Polyanionic stretch-deleted histone chaperone cia1/Asf1p is functional both in vivo and in vitro

BACKGROUND: CIA, an interactor of the CCG1 histone acetyltransferase subunit of TFIID, was identified as a human histone chaperone. The Saccharomyces cerevisiae orthologue ASF1, when it was over-expressed, was reported to cause de-repression of silent loci; however, the involvement of Asf1p in the alteration of nucleosomal structures remained unknown. Curiously, there is a polyanionic stretch, a structural motif characteristic of histone chaperones, in S. cerevisiae Asf1p, but not in human CIA. We investigated how CIA/Asf1p utilizes its domain(s) for the alteration of nucleosomal structure. RESULTS: To characterize the relationships between the domain structures and nuclear functions of CIA, we isolated the gene for the CIA counterpart in Schizosaccharomyces pombe, designated cia1+, whose putative product contains a polyanionic stretch. Gene disruption of cia1+ was lethal, which is the distinct phenotype of viable S. cerevisiae asf1. The cia1- lethality was rescued by the introduction of S. cerevisiae ASF1, but not by the introduction of human CIA cDNA. To our surprise, the construct that produces Asf1p, lacking the polyanionic stretch, is capable of rescuing the lethality caused by the cia1+ deletion, while the highly conserved N-terminal region of Asf1p is essential for the complementation of cia1- growth defects. The polyanionic stretch-deleted Asf1p is sufficient both for interaction with histones H3/H4 and for nucleosome assembly in vitro, as well as for telomeric de-repression in vivo. CONCLUSION: These findings suggest that the areas responsible for both the conserved and species-specific functions of CIA/cia1/Asf1p are within their highly conserved regions and that the yeast-specific polyanionic stretch of cia1/Asf1p is not necessary for viability, histone binding, nucleosome assembly, or anti-silencing.     

Characterization of spontaneous mutation in the delta soxR and SoxS overproducing strains of Escherichia coli

To examine the role of the soxRS regulon in mutagenesis, we characterized the spontaneous mutations occurring in the endogenous tonB gene in the delta soxR strain and the SoxS overproducing strain of Escherichia coli. Neither the delta soxR strain nor the SoxS overproducing strain led to an enhancement or diminishment of the spontaneous mutation frequency. By DNA sequencing, we determined 50 spontaneous mutants from the delta soxR strains, and found that 36% were both base substitutions and IS insertions, 14% frameshifts and 10% deletions. Among the base substitutions, G:C-->T:A transversions and G:C-->A:T transitions predominated, followed by A:T-->T:A transversions. We determined 54 spontaneous mutants from the SoxS overproducing strains, and found that 37% were IS insertions, 31% base substitutions, 17% frameshifts, 9% deletions and 6% duplications. Among the base substitutions, G:C-->T:A transversions dominated, followed by A:T-->T:A transversions and G:C-->A:T transitions. These results were similar to those from the soxRS+ strains. Thus, it is suggested that the soxRS-regulated genes do not play a significant role in the defense against spontaneous mutagenesis.     

Analysis with magnetic resonance imaging of lesions in cerebral achromatopsia

PURPOSE: To localize the lesions associated with cerebral achromatopsia. METHODS: We examined 20 patients with homonymous hemianopsia caused by cerebral infarction(17 men and 3 women aged 49 to 81 years; mean age, 65.1 years). Ishihara plates, standard pseudoisochromatic plates(part 2) and the panel D-15 test were used to examine color perception. Color matching tasks and color naming tasks were used to test color recognition. We tried to apply functional magnetic resonance imaging(fMRI) to lesion analysis in the brain. RESULTS: Cerebral achromatopsia was diagnosed in four patients. The analysis showed that lesions in the infracalcarine area(Brodmann's area 18 and 19) were associated with cerebral achromatopsia. Additionally, the lesions associated with failure of the panel D-15(PD-15) test were located more anterior than the lesions associated with failure of Ishihara plates. CONCLUSION: We show evidence that lesions in the anterio-ventral temporo-occipital area are associated with cerebral achromatopsia. This result is in accord with past observations(autopsy, fMRI and positron emission tomography).     

Mechanism in inhibitory effects of vitamin K2 on osteoclastic bone resorption: in vivo study in osteopetrotic (op/op) mice

Osteoclast deficiency in op/op mice was cured by a single injection of 5 micrograms recombinant human macrophage colony-stimulating factor (M-CSF). On d 5, the osteoclast number reached a maximum value. By d 15, the osteoclast number had decreased to about 70% of the maximum level. Moreover, by d 20, the osteoclast number had decreased to about 30% of its maximum level. On d 5, the osteoclast number of vitamin K2 12 h previously had decreased to about 30% of the M-CSF-only injected mice. Moreover, on d 5, the osteoclast number of the mice receiving a single injection of vitamin K2 24 h previously had decreased to about 15% that of mice injected only with M-CSF. These results indicate that vitamin K2 inhibits in vivo osteoclast formation. On d 20, the osteoclast number of the mice injected with a single dose of vitamin K2 12 or 24 h previously had decreased to 0% compared with those receiving only M-CSF. The present results suggest that the vitamin K2 "causes cell death" to mature osteoclasts and inhibits in vivo osteoclast formation.     

