Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 feeding enhances humoral immune responses, which are suppressed by the antiviral neuraminidase inhibitor oseltamivir in influenza A virus-infected mice.

Antiviral neuraminidase inhibitors, such as oseltamivir, zanamivir, and peramivir, are widely used for treatment of influenza virus infection. We reported previously that oseltamivir inhibits the viral growth cycle, ameliorates symptoms, and reduces viral antigen quantities. Suppressed viral antigen production, however, induces a reduction of acquired antiviral humoral immunity, and increases the incidence of re-infection rate in the following year. To achieve effective treatment of influenza virus infection, it is necessary to overcome these adverse effects of antiviral neuraminidase inhibitors. Feeding of yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) OLL1073R-1 is reported to have immune-stimulatory effects on influenza virus infection in mice and humans. In the present study, we assessed the effect of feeding L. bulgaricus OLL1073R-1 yogurt cultures (YC) on local and systemic humoral immune responses, which were suppressed by oseltamivir treatment, in mice infected with influenza A virus. Yogurt culture (1.14 × 108 cfu/0.4 mL per mouse per day) or sterile water (vehicle) was administered by intragastric gavage for 35 d. At d 22, influenza A virus/Puerto Rico/8/34 (H1N1) (PR8; 0.5 pfu/15 µL per mouse) was instilled intranasally, followed immediately by oral administration of oseltamivir (50 µg/100 µL per mouse, twice daily) or 5% methylcellulose (100 µL/mouse) as a vehicle for 13 d. Titers of anti-PR8-specific IgG and IgA in serum and mucosal secretory IgA (S-IgA) and IgG in bronchoalveolar lavage fluid (BALF) were analyzed by ELISA at 14 d after infection. Oseltamivir significantly suppressed the induction of anti-PR8-specific IgG and IgA in serum and S-IgA and IgG in BALF after infection. Feeding YC mildly but significantly stimulated production of PR8-specific IgA in serum, S-IgA in BALF, and IgG in serum without changing the IgG2a:IgG1 ratio. We analyzed the neutralizing activities against PR8 in serum and BALF and found that oseltamivir also reduced protective immunity, and YC feeding abrogated this effect. The immune-stimulatory tendency of YC on anti-PR8-specific IgA and IgG titers in serum and BALF was also detected in mice re-infected with PR8, but the effect was insignificant, unlike the effect of YC in the initial infection.

Negative-U system of carbon vacancy in 4H-SiC.

Using electron paramagnetic resonance (EPR), energy levels of the carbon vacancy (V(C)) in 4H-SiC and its negative-U properties have been determined. Combining EPR and deep-level transient spectroscopy we show that the two most common defects in as-grown 4H-SiC--the Z(1/2) lifetime-limiting defect and the EH(7) deep defect--are related to the double acceptor (2-|0) and single donor (0|+) levels of V(C), respectively.

Numerical and Experimental Study to Fabricate the New Type Compact NMR Device Using Stacked HTS Bulks.

In fabrication of a compact NMR (Nuclear Magnetic Resonance) magnet which consists of a stacked HTS (High Temperature Superconducting) bulk annuli, generally there are three key issues: spatial homogeneity, temporal stability, strength of trapped magnetic fields. This paper presents a study on the effects of axial gap length between stacked HTS bulks on the three key issues of a bulk HTS magnet for compact HTS NMR applications. The HTS bulk magnet of which the ID and OD are 20 and 60 mm respectively has a 50 and 80 mm heights depending on the axial gap lengths between HTS bulks. The gap length between each HTS bulk varied from 0 mm to 10 mm and were used as parameters to optimize, analytically as well as experimentally, the overall field homogeneity of the HTS bulk magnet. The optimized axial gap length was obtained by analytical results, and the better magnetic field homogeneity and temporal stability of trapped magnetic field were achieved by lower magnetization field. The improved spatial homogeneity and strength of generated magnetic field by a new compact NMR magnet will be presented.

The effects of displacement threshold irradiation energy on deep levels in p-type 6H-SiC.

