Major Polypeptides Research Articles

Isolation and partial characterization of a novel paramyxovirus from the gills of diseased seawater-reared Atlantic salmon (Salmo salar L).

A formerly undescribed virus has been isolated from the gills of farmed Atlantic salmon post-smolts in Norway suffering from gill disease. Cytopathic effects appeared in RTgill-W1 cells 9 weeks post-inoculation with gill tissue material. Virus production continued for an extended period thereafter. Light and electron microscopic examination revealed inclusions and replication in the cytoplasm. The viral nucleocapsid consisted of approximately 17 nm thick filaments in a herringbone pattern. Certain areas of the plasma membrane were thickened by the alignment of nucleocapsids on the internal surface and projections of 10 nm long viral glycoprotein spikes on the external surface. Virus assembly and release was achieved by budding through the modified plasma membrane. Negatively stained virions were spherical and partly pleomorphic with a diameter of 150-300 nm as seen by electron microscopy. The virus was sensitive to chloroform, heat and low and high pH, and replication was not inhibited by Br-dU or IdU indicating an RNA genome. Both haemagglutination and receptor-destroying enzyme activity were associated with the virions and the formation of syncytia in infected cultures indicated fusion activity. The receptor-destroying enzyme was identified as neuraminidase. The virus contained five major structural polypeptides with estimated molecular masses of 70, 62, 60, 48 and 37 kDa. Its buoyant density was 1.18-1.19 g ml(-1) in CsCl gradients. From the observed properties we conclude that this new virus belongs to the Paramyxoviridae and suggest the name Atlantic salmon paramyxovirus (ASPV). Furthermore, replication occurred at 6-21 degrees C, suggesting a host range confined to cold-blooded animals.

Leishmania ( Leishmania) amazonensis: differential expression of proteinases and cell-surface polypeptides in avirulent and virulent promastigotes

A comparative study of proteolytic enzymes and cell-surface protein composition in virulent and avirulent Leishmania ( Leishmania) amazonensis promastigote forms was carried out using one- and two-dimensional dodecyl sulfate sodium–polyacrylamide gel electrophoresis (SDS–PAGE). The surface iodinated protein profiles showed two major polypeptides of 65–60 and 50–47 kDa that were expressed in both virulent and avirulent promastigote forms. However, minor quantitative differences were observed in the cell-surface profile between the avirulent and virulent promastigotes. These included polypeptides of 115, 52, 45, 32, and 25 kDa that were preferentially expressed in the virulent forms. Two-dimensional SDS–PAGE showed an accentuated expression of acidic polypeptides; some of them differentially expressed in the promastigote forms analyzed. Live parasites treated with glycosylphosphatidylinositol (GPI)-specific phospholipase C (PLC) from Trypanosoma brucei and immunoprecipitated with the cross-reacting determinant (CRD) antibody recognized three major polypeptides of 65–60, 52, and 50–47 kDa, hence suggesting that these peptides were anchored to the plasma membrane domains through GPI anchor. Moreover, the polypeptides of 65–60 and 52 kDa were also recognized by the gp63 antiserum. Several metalloproteinase activities were similar in both virulent and avirulent promastigote forms, whereas cysteine proteinase activities, sensitive to E-64, were preferentially expressed in virulent promastigotes. These results suggest that cell-surface polypeptides and intracellular cysteine proteinases might play an important role in the virulence of L. ( L.) amazonensis. Index Descriptors and Abbreviations: cell-surface protein, Leishmania ( L.) amazonensis; proteinases; promastigotes, trypanosomatid, virulence; BSA, bovine serum albumin; CRD, cross-reacting determinant; DTT, dithiothreitol; E-64, trans-epoxysuccinyl l-leucylamido-(4-guanidino) butane; EDTA, ethylenediaminetetraacetic acid; GPI, glycosylphosphatidylinositol; kDa, kilodalton; LPG, lipophosphoglycan; PLC, phospholipase C; PMSF, phenylmethylsulphonyl fluoride; SDS–PAGE, dodecyl sulfate sodium–polyacrylamide gel electrophoresis; VSG, variant surface glycoprotein

