Specific Antisera Research Articles

Prevalence of E. coli serotypes among diarrheal patients in Hilla City, Iraq

Escherichia coli was among the most common pathogens that causes intestinal and extra-intestinal diseases worldwide. One hundred fifty (150) stool samples were collected from patients suffering from significant diarrhea during the period of October 2017 to the end of December 2017 from three main hospitals in Hilla city, Iraq, about 46 isolates were identified as E.coli. All stool samples were cultured on MacConkey and blood agar plates. E.coli positive cultures were set further serological test by using slide agglutination test with specific antisera. The high prevalence of E.coli serotypes among diarrhea patient in Hilla city was with ETEC that about 16(34.8%). The high prevalence serotypes in ETEC serogroups are the O6: K15. Also subdivided the results of serotypes according to the age and sex factors. We detect the ETEC in age factor are 8 adults and 8 children while according to sex factor were 8 male and 8 female.

Standardization and application of immunofluorescence and immuno- peroxidase tests for detection of bluetongue virus antigen

India is enzootic for bluetongue (BT), a predominant disease of small ruminants. The most important task in the control of disease is rapid and sensitive detection of virus. The present study was undertaken to standardize immunofluorescence (IFT) and immunoperoxidase tests (IPT) employing BTV serogroup specific VP7 monoclonal antibodies (MAbs), polyclonal homologous, and polyclonal heterologous antisera against specific serotypes of BTV for detection of BTV antigen. Serial tenfold dilutions of BTV-9 were tested for limit of detection (LoD) of IFT, IPT, and molecular assays by using MAbs against VP7, homologous anti-BTV-9 serum, and heterologous anti-BTV-16 serum. LoD of IFT was found to be 101 TCID50/mL using MAbs against VP7, anti BTV-9 serum, and anti BTV-16 serum. LoD of IPT was found to be 101 TCID50/mL, 102 TCID50/mL, and 102 TCID50/mL using MAbs against VP7, anti BTV-9 serum and anti BTV-16 serum, respectively. LoD of RT-PCR was 101 TCID50/mL and that of real time PCR was 100 TCID50/mL. This standardized assay was then applied for BTV detection in BTV suspected field samples collected from BT outbreaks followed by confirmation with virus isolation and NS3 group specific PCR. The current study shows that IFT and IPT are specific tests for group specific BTV identification. For IFT, monoclonal and polyclonal (homologous and heterologous) source of antibodies had similar sensitivity in the ability of BTV detection; whereas the most sensitive mode of detection by IPT was with MAbs.

Immunolocalization suggests a role of the histamine-gated chloride channel PxHCLB in spectral opponent processing in butterfly photoreceptors.

Spectrally opponent responses, that is, wavelength-dependent inversions of response polarity, have been observed at the level of photoreceptors in butterflies. As inter-photoreceptor connections have been found in the butterfly Papilio xuthus, and histamine is the only neurotransmitter so far identified in insect photoreceptors, we hypothesize that histaminergic sign-inverting synapses exist in the lamina between different spectral receptors as a mechanism for spectral opponency as in the medulla of Drosophila. Here, we localized two histamine-gated chloride channels, PxHCLA (Drosophila Ort homolog) and PxHCLB (Drosophila HisCl1 homolog), in the visual system of Papilio xuthus by using specific antisera. The antiPxHCLA labeling was associated with the membrane of nonphotoreceptor cells that are postsynaptic to photoreceptors, while the antiPxHCLB labeling overlapped with photoreceptor axons, indicating that PxHCLB is expressed at inter-photoreceptor synapses: PxHCLB is likely involved in producing spectral opponency at the first visual synapses. Color processing in Papilio may appear earlier than previously hypothesized in insect visual systems.

