Seroepidemiology of human parechovirus types 1, 3, and 6 in Yamagata,Japan, in 2014

To clarify the seroepidemiology of human parechovirus type 1 (HPeV1), 3 and 6, neutralizing antibodies (NT Abs) were measured in 214 serum specimens collected in 2014 in Yamagata, Japan. The seroprevalence against HPeV1 was 100% in all age groups, while that against HPeV3 and HPeV6 was 79.4% and 66.8%, respectively, overall. The geometric mean titers of NT Abs against HPeV1, 3 and 6 were 755.2, 255.0 and 55.9, respectively, overall. Our findings indicate that HPeV1 is the most prevalent HPeV circulating in Yamagata, followed by HPeV3 and HPeV6.     

Deterioration of Antarctic Krill Muscle during Freeze Storage

The effects of freezing on the heat-induced gelation, Ca²⁺-ATPase activity and myofibril structure of Antarctic krill muscle were investigated. Muscle from freshly caught krill was immediately stored at-20°C in the presence (to prevent freezing) (glycerol krill) and absence (frozen krill) of glycerol. Several protease inhibitors, monosodium glutamate and Ca²⁺ were individually added to glycerol krill to inhibit endogenous proteolysis. The examinations described above were carried out after about 3-month storage at-20°C. In glycerol krill (unfrozen state), viscoelastic parameters of the heat-induced gels and Ca²⁺-ATPase activities of all the krill samples were similar to those of “surimi” (raw fish meat paste) of Alaska pollack which gave gel of good quality, although the micro structure (Z-lines) of myofibrils was different among the glycerol krill samples. In frozen krill, however, the parameters of the gel were different from those of “surimi”, the ATPase activity was completely lost and disruption of the myofibril structure occurred. Refreezing (-20°C) of glycerol krill after removal of glycerol resulted in a marked decrease in the gelation ability. These results suggest that freezing of krill muscle causes deterioration of the gelation ability.     

Classification of Proteases in Antarctic Krill

The species of endogenous proteases in Antarctic krill, Euphausia superba, were investigated using the homogenate of krill and the active fractions after gel filtration of the homogenate as to the following criteria: Substrate specificity (benzyloxycarbonyl (Z)-Phe-Ala, Z-Glu-Tyr, hippuryl-Arg, hippuryl-Phe, benzoyl Arg-p-nitroanilide, Leu-p-nitroanilide, ¹⁴C-hemoglobin), sensitivity to protease inhibitors (diisopropyl fluorophosphate, iodoacetamide, EDTA, soybean trypsin inhibitor and pepstatin), molecular weight and isoelectric point.

From the experimental results, we found that the carboxypeptidase A and B, aminopeptidase, trypsin and cathepsin A types of proteases were present in krill.     

Activation of Rice Aminopeptidase by Anions and Some Properties of the Enzyme

In order to determine which proteases are responsible for the autolysis of krill, the effects of several protease inhibitors on the autolysis and protease activities of krill were investigated.

Homogenates of whole bodies, and the cephalothorax and abdomen parts of frozen krill were equilibrated at 37°C at different pHs between 2 to 10 and allowed to stand for 16 hr, following which the increase in the TCA soluble fraction was monitored. ¹⁴C-Hemoglobin (¹⁴C-Hb) hydrolyzing activity was also measured using each homogenate as a crude enzyme preparation. The degree of autolysis and the ¹⁴C-Hb hydrolyzing activity were maximum at pH 5 ~ 8 for the parts studied. The hydrolytic activity was highest in the cephalothorax, followed by that in the whole body and then the abdomen.

The effects of inhibitors on the ¹⁴C-Hb hydrolyzing activity were examined, and it was seen that soybean trypsin inhibitor (STI), diisopropyl fluorophosphate (DFP) and leupeptin significantly inhibited the activity at neutral pH, and pepstatin, monoiodoacetic acid (IAAcid) and leupeptin were effective at acidic pH for all the parts. Investigation of the effects of inhibitors on the autolysis at 20°C at pH 4 and 7 by SDS–polyacrylamide gel electrophoresis indicated that the autolysis of the cephalothorax and whole body at pH 7 was suppressed a little by STI and the autolysis of the abdomen and whole body at pH 4 was significantly inhibited by iodoacetamide (IAA) and leupeptin.

