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.

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.

Mouse peritoneal lymphocytes, a new target for analyzing induction of sister chromatid exchanges on in vivo exposure to a genotoxic agent.

The availability of use of mouse peritoneal lymphocytes as target cells for analyzing sister chromatid exchanges (SCE) upon exposure to a genotoxic drug, cyclophosphamide, was investigated using female ICR mice. Use of these cells overcame the difficulty in use of mouse lymphocyte cultures, recovering sufficient metaphase cells. The greatest advantage of use of peritoneal lymphocytes was that about 1-2 X 10(6) lymphocytes/mouse could easily be recovered from the peritoneal cavity in high purity. Their mitogenic responses were good when Escherichia coli lipopolysaccharide, in combination with 2-mercaptoethanol, was used as mitogens, but they were less when purified phytohemagglutinin was used. In the presence of lipopolysaccharide (60 micrograms/ml) and 2-mercaptoethanol (22-88 microM), the maximum incidence of second division metaphases (greater than 50%) and the highest mitotic index (greater than 4%) were observed 36-40 h after stimulation. Under these conditions, the base-line SCE showed the constant level. The range of intrastrain variations in the base-line SCE was 0.24-0.36/chromosome. The distribution histograms of SCE/chromosome did not fit a single Poisson model, suggesting that these cells are heterogeneous with respect to the base-line SCE. Single s.c. injections 1 h before harvest of doses of 0.75-3.0 mg of cyclophosphamide per kg evoked positive responses, and injections of over 0.375 mg/kg had linear dose-dependent effects. On harvest of cells for up to 192 h after the injection, the maximal induction of SCE attained 1 h after exposure was found to return time dependently to the control level at 192 h. After the initial rapid reduction in the cell number, cellular recovery, measured as the mitotic index and the number of peritoneal exudate cells recovered, returned to the control level within 48 h, without a significant increase thereafter. After maintaining cells under the liquid-holding experiment for various times in vitro following a single exposure to cyclophosphamide for 1 h in vivo, the reduction of their SCE and recovery of their mitotic index were more rapid than those of cells in the time-course experiment. These findings suggest that the association of the recruitment of less- and/or nondamaged cells from their precursors with reduction of the SCE is slight. Repair(s) and, to a lesser extent, selective loss of more damaged cells may be the main factors contributing to the early reduction response of the SCE frequency. The relations of these factors are discussed.

Comparison of 6-thioguanine-resistant mutation and sister chromatid exchanges in Chinese hamster V79 cells with forty chemical and physical agents.

The induction of sister chromatid exchanges (SCE) and mutation at the hypoxanthine-guanine phosphoribosyl transferase locus and toxicities of 40 different chemical and physical agents were examined on Chinese hamster V79 cells. These agents included mono-, di-, tri-, and polyfunctional alkylating agents, intercalators, gamma-rays, and UV light irradiation. Mutation was measured as resistance to 6-thioguanine and toxicity as loss of cell-plating efficiency. SCE were examined 29 hr after treatment. With the agents examined, a highly positive correlation (r = 0.89) existed between SCE-inducing and mutagenic potencies, when expressed as increase in the number per a unit dose over the control values. But the great difference of the ratios of mutagenic potencies versus SCE-inducing potencies among agents was observed, the maximal difference in the ratios being about 200-fold. The agents that showed the higher values of the ratio (agents producing more mutations than SCE) were bleomycin, cobalt-60 gamma-rays, all ethylating agents (N-ethyl-N-nitrosourea, N-ethyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and diethylsulfate), N-propyl-N-nitrosourea, N-butyl-N-nitrosourea, isopropyl methanesulfonate, intercalating acridine compounds (2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino]-acridine X 2HCl and 2-methoxy-6-chloro-9-[3-(chloroethyl)-aminopropylamino]acridine 2HCl) and UV light at 254 nm. The agents that showed the lower values (agents producing more SCE than mutations) were platinum compounds (cis-diamminedichloro-platinum and trans-diamminedichloroplatinum), epoxides (epichlorohydrin, styrene oxide, and diepoxybutane) and aziridines (mitomycin C, decarbamoyl mitomycin C, tris(1-aziridinyl)phosphine sulfide, triethylenemelamine, and carboquone). The agents that showed the intermediate values included all methylating agents (N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine, methyl methanesulfonate, and dimethyl sulfate), N-(2-hydroxyethyl)ethyleneimine, beta-propiolactone, treatment of 8-methoxypsoralen plus near-UV light irradiation at 352 nm, 4-nitroquinoline-1-oxide, quinacrine mustard, sodium sorbate, cigarette tar, and diesel tar. For most agents that induced SCE, the toxicity dependency of induced SCE was rather biphasic; increase in SCE was steep at low to moderate toxicity and less at moderate to high toxicity. At equitoxic doses, the agents showed great difference in induction of SCE.

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.

