Roles of histone H3.5 in human spermatogenesis and spermatogenic disorders.

Histone H3.5 (H3.5) is a newly identified histone variant highly expressed in the human testis. We have reported the crystal structure, instability of the H3.5 nucleosome and accumulation around transcription start sites, mainly in primary spermatocytes, but its role in human spermatogenesis remains poorly understood. Testicular biopsy specimens from 30 men (mean age: 35 years) with non-obstructive azoospermia (NOA) who underwent microdissection testicular sperm extraction and 23 men with obstructive azoospermia (OA) were included. An H3.5-specific mouse monoclonal antibody recognizing an H3.5-specific synthetic peptide was generated, and immunohistological staining for H3.5 and proliferating cell nuclear antigen (PCNA) was performed on Bouin's solution-fixed sections. Expression and localization of H3.5 were compared with patient background, germinal stage, and PCNA expression. In testes of patients with normal spermatogenesis, differentially expressed H3.5 was specifically localized in either spermatogonia or preleptotene/leptotene-stage primary spermatocytes, especially during germinal stages VI-X. In NOA testes, mRNA expression of H3.5 (H3F3C) was significantly reduced compared with other H3 histone family members, and expression of H3.5 was significantly lower than that in OA. Additionally, the number of H3.5-positive germ cells was higher in hypospermatogenesis or late maturation arrest than in early maturation arrest in NOA testes (p < 0.01). A significant positive correlation was observed between H3.5 and PCNA expression (p < 0.05) but not TUNEL-positive cells, and expression of H3.5 was enhanced after hCG-based salvage hormonal therapy. Different from other testis-specific histones, which are often expressed during the histone-to-protamine transition during meiosis, H3.5 was expressed mainly in immature germ cells. H3.5 may play roles in DNA synthesis, but not apoptosis, and its expression is regulated by gonadotropins, indicating that such epigenetic regulations are important in normal spermatogenesis and spermatogenic disorders.

ß-Catenin signaling regulates Foxa2 expression during endometrial hyperplasia formation.

The Wnt/ß-catenin signaling is essential for various organogenesis and is often implicated during tumorigenesis. Dysregulated ß-catenin signaling is associated with the formation of endometrial adenocarcinomas (EACs), which is considered as the common form of endometrial cancer in women. In the current study, we investigate the downstream target of Wnt/ß-catenin signaling in the uterine epithelia and the mechanism leading to the formation of endometrial hyperplasia. We report that conditional ablation and activation of ß-catenin in the uterine epithelia lead to aberrant epithelial structures and endometrial hyperplasia formation, respectively. We demonstrate that ß-catenin regulates Foxa2 with its candidate upstream region for the uterine epithelia. Furthermore, knockdown of Foxa2 leads to defects in cell cycle regulation, suggesting a possible function of Foxa2 in the control of cell proliferation. We also observe that ß-catenin and Foxa2 expression levels are augmented in the human specimens of complex atypical endometrial hyperplasia, which is considered to have a greater risk of progression to EACs. Thus, our study indicates that ß-catenin regulates Foxa2 expression, and this interaction is possibly essential to control cell cycle progression during endometrial hyperplasia formation. Altogether, the augmented expression levels of ß-catenin and Foxa2 are essential features during the formation of endometrial hyperplasia.

[Report on the 89th Scientific Assembly and Annual Meeting of the Radiological Society of North America--analysis of radiation dose by a change of a scan parameter of multi-slice computed tomography by a film method].

PURPOSE: It is for a purpose of this study to measure radiation dose by analyzing a dose profile of multi-slice computed tomography varying with helical pitch and a row slice thickness difference complicatedly. MATERIALS AND METHODS: We used multi-slice computed tomography, and helical pitch and row slice thickness change and scanned the helical scan. I used CTDI phantom of a diameter of 25 cm and I inserted roentgen diagnosis use film UR-2(new) which I put between my own phantom in center and 1 cm away from the outer surface and scanned it. And the provided level profile was converted into a dose profile with the dose-density curve which I made beforehand. I analyzed radiation dose than the dose profile. RESULT: In multi-slice computed tomography, radiation dose varied with assembly of row slice thickness and helical pitch. The change of a dose profile changed in a phantom surface part complicatedly. The maximum dose by the measurement of this time was 29 mGy in row slice thickness 0.5 mm, assembly of helical pitch 2.5. In addition, the minimum dose was 6.8 mGy in row slice thickness 3.0 mm, assembly of helical pitch 5.5. And, as for the difference of maximum dose in the same dose profile and the smallest dose, there were about 20 % in row slice thickness 1.0 mm, assembly of helical pitch 5.5. CONCLUSION: The dosimetry of multi-slice computed tomography by a film method enabled it to measure a change of a dose profile by a difference of a scan parameter by high interest solution ability. In addition, it is a method more superior in dosimetry of multi-slice computed tomography spreading through a Z-axis direction broadly than determination by computed tomography use ionization chamber dosimeter. Because radiation dose increases by a scan in thin row slice thickness and small helical pitch, care is necessary.