Purification and biochemical characterization of a vitellogenin-like protein from sea urchin

The major yolk protein in sea urchins is a transferrin-like protein. In this report, a new component was detected in gonad extracts of Strongylocentrotus intermedius and Mesocentrotus nudus, which cross-reacts with antiserum against egg yolk proteins. We tentatively named them egg yolk-related proteins siYRP and mnYRP. The siYRP was purified from testis of S. intermedius by ammonium sulfate fractionation, anion exchange chromatography, affinity chromatography and gel filtration. Purified siYRP was > 900 kDa in size. The siYRP purified on SDS-PAGE under reducing conditions gave seven bands, corresponding to 93, 213 and > 250 kDa. Purified mnYRP displayed similar structural characteristics as siYRP. Purified siYRP and mnYRP were identified by tandem mass spectrometry and renamed as siVitellogenin (Vtg)-like and mnVtg-like proteins, respectively. Both Vtg-like proteins were confirmed to be lipoglycoproteins by staining with Sudan black and periodic acid-Schiff. A specific antiserum against the siVtg-like protein was raised in rabbit. Antiserum against the siVtg-like protein immunostained siVtg-like and mnVtg-like proteins. Immunochemical methods using the antiserum revealed that siVtg-like and mnVtg-like proteins were present in the ovary, testis and unfertilized eggs of both species. These results indicated that Vtg-like proteins have important physiological functions for gonadal growth and gametogenesis in sea urchins.

Development of a Rhinovirus Inoculum Using a Reverse Genetics Approach

BackgroundExperimental inoculation is an important tool for common cold and asthma research. Producing rhinovirus (RV) inocula from nasal secretions has required prolonged observation of the virus donor to exclude extraneous pathogens. We produced a RV-A16 inoculum using reverse genetics and determined the dose necessary to cause moderate colds in seronegative volunteers.MethodsThe consensus sequence of RV-A16 from a previous inoculum was cloned, and inoculum virus was produced using reverse genetics techniques. After safety testing, volunteers were inoculated with either RV-A16 (n = 26) or placebo (n = 10), Jackson cold scores were recorded, and nasal secretions were tested for shedding of RV-A16 ribonucleic acid.ResultsThe reverse genetics process produced infectious virus that was neutralized by specific antisera and had a mutation rate similar to conventional virus growth techniques. The 1000 median tissue culture infectious dose (TCID50) dose produced moderate colds in most individuals with effects similar to that of a previously tested conventional RV-A16 inoculum.ConclusionsReverse genetics techniques produced a RV-A16 inoculum that can cause clinical colds in seronegative volunteers, and they also serve as a stable source of virus for laboratory use. The recombinant production procedures eliminate the need to derive seed virus from nasal secretions, thus precluding introduction of extraneous pathogens through this route.

A new PCR assay based on the new gene-SPUL_2693 for rapid detection of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum.

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) and biovar Pullorum (S. Pullorum) are gram-negative bacteria, members of the most important infectious pathogens, and have caused common problems in the poultry industry, especially in the developing countries. O- and H-antigen specific anti-sera are commonly for slide and tube agglutination tests to identify Salmonella serovars. However, it is both labor intensive and time consuming, so there is an urgent need for a new technique for the rapid detection of the major Salmonella serovars. In this study, we developed a 1-step PCR assay to identify the serovar Gallinarum. This PCR-based assay was based on the SPUL_2693 gene, which was located in SPI-19 and found by comparing the genomes of the S. Pullorum and S. Gallinarum in the whole data of NCBI. The specificity of this gene was evaluated by bioinformatics analysis, and the results showed that the SPUL_2693 gene exists in all serovar Gallinarum. The specificity and sensitivity of this PCR assay were evaluated in our study. The developed PCR assay was able to distinguish the serovar Gallinarum from 27 different Salmonella serovars and 5 different non-Salmonella pathogens. The minimum limit of genomic DNA of S. Pullorum for PCR detection was 2.143 pg/μL, and the minimum limit number of cells was 6 CFU. This PCR assay was also applied to analyze Salmonella strains isolated from a chicken farm in this study. The PCR assay properly identified the serovar Gallinarum from other Salmonella serovars, and the results were in agreement with the results of a traditional serotyping assay. In general, the newly developed PCR-based assay can be used to accurately judge the presence of the serovar Gallinarum and can be combined with traditional serotyping assays, especially in the case of large quantities of samples.

