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Several crustaceans including shrimps change the amount of specific free amino acids to regulate the osmotic pressure in their bodies. Kuruma shrimp Penaeus japonicus also increases the concentration of alanine (Ala) in the abdominal muscle following the increase of environmental salinity. In the present study, to elucidate the mechanisms of changes in Ala accumulation of kuruma shrimp depending on salinity, we cloned the gene encoding alanine aminotransferase (ALT), an enzyme involved in Ala biosynthesis, and examined its expression profile. It was found that the full-length kuruma shrimp ALT1 cDNA consisted of 3,301 bp, encoding 514 amino acids, and that all amino acid residues important for ALT activity were conserved. Phylogenetic analysis also indicated that the ALT gene cloned in this study was classified as ALT1. Moreover, we examined the expression levels of the ALT1 gene in the abdominal muscle and the hepatopancreas of kuruma shrimp acclimated at 17‰, 34‰, and 40‰ salinities, resulting that the mRNA levels of the ALT1 genes in both tissues of the shrimp acclimated at 40‰ were significantly higher than those at 17‰ for 12 h (p < 0.05). The mRNA levels of the ALT1 gene in the abdominal muscle of the shrimp acclimated for more than 24 h tended to increase following the increase of environmental salinity. These results indicate that ALT1 is responsible for the increase of free Ala concentration in the abdominal muscle of kuruma shrimp to regulate osmotic pressure at high salinity., (© 2024 Wiley Periodicals LLC.)

Type V collagen is considered to be a crucial minor collagen in fish skin with unique physiological functions. In this research, the cDNAs of three procollagens (Tacol5a1, Tacol5a2, and Tacol5a3) in type V collagen were cloned from the skin of shortbill spearfish ( Tetrapturus angustirostris ). The open reading frames (ORFs) of Tacol5a1, Tacol5a2, and Tacol5a3 contained 5991, 4485, and 5607 bps, respectively, encoding 1997, 1495, and 1869 amino acid residues. Each of the deduced amino acid sequences of procollagens contained a signal peptide and a fibrillar collagen C-terminal domain (COLFI). A conserved thrombospondin-like N-terminal domain (TSPN) was found at the N-terminus of Tacol5a1 and 5a3 procollagens, whereas a von Willebrand factor (VWC) was found at the N-terminus of Tacol5a2 procollagen. Tacol5a1, Tacol5a2, and Tacol5a3 had their theoretical isoelectric points of 5.06, 6.75, and 5.76, respectively, and predicted molecular weights of 198,435.60, 145,058.48, and 189,171.18, respectively. The phylogenetic tree analysis revealed that Tacol5a1 of shortbill spearfish clustered with that of yellow perch ( Perca flavescens ) instead of broadbill swordfish ( Xiphias gladius ). In addition, type V collagen was extracted from the shortbill spearfish skin. The in silico method demonstrated that shortbill spearfish type V collagen has a high potential for angiotensin-converting enzyme (ACE) inhibition activity (79.50%), dipeptidyl peptidase IV inhibition (74.91%) activity, and antithrombotic activity (46.83%). The structural clarification and possible functional investigation in this study provide the foundation for the applications of exogenous type V collagen derived from fish sources.

Akanes are fluorescent proteins that have several fluorescence maxima. In this report, Akane1 and Akane3 from Scleronephthya gracillima were selected, successfully overexpressed in Escherichia coli and purified by affinity chromatography. Fluorescence spectra of the recombinant Akanes matured in darkness, or ambient light were found to have several fluorescence peaks. SDS-PAGE analysis revealed that Akanes matured in ambient light have two fragments. MS/MS analysis of Akanes digested with trypsin showed that the cleavage site is the same as observed for the photoconvertible fluorescent protein Kaede. The differences between the calculated masses from the amino acid sequence of Akane1 and the measured masses of Akane1 fragments obtained under ambient light coincided with those of Kaede. In contrast, a mass difference between the measured N-terminal Akane3 fragment and the calculated mass indicated that Akane3 is modified in the N-terminal region. These results indicate that numerous peaks in the fluorescent spectra of Akanes partly arise from isoproteins of Akanes and photoconversion. Photoconversion of Akane1 caused a fluorescence change from green to red, which was also observed for Akane3; however, the fluorescent intensity decreased dramatically when compared with that of Akane3. [ABSTRACT FROM AUTHOR]

