Expression of Bcl-2 in feline lymphoma cell lines

BACKGROUND: The Bcl-2 gene is a member of the rapidly expanding Bcl-2 family of genes that regulate apoptosis. Bcl-2 has been shown to repress cell death triggered by a diverse array of stimuli, including chemotherapy and gamma irradiation. OBJECTIVE: The purpose of this study was to determine feline Bcl-2 expression level in feline lymphoma cells using an immunoblot assay with anti-human and anti-canine Bcl-2 monoclonal antibodies. METHODS: About 708 base pairs containing the coding sequence of the feline Bcl-2 gene were transformed into Escherichia coli. The recombinant Bcl-2 was used as a positive control for an immunoblot assay using mouse monoclonal antibodies against human and canine Bcl-2. An immunoblot assay using the monoclonal antibodies was carried out to determine the level of feline Bcl-2 expression in lymphoma and lymphocytic leukemia cell lines. RESULTS: The recombinant feline Bcl-2 protein produced in E. coli had a molecular weight of about 26 kDa and was detected by immunoblot assay by using anti-human Bcl-2 mouse monoclonal antibody. Feline Bcl-2 expression was high in lymphoma cell lines (FL-74-UDC-1 and FT-1) and low in the cell line from peripheral blood mononuclear cells from a healthy cat (FeTJ-1) but not low in freshly isolated peripheral blood mononuclear cells from a healthy cat. The anti-human Bcl-2 mouse monoclonal antibody was found to cross-react with feline Bcl-2. CONCLUSIONS: These results confirm the expression of Bcl-2 in T-cell lymphoma cell lines and indicate that it is suitable to detect feline Bcl-2 using an immunoblot assay. Pending further evaluation, Bcl-2 expression might be useful in the differential diagnosis of feline tumors.     

Cloning of various promoters for foreign gene expression in Erwinia ananas

We constructed a promoter-trap plasmid, pEGFP-V1, to isolate various promoters for foreign gene expression in the leaf-colonizing bacterium Erwinia ananas NR-1. A library was constructed in pEGFP-V1 by introducing genomic DNA fragments upstream of the promoterless EGFP gene to transform E. ananas cells. The library, which consisted of 3500 E. ananas transformants was screened for GFP expression. We found nine strong GFP-expressing clones from the library. Furthermore, we characterized the clones by restriction analysis, sequencing, primer extension analysis, and then quantification of promoter activity. Selected promoters, specifically two (PCF9 and PCF53), gave strong gene expression in E. ananas. Our results indicate that pEGFP-V1 is a useful tool for screening DNA fragments with strong promoter activity in E. ananas.     

The use of CPPU for efficient propagation of pineapple

The use of forchlorfenuron (N-(2-chloro-4-pyridyl)-N′-phenylurea) (CPPU) for efficient propagation of pineapples was investigated. About 85% of axillary buds can be forced to sprout by soaking defoliated stem pieces (12 cm in length) in a 2.5 or 5.0 mg l⁻¹ CPPU solution for more than 3 h. The use of old stems taken from the third or fourth ratoon plants had the advantage of less liability to fungal decay, as compared to young stems from the first crop plants. The CPPU treatment combined with the removal of shoots from stems at monthly intervals significantly increased the number of shoots per stem piece (about 15 shoots per piece at 5.0 mg l⁻¹ CPPU), and resulted in a more uniform shoot size (the percentage of shoots within a range of 5–15 cm in length was about 90% at 5.0 mg l⁻¹ CPPU). The rooting of shoots was easily promoted within 1 month by treating the basal portion of shoots with 20 mg l⁻¹ indole-3-butyric acid (IBA) for 15 min. The CPPU method required about 5 months for plantlet propagation. From these results, we found that pineapple plantlets could be efficiently propagated by the following method: (1) soaking defoliated stems in a 2.5–5.0 mg l⁻¹ CPPU solution for more than 3 h; (2) harvest of developed shoots from the stems at regular intervals; and (3) promotion of rooting on the shoots at 20 mg l⁻¹ IBA for 15 min.     