Variations of Garlic Common Latent Virus and Shallot Latent Virus Concentration on Shallot and Garlic

Infection of Garlic common latent virus (GCLV) and Shallot latent virus (SLV) on shallot and garlic has been reported in Indonesia. Both viruses are known to infect the plants in the field in high incidence and can be transmitted through the bulbs. A research was conducted to determine the potency of infected bulbs as source of disease inoculum by measuring virus titer throughout bulb growing stadia. Two kinds of shallot’s and garlic’s bulbs was used, i.e. shallot seed bulb (cv. Bima curut), garlic seed bulb (cv. Sembalun), consumption shallot and garlic bulb (unknown cultivar). The experiment was conducted using completely randomized design. Ten samples with three repetition of each stadia, i.e. adventitious shoot, shoot, leaf on 7 days after planting (DAP), and 14 DAP were analyzed by enzyme-linked immunosorbent assay using specific antisera to GCLV and SLV. Infection of GCLV and SLV was found higher on garlic than on shallot. Both viruses were detected in high incidence from adventitious shoot and 14 DAP-foliar leaves. Virus titer is higher on consumption bulbs than those on seed bulbs for both shallot and garlic. This study indicated that infected bulbs is very potential as the primary source of disease in the field.

First Report of Tobacco Mosaic Virus on Cucumber [Cucumis sativus (L.)] in Java, Indonesia

A mosaic and mottle symptom was observed on several cucumber cultivations in Java, Indonesia. The study was aimed to characterize the causal virus of typical Tobamovirus-like symptoms. Symptomatic leaves were collected randomly from each location in Nganjuk, Kediri, Tulungagung (East Java), Brebes and Klaten (Central Java), Kulon Progo (Yogyakarta), and Subang, Indramayu, Bogor (West Java). The diseases frequency was determined by dot blot immuno binding assays (DIBA) test using specific antiserum of Cucumber green mottle mosaic virus (CGMMV). Furthermore, the Tobamovirus was detected by RT-PCR, then cloned into TA-cloning vector, and the DNA was sequenced. The disease frequency of CGMMV in East Java, Central Java, Yogyakarta, and West Java was up to 24.44%, 9.09%, 0%, and 5.49%, respectively. CGMMV was unable to be amplified by specific primer, however it was able to be amplified by universal primer of Tobamovirus with sized ± 800 bp. Nucleotide sequence analysis of samples which gave positive reaction against CGMMV antisera were identified as Tobacco mosaic virus (TMV). The homology of nucleotide and amino acid sequences analysis ranged from 95.0-97.7% and 98.1-99.4%, respectively. This is the first report of TMV infection on cucumber in Indonesia.

Characterization of a serologically atypical Shigella flexneri Z isolated from diarrheal patients in Bangladesh and a proposed serological scheme for Shigella flexneri.

