by Great Cormorant (Phalacrocorax carbo sinensis), which is distributed mainly on the Qinghai Lake area. Cormorants are considered as one of the main NDV-reservoir. We conducted the study for the presence of antibodies to Newcastle disease virus by hemagglutination inhibition test in yolks. We got 50% of seropositive yolks to Newcastle disease virus. These results show that NDV circulates in the Qinghai Lake population of cormorants. We first used the technique of detection of antibodies to Newcastle disease virus in the egg yolk for study the circulation of the virus in cormorants and demonstrated its effectiveness. We should carefully monitor cases of pneumonia in the population of people living around the lake and assess the causes of the disease.

Comparative Study of Serologic Results Obtained by HI Test of Avian Influenza and Newcastle Disease Performed in Four Mexican Laboratories

In the poultry industry, it is common to evaluate the humoral immune response associated to the vaccination calendar used for the different zootechnical purposes. When the vaccine against avian influenza and Newcastle disease currently used in a farm is going to be changed, an important parameter observed to choose a product over the others is based on the antibody titers reached by the application of the new vaccine. This study aimed to compare the serologic results obtained by hemagglutination inhibition （HI） test of avian influenza and Newcastle disease reported in four different national laboratories. One-day-old Ross broiler chickens were kept in Horsfall-Bauer isolation units and were vaccinated subcutaneously for the prevention of avian influenza and Newcastle disease. Then, serum of all the birds was extracted at three, six and seven weeks old and sent to four different national diagnostic laboratories, where HI test was performed for avian influenza and Newcastle disease. The treatments were designed in 4 × 3 factorial. Data showed significant statistical differences between laboratory results （up to six logarithms for influenza at six and seven weeks）. This study confirms that the results of the HI test can vary from one laboratory to another, thus it is important to consider this, when the vaccines against avian influenza and Newcastle disease are evaluated at field.

Evidence of Synergistic Activity of Medicinal Plant Extracts against Neuraminidase Inhibitor Resistant Strains of Influenza Viruses

The frequent emergence of drug resistant influenza viral strains emphasizes the urgent and continual need to develop new antiviral drugs. Given the encouraging findings of previous studies on antiviral compounds from plant sources, this study focused on medicinal plants from Borneo that were traditionally used to treat symptoms of influenza infection. Following the promising results of earlier investigations, four plant extracts that demonstrated multiple modes of viral inhibition were studied against wild-type and neuraminidase (NA) inhibitor-resistant strains of Types A and B influenza viruses. The extracts exhibited more pronounced activities against the wild-type viruses than the NA inhibitor-resistant strains. Variations in the antiviral potential of the extracts collected from different parts of the same plant were also evidenced in the in vitro micro-inhibition assays. Even though all plant extracts affected NA activity of all viruses, only two extracts demonstrated hemagglutination inhibitory (HI) activities against Type A pandemic H1N1 and Type B viruses. Furthermore, Receptor Destroying Enzyme (RDE) treatments of extracts exhibiting HI activities indicated the presence of sialic acid (SA)-like component(s) that may be responsible for HI activity. Since the antiviral potential of extracts was not completely suppressed by RDE, the possibility of non SA-like antiviral components cannot be ruled out. Therefore, synergistic activity between SA-like and non SA-like components contained in the plant extracts may be responsible for the demonstrated antiviral potential. The results also indicated the presence of non SA-like components that may act against other viral proteins apart from hemagglutinin (HA) and NA. Hence, this study supports the presence of multiple antiviral components that act against different viral proteins or interfere with different stages of viral replication. Our results suggest that these plant extracts have the potential to be developed as therapeutic agents for the treatment of influenza and could be a solution to the global occurrence of viral strains resistant to NA inhibitors.

