Isolation and characterization of aminopeptidase (Jc-peptidase) from Japanese cedar pollen (Cryptomeria japonica)

An aminopeptidase, Jc-peptidase, was purified from Japanese cedar pollen by seven steps, including precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration, hydrophobic interaction chromatography on phenyl-agarose, and high-performance liquid chromatography. Purified Jc-peptidease has a molecular weight of 42 kDa and hydrolyzes the synthetic substrates of L-phenylalanyl-4-methylcoumaryl-7-amide (Phe-MCA) with Km = 5 x 10(-5) M, Tyr-MCA with Km = 7 x 10(-4) M, Leu-MCA with Km = 1 x 10(-3) M, and Met-MCA with Km = 1 x 10(-3) M. Other MCA analogues such as Arg-MCA or Glu-MCA failed to serve as its substrates. The activity was inhibited in the presence of phebestin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl]-L-phenylalanine, with Ki = 4.7 x 10(-5) M, or bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, with Ki = 1.1 x 10(-4) M. According to amino acid sequence analysis, the N-terminal amino group seems to be blocked. The physiological function of the aminopeptidase (Jc-peptidase) has not been clarified in vivo.     

Improvement of the emulsifying properties of porcine sarcoplasmic proteins with a low-molecular weight surfactant by the two-step emulsification method

The emulsifying properties of porcine sarcoplasmic protein (SP) with or without low-molecular weight surfactants were examined. The emulsifying activity (EA) of SP was weaker than that of sodium caseinate (CA), blood plasma (BP) and whey protein isolates at a protein concentration of 0.5–3%. Sodium chloride decreased the EA of SP. The EA of SP was about one quarter that of CA in the presence of 3% sodium chloride. As homogenization time increased, the EAs of SP and BP decreased. The EA of SP was not influenced by sucrose stearate ester (SE) with a hydrophilic-lipophilic balance (HLB) value of 16 (SE-16) but was decreased by those with HLB values of 5 and 1. Low-molecular weight surfactants such as SE-16, Tween 80 and sodium cholate improved the EA of SP with the two-step emulsification method: corn oil was previously emulsified with the surfactant before addition of SP and homogenization. In the case of the addition of SE-16 as primary emulsifier, the EA was about twice as high as that of SP alone. The same effect was shown in the emulsions formed by CA and soy protein isolate. These results indicate that the emulsifying properties of meat protein would be improved by the addition of a low-molecular weight surfactant in a two-step emulsification.     

Humoral immune responses of cats to feline infectious peritonitis virus infection.

Immunoperoxidase antibody (IPA) method as a titrating method of feline infectious peritonitis (FIP) virus (FIPV) was developed for titrating antibody to FIPV (IPA-titer). By this method the immune responses of the cats that had been infected with FIPV, were traced. The infected cats could be grouped into three types by their immune response to FIPV and clinical appearances. Type I cats lived for a long time, formed a major group among infected cats, had 160 to 1 x 10(4) IPA-titers, and showed healthy appearances without any changes both on autopsy and histopathologically. From among type I cats, type II cats appeared sporadically with rapid elevation of IPA titers to 3.2 x 10(5) and showing clinical signs of FIP, and died. Type III cats lived healthily for a long time with gradual elevation of IPA-titers to a plateau of about 1 x 10(5), then showed neuronal disorder of hind leg paralysis with the descending IPA-titers to 2 x 10(4), and died. Thus, typical FIP appeared as a hyper-immune disease. Other related problems are discussed.     

Pregnancy and lactation affect the microvasculature of the mammary gland in mice.

Microvascular changes in the mammary gland in mice during pregnancy and lactation were investigated by scanning electron microscopy (SEM) with a corrosion cast method, transmission electron microscopy (TEM) and morphometry. By SEM, duct-associated capillary plexuses were sparsely distributed to branch into adipocytes during virgin period. With advance in pregnancy, both branches from the capillary plexuses and branches from the vessels surrounding adipocytes extended further to form capillary networks. The basket-like architecture was completed by day 18 of pregnancy. These findings may indicate that angiogenesis occurs frequently during this period. During lactation, the basket-like architecture still remained and the capillaries surrounding alveoli meandered. After weaning, the regression of microvasculature followed the degeneration of alveoli. By TEM and morphometry, the density of pinocytotic vesicles (PVs) (number of PVs per microns 2 of endothelium cytoplasm) increased twofold from day 18 of pregnancy to day 5 of lactation, furthermore increased threefold from days 10 to 20 of lactation, and subsequently decreased after weaning. Marginal folds and microvillous processes gradually increased in length with advance in pregnancy, reached the maximum from days 5 to 15 of lactation, and thereafter decreased. In addition, the capillaries with thinner walls were in close contact with alveoli during the late stage of pregnancy and during lactation. Furthermore, the alveolar epithelial cells had well-developed basal infoldings during lactation. These findings suggest that the capillaries play an important role in transporting materials necessary for milk production.     

Fine surface structure of bovine acrosome-intact and reacted spermatozoa observed by atomic force microscopy

The atomic force microscope (AFM) provides nanometer resolution, topographic data of the natural surface structure of materials. We studied the topology of the surface structure of bovine sperm heads during the acrosome reaction by AFM. In addition, we numerically analyzed the areas of the median sagittal plane of the sperm heads. Bovine frozen-thawed spermatozoa were washed, capacitated by heparin, and incubated with lysophosphatidylcholine (LPC) to induce the acrosome reaction, smeared on a cover glass, air-dried, and observed with AFM using the dynamic force (tapping) mode. AFM analysis of spermatozoa showed the clear surface structure of acrosomes, equatorial segments, postacrosomal regions and necks. Although AFM images of spermatozoa capacitated by heparin had complete acrosomes, most spermatozoa treated with LPC had no acrosomal caps as shown by AFM. These observations coincided with those obtained by light microscopy after staining with naphthol yellow S and erythrosin B. Furthermore, numerical analysis of AFM images indicated that areas of the median sagittal plane of the anterior portions of acrosome-reacted sperm heads (2679 +/- 616 pixels) were approximately 40% less than those of intact heads (4535 +/- 174 pixels, P<0.05). These results indicate that AFM can usefully observe and numerically analyze the fine surface structures of bovine spermatozoa.     

Epidemiological study on periodontal diseases and some other dental disorders in dogs

The prevalence of dental disorders in dogs was studied by applying index systems for human with some modifications. A total of 251 mongrel dogs including 143 stray dogs kept in the Animal Protection Offices in Tokyo and Hokkaido and 108 pet dogs visiting veterinary clinicians in Chiba Prefecture and Hokkaido were used. Periodontitis was prevalent among these dogs regardless of their sources and its incidence was increased with age. The lesion was more severe and more frequent in the premolar and molar regions than in the maxillary and mandibular incisor regions. Missing of teeth was observed at a high and increasing incidence with age. The tooth most commonly lost was the first premolar, followed by the other premolars and molars, where severe periodontitis was frequently found. Calculus was seen on many teeth, and aging agravated its prevalence and severity. Dental caries was observed in stray dogs, but neither to a serious degree nor at a significant level. These findings emphasize the necessity of dental hygiene, proper dental care and continuous periodical survey for dogs.