Complementary DNA encoding a part of myosin heavy chain was cloned from fast skeletal muscle of Japanese codling Physiculus japonicus. Japanese codling, a frequent catch off the Pacific coast of northeastern Japan, is a typical underutilized fish species, especially in summer. Phylogenetic analysis of the deduced amino acid sequence of Japanese codling myosin heavy chain loop 2 region revealed that this fish belongs to a group comprising white croaker Pennahia argentata and walleye pollack Gadus chalcogrammus, which are commonly used as raw materials for surimi-based products. Thus Japanese codling meat was considered a promising candidate ingredient for surimi-based products. Salt-ground meat was prepared from Japanese codling in the presence of 0.5 M NaCl and subjected to a two-step heating procedure: pre-heating at 15–70 °C for 30 min, and secondary heating at 85 °C for 20 min. Thermal gels with a maximum breaking strength of > 10 N were produced by this process. The breaking strength was comparable to that of white croaker and walleye pollack. On the basis of the phylogenetic analysis of myosin heavy chains, and the properties of the heat-induced surimi gel, we conclude that Japanese codling is a promising raw material for the production of surimi-based products. [ABSTRACT FROM AUTHOR]

In our previous publication, we identified novel gene candidates involved in shell formation by EST analyses of the nacreous and prismatic layer-forming tissues in the pearl oyster Pinctada fucata. In the present study, 14 of those genes, including two known genes, were selected and further examined for their involvement in shell formation using the RNA interference. Molecular characterization based on the deduced amino acid sequences showed that seven of the novel genes encode secretory proteins. The tissue distribution of the transcripts of the genes, as analyzed by RT-PCR and in situ hybridization, was mostly consistent with those obtained by the EST analysis reported previously. Shells in the pearl oysters injected with dsRNAs targeting genes 000027, 000058, 000081, 000096, 000113 (nacrein), 000118, 000133 and 000411 (MSI60), which showed expression specific to the nacreous layer forming tissues, showed abnormal surface appearance in this layer. Individuals injected with dsRNAs targeting genes 000027, 000113 and 000133 also exhibited abnormal prismatic layers. Individuals injected with dsRNAs targeting genes 000031, 000066, 000098, 000145, 000194 and 000200, which showed expression specific to prismatic layer forming tissues, displayed an abnormal surface appearance in both the nacreous and prismatic layers. Taken together, the results suggest that the genes involved in prismatic layer formation might also be involved in the formation of the nacreous layers. [ABSTRACT FROM AUTHOR]

Abstract: The periostracum is a layered structure that is formed as a mollusk shell grows. The shell is covered by the periostracum, which consists of organic matrices that prevent decalcification of the shell. In the present study, we discovered the presence of chitin in the periostracum and identified a novel matrix protein, Pinctada fucata periostracum protein named PPP-10. It was purified from the sodium dodecyl sulfate/dithiothreitol-soluble fraction of the periostracum of the Japanese pearl oyster, P. fucata. The deduced amino acid sequence was determined by a combination of amino acid sequence analysis and cDNA cloning. The open reading frame encoded a precursor protein of 112 amino acid residues including a 21-residue signal peptide. The 91 residues following the signal peptide contained abundant Cys and Tyr residues. PPP-10 was expressed on the outer side of the outer fold in the mantle, indicating that PPP-10 was present in the second or third layer of the periostracum. We also determined that the recombinant PPP-10 had chitin-binding activity and could incorporate chitin into the scaffolds of the periostracum. These results shed light on the early steps in mollusk shell formation. [Copyright &y& Elsevier]

