Inhibition of spicule elongation in sea urchin embryos by the acetylcholinesterase inhibitor eserine

The activity of acetylcholinesterase (AchE) increases rapidly after the gastrula stage of sea urchin development. In this report, changes in activity and in the molecular differentiation of AchE were investigated. AchE activity increased slightly during gastrulation and rose sharply thereafter, and was dependent on new RNA synthesis. No activity of butyrylcholinesterase was found. Morphogenesis in sea urchin embryos was inhibited by the AchE inhibitor eserine, which specifically inhibited arm rod formation but not body rod formation. Spicule formation and enzyme activity in cultured micromeres were inhibited by eserine in a dose-dependent manner. During gastrulation, two molecular forms of AchE were detected with polyacrylamide gel electrophoresis. The appearance of an additional band on the gel was consistent with the occurrence of a remarkable increase in the enzyme activity. This additional band appeared as a larger molecular form in Anthocidaris crassispina, Hemicentrotus pulcherrimus, Stomopneustes variolaris, and Strongylocentrotus nudus, and as a smaller form in Clypeaster japonicus and Temnopleurus hardwicki. These results suggest that the change in the molecular form of AchE induced a change in enzymatic activity that in turn may play a role in spicule elongation in sea urchin embryos.     

A host-vector system for molecular study of the intracellular growth of Mycobacterium tuberculosis in phagocytic cells

The mechanisms by which Mycobacterium tuberculosis survives and persists in phagocytic cells remain poorly understood. To study the question, a convenient and safe host-vector system is indispensable. In this study it has been shown that, in contrast with M . smegmatis strain mc²155 which has been widely used for molecular analysis, M. smegmatis strain J15cs is able to survive even at day 6 post-infection in a murine macrophage cell line, J774. The survivability of J15cs was found to depend on the culture medium used for the bacteria prior to infection. Bacteria precultured on nutrient agar medium showed a high survivability and a characteristic cell wall ultrastructure. A plasmid vector, pYT923hyg, was developed from an Escherichia coli - mycobacterium shuttle vector pYT923 (previously constructed in our laboratory) to obtain three drug resistant genes (amp-, hyg- and km-resistant gene) and cloning sites in the km resistant gene. The vector pYT923hyg exerted no influence on in vitro growth of J15cs and intracellular survival in J774 cells, and was stably retained in J15cs after serial subculturing (three subcultures) in Luria-Bertani broth and at day 5 post-infection into J774 cells. Furthermore, using this system, the possibility of a relationship between some seemingly essential genes of M. tuberculosis and intracellular growth was demonstrated.
In this study, M. smegmatis strain J15cs and pYT923hyg were found to be capable of serving as an appropriate host-vector system for molecular study of the intracellular growth of M . tuberculosis in phagocytic cells; this system may be useful as a screening tool for M . tuberculosis genes.     

Agaritine purified from Agaricus blazei Murrill exerts anti-tumor activity against leukemic cells

Background

Mushrooms of the genus Agaricus are a common folk remedy against carcinoma. The active ingredients, polysaccharides and protein-polysaccharide complexes containing β-glucan, have been isolated and shown to have indirect tumor-suppressing activity via an immunological activation.

Methods

The diffusible fraction of a hot-water extract of Agaricus blazei Murrill (ABM) powder was fractionated by HPLC based on the anti-tumor activity against leukemic cells in vitro. The structure of the anti-tumor substance was determined by NMR and MS analyses.

Results

We purified a tumorcidal substance from the diffusible fraction of ABM and identified it as agaritine, β-N-(γ-l(+)-glutamyl)-4-(hydroxymethyl) phenylhydrazine, having a molecular mass of 267 Da. This compound inhibited the proliferation of leukemic cell lines such as U937, MOLT4, HL60 and K562 with IC₅₀ values of 2.7, 9.4, 13.0, and 16.0 μg/mL, respectively, but showed no significant effect on normal lymphatic cells at concentrations up to 40 μg/mL. Although agaritine has been suspected of having genotoxic or carcinogenic properties, agaritine did not activate the umu gene of Salmonella, which reacts to carcinogens.

