Study of the parentage of grape cultivars by genetic interpretation of GPI-2 and PGM-2 isozymes

Summary Two highly variable enzyme systems of glucosephosphate isomerase (GPI) and phosphoglucomutase (PGM) were used to investigate the parentages of grape cultivars. Of 35 parent/offspring combinations that we investigated, 30 combinations gave alleles in the offspring which were presented in the reported parents, whereas 5 combinations gave alleles in the offspring which were not extractable from the reported parents. The Gpi-2 genotype of Hiro Hamburg and the Pgm-2 genotype of Pione indicated that Koshu Sanjaku and Cannon Hall Muscat may not have been the paternal parent respectively. The Gpi-2 genotype of New Niagara and the Gpi-2 and Pgm-2 genotypes of Beniyamabiko indicated that Niagara and a hybrid from DxK151 x Delaware may not have been the maternal parent respectively. The Gpi-2 genotype of Cannon Hall Muscat grown in Japan indicated that this cultivar may not have originated as a tetraploid sport of Muscat of Alexandria.     

Synthesis of theaflavin from epicatechin and epigallocatechin by plant homogenates and role of epicatechin quinone in the synthesis and degradation of theaflavin

Oxidation products of (-)-epicatechin and (-)-epigallocatechin by treatment with homogenates of 62 plants belonging to 49 families were compared. Forty-six plants were capable of synthesizing theaflavin, a black tea pigment, regardless of whether they contained catechins. Loquat, Japanese pear, and blueberry had activities higher than that of fresh tea leaves after 5 h of treatment; furthermore, these plants oxidized theaflavin to theanaphthoquinone. An additional new metabolite, dehydrotheasinensin, was generated on treatment with fresh tea leaves, eggplant, and unripened Japanese orange. Evidence for the oxidation of epigallocatechin and theaflavin by electron transfer to epicatechin quinone was demonstrated in a time course study using bananas and trapping the quinone intermediates as glutathione conjugates.     

DNA- versus RNA-based denaturing gradient gel electrophoresis profiles of a bacterial community during replenishment after soil fumigation

We compared the responsiveness and sensitivity to soil fumigation of DNA- and RNA-based analyses of a bacterial community. We first established an improved RNA extraction method using DNA as an adsorption competitor, because it is extremely difficult to extract nucleic acids from clay-rich volcanic ash soil (Andisol), which adsorbs nucleic acids. This novel method facilitated RNA extraction from 500 mg of Andisol for molecular analyses. Then we monitored 16S rDNA PCR and 16S rRNA RT-PCR denaturing gradient gel electrophoresis (DGGE) profiles of samples collected from a chloropicrin (CP)-treated field over 2 months. The difference between untreated control and CP-treated plots was detected clearly both in DNA- and RNA-based DGGE profiles after treatment. The temporal changes in DGGE profiles, however, differed between DNA- and RNA-based analyses in CP-treated plots. RNA-based DGGE showed quicker and greater changes in the bacterial community after CP treatment than did DNA-based DGGE, which showed similar trends to RNA-based DGGE but with a time lag. The extent of decrease in the diversity index (H′) and the change in principal response curves was larger in RNA-based analyses. These results indicate that the rDNA PCR-DGGE method also detects DNA of microbes no longer alive after fumigation, and that rRNA provides a more responsive biomarker than rDNA.     

Development of a neurosurgical operating table for adult cattle and changes in intracranial pressure and blood pressure in adult cattle undergoing long-time isoflurane anesthesia

We developed a neurosurgical operating table for restraining adult cattle in the sternal recumbent position during long-time inhalation anesthesia, and examined intracranial pressure (ICP), blood pressure and blood gases during isoflurane anesthesia. We confirmed that the maintenance of inhalation anesthesia, the restraint of cattle in the sternal recumbent position and bringing the cattle out of anesthesia could all be carried out safely using the operating table we produced. For the purposes of the present experiment, the cattle were divided into 2 groups: the SR group, which underwent sternal recumbency for 8 hr under isoflurane anesthesia using the neurosurgical operating table, and the RR group, which underwent right lateral recumbency for 3 hr under isoflurane anesthesia on a standard operating table. The mean ICP was found to be significantly lower in the SR group than in the RR group during anesthesia, and PaO2 was significantly higher in the SR group. In the SR group, no complications such as regurgitation or ruminal tympany occurred for 8 hr after the induction of anesthesia, and recovery from anesthesia was uneventful. In contrast, all RR cattle showed ruminal tympany and regurgitated ruminal fluid at 3 hr after the induction of anesthesia. Thus, the neurosurgical operating table developed in the present study may be useful for long-time anesthesia and neurosurgery of adult cattle.     