We report on the electrical characterization, by means of deep level transient spectroscopy, of electron-irradiated Al-doped 6H-SiC epilayers. Samples were irradiated with either 116 keV, in order to displace only carbon atoms, or 400 keV. Seven deep traps, in the 0.1-1.6 eV range above the valence band, were found. The thermal stability of the detected levels was analyzed by performing an isochronal annealing series in the 100-1800 °C temperature range and the atomic structure of most of the detected traps was found to be related to C-displacement.

The structural and electronic properties of chiral SiC nanotubes: a hybrid density functional study.

Hybrid density functional theory was employed in investigating the structural and electronic properties of 14 chiral and 3 armchair SiC nanotubes (SiCNTs). The role of the tube diameter, as well as that of the chiral angle theta, was studied in detail by considering nanotubes of diameters varying from 2 to 9 A and chiral angles theta varying between 7 degrees and 30 degrees. The study revealed that all the investigated SiCNTs are semiconductors with a broad spectrum of bandgap values ranging from 0.2 to 2.9 eV and that the structural stability of the nanotubes increases with diameter. By analyzing the behavior of the molecular orbitals, an explanation of the mechanism by which theta affects the determination of such values is put forward.

Hippocampal synthesis of estrogens and androgens which are paracrine modulators of synaptic plasticity: synaptocrinology.

Hippocampal pyramidal neurons and granule neurons of adult male rats are equipped with a complete machinery for the synthesis of pregnenolone, dehydroepiandrosterone, testosterone, dihydrotestosterone and 17beta-estradiol. Both estrogens and androgens are synthesized in male hippocampus. These brain steroids are synthesized by cytochrome P450s (P450scc, P45017alpha and P450arom), hydroxysteroid dehydrogenases and reductases from endogenous cholesterol. The expression levels of enzymes are as low as 1/300-1/1000 of those in endocrine organs. Synthesis is dependent on the acute Ca(2+) influx upon neuron-neuron communication via NMDA receptors. Estradiol is particularly important because estradiol rapidly modulates neuronal synaptic transmission such as long-term potentiation via synaptic estrogen receptors. Xenoestrogens may also act via estrogen-driven signaling pathways.

High molecular weight organic compounds (HMW-OCs) in severe winter haze: Direct observation and insights on the formation mechanism.

High molecular weight organic compounds (HMW-OCs), formed as secondary organic aerosols (SOA), have been reported in many laboratory studies. However, little evidence of HMW-OCs formation, in particular during winter season in the real atmosphere, has been reported. In January 2013, Beijing faced historically severe haze pollution, in which the hourly PM2.5 concentration reached as high as 974 µg m-3. Four typical haze events (HE1 to HE4) were identified, and HE2 (Jan. 9-16) was the most serious of these. Based on the hourly observed chemical composition of PM2.5 and the daily organic composition analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), we found that abundant ion peaks in m/z 200-850 appeared on heavy haze days, whereas these were negligible on a clear day, indicating the existence of HMW-OCs in the wintertime haze. A negative nonlinear correlation between HMW-OCs and O3 suggested that gas oxidation was not likely to be the dominant mechanism for HMW-OCs formation. During the heavy haze events, the relative humidity and mass ratio of H2O/PM2.5 reached as high as 80% and 0.2, respectively. The high water content and its good positive correlation with HMW-OCs indicated that an aqueous-phase process may be a significant pathway in wintertime. The evidence that acidity was much higher during HE2 (0.37 µg m-3) than on other days, as well as its strong correlation with HMW-OCs, indicated that acid-catalyzed reactions likely resulted in HMW-OCs formation during the heavy winter haze in Beijing.

Calcium ion as a second messenger for o-nitrophenylsulfenyl-adrenocorticotropin (NPS-ACTH) and ACTH in bovine adrenal steroidogenesis.

