Construction of a genetic linkage map of the model legume Lotus japonicus using an intraspecific F2 population.

Among leguminous plants, the model legume Lotus japonicus (Regel) Larsen has many biological and genetic advantages. We have developed a genetic linkage map of L. japonicus based on amplified fragment length polymorphism (AFLP), simple sequence repeat polymorphism (SSRP) and derived cleaved amplified polymorphic sequence (dCAPS). The F2 mapping population used was derived from a cross between two L. japonicus accessions Gifu B-129 and Miyakojima MG-20. These parental accessions showed remarkable cytological differences, particularly with respect to size and morphology of chromosomes 1 and 2. Using fluorescence in situ hybridization (FISH) with BAC clones from Gifu B-129 and TAC (Transformation-competent Artificial Chromosome) clones from Miyakojima MG-20, a reciprocal translocation was found to be responsible for the cytological differences between chromosomes 1 and 2. The borders of the translocations were identified by FISH and by alignment toward the L. filicaulis x L. japonicus Gifu B-129 linkage map. The markers from the main translocated region were located on linkage groups 1 and 2 of the two accessions, Gifu B-129 and Miyakojima MG-20, respectively. The framework of the linkage map was constructed based on codominant markers, and then dominant markers were integrated separately in each linkage group of the parents. The resulting linkage groups correspond to the six pairs of chromosomes of L. japonicus and consist of 287 markers with 487.3 cM length in Gifu B-129 and 277 markers with 481.6 cM length in Miyakojima MG-20. The map and marker information is available through the World Wide Web at http://www.kazusa.or.jp/lotus/.

Involvement of eosinophils in the early-phase allergic reaction in a guinea pig rhinitis model.

BACKGROUND: Eosinophils are found in the nasal lavage fluid (NLF) and nasal biopsies of patients with allergic rhinitis after a nasal antigen challenge, and associated not only with a late-phase allergic reaction (LPR) but also an early phase allergic reaction (EPR). Numerous studies have been carried out to clarify the participation of eosinophils in LPR or airway hyperresponsiveness. However, there has been no published report describing in detail the role of eosinophils during EPR. To better understand the involvement of eosinophils in EPR, we studied the effects of repeated antigen challenges on nasal airway responsiveness and eosinophilic inflammation in EPR using a guinea pig rhinitis model. METHODS: Nasal airway responsiveness was measured as the nasal airway resistance (NAR) after nasal antigen provocation. Eosinophilic inflammation during EPR was assessed by nasal lavage and histopathological examination using two groups of animals: those in group 1 were subjected to a sensitization pretreatment only, and those in group 2 were subjected to a pretreatment of sensitization followed by repeated nasal challenges. RESULTS: Repeated antigen challenges induced nasal hyperresponsiveness as indicated by a decrease in the antigen provocation dose and a significant increase in NAR. Furthermore, significant increases in eosinophil counts, eosinophil peroxidase (EPO) activity and protein content in NLF during EPR were observed following antigen provocation in group 2. There were significant correlations between the levels of these parameters, and albumin was the most prevalent of the proteins in NLF. Histopathological examination showed that the degree of eosinophil infiltration into the lamina propria of the nasal mucosa of the animals in group 2 was significantly and apparently higher than in group 1. Particularly, epithelial disruption and mucosal edema were significantly elevated after antigen provocation in group 2. CONCLUSIONS: These results suggest that chronic eosinophil accumulation is induced by repeated antigen challenges in the nasal tissue, and that once antigen provocation occurs, eosinophils in the tissue are activated and responsible for the amplification of EPR such as vascular permeability and mucosal edema.

A novel role of IL-15 in the development of osteoclasts: inability to replace its activity with IL-2.