We confirmed the existence of growth-promoting substances in the conditioned media (CM) from the rotifer Brachionus plicatilis at an early exponential growth phase and isolated a novel protein with a growth-promoting activity from the crude extract (CE) of rotifer cells. CM was prepared from the culture media where rotifers had been cultured at an early exponential growth phase and filtered through a 0.22-μm filter membrane. The growth-promoting activity was determined using rotifers in CM for 5 days. As a result, the increase of rotifers added with CM was significantly higher than that of the control in artificial seawater ( P < 0.001). Moreover, the growth-promoting activity of CM was dose-dependent and inactivated by heat treatment at 80°C for 60 min. Meanwhile, CM filtered through a <10 kDa ultrafiltration membrane showed a low activity, whereas proteinase K treatment resulted in a complete inactivation. These results suggest that the rotifer secrets growth-promoting proteins into CM. CE also contained a protein with the activity and properties similar to those found in CM. Then, CE was subjected to purification of a growth-promoting protein for convenience using various types of chromatography after fractionation with 30-80% saturated ammonium sulfate. Subsequently, a protein with an approximate molecular weight of 25000 was isolated, and its N-terminal amino acid sequence was determined to be PAVVDFTAVWFGPLQMIKP. An orthologue was found in the EST database of B. plicatilis, the full sequence of which showed about 50% identity to the corresponding regions of thioredoxins from other organisms. [ABSTRACT FROM AUTHOR]

Tropomyosins from fish skeletal muscle show high amino acid sequence homology, although their thermal stability is clearly different among species. In order to determine the regions that are responsible for the stability of this protein, five synthetic peptides of 30mer were synthesized by Fmoc method, based on the sequence of walleye pollack Theragra chalcogramma fast skeletal muscle tropomyosin, namely, N terminal Met1-Lys30, the variable region Asp84-Leu113, the middle region Val128-Ala157, the region containing the conservative Cys (Leu176-Lys205), and C terminal Asp255-Ile284. The thermal stability of these peptides was measured by circular dichroism and differential scanning calorimetry. The helical contents of these peptides were decreased in a temperature-dependent manner, although they showed no clear melting temperature, suggesting that the enthalpy necessary for the complete denaturation of these peptides was low. Peptides Asp255-Ile284 and Asp84-Leu113 showed the highest and second highest α-helical contents, respectively, and the other peptides gave rise to lower α-helical contents. [ABSTRACT FROM AUTHOR]

A cDNA library constructed from the dorsal fast skeletal muscle of white croaker Pen-nahia argentata was screened for myosin heavy chain using antibody raised against carp fast skele-tal myosin. A full-length cDNA was further cloned by reverse transcription--PCR and 5-RACE using first-strand cDNA as a template together with appropriate sets of primers. The entire cDNA consisted of 5986 nucleotides (nt) with 64 nt 5-untranslated and 129 nt 3-untranslated regions. This full-length cDNA had an open reading frame encoding a polypeptide of 1930 amino acid residues. Amino acid alignment with myosins from various vertebrates revealed some striking differences between fish and mammalian sequences which could be due to their position in the vertebrate evolutionary process. Hydrophilicity analysis revealed two different features of the myosin molecule: S1 heavy chain showed a mixed profile of hydrophobicity and hydrophilicity, whereas rod had a profile of only hydrophilicity. Comparison of amino acid sequences in the rod region between white croaker and walleye pollack showed a markedly high identity of 92%. White croaker myosin rod had a charac-teristic seven-residue (heptad) repeat (a, b, c, d, e, f, g)n, where positions a and d were normally occupied by hydrophobic residues, and positions b, c and f by oppositely charged residues, which may lead to interhelical electrostatic attractions stabilizing the coiled-coil of a -helices. [ABSTRACT FROM AUTHOR]

Examines the occurrence of collagen proα-chain in the abalone Haliotis discus muscle. Composition of the fibril-forming collagens; Presence of cysteine residues in vertebrate collagens; N-terminal amino acid sequencing of collagens.