Environmental Conditions during Vegetative Growth Determine the Major Proteins That Accumulate in Mature Grapes

Despite recent reports in the literature that chitinases comprise approximately 50% of the soluble proteins present in mature Vitis vinifera L. (cv. Moscatel) grapes, with the other major proteins being thaumatin-like proteins, a careful inspection of the published data reveals inconsistencies as to which proteins actually accumulate in mature grapes. Mature Moscatel grapes were harvested in the same vineyard in two consecutive years, 1999 and 2000. The grapes exhibited widely distinct polypeptide patterns when analyzed by either FPLC cation exchange chromatography or two-dimensional electrophoresis: whrereas the 2000 grapes possessed a much higher protein content (1.96 versus 1.11 mg g(-)(1) of fresh weight), the 1999 grapes contained a greater heterogeneity of polypeptides. In addition, highly specific antibodies that recognize the pathogenesis-related proteins present in the grapes demonstrated that the 2000 harvest grapes had a wider variety of pathogenesis-related polypeptides. N-Terminal sequencing of the major polypeptides revealed differences in the relative abundance and amino acid sequence of thaumatin-like and osmotin-like polypeptides present in the 1999 and 2000 harvest Moscatel grapes and did not detect the presence of chitinase. As a whole, the data indicate that the expression and accumulation of the major proteins in grapes is essentially determined by the environmental and/or pathological conditions prevailing during grape development and maturation. The potential physiological and technological implications are discussed. The results of the present work suggest that it is not possible to base varietal differentiation of grapes on the profile of the pathogenesis-related polypeptides present in the mature berries.

Kinesin‐like proteins and transport of pollen tube organelles

Cell Biology InternationalVolume 27, Issue 3 p. 255-256 Kinesin-like proteins and transport of pollen tube organelles Silvia Romagnoli, Corresponding Author Silvia Romagnoli Dipartimento Scienze Ambientali ‘G. Sarfatti’, Università di Siena, Via Mattioli 4, 53100 Siena (I), ItalyCorresponding author. Tel.: +39-0577-232854; fax: +39-0577-232860. [email protected]Search for more papers by this authorGiampiero Cai, Giampiero Cai Dipartimento Scienze Ambientali ‘G. Sarfatti’, Università di Siena, Via Mattioli 4, 53100 Siena (I), ItalySearch for more papers by this authorMauro Cresti, Mauro Cresti Dipartimento Scienze Ambientali ‘G. Sarfatti’, Università di Siena, Via Mattioli 4, 53100 Siena (I), ItalySearch for more papers by this author Silvia Romagnoli, Corresponding Author Silvia Romagnoli Dipartimento Scienze Ambientali ‘G. Sarfatti’, Università di Siena, Via Mattioli 4, 53100 Siena (I), ItalyCorresponding author. Tel.: +39-0577-232854; fax: +39-0577-232860. [email protected]Search for more papers by this authorGiampiero Cai, Giampiero Cai Dipartimento Scienze Ambientali ‘G. Sarfatti’, Università di Siena, Via Mattioli 4, 53100 Siena (I), ItalySearch for more papers by this authorMauro Cresti, Mauro Cresti Dipartimento Scienze Ambientali ‘G. Sarfatti’, Università di Siena, Via Mattioli 4, 53100 Siena (I), ItalySearch for more papers by this author First published: 02 January 2013 https://doi.org/10.1016/S1065-6995(02)00320-7Citations: 6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume27, Issue3March 2003Pages 255-256 RelatedInformation

Further studies on the structural analysis of the cuticle of Litomosoides chagasfilhoi (Nematoda: Filarioidea).

