Involvement of NbNOA1 in NO production and defense responses in INF1-treated Nicotiana benthamiana

During defense responses, plant cells produce nitric oxide (NO), which may control many physiological processes. In a previous study, we reported that nitrate reductase (NR) is responsible in part for INF1 elicitor-induced NO production in Nicotiana benthamiana, but the possibility remains that other NO-generating system(s) contribute to NO production. In mammalian cells, NO production is catalyzed by NO synthase (NOS). However, NOS-like enzyme(s) have never been identified in plants, and only the gene for Arabidopsis thaliana nitric oxide-associated 1 (AtNOA1) has been identified as a putative regulator of NOS activity in plants. In this study, we cloned NbNOA1, a homolog of AtNOA1, from N. benthamiana and investigated its involvement in NO production induced by INF1. The NbNOA1 gene was silenced by a virus-induced gene-silencing (VIGS) technique. NbNOA1-silenced plants had yellowish leaves. Silencing NbNOA1 partially decreased INF1-induced NO production, while overexpressing NbNOA1 did not affect NO production. Silencing NbNOA1 suppressed INF1-induced PR1a gene expression and increased susceptibility to Colletotrichum lagenarium. These results suggest that NbNOA1 is involved in INF1-mediated NO production and is required for defense responses. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB303300.     

Isolation and characterization of macrophages from a mixed primary culture of bovine liver cells

Previously, we developed a simple and efficient method to isolate liver macrophages from a mixed primary culture of adult rat liver cells. To extend the applicability of this method, we isolated macrophages from mixed primary cultures of bovine liver cells. Macrophage cells proliferated on the cell sheet of mixed bovine liver cells after 8-16d of culture. These cells were detached by shaking of the culture flasks. Subsequent transfer and brief incubation in plastic dishes resulted in selective adhesion of macrophages. After rinses with PBS, attached macrophages were harvested. More than 10(6) cells could be harvested from the culture flask at intervals of 2-3d for more than three weeks. The isolated cells were strongly positive for bovine macrophage markers, such as CD68, CD172a and Iba-1. These cells exhibited functional properties of macrophages, including active phagocytosis of polystyrene microbeads, proliferative response to recombinant bovine granulocyte-macrophage colony-stimulating factor, upregulation of specific inflammatory cytokine genes upon stimulation with lipopolysaccharide, and formation of multinucleated giant cells. The shaking and attachment method provides a simple and efficient alternative to obtain bovine liver macrophages without requiring complex equipment or specialized technical skills.     

Expression of nestin and IGF-1 in rat pancreas after streptozotocin administration

The present study examines whether centroacinar (CA) and intercalated duct (ICD) cells can serve as stem cells, after administration of the diabetogenic agent streptozotocin (STZ). Thirty rats were divided into five experimental groups: (1) control, (2) 1 day after STZ (STZ-1), (3) 3 days after STZ (STZ-3), (4) 7 days after STZ (STZ-7) and (5) 14 days after STZ (STZ-14). Many small pancreatic islets were observed in the STZ-7 group than in the other experimental groups, and many of these small islets were in close contact with ICD and CA cells. A higher number of nestin, insulin-like growth factor-1 (IGF-1) and IGF-1-receptor positive ICD and CA cells were observed at STZ-3 and STZ-7 than at the others. These expression patterns coincided well with the proliferating cell nuclear antigen pattern. The results suggest that rat pancreatic endocrine cells after damage by STZ administration might be recovered from newly generated cells derived from ICD and CA cells.     

A cat with acute myeloblastic leukemia without maturation (M1) treated with combination chemotherapy

A 2-year-old domestic shorthair cat was presented to us with decreased activity and anorexia. Hematologic findings revealed a mild non-regenerative anemia, thrombocytopenia, and leukocytosis with an increase in blast cells. Bone marrow aspirates also revealed a marked increase of blasts. The blastic cells were shown to be positive for peroxidase. Acute myeloblastic leukemia without maturation (M1) was diagnosed according to the FAB classification. Chemotherapy was initiated with cyclophosphamide, vincristine, prednisolone, and cytosine arabinoside. The cat responded partially. In total, the cats were given 7 blood transfusions. The cat died 14 weeks after first being presented to us.     