Client anxiety from awareness of personal HIV risk and relation to taking HIV test at public health centers in Japan]

Clients taking HIV antibody tests at public health centers (PHCs) in Japan were surveyed in autumn of 1995 centered on 17 PHCs selected by a snow-ball sampling method in domestic PHCs. The anonymous self-administered questionnaires were given out by PHC's medical doctors or public health nurses after receiving informed consent of this survey's objectives following HIV post-test counseling. A total of 250 questionnaires were collected (71.8%). Valid questionnaires were 233 (67.0%) and 62.7% (146 people) were male, 37.3% (87 people) were female, with differences in mean age by sex being significant (p < .001). The greatest motivating factor for getting HIV testing was concerning secondary infection to partners and extreme anxiety relating to one's own possible infection. This motivating factor was greater than having experienced previous HIV infectious high-risk behavior (i.e. condomless sex). Repeat (more than twice) visitors totaled 49 (21.0%), with 75% having experienced HIV infectious high-risk behavior since the last HIV test. "Having being advised or having received from partners or friends" affected decision making to get tested. About half of subjects vacillated before coming to the PHC for taking anonymous the HIV antibody test. Through multiple-logistic regression analysis factors causing vacillation were: greater self-awareness of possibility of having HIV infection and the fact that HIV infected people must retire from their work (or studies) despite their current health condition being fine. While HIV medical/clinical treatment progress continues, the stigma toward HIV/AIDS and the resulting discrimination is not changing in terms of the general citizen's knowledge and attitudes. This result will likely curtail HIV antibody testing behavior.     

Suppression of solid tumor growth by a monoclonal antibody against tumor vasculature in rats: involvement of intravascular thrombosis and fibrinogenesis.

We have reported that immunization of rat tumor-derived endothelial cells (TEC) isolated from KMT-17 solid tumors results in the generation of several monoclonal antibodies (MAbs). TES-23, one of these MAbs, recognizes a naturally occurring 80-kDa antigen expressed on endothelial cells of tumor blood vessels. To determine whether such MAbs can suppress solid tumor growth in vivo by impairment of endothelial cells in tumors following direct binding, we tested the biodistribution of (125)I-labeled TES-23 in rats bearing KMT-17 solid tumors. We also examined the effect of treatment using unconjugated TES-23 on tumor growth and histo-pathological changes in tumor tissues. Biodistribution studies showed localization of TES-23 into tumor tissues 60 min after intravenous injection. TES-23 suppressed significantly the growth of KMT-17 solid tumors following administration for 5 days. Histo-pathological examination showed that TES-23 caused degeneration, apoptosis and/or necrosis and denudation of endothelial cells in viable tumor areas following local aggregation and adhesion of lymphocytes, with subsequent intravascular thrombus formation by platelets and fibrin. Our results indicate that TES-23, which recognizes TEC, can target endothelial cells of solid tumor vasculature directly, resulting in growth suppression in vivo by reduction of blood flow due to intravascular thrombosis. Our results also suggest that targeting tumor vasculature is a potentially attractive approach for the treatment of solid tumors.     

Interaction between purinoceptor subtypes on hippocampal serotonergic transmission using in vivo microdialysis.

The effects of purinoceptor subtypes on hippocampal extracellular serotonin levels were determined by using in vivo microdialysis. Perfusion with adenosine-5'-triphosphate (ATP) for 20 min produced concentration-dependent changes in hippocampal extracellular serotonin levels, which consisted of an initial rise phase, with levels increasing to 309% of control with 100 microM ATP, followed by a later rebound reduction phase, with levels decreasing to 6% of control. The P2X1-7 active P2 purinoceptor agonist, 2-methylthioATP (2-MeSATP: 100 microM) increased the extracellular serotonin level drastically (638%), while the P2X1,3 active P2 purinoceptor agonist, alpha, beta-methylene-L-ATP (alpha, beta-meATP: 100 microM) produced a small increase (132%) in the serotonin level. The P2X1,2,3,5,7 active P2 purinoceptor antagonist, suramin (100 microM), reduced the basal serotonin level (86%) and the ATP-evoked initial rise phase (from 309 to 254%) without affecting the late reduction phase. The adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT: 50 microM) potentiated the rising phase (167%) and abolished the subsequent ATP-evoked reduction phase. Perfusion with CPT and an adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX: 10 microM), reduced the ATP-evoked initial rise (to 181%) and abolished the late reduction phases of serotonin release. These results indicate that ATP-evoked hippocampal serotonin release is composed of an initial rise phase and a later reduction phase. The ATP-evoked initial rise phase might be produced by an activation of P2X purinoceptor function, whereas the late reduction phase was modulated by the activation of adenosine A1 receptor function by adenosine, metabolized from ATP in the synaptic cleft.     