[Aortic root redissection associated with the use of gelatin-resorcin-formalin glue following surgical treatment for acute type A aortic dissection; report of a case].

A late redissection case of the aortic root after total arch replacement for acute Stanford type A aortic dissection was reported. A 55-year-old male was treated with total arch replacement for acute Stanford type A aortic dissection. The aortic valve was bicuspid valve, and the right coronary leaflet was prolapsed because of the dissection of right and non coronary cusp. Resuspension of the commissure and the fixation of the dissected aortic wall with gelatin-resorcin-formalin (GRF) glue was performed during the operation. The initial postoperative course was uneventful and the patient discharged 52 days after the operation. Redissection of aortic root was pointed out on the computed tomography (CT) 3.5 years after the operation. As the second operation, the aortic root replacement was performed. Coronary artery bypass for right coronary artery was simultaneously performed with right internal thoracic artery because the right coronary ostium was stenotic and showed ischemic change on electrocardiogram monitor during the operation. The redissection was seen on the right coronary sinus, which was fixed by the GRF glue during the first operation. The pathological study showed the migration of macrophages and the tear of the fibrous tissue. These findings was thought to be associated with the use of the GRF glue. Careful use of the GRF glue for the fixation of the dissected aorta during the surgical treatment for the Stanford type A aortic dissection was thought to be important.

[Surgical treatment for ruptured descending thoracic aortic aneurysm with the left lung hematoma; report of a case].

An 80-year-old female was admitted with sudden onset of back pain and hemoptysis. The diagnosis was ruptured descending aortic aneurysm with the left lung hematoma. Emergency operation was performed. The graft replacement of the ruptured descending thoracic aneurysm and left lower lobectomy was done. She was extubated on the 1st postoperative day. The postoperative course was uneventful without pulmonary and graft complications. We thought that concomitant left lower lobectomy was useful in this patient.

Enhancement of metabolizing herbicides in young tubers of transgenic potato plants with the rat CYP1A1 gene.

A rat P450 monooxygenase gene ( CYP1A1) was introduced into potato plants to enhance the metabolism of the environmental contaminants in subterranean organs. The CYP1A1 gene was kept under the control of the potato patatin promoter to enhance tuber-specific expression. A total of 106 transgenic plants (PAT1A1 plants) were obtained following selection by a resistance test to kanamycin and PCR analysis. PAT1A1 plants treated with 10% exogenous sucrose showed a higher activity of monooxgenase in the leaves than the non-transgenic plants. This indicated that the activity enhanced by 10% sucrose was due to the patatin promoter containing the sucrose-inducted elements. One representative transgenic plant, Ag2197, was selected on the basis of monooxgenase activity in the leaves and Western blot analysis. Ag2197 was found to accumulate a large amount of CYP1A1 mRNA and protein in the developing tuber but not in the mature tuber. The residual herbicides, atrazine and chlortoluron, were analyzed in the micro-tubers of Ag2197 and non-transgenic plants. The amount of residual herbicides in Ag2197 was much lower than that in the non-transgenic plant, indicating that the transgenic plant metabolized the herbicides to a detoxified form. The transgenic plants produced in this study might be useful for the phytoremediation of chemical pollution in the soil.

Inducible cross-tolerance to herbicides in transgenic potato plants with the rat CYP1A1 gene.