In order to obtain further information on the structural organization of the cuticle of nematodes, this structure was isolated from adult forms of the filariid Litomosoides chagasfilhoi. The purity of the fraction was determined by light and transmission electron microscopy, deep-etching, high resolution scanning electron microscopy, atomic force microscopy, immunocytochemistry, gel electrophoresis (SDS-PAGE) and Western blot. The epicuticle presented a rugous surface with parallel rows and several globular particles that could be involved in the absorption of nutrients and secretion of products. Analysis by SDS-PAGE of purified cuticles revealed five major polypeptides corresponding to 151, 41, 28, 13 and 11 kDa. A polyclonal antibody against a synthetic 18 amino-acid peptide that corresponds to the sequence of domain E of the Haemonchus contortus3A3 collagen gene recognized several protein bands on the Western blot of purified cuticle, and labeled all cuticular layers, as shown by immunocytochemistry.

Identification of CfNek, a novel member of the NIMA family of cell cycle regulators, as a polypeptide copurifying with tubulin polyglutamylation activity in Crithidia.

Post-translational glutamylation of tubulin plays an important role in regulating the interaction between microtubules and associated proteins, but so far the enzymes involved in this process have not been cloned from any cellular source. Using a modified purification scheme that employs a hydroxyapaptite chromatography as the final step we identified a 54 kDa band as the major polypeptide copurifying with tubulin polyglutamylation activity from the trypanosomatid Crithidia fasciculata. Based on peptide sequence information we have cloned the corresponding cDNA and identify Crithidia p54 as a novel member (termed CfNek) of the NIMA family of putative cell cycle regulators. CfNek is a protein of 479 amino acids that contains an unusual protein kinase domain that lacks the glycine-rich loop in subdomain I. The protein also harbours a PEST sequence and a pleckstrin homology domain. The tubulin polyglutamylase preparation displays the beta-casein phosphorylation activity typical for NIMA related kinases. Recombinant His-tagged CfNek expressed in Crithidia localises to the flagellar attachment zone/basal body of the parasite. After purification on a Ni(2+)-column the recombinant enzyme preparation displays ATP-dependent tubulin polyglutamylation activity as well as casein-phosphorylation activity.

Characterisation of Midgut Antigens of Hyalomma anatolicum anatolicum Koch, 1844 (Acari: Ixodidae)

The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of gut supernate antigen (GS Ag) derived from partly fed Hyalomma anatolicum anatolicum Koch, 1844 (Acari: Ixodidae) adult females revealed 26 discrete polypeptide bands of molecular weight ranging from 25–208 kDa. Out of these, seven bandswere of major polypeptides with molecular weights of between 25.2 and 185.8 kDa. On immunoblotting with antisera raised in rabbits against gut supernate (crude extract), eight immunogenic polypeptides with molecular weights of between 51.7 and 185.8 were identified. The implications of these findings for the potential development of anti-tick vaccine are discussed.

Isolation of H +-translocating ATPase in tonoplast of Tradescantia virginiana L. leaf cells

The tonoplast of Tradescantia virginiana L. was prepared from leaf cells and then solubilized with deoxycholate (DOC) and n-octyl-β- d-glucoside ( n-OG). Three major polypeptides (68, 60, 16 kDa) and several other minor components were isolated. These polypeptides were reconstituted in soybean phospholipids (asolectin). The H + pump activity was investigated with the reconstituted system as well as with the tonoplast. In both cases, the quinacrine–fluorescence quenching was observed in the presence of ATP–Mg 2+, indicating the H + pumping. The H + pump activity was inhibited by gramicidin D, a channel-forming ionophore, and by KNO 3, an inhibitor specific to tonoplast-type (V-type) H +-ATPase.

Yolk Protein Immunoassays (YP-ELISA) to Assess Diet and Reproductive Quality of Mass-Reared Orius insidiosus (Heteroptera: Anthocoridae)

