Developing Transposable Element Marker System for Molecular Breeding.

Transposable element (TE) marker system was developed considering the useful properties of the transposable elements such as their large number in the animal and plant genomes, high rate of insertion polymorphism, and ease of detection. Various methods have been employed for developing a large number of TE markers in several crop plants for genomics studies. Here we describe some of these methods including the recent whole genome search. We also review the application of TE markers in molecular breeding.

Novel QTLs for photoperiodic flowering revealed by using reciprocal backcross inbred lines from crosses between japonica rice cultivars.

The rice japonica cultivars Nipponbare and Koshihikari differ in heading date and response of heading to photoperiod (photoperiod sensitivity). Using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers, we conducted quantitative trait locus (QTL) analyses for heading date in a set of reciprocal backcross inbred lines (BILs) from crosses between Nipponbare and Koshihikari. Under natural-day conditions, transgressive segregation in days to heading (DTH) toward both early and late heading was observed in both BIL populations. QTL analyses revealed that two QTLs--on chromosomes 3 and 6--were involved in the difference in heading date between the parental cultivars. The Nipponbare allele at the QTLs on chromosomes 3 and 6 showed, respectively, increasing and decreasing effects on DTH in both BIL populations. The transgressive segregation observed in the BILs could be accounted for mainly by the complementary action of a set of alleles with opposing effects. Both QTLs were finely mapped as single Mendelian factors in secondary mapping populations (BC2F2 plants/BC2F3 lines). The QTL on chromosome 3 was mapped in the 1,140-kb interval between 94O03-4 (SSR) and OJ21G19-4 (SNP) and was designated Hd16. The QTL on chromosome 6 was mapped in the 328-kb interval between P548D347 (SSR) and 0007O20 (SSR) and was designated Hd17. Both Hd16 and Hd17 were involved in photoperiod sensitivity, as revealed by observation of the DTH of nearly isogenic lines of Nipponbare under short- and long-day conditions, suggesting that allelic differences in both Hd16 and Hd17 account for most of the difference in photoperiod sensitivity between the parental cultivars.

Dot-blot-SNP analysis for practical plant breeding and cultivar identification in rice.

We report dot-blot hybridization with allele-specific oligonucleotides for single nucleotide polymorphisms (SNPs) analysis to be applicable for practical plant breeding and cultivar identification. Competitive hybridization of a digoxigenin-labeled oligonucleotide having the sequence of a mutant allele (or a wild-type allele) together with an unlabeled oligonucleotide having the sequence of a wild-type allele (or a mutant allele) was highly effective to reduce background signals in dot-blot hybridization. All 100 tested genes (200 alleles) in rice having SNPs or insertions/deletions were detected in an allele-specific manner. Genotypes of 43 rice cultivars were identified by this technique, and eight SNP markers were found to be sufficient for distinguishing all the cultivars from each other. Dot-blot analysis was also applied to genotyping of Wx and Sd1 of F4 plants in a conventional breeding program. Since dot-blot analysis with competitive hybridization provides a highly reliable, simple, and cost-effective technique for SNP analysis of a large number of samples, this technique is expected to realize the practical use of a novel breeding method, in which plants or breeding lines are selected by SNP analyses of many genes in a laboratory.

Extended surgery with en bloc resection of the right common iliac vessels for lymph node metastasis of mucinous colon carcinoma: report of a case.

We report herein the case of a 63-year-old woman who underwent surgery for recurrent mucinous carcinoma of the cecum. Recurrent metastatic lymph nodes had invaded the right common iliac vessels and right ureter, but she had no distant metastases and no peritoneal dissemination. Extended surgery with en bloc resection of the right iliac vessels and right ureter, and femorofemoral bypass were performed. Postoperatively, several complications developed which were successfully treated by further operations. By 1 year after surgery, she had no recurrent tumors on radiological examination, suggesting that our aggressive surgery with resection of the invaded regional vessels had effectively removed the recurrent tumors. This procedure may therefore significantly prolong the survival time and improve the quality of life of such patients.

Radiotherapy for liposarcoma of the vulva.

BACKGROUND: Primary liposarcoma of the vulva is extremely rare. We report a case of liposarcoma of the vulva which was treated with local excision and postoperative radiotherapy. CASE: A 21-year-old woman complained of a painless lump in her labium majus which she first noticed 3 years earlier. An initial diagnosis of lipoma of the vulva was made. The patient was treated by surgical removal. On examination of the surgical specimen, the final pathological diagnosis was well-differentiated liposarcoma and the stalk was not excised completely. Radiotherapy was initiated because of the uncertainty of the patient's prognosis. Eighteen months after radiotherapy, no evidence of local recurrence, metastasis, and late complication has been seen. CONCLUSION: This case is suggestive of the contribution of postoperative radiotherapy, although not conclusive, and continued monitoring is necessary because recurrence or metastasis can occur years later.