IL-15 shares many activities with IL-2 on stimulating lymphocytes, hematopoietic progenitor cells, and macrophages. However, the role of IL-15 in osteoclastogenesis has not been elucidated. The recent finding of abundant IL-15 in rheumatoid arthritis synovial fluids suggested a possible role for this cytokine in the pathological destruction of bone and prompted us to determine whether IL-15 stimulates osteoclast formation. IL-15 stimulated the formation of multinucleated osteoclast-like cells in rat bone marrow cultures. In stroma-free cultures, IL-15 increased the number of mononuclear preosteoclast-like cells in the early stage of osteoclast formation. The stimulation was observed even after treatment with IL-15 for only 24 or 48 h of culture. Moreover, low IL-15 concentration (0.1 ng/ml) strongly increased the level of calcitonin receptor mRNA of mononuclear preosteoclast-like cells. Although IL-15 is known as a potent stimulator of TNF-alpha, its activity was not abolished by addition of anti-TNF-alpha Ab. Interestingly, IL-2 and IL-7, which utilize some IL-15R components, had no effect on osteoclast differentiation, but pretreatment with IL-2 or IL-7 of bone marrow cells before the addition of IL-15 inhibited the enhancing activity of IL-15. In summary, IL-15 has a novel activity to stimulate the differentiation of osteoclast progenitors into preosteoclasts, which cannot be replaced by IL-2 but may use components in common with IL-2R to mediate its effects.

Multicenter experience with maxillary prostheses supported by Brånemark implants: a clinical report.

This retrospective study involved Japanese patients with prostheses supported by Brånemark implants following maxillectomy. Questionnaires were sent to 75 institutions, and data on 19 patients were collected from 8 institutions. The mean age of patients at the time of implant placement was 64.2 years (range 22 to 82 years). The mean follow-up time was 27.6 months. Of the 81 implants placed, 16 were lost for an implant survival rate of 80.2%. The effects on implant survival rate of radiotherapy, chemotherapy, hyperbaric oxygen therapy, and the support system of the prosthesis were analyzed, but no significant differences were observed.

Prediction of drug responses in schizophrenia: a method using a test dose of chlorpromazine.

Thirty-seven newly admitted schizophrenic patients were treated with an open and flexible dosage of chlorpromazine for 3 months after receiving a test dose. Levels of chlorpromazine, demethylated chlorpromazine and chlorpromazine sulfoxide 3 h after the test dose were measured. Twenty-three patients responded to long-term chlorpromazine treatment but 14 did not, a rate of 62.2%. A discriminant function analysis was performed using variables relating to the patients, backgrounds added to the ratios of plasma drug levels separately by sex to increase predictability over the level of previous studies. The obtained equations were applied to 23 newly admitted schizophrenic patients, with the prescription of chlorpromazine for designated responders and haloperidol for designated non-responders for 4 weeks. The patients in the latter study responded better than those of the former with chlorpromazine alone; 71.4 and 88.9% of chlorpromazine- and haloperidol-treated groups improved, respectively, for an overall rate of 78.3%. However, the chlorpromazine-treated group had a lower level of positive symptoms than the haloperidol-treated group before treatment and this and other differences between the groups should be further examined.

Changes in the initial distribution volume of glucose and plasma volume following volume challenge in dogs.

OBJECTIVE: This study aimed to test the hypothesis that the initial distribution volume of glucose (IDVG) is correlated with plasma volume determined by indocyanine green (ICG) following colloidal fluid challenges. The IDVG was also compared with cardiac output (CO) as measured by the conventional thermodilution method. DESIGN AND SETTING: The study was prospective and was conducted in the animal laboratory, University of Hirosaki School of Medicine, after approval of the institutional animal experiment committee. PARTICIPANTS: Thirteen mongrel dogs of either sex, weighing 9.0-14.5 kg, were the subjects of the study. INTERVENTIONS: They were anesthetized with pentobarbital and paralyzed with pancuronium. 30 ml x kg(-1) of 3% dextran in lactated Ringer's solution was given twice to induce a hypervolemic state. MEASUREMENTS AND RESULTS: In each stage of the fluid loading, the IDVG, plasma volume and CO were measured simultaneously. The IDVG was measured following an injection of glucose (100 mg x kg(-1)) by repeated determinations of plasma glucose (3-7 min) with a one-compartment model, and plasma volume was assessed similarly following ICG (0.5 mg x kg(-1) injection. A closer correlation was observed between the IDVG and plasma volume (r = 0.79, p < 0.001) than between the IDVG and CO (r = 0.48, p < 0.01). CONCLUSION: The IDVG correlates with plasma volume following volume challenge in dogs. CO did not show a consistent change following volume challenge. The IDVG may have the potential as an indicator to presume the fluid volume in the body, even though the IDVG cannot be used directly to estimate plasma volume.