o-Nitrophenyl sulfenyl-modified ACTH (NPS-ACTH) stimulated steroidogenesis acutely in bovine fasciculata-reticularis cells without increase in cellular cAMP synthesis. Application of NPS-ACTH to the cultured cells induced Ca2+ signals in individual cells as detected by video-enhanced microscopic fluorescence measurements. The percentage of Ca2+ signaling cells corresponded well with the increase of steroidogenesis induced by NPS-ACTH below 1 nM. Treatment of the cells with nicardipine, a Ca2+ channel blocker, suppressed the Ca2+ signals except for the transient increase just after the addition of NPS-ACTH and also blocked completely the stimulative effect on the steroidogenesis of NPS-ACTH below 1 nM. At a dosage of NPS-ACTH higher than 10 nM, the stimulative effect of steroidogenesis was partly suppressed by nicardipine and also by AA-861, a lipoxygenase inhibitor. The action of NPS-ACTH might be mediated by both Ca2+ and lipoxygenase metabolite(s) of arachidonic acid as dual second messengers. The effect of ACTH in pM range on the steroidogenesis was suppressed completely by the treatment with nicardipine and AA-861 at the same time, indicating that the action was mediated by both Ca2+ and the lipoxygenase metabolite(s) but not by cAMP. cAMP plays a significant role as a second messenger for ACTH action only at ACTH concentrations greater than 10 pM.

Neurosteroid synthesis by cytochrome p450-containing systems localized in the rat brain hippocampal neurons: N-methyl-D-aspartate and calcium-dependent synthesis.

Neurosteroidogenesis has not been well elucidated due to the very low level of steroidogenic proteins in the brain. Here we report the first demonstration of the neuronal localization of neurosteroidogenic systems as well as the regulation of neurosteroidogenic activity in the adult rat hippocampus. Significant localization of cytochrome P450scc was observed in pyramidal neurons and granule neurons by means of immunohistochemical staining of slices. We also observed the colocalization, in hippocampal neurons, of P450scc with redox partners, hydroxysteroid sulfotransferase and steroidogenic acute regulatory protein. The distributions of astroglial cells and oligodendroglial cells showed very different patterns from that of the P450scc-containing cells. The expression of P450scc, redox partners, the sulfotransferase, and steroidogenic acute regulatory protein was also confirmed by Western blot analysis. The process of active neurosteroidogenesis was stimulated by exposing neurons to N-methyl-D-aspartate. Upon stimulation with N-methyl-D-aspartate, Ca(2+) influx through the N-methyl-D-aspartate subtype of glutamate receptors occurred, and significant net production of pregnenolone and pregnenolone sulfate was observed in the hippocampus. This neurosteroid production was considerably suppressed by the addition of antagonists of N-methyl-D-aspartate receptors, by Ca(2+) depletion, or by the addition of an inhibitor of P450scc. Upon stimulation with N-methyl-D-aspartate, the processing of full-length steroidogenic acute regulatory protein (37-kDa) to the truncated 30-kDa steroidogenic acute regulatory protein was observed. Taken together, these observations imply that hippocampal neurons synthesize neurosteroids. This synthesis may be stimulated and regulated by glutamate-mediated synaptic communication.

The salutary effect of FK506 in ischemia-reperfusion injury of the canine liver.

The present study was designed to elucidate the effect of FK506 on 90 min of warm ischemia of the liver and reperfusion in 30 dogs. Three groups of animals were studied. Group 1 animals received FK (0.15 mg/kg/day) for three days prior to the ischemia and group 2 animals got 2 ml of saline solution for three days instead of FK and were considered controls. In group 3 FK (0.15 mg/kg/day) was injected immediately upon reperfusion and two days thereafter. Evaluation of the effectiveness of the drug was monitored by measuring the serum activities of AST, ALT, LDH, serum total bilirubin, malondialdehyde, and by histopathological examinations of the liver specimens and survival of the animals for 7 days after reperfusion. The 7 day survival of the animals in group 1 (80%) was significantly (P < 0.05) improved compared with those in group 2 (30%) and group 3 (20%). The serum activities of AST, ALT, and LDH and total bilirubin were significantly lower in group 1 than in group 2 and group 3. FK pretreatment significantly prevented hepatocellular necrosis and neutrophilic infiltration in group 1 in comparison with those in group 2 and group 3. Although the malondialdehyde level in hepatic venous blood was relatively lower in group 1, this difference was not statistically significant. Three days FK pretreatment prevented hepatocellular injury and enzyme leakage after 90 min of hepatic ischemia, whereas FK treatment immediately upon reperfusion failed to do so. In conclusion, donor organ pretreatment with FK may become a promising strategy for improved allograft survival in liver transplantation.

Nucleotide sequences of the matrix protein gene of subacute sclerosing panencephalitis viruses compared with local contemporary isolates from patients with acute measles.