Transpedicular fixation with Zielke instrumentation in the treatment of thoracolumbar and lumbar injuries.

STUDY DESIGN: Sixty-five patients who underwent transpedicular fixation for thoracolumbar and lumbar injuries were studied for type of injury, the severity of paralysis, the degree of postoperative correction, and instrumentation failures. OBJECTIVES: To evaluate the surgical approaches and the selection of instrumentation to determine indications for using the transpedicular fixation procedure. SUMMARY OF BACKGROUND DATA: Various transpedicular fixation devices have been used for different type of injuries, and satisfactory postoperative results were not obtained in some studies. METHODS: Forty patients had burst fractures, 19 had fracture dislocations, and six had chance-type fractures. An anterior decompression procedure was used for most cases of burst fracture and some cases of fracture dislocation where anterior compression factors were present. The Zielke or modified Zielke system was used as an internal fixator for posterior segmental fixation. RESULTS: No patient had neurologic deterioration after surgery. Twenty of 28 patients with incomplete lesions improved postoperatively according to Frankel grades. The instrumentation failed in only one patient, in whom a nonunion developed. CONCLUSION: With transpedicular fixation, it is possible to provide solid internal fixation that is circumscribed to the injured vertebral segments. The elasticity of the Zielke rod makes it an excellent transpedicular fixation device because it is easily attached and reduction is easily performed. Anterior decompression with fusion needs to be used with transpedicular fixation in the treatment of injuries (especially burst fractures).

[Quality assurance for histodiagnosis and cytodiagnosis with a model of proficiency testing].

A quality assurance (QA) program for histopathology and cytology has not yet been completed although an inhouse quality control for technical standardization is being tested. The fact that a strict Performance Improvement Program of CAP (College of American Pathologists) for cytology is required separately from other laboratory tests emphasizes the importance of cytology, since erroneous cytology reports would directly cause incorrect clinical diagnosis. The results of questionnaires gathered by the QA Committee of the Japan Association of Registered Clinical Laboratories on preparation techniques, workload limits of pathologists and technologists, double diagnostic systems, error detection programs, and specialization of personnel are presented. In addition to pathology and cytology proficiency testing provided by CAP survey, a model of blind QC (quality control) in our laboratory is discussed; for diagnostic standardization of histopathology, reexamination of randomly selected cases by another pathologist and reexamination of previously diagnosed cases by the same doctor are periodically performed. For error detection of cytoscreening, cytologists are obligated to reexamine random samples of 1 to 10% of negative gynecological slides. Evaluation of sufficiency of slides as required by the Bethesda System is referred to diagnostic interpretation.

Tryptophan metabolism by the isolated rat liver cells--effects of leucine and its metabolites.

1. The effects of leucine and its metabolites, such as alpha-ketoisocaproate and ketone bodies, on the metabolic fluxes of tryptophan were investigated in isolated rat liver cells using [benzene ring-U-14C]- or [methylene-14C]-tryptophan. 2. Tryptophan metabolized through the kynureninase flux decreased while that metabolized to acetyl CoA remained unchanged in the presence of leucine or its metabolites. Accordingly an amount of tryptophan metabolized through the quinolinate-NAD pathway, which was estimated by subtracting an amount of tryptophan metabolized via the acetyl CoA flux from that via the kynureninase flux, was decreased in the presence of leucine or its metabolites. 3. Less quinolinate accumulated during incubation with metabolites of leucine, however, the amount was still sufficient to saturate quinolinate phosphoribosyltransferase (EC 2.4.2.19). 4. Leucine and its metabolites added in vitro at 1 mM level did not inhibit quinolinate phosphoribosyltransferase activity in rat liver homogenate. 5. The results indicate that a decrease in NAD biosynthesis from tryptophan caused by metabolites of leucine in the isolated rat liver cells was neither due to insufficient supply of quinolinate nor to direct inhibitory effect on quinolinate phosphoribosyltransferase.

Acute leukemia of megakaryocyte lineage with tumor formation. An autopsy case of patient with Down's syndrome.

In this report we present the autopsy findings of acute megakaryoblastic leukemia with tumor formation in a 2-year-old female infant with Down's syndrome. Chromosomal analysis of blast cells revealed constitutional anomaly of trisomy 21 and two other related types of abnormal clones. Flow cytometric examination revealed blast cells expressing Ia-like or HLA-DR antigens. Postmortem examination showed extensive infiltration of leukemic cells in most of the examined organs, including the bone marrow with myelofibrosis. Tumor masses in the maxillary, frontal and femoral bones and the atria of the heart had undergone massive infiltration of atypical blast cells with an increase in the reticulin network. The final diagnosis was confirmed by ultrastructural cytochemistry of the platelet peroxidase reaction as well as by immunological staining utilizing anti-platelet glycoprotein IIb/IIIa, antiplatelet factor 4 and anti-beta-thromboglobulin antibodies for the blast cells. It seems likely that platelet-derived growth factor, secondary to an increase in the reticulin network, plays a major role in myelofibrosis of acute megakaryoblastic leukemia with tumor formation.