A yolk protein enzyme-linked immunosorbent assay (YP-ELISA) was developed for the predator Orius insidiosus (Say). The YP-ELISA is intended to assess reproductive response to dietary and other rearing conditions, and to assist in quality control and diet development for mass rearing. Hybridomas and monoclonal antibodies were produced against homogenates of eggs dissected from females. Hybridomas were selected for secretion of IgG that reacted with extracts of both females and their eggs, and that did not react with male extracts. Each cloned hybridoma produced a monoclonal antibody that specifically reacted on western blots against one of the two major yolk polypeptides, apoVn-I (180,000 molecular weight) or apoVn-II (40,000). Yolk protein ELISAs were developed with these antibodies to assess yolk protein content of female O. insidiosus as a measure of reproductive fitness and as a potential predictor of fecundity. Protocols for an indirect antigen ELISA and double antibody sandwich ELISA were developed to assess yolk protein contents of eggs and total contents in whole body homogenates. ELISA standards consisted of homogenates of eggs collected 0–24 h following oviposition. As determined with the sandwich ELISA, yolk protein contents of eggs declined with age before hatch, with a half-life of 32–34 h. Results were similar whether the detecting antibody-enzyme conjugate was anti-apoVn-I or anti-apoVn-II. Optimal conditions and sampling parameters were developed for the sandwich ELISA, which demonstrated minimal nonspecific interference in whole-insect extracts. In an initial application of the YP-ELISA, oviposition rates over a 10-d period were compared with yolk protein contents at the end of that period, dependent on diets of differing nutritional composition and quality. High and low yolk protein contents correlated with oviposition rates on respective diets, though oviposition showed more graded response to diets than did yolk protein. Improvements in sampling methods are discussed.

Yolk protein immunoassays (YP-ELISA) to assess diet and reproductive quality of mass-reared Orius insidiosus (Heteroptera: Anthocoridae).

A yolk protein enzyme-linked immunosorbent assay (YP-ELISA) was developed for the predator Orius insidiosus (Say). The YP-ELISA is intended to assess reproductive response to dietary and other rearing conditions, and to assist in quality control and diet development for mass rearing. Hybridomas and monoclonal antibodies were produced against homogenates of eggs dissected from females. Hybridomas were selected for secretion of IgG that reacted with extracts of both females and their eggs, and that did not react with male extracts. Each cloned hybridoma produced a monoclonal antibody that specifically reacted on western blots against one of the two major yolk polypeptides, apoVn-I (180,000 molecular weight) or apoVn-II (40,000). Yolk protein ELISAs were developed with these antibodies to assess yolk protein content of female O. insidiosus as a measure of reproductive fitness and as a potential predictor of fecundity. Protocols for an indirect antigen ELISA and double antibody sandwich ELISA were developed to assess yolk protein contents of eggs and total contents in whole body homogenates. ELISA standards consisted of homogenates of eggs collected 0-24 h following oviposition. As determined with the sandwich ELISA, yolk protein contents of eggs declined with age before hatch, with a half-life of 32-34 h. Results were similar whether the detecting antibody-enzyme conjugate was anti-apoVn-I or anti-apoVn-II. Optimal conditions and sampling parameters were developed for the sandwich ELISA, which demonstrated minimal nonspecific interference in whole-insect extracts. In an initial application of the YP-ELISA, oviposition rates over a 10-d period were compared with yolk protein contents at the end of that period, dependent on diets of differing nutritional composition and quality. High and low yolk protein contents correlated with oviposition rates on respective diets, though oviposition showed more graded response to diets than did yolk protein. Improvements in sampling methods are discussed.

Characterization of the Cyno-EBV LMP1 homologue and comparison with LMP1s of EBV and other EBV-like viruses

EBV latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation and has been associated with several cases of malignancies. EBV-like viruses in Cynomolgus monkeys ( Macaca fascicularis) have been associated with high lymphoma rates in immunosuppressed monkeys. In the study, the entire coding region of the Cyno-EBV LMP1 gene was cloned, sequenced and expressed in human embryonic kidney (HEK) cells 293. The Cyno-EBV LMP1 homologue sequence predicted a 588 amino acid (a.a.) protein with a short 19 a.a. N-terminus, six transmembrane domains and a long carboxy tail of 404 a.a. The protein contained a series of seven 9 a.a.-tandem repeats and two 20 a.a.-repeats, which harbored two potential TRAF binding motifs, PxQxT/S. These repeats shared no homology with the repeats in any other LMP1. However, the proline-rich sequence GPxxPx 6 found within the 11 a.a.-repeats of EBV LMP1 was conserved in Cyno-EBV carboxy tail and contained two consensus JAK/STAT sequences PxxPxP. A cluster of eight histidine residues was found in proximity to the last transmembrane domain of Cyno-EBV LMP1 and was exploited as a natural protein tag in expression studies. Western blot analysis revealed a major polypeptide of 110 kDa. Comparative functional studies showed that Cyno-EBV LMP1 expressed in HEK 293 cells shares the same ability as EBV LMP1 to induce NFκB driven CAT activity.