[Production of a monoclonal antibody against Cry j 2].

The peptide possessing N-terminal 10 amino acid sequence of Cry j 2 was chemically synthesized and conjugated to KLH-carrier protein. Rabbits were immunized with this haptenized protein and the antibody (N-10 antibody) was obtained. Cross-blot analysis revealed that N-10 antibody recognized the hapten moiety. Purification of Cry j 2 was carried out with monitoring its reactivity with N-10 antibody. The substance reactive to N-10 antibody existed in the DEAE-Sephadex unadsorbed fraction. The substance reactive to N-10 antibody was subjected to CM-Sephadex equilibrated with 10 mM acetate buffer (pH 5.0). In contrast to the previous report on Cry j 2, the substance reactive to N-10 antibody existed in the CM-Sephadex unadsorbed fraction. The CM-Sephadex unadsorbed fraction was concentrated with 80% saturated ammonium sulfate, and applied to a Superdex pg 200 column. The major peak of protein was regarded as the final preparation and was subjected to SDS-PAGE. The substance reactive to N-10 antibody had a MW of 40 kDa under reducing and 37 kDa under non-reducing conditions, respectively. Furthermore, the substance reactive to N-10 antibody showed potent allergenic activity. These data completely agreed with the previous data on Cry j 2 and strongly suggested that the final preparation contained a large amount of Cry j 2. BALB/c mice were immunized intraperitoneally with the final preparation to produce a monoclonal antibody against Cry j 2. Eleven clones reacting to the final preparation were obtained, and the antibodies produced from these clones did not react with purified Cry j 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Does the initial distribution volume of glucose reflect plasma volume after haemorrhage in dogs?

To test the hypothesis that the initial distribution volume of glucose (IDVG) reflects plasma volume, the relationship between the IDVG and indocyanine green (ICG) assessments of plasma volume (Vd-ICG) were evaluated simultaneously both before and after induced haemorrhage (30 ml.kg-1) in eight mongrel dogs. The IDVG and the Vd-ICG were calculated with a one-compartment model from repeated measurements of plasma glucose three to seven minutes, and of plasma ICG three to nine minutes after simultaneous infusions of both glucose 100 mg.kg-1 and ICG 0.5 mg.kg-1. The IDVG calculated with a one-compartment model (IDVG-OCM) was also compared with a two-compartment model within 15 min (IDVG-TCM) on nine occasions among a total of 12 determinations. Using Bland and Altman analysis to compare the two analytical models, the IDVG-OCM tends to overestimate the IDVG-TCM by an average of 0.04 L. Although the IDVG-OCM was two to three times larger than the Vd-ICG at each corresponding point, a correlation was obtained between the IDVG-OCM and the Vd-ICG before and after induced haemorrhage (r = 0.85, n = 16, P < 0.001). We conclude that the IDVG reflects plasma volume in normal and hypovolaemic dogs, although the IDVG cannot be used directly to estimate plasma volume.

[Changes in rectal temperature during tympanoplasty under general anesthesia].

We measured rectal temperature of 35 patients who underwent tympanoplasty under various types of general anesthesia. They were allocated at random to three groups according to the types of general anesthesia; 10 patients of enflurane-N2O (enflurane group); 10 patients of neuroleptic anesthesia with droperidol, pentazocine-N2O (NLA group); 15 patients of total intravenous anesthesia with droperidol, fentanyl and ketamine (DFK group). After the induction of anesthesia, their rectal temperature was continuously monitored with an electric thermometer of NEC San-ei throughout the surgical procedure. Increase in rectal temperature was observed in all three groups, and increase in DFK group was significant compared with other two groups. It is possible that the significant increase in rectal temperature in DFK group is supposedly due to normally maintained hypothalamic thermoregulatory function as well as direct surgical stimulation to central nervous system.

Antigenic relationship between the house dust mite Dermatophagoides farinae and the predacious mite Phytoseiulus persimilis.