Studies on the initial reactive sites for platelet activation. Cationized ferritin- and wheat germ agglutinin-binding sites on the cells.

The binding sites for cationized ferritin or ferritin-conjugated wheat germ agglutinin on the cell surface were studied on platelets before or after fixation in glutaraldehyde. The effects of neuraminidase on the binding sites were also demonstrated after fixation of the platelets. Changes in the binding sites and distribution pattern due to exposure to these ligands were further investigated in the unfixed platelets under a variety of conditions such as incubation time and medium. The fixed platelets incubated with either ligand showed an even and continuous distribution of particles on the cell surface. In the unfixed platelets, the ligands were rapidly moved and aggregated probably by lateral migration after binding to the cell surface. The ligands were also bound to the membrane surface and simultaneously appeared in the interior of the open canalicular system. As the binding sites were moved on the cell surface as well as into the open canalicular system, morphological changes suggestive of platelet secretion occurred. The binding sites of either ligand were redistributed on the platelet cell surface. Glycoprotein Ib, thought to be the receptor site for wheat germ agglutinin on the cell surface, contains sialic acids that contribute to the negative charge of platelets. Therefore, glycoprotein Ib may play an important role as the initial reactive site for thrombotic stimuli.

[Anti-tumor activity of peripheral lymphocytes of tumor-bearing rats by in vitro culture].

This basic study was performed to determine whether the anti-tumor effect of lymphocytes obtained from the peripheral blood of a tumor bearing host can be increased when cultured in vitro with Mitomycin C (MMC)-treated AH109A tumor cells, using interleukin-2 (IL-2). Donryu rats were used as tumor bearing hosts. The following results were obtained. Weak anti-tumor activity of lymphocytes was noted 7 days after lymphocytes were cultured in the medium to which only IL-2 was added. Anti-tumor activity was augmented by adding antigen (MMC treated tumor cells) to the above (1). Anti-tumor activity was further augmented by adding to (2) above antigen presenting cells such as intraperitoneal exudate macrophages or peripheral hole leukocytes. Anti-tumor activity of lymphocytes was detected when IL-2 and MMC treated antigen were added to the peripheral hole leukocytes containing the lymphocytes.

[Experimental studies of plasma exchange therapy for malignant tumor].

We conducted the experiment to evaluate the effect of plasma exchange in treatment of cancer. Donryu rats of 8-10 week old female were used: 1 X 10(6) AH109A were inoculated subcutaneously in the back of rats. We performed comparative study on four groups: 1) untreated group; 2) plasma exchanged group; 3) MMC treated group; and 4) plasma exchange + MMC treated group. Plasma exchange was conducted from 8th day of the tumor inoculation for three consecutive days. The plasma contained in 6ml blood was taken out from the tumor-bearing rats and same volume of fresh frozen normal serum was replaced. MMC (0.5 mg/kg/day) was administered intraperitoneally from 13th day of the tumor inoculation for five consecutive days. Efficacy of plasma exchange therapy was evaluated by tumor inhibition effect as well as by 50% survival time. Plasma exchanged group showed no tumor inhibition effect, however a considerable extention of survival of 36 days was observed compared with the untreated group (29.3 days). Five out of 14 cases of MMC treated group showed tumor inhibition effect and survived for 36.5 days. Six out of 10 cases of plasma exchange + MMC treated group showed tumor inhibition effect and the survival time was 49 days. The above findings indicate: plasma exchange therapy has effects on prolongation in survival time, but has no effects on tumor inhibition and; a combination of plasma exchange and chemotherapy (MMC) produces greater tumor inhibition effect and longer survival compared to plasma exchange therapy alone and chemotherapy alone.

Simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine by high-performance liquid chromatography using electrochemical detection.

A rapid, selective and sensitive method for the simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine has been developed using high-performance liquid chromatography with electrochemical detection. The unchanged drugs and internal standard extracted from plasma and urine were separated by reversed-phase high-performance liquid chromatography. The influence of acetonitrile concentration and of the pH of the mobile phase were investigated. The detection limits were 100 pg for chlorpromazine and for levomepromazine. In comparison with three other detection systems this was found to be the most sensitive method. This method was successfully applied to the simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine for pharmacokinetic studies.

Type III glycogenosis with deposition of urate and amyloid.

