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To effectively utilize the lizardfish viscera generated as a by-product of surimi manufacturing, the properties of endogenous proteases in the viscera were investigated. Two protease activity groups were identified from lizardfish viscera, and their optimal temperature and pH were 55oC and 7.0, and 45oC and 11.0, respectively. The results of protease inhibitory and fluorogenic substrate specificity assays showed that both dominant proteases belong to the serine protease family. Also, the effects of NaCl on the catalytic activity and thermal stability of proteases from viscera were clarified. The catalytic activity of protease would increase after incubation in NaCl solutions (5-25%) for 30 days and the thermal stability of proteases was also enhanced markedly in 15% NaCl solution. All the results demonstrated the salt-dependent behavior of proteases in NaCl solution and that NaCl-dependent changes in catalytic activity and thermal stability have potential applications for the production of fermented food. [ABSTRACT FROM AUTHOR]

The efficacy of porcine recombinant interleukin-12 (prIL-12) as an adjuvant to stimulate immunity was investigated in pigs vaccinated against porcine reproductive and respiratory syndrome virus (PRRSV). Porcine recombinant IL-12 was cloned and expressed in the insect baculovirus system. Thirty pigs were randomly allocated to five groups, and individual pigs were injected with PRRSV modified live virus with and without coadministration of prIL2; PRRSV killed virus with and without coadministration of prIL-12; or prIL-12 only (control). The effect of prIL-12 was evaluated through monitoring of humoral and cell-mediated immune (CMI) responses. Results indicated that the killed virus given in conjunction with prlL-12 induced humoral and CMI responses of similar magnitude to those of the modified live virus. However, without prIL-12, the immunologic response to vaccination with the killed virus vaccine was poor and similar to that of controls.

A novel myofibril-bound serine proteinase inhibitor (MBSPI) was purified to homogeneity from the skeletal muscle of lizard fish (Saurida wanieso). Purification was carried out by ammonium sulfate fractionation, followed by column chromatographies on DEAE-Sephacel, SP-Sepharose and Sephadex G-150. MBSPI was purified 7.7-fold starting from the DEAE-Sephacel fraction, with a yield of 0.2%. It is a monomeric protein with the molecular mass of 50 kDa as estimated by SDS-PAGE and gel filtration. MBSPI reveals high inhibition specificity toward a myofibril-bound serine proteinase (MBSP) purified from lizard fish muscle. No inhibition is detected toward bovine trypsin, bovine chymotrypsin, two trypsins from carp hepatopancreas and a serine proteinase isolated from the sarcoplasmic fraction of white croaker muscle. It does not exert any inhibitory activity toward a myofibril-bound serine proteinase from carp muscle.

A novel serine proteinase inhibitor has been purified to homogeneity from the skeletal muscle of white croaker (Argyrosomus argentatus). The purification was carried out by ammonium sulfate fractionation, DEAE-Sephacel, heating treatment followed by column chromatographies on SP-Sepharose, Sephadex G-150 and gel-filtration high performance liquid chromatography. The molecular mass of the inhibitor was 55 kDa as estimated by SDS-PAGE and gel filtration. It specifically inhibited a myofibril-bound serine proteinase (MBSP) isolated from the skeletal muscle of lizard fish (Saurida wanieso). No inhibition, however, was detected toward other serine proteinases such as bovine trypsin, bovine chymotrypsin and a myofibril-bound serine proteinase from carp (Cyprinus carpio) muscle. Interestingly, the sequences of tryptic digested peptide fragments of MBSPI revealed high identity to that of porcine phosphoglucose isomerase (PGI) (76%) and other PGIs. Furthermore, purified MBSPI exhibits PGI activity, suggesting the inhibitor is a protein closely related to PGI. When rabbit muscle PGI was investigated, it also specifically suppressed the activity of MBSP. It thus strongly suggests that MBSPI is actually PGI and conversely, PGI is a specific inhibitor toward myofibril-bound serine proteinase(s)., (Copyright 2000 Academic Press.)