We have examined the antigenic relationship between the house dust mite Dermatophagoides farinae and the predacious mite Phytoseiulus persimilis. Immunoblotting analysis demonstrated that there was a very weak antigenic cross-reactivity between these different suborder of mites but that this cross-reactivity was not attributed to D. farinaes major allergen's, Der fI and Der fII. These results suggest that P. persimilis might scarcely provoke allergic symptoms in patients sensitized to house dust mites.

All-or-none like responses in increment of cytoplasmic free calcium concentrations to histamine in single smooth muscle cells of guinea pig trachea.

Single smooth muscle cells were isolated from guinea pig trachea and cultured for 48 hours. The cells were then loaded with a fluorescent-free calcium indicator, fluo-3. Concentrations of cytoplasmic-free calcium ([Ca2+]i) were determined by using a confocal laser scanning fluorescence imaging system. Cells exposed to histamine at concentrations less than 1.0 microM did not indicate any changes of the fluorescence intensities, but stayed at the basal level of [Ca2+]i. Cells exposed to histamine at a concentration in the range of 1.5 to 10 microM indicated marked increases in [Ca2+]i. A further increase in [Ca2+]i by application of higher concentrations of histamine was not observed. The threshold concentration of histamine to increase [Ca2+]i and peak [Ca2+]i varied from cell to cell. In addition, half-maximal response time was shortened with increasing concentrations of histamine from 1.5 to 100 microM. These results indicate that [Ca2+]i responses to histamine is an all-or-none type in each cell. The heterogeneity in the required threshold concentration of histamine to increase in [Ca2+]i and the concentration dependency in half-maximal response time of histamine-induced [Ca2+]i increase may be related to the graded responses of histamine-induced contractions in preparations of the tracheal tissue.

[Effect of pirenzepine on gastric secretion during anesthesia and surgery].

Effect of pirenzepine on gastric secretion during anesthesia and surgery was evaluated in 46 surgical patients ranged in age from 18 to 68 years. The patients underwent orthopedic, ophthalmic, ENT, plastic or non-abdominal general surgery under neuroleptanesthesia except two patients who had enflurane or isoflurane anesthesia. They received either pirenzepine 10 mg, 20 mg or the combination of pirenzepine 10 mg and famotidine 20 mg intravenously just before the induction of anesthesia. Volume and acidity of gastric juice were measured for 3 hours after the administration of these agents. Decrease in volume and acidity of gastric juice after pirenzepine 10 mg as well as after pirenzepine 20 mg continued for more than 3 hrs after the administration of the agents. Efficacy of the combination of pirenzepine and famotidine on gastric secretion was more prominent than that of pirenzepine alone in a double dose.

Immunocytochemical studies of major gap junction proteins in rat salivary glands.

We examined protein components of the gap junctions between acinar cells of the parotid, sublingual and submandibular glands of the rat, using type-specific antibodies directed against major gap junction proteins, connexin32 (Cx32) and connexin26 (Cx26). Double-immunofluorescence microscopy revealed that fluorescent spots of both connexins in the parotid and sublingual glands were distributed between the apposed regions of acinar cells. They appeared together, or were co-localized. The intensity of the Cx26-associated fluorescent signals was relatively weak in the submandibular glands compared with the other glands and was absent from some acini. When present, these spots were always co-localized with Cx32 immunoreactive positive spots. The results suggest that Cx32 and Cx26 in rat salivary glands are colocalized within the same gap junctional plaques when simultaneously expressed by the same acinar cells.

[Clinical study on intraoperative hyperketonemia in non-diabetic surgical patients under general anesthesia].

We measured plasma 3-hydroxybutyrate (3-OHBA) concentrations in 925 non-diabetic surgical patients who underwent various surgical procedures under various types of general anesthesia. Lactated Ringer's solution only was used as an intraoperative fluid. Among them, 46 patients (4.9%) developed high 3-OHBA levels of over 500 microM.l-1 during surgery. The causes of observed hyperketonemia would be surgical stress and preoperative fasting irrespective of 8 types of general anesthesia. Blood glucose, lactic acid, base excess and pH were not influenced by hyperketonemia. Prolonged recovery from anesthesia was not observed in any patients with hyperketonemia. Without any specific treatments, plasma 3-OHBA levels were unchanged or rather decreased in 70% of the patients whose 3-OHBA levels were over 500 microM. The results suggest that hyperketonemia below 500 microM.l-1 is not always disadvantageous in nondiabetic patients under general anesthesia.