General significance

The results indicate that agaritine from ABM has direct anti-tumor activity against leukemic tumor cells in vitro. This is in contrast to the carcinogenic activity previously ascribed to this compound. Our results also show that this activity is distinct from that of β-glucan, which indirectly suppresses proliferation of tumor cells.     

Hedgehog Promotes Neovascularization in Pancreatic Cancers by Regulating Ang-1 and IGF-1 Expression in Bone-Marrow Derived Pro-Angiogenic Cells

Background

The hedgehog (Hh) pathway has been implicated in the pathogenesis of cancer including pancreatic ductal adenocarcinoma (PDAC). Recent studies have suggested that the oncogenic function of Hh in PDAC involves signaling in the stromal cells rather than cell autonomous effects on the tumor cells. However, the origin and nature of the stromal cell type(s) that are responsive to Hh signaling remained unknown. Since Hh signaling plays a crucial role during embryonic and postnatal vasculogenesis, we speculated that Hh ligand may act on tumor vasculature specifically focusing on bone marrow (BM)-derived cells.

Methodology/Principal Findings

Cyclopamine was utilized to inhibit the Hh pathway in human PDAC cell lines and their xenografts. BM transplants, co-culture systems of tumor cells and BM-derived pro-angiogenic cells (BMPCs) were employed to assess the role of tumor-derived Hh in regulating the BM compartment and the contribution of BM-derived cells to angiogenesis in PDAC. Cyclopamine administration attenuated Hh signaling in the stroma rather than in the cancer cells as reflected by decreased expression of full length Gli2 protein and Gli1 mRNA specifically in the compartment. Cyclopamine inhibited the growth of PDAC xenografts in association with regression of the tumor vasculature and reduced homing of BM-derived cells to the tumor. Host-derived Ang-1 and IGF-1 mRNA levels were downregulated by cyclopamine in the tumor xenografts. In vitro co-culture and matrigel plug assays demonstrated that PDAC cell-derived Shh induced Ang-1 and IGF-1 production in BMPCs, resulting in their enhanced migration and capillary morphogenesis activity.

Conclusions/Significance

We identified the BMPCs as alternative stromal targets of Hh-ligand in PDAC suggesting that the tumor vasculature is an attractive therapeutic target of Hh blockade. Our data is consistent with the emerging concept that BM-derived cells make important contributions to epithelial tumorigenesis.     

Gender-related differences in expression and function of hepatic P-glycoprotein and multidrug resistance-associated protein (Mrp2) in rats

To clarify whether gender-related differences exist in the expression and function of hepatic P-glycoprotein- and/or multidrug resistance-associated protein (Mrp2), we measured the hepatobiliary excretion of doxorubicin and their protein levels in male and female Sprague-Dawley rats. When rats received a single intravenous injection of doxorubicin (5 mg/kg), a delay in the disappearance of doxorubicin from plasma was observed in male rats. When rats received a constant-rate infusion of doxorubicin, no significant gender-related differences in the apparent biliary clearance of doxorubicin based on the steady state plasma concentrations were observed between male and female rats. However, the net biliary clearance of doxorubicin based on the liver concentration, which represents the actual function of P-glycoprotein and/or Mrp2, was higher in female rats than in male rats. These results suggest that the actual function of the hepatobiliary transport of doxorubicin is greater in female than in male rats. Western blot analysis revealed that the expression of P-glycoprotein and Mrp2 in the liver of female rats was significantly higher than in male rats, similar to results of hepatobiliary excretion experiments. The expression of hepatic cytochrome P450 (CYP) 2B1, which is involved in the metabolism of doxorubicin, was significantly higher in male than in female rats. By pretreatment with testosterone (10 mg/day for 7 days), the actual biliary clearance of doxorubicin in female rats was nearly that of male rats. The protein levels of P-glycoprotein and Mrp2 in female rats were also lowered by treatment with testosterone so as to be nearer those in male rats. These results suggest that gender-related differences exist in P-glycoprotein- and Mrp2-mediated hepatobiliary transport and that these two transporters may be regulated by sex hormones.     