A novel gene encoding a thrombin inhibitory protein in a cDNA library from Haemaphysalis longicornis salivary gland

A novel thrombin inhibitory protein coding gene was identified from a cDNA library derived from salivary gland of partially-fed Haemaphysalis longicornis (hard tick). The gene encoded a 93-amino acid protein, designated chimadanin, which had a signal peptide sequence and was predicted to be a secretory protein. It showed no similarity to any other previously identified proteins or conserved domain sequences. The gene was expressed during blood feeding and suggested to be expressed mainly in the salivary gland. The predicted mature region of chimadanin was expressed in Escherichia coli and characteristics of the recombinant chimadanin were determined. The activated partial thromboplastin time and the prothrombin time in sheep plasma were significantly prolonged by chimadanin in a dose dependent manner. Amidolytic activity of thrombin was also inhibited by chimadanin in a dose dependent manner and it suggested that chimadanin was an anticoagulant with thrombin inhibitory activity. This newly identified thrombin inhibitor may play an important role in tick blood feeding.     

Expression of Membrane Complement Regulatory Proteins Crry and CD55 in Normal Rats

Some anticancer therapeutic antibodies are designed to act through complement-dependent cytotoxicity (CDC). It has been reported that there are many membrane complement regulatory proteins (mCRPs) that inhibit CDC. In the present study, we examined the expression of two mCRPs, the complement receptor 1-related gene/protein Y (Crry) and the decay-accelerating factor CD55, in three normal rats by immunohistochemistry. Crry and CD55 were detected widely in rat organs and tissues. Crry was found mainly in the urinary, digestive, respiratory, immunohematopoietic, circulatory and neuroendocrine systems. CD55 was found in the urinary, digestive and neuroendocrine systems. However, the two molecules were expressed in separate cells within the same organ. These results suggest that the distribution of mCRPs is related to the strict regulation of CDC activation in these organs and tissues and that the two molecules have a nonoverlapping expression pattern, a fact indicating specific roles in CDC regulation.     

Isolation, characterization, and surfactant properties of the major triterpenoid glycosides from unripe tomato fruits

Various triterpenoid glycosides were extracted from whole unripe tomato fruits ( Lycopersicon esculentum cv. Cedrico), using aqueous 70% (v/v) ethanol to study their surfactant properties. Cation-exchange chromatography using a Source 15S column and subsequent semipreparative HPLC using an XTerra RP18 were employed to purify individual triterpenoid glycosides from the extract. The structure of the purified compounds was established by mass spectrometry and nuclear magnetic resonance spectroscopy. The furostanol glycoside tomatoside A (749 mg/kg of DW) and the glycoalkaloids alpha-tomatine (196 mg/kg of DW) and esculeoside A (427 mg/kg of DW) were the major triterpenoid glycosides present. Furthermore, minor amounts of a new dehydrofurostanol glycoside, dehydrotomatoside, were found. The critical micelle concentrations of the major triterpenoid glycosides, alpha-tomatine, tomatoside A, and esculeoside A, were determined as 0.099, 0.144, and 0.412 g/L, respectively. The results show that tomatoside A, and not the more well-known alpha-tomatine, is the predominant triterpenoidal surfactant in unripe tomato fruits.     

Stimulation of Adrenal Chromaffin Cell Proliferation by Hypercalcemia Induced by Intravenous Infusion of Calcium Gluconate in Rats

Increased incidence of adrenal pheochromocytoma is frequently encountered in rat carcinogenicity studies. In some of the studies, the finding is judged to be due to a rat-specific mechanism of carcinogenesis caused by a disturbance of calcium homeostasis. However, direct evidence that the proliferation of chromaffin cells in the adrenal medulla is induced solely by hypercalcemia is not available. In this study, calcium gluconate was intravenously infused for 7 days to rat chromaffin cells by a tail cuff method, and cumulative labeling with bromodeoxyuridine (BrdU) was carried out to evaluate the proliferative activity. The serum calcium concentration was dose-dependently increased, and a high calcium concentration was stably sustained from day 2 to 7. In the adrenal medulla, BrdU-positive chromaffin cells increased in the calcium gluconate-treated animals, and the BrdU-labeling index increased in a dose-dependent manner. In addition, an increased BrdU-labeling index of chromaffin cells was shown to correlate with the serum calcium concentration. Our results demonstrate that hypercalcemia directly enhances the proliferative activity of chromaffin cells and that the proliferative activity is correlated with the serum calcium concentration.     

Efficacy of soluble recombinant FliC protein from Salmonella enterica serovar enteritidis as a potential vaccine candidate against homologous challenge in chickens

FliC, the flagellin antigen of Salmonella Enteritidis, was tested as a vaccine candidate for protective effect against a homologous challenge in chickens. After immunization with recombinant FliC (rFliC) or administration of phosphate-buffered saline (PBS) at 56 days old, the chickens were challenged with 10(9) colony-forming units of Salmonella Enteritidis at 76 days old. The vaccinated birds showed significantly decreased bacterial counts in the liver and cecal contents compared to those administered PBS at 7 days postchallenge, but the protection was partial. The replication experiment also showed a similar result. In both experiments, vaccination induced an increased level of serum anti-rFliC IgG, which was also reactive to the native flagella. The intestinal IgA level was slightly higher in the vaccinated birds than in the control. However, neither the proliferative response nor interferon-gamma secretion of splenic cells upon stimulation with rFliC was induced. Therefore, the effect of rFliC as a vaccine is limited, and further improvement is needed.