Effects of environmental Ca2+ levels on branchial chloride cell morphology in freshwater-adapted killifish Fundulus heteroclitus

ABSTRACT: To examine the involvement of chloride cells in the uptake of Ca²⁺ in freshwater (FW) killifish, chloride cell morphology was compared in fish acclimated to different defined FW environments with Ca²⁺ concentrations of either 0.1 mM, 0.5 mM, or 2.5 mM. Numerous chloride cells were detected in whole-mount preparations of the gill filaments, which were stained with an antiserum specific for Na⁺, K⁺-ATPase. Chloride cells, located mostly on the afferent–vascular edge of the filaments, were larger at lower Ca²⁺ concentrations. Electron microscopic observations showed that in the 0.1 mM and 0.5 mM Ca²⁺ experimental groups, the apical membrane of chloride cells were flat or slightly projecting and equipped with numerous microvilli. In the 2.5 mM Ca²⁺ group, some chloride cells formed an apical pit, whereas other cells were similar to those observed in the 0.1 mM and 0.5 mM Ca²⁺ groups. Plasma osmolality decreased with decreasing ambient Ca²⁺ concentration, suggesting that environmental Ca²⁺ affects the permeability of the body surfaces. Gill Na⁺, K⁺-ATPase activity in the 0.1 mM and 0.5 mM Ca²⁺ groups were significantly higher than that in the 2.5 mM Ca²⁺ group, implying the involvement of the Na⁺–Ca²⁺ exchanger in Ca²⁺ uptake in the gills. These findings suggest that chloride cells function as the site for Ca²⁺ uptake in killifish acclimated to low Ca²⁺ environments.     

Oversight probability in presence–absence sampling for detection of fish eggs

ABSTRACT:   Presence–absence sampling (PAS) is a sampling technique that observes only the presence or absence of objects, such as eggs, in sampling units. The purpose of the present paper was to study the oversight probability in a PAS study for detecting fish eggs in connection with the spatial distribution of eggs. It was shown that the oversight probability of presence of eggs under no-eggs observation is a monotone decreasing function of the parameter of the degree of contagion.     

Isolation and characterization of the interspecific fusants from Streptomycetes obtained using a liquid regeneration method

ABSTRACT: Protoplast fusion between different species of Streptomyces was performed using a liquid regeneration method developed for a rapid and simple preparation of the fusants. Consequently, new clones, which could not be obtained using the conventional agar regeneration method, were obtained. In the crosses between S. griseus and S. durhamensis , and between S. californicus and S. catenulae , eight and two recombinants, respectively, were obtained using the liquid regeneration method. Conversely, in the case of crosses between S. ornatus and S. catenulae , and between S. ornatus and S. vendargensis , seven recombinants each were obtained using only the agar method. The physiological characteristics, such as the assimilation of carbohydrate and antibiotic resistance, of these fusants differed considerably from those of their parental strains. Using the proposed liquid regeneration method, a simpler and quicker procedure for protoplast fusion is described.     

Improved solubility and stability of carp myosin by conjugation with alginate oligosaccharide

ABSTRACT:   Carp myosin was conjugated with alginate oligosaccharide (AO) through the Maillard reaction under low relative humidity, and the functional properties of the myosin-AO conjugate were investigated to clarify the role of myosin in the functional improvement of fish myofibrillar proteins (Mf) by the glycosylation. The findings were as follows. First, myosin became highly solubilized at lower NaCl concentrations by conjugation with AO and NaCl-dependence of the solubility was lost when > 12% of the available lysine residues were reacted with AO and 50 µg/mg of AO was attached to myosin. Second, the thermal stability of myosin was effectively improved by conjugation with AO. Heat-treatment at 50°C for 6 h has no effect on the solubility of the myosin-AO conjugate regardless of the NaCl concentration. Third, the improved functionalities of myosin conjugated with AO remained even at a nearly isoelectric point. The improving effect of AO-conjugation on the characteristics of myosin was almost the same as Mf reacted with AO. Therefore, it is apparent that that improved functionalities of the glycosylated Mf reflect the functional changes of myosin.     

Reactivity of serum immunoglobulin E to bullfrog Rana catesbeiana parvalbumins in fish-allergic patients

ABSTRACT:   Parvalbumin, a calcium-binding sarcoplasmic protein of approximately 12 kDa, represents the cross-reactive, major allergen in fish. In consideration of the fact that parvalbumin is contained at high levels not only in fish muscle but also in frog muscle, the present study was undertaken to clarify whether fish-allergic patients react to two parvalbumins (α- and β-parvalbumins) purified from the bullfrog Rana catesbeiana , which is sometimes consumed as a delicacy in Japan. In enzyme-linked immunosorbent assays (ELISA), sera from 12 of the 14 patients tested reacted equally to both parvalbumins purified from the Pacific mackerel Scomber japonicus and the bigeye tuna Thunnus obesus . Of the 12 sera positive to fish parvalbumins, eight sera also reacted to α- and β-parvalbumins of the bullfrog with different spectra: one serum reacted strongly to α-parvalbumin, six sera reacted strongly to β-parvalbumin and one serum reacted equally to both α- and β-parvalbumins. In addition, inhibition ELISA experiments revealed cross-reactivity between fish and bullfrog parvalbumins. Based on these results, it is proposed that fish-allergic patients should avoid the consumption of frog meat unless they are accurately diagnosed as lacking immunoglobulin E against frog.     