A gene of the enzyme involved in xenobiotic metabolism in mammalian liver was introduced into potato to confer inducible herbicide tolerance. A rat cytochrome P450 monooxygenase, CYP1A1 cDNA, was kept under the control of the tobacco PR1a promoter in order to apply the system of chemical inducible expression using the plant activator Benzothiadiazole (BTH). Transgenic plants were obtained based on the kanamycin resistance test and PCR analysis. Northern-blot analysis revealed the accumulation of mRNA corresponding to rat CYP1A1 in the transgenic plants treated with BTH (3.0 micro mol/pot), whereas no accumulation of the corresponding mRNA occurred without BTH treatment. These transgenic plants also produced a protein corresponding to CYP1A1 in the leaves by BTH treatment. The transgenic plants with BTH application showed a much-higher tolerance to the phenylurea herbicides chlortoluron and methabenzthiazuron than non-transgenic plants. These findings indicated that the ability of metabolizing the two herbicides to less-toxic derivatives was displayed in the transgenic plants after BTH treatment. Transgenic plants harboring the CYP1A1 cDNA fused with the yeast P450 reductase (YR) gene under the control of PR1a were also produced. Although the plants showed a lower expression level of the fused gene than transgenic plants with CYP1A1 cDNA alone, they were tolerant to herbicides. These facts suggested that the CYP1A1 enzyme fused with YR showed a higher specific activity than CYP1A1 alone. This study demonstrated that the mammalian cDNA for the de-toxification enzyme of herbicides under the control of the PR1a promoter conferred chemical-inducible herbicide tolerance on potato.

Phenotypic modulation of vascular smooth muscle cells induced by unsaturated lysophosphatidic acids.

The phenotypic modulation of vascular smooth muscle cells (VSMCs) from the differentiated state to the dedifferentiated one is critically involved in the development and progression of atherosclerosis. Although many cytokines and growth factors have been reported as atherogenic factors, the critical pathogens for inducing atherosclerosis remain unknown, largely because proper examining systems of them have not been developed. We recently established primary culture systems for visceral SMCs and VSMCs in which both SMCs, when cultured on laminin with insulin-like growth factor-I, show a differentiated phenotype, as indicated by a spindle-like shape, ligand-induced contractility, and a high level of SMC differentiation marker gene expression. In this study, we searched for critical dedifferentiation factors for these SMCs using our culture system. We found that polar lipids extracted from human serum markedly induced VSMC dedifferentiation, and this activity was solely present in the lysophosphatidic acid (LPA) fraction. Among several LPA species detected in human serum lipids, unsaturated LPAs were identified as major contributors to the induction of VSMC dedifferentiation. Signaling and phenotype analyses revealed that unsaturated LPA-induced VSMC dedifferentiation is mediated through the coordinated activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase. Thus, this report demonstrates the first finding that unsaturated LPAs, but not saturated LPAs, specifically induce VSMC phenotypic modulation, suggesting that these molecules could function as atherogenic factors.

Effects of 4-hydroxyantipyrine and its 4-O-sulfate on antipyrine as biodistribution promoter.

The effects of 4-hydroxyantipyrine (4-OH), a major metabolite of antipyrine, and its 4-O-sulfate (4-S) on the pharmacokinetics of antipyrine were investigated in rats. Plasma elimination of intravenously administered antipyrine was significantly decelerated under a steady-state concentration of 4-OH but not under that of 4-S. Tissue-to-plasma concentration ratio (Kp) of antipyrine under its steady-state concentration was significantly increased in the brain and heart by the concomitant use of 4-OH, while similar use of 4-S had no effect. The enhancement of the blood-brain barrier (BBB) permeability of antipyrine caused by the concomitant use of 4-OH was believed to be concerned with the increase of the Kp value of antipyrine in the brain. These results suggested that 4-OH could be used as a biodistribution promoter.

Study on glutathionesulphonic acid as biodistribution promoter: concomitant use effect on verapamil hydrochloride and tegafur.

The effect of glutathionesulphonic acid (N-(N-gamma-L-glutamyl-L-beta-sulphoalanylglycine, GSO3H), which is one of the minor metabolites of glutathione (GSH), on the pharmacokinetics of verapamil hydrochloride (verapamil x HCl) and tegafur was investigated in rats. GSO3H was concomitantly used as sodium salt (GSO3Na). No significant change by the concomitant use of GSO3Na was recognized in the pharmacokinetics parameters of verapamil x HCl and tegafur, and plasma elimination of both substances was not affected by GSO3Na. The tissue-to-plasma concentration ratio (Kp) of verapamil x HCl in the lung 5 min after its administration under concomitant use of GSO3Na rose significantly, however, this effect disappeared 120 min after administration. No significant change was recognized in other organs. On the other hand, a significant difference of Kp of tegafur under a steady state concentration of GSO3Na was not recognized in any organs. It seemed that the elevation of a lipid solubility (oil water partition coefficient) of verapamil x HCl by the concomitant use of GSO3Na was related to the increase of the Kp value of verapamil x HCl in the lung. The partition coefficient of GSO3Na itself decreased when it was used concomitantly with verapamil x HCl.