Deals with the study that determined the N-terminal amino acid sequences of the white croaker tropomyosin. Procedures involved in the preparation of the tropomyosin; Composition of the complementary DNA clone obtained; Amino acid sequences of the α-chymotryptic peptides deduced from the tropomyosin gene.

Abstract: Structural stabilities of myoglobin (Mb) from several tuna fish species significantly differ from each other, although the amino acid sequence identities are very high (>95%), suggesting that limited number of substitutions greatly affect the stability of Mb. To address this hypothesis, attempts were made to elaborate recombinant tuna Mbs with point mutations on the different residues among fish Mbs. The expression plasmid constructs were based on bigeye tuna Mb cDNA sequence, and the recombinant proteins were expressed as GST-fusion proteins in Escherichia coli. After removal of the GST segment and affinity purification, the stability of five Mb mutants, namely, A49G, T91K, K92Q, V108A, and H112Q, together with the wild type (WT) were measured, taking temperature dependency of α-helical content and denaturant (urea and guanidine-HCl) concentration dependency of Soret band absorbance as parameters. As a result, the mutant H112Q showed much higher stability than WT, while the structures of K92Q, T91K and A49G mutants were destabilized. No essential change in helical content was observed for V108A, but the mutant was found to be destabilized easier by the denaturants. These findings suggested that the highly conserved residues among tuna species are responsible for their stability of Mbs, but a few non-conserved residues dramatically give rise to the differences in stability of Mbs among species. [Copyright &y& Elsevier]

Abstract: Fast skeletal muscle tropomyosin (TM) of tunas is composed of nearly equimolar amount of two isoforms designated α-TM and β-TM expediently based on their migration behavior in SDS–PAGE, whereas corresponding TMs from the other fish species are homogenous (α-type). The presence of β-TM is thus specific to tunas so far. The amino acid sequence of β-TM from bluefin tuna Thunnus thynnus orientalis, which has not been revealed to date unlike α-TM, was successfully obtained in this study by cDNA cloning. The coding region of β-TM cDNA comprised of an open reading frame of 855bp encoding 284 amino acid residues, like most of the TMs. Unexpectedly, the sequence of β-TM showed high similarity to those of other vertebrate α-type TMs including tuna α-TM. Phylogenetic analysis also showed that β-TM has the closest relationship with α-TM of tuna. This fact was quite unlike the relation of mammalian α- and β-TMs. Based on the distribution of amino acid substitutions, it was suggested that tuna TM isoforms are the products of different genes. By thermodynamic analysis of native and reconstituted TMs, it was demonstrated that β-TM is less thermostable than α-TM. Proteolytic digestion also supported the lower stability of the former. [ABSTRACT FROM AUTHOR]

Abstract: Three types of myosin heavy chain cDNA clone named 10 °C, intermediate and 30 °C types were isolated from fast skeletal muscles of thermally acclimated grass carp Ctenopharyngodon idellus. Three clones encompassing parts of 3′-translated and entire 3′-untranslated regions showed high heterogeneities in their nucleotide sequences in the 3′-untranslated region. The comparison in the deduced amino acid sequence of the 10 °C-type clone with those of the intermediate- and 30 °C-type clones showed 88% and 89% identities, respectively. By contrast, the deduced amino acid sequence of the intermediate-type clone shared much higher identity of 97% with its 30 °C-type counterpart. Northern blot analysis demonstrated that the 10 °C- and 30 °C-type clones were predominantly expressed in grass carp acclimated to 10 and 30 °C, respectively. The intermediate type was expressed both in grass carp acclimated to 20 and 30 °C. Furthermore, expression patterns of the three myosin heavy chain genes were altered in accompaniment with seasonal temperature fluctuation. In autumn and winter grass carp expressed the 10 °C-type gene almost exclusively, whereas it was completely replaced by the intermediate- and 30 °C-type genes in spring and summer. These results suggest that tetraploid grass carp also undergo an adaptation to fluctuating environmental temperatures by selectively expressing fast skeletal myosin heavy chain isoforms as do diploid common carp previously reported. [Copyright &y& Elsevier]