Quantification of vitellogenesis and its control by 20-hydroxyecdysone in the ixodid tick, Amblyomma hebraeum

Ovaries of the ixodid tick, Amblyomma hebraeum Koch, grew rapidly after engorgment as a result of yolk uptake. At 26 °C, oviposition began by day 10 post-engorgement, plateaued on days 16–18, and ended by day 38. Vitellin (Vt) was partially purified from ovaries of day 10 engorged ticks by gel filtration and ion exchange chromatography. This Vt comprises seven major and several minor polypeptides. Two polypeptides (211 and 148 kD) from haemolymph of engorged female ticks corresponded to minor polypeptides of similar molecular weight in the ovary. The haemolymph titre of the 211 and 148 kD polypeptides increased up to the onset of oviposition. These polypeptides were absent in males and non-vitellogenic females (day 0 engorged or day 10 partially-fed females), and were thus designated as vitellogenin (Vg). Antibodies raised against haemolymph Vg211 and 148 recognized these polypeptides in partially purified Vt, as well as six of the seven major polypeptides. Using these antibodies we developed an indirect, competitive ELISA to quantify Vg. Rise in haemolymph Vg-concentration lagged slightly behind the rise in haemolymph ecdysteroid (ES)-concentration, and Vg-synthesis was stimulated by injections of 20E into non-vitellogenic females. These observations indicate that an ES is the vitellogenic hormone in A. hebraeum.

Entamoeba histolytica: purification and characterization of ornithine decarboxylase

Ornithine decarboxylase, a rate-limiting enzyme in polyamine biosynthesis in eukaryotes, was stabilized and purified from trophozoites of the parasite protozoan E. histolytica. Analytical electrophoresis revealed the presence in the purified preparations of a major polypeptide of 45 kDa and barely detectable amounts of two other proteins of 70 and 120 kDa. Both the 45 and 70 kDa polypeptides were recognized by a mouse anti-ODC monoclonal antibody. The major polypeptide exhibited amino terminal sequence homology in the range of 40–73% with ODCs from other organisms. The immunoreactive polypeptide of 70 kDa was not identified. The molecular masses of 216 and 45 kDa determined for the native enzyme by gel filtration and for the major polypeptide by SDS–PAGE, respectively, suggest that the amoeba ODC is a homopentamer. Dialysis against hydroxylamine rendered the enzyme activity fully dependent on pyridoxal 5 ′-phosphate (PLP). As expected for an oligomeric enzyme, ODC activity exhibited sigmoidal kinetics when it was measured as a function of increasing concentrations of l-ornithine and PLP yielding S 0.5 values of 0.45 and 0.18 mM, respectively. Purified ODC was inhibited by 1,3-diaminopropane and 2,4-diamino-2-butanone but was largely insensitive to inhibition by α-difluoromethylornithine (DFMO), indicating that the enzyme may not be a suitable target for this anti-parasitic drug. Other features of the amoeba ODC were common with the enzyme from prokaryotes and eucaryotes. Index Descriptors and Abbreviations: Entamoeba histolytica; polyamine metabolism; ornithine decarboxylase purification; BAC, N, N ′-bis(acryloyl)-cystamine; DAB, 2,4-diamino-2-butanone; DAP, 1,3-diaminopropane; DFMO, α-difluoromethylornithine; DTT, dithiothreitol; ODC, ornithine decarboxylase; PLP, pyridoxal 5 ′-phosphate.

New creatine transporter assay and identification of distinct creatine transporter isoforms in muscle.