A myofibril-bound serine proteinase (MBSP) from the skeletal muscle of lizard fish (Saurida wanieso) was purified to homogeneity by a heating treatment followed by a series of column chromatographies on DEAE-Sephacel, Sephacryl S-200, Q-Sepharose, Hydroxyapatite and Benzamidine-Sepharose 6B, and characterized enzymatically. On SDS-poly-acrylamide gel electrophoresis (SDS-PAGE), the purified enzyme showed a band with molecular mass of approximately 29 kDa under reducing conditions, while 60 kDa under non-reducing conditions. The optimum temperature of the enzyme was 50 degrees C using t-butyloxycarbonyl-Phe-Ser-Arg-4-methylcoumaryl-7-amide (Boc-Phe-Ser-Arg-MCA) as a substrate. Substrate specificity analysis both using MCA-substrates and peptides showed that MBSP specifically cleaved at the carboxyl side of the arginine residue. Inhibitor susceptibility analysis revealed that MBSP was inhibited effectively by Pefabloc SC, soybean trypsin inhibitor (STI) and aprotinin, indicating the characteristic of a serine proteinase. When myofibril was incubated with the enzyme, it optically degraded myosin heavy chain at 55-60 degrees C, while alpha-actinin and actin were not at all hydrolyzed as detected by immunoblotting. The N-terminal amino acid sequence of MBSP was partially determined as IVGGAEXVPY- and was very homologous to other serine proteases.

Previously, we reported the purification and characterization of a myofibril-bound serine proteinase (MBP) from carp muscle (Osatomi K, Sasai H, Cao M-J, Hara K, Ishihara T. Comp Biochem Physiol 1997;116B:159-66). In the present study, the N-terminal amino acid sequence of the enzyme was determined, which showed high identity with those of other trypsin-like serine proteases. The cleavage specificity of MBP for dibasic and monobasic residues was investigated using various fluorogenic substrates and peptides. Analyses of the cleaved peptide products showed that the enzyme hydrolyzed peptides both at monobasic and dibasic amino acid residues. Monobasic amino acid residues were hydrolyzed at the carboxyl side; dibasic residues were cleaved either at the carboxyl side of the pair or between the two basic residues and the enzyme showed a cleavage preference for the Arg-Arg pair. Unexpectedly, MBP hydrolyzed lysyl-bradykinin and methionyl-lysyl-bradykinin at the carboxyl side of Gly fairly specifically and efficiently displaying a unique cleavage. Because MBP also degraded protein substrates such as casein and myofibrillar proteins, the substrate specificity of MBP appeared not to be strictly specific.

In order to solve the loosening problem of endoprosthesis, a titanium-based alloy bone and joint prosthesis with plasma-sprayed ceramic coating was developed by the authors. By the fabrication powdered alumina or zirconium oxide were melted in an electric arc by a temperature of 10,000 K and sprayed onto the metallic prosthesis. The coating was 0.3 to 0.6 mm in thickness and had innumerable micropores. The pores were 50 to 200 microns in diameter. The metallic bone and joint prosthesis with plasma-sprayed ceramic coating exhibits characteristics of metal and ceramic, such as high mechanical strength, high resistance to corrosion and elution and good biocompatibility. According to our experimental study, the plasma-sprayed ceramic coating allows tissue ingrowth into its micropores and prevents the loosening of the prosthesis. Successful cases have been experienced through 10 years clinical application.

The authors developed a titanium-based alloy artificial total femur incorporating its total hip and total knee joints with plasma-sprayed ceramic coating 0.3 to 0.6 mm in thickness and a myriad of micropores of 50-200 microns in diameter. On the basis of reliable data obtained in the animal experiments and physical testing, the prosthesis was applied clinically with satisfactory results. This paper reports the results of two patients with a follow-up of 42-70 months.

A method of prosthetic replacement using total femur incorporating its total hip and knee joints is reported. On the surface of the titanium endoprosthesis was plasma-sprayed a ceramic coating to prevent loosening of the prosthesis. It can preserve the limb and restore the function in a short period. It can be used in certain non-malignant tumours and diseases of the bone with extensively destructive lesions of the entire femur. This method was used in 3 patients with good results. The operated patients were followed-up for 32-70 (mean 54) months. Types of procedures and advantages of the operation are presented.

A method of prosthetic replacement using total femur with total hip and knee joints is reported. On the surface of the titanium endoprosthesis a ceramic coating was plasma-sprayed to prevent loosening of the prosthesis. By this method the limb can be preserved and the function restored within a short period. It can be used in certain cases of non-malignant tumors and diseases of the bone with extensive destructive lesions of the entire femur. This method was used on 3 patients with good results. The patients operated on were followed up for 32-70 (mean 54) months. The procedures and advantages of the operation are presented.