These results suggest that the main proteases responsible for the autolysis of krill are trypsin like-proteases at neutral pH and cathepsins (B, H and L types) at acidic pH.     

Tissue-specific and non-tissue-specific heavy-chain isoforms of myosin in the brain as revealed by monoclonal antibodies.

Four types of monoclonal antibody (BM-1, BM-2, BM-3 and BM-4) each having distinctive tissue specificity were obtained by immunizing mice with purified bovine cerebrum myosin. Both BM-1 and BM-2 reacted most efficiently with cerebrum myosin and less efficiently with myosins from other limited nonmuscle tissues, the tissue specificity of BM-1 being much narrower than that of BM-2. BM-3 reacted more efficiently with several other nonmuscle myosins than with cerebellar or cerebral myosin. BM-4 recognized various nonmuscle and smooth muscle myosins with a nearly equal efficiency. Cerebral myosin as well a cerebellar myosin contained two or more electrophoretic variants of the heavy chains. BM-1 and BM-3 as well as BM-2 and BM-3 were found to recognize selectively these distinct heavy-chain isoforms. The antigenic sites of the three tissue-specific antibodies (BM-1, BM-2 and BM-3) were all localized near the head/tail junction of the myosin molecules, while that of non-tissue-specific antibody BM-4 was near the center of the tail. These and additional results indicate that mammalian brain tissues as well as several other nonmuscle tissues contain multiple heavy-chain isoforms of myosin, the levels of which differed considerably from one tissue to another.     

Characterization of reconstituted high-density lipoprotein particles formed by lipid interactions with human serum amyloid A

The acute-phase human protein serum amyloid A (SAA) is enriched in high-density lipoprotein (HDL) in patients with inflammatory diseases. Compared with normal HDL containing apolipoprotein A-I, which is the principal protein component, characteristics of acute-phase HDL containing SAA remain largely undefined. In the present study, we examined the physicochemical properties of reconstituted HDL (rHDL) particles formed by lipid interactions with SAA. Fluorescence and circular dichroism measurements revealed that although SAA was unstructured at physiological temperature, α-helix formation was induced upon binding to phospholipid vesicles. SAA also formed rHDL particles by solubilizing phospholipid vesicles through mechanisms that are common to other exchangeable apolipoproteins. Dynamic light scattering and nondenaturing gradient gel electrophoresis analyses of rHDL after gel filtration revealed particle sizes of approximately 10 nm, and a discoidal shape was verified by transmission electron microscopy. Thermal denaturation experiments indicated that SAA molecules in rHDL retained α-helical conformations at 37 °C, but were almost completely denatured around 60 °C. Furthermore, trypsin digestion experiments showed that lipid binding rendered SAA molecules resistant to protein degradation. In humans, three major SAA1 isoforms (SAA1.1, 1.3, and 1.5) are known. Although these isoforms have different amino acids at residues 52 and 57, no major differences in physicochemical properties between rHDL particles resulting from lipid interactions with SAA isoforms have been found. The present data provide useful insights into the effects of SAA enrichment on the physicochemical properties of HDL.     

Expression profile of active genes in human periodontal ligament and isolation of PLAP-1, a novel SLRP family gene

Periodontal ligament (PDL) is one of the most important tissues in maintaining the homeostasis of tooth and tooth-supporting tissue, periodontium. In this study, we investigated the expression profile of active genes in the human PDL obtained by collecting sequences with a 3'-directed cDNA library, which faithfully represents the composition of the mRNA population. We succeeded in obtaining a total of 1752 cDNA sequences by sequencing randomly selected clones and identified a total of 1318 different species as gene signatures (GS) by their sequence identity, 344 of which were known genes in the GenBank, and 974 of which were new genes. The resulting expression profile showed that collagen type I and type III were the most abundant genes and that osteogenesis-related proteins, such as SPARC/osteonectin and osteoblast specific factor 2, were highly expressed. By comparing the expression profile of PDL with 44 profiles similarly obtained with unrelated human cell/tissue, nine novel genes, which are probably expressed specifically in PDL, were discovered. Among them, we cloned a full-length cDNA of GS5096, which is frequently expressed in freshly-isolated periodontal tissue. We found that it encodes a novel protein, which is a new member of the class I small leucine-rich repeat proteoglycan family, and designated it PLAP-1 (periodontal ligament associated protein-1). PLAP-1 mRNA expression was confirmed in in vitro-maintained PDL cells and was enhanced during the course of the cytodifferentiation of the PDL cells into mineralized tissue-forming cells such as osteoblasts and cementoblasts. These findings suggest the involvement of PLAP-1 in the mineralized matrix formation in PDL tissues.     