Measles viruses isolated from brain cells of patients with subacute sclerosing panencephalitis (SSPE) have numerous mutations, especially in the matrix protein (M) gene. To find whether the M genes of these SSPE viruses were mutated randomly or in a pattern, we sequenced this gene from three strains of defective measles virus isolated in Osaka, Japan. We could deduce the sequence of the possible progenitor measles virus for each patient by comparison of the isolate with measles viruses prevailing at roughly the same time and place as the primary infection. Biased hypermutation affected the M genes of all three SSPE viruses, although the molecular mechanisms for the mutations might be various. Replacements of U with C in the plus strand accounted for 76% of all mutations in two of the strains, but in the other strain, replacements of A with G accounted for 52% of the mutations, and the U residues were unchanged.

Potentiation of cytotoxic effects of 5-fluorouracil by inosiplex on cancer cells.

The antitumor effect of 5-fluorouracil (5-FU) was significantly enhanced by inosiplex which has been developed as a drug possessing antiviral activity. The enhancement of antitumor effect of 5-FU was demonstrated by experiments both in vitro and in vivo, viz. depression of the colony formation rate in cultures of HeLa cells (an established cell line of human cervical carcinoma), and prolongation of the survival of mice bearing transplanted Ehrlich ascites tumor of murine mammary carcinoma origin. The HeLa cell colony formation was synergistically decreased in the presence of 0.5-2.0 micrograms/ml of 5-FU combined with 100 micrograms/ml of inosiplex. Inosiplex did not cause any appreciable inhibition of cell growth at this concentration when added alone to the culture. The mean duration of survival of tumor-bearing mice was 18.2, 20.3, 31.9 and 47.1 days in the control group and groups receiving inosiplex, 5-FU, or a combination of 5-FU and inosiplex, respectively; hence significantly prolonged in the combined therapy regimen group as compared with the control and the 5-FU treated group (P less than 0.01).

Comparison of major cytoskeletons among normal human fibroblasts, immortal human fibroblasts transformed by exposure to Co-60 gamma rays, and the latter cells made tumorigenic by treatment with Harvey murine sarcoma virus.

Immortally transformed human fibroblasts in general acquire an epithelial shape, while normal human fibroblasts demonstrate a spindle-shaped feature. In order to investigate this difference, three types of major cytoskeletal elements, namely, F-actin, tubulin, and vimentin of immortal human fibroblasts were morphologically compared with those of normal human fibroblasts. As a result, a significant difference was observed in the distribution and in the number of F-actin fibers between immortal and normal fibroblasts. The cells of three immortally transformed fibroblast lines, KMST-6, WI-38 VA-13, and SUSM-1, showed a striking reduction in the number, and an altered pattern of organization, of actin fibers. On the other hand, in the normal fibroblasts, actin fibers ran parallel to each other along the long axis of the cells. Tubulin and vimentin showed no significant difference between the immortal and normal cells. Our present data show that the morphological changes seen in the immortally transformed cells are due to the disorganization and the decrease in number of actin fibers. Interestingly, both the immortal cells (KMST-6), which were not tumorigenic, and the Harvey murine sarcoma virus-transformed KMST-6 cells, which were tumorigenic and demonstrated an enhanced expression of the ras gene, revealed an overall similarity in the organization pattern and the number of actin fibers. These findings seem to indicate that the immortally transformed cells have already acquired some cancer characteristics.

Diverse mechanisms of osteoblast spreading on hydroxyapatite and titanium.

Hydroxyapatite (HA) is an osteoconductive implant material. We previously demonstrated that RGD peptides regulate the spreading of HOS cells on HA but not on titanium, speculating that the osteoconductivity of HA might be attributed to this RGD domain-dependent spreading of osteoblasts. To confirm this hypothesis, the molecules which regulate the spreading of HOS cells on HA and on titanium were investigated. The 50% effective dose (ED50) of RGD peptide for the spreading on HA was five fold lower comparing to titanium. Anti-alphaV integrin antibody, vitronectin, and fibronectin inhibited the spreading on HA but not on titanium. In Western blot analysis, vitronectin and fibronectin were found in components adsorbed to HA but not to titanium. Taken together, the spreading of HOS cells on HA but not on titanium requires the interaction of alphaV integrin and its ligands. The ED50 of the RGD peptides on titanium but not on HA was remarkably reduced by neuraminidase treatment, that by itself could not inhibit the spreading on both materials. This phenomenon suggests that RGD domain and sialic acid cooperatively but not independently mediate the spreading of HOS cells on titanium. Collectively, the molecules regulating the spreading on HA are apparently different from those on titanium. The spreading of osteoblasts mediated by RGD domain of vitronectin and fibronectin might contribute to the osteoconductive ability of HA.