BackgroundAtypical Shigella flexneri Z variant, that agglutinate with E1037 group factor specific monoclonal antisera against Shigella flexneri IV-I but not with other group or type specific antisera, has continuously being isolated in Bangladesh since 1997. Later this serotype has been reported in Indonesia, China and Argentina. Despite being a provisional serotype, continuous isolation of these strains in diverse geographical regions implicated a great necessity to study the overall characteristics of these strains. Therefore, we extensively characterized S. flexneri Z strains using various phenotypic and molecular tools.MethodOf 3569 S. flexneri isolated between 1997 and 2015, 95 strains were identified as S. flexneri Z using a panel of polyvalent absorbed antisera and monoclonal antisera of S. flexneri (MASF). Of them, randomly selected 65 strains were molecular O-serotyped using multiplex PCR and characterized using different phenotypic and molecular techniques (i.e.biotyping, plasmid profile, virulence marker and PFGE) to determine relationship with other subserotypes of S. flexneri.ResultsAll these atypical S. flexneri Z strains were agglutinated with MASF B and IV-I antisera. Concordantly, these strains were positive to opt-gene, responsible for MASF IV-I sero-positive phenotype. However, molecular O-serotyping of all 65 strains could not differentiate between Z and Yb giving similar amplification products (wzx1-5 and opt). Contrarily, MASF based serotypic scheme distinguished among Z and Yb as well as Ya. All these S. flexneri Z showed typical biochemical reaction of S. flexneri, harboured a 140 MDa virulence plasmid and virulence markers namely ipaH, ial, sen, sigA and sepA genes. Along with the virulence plasmid, small plasmids (2.6, 1.8 and 1.6 MDa) were present as core plasmid. Moreover, a middle ranged plasmid and a 4.0 MDa sized plasmid were observed in 65% and 20% strains, respectively. Analysis of PFGE on XbaI-digested chromosomal DNA of Bangladeshi strains showed that S. flexneri Z had a close relatedness with Ya and Yb but completely different than the strains of Xa, Xb, 2a and 2b. This observation was found to be unequivocal while the overall result of biotyping, plasmid profile, and virulence factors was compared. Therefore, we conclude that these atypical serotype Z isolated in Bangladesh had a clonal relationship with Ya and Yb of Bangladesh and the opt gene played an important role in serotypic switching among them. Current serotyping scheme of S. flexneri strains fails to place many such atypical strains (1c, 1c+6, 1d, type 4, and 4c) including S. flexneri Z isolated from different parts of the world. Therefore, an updated serotyping scheme for identification of subserotypes of S. flexneri has been proposed to avoid multiple naming of the same subserotype having similar agglutination pattern.

Prevalence and Antibiotic Susceptibility Patterns of Salmonella and Shigella Species Isolated from Pediatric Diarrhea in Tehran

Background: Salmonella and Shigella infections are important public health concerns among children. The emergence of antibiotic resistance amongst Salmonella and particularly Shigella isolates has posed serious problems in antimicrobial treatment worldwide. Data on local antibiotic resistance patterns are essential to design suitable antibiotic treatment guidelines. Objectives: The aim of the present study was to determine the prevalence and drug susceptibility patterns of Shigella and Salmonella species in addition to the detection of extended-spectrum β-lactamase producing isolates among diarrhea samples of pediatric patients. Methods: A total of 5300 diarrheic samples from children were examined for the presence of Salmonella and Shigella species. Biochemical and microbial tests, as well as specific polyvalent antisera, were used for the identification of the bacterial species. Antibiotic susceptibility tests and extended-spectrum β-lactamase detection were conducted by disc diffusion and combination disc methods, respectively. Results: A total of 371 (7%) and 472 (8.9%) samples were positive for Salmonella and Shigella species, respectively. The most prevalent Salmonella isolate was serogroup D (n: 176, 47.5%). Of the Shigella isolates, 60.8% were found as Shigella sonnei and 39.2% as Shigella flexneri. The highest level of antibiotic resistance was noted among Shigella flexneri isolates. Overall, 35.7% of the Shigella flexneri isolates and 31% of the Shigella sonnei isolates produced extended-spectrum β-lactamases. Conclusions: This study provided information on the prevalence and antimicrobial susceptibility patterns of Salmonella and Shigella isolates in Iran. It also indicated a high-level resistance among Shigella isolates to co-trimoxazole and ampicillin and among Salmonella isolates to nalidixic acid.

