The shelf life of Japanese pear fruit is determined by its level of ethylene production. Relatively high levels of ethylene reduce storage potential and fruit quality. We have identified RFLP markers tightly linked to the locus that determines the rate of ethylene evolution in ripening fruit of the Japanese pear. The study was carried out using sequences of two types of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase genes (PPACS1 and pPPACS2) and a ACC oxidase gene (PPAOX1) as probes on 35 Japanese pear cultivars expressing different levels of ethylene (0.0 to approximately 300 microl/kg fresh weight/h) in ripening fruit. When total DNA was digested with HindIII and probed with pPPACS1, we identified a band of 2.8 kb which was specific to cultivars having very high ethylene levels (> or = 10 microl/kg f.w./h) during fruit ripening. The probe pPPACS2 identified a band of 0.8 kb specific to cultivars with moderate ethylene levels (0.5 microl/kg f.w./h-10 microl/kg f.w./h) during fruit ripening. The cultivars that produce high levels of ethylene possess at least one additional copy of pPPACS1 and those producing moderate levels of ethylene have at least one additional copy of pPPACS2. These results suggest that RFLP analysis with different ACC synthase genes could be useful for predicting the maximum ethylene level during fruit ripening in Japanese pear.

The proteinase responsible for bone collagen degradation in osteo-resorption was examined. The bone pit formation induced by parathyroid hormone (PTH) was markedly suppressed by leupeptin, E-64 and cystatin A, while no inhibition was observed by CA-074, a specific inhibitor of cathepsin B. Pig leucocyte cysteine proteinase inhibitor (PLCPI), a specific inhibitor of cathepsin L, and chymostatin, a selective inhibitor of cathepsin L, completely inhibited the pit formation. Cathepsin L activity in osteoclasts was much higher than the other cathepsin activities. Serum calcium in rats placed on a low calcium diet was decreased by treatment of E-64 or cystatin A, but not by CA-074. These findings suggest that cathepsin L is the main proteinase responsible for bone collagen degradation.

85 Vibrio phages, 84 of them tailed and 1 filamentous, were surveyed. The tailed phages belonged to six basic morphotypes and to the Myoviridae, Siphoviridae, or Podoviridae families. 63 phages were classified into 18 species. The filamentous phage is a member of the Inovirus genus of the Inoviridae family. Vibrio phages are very heterogenous and include some morphologically interesting viruses. Several Vibrio phages closely resemble phages of other gram-negative bacteria, possibly indicating phylogenetic relationships between their hosts.

The presence of regular arrays (RAs) in the cell walls of strains of the genus Lactobacillus was examined by electron microscopy. The RAs were found in 6 species including L. bulgaricus, L. helveticus, L. acidophilus, L. fermentum, L. brevis and L. buchneri. The RAs were composed of a protein with an apparent Mr ranging from about 41000 to 55000, depending on the species upon sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The amino acid composition of the RA proteins was shown to be acidic and hydrophobic. The antigenicity of the RA protein from L. buchneri appeared to be specific but not common among the RA proteins from the other lactobacilli.

A broad-host-range vibriophage, KVP40, was isolated from sea water by using Vibrio parahaemolyticus 1010 (EB101) as the indicator host. The host range of KVP40 extended over at least 8 Vibrio and 1 Photobacterium species. KVP40 was a large tailed phage containing double-stranded DNA and belonged to Ackermann's morphotype A2. KVP40 DNA was cleaved by 11 different type II restriction endonucleases including EcoRI and HindIII, but not by 17 other enzymes including BamHI, Kpnl and Sall. Many vibriophages have been described (1,4,(7)(8)(9)(10)(11)(12)16). Most of them are for Vibrio cholerae, some for V. parahaemolyticus, and a few for other Vibrio species. These phages are generally specific for a single Vibrio species on which the individual phage was isolated. This report describes a unique vibriophage isolated from sea water, which showed a relatively broad host specificity for species of family Vibrionaceae. This phage, designated KVP40, belongs to a rather rare morphotype A2 according to Ackermann's classification (1).Bacterial strains, for which the host range of KVP40 was determined, are listed in Table 1. The following culture media were used for experiments. YPH, YP, and YPL media (pH 8.0) contained 0.3% Yeast Extract (Difco Lab., Detroit, Mich., U.S.A.) and 1% Polypepton (Daigo Eiyo-Kagaku, Osaka, Japan) as basal constituents, and were supplemented with 5, 3, and 0.2% NaCl, respectively. Two percent agar was added to the media for solid culture, but 0.5% for soft agar media. Phage KVP40 was isolated from a sample of highly polluted sea water collected at Urado Bay, Kochi, Japan, with V. parahaemolyticus 1010 as the host. Phage lysates (ca. 109 PFU/ml) were prepared by growing strain 1010 with KVP40 in YP medium at 30 C until lysis occurred. Plaque formation of KVP40 on various bacterial species was examined by either the conventional soft-agar-overlay method or the spot test using YP agar medium with the exception that plaque formation on V. costicola was determined with YPH agar. In some experiments, YPL agar was also used. Plates were incubated for 20 hr at 30 C.Host range of phage KVP40 is shown in