Pulmonary carcinogenesis induced by ferric nitrilotriacetate in mice and protection from it by Brazilian propolis and artepillin C.

In experiments using the renal carcinogen ferric nitrilotriacetate (Fe-NTA) in male ddY mice, primary pulmonary cancers were also induced in bronchiolar and alveolar tissues. 4-Hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2'-deoxyguanosine (8-OHdG), products of oxidative processes, increased in bronchiolar and alveolar cells after administration of Fe-NTA. These substances disappeared after oral administration of propolis or artepillin C, as shown histochemically, and correlated with an anticancer prophylactic effect of propolis and artepillin C. From our investigation, lipid peroxidation seems to play an important role in pulmonary carcinogenesis. Malignant progression from adenoma of bronchiolar or alveolar origin to malignant tumors has been proposed to involve a stepwise transformation. In our study, adenomas developed into adenocarcinomas and large cell carcinomas after treatment with Fe-NTA. In contrast, after oral administration of propolis or artepillin C, adenomas did not progress to carcinomas. Instead of developing into large cell cancers, as induced by Fe-NTA in control mice, adenomas showed remarkable proliferation of macrophages and local anti-oxidant activity after treatment with either propolis or artepillin C. Propolis and artepillin C therefore appear to inhibit lipid peroxidation and the development of pulmonary cancers.

Reverse transcription-polymerase chain reaction detection and sequence analysis of small round-structured viruses in Japan.

Between 1985 and 1995, mass outbreaks of acute gastroenteritis caused by small round-structured virus (SRSV), occurred in eight prefectures in Japan. Fecal samples from 59 patients ill during these outbreaks were recently examined in our laboratory by electron microscopy (EM) and by reverse transcription-polymerase chain reaction (RT-PCR). For RT-PCR, we prepared two sets of primers, a set corresponding to the polymerase region of open reading frame 1 (ORF-1) and a set corresponding to the capsid region of ORF-2 of Norwalk virus (NV). The SRSV nucleic acid detection rate with these primers was more than double that achieved with EM. Most samples found by EM to contain virus particles were also positive by PCR. When the two sets of primers were used separately, the virus detection rate differed depending on the primer used, suggesting that the viral strains examined were not genetically not homogeneous. We then selected nine strains of the virus, cloned their PCR products and analyzed their base sequences. The base sequences of these strains were compared with those of reference strains including prototype NV and Snow Mountain agent (SMA). This comparison yielded the following findings: (1) SRSVs that cause mass outbreaks of gastroenteritis in Japan are genetically variable; (2) SRSV strains that are genetically similar to SMA and SRSV-OTH 25/89/J(OTH25) are dominant in Japan, but strains similar to NV are also present in this country; and (3) a strain (MI1/94) which is genetically identical to Southampton virus (SHV) was detected. Detection of SRSV using sensitive RT-PCR and analysis of the sequences of the amplification products seems to provide a useful means of studying the molecular epidemiology of SRSV.

Difference in cholesterol-binding and cytolytic activities between listeriolysin O and seeligeriolysin O: a possible role of alanine residue in tryptophan-rich undecapeptide.

We have constructed recombinant listeriolysin O (rLLO) and seeligeriolysin O (rLSO) from Listeria monocytogenes and Listeria seeligeri, respectively. In hemolysis and cholesterol-binding assays, the specific activity of recombinant toxin was lower for LSO as compared to LLO. To understand the molecular basis of this difference, in particular with respect to the conserved Trp-rich undecapeptide, a naturally occurring Ala to Phe substitution in LSO was introduced into rLLO. The rLLO:A488F hemolysin exhibited a reduced activity in both hemolysis and cholesterol-binding. The reverse mutation, inserted into rLSO, also increased the hemolytic activity of this mutant LSO. These results suggested that the natural replacement of Ala to Phe is involved in the weak cytolytic activity of LSO.

