Maternal Transfer and Protective Role of the Alternative Complement Components in Zebrafish Danio rerio

Embryos of most fish develop externally and are exposed to an aquatic environment full of potential pathogens, whereas they have little or only limited ability to mount an efficient and protective response. How fish embryos survive pathogenic attacks remains poorly defined. Here we demonstrate that the maternal immunization of female zebrafish with formalin-killed Aeromonas hydrophila causes a significant increase in C3 and Bf contents in the mother, a corresponding rise in the offspring, and induces a remarkable increase in the hemolytic activities in both the mother and offspring. In addition, the embryos derived from the immunized mother are significantly more tolerant to A. hydrophila challenge than those from the unimmunized fish, and blocking C3 and Bf activities by injection of the antibodies against C3 and Bf into the embryos render them more susceptible to A. hydrophila. These results clearly show that the protection of zebrafish embryos against A. hydrophila can be achieved by the maternally-transferred immunity of the complement system operating via the alternative pathway. This appears to be the first report providing in vivo evidences for the protective role of the alternative complement components in the early embryos of zebrafish, paving the way for insights into the in vivo function of other maternally-transferred factors in fish.     

An aerodynamic valve in the avian primary bronchus.

The segmentum accelerans in geese is a constriction in the caudal end of the primary bronchus. Experimental evidence suggests that this part of the airway functions as an inspiratory aerodynamic valve, accelerating the incoming airstream past the ventrobronchial openings. The luminal diameter of the segmentum accelerans dilates in the presence of elevated CO2 levels, probably through relaxation of smooth muscle. Physiological control of the segmentum accelerans may permit inspiratory aerodynamic valving to be maintained throughout a wide range of ventilatory flows.     

Acid-mediated mutagenicity of tobacco snuff: its possible mechanism

Polar solvent extracts of tobacco snuff under acidic conditions were mutagenic in Salmonella typhimurium. Using the Griess reagent test, nitrite ranging from approximately 1.8 to 5.4 mg/g of snuff was found in the polar fraction of extracts. After acid treatment, nitroso compounds in the amount corresponding to the nitrite concentration were detected. The mutagenic potency of the acid-treated extracts was consistent with the content of nitroso compounds generated. Formation of nitroso compounds and the mutagenic activity under acidic conditions was inhibited by ascorbic acid. The results indicate that a nitrosation process was involved in snuff extracts during acid treatment. Studies related to the source of nitrite in tobacco snuff demonstrated that snuff contained bacteria which were able to reduce nitrate to nitrite and that the amount of nitrite in snuff extracts could be further increased by incubation of the extracts with the bacteria. Since snuff contains a considerable amount of nitrate, it seems that reduction of nitrate in snuff to nitrite by bacteria, and nitrosation of certain constituents in snuff by nitrite under acidic conditions to form mutagenic nitroso compounds are possible mechanisms responsible for the acid-mediated mutagenicity of snuff extracts.     

Immunocytochemical studies on prolactin cells in the adenohypophysis of the golden hamster

Mammotrophs or prolactin (PRL) cells were identified in the adenohypophysis of adult golden hamsters by immunocytochemical techniques with a polyclonal anti-PRL, that was proved to be specific to PRL by the dot immunoblotting test. Postembedding immunostaining was performed on Araldite thin sections by immunoperoxidase and immunogold methods. PRL cells were classified into three types according to the different size of the secretory granules. The Type A cells were usually small and angular or oval in shape, and had secretory granules ranging in diameter from 100-230 nm, and showed poorly developed organelles. The Type B and C cells were larger and round or ovoid in shape, contained larger granules, 230-280 nm and 280-570 nm, respectively, and displayed well developed organelles. Immunoreactive PRL cells in the male pituitaries were far less numerous than in the nonpregnant female glands, and were mostly of the Type A and B, whereas in the female the Type C and B cells predominated. In pregnant females, Type C cells became activated and increased in number, while the other two types decreased in proportion. In lactating females, Type A and B cells significantly increased in number at the expense of the Type C cells; meanwhile, the exocytosis of secretory granules was frequently found in all types of PRL cells. The present findings suggest that Type C and B PRL cells, especially the former, are potent in producing and releasing PRL and highly responsive to various physiological stimuli, while Type A cells are probably relatively inert in synthetic activity.     

