A new DNA marker for sex identification in purple asparagus

Garden asparagus (Asparagus officinalis L.) is an economically-important perennial crop. This plant is dioecious, as there are both male and female individuals; male individuals are preferred over females for agricultural production. To reduce the time required for garden asparagus breeding, various male-specific DNA markers are utilized. Male-specific DNA markers, such as Asp1-T7sp and MSSTS710, are currently available for sex identification in many asparagus cultivars. In the current study, we found that these markers are not suitable for sex identification in the purple asparagus cultivar ‘Pacific Purple’, as male-specific amplification of this marker was detected in some male individuals of this cultivar but not in other males. The Asp1-T7sp marker is suitable for use in sex identification in various Asparagus species related to A. officinalis, indicating that the region around this marker is conserved among these species. Thus, we isolated a DNA fragment around this marker by inverse PCR and produced a new DNA marker, MspHd, based on this sequence. However, like Asp1-T7sp and MSSTS710, MspHd was not suitable for sex identification in the cultivar ‘Pacific Purple’. Since all ‘Pacific Purple’ males have morphologically similar male flowers with functional stamens, we produced a new male-specific marker based on the sex determination gene, MSE1/AspMYB35/AspTDF1, which is responsible for stamen development. This marker, named AspMSD, is suitable for sex identification in ‘Pacific Purple’. In addition, this marker can be utilized for sex identification in various asparagus cultivars and some related Asparagus species.     

Analysis by enzyme-linked immunosorbent assay and 2-dimensional electrophoresis of haptoglobin in the high-density lipoprotein fraction in cows

Haptoglobin (Hp) is a hemoglobin (Hb)-binding acute-phase protein. Besides its relevance in inflammation, Hp is involved in the regulation of lipid metabolism. In cattle, in addition to the lipoprotein-deficient fraction, Hp is distributed in high-density lipoprotein (HDL) and very high-density lipoprotein (VHDL) fractions. The purpose of this study was to determine Hp concentrations in the lipoprotein fractions using an enzyme-linked immunosorbent assay (ELISA) based on the affinity with Hb, and also to detect structural differences of HDL Hp from that in the lipoprotein-deficient fraction using 2-dimensional electrophoresis. When purified Hp was used as the antigen for the ELISA, the detection limit was 7.4 ng/ml and linearity was obtained from 14.8 to 475 ng/ml. The correlation coefficient between the ELISA and single radial immunodiffusion was 0.884. The ELISA was shown to be applicable to evaluate Hp concentrations in the lipoprotein fractions. Hp concentrations in the lipoprotein fractions were in the range of 0.94 to 8.77 microg of Hp/ml (n = 4), and concentration ratios were 0.2 to 0.3% of whole serum Hp. Of the lipoprotein fractions, Hp was most abundant in HDL, moderate in VHDL and faint in chylomicrons, the very low-density lipoprotein fraction and low-density lipoprotein fraction. By 2-dimensional electrophoresis, alpha- and beta-chains of serum Hp were each separated into 5 spots, and their isoelectric point (pI) values were from 5.05 to 6.28 in the alpha-chain and from 5.92 to 6.95 in the beta-chain. The pI values of HDL Hp were indistinguishable from those of serum Hp. These results indicate that the ELISA based on the affinity with Hb is useful for evaluating Hp concentrations in lipoprotein fractions, and also suggest that HDL Hp is structurally similar to that in the lipoprotein-deficient fraction.     

Detection of natural infection of Boophilus microplus with Babesia equi and Babesia caballi in Brazilian horses using nested polymerase chain reaction

The potential role of Boophilus microplus as a natural tick vector of Babesia equi and Babesia caballi in Brazilian horses was assessed using nested polymerase chain reaction (PCR)-based marker assay. B. equi merozoite-specific 218bp gene fragment was detected in almost 96% of horse blood samples, and 45.3-62.5% of females, eggs, larvae, and nymphs of B. microplus collected from 47 horses at Campo Grande in the State of Matto Grosso, Brazil. Except for the partially-fed female ticks, the B. caballi-specific 430bp gene fragment was amplified from horse blood samples, and all developmental stages. Parasite DNA from both species was detected in horse blood samples and B. microplus, with the preponderance of B. equi DNA. No DNA samples were positive solely for B. caballi parasite. Only 32% of the Giemsa-stained thin blood smears were positive for Babesia parasites, as against detection of B. equi parasite DNA in 95.7% of the blood samples by nested PCR. We have obtained molecular evidence that strengthens earlier experimental and ultrastructural studies in Brazil incriminating B. microplus as a natural vector of B. equi, and possibly of B. caballi. The detection of B. equi and B. caballi DNA in eggs and larvae of B. microplus is likewise suggestive of the possibility of both transovarial and transstadial parasite transmission in this tick vector.     

Vitelline cyst in the rat ileum

Congenital vitelline duct anomalies other than Meckel’s diverticulum are rare in animals. A cyst of approximately 8 mm in diameter was observed on the antimesenteric surface of the ileal serosa in a 10-week-old female Crl:CD(SD) rat. Microscopically, the cyst closely resembled the ileum, but it did not communicate with the ileal lumen. We diagnosed this case as a vitelline cyst derived from the vitelline duct based on the location where it developed and its histological behavior. In rats, only Meckel’s diverticulum has been reported with a congenital anomaly of the vitelline duct, and no other spontaneous anomalies including a vitelline cyst have been reported. This case may be the first report concerning a vitelline cyst in the rat ileum.     

