Comparison of 5′-inosine monophosphate and p-nitrophenyl phosphate degrading activities among red,pink, and white muscle fibers of cultured carp

ABSTRACT:   As part of a study to clarify the differences in the temporal change in K -value among fish species, the temporal change in K -value and the 5'-inosine monophosphate (5'-IMP) and p-nitrophenol phosphate (p-NPP) degrading activities in the red, pink, and white muscle fibers in the dorsal muscle of the carp were compared. The temporal change in K -value was fastest in red, followed by pink, and white muscle fibers, at both 0°C and 32°C. Moreover, the 5'-IMP and p-NPP degrading activities were highest in red, followed by pink, and white muscle fibers at near optimum pH concentrations. The 5'-IMP degrading activity at pH 7.0 had a positive correlation with the increasing rate of K -value at 32°C for all types of muscle fibers. These results suggest that differences in increasing rates of K -values between red, pink, and white muscle fibers corresponded to the 5'-IMP degrading activities.     

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.     

Effect of Garcinia cambogia extract on serum leptin and insulin in mice

In this study we examined the effects of 3.3% Garcinia cambogia extract on 10% sucrose loading in mice for 4 weeks. Treatment was found to have no effect on body weight, fat pad weight or serum glucose level. On the other hand, serum total cholesterol, triglycerides, NEFA were observed. Levels of serum insulin and leptin, as well as the leptin/WAT ratio, were lower in the treated mice than in the control. These findings suggested that G. cambogia extract efficiently improved glucose metabolism and displayed leptin-like activity.     

Balenine,imidazole dipeptide,induces activation of superoxide dismutase in myotubes

Balenine is one of the endogenous imidazole dipeptides. It is composed of beta-alanine and 3-methyl-l-histidine, which exist mainly in the muscles and the brain. The exact biological properties of balenine are still not well known, although the antioxidant activity of carnosine, another imidazole dipeptide, is known. In this study we investigated whether balenine exhibits antioxidant activity. It was found to decrease the superoxide anion (O₂⁻) and increased hydrogen peroxide (H₂O₂) generation. We found that SOD activity increased in balenine-treated C2C12 myotubes, although balenine did not increase expression of SOD mRNA. On the other hand, there were no changes in other antioxidant enzymes, CAT and GPX activity, and mRNA levels. In an in vitro assay, the direct activation of SOD treated by balenine was significantly higher than with carnosine treatment. Moreover, balenine constituent amino acids did not have the ability to activate SOD. Our results suggest that balenine contributes to antioxidant effects through activation of SOD.

     

Mung bean trypsin inhibitor is effective in suppressing the degradation of myofibrillar proteins in the skeletal muscle of blue scad (Decapterus maruadsi)

Mung bean trypsin inhibitor (MBTI) of the Bowman-Birk family was purified to homogeneity with a molecular mass of approximately 9 kDa on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 8887.25 Da as determined by matrix-assisted laser desorption/ionization-quadrupole ion trap-time-of-flight mass spectrometry (MALDI-QIT-TOF MS). Using blue scad myofibrillar proteins as targets, it was found that, in the absence of MBTI, proteolysis of myofibrillar proteins, especially myosin heavy chain (MHC), could be identified after incubation at 55 °C for 2 h, while in the presence of MBTI, with a final concentration of 25 ng/mL, proteolysis of these proteins was greatly suppressed even after incubation for 3 h. Although cysteine proteinase inhibitor E-64 was also effective in preventing protein degradation, inhibitors for metallo- and asparatic proteinases did not reveal obvious inhibitory effects. Our present results strongly suggested that the naturally occurring legume bean seed protein MBTI can be used as an effective additive in preventing marine fish blue scad surimi gel softening, which is quite possibly caused by myofibril-bound serine proteinase (MBSP).     

Pavoninins: shark-repelling ichthyotoxins from the defense secretion of the pacific sole

A series of ichthyotoxic and hemolytic steroid aminoglycosides, pavoninins-1, to -6, has been isolated from the defense secretion of the sole Pardachirus pavoninus, and their respective chemical structures have been established by spectroscopic studies and chemical conversions. The pavoninins exert repellent activity against sharks and are considered to be the factors responsible for the predator-repelling property of the sole.     

A broad spectrum,one-step RT-PCR to detect <Emphasis Type="Italic">Satsuma dwarf virus</Emphasis> variants using universal primers targeting both segmented RNAs 1 and 2

Universal primers to detect Satsuma dwarf virus (SDV), including distantly related strains Citrus mosaic virus (CiMV), Navel orange infectious mottling virus (NIMV), and Hyuganatsu virus (HV), were tested in a convenient one-step RT-PCR assay. SDV was the most broadly detected using uSDVup/uSDVdo primers that specifically targeted a nucleotide sequence in the 3′-noncoding region that is conserved in both segmented RNAs 1 and 2 of SDV among the tested primers. Nucleotide sequence analysis confirmed that the amplified RT-PCR products could be derived from RNAs 1 or 2 of SDV variants, some of which had interesting genetic diversity.     

