Isolation of transposon Tn5-induced hydrophobic mutants of a Bradyrhizohium japonicum strain with improved competitive nodulation abilities

Abstract

Hydrophobic mutants of the Bradyrhizohium japonicum strain 138NR were obtained by transposon Tn5 mutagenesis followed by replica-plating on polystyrene plates. Fifteen mutants were isolated at a frequency of 10⁶. Gel-filtration analysis of the exopolysaccharides revealed that the hydrophobic mutants produced a significantly smaller amount of low-molecular-weight polysaccharides than the parent. Four of the isolated mutants formed a larger number of nodules on soybean (Glycine max L. Merr. cv. Tamahomare) than the parent, two were superior in symbiotic nitrogen fixation, and two were symbiotically defective on soybean. Competitive nodulation abilities of the mutants were examined by inoculating them to soybean with B. japonicum strain 123ET as a competitor and determining the nodule occupancy based on the antibiotic resistance. All the mutants tested except for the symbiotically defective ones were superior in their competitive nodulation ability to the parent strain. When inoculated 24 h before the inoculation with 123ET, the mutants exhibited an increased nodule occupancy (44–93% compared to 9% by the parent).     

Corneal Mineralization in Wistar Hannover Rats

We have recently started using Wistar Hannover rats in Japan and are now collecting background data. We have been frequently observing corneal mineralization in Wistar Hannover rats of both the RccHan^TM:WIST and Crl:WI (Han) strains. In this study, details of corneal mineralization in Wistar Hannover rats were histopathologically and ultrastructurally investigated. According to the results, Wistar Hannover rats had a much higher incidence of corneal mineralization compared with Sprague-Dawley rats. The incidence of corneal mineralization was higher in males than females. According to the histological examination, mineral deposits were positive for calcium by von Kossa’s method. Furthermore, in response to mineralization, keratocytes probably become active to play an important role against the mineralized substance.     

Efficient haploid and doubled haploid production from unfertilized ovule culture of gentians (Gentiana spp.)

Factors affecting reliable plant regeneration from unfertilized ovule culture of gentians (Gentiana spp.) were examined. Cold pretreatment (4°C) of flower buds enhanced or maintained production of embryo-like structure (ELS). When 43 genotypes were surveyed in two different labs, 40 of them produced ELSs ranging from 0.01 to 26.5 ELSs per flower bud. No ELSs could be obtained in three genotypes. A significant correlation (r = 0.64) was observed between the number of ELS per flower and the frequency of responding flower buds. Eight genotypes of G. triflora, which were used as common materials in two different labs, produced ELSs in both labs. The ploidy levels of a total of 1,515 regenerated plantlets were determined, revealing that the majority of these plants consisted of haploids (57.9%) and diploids (34.3%). However, the frequency of haploids and diploids was different between G. triflora and G. scabra, and G. triflora showed higher frequencies of haploids than G. scabra. When haploids were treated with oryzalin for chromosome doubling, diploids and tetraploids were obtained. These results demonstrate that the unfertilized ovule culture technique of gentians is a powerful tool for obtaining haploids and DHs because of its reproducible and reliable nature and application to a wide range of genotypes.     

Malignant Mesothelioma in the Thoracic Cavity of a Crj:CD(SD) Rat Characterized by Round Hyalinous Stroma

Spontaneous malignant mesothelioma was found in a 104-week-old male Crj:CD(SD) rat. The tumor was scattered on the surface of the lung, heart, mediastinal pleura and thoracic wall and metastasized to the alveolar septa. Histopathologically, small flattened or cuboidal tumor cells proliferated with stroma, formed almost normal papillary structures and reacted positively to colloidal iron stain and immunohistochemical staining for mesothelin. Round hyalinous stromata were pronounced, which is a characteristic feature, and the possible reason for this is as follows; at first, a small amount of collagen fibers was formed in the center of the clusters of several tumor cells, and then the cell clusters expanded like balloons with an increase in the collagen fibers.     

A case of renal oxalosis in a 3-month-old cat raised under controlled conditions

The kidneys of a 3-month-old female cat were examined. The cat which had been raised under controlled conditions with no history of any poisoning showed progressive weight loss with increases in blood BUN and creatinine concentrations. At necropsy, both kidneys were firm in consistency with formation of focal scars. Histopathologically, widespread deposition of crystals was observed in the renal tubules (in both dilated lumina and degenerative epithelia) accompanying mild interstitial fibrosis with lymphocyte infiltration. The crystals were colorless or basophilic on the hematoxilin and eosin-stained section and could be visualized with polarized light as doubly fractile crystals. The crystals were identified as calcium oxalate crystals by histochemical examinations using von Kossa stain and alizarin red S stain under different conditions and by ultrastructural examination. Judging from the above-mentioned findings, the present renal lesion detected in an infant cat was diagnosed as renal oxalosis which was suspected to be hereditary in nature.     

Chronic cadmium treatment induces islet B cell injury in ovariectomized cynomolgus monkeys

In an attempt to establish a primate model of chronic cadmium (Cd) toxicosis, we ovariectomized cynomolgus monkeys and treated with CdCl2 by repeated intravenous injections for 13 to 15 months. The animals showed an increase in blood glucose from Month 10 and a decrease in blood insulin at Month 11of the Cd-treatment. Histopathological examination of the Cd-treated animals revealed islet atrophy with reduction in islet number and vacuolation of the islet cells, whereas there was no remarkable change in the acinar cells of the exocrine pancreas. In histomorphometrical examination, insulin-positive areas in the islets were significantly decreased, accompanying a relative increase of glucagon-positive areas. Large amounts of Cd accumulated in the pancreas, and metallothionein (MT), a Cd binding protein, was localized in the islets of Cd-treated animals. The present study demonstrated that the chronic intravenous injection of Cd to cynomolgus monkeys induced the accumulation of the metal in the pancreas, degeneration of islet B cells and the diabetic clinical signs. Therefore the islet B cell is one of the major targets of the chronic Cd poisoning in monkeys.     

Residues of aflatoxins in the liver,muscle and eggs of domestic fowls

The residue of aflatoxins in the liver, muscle and eggs of laying ducks, hens and quails and in broiler chickens was examined by conducting 7-day feeding experiments with a diet containing 3 ppm Aflatoxin B1 (AFB1). Birds were sacrificed on the 8th or 11th day of AFB1 feeding. AFB1 and its metabolites in the tissues and eggs were determined by HPLC. The tissue levels of AFB1 and its metabolites were higher in quail than in the other birds. The levels of AFB1 and its metabolites, including acid-hydrolyzable metabolites, were more than 10-fold higher in the liver than in the muscle in all the species. The ratio of AFB1 in the feed to the residual level in the liver was 383 in quail, but was > or = 5769 in the other birds. The corresponding ratios in the egg yolk and albumen of AFB1 were 4615 and 3846, respectively, in chicken hens, and these values were higher than those in the other birds.     

酒制熟地黄炮制过程中梓醇的变化趋势及变化机理

为研究酒制熟地黄炮制工艺过程中梓醇的变化趋势和变化机理，利用HPLC检测酒制熟地黄炮制过程中不同时间点梓醇的含量，并利用FT-IR结合变温附件获取梓醇在不同温度点的红外光谱信息。结果表明，酒制熟地黄炮制过程中，梓醇含量整体呈下降趋势，但不同时间段梓醇含量下降率不同。梓醇含量下降的原因是其热稳定性较低，在温度90～100℃之间，其结构就发生变化。所以，生地黄产地加工焙干时，温度不能太高，以免梓醇含量下降；熟地黄炮制过程中，时间不能太短，以免“火候”不到。