A case of a 44-year-old man with hepatic form of glycogenosis was presented. The patient had abdominal distension and muscular weakness. The glucose tolerance test showed a diabetic pattern, though he had hypoglycemia in fasting state. The fructose tolerance test showed an ability of conversion from fructose to glucose. The double glucagon test showed no rise of blood glucose in fasting state but a rise 2 hours after meal. These symptoms and laboratory data supported the clinical diagnosis of type III glycogenosis. At autopsy, glycogen was markedly deposited in the liver, and slightly in the kidneys and heart. The glycogen pooled in the hepatic cells histochemically showed a normal reaction to several glycogen stainings. Electron microscopy by using Thiéry's method revealed that the pooled glycogen particles were clearly arranged as rosettes measuring 1,000A in largest diameter composed of clustered monoparticulates. There were marked hyalinization of the islets of Langerhans containing amyloid. As to its pathogenesis, this change can be interpreted as a morphological expression of the hypofunction of beta-cells ascribed to long-standing hypoglycemia.

Widespread eczema vaccinatum acquired by contacts. A report of an autopsy case.

A 4-month-old male infant predisposed to allergic dermatitis acquired wide-spread eczema vaccinatum by contacts with a recently vaccinated sibling. He died of acute purulent peritonitis following a perforation of multiple duodenal ulcers. Fluorescence immunocytochemical and electron microscopic studies on the skin lesions revealed the presence of viral antigens and numerous virus particles compatible morphologically with those of the mature form from the same batch of smallpox vaccine given to the sibling. A large number of virus particles in the developmental form were also predominantly scattered in the cytoplasm of cells at the stratum malpighii of the epidermis as well as in neutrophils and macrophages in the skin lesions. The virus isolation from the skin lesions was done by using the HeLa cells and the human embryonic lung fibroblasts. No abnormal laboratory data were noted in immunoglobulins. On the basis of atrophy of the thymus and other lymphatic tissues and an appearance of large pyroninophilic cells in association with blastoid transformation, the authors discussed a possible participation of the disturbance of cellular immunity secondary to the virus infection in the development of the disease.

Familial medullary carcinoma of the thyroid through 3 generations.

A family with high genetic penetrance of medullary carcinoma of the thyroid was reported. Seven proven (6 patients and one autopsy case) and 2 probable cases of medullary carcinoma were present in 25 members through 3 generations. An endogemy had intervened in the prior generation of these cases. Preliminary results in clinicopathological examinations of 7 proven cases were as follows; clinically, all of the cases showed B type blood group. Glycosuria was found in 2 cases, and diarrhea in one case. Serum thyrocalcitonin being estimated in two cases showed high levels. The autopsy cases coexisted with medullary carcinoma of the thyroid and pheochromocytoma of the right adrenal. Pathologically, the majority of tumors occurred in both thyroid lobes, and were present from the middle to upper portion of the thyroid. The tumor showed a variety of histological features even in the same tumor. In the tumor cell, numerous membrane-limited granules were seen with an electron microscope. Amyloid was demonstrated only in the tumor tissue.

Localization of ferritin-conjugated anti-fibrin-fibrinogen in platelet aggregates produced in vitro.

A ferritin-conjugated anti-fibrin/fibrinogen was localized by means of light and electron microscopy in artificial in vitro thrombi formed in the presence of the labeled antibody, and in preformed ADP-induced platelet aggregates. The ferritin was distributed throughout the central and peripheral regions of the columns of aggregated platelets in the thrombi. In the preformed ADP aggregates, ferritin was deposited only by infiltration from surrounding plasma and was confined to the periphery of the columns. The even distribution of ferritin in the central zone of the platelet columns of the thrombi indicated a specific reaction had occurred before or during thrombus formation unrelated to infiltration of plasma. In the artificial thrombi, the ferritin-labeled antibody was localized on the surface layer of platelets and on the bridging structures composed of the combined surface layers in the narrow spaces between cohesive platelets. Vesicles and alpha granules within the platelets also were tagged. The absence of obvious fibrin between narrow interspaces and within the platelets indicated that the antibody had reacted with fibrinogen or partly polymerized fibrin at these sites. Many invaginations of the platelet membrane containing dense fibrillar material were interpreted to be alpha granules discharging their contents during the "release reaction" at the time of aggregation. This material, which was tagged by the ferritin-conjugated antibody, merged with the interplatelet bridges to suggest that released fibrinogen from within the platelet contributed to the structural bond and strengthened it. A layer of dense fibrin and altered platelets in the periphery of the columns of aggregated platelets in the artificial thrombi contained the platelets and limited further growth of the aggregates. The fibrin was thought to be derived from infiltrated plasma as well as from released intraplatelet fibrinogen. Platelet fibrinogen thus appeared to take part both in the cohesion of aggregated platelets and in the stabilization of the aggregates formed.