Application of 4-hydroxyantipyrine and acetaminophen O-sulfate as biodistribution promoter.

The effects of 4-hydroxyantipyrine (4-OH), a major metabolite of antipyrine and its sulfate, 4-hydroxyantipyrine O-sulfate (4-S), on the pharmacokinetics of citicoline and thiopental sodium were investigated in rats. The concomitant use of 4-OH increased significantly the tissue-to-plasma concentration ratio (Kp) of citicoline in the brain and liver and that of thiopental sodium in the brain, liver, and heart, while 4-S did not affect them. The permeability clearance of blood-brain barrier (BBB) (Kin) and the total distribution volume (Vdbr) of citicoline were not affected by either 4-OH or 4-S. However, those of thiopental sodium were significantly increased by not only 4-OH but also by 4-S. On the other hand, the plasma concentration of antipyrine was significantly decreased by the intravenous bolus coadministration of N-acetyl-p-aminophenyl O-sulfate (APAPS) at steady-state plasma concentration of antipyrine. A similar reduction was not observed with the intravenous coadministration of acetaminophen (APAP). The Kp value of antipyrine was significantly increased in the brain by the coadministration of APAPS, but was not affected by APAP. The increment in the drug distribution to the brain with the concomitant use of 4-OH (or APAPS) observed in this study is useful information for the application of drug combinations as biodistribution promoters.

[A case of mesoblastic nephroma in an adult patient].

We treated a rare case of adult mesoblastic nephroma. The patient was a 52-year-old Japanese man with the chief complaint of intermittent gross hematuria and left lumbar pain. Abdominal ultrasonography, computed tomography, excretory urography, retrograde pyelography and angiography revealed a left renal tumor suspected to be a left pelvic tumor. A left nephroureterectomy was performed. The histologic examination showed a mesoblastic nephroma. A total of 38 adult mesoblastic nephroma cases were reviewed.

[Clinical study of aortic root replacement].

From 1990 to 1999, 19 patients underwent aortic root replacement. Annulo-aortic ectasia was observed in 14 patients, aortic dissection in 4, and aortitis in 1. Mean aortic cross-clamp times and cardiopulmonary bypass times were 163 +/- 44 and 247 +/- 99 min, respectively. Concomitant procedures were coronary artery bypass grafting in 3 patients, aortic arch replacement in 1 and aortic arch replacement and elephant trunk method in 2. There were two hospital deaths due to cardiac failure. There was one late death because of pharyngeal cancer 1.7 years after the operation. Seventeen survivors were followed for a mean duration of 4.4 years (0.6-9.3 years). There were no complications during this period. In conclusion, good results were obtained by modified Bentall operation.

Six months of maintenance chemotherapy after intensified treatment for acute lymphoblastic leukemia of childhood.

PURPOSE: We postulated that intensification of chemotherapy immediately after remission induction might reduce the leukemic cell burden sufficiently to allow an abbreviated period of antimetabolite therapy. PATIENTS AND METHODS: Three hundred forty-seven children (ages 1 to 15 years) with previously untreated acute lymphoblastic leukemia (ALL) were enrolled onto the Tokyo L92-13 study, which excluded patients with mature B-cell ALL and patients less than 1 year old. One hundred twenty-four patients were classified as standard risk, 122 as high risk, and 101 as extremely high risk, according to age, peripheral-blood leukocyte count, selected genetic abnormalities, and immunophenotype. All subjects received four drugs for remission induction, followed by a risk-directed multidrug intensification phase and therapy for presymptomatic leukemia in the CNS. Maintenance chemotherapy with oral mercaptopurine and methotrexate was administered for 6 months, with all treatment stopped by 1 year after diagnosis. RESULTS: The mean (+/- SD) event-free survival (EFS) and overall survival rates for all patients were 59.5% +/- 3.4% and 81.5% +/- 2.2%, respectively, at 5. 5 years after diagnosis. EFS rates by risk category were similar (60. 2% +/- 6.0% for standard risk, 57.7% +/- 5.6% for high risk, and 62. 5% +/- 5.7% for extremely high risk), whereas overall survival rates differed significantly (91.2% +/- 2.7%, 80.0% +/- 4.1%, and 72.1% +/- 4.5%, respectively, P <.0001 by the log-rank test). There were 107 relapses. Eighty-five (79.4%) of these 107 patients achieved second complete remissions, with subsequent EFS rates of 61.5% +/- 7. 9% (standard risk), 42.6% +/- 8.1% (high risk), and 9.6% +/- 6.4% (extremely high risk). Of the five risk factors analyzed, only the response to prednisolone monotherapy among extremely high-risk patients proved important. CONCLUSION: Early treatment intensification did not compensate for a truncated phase of maintenance chemotherapy in children with standard- or high-risk ALL. However, 6 months of antimetabolite treatment seemed adequate for extremely high-risk patients who were good responders to prednisolone and received intensified chemotherapy that included high-dose cytarabine early in the clinical course.