Blueberry scorch virus Detected on Blueberry Plants Imported Into China

Blueberry scorch virus (BlScV), a member of the Carlavirus genus in the Betaflexiviridae family, was first reported in the United States. Scorch caused by BlScV is a serious disease of blueberry and can lead to great loss of yield. At present, the disease has also been reported in Germany, Canada, Italy, and the Netherlands (Ciuffo et al. 2005; Kalinowska et al. 2013; Wegener and Punja 2004). However, the disease had never been reported on blueberry in China. Symptoms caused by BlScV range from asymptomatic to severe blighting of flowers and leaves depending on blueberry cultivars and virus strain. In 2015, 57 imported samples of highbush blueberry cultivars showing virus-like symptoms in an isolated blueberry orchard in Fujian Province (south China) were collected and tested for BlScV by DAS-ELISA using specific antiserum according to the manufacturer’s protocol (Agdia, U.S.A.). Among the samples, one was positive for BlScV in DAS-ELISA. To confirm the presence of the virus, RT-PCR was performed using BlScV-specific primers. A set of BlScV-specific primers, forward primer (5′-CTCAAGCCGCAGCGTCACA-3′) and reverse primer (5′-CACCAGTGTACTCCGTTTCGAGA-3′), was designed to amplify a 667-bp fragment of the coat protein gene of BlScV. Total RNA was extracted from the extracts of ELISA-positive sample using an RNA extraction kit (Qiagen, Germany). The first-strand cDNA was synthesized using cDNA synthesis kit (Promega, U.S.A.) according to the manufacturer’s instructions with the N6 random primer (Promega, U.S.A.). PCR was carried out using GoTaq Master Mix following the manufacturer’s protocol (Promega, U.S.A.). The program used for PCR amplification was 94°C for 3 min, followed by 35 cycles at 94°C for 30 s, 53°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 10 min. The expected 667-bp fragment was purified, ligated to pGEM-T easy vector (Takara, Japan), and sequenced. The obtained sequence was deposited in GenBank (accession no. KX977562) and analyzed. Further sequence comparison revealed that the amplicon nucleotide sequence was 97.6% identical to the sequence of an isolate of BlScV from the United States (KP232989), which confirmed the presence of BlScV. To our knowledge, this is the first report of BlScV in blueberries in China. In recent years, a number of blueberry cultivars from abroad have been introduced to China, and thus BlScV-infected blueberry has the potential to be an important long-distance vector. Considering that BlScV could become an important threat to blueberry production, all the imported blueberry bushes in the orchard were destroyed to prevent the spread of the virus.

Optimization of real-time multiplex polymerase chain reaction for the diagnosis of acute bacterial meningitis and Neisseria meningitidis serogrouping

Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae type B are responsible for about 80-85% of acute bacterial meningitis cases among adults and in our country it is mandatory to report bacterial meningitis cases caused by these agents. In recent years, disease control programs have focused especially on these three pathogens in our country. It is very important to know the causative agent of meningitis for prevention/control measures, choice of treatment regimen, and prediction of the severity and the course of the disease. Rapid diagnosis of meningococcal meningitis is especially vital. In addition, the identification of N.meningitidis serogroups in the community is also important for the decision of the type and combination of vaccine to be administered. The aim of this study was to optimize the real-time multiplex polymerase chain reaction (Rt-multiplex PCR) for the diagnosis of the disease and serogrouping of N.meningitidis in clinical samples in a direct, quick and reliable way. In this study, three Rt-multiplex PCR assays were optimized and standardized for the detection of N.meningitidis, H.influenzae and S.pneumoniae (NHS mix) and for the serogrouping of N.meningitidis (BCY mix and AWX mix) in our laboratory. Sensitivity of the Rt-multiplex PCR method was detected by reference strains and simulation studies. Forty three samples (41 cerebrospinal fluid (CSF), 1 serum and 1 clinical isolate) with the suspicion of acute bacterial meningitis and six nasopharyngeal isolates sent to National Reference Laboratory for Molecular Microbiology between July 2012-May 2014 were included in the present study. All samples were examined with the Rt-multiplex PCR methods. Clinical isolates were studied by both conventional and Rt-multiplex PCR methods. Oxidase, catalase test, Gram stain, API-NH and agglutination tests with specific antisera were performed in the National Respiratory Pathogens Reference Laboratory. The detection limit of the method, which was optimized and standardized in our laboratory was determined as 102 cfu/ml. The CT (threshold cycle) value in this dilution was detected approximately as 35. N.meningitidis was detected in 14 of the 41 CSF samples by the NHS mix. However, only 10 of the positive samples could be typed with BCY mix and AWX mix. Eight (80%) of them were serogroup B, one of each was (10%) serogroup A and serogroup W135, respectively. All the isolates (six nasopharyngeal and one clinical specimen) were identified as N.meningitidis serogroup B by Rt-multiplex PCR and the isolates were also confirmed by conventional methods. The CSF specimens with CT value > 35 could not be typed. We concluded that the Rt-multiplex PCR method is a rapid and reliable test for the direct diagnosis of acute bacterial meningitis due to NHS and serogrouping of N.meningitidis. Rapid diagnosis plays an important role for the treatment and control of the disease, and serogrouping of the agent plays an important role in terms of prevention/control and vaccination policies.