Characterization of CD34+ cells from canine umbilical cord blood, bone marrow leukocytes, and peripheral blood by flow cytometric analysis

Characterization of CD34+ cells in canine bone marrow, umbilical cord blood, and peripheral blood was performed by flow cytometric analysis. The ratio of CD34+CD45hi cells, which are absent in human blood, was high in the CD34+ cell fraction, but 98% of these was suggested B-cells. The remaining CD34+CD45lo cells may comprise canine hematopoietic progenitor cells, and these cells accounted for 0.23 +/- 0.07% of the fraction in cord blood, 0.30 +/- 0.07% in bone marrow, and 0.02 +/- 0.01% in peripheral blood.     

Search for an index for the taste of Japanese Black cattle beef by panel testing and chemical composition analysis

To search for an index for chemical composition related to superior taste in Japanese Black beef, we conducted panel tests and analyzed the chemical composition of seven beef brands. Thirty‐five sirloin beefs from five heifers were used in this study, sold under seven beef brands graded as more than A4 on the Japanese Meat Grade scale. The chemical composition analyses assessed both raw and roasted meat, the latter of which was roasted under the same conditions as those used for the panel test. Results of the panel test and chemical composition analyses revealed that fatty acid composition, sugar content, adenosine triphosphage (ATP)‐related compounds, amino acid composition and odor composition in the sirloin meat differed among beef brands. Furthermore, the correlations of chemical compositions between roasted and raw meat were significantly high. Sugar content and ATP‐related compounds in roasted meat were significantly correlated with the item ‘overall evaluation’ of the panel test. ATP‐related compounds, such as inosinic acid, carnosine and taurine, in roasted and raw meat were correlated significantly with the item ‘umami intensity’ of the panel test. These results suggest that the composition of these components is important for an index related to the overall evaluation of beef.     

Simple method to reduce interference from excess magnesium in cadmium immunoassays

In order to develop a rapid inexpensive test for cadmium in rice, we identified an antibody specific for cadmium-EDTA complexes; this antibody binds to cadmium-EDTA with a Kd of approximately 10(-8) M. Although the antibody's cross reactivity to magnesium was minimal (Kd approximately 10(-5) M), the high toxicity of cadmium coupled with the high natural occurrence of magnesium in rice resulted in a situation where magnesium interfered with cadmium determination and resulted in falsely elevated estimates of cadmium. Fortunately, the formation constant of EDTA for cadmium is approximately 5 x 10(7) times higher (at pH 7) than the formation constant of EDTA for magnesium, and we were able to eliminate the magnesium interference by judicious selection of the EDTA concentration used in the assay. The resulting equilibria are complex, but we show that a relatively simple two-step model in which cadmium and magnesium compete for EDTA followed by cadmium-EDTA and magnesium-EDTA competing for antibody provided a good fit to the measured data. These analyses enabled appropriate selection of the optimum EDTA concentration for an immunoassay with improved selectivity.     

Plant growth inhibitors in turmeric (Curcuma longa) and their effects on Bidens pilosa

Turmeric (Curcuma spp.) has numerous biological activities, including anticancer, antibacterial, antifungal and insecticidal properties. Here, we evaluated the plant growth‐inhibitory activities of two cultivars of Curcuma longa (C. longa ; Ryudai gold and Okinawa ukon) against radish, cress, lettuce and Bidens pilosa (B. pilosa ). The methanol extracts of both turmeric varieties inhibited the seed germination and seedling growth of all the tested plants. Ryudai gold had a significantly higher inhibitory effect on the seed germination and root and shoot growth of the plants than Okinawa ukon. Therefore, Ryudai gold was chosen for the isolation of plant growth‐inhibitory compounds using a silica gel column and high‐performance liquid chromatography. The structural identification of the compounds was carried out using ¹H NMR, ¹³C NMR and liquid chromatography–tandem mass spectrometry. The growth inhibitors were identified as four curcuminoids; dihydrobisdemethoxycurcumin ( 1 ), bisdemethoxycurcumin ( 2 ), demethoxycurcumin ( 3 ) and curcumin ( 4 ). The IC₅₀ of the curcuminoids against the root and shoot growth of B. pilosa ranged from 8.7 ± 1.7 to 12.9 ± 1.8 and from 15.5 ± 1.8 to 38.9 ± 2.8 μmol L^?1, respectively. Compound 1 showed the lowest IC₅₀ against the root and shoot growth of B. pilosa . These results suggested that Ryudai gold has a potential growth‐inhibitory effect against B. pilosa .     