Trypsin-like protease of mites: purification and characterization of trypsin-like protease from mite faecal extract Dermatophagoides farinae. Relationship between trypsin-like protease and Der f III.

A serine protease from mite faecal extract, Dermatophagoides farinae, was purified using DEAE-Sephacel anion exchange chromatography and Superdex 75 pg gel chromatography. The molecular weight of this protease was 34 kD on SDS-PAGE under reducing conditions. The optimal pH and temperature of the protease were 8.0 and 47 degrees C, respectively. In addition, this protease cleaved arginyl or lysyl residue containing substrates selectively and was only inhibited by aprotinin, FUT-175, and soy bean trypsin inhibitor and not by chymostatin, E-64 and iodoacetic acid. These results show that our purified serine protease belongs to the trypsin-type. Purified trypsin-like protease was shown to be allergenic by enzyme-linked immunosorbent assay. Antigenicity of trypsin-like protease was completely different from those of Der f I and Der f II. Both, 20 N-terminal amino acid sequence and amino acid compositions of the purified protease were very similar to those of Der f III. Good similarities were found between trypsin-like protease and Der f III concerning physicochemical properties such as molecular weight on SDS-PAGE and ammonium sulphate solubility. Summarizing the above data, it can be concluded that a trypsin-like protease from mite faecal extract is actually the Der f III allergen and that it may be involved in the digestive process of the mite as it was found not in mite body but in mite faeces.

[Clinical study on total intravenous anesthesia with droperidol, fentanyl, and ketamine--18. Effect on peripheral circulation as judged by core-peripheral temperature gradient].

Effect of total intravenous anesthesia with droperidol, fentanyl and ketamine (DFK) on peripheral circulation was studied by examining core-peripheral temperature gradient in twenty five patients who underwent abdominal surgery. A core temperature probe was attached on the forehead and peripheral probe on the palm of the hand of the side on which the blood pressure cuff was not applied. The temperature gradient was less than three degrees centigrade in 60% of the patients and the gradient was significantly less as compared with that of isoflurane anesthesia even at 300 minutes after the start of surgical operation. This advantage would have been caused by such factors as circulatory stimulating effect of ketamine, sympathetic blocking effect by droperidol and adequate postoperative analgesia by fentanyl and norketamine, a metabolite of ketamine. The results suggest that DFK would exert a beneficial effect on peripheral circulation, particularly during prolonged surgical procedures.

Is a trypsin-like protease of mites a Der f III allergen?

A trypsin-like protease was purified from mite (Dermatophagoides farinae) fecal extract. In SDS-PAGE, the mite trypsin-like protease showed a single band at 34 kD. The purified trypsin-like protease possessed potent allergenic activity. Both the twenty N-terminal amino acid sequence and the amino acid composition of the purified protease were very similar to those of Der f III. These data strongly suggest that the trypsin-like protease in the mite is a Der f III allergen.

Effect of acute administration of cadmium on distribution of zinc in the hamster.

Acute administration of sc doses of Cd (1mg/kg . d, 3 or 6 d) to male golden hamsters resulted in a remarkable dose-dependent increase of Zn in the liver and kidney. In contrast, Zn contents in the heart and testes showed a significant dose-dependent decrease. No change was found in Zn contents of the bone. The correlation coefficients between contents of Cd and Zn in the liver and kidney were much higher in metallothionein fractions than in the corresponding whole organs. These results suggest that Cd induces the synthesis of metallothionein in the liver and kidney, leading to simultaneous accumulation of Cd and Zn in the organs; this in turn decreases the Zn contents in other organs, where weak or no induction of metallothionein synthesis takes place. Therefore Cd might induce Zn deficiency in humans or animals whose pool size and intake of Zn are marginal.