Mutational Analysis of BRCA1 Gene in Ovarian and Breast-ovarian Cancer Families in Japan

We analyzed the alteration of BRCAI in DNA obtained from 83 individuals of 13 Japanese site-specific ovarian cancer families and 6 breast-ovarian cancer families. Six germline mutations were detected in 7 families, which consisted of 4 breast-ovarian cancer and 3 site-specific ovarian cancer families, by single-strand conformation polymorphism analysis, followed by direct sequence determination. The mutations included three framcshifts, two nonsense mutations, and one missense mutation causing loss of a zinc-binding motif. The frequency of loss of heterozygosity at the microsatellite markers on the BRCAI gene was 57% (8 of 14 cases) in site-specific ovarian cancer families, and 100% (6 of 6 cases) in breast-ovarian cancer families. All tumors of the patients carrying a mutation of BRCAI showed deletion of wild-type alleles, implicating BRCAI as a tumor suppressor gene. Tbese results suggest tbat germline mutations of the BRCAI gene play an important role in the carcinogen-esis of breast and/or ovarian cancer in a majority of breast-ovarian cancer families and in some site-specific ovarian cancer families.     

Chemoprevention of N-Nitroso-N-methylurea-induced Rat Mammary Cancer by Miso and Tamoxifen, Alone and in Combination

We examined the effects of a Japanese fermented soybean product, miso, and tamoxifen (TAM), alone and in combination, on N -nitroso- N -methylurea (MNU)-induced rat mammary cancer. Seven-week-old female CD/Crj rats received a single i.v. dose (50 mg/kg body weight) of MNU. After administration of MNU, the rats were divided into 4 groups: regular diet (control), 10% miso diet, regular diet+TAM, and 10% miso diet+TAM. TAM was implanted s.c. in the form of pellets containing 2.5 mg at the same time as MNU was administered. All rats were observed for 18 weeks after MNU administration. Incidence (percentage of rats with tumors) and multiplicity (mean tumors/rat) of mammary tumors were 91% and 4.5 in the control, 77% and 2.4 ( P <0.05) in the 10% miso group, 68% and 1.4 ( P <0.01) in the TAM group, and 10% ( P <0.0001 or less) and 0.2 ( P <0.0001) in the 10% miso+TAM group. In the second experiment, the effect of the combination of miso and TAM on established rat mammary tumors was investigated. When the mammary tumors induced by MNU reached 10 to 25 mm, the rats were divided into 3 treatment groups: regular diet, regular diet+TAM, and 10% miso diet+TAM. At 6 weeks after the start of treatment, the mean tumor size in the control and TAM groups was 160% and 141% of the pretreatment value, but a decrease to 85% of the pretreatment value was produced by the combination of miso and TAM, and this was significantly different from both the control and TAM groups ( P <0.01 and P <0.05, respectively). These results indicate that miso is useful in protecting against mammary cancer and it can be expected to have a potent antitumor effect, especially when used in combination with TAM.     

Chemoprevention of N-Nitroso-N-methylurea-induced Rat Mammary Carcinogenesis by Soy Foods or Biochanin A

We examined the effects of soybeans, a soy product (miso) and biochanin A, an isoflavone derivative, on N -nitroso- N -methylurea (MNU)-induced rat mammary carcinogenesis. Seven-week-old female CD/Crj rats received a single i.v. dose (40 mg/kg body weight) of MNU. After administration of MNU, rats were fed diet containing 0% (control), 2% or 10% soybeans, or 10% miso as a soy-supplemented diet, or 10 or 50 mg/kg biochanin A. All rats were observed for 18 weeks after MNU administration. At 18 weeks, the multiplicity (mean tumors/rat) of palpable mammary tumors was significantly decreased in the 10% soybean (1.1) and 10% miso (1.2) diet groups compared to the control (2.2) ( P <0.05, respectively). In the biochanin A-supplemented diet groups, the incidence (percentage of rats with tumors) was significantly decreased in the 50 mg/kg (32%) diet group compared to the control (80%) ( P <0.01), and the multiplicity was significantly decreased in both the 10 mg/kg (0.7) and 50 mg/kg (0.5) diet groups compared to the control (2.2) ( P <0.01 and P <0.001, respectively). The proliferative cell nuclear antigen labeling index of mammary tumors was significantly decreased in both biochanin A-supplemented diet groups compared to the control. The present results indicate that soybeans, miso, and biochanin A are useful for the prevention of mammary cancer.