Continuous administration of basic fibroblast growth factor (FGF-2) accelerates bone induction on rat calvaria--an application of a new drug delivery system.

Some studies have shown that locally applied basic fibroblast growth factor (FGF-2) enhances bone regeneration at a fracture site, while others have not been in agreement. We developed a new continuous FGF-2 delivery system designed to accelerate cytokine-induced new bone formation. A subperiosteal pocket was surgically formed in 36 eight-week-old male Wistar rats. The rats were administered 0, 1, 10, or 100 ng of FGF-2 contained in a collagen minipellet, mixed with allogeneic demineralized bone matrix in a dome-shaped Millipore filter and then placed into the pocket. New bone formation in the dome was evaluated at 2, 4, and 8 wks after placement. Soft x-ray radiographs disclosed an apparently larger radiopaque region in the 1-ng group at 4 wks compared with those in the other groups. Morphometrical analysis revealed that the new bone area in the 1-g group was significantly larger than that in the 0-g group (p<0.01). In the 100-ng FGF-2 group, new bone formation seemed suppressed. We concluded that continuous slow administration of a small amount of FGF-2 accelerates bone-derived osteogenic cytokine-induced new bone formation.

Multistep carcinogenesis of normal human fibroblasts. Human fibroblasts immortalized by repeated treatment with Co-60 gamma rays were transformed into tumorigenic cells with Ha-ras oncogenes.

Two normal mortal human fibroblast cell strains were transformed into immortal cell lines, SUSM-1 and KMST-6, by treatment with 4-nitroquinoline 1-oxide (4NQO) and Co-60 gamma rays, respectively. These immortalized cell lines showed morphological changes of cells and remarkable chromosome aberrations, but neither of them grew in soft agar or formed tumors in nude mice. The immortal cell line, KMST-6, was then converted into neoplastic cells by treatment with Harvey murine sarcoma virus (Ha-MSV) or the c-Ha-ras oncogene derived from a human lung carcinoma. These neoplastically transformed cells acquired anchorage-independent growth potential and developed tumors when transplanted into nude mice. All the tumors grew progressively without regression until the animals died of tumors. In addition, the tumors were transplantable into other nude mice. Normal human fibroblasts, on the other hand, were not transformed into either immortal or tumorigenic cells by treatment with Ha-MSV or c-Ha-ras alone. Our present data indicate that (1) the chemical carcinogen, 4NQO, or gamma rays worked as an initiator of carcinogenesis in normal human cells, giving rise to immortality, and (2) the ras gene played a role in the progression of the immortally transformed cells to more malignant cells showing anchorage-independent growth and tumorigenicity. In other words, the immortalization process of human cells seems to be a pivotal or rate-limiting step in the carcinogenesis of human cells.

Prevention of azoxymethane-induced intestinal tumors by a crude ethyl acetate-extract and tryptanthrin extracted from Polygonum tinctorium Lour.

The effect of a crude ethyl acetate (AcOEt)-extract and tryptanthrin extracted from the Indigo plant (Polygonum tinctorium Lour.) on azoxymethane (AOM)-induced intestinal tumors was examined in F344 rats. The rats were given subcutaneous (s.c.) injections of either AOM (15 mg/kg body weight (b.w.)) once a week for 3 weeks to induce atypical crypt foci (ACF) as a known cancer precursor, or AOM (7.5 mg/kg b.w.) once a week for 10 weeks to induce intestinal tumors. The rats were also administered the AcOEt-extract (500 mg/kg b.w.) or tryptanthrin (50 mg/kg b.w.) orally, 5 days a week, for 7 or 30 weeks, starting two days before the first administration of AOM. All rats were killed 4 or 20 weeks after the last treatment. In the short-term experiment, the incidence of ACE and atypical crypts (AC) in the groups receiving the AcOEt-extract and tryptanthrin was significantly lower than in the control group. In the tumor-inducing experiment, intestinal tumor incidence in the tryptanthrin group was lower than in the AOM-control group (5% versus 26%), and small intestine tumor incidence in the AcOEt-extract and tryptanthrin groups were lower than in the AOM-control group (0% and 0% versus 23%). These results show that the AcOEt-extract of Indigo and tryptanthrin have cancer chemopreventive activity.