Kisaran Inang Cowpea mild mottle virus dan Respons Varietas Kedelai

Cowpea mild mottle virus (CPMMV) is one of important virus which infects soybean and become an endemic disease since the first time reported in Java and Sumatra. Research was conducted to study the host range of CPMMV and to evaluate response of new type soybean varieties to CPMMV specific isolate. Host range study of CPMMV CR16 isolate was conducted by sap transmission method to 11 species of indicator plants, whereas evaluation of soybean response involved 10 varieties. The resistance response of soybean variety was based on incubation period, and of either the disease incidence or severity. Virus infection on test plants were confirmed by dot immunobinding assay (DIBA) using specific CPMMV antisera. The CPMMV CR16 isolate was able to infect systemically 8 plant species belongs to Leguminoceae, and Solancaeae; while the virus caused local infection on Amaranthaceae. All plant species infected systemically proved to become sources of inoculum for CPMMV when they used in back inoculation to soybean. Response of soybean varieties to CPMMV CR16 isolate can be categorized into susceptible (Detam 1, Detam 2, Detam 3, Anjasmoro, Wilis), moderated (Detam 4, Malika, Dena 1) and resistant (Argomulyo and Grobogan).

AtA6PR1 and AtA6PR2 encode putative aldose 6-phosphate reductases that are cytosolically localized and respond differentially to cold and salt stress in Arabidopsis thaliana

Sorbitol is synthesized in Rosaceae species, especially in source organs, in a pathway involving aldose 6-phosphate reductase (A6PR, EC 1.1.1.200). As compatible solutes, sorbitol and other sugar alcohols assist in the ability of the plant to withstand abiotic stress conditions. Here, we identify two tandemly-duplicated genes in a non-Rosaceae species (Arabidopsis thaliana L.), and show that the proteins encoded by At2g21250 (AtA6PR1) and At2g21260 (AtA6PR2) possess the molecular characteristics of A6PRs. Consistent with bioinformatic predictions, we determined that green fluorescent protein-tagged versions of AtA6PR1 and AtA6PR2 are cytosolically localized, a finding supported by immunoblotting using a specific anti-AtA6PR1 antisera after subcellular fractionation of Arabidopsis leaves. We also show that under standard growth conditions, both genes are widely-expressed, whilst AtA6PR1 protein accumulates in both source and sink organs. Under short term (24 h) cold (4 °C) or saline (150 mM NaCl) stress, both genes respond differentially and reciprocally in leaf and root tissues, suggesting a sub-functionalization of their roles. The identification of homozygous T-DNA insertional ata6pr1-mutants will facilitate the functional characterization of this gene, and help to determine its role under abiotic stress conditions.