Rho-kinase mediates TNF-α-induced MCP-1 expression via p38 MAPK signaling pathway in mesangial cells

Macrophage accumulation has been implicated in the pathogenesis of inflammatory glomerular disease. Monocyte chemoattractant protein-1 (MCP-1) plays a central role in recruiting monocytes to the glomeruli. Tumor necrosis factor-α (TNF-α) has been shown to induce MCP-1 expression in mesangial cells, although the precise mechanisms remain unclear. We previously demonstrated that RhoA and its effector, Rho-kinase (Rho-associated coiled-coil containing protein kinase, ROCK), are involved in the pathogenesis of diabetic nephropathy. However, its role in MCP-1 induction by TNF-α has not been elucidated. In the present study, we investigated whether the Rho/Rho-kinase signaling pathway regulates the TNF-α-mediated induction of MCP-1 in mesangial cells. Exposure of mouse mesangial cells (MES-13) to TNF-α resulted in an increase of MCP-1 expression (by RT-PCR) and secretion into the medium (by ELISA). Pull down and Western blot analysis revealed that TNF-α activated RhoA and Rho-kinase. Based on these observations, we speculated that the Rho/Rho-kinase signaling pathway may be involved in MCP-1 induction by TNF-α. In agreement with this concept, Y-27632, a specific Rho-kinase inhibitor, attenuated TNF-α-mediated induction of MCP-1. We demonstrated that Y-27632 inhibited TNF-α-mediated monocyte migration and attenuated TNF-α-mediated p38 MAPK activation. Based on these data we infer that Y-27632 inhibits TNF-α-induced MCP-1 expression, secretion and function through inhibition of Rho-kinase and p38 MAPK activity. Our study suggests that Rho/Rho-kinase is an important therapeutic target of monocyte recruitment and accumulation within the glomerulus in inflammatory renal disease.     

Efficient production of offspring from Japanese wild-derived strains of mice (Mus musculus molossinus) by improved assisted reproductive technologies

Because the genetic diversity of the laboratory mouse (Mus musculus) is very limited, wild-derived strains from this genus could provide invaluable experimental models for studies of mouse genetics and epigenetics such as quantitative trait locus analysis. However, such strains generally show poor reproductive performance under conventional husbandry conditions, so their use for large-scale analyses has been limited. This study was undertaken to devise assisted reproductive technologies (ARTs) for the efficient production of offspring in two wild-derived strains, MSM/Ms and JF1/Ms (Mus musculus molossinus). First, as females of these strains are poor responders to equine chorionic gonadotropin (eCG) stimulation, we examined the efficiency of superovulation by injecting anti-inhibin serum followed by human chorionic gonadotropin (hCG). Approximately four to six times more oocytes were ovulated than with eCG-hCG treatment in both strains, reaching ～25-30 oocytes per female. Consequently, the procedures for in vitro fertilization using these superovulated oocytes and cryopreservation of embryos and spermatozoa could be optimized for both of the wild-derived strains. However, MSM/Ms embryos but not JF1/Ms embryos failed to develop to term after embryo transfer because of intrauterine death at mid to late gestation. We were able to overcome this obstacle by cotransfer of these embryos with those from laboratory strains combined with treatment of recipient females with an immunosuppressant (cyclosporin A). Thus, a series of ARTs essential for efficient production and preservation of the wild-derived strains were successfully devised. These technologies will facilitate systematic studies of mouse genetics and epigenetics using a wider range of genetic diversity than currently available in the genus Mus.