Philadelphia-chromosome-positive, monosomy 7 biphenotypic acute mixed lineage leukemia in adults: a pluripotent stem cell disorder.

Two adult patients with acute mixed lineage leukemia (AMLL) having combined Philadelphia chromosome (Ph1) positivity and monosomy 7 are presented. The phenotypes of leukemic blasts from both cases were almost same (early B-lymphoid lineage and myeloid lineage); CD10+, CD13+, CD19+. HLA-DR+, and dual-color analysis showed simultaneous expression of CD10 (CD19) and CD13 antigens in individual blasts (biphenotypic) in both cases. On molecular analysis, the leukemic blasts showed rearrangement in the first intron of the BCR gene with breakpoint just outside of 3' end of m-BCR-2 (bcr 3) in case 1, and in the M-BCR in case 2. Immunoglobulin heavy chain gene (IgH) rearrangement was noted in both cases, but rearrangement of the T-cell receptor beta-chain gene (TCR beta) was detected only in case 1. Clinically, both cases achieved complete remission by the combination chemotherapy consisting of L-asparaginase, doxorubicin, vincristine, and prednisolone (L-AdVP). In remission, all these molecular abnormalities disappeared in both patients. These results suggest that the Ph1-positive and monosomy 7 AMLL in adults is de novo acute leukemia with both early B-lymphoid and myeloid phenotypes and may arise from malignant transformation of pluripotent stem cell, and expresses a heterogenous rearrangement pattern of the BCR gene.

Retinoic acid and butylated hydroxyanisole inhibit promoter-enhanced transformation in vitro.

The inhibitory effects of some antipromoters were studied using a two-stage transformation assay system in vitro with 3-methylcholanthrene (3-MC)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in BALB 3T3 cells. Butylated hydroxyanisole (BHA), a phenolic antioxidant, inhibited TPA-enhanced transformation in a dose-dependent manner, but butylated hydroxytoluene (BHT) did not. Among the three antipromoters tested, retinoic acid (RA) was the most effective inhibitor.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-enhanced transformation in vitro by radical scavengers.

The inhibitory effects of some scavengers of oxygen radicals were studied, using a two-stage transformation assay system in vitro 3-methylcholanthrene (3-MC)-initiation and 12-O-tetradecanoylphorbol-13-acetate-(TPA)-promotion in Balb 3T3 cells. Mannitol, a scavenger for hydroxyl radicals, strongly inhibited the TPA-enhanced transformation in a dose-dependent manner. Superoxide dismutase (SOD) and catalase, which are specific scavengers for O2-. and H2O2, respectively, also inhibited the transformation. These results suggest that oxygen radicals may play an important role in the TPA-enhanced transformation in Balb 3T3 cells.

Induction of nitroblue tetrazolium reduction in mouse peritoneal macrophages by tumour promoters and inhibition of the induced nitroblue tetrazolium reduction by some inhibitors.

Two polyacetates, aplysiatoxin and debromoaplysiatoxin, as well as 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein and teleocidin enhance nitroblue tetrazolium (NBT) reduction in mouse peritoneal macrophages in vitro. The ED50 values for NBT reduction of these 5 TPA-type tumor promoters were 4.2 ng/ml for TPA, 36 ng/ml for mezerein, 0.53 ng/ml for teleocidin, 1.5 ng/ml for aplysiatoxin and 108 ng/ml for debromoaplysiatoxin. The NBT reduction induced by the 5 tumor promoters is inhibited by 2 inhibitors of tumor promotion, retinoic acid and dibromoacetophenone. The possibility that tumor promotion by TPA-type tumor promoters involves similar mechanisms such as superoxide anion radicals release in cell membranes is discussed.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced nitroblue tetrazolium reduction in mouse peritoneal macrophages by various tumor promotion inhibitors.

The effects of various inhibitors on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced reduction of nitroblue tetrazolium (NBT) in mouse peritoneal macrophages were investigated. The reduction was inhibited by phospholipase A2 inhibitors, such as dibromoacetophenone, the lipoxygenase inhibitor nordihydroguaiaretic acid, an NADH-dehydrogenase inhibitor, the microfilament inhibitor cytochalasin B, oxygen radical scavengers such as superoxide dismutase, antioxidants such as butyl hydroxyanisole and non-specific inhibitors such as retinoic acid. The reduction was not affected by the cyclooxygenase inhibitor indomethacin or the H2O2 scavenger catalase.

A rapid, simple screening method for skin-tumor promoters using mouse peritoneal macrophages in vitro.

The enhancing effects in vitro of a potent skin-tumor promoter, phorbol myristate acetate (PMA), and its derivatives on nitroblue tetrazolium (NBT) reduction, phagocytosis and cell spreading of mouse peritoneal macrophages were compared. The enhancing effects on these markers of macrophage function, especially NBT reduction, were found to correlate well with the skin-tumor promoting activities of the phorbol esters. Another potent promoter, teleocidin, strongly enhanced NBT reduction, although teleocidin is structurally different from phorbol esters. Other tumor promoters, namely saccharin, phenobarbital, cantharidin and Tween 60 did not enhance NBT reduction by macrophages. These data indicate that this test system is appropriate for screening of PMA-type tumor promoters such as phorbol esters and teleocidin.