Cerebral atrophy in multiple system atrophy by MRI.

Cranial magnetic resonance images (MRI) of the cerebral areas of 40 patients with multiple system atrophy (MSA) and of 61 age-matched controls were analyzed. The cerebral area of MSA patients was 131. 95+/-15.89 cm(2) (mean+/-S.D.), which was significantly smaller than that of normal controls at 149.01+/-10.93 cm(2) (P<0.0001). All 23 MSA cases subjected to the MRI study over a 1-year period showed progressive cerebral atrophy, and the atrophy rate was 2.46+/-1. 66%/year. There were no significant differences within the MSA subtypes or between gender. The progression of cerebral atrophy in MSA correlated more with duration (r=-0.634) than age (r=-0.421). We conclude that MRI findings throughout the course of MSA suggest progressive cerebral atrophy, which is common in all subtypes and reflects duration of the disease rather than age.

[A case of coronary artery bypass grafting in a patient who has a rosary-like coronary lesion with Buerger's disease].

We report a case of coronary artery bypass grafting using left intra thoracic artery (LITA) in a 63-year-old male with Buerger's disease. His coronary angiography showed a long lesion seemed rosary in the left anterior descending (LAD). At operation, the surface of the coronary artery was dark red with white speckles. The LAD was opened distal end of disease, the intima of the coronary artery made much soft thin fold. The disease was never atheromatous. His coronary angiography showed a very rarely finding that seemed rosary.

Caspase-3 activation during apoptosis caused by glutathione-doxorubicin conjugate.

Glutathione-doxorubicin (GSH-DXR) effectively induced apoptosis in rat hepatoma cells (AH66) at a lower concentration than DXR. After 24 h of drug treatment, DNA fragmentation of the cells was observed at the concentration of 1.0 microM DXR or 0.01 microM GSH-DXR. Increase in caspase-3 activity and DNA fragmentation were observed within 12 h and 15 h after treatment with either drug. Intracellular caspase-3 activity was increased in a dose-dependent manner after treatment with DXR or GSH-DXR, and caspase-3 activity correlated well with the ability to induce DNA fragmentation. When the cells were treated with either DXR or GSH-DXR for only 6 h, apoptotic DNA degradation and caspase-3 activation occurred 24 h after treatment. DNA fragmentation caused by these drugs was prevented completely by simultaneous treatment with the caspase-3 inhibitor, acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), at 10 microM. By contrast, DNA fragmentation was not prevented by the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO), at the same concentration as DEVD-CHO, and caspase-1 was not activated at all by the treatment of AH66 cells with both DXR and GSH-DXR. These results demonstrate that DXR and GSH-DXR induce apoptotic DNA fragmentation via caspase-3 activation, but not via caspase-1 activation, and that GSH-DXR enhances the activation of caspase-3 approximately 100-fold more than DXR. Moreover, the findings suggested that an upstream apoptotic signal that can activate caspase-3 is induced within 6 h by treating AH66 cells with the drug.

Safety assessment of transgenic potatoes with soybean glycinin by feeding studies in rats.

Feeding studies of transgenic potatoes with native and designed soybean glycinins in rats were done for four weeks. The designed glycinin has four additional methioninyl residues in the middle of the glycinin molecule. Rats were divided into four groups fed (I) only a commercial diet, (II) the diet plus non-transgenic potatoes, (III) the diet plus transgenic potatoes with native glycinin, and (IV) the diet plus transgenic potatoes with designed glycinin. Rats were fed 2,000 mg/kg-weight potatoes every day by oral administration. During the period tested, rats in each group (groups II, III, and IV) grew well without marked differences in appearance, food intake, body weight, or in cumulative body weight gain. No significant differences were also found in blood count, blood composition, and in internal organ weights among the rats after feeding potatoes (groups II, III, and IV) for four weeks. Necropsy at the end of experiment indicated neither pathologic symptoms in all rats tested nor histopathological abnormalities in liver and kidney. Judging from these results, the transgenic potatoes with glycinins are confirmed to have nearly the same nutritional and biochemical characteristics as non-transgenic one.