Despite the pivotal role of creatine (Cr) and phosphocreatine (PCr) in muscle metabolism, relatively little is known about sarcolemmal creatine transport, creatine transporter (CRT) isoforms, and subcellular localization of the CRT proteins. To be able to quantify creatine transport across the sarcolemma, we have developed a new in vitro assay using rat sarcolemmal giant vesicles. The rat giant sarcolemmal vesicle assay reveals the presence of a specific high-affinity and saturable transport system for Cr in the sarcolemma (Michaelis-Menten constant 52.4 +/- 9.4 microM and maximal velocity value 17.3 +/- 3.1 pmol x min(-1) x mg vesicle protein(-1)), which cotransports Cr into skeletal muscle together with Na(+) and Cl(-) ions. The regulation of Cr transport in giant vesicles by substrates, analogs, and inhibitors, as well as by phorbol 12-myristate 13-acetate and insulin, was studied. Two antibodies raised against COOH- and NH(2)-terminal synthetic peptides of CRT sequences both recognize two major polypeptides on Western blots with apparent molecular masses of 70 and 55 kDa, respectively. The highest CRT expression occurs in heart, brain, and kidney, and although creatine kinase is absent in liver cells, CRT is also found in this tissue. Surprisingly, immunofluorescence staining of cultured adult rat heart cardiomyocytes with specific anti-CRT antibodies, as well as cell fractionation and cell surface biotinylation studies, revealed that only a minor CRT species with an intermediate molecular mass of approximately 58 kDa is present in the sarcolemma, whereas the previously identified major CRT-related protein species of 70 and 55 kDa are specifically located in mitochondria. Our studies indicate that mitochondria may represent a major compartment of CRT localization, thus providing a new aspect to the current debate about the existence and whereabouts of intracellular Cr and PCr compartments that have been inferred from [(14)C]PCr/Cr measurements in vivo as well as from recent in vivo NMR studies.

Antitrichomonal strains of Bacillus thuringiensis.

Parasporal inclusion proteins from a total of 816 Bacillus thuringiensis strains isolated in Japan were examined for antitrichomonal activity against Trichomonas vaginalis. Ten strains of B. thuringiensis inhibited the growth of T. vaginalis in 48 h cultures at 37 degrees C. Moreover, the B622 and B626 strains clearly showed trichomonacidal effects against T. vaginalis. The H antigen serotypes of both strains were identified as H13/29 (pakistani/amagiensis). The parasporal inclusion protein from both strains consisted of three major polypeptides of 77, 45 and 25 kDa.

Epitope analysis of the FanC subunit protein of the K99 (F5) fimbriae of enterotoxigenic Escherichia coli using a recombinant fusion technique.

We have used a recombinant approach to characterise the B- and T-cell epitopes of FanC, the major subunit polypeptide of K99 (F5) fimbriae of enterotoxigenic Escherichia coli strains. This involved the fusion of FanC and its carboxy-terminal truncated derivatives to a reporter, the E. coli alkaline phosphatase (PhoA), generating stable, recombinant fusions. The B-cell epitopes of FanC were characterised by Western blotting of FanC::PhoA fusion proteins with a polyclonal mouse antiserum directed against K99 fimbrial antigen, and with a panel of monoclonal antibodies generated to the K99 antigen. An attempt to characterise the T-cell epitopes of the fimbrial subunit was made by standard in vitro T-cell proliferation assay. Our results suggest that the B-cell epitopes of FanC are likely to be continuous, with a potentially immunodominant epitope at the carboxy-terminus. However, T-cell proliferation assays with the FanC::PhoA fusion proteins did not indicate any immunodominant T-cell epitope(s). We hypothesise that fusion of FanC peptides to PhoA had resulted in altered folding of the peptides for antibody and T-cell recognition, highlighting the potential problems and drawbacks of the recombinant fusion technique in defining the epitopes of certain proteins.

