Properties of cod metallothionein,its presence in different tissues and effects of Cd and Zn treatment

One isoform of the low-molecular-weight metal-binding protein metallothionein (MT) has been isolated from the liver of Atlantic cod by size-exclusion and ion-exchange chromatography. Cod MT contained 33% cysteine, no aromatic amino acids or arginine. As is the case for other piscine MTs, the N-terminus of cod MT lacked the asparagine in position 4 which is present in mammalian MTs. In addition, cod MT differed from all other vertebrate MTs described in that the N-terminal methionine was not acetylated. Antibodies were raised in rabbits against hepatic MT from cod by repeated injections of native protein mixed with adjuvant. Anti-cod MT antisera cross reacted with similarly-sized proteins in liver, brain, anterior kidney, posterior kidney, spleen, intestine, gills and ovaries. The putative MT in cod brain migrated differently to that of the other tissues in native gel electrophoresis. Intraperitoneally injected Cd (1 mg/kg) was nearly entirely associated with the MT-peak in hepatic and renal cytosols, whereas a single injection of Zn (10 mg/kg) resulted in increases in all cytosolic Zn pools of the liver and no apparent change in cytosolic Zn, Cu, Ni or Cd in kidney.     

Characterization of the pattern of cytokeratin proteins in the epidermal cells of loach,Misgurnus anguillicaudatus (Cyprinformes)

The pattern of cytokeratin proteins in the epidermal cells of loach was studied by immunotechniques and partial separation of the epidermal cells. Two monoclonal antibodies, namely 8F7 and 1C45, against the cytokeratin proteins of the loach epidermis were prepared. these two monoclonal antibodies exhibit distinctive results in immunohistochemical staining. The 8F7 monoclonal antibody stains mainly with the epithelial cells, while the 1C45 monoclonal antibody stains specifically with the club cells. The pattern of cytokeratin proteins in the club cells and the epithelial cells of various epidermal layers was further determined by partial separation of these cells. Immunoblotting analysis of the cell fractions confirms the cytokeratin proteins to be differentially expressed in the club cells and the epithelial cells. However, the cytokeratin proteins expressed in the epithelial cells of the basal, middle and outer layers are same. The results indicate that differentiation of the epithelial cells seems limited during their translocation from basal to upper layers, but in those cells that do differentiate into club cells, the cytokeratin pattern changes.     

Purification and characterization of two anionic trypsins from the hepatopancreas of carp

SUMMARY: Two trypsins, designated as trypsin A and trypsin B, have been purified from the hepatopancreas of carp. The purification procedures consisted of ammonium sulfate fractionation, and chromatographies on DEAE-Sephacel, Ultrogel AcA54 and Q-Sepharose. Trypsin A was purified to homogeneity with the molecular mass of approximately 28 kDa, while trypsin B gave two close bands of 28.5 kDa and 28 kDa on sodium dodecylsulfate polyacrylamide gel electrophoresis both under reducing and non-reducing conditions. On native-PAGE, both trypsin A and trypsin B showed a single band. Trypsin A and trypsin B revealed optimum temperature of 40°C and 45°C, respectively, and shared the same optimum pH 9.0 using Boc-Phe-Ser-Arg-MCA as substrate. Both enzymes were effectively inhibited by trypsin inhibitors and their susceptibilities were similar. The NH₂-terminal amino acid sequences of trypsin A and trypsin B were determined to 37th and 40th amino acid residue, respectively. Their sequences were very homologous, but not identical to that of a trypsin-type serine proteinase from carp muscle and these of other trypsins. Immunoblotting test using the antibody raised against trypsin A cross-reacted with trypsin B positively.     

检测肥育猪CAST蛋白的免疫印迹方法研究

为了研究3个不同蛋白质水平(10%、18%、26%)饲粮对肥育猪的钙蛋白抑制蛋白酶(CAST)的影响,对CAST蛋白提取、电泳、转膜等条件进行优化。试验表明:CAST被激活的条件是将蛋白提取液于上样缓冲液95℃煮15min后,立即在冰块上冷却,防止蛋白降解;采用分离胶浓度为10%进行电泳,可以明显检测到68kD、107 kD的CAST;采用预染Marker和DAB染色液可针对性地检测到目的蛋白的位置;利用上述程序检测到了猪CAST两种类型的表达,取得了较满意的结果。     

Identification of immunogenic proteins of Flavobacterium columnare by two‐dimensional electrophoresis immunoblotting with antibacterial sera from grass carp,Ctenopharyngodon idella (Valenciennes)

Flavobacterium columnare is a Gram‐negative bacterium causing columnaris disease of freshwater fish worldwide, and development of efficacious vaccines has been a continuous challenge in aquaculture. In this study, 14 proteins were identified from cellular components of F. columnare using an immunoblotting approach in two‐dimensional electrophoresis map gels with antibacterial sera from grass carp, Ctenopharyngodon idella (Valenciennes), and then anti‐grass carp‐recombinant Ig (rIg) polyclonal antibodies. These proteins were characterized conclusively by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF/TOF MS). The 14 proteins are immunogenic molecules of F. columnare, including chaperonins DnaK, GroEL and trigger factor, and translation elongation factor G, translation elongation factor Tu, 30S ribosomal subunit protein S1, dihydrolipoamide succinyltransferase, succinyl‐CoA synthetase, SpoOJ regulator protein, alcohol dehydrogenase, fructose‐bisphosphate aldolase, 3‐hydroxybutyryl‐CoA dehydrogenase and two conserved hypothetical proteins. These identified immunogenic proteins may provide candidate molecules for the development of vaccines against columnaris disease.     