The S layer of Cfostridium di,,cife GAI 0714 was shown to be composed of two proteins, of 32 kDa and 45 kDa, as determined by SDS-PAGE. The two proteins were extracted with 8 M-urea (pH 8-3) from a cell wall preparation and purified by DEAE-Sepharose CL-6B chromatography followed by HPLC gel filtration. When solubilized in 0.1 M-urea, both proteins appeared to exhibit dimeric forms, with respective molecular masses o f about 61 kDa and 99 kDa, upon HPLC. Although the amino acid compositions of the two proteins differed from each other, both proteins had a high content of acidic amino acids, very low contents of histidine and methionine, and no cysteine.The 32 kDa protein exhibited multiple isoelectric forms (PI 3-7-3.9), whereas the 45 kDa protein had a single form (PI 3.3). Radioiodination and immunogold labelling revealed that both proteins were exposed evenly over the entire cell surface. Based on immunodiffusion analysis using monospecific antiserum raised to the individual proteins, there was no antigenic relationship between the two proteins. Furthermore, immunoblot analysis showed that the antigenicity of the 32 kDa protein appeared to be strain specific, whereas that of the 45 kDa protein appeared to be group specific.

The outer sheath carrying a polygonal array was isolated from an oral treponeme, Treponema sp. strain E-21, by disruption of cells by means of repeated freeze-thawing and by removal of flagella under acidic conditions followed by linear sucrose density gradient centrifugation. Electron microscopy revealed that the outer sheath was isolated as a triple-layered vesicle having a polygonal array, free of flagella and wall membrane complex. Using optical diffrction, negatively stained preparations of the outer sheath fiagments showed that the polygonal array appeared to be composed of a hexagonal pattern with a predominant spacing of about 16.3 nm. The isolated outer sheath contained 49.7% protein, 30.8% total lipid, and 11.0% carbohydrate. Phospholipid comprised about 95% of the total lipid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the outer sheath was composed primarily of one major protein with an apparent molecular weight of about 62,000. The material from the isolated outer sheath solubilized with 1% sodium deoxycholate was reassembled into vesicles having a roughly polygonal array upon removal of the detergent by dialysis against 10 mM Tris-hydrochloride buffer with or without Mg2+.Spirochetes possess an outer sheath which surrounds the coiled protoplasmic cylinder and flagella (axial filaments) (6, 12). Adequate information concerning the ultrastructural organzation, as well as the chemical composition and physicochemical nature, is meager for an accurate assessment of its function. Johnson and his colleagues isolated the outer sheath from some species of Leptospira (2), Treponema (13, 32), and Borrelia (17) by using the procedure involving solubilization of the outer sheath with low concentrations of sodium dodecyl sulfate (SDS). The outer sheath was also isolated and purified from Leptospira interrogans by exposure to osmotic shock in a hypotonic buffer followed by subsequent centrifugation in KBr and CsCl density gradients (36).In some species of Spirochaeta and Treponema, the outer sheath consists of a morphologically complex layer of polygonally arranged subunits (6). The presence of a polygonal array in the outer sheath was also revealed in several cultivable treponemes by Hovind-Hougen (7-9). However, the chemistry and morphological organization of the polygonally arranged subunits and the specific interaction between the subunits and the other components of the outer sheath are unknown. Treponema sp. strain E-21, isolated from a human oral cavity with pyorrhea (29), has been found to possess an outer sheath carrying a reticular structure (33). In this paper, we describe a procedure for the isolation of the outer sheath from an oral treponeme. Morphological and chemical properties, as well as reassembly of the isolated outer sheath, are also presented.MATERIALS AND METHODS Orpnim and growth. Treponema sp. strain E-21, which was isolated from the oral pus of a patient with pyorrhea (29) and kindly supplied

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