Solid-phase synthesis of mimosine tetrapeptides and their inhibitory activities on neuraminidase and tyrosinase

Neuraminidase is a rational target for influenza inhibition, and the search for neuraminidase inhibitors has been intensified. Mimosine, a nonprotein amino acid, was for the first time identified as a neuraminidase inhibitor with an IC(50) of 9.8 ± 0.2 μM. It was found that mimosine had slow, time-dependent competitive inhibition against the neuraminidase. Furthermore, a small library of mimosine tetrapeptides (M-A(1)-A(2)-A(3)) was synthesized by solid-phase synthesis and was assayed to evaluate their neuraminidase and tyrosinase inhibitory properties. Most of the tetrapeptides showed better activities than mimosine. Mimosine-FFY was the best compound, and it exhibited 50% neuraminidase inhibition at a low micromolar range of 1.8 ± 0.2 μM, whereas for tyrosinase inhibition, it had an IC(50) of 18.3 ± 0.5 μM. The kinetic studies showed that all of the synthesized peptides inhibited neuraminidase noncompetitively with K(i) values ranging from 1.9 -to 7.2 μM. These results suggest that mimosine could be used as a source of bioactive compounds and may have possibilities in the design of drugs as neuraminidase and tyrosinase inhibitors.     

Estimation of mean tree height using small-footprint airborne LiDAR without a digital terrain model

In order to estimate mean tree height using small-footprint airborne light detection and ranging (LiDAR) data, a digital terrain model (DTM), which is a continuous elevation model of the ground surface, is usually required. However, generating accurate DTMs in mountainous forests using only the LiDAR data is laborious and time consuming, because it requires human-assisted methods, especially in the forests with poor laser penetration rates. Based on our previous finding that a hypothetical continuous surface model passing through the predominant tree tops (hereafter, called the “top surface model” or TSM) might be nearly parallel to a DTM, we assumed that the vertical difference between the TSM and the ground return was the mean tree height. According to this assumption, we propose a new methodology that does not require a DTM to estimate mean tree height. This method completely, automatically, and directly estimates mean tree height (MTH _E) from the LiDAR data without requiring a regression analysis using reference data. From the relationships between the MTH _E and the observed mean tree height (MTH _O) in different hinoki cypress forests, we demonstrate that this method effectively estimates the mean tree height with nearly 1-m accuracy.     

Relationships between urine pH and electrolyte status in cows fed forages

Data of 20 balance measurements from Holstein dairy cows and urine samples from 24 Japanese Black beef cows were collected to evaluate the relationships between urine pH and electrolyte status in cows fed forages. The ratio of forages in the diet was 70–100% in dairy cows and beef cows were fed Italian ryegrass silage and wheat bran. Mean urine pH in dairy cows was 8.10, ranging from 7.27 to 8.71, and that in beef cows was 7.73, ranging from 7.42 to 8.12. There were positive correlations between urine pH and urinary K contents (P = 0.0012) or K intake (P = 0.019) in dairy cows, although plasma Na, Cl and K had no effect on urine pH. There was a weak negative correlation (P = 0.039) between urine pH and urinary Na content in dairy cows. However, there were no significant correlations between urine pH and urinary Na, Cl and K contents in beef cows. These results indicate that the concentrated urinary K due to the increased K intake may directly enhance urine pH in dairy cows fed mainly forages.     

Biological control of rice blast by the epiphytic bacterium <Emphasis Type="Italic">Erwinia ananas</Emphasis> transformed with a chitinolytic enzyme gene from an antagonistic bacterium, <Emphasis Type="Italic">Serratia marcescens</Emphasis> strain B2

The gene chiA, encoding for the endochitinase ChiA, was cloned from Serratia marcescens strain B2, a tomato epiphytic bacterium, and introduced into the epiphytic bacterium Erwinia ananas NR-1, isolated from rice phylloplane. The gene chiA was introduced under the control of two types of promoter into a broad-host-range plasmid vector. The vector contained various fragments with promoter activity isolated from E. ananas chromosomal DNA. The constructed vectors were designated pchiA-V1pcf9 and pchiA-V1pcf53 for their respective promoters. E. ananas NR-1 transformed with either of these vectors produced and secreted ChiA. The antifungal activity of ChiA produced by transformed E. ananas NR-1 was demonstrated in vitro by the inhibition of Pyricularia oryzae germ tube elongation such as bursting of the hyphal tip. Transformed E. ananas NR-1 suppressed the incidence of rice blast caused by P. oryzae under greenhouse conditions; however, the magnitude of the suppressive effect depended on which promoter was used. Both transformants and the nontransformant E. ananas NR-1 survived on rice phylloplane. It is expected that the rice epiphytic bacterium E. ananas NR-1 carrying a chitinolytic enzyme gene is an efficient biological control agent against rice blast.     

Homogeneous cell suspension of Malassezia pachydermatis obtained with an ultrasonic homogenizer

It is difficult to produce homogeneous cell suspensions of Malassezia pachydermatis, since yeast cells paste up and form many clumps. However, homogeneous fungal suspensions are required for susceptibility examinations and biochemical analyses. Although several types of trials have been carried out using glass homogenizers and many types of agents to obtain homogeneous fungal suspension. They have not yielded good results. We therefore attempted to use an ultrasonic homogenizer to separate clumps of yeast cells into separate individual cells. We succeeded in this fashion in producing homogeneous cell suspensions of M. pachydermatis. These results indicate that an ultrasonic homogenizer can be used to prepare homogeneous fungal suspensions of M. pachydermatis.     

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.