Antiallergic tea catechin, (-)-epigallocatechin-3-O-(3-O-methyl)-gallate,suppresses FcepsilonRI expression in human basophilic KU812 cells

We previously found that the O-methylated derivative of (-)-epigallocatechin-3-O-gallate (EGCg), (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG' '3Me), has potent antiallergic activity. The high-affinity IgE receptor, FcepsilonRI, is found at high levels on basophils and mast cells and plays a key role in a series of acute and chronic human allergic reactions. To understand the mechanism of action for the antiallergic EGCG' '3Me, the effect of EGCG' '3Me on the cell surface expression of FcepsilonRI in human basophilic KU812 cells was examined. Flow cytometric analysis showed that EGCG' '3Me was able to decrease the cell surface expression of FcepsilonRI. Moreover, immunoblot analysis revealed that total cellular expression of the FcepsilonRI alpha chain decreased upon treatment with EGCG' '3Me. FcepsilonRI is a tetrameric structure comprising one alpha chain, one beta chain, and two gamma chains. The level of mRNA production of each subunit in KU812 cells was investigated. EGCG' '3Me reduced FcepsilonRI alpha and gamma mRNA levels. The cross-linkage of FcepsilonRI causes the activation of basophils, which leads to the secretion of inflammatory mediators including histamine. EGCG' '3Me treatment inhibited the FcepsilonRI cross-linking-induced histamine release. These results suggested that EGCG' '3Me can negatively regulate basophil activation through the suppression of FcepsilonRI expression.     

Xenografting of bovine secondary follicles into ovariectomized female severe combined immunodeficient mice

Xenografting of ovarian tissue into immunodeficient mice has been used as a model to study the dynamics of follicular development and provides an alternative method for the production of mature oocytes. In a previous experiment, we demonstrated that xenografted bovine secondary follicles developed to the antral stage in severe combined immunodeficient (SCID) mice. In the present study, we examined the development of bovine secondary follicles (140-190 microm in diameter) grafted into ovariectomized mice in comparison with intact female mice as a control. At 4 weeks after grafting, several antral follicles ranging from 350 to 550 microm (457.6 +/- 50.8 microm) in diameter were found in the control mice, while a single large (larger than 2.5 mm) antral follicle and other small follicles were observed in every ovariectomized mouse. At 6 weeks after grafting, the mean diameter of morphologically normal follicles had further increased in the control group (591.8 +/- 132.0 microm). In ovariectomized mice, however, the mean diameter of follicles decreased (4 weeks: 864.2 +/- 988.2 microm; 6 weeks: 496.5 +/- 137.6 microm), since the single large antral follicle observed at 4 weeks had degenerated by 6 weeks. In control mice, more than 70% of follicles were morphologically normal and formed an antrum, and most of the follicles contained morphologically normal oocytes which grew to 122.5 +/- 2.2 microm. In ovariectomized mice, morphologically normal oocytes also grew larger than before grafting, but their survival rate was significantly lower than that in control mice. These results suggest that ovariectomy of host mice alters the developmental pattern of xenografted bovine secondary follicles to accelerate a single follicle to develop in the graft.     

Simultaneous observation of the GnRH pulse generator activity and plasma concentrations of metabolites and insulin during fasting and subsequent refeeding periods in Shiba goats

The time course of GnRH pulse generator activity and plasma concentrations of energy substrates and insulin were simultaneously observed in female goats during 4-day fasting and subsequent refeeding in the presence or absence of estrogen for a better understanding of the mechanism of energetic control of gonadotropin secretion in ruminants. The GnRH pulse generator activity was electrophysiologically assessed with the intervals of characteristic increases in multiple-unit activity (MUA volleys) in the mediobasal hypothalamus. In estradiol-treated ovariectomized (OVX+E2) goats, the MUA volley intervals increased as fasting progressed. Plasma concentrations of non-esterified fatty acid and ketone body increased, while those of acetic acid and insulin decreased during fasting. The MUA volley intervals and plasma concentrations of those metabolites and insulin were restored to pre-fasting levels after subsequent refeeding. In ovariectomized (OVX) goats, changes in plasma metabolites and insulin concentrations were similar to those in OVX+E2 goats, but the MUA volley intervals were not altered. The present results demonstrated that fasting suppressed GnRH pulse generator activity in an estrogen-dependent manner. Changes in plasma concentrations of energy substrates and insulin during fasting were associated with the GnRH pulse generator activity in the presence of estrogen, but not in the absence of the steroid in female goats.