Total one-stage repair of interrupted aortic arch associated with aortic septal defect and patent ductus arteriosus.

Successful total repair in one stage was performed in a 3-year-old girl who had interrupted aortic arch associated with aortic septal defect and patent ductus arteriosus. Surface-induced deep hypothermia and interrupted perfusion were used. The results of postoperative catheterization and angiocardiographic studies are analyzed, and the literature and results of previous surgical attempts at correction are reviewed.

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.

Surgical treatment of congenital distal tracheal stenosis involving the carina.

Untreated congenital stenosis of the distal trachea frequently results in lethal airway obstruction. A 3-year-old boy with segmental stenosis of the distal trachea and a 2-year-old girl with segmental stenosis involving the carina and the right main bronchus were treated successfully with resection and reanastomosis. Operative techniques, anesthetic management, postoperative care, and tracheal growth after anastomosis are discussed.

Surgical treatment of anomalous origin of the left coronary artery from the pulmonary artery associated with tetralogy of Fallot.

An 8-year-old boy with anomalous origin of the left coronary artery from the pulmonary artery associated with tetralogy of Fallot, which was definitely diagnosed preoperatively, was operated on with success. Direct implantation of the left coronary artery into the aorta following division of the left coronary artery from the pulmonary artery and, concomitantly, total repair for tetralogy of Fallot using an external valved conduit were performed. Postoperative cineangiogram revealed a hemodynamically well-repaired intracardiac condition and anterograde filling of the left coronary artery, compared with retrograde left coronary flow from intercoronary collateral vessels preoperatively. To the best of our knowledge, there is not a previously published report of anomalous origin of the left coronary artery from the pulmonary artery associated with tetralogy of Fallot that was treated surgically with success.

Anticoagulant therapy in recurrent cerebral embolism: a retrospective study in non-valvular atrial fibrillation.

For the prevention of recurrent embolic stroke, 23 elderly patients with non-valvular atrial fibrillation (NVAF) were treated with oral anticoagulants (warfarin) during a mean period of 3.8 years. Only one patient suffered recurrent embolism, and another had acute myocardial infarction. There was no cerebral haemorrhage during the treatment. In an untreated control group (from an autopsy series), recurrent embolic strokes occurred in 18 of 70 NVAF patients (26%) during a mean period of 1.3 years. Long-term anticoagulant therapy appears to be effective in the prevention of recurrent embolic stroke in elderly patients with NVAF.

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.

Human papilloma virus (HPV) type 16 and 18 detected in head and neck squamous cell carcinoma.

BACKGROUND: Recently the HPV genome has been detected in 75-80% of uterine cervical squamous cell carcinomas. High-risk HPV 16,18,31,33, and 45 are frequently associated with invasive carcinoma of the oral cavity, oropharynx, larynx, and nasal cavity. In this study we investigated the association of HPV16 and 18 in head and neck squamous cell carcinomas (HNSCC) using PCR-based methods. MATERIALS AND METHODS: Ninety-eight fresh frozen tissues from HNSCC were collected. The samples were consisted of 26 cases from the larynx, 19 from the nasal and paranasal sinus, 16 the hypopharynx, 14 the oral cavity, 13 the oropharynx, and 10 from the nasopharynx. The presence of HPV genome was examined by PCR using HPV16 and 18 specific primers encoding E7 ORF. RESULTS: HPV16-DNA was detected in 23% of all cases (23/98), 31% (8/26) larynx, 16% (3/19) nasal and paranasal sinus, 19% (3/16) hypopharynx, 21% (3/14) oral cavity, 38% (5/13) oropharynx, and 10% (1/10) nasopharynx. HPV18-DNA was detected in 4% of all cases (4/98), 8% (2/26) larynx, and 15% (2/13) oropharynx. 54% (7/13) in oropharynx and 38% (10/26) in larynx showed rather high prevalence in the head and neck. CONCLUSIONS: HPV 16 and 18 play an important role in carcinogenesis of the head and neck, especially, in the oropharynx and larynx.

Screening for skin-tumor promoters.

Since attention has been drawn to the role of oxygen radicals in tumor promotion, we investigated whether or not the nitroblue tetrazolium (NBT) reduction test using mouse peritoneal macrophages was useful as a rapid screening method for skin-tumor promoters. On the basis of our results, we concluded that this test system was appropriate for the screening of 12-0-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters and bore comparison with other screening methods for tumor promoters. A large number of skin-tumor promoters, such as active phorbol esters, teleocidin and aplysiatoxin, appear to act indirectly through a free radical mechanism by binding to a membrane receptor.