Genomic Recombination Enhances Pathogenic Factors in the Periodontopathogenic Bacterium <i>Eikenella corrodens</i>

We reported previously that plasmid-mediated genomic recombination at the pilin gene locus increased hemagglutination activity, growth rate, biofilm formation, hemolytic activity, and adherence to epithelial cells in Eikenella corrodens 23834. To determine whether these enhancements were common in this bacterium, we introduced the recombinase gene ORF4 into seven clinically isolated strains. Genomic recombination at the type IV pilin gene locus was observed in strains 1080, L9B6, L8Ao3, and RV2 (group A), but not in strains 261-2, 612-L, and 257-4 (group B). Similarly, group A strains displayed changed colony morphology following loss of type IV pili, which was not observed in group B. Group A strains showed also enhanced hemagglutination activity, growth rate, hemolytic, activity and biofilm formation. These results suggest that ORF4-induced genomic recombination at the pilin gene locus is a general phenomenon in a part of E. corrodens, which likely stimulates patho-genicity and virulence.

Evaluation of Usefulness of Three Serological Tests Using Native Crude Antigen in Diagnosis of Hepatic Cystic Echinococcosis Patients

Objective: To evaluate three different serological tests [Indirect Hemaglutination (IHA), Enzyme Linked Immunosorbent Assay (ELISA) and Western Blotting (WB)] using native crude antigen for diagnosis of hepatic cystic echinococcosis (HCE) patients. Materials and Methods: Sheep hydatid fluid (HF) was collected from fertile cysts obtained from a slaughterhouse and used as an antigen. Forty patients who were attended the Dr. Ersin Arslan Training and Research Hospital in Gaziantep, Turkey, were investigated. Serum samples were obtained from surgically confirmed CE patients. Healthy Turkish people and 16 patients with other helminthic infections were included as a control group. Results: Of the 40 analyzed patients, 10 (25%) were men and 30 (75%) were female. The average age was 46.97 years (s.d.;18.95). The majority of the patients had a single cystic lesion situated in one lobe of the liver (usually in the right lobe) (55%), 32.5% of patients had two cystic lesions and 12.5% of patients had multiple cyst formations with various numbers. In all cases, ultrasound (US) examinations were positive and the size of cysts was between 2.1 - 12.7 cm. Twenty-three patients of the total 40 patients were classified according to the WHO classification system based on US findings. According to the results of WB analysis, molecular weights of 8 kDa (80%), 12 kDa (80%), 22 - 24 kDa (97.5%), 26 kDa (97.5%), 34 kDa (100%), 36 - 38 kDa (90%), 45 - 50 - 55 kDa (97.5%), and 60 - 75 kDa (97.5%) bands were identified. But 34, 50, and 55 kDa bands were also found in other helminthic diseases. Conclusion: The specificity and sensitivity of three serological tests (IHA, ELISA and WB) using crude antigen were compared by diagnosing hepatic cystic echinococcosis patients. IHA and ELISA showed high sensitivity but low specificity. Western blotting showed low sensitivity but high specificity.

New model for cardiomyocyte sheet transplantation using a virus-cell fusion technique