Epitope analysis of the FanC subunit protein of the K99 (F5) fimbriae of enterotoxigenic Escherichia coli using a recombinant fusion technique

We have used a recombinant approach to characterise the B- and T-cell epitopes of FanC, the major subunit polypeptide of K99 (F5) fimbriae of enterotoxigenic Escherichia coli strains. This involved the fusion of FanC and its carboxy-terminal truncated derivatives to a reporter, the E. coli alkaline phosphatase (PhoA), generating stable, recombinant fusions. The B-cell epitopes of FanC were characterised by Western blotting of FanC::PhoA fusion proteins with a polyclonal mouse antiserum directed against K99 fimbrial antigen, and with a panel of monoclonal antibodies generated to the K99 antigen. An attempt to characterise the T-cell epitopes of the fimbrial subunit was made by standard in vitro T-cell proliferation assay. Our results suggest that the B-cell epitopes of FanC are likely to be continuous, with a potentially immunodominant epitope at the carboxy-terminus. However, T-cell proliferation assays with the FanC::PhoA fusion proteins did not indicate any immunodominant T-cell epitope(s). We hypothesise that fusion of FanC peptides to PhoA had resulted in altered folding of the peptides for antibody and T-cell recognition, highlighting the potential problems and drawbacks of the recombinant fusion technique in defining the epitopes of certain proteins.

Characterization, pathogenicity and neutralization studies of a nervous necrosis virus isolated from grouper, Epinephelus tauvina, in Singapore

A virus isolated from diseased marine fish, Grouper, Epinephelus tauvina, was cultured in sea bass (SB) cell line, characterized and its pathogenicity and neutralization studies were carried out. This isolated virus is an icosahedral virus with a mean diameter of 28–30 nm and has buoyant density of 1.30–1.35 g/ml. It replicates exclusively in the cytoplasm and forms paracrystalline array and inclusion bodies in the infected cells. SDS-PAGE analysis of purified virus structural proteins resolved one major polypeptide of approximately 42 kDa. This virus induces cytopathic effects such as rounding and granulation of cells, localized cell death and detachment of cells within 3–5 days postinfection on sea bass larval cell line. Typical histopathological changes in the virus-infected sea bass larvae under experimental conditions showed vacuolation of nervous tissue. Polyclonal antisera raised against the purified virus and the coat protein of this virus effectively neutralized virus infectivity in vitro suggesting the use of coat protein as a vaccine against this viral infection.

Changes of protein tyrosine phosphorylation in third instar larval integument of the Mediterranean fruit fly, Ceratitis capitata.

Developmental analysis of the tyrosine protein phosphorylation levels in larval integument and partial characterization of the endogenous protein tyrosine kinase activity (PTK) in Ceratitis capitata are described in this study. Larval integument contains high levels of PTK activity at the early stages of the third instar, which progressively declines to low levels in the white pupal stage. An integumental 90-kDa polypeptide was identified to have prominent endogenous PTK activity and follow a similar developmental pattern. The major integumental phosphotyrosine-containing polypeptides have apparent molecular weights of 30, 41, 44, 46, and 54 kDa, respectively. Polypeptides with molecular weights of 62 and 73 kDa were identified as Ser/Thr-containing phosphoproteins and were shown to exhibit high levels of phosphorylation at the middle stage of larval development. These differences are likely to be due to the higher activation state of the protein tyrosine kinase(s) at the early stages of larval development.

Nitration of cytoskeletal proteins in the chicken embryo chorioallantoic membrane

Protein tyrosine nitration is one of the post-translational modifications that alter the biological function of proteins. Two important mechanisms are involved: peroxynitrite formation and myeloperoxidase or eosinophil peroxidase (EPO) activity. In the present work we studied the nitration of proteins in the in vivo system of chicken embryo chorioallantoic membrane (CAM). 3-Nitrotyrosine was detected only in the insoluble fraction of the CAM homogenate. By immunoprecipitation, Western blot analysis, and double immunofluorescence, we identified two major polypeptides that were nitrated: actin and α-tubulin. Quantification of actin and α-tubulin nitration revealed that they are differentially nitrated during normal development of the chicken embryo CAM. After irradiation, although they were both increased, they required different time periods to return to the physiological levels of nitration. It seems that both peroxynitrite formation and EPO activity are involved in the in vivo tyrosine nitration of cytoskeletal proteins. These data suggest that tyrosine nitration of cytoskeletal proteins has a physiological role in vivo, which depends on the protein involved and is differentially regulated.