Immunochemical relationships of cytochrome P4503A-like proteins in teleost fish

Multiple P450 proteins have been purified from several teleost species, including rainbow trout (Oncorhynchus mykiss), scup (Stenotomus chrysops) and Atlantic cod (Gadus morhua). Identity, relationships and/or functions have been established in these fish species for the cytochrome P4501 As. Information about the structure, function, regulation and relationships of other piscine cytochrome P450 (CYP) proteins is sparse. In the present study we have focused on constitutively expressed CYP forms, P450con and LMC5 isolated from rainbow trout, P450A from scup, and P450b from Atlantic cod, and we consider evidence for the relationship of these proteins to mammalian members of the CYP3A subfamily. Reciprocal western blot analysis shows that P450con and LMC5, isolated from rainbow trout in two different laboratories, are closely related and ostensibly identical proteins. These trout proteins show specific reciprocal cross-reactivity with scup P450A, and polyclonal antibodies (PAb) to the trout and scup proteins both recognize cod P450b, indicating that rainbow trout P450con/LMC5, scup P450A and cod P450b are immunochemically-related proteins. In analyses of liver microsomes of trout, scup and cod, PAb to trout P450con/LMC5 and scup P450A recognize only bands that are identical in migration to the CYP proteins purified from these species, and which were used as immunogens. These CYP proteins purified from fish are each immunochemically-related to mammalian CYP3A proteins, showing recognition by PAb to human CYP3A4 and to rat CYP3A1. PAb to the mammalian CYP3As also recognize the same bands in liver microsomes from these fish species as seen by PAb to the fish proteins. These results strongly suggest that these fish proteins are members of theCYP3 gene family and probably theCYP3A subfamily. Although sequence analysis is required before their designation in the CYP3A subfamily can be confirmed and specified, we refer to these as CYP3A-like. Immunoblot analyses of hepatic microsomes from other fish species with PAb to scup P450A and trout P450con show that multiple CYP3A-like proteins are expressed in liver of several species, including killifish (Fundulus heteroclitus) and winter flounder (Pleuronectes americanus). Important questions still remain to be addressed concerning CYP3A structure, multiplicity, physiological function, regulation and metabolism of endogenous as well as exogenous substrates in fish.Part of this study was presented at the 10th International Symposium on Microsomes and Drug Oxidations. Toronto, Canada, July 18–21, 1994.     

小肠结肠耶氏菌O:9血清型与布氏杆菌M_5株共同抗原的免疫印迹分析

应用SDS-聚丙烯酰胺凝胶电泳和免疫印迹技术,对经超声波打碎的小肠结肠耶氏菌O:9血清型和布氏杆菌M5株全菌体蛋白成分进行了分子量测定及抗原分析。结果表明,小肠结肠耶氏菌O:9血清型Y15株与布氏杆菌M5株存在一条发生交叉反应的蛋白质共同抗原带,其分子量为11400。Y15株与M5株有多个分子量相同的条带,但不发生交叉反应。Y15株与M5株皆有多个各自特异的条带。     

Identification of highly specific and cross-reactive antigens of Leishmania species by antibodies from Leishmania (Leishmania) chagasi naturally infected dogs

The Leishmania species present a genetic homology that ranges from 69 to 90%. Because of this homology, heterologous antigens have been used in the immunodiagnosis and vaccine development against Leishmania infections. In the current work, we describe the identification of species-specific and cross-reactive antigens among several New World Leishmania species, using symptomatic and asymptomatic naturally Leishmania chagasi-infected dog sera. Soluble antigens from five strains of New World Leishmania were separated by electrophoresis in SDS-PAGE and immunoblotted. Different proteins were uniquely recognized in the L. chagasi panel by either symptomatic or asymptomatic dog sera suggesting their use as markers for the progression of disease and diagnosis of the initial (sub-clinical) phase of the infection. Cross-reactive antigens were identified using heterologous antigenic panels (L. amazonensis strains PH8 and BH6, L. guyanensis and L. braziliensis). L. guyanensis panel showed the highest cross-reactivity against L. chagasi specific antibodies, suggesting that proteins from this extract might be suitable for the diagnosis of visceral canine leishmaniasis. Interestingly, the 51 and 97 kDa proteins of Leishmania were widely recognized (77.8% to 100%) among all antigenic panels tested, supporting their potential use for immunodiagnosis. Finally, we identified several leishmanial antigens that might be useful for routine diagnosis and seroepidemiological studies of the visceral canine leishmaniasis.     

Presence of a lethal protease in the extracellular products of Vibrio splendidus-Vibrio lentus related strains

The presence of a lethal extracellular 39-kDa protease, a virulence determinant of a Listonella pelagia strain which produces vibriosis in turbot, was determined in the extracellular products (ECP) of 33 Vibrionaceae strains. Both immunological and enzymatic techniques distinguished this specific protease from other Vibrionaceae proteins. It was detected in 15% (5/33) of the ECPs assayed belonging to strains of the Vibrio splendidus-V. lentus related group isolated in Galician aquaculture systems (NW Spain). As these strains were associated with diseased octopus and cultured turbot, were able to colonize the internal organs of fish and produced a lethal ECP for fish, they are a potential risk for the health of reared aquatic organisms.