Development of an opsonophagocytic killing assay for group a streptococcus

Group A Streptococcus (GAS) or Streptococcus pyogenes is responsible for an estimated 500,000 deaths worldwide each year. Protection against GAS infection is thought to be mediated by phagocytosis, enhanced by bacteria-specific antibody. There are no licenced GAS vaccines, despite many promising candidates in preclinical and early stage clinical development, the most advanced of which are based on the GAS M-protein. Vaccine progress has been hindered, in part, by the lack of a standardised functional assay suitable for vaccine evaluation. Current assays, developed over 50 years ago, rely on non-immune human whole blood as a source of neutrophils and complement. Variations in complement and neutrophil activity between donors result in variable data that is difficult to interpret. We have developed an opsonophagocytic killing assay (OPKA) for GAS that utilises dimethylformamide (DMF)-differentiated human promyelocytic leukemia cells (HL-60) as a source of neutrophils and baby rabbit complement, thus removing the major sources of variation in current assays. We have standardised the OPKA for several clinically relevant GAS strain types (emm1, emm6 and emm12) and have shown antibody-specific killing for each emm-type using M-protein specific rabbit antisera. Specificity was demonstrated by pre-incubation of the antisera with homologous M-protein antigens that blocked antibody-specific killing. Additional qualifications of the GAS OPKA, including the assessment of the accuracy, precision, linearity and the lower limit of quantification, were also performed. This GAS OPKA assay has the potential to provide a robust and reproducible platform to accelerate GAS vaccine development.

Facile Chemoenzymatic Synthesis of O‐Mannosyl Glycans

AbstractO Mannosylation is a vital protein modification involved in brain and muscle development whereas the biological relevance of O‐mannosyl glycans has remained largely unknown owing to the lack of structurally defined glycoforms. An efficient scaffold synthesis/enzymatic extension (SSEE) strategy was developed to prepare such structures by combining gram‐scale convergent chemical syntheses of three scaffolds and strictly controlled sequential enzymatic extension catalyzed by glycosyltransferases. In total, 45 O‐mannosyl glycans were obtained, covering the majority of identified mammalian structures. Subsequent glycan microarray analysis revealed fine specificities of glycan‐binding proteins and specific antisera.

Facile Chemoenzymatic Synthesis of O-Mannosyl Glycans.

O Mannosylation is a vital protein modification involved in brain and muscle development whereas the biological relevance of O-mannosyl glycans has remained largely unknown owing to the lack of structurally defined glycoforms. An efficient scaffold synthesis/enzymatic extension (SSEE) strategy was developed to prepare such structures by combining gram-scale convergent chemical syntheses of three scaffolds and strictly controlled sequential enzymatic extension catalyzed by glycosyltransferases. In total, 45 O-mannosyl glycans were obtained, covering the majority of identified mammalian structures. Subsequent glycan microarray analysis revealed fine specificities of glycan-binding proteins and specific antisera.

Distribution and co-localization of diversified natriuretic peptides in the eel heart.

Atrial and B-type natriuretic peptides (ANP and BNP) are cardiac hormones important for cardiovascular and body fluid regulation. In some teleost species, an additional member of the natriuretic peptide family, ventricular NP (VNP), has been identified. In this study, we examine tissue distribution of these three NPs in the eel heart. Quantitative real-time PCR showed that anp is almost exclusively expressed in atria, bnp equally in atria and ventricles and vnp three-fold more in ventricles than in atria. The amount of bnp transcript overall in the heart was 1/10 those of anp and vnp. There was no difference in transcript levels between freshwater and seawater-acclimated fishes. Immunohistochemistry using specific antisera and in situ hybridization using gene-specific probes showed that NP signals were detected in most atrial and ventricular myocytes with some regional differences in density. Because of high sequence similarity of the three NPs, each of the three NP antisera individually was pre-incubated with 10-8M of the other two non-targeted cardiac NPs to increase the specificity. A few atrial myocytes contained all three NPs in the same cell. Immuno-electron microscopy identified many dense-core vesicles containing ANP in atria and VNP in ventricles and some vesicles contained both ANP and VNP as demonstrated using pre-absorbed antisera. Based on these data and those of previous studies, we suggest that in eels ANP is secreted from atria in a regulatory pathway and VNP from ventricles in a constitutive pathway. In addition, VNP, not BNP, is the principal ventricular hormone in eels.