Biosynthesis of estradiol-17β in the ovarian follicles of the red seabream Pagrus major during vitellogenesis

ABSTRACT: The red seabream Pagrus major is a useful experimental fish for studying the endocrine control of oogenesis in teleosts. This study investigated the steroidogenic pathway for estradiol-17β (E2) biosynthesis in the ovarian follicles of red seabream. Intact follicles were isolated during vitellogenesis and incubated in vitro with different radiolabeled steroid precursors. When 17-hydroxyprogesterone (17-P), dehydroepiandrosterone (DHEA), or androstenedione (AD) were used as precursors, both testosterone (T) and estrone (E1) were synthesized by follicles, leading to estradiol-17β (E2) production. Serum steroid levels measured by enzyme-linked immunosorbent assay showed that T, E1, and E2 were present in the circulation at levels ranging from 1 ng/mL to 2 ng/mL throughout the day during the spawning season. In vitro conversion of E1 into E2, however, was 15.8-fold greater than T conversion into E2, suggesting that E2 is synthesized mainly via E1 rather than T. The results showed that E2 was synthesized from pregnenolone via 17-hydroxypregnenolone, DHEA, AD, and E1. Thus, the study demonstrated the complete steroidogenic E2 synthesis pathway in the ovarian follicles of red seabream, and revealed that E1 is the major precursor of E2.     

Neurotoxin tetrodotoxin as attractant for toxic snails

ABSTRACT:   The attracting effect of a small dose of tetrodotoxin (TTX) on eight toxic snail species ( Polinices didyma, Natica lineata, Natica vitellus, Zeuxis sufflatus, Niotha clathrata, Oliva miniacea, Oliva mustelina and Oliva hirasei ) and two non-toxic species ( Pomacea canaliculata and Satsuma bairdi ) was investigated. Each toxic snail species was determined to contain TTX. The minimum lethal dose of TTX for most toxic snails was estimated to be >44.5 µg TTX/20 g body weight, but for non-toxic snails it was <3.6 µg TTX/20 g body weight. After the attracting test, it was found that for all toxic snails there was a significantly positive relationship between the comparative attracting variation and the toxicity reported, and this relationship was linear ( y  = 5.895 x  + 3.443, r  = 0.806). The relationship between TTX resistance ability and toxicity also had a positive correlation ( y  = 0.113 x  + 52.447, r  = 0.814). However, non-toxic species showed a negative response. The more toxic snails appeared to prefer TTX, indicating that TTX is an attractant for toxic snails.     

Mass spectrometric detection of proopiomelanocortin (POMC)-related peptides following molecular cloning of POMC cDNA in bigeye tuna Thunnus obesus

Several pituitary hormones, including adrenocorticotropin (ACTH), melanotropin (MSH) and β-endorphin, are generated from a common precursor protein, proopiomelanocortin (POMC). In fish, in addition to steroidogenesis of ACTH and melanogenesis of MSH, immunomodulating activity has been found in some POMC-related peptides. To investigate the functions of these peptides in the homologous system, it is necessary to establish a convenient detection method for the peptides. The present study aimed to establish a method for the detection of POMC-related peptides in bigeye tuna Thunnus obesus using a small amount of tissue sample, but not requiring peptide purification. We first determined the nucleotide sequence of tuna POMC cDNA. The cDNA was composed of 1084 base pairs (excluding the poly A tail) that encoded POMC consisting of 222 amino acids. We then fractionated an acid-acetone extract of one pituitary by reverse-phase high performance liquid chromatography (HPLC) and determined the molecular weight of each separated peptide by mass spectrometry. Consequently, we detected eight POMC-related peptides by comparing the values to the deduced amino acid sequence. Thus, the present study enabled the detection of POMC-related peptides from a small amount of tissue without the use of several purification steps.     

Structural and thermodynamic characterization of tropomyosin from fast skeletal muscle of bluefin tuna

ABSTRACT:   Tuna tropomyosin is a mixture of nearly equimolar amounts of two isoforms (designated α and β). cDNA encoding the α form was cloned from bluefin tuna Thunnus thynnus fast skeletal muscle. The full-length cDNA contained 1220 bp, comprising an open reading frame of 855 bp encoding 284 amino acid residues, flanked by 5'-untranslational regions (156 bp) and 3'-untranslational regions (209 bp). The deduced amino acid sequence showed considerably high homology in a range of 93.7–98.6% to those of other vertebrate α-type tropomyosins. In phylogenetic analysis, bluefin tuna tropomyosin showed the closest relationship with the white croaker counterpart. The predicted mass was 32 919 Da, and isoelectric point was 4.50, assuming acetylation of the N-terminus. By differential scanning calorimetry, bluefin tuna tropomyosin gave two major endothermic peaks at 29.3 and 41.5°C, probably caused by the presence of two isoforms. Circular dichroism spectra supported such a unique denaturation profile.