Characterization of the Erwinia carotovora pelB gene and its product pectate lyase.

The pelB gene encodes pectate lyase B, one of three pectate lyases identified in Erwinia carotovora EC. Pectate lyase B was purified from Escherichia coli containing the pelB gene on a recombinant plasmid. The activity of the protein was optimal at a pH of 8.3. The amino acid composition, N-terminal amino acid sequence, and C-terminal peptide sequence were determined and compared with the polypeptide sequence deduced from the DNA sequence of pelB. Purified pectate lyase B started at amino acid 23 of the predicted sequence, suggesting that a 22-amino-acid leader peptide had been removed. Pectate lyase B of E. carotovora EC and pectate lyase B of E. chrysanthemi EC16 contain 352 and 353 amino acids, respectively (N. T. Keen, S. Tanaki, W. Belser, D. Dahlbeck, and B. Staskawicz, J. Bacteriol. 168:595-606, 1986). The two proteins are 72% homologous on the basis of DNA sequence data, and 75% of the amino acids are identical.     

Cross-linking study of the quaternary fine structure of mitochondrial F1-ATPase

A method has been developed for exploring the quaternary fine structure of oligomeric proteins by crosslinking studies and applied to bovine heart mitochondrial F1-ATPase. The F1 was first labeled with 1-fluoro-2,4-dinitro-[14C]benzene, subsequently reduced with sodium hydrosulfite, and finally cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Gel electrophoresis in the chemically modified protein in the presence of sodium dodecyl sulfate and mercaptoethanol showed the existence of a 105-115-kilodalton molecular species in addition to the five monomeric subunits of F1. This cross-linked species could be alpha 2, alpha beta, or beta 2. Isolation of the cross-linked species and titration with 5,5'-dithiobis-(2-nitrobenzoic acid) showed the absence of sulfhydryl group. Therefore, the cross-linked species must be the dimer beta 2. After digestion of the purified beta 2 with pepsin, a single radioactive peptide was isolated. Determination of the amino acid sequence of this peptide and comparison of its radioactivity with the total radioactivity on beta-subunits show that it was formed exclusively by cross-linking Lys162 of one beta-subunit with Glu199 of another beta-subunit. The observation that two beta-subunits can be cross-linked by a rigid phenylenediamine bridge of 5.7- or 4.3-A length is difficult to reconcile with the widely assumed structure of F1 with the alpha- and beta-subunits occupying alternate corners of a planar hexagon, but is consistent with the structure in which a triangular set of three beta-subunits sits above a triangular set of three alpha-subunits in a staggered conformation.     

Effect of phosphorylation by cyclic AMP-dependent protein kinase on the smooth muscle actomyosin Mg2+-ATPase stimulatory activity of fodrin

Fodrin, a non-erythrocyte spectrin-like protein, has been purified from bovine brain and found to be phosphorylated by the cyclic AMP-dependent protein kinase with a maximal stoichiometry of 1.02 +/- 0.06 mol of phosphate/mol of fodrin dimer (n = 4). This phosphorylation was not affected by the presence of actin and calmodulin. The phosphorylation of fodrin was found to occur exclusively at serine residues on the beta subunit. Two-dimensional thin layer electrophoresis and chromatography of a tryptic digest of phosphorylated fodrin showed one major phosphopeptide and a few minor ones. We have previously reported that nonphosphorylated fodrin is capable of stimulating the smooth muscle actomyosin Mg2+-ATPase by 50-70% under a well-defined set of conditions such as a critical fodrin concentration and an optimal preincubation time (Wang, C., Ngai, P.K., Walsh, M.P., and Wang, J.H. (1987) Biochemistry 24, 1110-1117). We now report that phosphorylation of fodrin completely eliminates this stimulatory effect. However, phosphorylation of fodrin was able to compete with nonphosphorylated fodrin to result in the abolition of the stimulatory effect. Similarly, nonphosphorylated fodrin could overcome the inhibitory effect created by phosphorylated fodrin. The present results support the suggestion that the stimulation of the smooth muscle actomyosin Mg2+-ATPase by fodrin may be a physiological phenomenon and cyclic AMP may serve as a regulator for this effect.