Eosinophilic substance is "not amyloid" in the mouse nasal septum

An eosinophilic substance (ES) is usually observed in the mouse nasal septum and increases in volume with aging. It has been described as amyloid in textbooks and one report. However, it has been described as "not amyloid" in other reports because there was a negative reaction to Congo red. In this study, the ES was investigated histopathologically and electron microscopically to determine whether it was amyloid or not. The ES was only observed at the interstitium of clear HE-stained nasal glands in the septum, in which 2 kinds of glands were present (dark and clear stained by HE). The volume of the ES was small in young mice and large in older ones. Neither nasal gland degeneration nor inflammation resulted, even if a large amount of the ES was observed. The ES reacted negatively to Congo red but was strongly positive to periodic acid-Schiff reaction with prior diastase treatment. In the electron microscope observation, the ES consisted of amorphous material and collagen, but no nonbranching fibrils. Similar amorphous material was also observed in the nasal gland epithelial cells and was connected to the material in the rough endoplasmic reticulum. The above-mentioned findings indicated that the ES was not amyloid and suggested the ES might consist of not only collagen but also complex carbohydrate, which was produced by the nasal gland epithelial cells.     

An outbreak of teat papillomatosis in cattle caused by bovine papilloma virus (BPV) type 6 and unclassified BPVs

Out of 700 heifers at a local farm in Hokkaido, the Northern island of Japan, 560 (80%) were found to have benign teat tumors. All of the analyzed tumors were macroscopically of the flat-and-round type, and no other types such as rice-grain or frond epithelial type were found. The lesions were characterized by epithelial hyperplasia, acanthosis and hyperkeratosis. Unlike in typical fibropapilloma, fibroplasia of the underlying dermis was not observed. Bovine papilloma virus (BPV) capsid antigen and virus particles were found in basophilic intranuclear inclusions of the stratum granulosum of the epidermis by immunohistochemistry and electron microscopy, respectively. BPV-specific DNA was also detected in the lesions. By means of the polymerase chain reaction (PCR) and DNA sequencing of the PCR products, the viruses causing this outbreak were identified mainly as BPV-6 (64%), partly as unclassified BPVs (14%) and their co-infections (21%). Our findings suggest that this outbreak of benign teat tumors was associated with several BPV types.     

Dispersion of humic allophane soils with supersonic vibration

The difficulties in dispersing volcanic ash soils of Japan and New Zealand have been considered to be due to the association or aggregation of allophane (3, 5, 7–10). In particular, MIYAZAWA (9) has obtained evidence that stable microaggregates of Humic Allophane soils derived from volcanic ash have been formed by dehydration of allophane. Recently, several investigators (2, 4, 6, 8, 9, 12) have pointed out that ultrasonic vibration is effective in dispersing the fine particles of soils. With respect to the applicability of supersonic vibration to the particle-size distribution analysis of Humic Allophane soils, MIYAZAWA (9) stated that the maximum dispersion, as measured by the clay content, was obtained only with supersonic vibration using an acidic medium. KOBO and OBA (8) reported that calgon (sodium hexametaphosphate) as a dispersing agent was successfully applicable to most Humic Allophane soils, but the use of HCI was necessary for some highly allophanic subsoils, and that the effect of supersonic vibration on dispersion of the soils is attributed to the breakdown of aggregates larger than 20 microns in diameter. They also recommended a mixture of 10g of soil and 50 ml of water and 20 min. exposure for a supersonic vibrator (10 Kc, 300 W). With respect to the applicability of vibration treatment in the particle-size distribution analysis of Humic Allophane soils containing volcanic glasses in abundance, some apprehension may be entertained about the breakdown of primary minerals, especially of volcanic glasses (9, 10).     

Emissions of trace gases (CO2, CO,CH4, and N2O) resulting from rice straw burning

Emissions of trace gases (CO₂, CO, CH₄, N₂O) resulting from rice straw burning were measured by using the open chamber method. The carbon contained in rice straw was mainly released to the atmosphere as CO₂. The percentage of CO₂-C emitted in total C in rice straw was in the range of 57–81%, followed by CO-C (5–9%). The percentages of CH₄-C and N₂O-N in total C and N in rice straw were in the range of 0.43–0.90 and 1.16–1.50%, respectively. In the case of the rice straw which had been left in the field for a period of one month after harvest, emission of imperfect combustible gases such as CO and CH₄ during burning increased slightly, while that of perfect combustible gas, CO₂, was reduced. The amount of CH₄ emission from rice straw burning was comparable to that from paddy fields.     

Genesis and characteristics of a red-yellow soil derived from Gabbro,Northern Kyushu

There are few pedological investigations of the soils derived from basic rocks in Japan. The object of this paper is to offer fundamental information concerning the genesis and analytical characteristics of a Red-Yellow soil derived from a basic rock (gabbro) of which silica content is approximately 44 per cent.