Target identification reveals protein arginine methyltransferase 1 is a potential target of phenyl vinyl sulfone and its derivatives.

Phenyl vinyl sulfone (PVS) and phenyl vinyl sulfonate (PVSN) inactivate protein tyrosine phosphatases (PTPs) by mimicking the phosphotyrosine structure and providing a Michael addition acceptor for the active-site cysteine residue of PTPs, thus forming covalent adducts between PVS (or PVSN) and PTPs. We developed a specific antiserum against PVS. This antiserum can be used in general antibody-based assays such as immunoblotting, immunofluorescence staining, and immunoprecipitation. Target identification through immunoprecipitation and mass spectrometry analysis reveals potential targets of PVS, mostly proteins with reactive cysteine residues or low-pKa cysteine residues that are prone to reversible redox modifications. Target identification of PVSN has been conducted because the anti-PVS antiserum can also recognize PVSN. Among the targets, protein arginine methyltransferase 1 (PRMT1), inosine-5′-monophosphate dehydrogenase 1, vimentin, and glutathione reductase (GR) were further confirmed by immunoprecipitation followed by immunoblotting. In addition, PVSN and Bay11-7082 inhibited GR activity, and PVS, PVSN, and Bay 11-7082 inhibited PRMT1 activity in in vitro assays. In addition, treatment of PVSN, Bay11-7082, or Bay 11-7085 in cultured HeLa cells can cause the quick decline in the levels of protein asymmetric dimethylarginine. These results indicate that the similar moiety among PVS, PVSN, Bay 11-7082, and Bay 11-7085 can be the key structure of lead compounds of PRMT1. Therefore, we expect to use this approach in the identification of potential targets of other covalent drugs.

Isotocin Regulates Growth Hormone but Not Prolactin Release From the Pituitary of Ricefield Eels.

The neurohypophyseal hormone oxytocin (Oxt) has been shown to stimulate prolactin (Prl) synthesis and release from the adenohypophysis in rats. However, little is known about the functional roles of Oxt-like neuropeptides in the adenohypophysis of non-mammalian vertebrates. In this study, cDNAs encoding ricefield eel oxytocin-like receptors (Oxtlr), namely isotocin (Ist) receptor 1 (Istr1) and 2 (Istr2), were isolated and specific antisera were generated, respectively. RT-PCR and Western blot analysis detected the presence of both Istr1 and Istr2 in the brain and pituitary, but differential expression in some peripheral tissues, including the liver and kidney, where only Istr1 was detected. In the pituitary, immunoreactive Istr1 and Istr2 were differentially distributed, with the former mainly in adenohypophyseal cell layers adjacent to the neurohypophysis, whereas the latter in peripheral areas of the adenohypophysis. Double immunofluorescent images showed that immunostaining of Istr1, but not Istr2 was localized to growth hormone (Gh) cells, but neither of them was expressed in Prl cells. Ist inhibited Gh release in primary pituitary cells of ricefield eels and increased Gh contents in the pituitary gland of ricefield eels at 6 h after in vivo administration. Ist inhibition of Gh release is probably mediated by cAMP, PKC/DAG, and IP3/Ca2+ pathways. In contrast, Ist did not affect either prl gene expression or Prl contents in primary pituitary cells. Results of this study demonstrated that Ist may not be involved in the regulation of Prl, but inhibit Gh release via Istr1 rather than Istr2 in ricefield eels, and provided evidence for the direct regulation of Gh cells by oxytocin-like neuropeptides in the pituitary of non-mammalian vertebrates.