Rice yield and soil carbon dynamics over three years of applying rice husk charcoal to an Andosol paddy field

Rice husk charcoal (RC) produced from the pyrolysis of rice husk (RH) can be one of the cost-effective biochars for use in rice-based farming systems. This study investigated changes in rice yield and soil carbon sequestration over three years of RC application to an Andosol paddy field. The treatments were RC application at 0.02, 0.2, and 2 kg m^?2 (RC0.02, RC0.2, and RC2, respectively), RH application at 0.2 kg m^?2 (RH0.2), and a control with no RC or RH application (CONT). The results showed that RC2 increased culm length by 4% and straw weight by 14% on average over the three years. These increases in plant growth coincided with a higher level of silicon uptake by the rice plants, although they did not significantly affect grain yield. The soil carbon content was progressively increased by RC2 over the three years, whereas it was not significantly affected by RC0.02 or RC0.2. A considerable amount (>72%) of the applied carbon with RC2 remained in the soil by taking account of its downward movement below the 10 cm layer of the paddy field after three consecutive years of RC application. We conclude that rice husk charcoal application to Andosol paddy fields is an effective option for increasing carbon sequestration. Furthermore, the increase in silicon uptake by rice plants suggests that rice husk charcoal can also be functioning as a silicon fertilizer.     

Relationship between watercore and maturity or sorbitol in apples affected by preharvest fruit temperature

The effect of preharvest fruit temperature on the relationships between development of watercore and ethylene evolution, flesh firmness, membrane permeability and sorbitol level in two watercore-susceptible apple (Malus domestica Borkh.) cultivars, ‘Himekami' and ‘Fuji', was investigated at ambient or modified temperatures of 10, 15 or 25°C. Watercore occurrence was completely inhibited at 25°C in both cultivars whereas fruit subjected to 10 or 15°C exhibited significantly higher watercore ratings after two or three weeks of the treatment than those at 25°C and ambient temperatures. Ethylene production of ‘Himekami' apples increased at 25°C and ambient temperature but not at 15°C. Ethylene production was not affected by treatment in ‘Fuji' apples. Flesh firmness of ‘Himekami' tended to maintain a higher level at 15°C than at 25°C, while there was no clear difference among the treatments in ‘Fuji'. The rate of potassium (K) ion elution from the flesh tissues of either cultivar was not affected by fruit temperature treatment. Sorbitol content at 15°C maintained significantly higher level than that at 25°C during the treatment in ‘Himekami' apples although no sorbitol difference was observed among the temperature treatments in ‘Fuji' apples.     

Reaction of manganese peroxidase ofBjerkandera adusta with synthetic lignin in acetone solution

The reaction of manganese peroxidase (MnP) of the white-rot fungusBjerkandera adusta with synthetic lignin dehydrogenation polymer, DHP) in acetone medium was investigated. Gel-permeation chromatography of the DHP treated by MnP demonstrated depolymerization of syringyl DHP in the reaction mixture containing 70% acetone; moreover, concomitant repolymerization occurred to give highly polymerized products. Guaiacyl DHP was only repolymerized by MnP in the same acetone solution without giving degradation products. Addition of ascorbic acid to reaction mixtures containing acetone resulted in preferential depolymerization of syringyl DHP.Part of this report was presented at the meeting of Kansai Branch, Japan Society for Bioscience, Biotechnology, and Agrochemistry in Kagawa, October 1996