Composites of wood and trans-1,4-isoprene rubber II: Processing conditions for production of the composites

 The occurrence of grafting between WF and MTIR through esterification, the processing conditions for production of the composites, and the effect of MAH concentration on some physical and mechanical properties as well as the biodegradability of the composites have been studied. The FTIR measurement demonstrated that grafting through esterification had been attained between OH-groups of WF and acid anhydride groups of MTIR during kneading. The optimum kneading conditions for preparing the composites were 120 °C, 30 ∼ 90 rpm for 10 min. The tensile properties of the composites with WF based filler are greater than those with CS (corn starch) based filler. Addition of 10% of MTIR•10 and 8% MAH prepared using the two-step and one-step process, respectively to the TIR-WF composites gave the best result of tensile properties. The presence of MAH in the TIR-WF composites also improved the dimensional stabilization of the resulting composites. The tensile strengh of the composites prepared using the one-step process was about 90% of the composite prepared using the two-step process. The composites of TIR-WF with MTIR compatibilizer had slightly less resistance against subterranean termite (C. formasanus), brown-rot fungus (T. palustris), and white-rot fungus (C. versicolor) and slightly more resistance against bacteria (Bacillus sp.), within the experimental conditioned adopted. Received 8 February 1999     

ESR studies of radicals generated by ultrasonic irradiation of lignin solution. An application of the spin trapping method

 An electron spin resonance (ESR) method combined with a spin trapping reagent was successfully applied to trap and characterize unstable radicals which were generated by ultrasonic irradiation of dimethylsulfoxide (DMSO) solution of hardwood, Fagus crenata lignin. It was found, consequently, that a secondary carbon radical, ∼CH· was trapped as the nitroxide spin adduct when the DMSO solution was subjected to ultrasonic irradiation in the presence of a spin trapping reagent: 2, 4, 6-tri-tert-butylnitrosobenzene (BNB) at 50 °C for 60 minutes under vacuum. This means that the alkyl phenyl ether bonds known as lignin linkage bonds were homolytically scissoned by the ultrasonic irradiation, although the phenoxy radical, Ph-O· as the counter radical of the secondary carbon radical was not trapped by the BNB spin trap. Further, the data showed that the primary carbon radical, ∼OCH₂ · is trapped by the BNB to form the corresponding spin adduct, indicating that the hydrogen abstraction from the ortho methoxy group in the syringyl and/or guaiacyl moiety is induced by the secondary carbon radical when irradiated. Based on these findings it was concluded that the Ph-O· radicals produced by the homolytic rupture of the alkyl phenyl ether bonds were not trapped by the BNB spin trap. This suggests that large steric hindrances operate between the syringyl with two methoxy moieties and/or guaiacyl with a methoxy moiety at the ortho position, and the BNB molecule bearing two bulky ortho tert-butyl groups. Received 5 January 1999     

Preparation and characterization of the phenolated wood using hydrochloric acid (HCl) as a catalyst

Summary Birch (Betula maximowicziana Regel) wood meal was liquefied in the presence of phenol using hydrochloric acid (HCl) as a catalyst at a temperature of 150 °C for 2 h in an autoclave. It was found out that HCl acid could be used as an effective catalyst for the hydroxy phenylation of wood under the experimental conditions. In this study the effect of the concentration of the acid catalyst and the phenol-to-wood ratio on the liquefaction were investigated. The results showed that the phenol-to-wood ratio and the concentration should be increased to a certain degree in order to achieve a less residual rate and sufficient amount of combined phenol. The phenolated woods had apparent flow temperatures in the range of 134.4 to 199.8 °C, being higher than that of a commercial novolak resin. Furthermore, increases in the HCl concentration during liquefaction reaction led to increase in the apparent flow temperature of the resulting phenolated woods. However, the changes in the liquid ratio did not bring about evident changes. The relationship between shear stress (τ) and shear rate showed that the phenolated wood resin melts were shear thinning fluids. The dependences of the apparent melt-viscosities (η) of the phenolated woods and a commercial novolak resin on the shear rates ( ) have the similar tendencies, however, it was found the viscosities of the phenolated woods are about one order higher than that of commercial novolak resin.     