AIM: To facilitate close contacts between transplanted cardiomyocytes and host skeletal muscle using cell fusion mediated by hemagglutinating virus of Japan envelope(HVJ-E) and tissue maceration. METHODS: Cardiomyocytes(1.5 × 106) from fetal rats were first cultured. After proliferation, some cells were used for fusion with adult muscle fibers using HVJ-E. Other cells were used to create cardiomyocyte sheets(area: about 3.5 cm2 including 2.1 × 106 cells), which were then treated with Nile blue, separated, and transplanted between the latissimus dorsi and intercostal muscles of adult rats with four combinations of HVJ-E and/or Na OH maceration: G1: HVJ-E(+), Na OH(+), Cardiomyocytes(+); G2: HVJ-E(-), NaO H(+), Cardiomyocytes(+); G3: HVJ-E(+),Na OH(-), Cardiomyocytes(+); G4: HVJ-E(-), Na OH(-), Cardiomyocytes(-). At 1 and 2 wk after transplantation, the four groups were compared by detection of beating domains, motion images using moving target analysis software, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerase chain reaction, hematoxylin-eosin staining, and immunostaining for cardiac troponin and skeletal myosin.RESULTS: In vitro cardiomyocytes were fused with skeletal muscle fibers using HVJ-E. Cardiomyocyte sheets remained in the primary transplanted sites for 2 wk. Although beating domains were detected in G1, G2, and G3 rats, G1 rats prevailed in the number, size, motion image amplitudes, and action potential compared with G2 and G3 rats. Close contacts were only found in G1 rats. At 1 wk after transplantation, the cardiomyocyte sheets showed adhesion at various points to the myoblast layer in the latissimus dorsi muscle. At 2 wk after transplantation, close contacts were seen over a broad area. Part of the skeletal muscle sarcoplasma seemed to project into the myocardiocyte plasma and some nuclei appeared to share both sarcoplasmas.CONCLUSION: The present results show that close contacts were acquired and facilitated the beating function, thereby providing a new cellular transplantation method using HVJ-E and NaO H maceration.

Characterization of a new lectin involved in the protoplast regeneration of Bryopsis hypnoides

A group of coenocytic marine algae differs from higher plants,whose totipotency depends on an intact cell(or protoplast).Instead,this alga is able to aggregate its extruded protoplasm in sea water and generate new mature individuals.It is thought that lectins play a key role in the aggregation process.We purified a lectin associated with the aggregation of cell organelles in Bryopsis hypnoides.The lectin was ca.27 kDa with a pI between pH 5 and pH 6.The absence of carbohydrate suggested that the lectin was not a glycoprotein.The hemagglutinating activity(HA) of the lectin was not dependent on the presence of divalent cations and was inhibited by N-Acetylgalactosamine,N-Acetylglucosamine,and the glycoprotein bovine submaxillary mucin.The lectin preferentially agglutinated Gram-negative bacterium.The HA of this lectin was stable between pH 4 to pH 10.Cell organelles outside the cytoplasm were agglutinated by the addition of lectin solution(0.5 mg ml-1).Our results suggest that the regeneration of B.hypnoides is mediated by this lectin.We also demonstrated that the formation of cell organelle aggregates was inhibited by nigericin in natural seawater(pH 8.0).Given that nigericin dissipates proton gradients across the membrane,we hypothesize that the aggregation of cell organelles was proton-gradient dependent.

Geographically Distinct Expression Profile of Host Defense Peptides in the Skin of the Chinese Odorous Frog, Odorrana margaretae

Odorrana margaretae （Anura： Ranidae） is widely distributed in the southern provinces of China. Previously, 72 antimicrobial peptides （AMPs） belonging to 21 families were identified from the skin of O. margaretae, which were captured in the Hunan province. In the present study, five O. margaretae frogs were captured from the Guizhou province and a total of 28 cDNAs encoding 17 host defense peptides （HDPs） belonging to 14 families were cloned from the skin cDNA library of O. margaretae. Among the 17 HDPs, only one （brevinin-1-Omar5） had been characterized. The distinct HDP expression profiles for O. margaretae in the previous and present study may be attributed to the environmental differences between the sampling locations and the genetic divergence among O. margaretae populations. Besides, 11 of the 17 HDPs identified in the present study were novel for ranids. In order to understand their roles in host defense reactions, three HDPs （odorranain-H-OM1, odorranain-M-OM and ranatuerin-2-OM）, which possess low sequence similarity with the known amphibian HDPs, were selected for further chemical synthesis and functional analysis. Odorranain-H-OM1 showed direct antimicrobial activity against bacteria and fungi. Odorranain-M-OM exhibited concentration-dependent anti-oxidant activity. Ranatuerin-2-OM showed lectin-like activity and could strongly hemagglu -tinate human intact erythrocytes with or without the presence of Ca2＋. The diverse activities of HDPs implied that they may play different roles in host defense reactions of O. margaretae.