Heregulin-alpha and heregulin-beta expression is linked to a COX-2-PGE2 pathway in human gastric fibroblasts

We have previously shown heregulin (HRG)-alpha expression in human gastric fibroblasts and its stimulation of gastric epithelial cell growth. Although cyclooxygenase (COX)-2 has also been shown to stimulate growth factor production in these cells, the interaction between COX-2 and HRG remains unknown. Conditioned media (CM) from gastric fibroblasts incubated with PGE(2) or interleukin (IL)-1beta, a well known COX-2 inducer, were analyzed for their effect on erbB3 tyrosine phosphorylation in MKN28 gastric epithelial cells. HRG protein expression in fibroblast lysates and CM was also examined by western blot. HRG-alpha and HRG-beta mRNA expression in gastric fibroblasts and human gastric tissue was examined by real-time quantitative PCR. HRG and COX-2 expressions in surgical resections of human gastric ulcer tissue were examined immunohistochemically. CM from fibroblasts incubated with PGE(2), or IL-1beta, stimulated erbB3 phosphorylation in MKN28 cells. Preincubation of the fibroblasts with celecoxib, a selective COX-2 inhibitor, suppressed CM-induced erbB3 phosphorylation. This inhibition was reversed by exogenous PGE(2). As with erbB3 phophorylation, IL-1beta stimulated both HRG-alpha and HRG-beta mRNA expression, as well as HRG release into gastric fibroblast CM. IL-1beta-stimulated HRG expression and release were also inhibited by celecoxib, and exogenous PGE(2) restored this inhibitory effect, suggesting the activation of an IL-1beta-COX-2-PGE(2) pathway that culminates in the release of HRG from fibroblasts. HRG-alpha and HRG-beta mRNA levels were significantly higher in gastric ulcer tissue than in normal gastric mucosa. HRG immunoreactivity was found in interstitial cells of the gastric ulcer bed and coexpressed with COX-2. These results suggest that HRG might be a new member of the growth factor family involved in the COX-2-dependent ulcer repair process.     

Antigen chemically coupled to the surface of liposomes are cross-presented to CD8+ T cells and induce potent antitumor immunity

We have previously demonstrated that liposomes with differential lipid components display differential adjuvant effects when Ags are chemically coupled to their surfaces. In the present study, Ag presentation of liposome-coupled OVA was investigated in vitro, and it was found that OVA coupled to liposomes made using unsaturated fatty acid was presented to both CD4+ and CD8+ T cells, whereas OVA coupled to liposomes made using saturated fatty acid was presented only to CD4+ T cells. Confocal laser scanning microscopic analysis demonstrated that a portion of the OVA coupled to liposomes made using unsaturated, but not saturated fatty acid, received processing beyond the MHC class II compartment, suggesting that the degradation of OVA might occur in the cytosol, and that the peptides generated in this manner would be presented to CD8+ T cells via MHC class I. The ability to induce cross-presentation of an Ag coupled to liposomes consisting of unsaturated fatty acid was further confirmed by in vivo induction of CTL and by the induction of tumor eradication in mice; E.G7 tumors in mice that received combined inoculation with OVA(257-264)-liposome conjugates, CpG, and anti-IL-10 mAbs were completely eradicated. In those mice, the frequency of CD8+ T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased. These results suggested that, by choosing lipid components for liposomes, surface-coupled liposomal Ags might be applicable for the development of tumor vaccines to present tumor Ags to APCs and induce antitumor responses.     

HIV+/AIDS related bioethical issues in Japan

Annual and cumulative incidences of HIV+ and AIDS in patients reported by the AIDS Surveillance Committee of the Ministry of Health and Welfare are cited to illustrate some characteristics in Japan: nearly 59% of either HIV+ or AIDS patients were infected through injection of blood products or by blood transfusion. A number of plaintiffs have sued the Japanese government and pharmaceutical companies since 1989, but no judicial decisions have yet been made. The incidence of HIV decreases for each of the following routes of infection: the second highest route of transmission being heterosexual, followed by bisexual and unconfirmed or unknown cases, and less frequently homosexual, drug use and lastly via vertical transmission. The most serious bioethical problems with HIV+ and AIDS patients in Japan is the social segregation of these patients. There are many emotional fears among the general public and medical personnel about AIDS because of their inadequate scientific knowledge of this disease. The Japan Hospital Association is doing The Stop AIDS Campaign and made several surveys. Japan has become increasingly aware of the importance of AIDS education and a great deal of effort is being made to enhance bioethical consideration.