Lateral female strobili production in a Japanese red pine (Pinus densiflora Sieb. et Zucc.) clone by exogenous cytokinin application

Application ofN ⁶-benzylaminopurine (BAP) and gibberellin (GA₃) to a grafted Japanese red pine (Pinus densiflora Sieb. et Zucc.) clone promoted flowering. BAP (250 mg/l) was sprayed five times to the top of branches in July or September 1992. GA₃ (100 or 500 mg/l) was applied five times to the whole plants by spray treatment during the above period, or GA₃ solution of the same concentration was injected once into a small incision made in the main stem of clone trees at the beginning of July or the middle of September 1992. In May 1993, no apical female strobili were observed at the top of the new shoots in any treatment. However, lateral female and bisexual strobili were produced in the lower part of new shoots by BAP application in September, regardless of GA₃ application. BAP applied in September without GA₃ application was sufficient to produce lateral female and bisexual strobili. In such treatment, production of female and bisexual strobili was 47 and 25, and they occurred in 16 out of 68 new shoots, 11 out of 15 treated branches and 5 out of 5 treated trees. The average number of female and bisexual strobili per new shoot was 1.06. BAP treatment either in July or in September significantly decreased (p<0.05) the production of male strobili. These results indicate that BAP application induces the development of lateral female strobili in Japanese red pine when applied during the period of floral differentiation. A part of this paper was presented at the 105th annual meeting of the Japanese Forestry Society.     

Newly developed nanocomposites from cellulose acetate/layered silicate/poly(ε-caprolactone): Synthesis and morphological characterization

New biodegradable cellulose acetate/layered silicate grafted poly(ε-caprolactone) [(CA/layered silicate)-g-PCL] nanocomposites were prepared by in situ polymerization of ε-caprolactone in the presence of cellulose acetate (CA) and organically modified layered silicate (OMLS). The structures of the resulting composites were investigated. X-ray diffractometry was carried out to survey general structural features of (CA/OMLS)-g-PCL nanocomposites, and revealed that OMLSs having hydroxyl groups in the organic modifiers greatly altered the layered silicate structure by monomer intercalation and successive exfoliation through its polymerization. Two of the representative cases were characterized by wide-angle and small-angle X-ray scattering analyses with a synchrotron source. The morphology of these nanocomposites was further examined by transmission electron microscopy. When SPN, one of OMLSs having one hydroxyl group in its modifier, was used, the silicate layers could not be dispersed thoroughly, but existed as aggregates consisting of several silicate layers. Among them, the crystal growth of PCL developed by transcrystallization, where the crystal growth was restricted in the confined space. When Cloisite 30B, having two hydroxyl groups within the modifier, was used, the silicate layers forming the clay were dispersed completely in the composite and random orientation of the OMLS was observed.     

Effects of feeding calves genetically modified corn bt11: a clinico-biochemical study

Genetically modified corn Bt11 is insect-resistant and expresses Cry1Ab toxin, an insecticidal protein, in kernels. Although Bt11 corn is considered safe based on animal performance, there are no reports available on the clinico-biochemical effects of feeding it to cattle. In this study, we evaluated the effects of feeding Bt11 to calves, using blood and ruminal clinico-biochemical parameters. Our three-month-long feeding experiment demonstrated that calves (n=6), fed with a ration containing 43.3% of Bt11 corn kernels as dry matter, did not develop any discernible clinical, hematological, biochemical, or ruminal abnormalities as compared with control calves (n=6) fed non-Bt11 corn. The results suggest that the transgenic Bt11 has no negative clinico-biochemical effects on calves.