Characterization of a thermostable and acidic-tolerable beta-glucanase from aerobic fungi Trichoderma koningii ZJU-T.

An extreme thermostable and acidic tolerable beta-glucanase was isolated and characterized from aerobic fungi Trichoderma koningii ZJU-T. The optimal reaction temperature and pH for the beta-glucanase were 100 degrees C and pH 2.0, respectively. The beta-glucanase showed increased stability at higher temperatures and lower pH values when compared to other beta-glucanases. The optimum conditions for the beta-glucanase stability were found to be pH 4.0 and 80 degrees C. Even subjected to 100 degrees C for 3 h, beta-glucanase activity did not show significant reduction. Moreover, K(+) significantly enhanced beta-glucanase activity at the concentration of 1 mM, while EDTA and other metal ions such as Mg(2+), Mn(2+), Zn(2+), Ca(2+), Fe(2+), Pb(2+), and Fe(3+) inhibited beta-glucanase activity. Denaturants, including sodium dodecyl sulfate (SDS) and mercaptoethanol, also inhibited beta-glucanase activity at a concentration of 5%. However, in the presence of 7 M urea, residual activity of the beta-glucanase still remained 14.5%.

Different effects of five catechins on 6-hydroxydopamine-induced apoptosis in PC12 cells.

Five catechins [(-)-epigallocatechins gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC), and (+)-catechin (C)] were compared with regard to their effects on 6-hydroxydopamine (OHDA)-induced apoptosis in PC12 cells--the vitro model of Parkinson's disease. Measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 6-OHDA inhibited cell viability in a time- and concentration-dependent manner. When PC12 cells were pretreated with the five catechins for 30 min before exposure to 250 microM 6-OHDA, MTT results showed that the five catechins had different effects: EGCG and ECG had obvious concentration-dependent protective effects at 50-400 microM; EC and (+)-C had almost no effects; and EGC especially decreased cell viability. Catechins also had different effects on apoptotic morphology. Only 200-400 microM EGCG and ECG kept cells adhering well. When pretreated with other catechins at any concentration, PC12 cells became round and some of them were detached as when treated with 6-OHDA. In addition, typical apoptotic characteristics of PC12 cells were determined by fluorescence microscopy, flow cytometry, and DNA fragment electrophoresis after the cells were treated with 250 microM 6-OHDA for 24 h or pretreated with catechins before it. Preincubation with 200-400 microM EGCG and ECG led to significant inhibitory effects against PC12 cell apoptosis, as shown by flow cytometry. The other catechins have little protective effect. Therefore, at 200-400 microM, the classified protective effects of the five catechins were in the order ECG > EGCG >> EC > (+)-C > EGC. The data also indicated that EGCG and ECG might be potent neuroprotective agents for Parkinson's disease. The results of fluorescence microscopy and DNA fragment analysis supported the conclusion.

Proteomic detection of changes in protein synthesis induced by NGX6 transfected in human nasopharyngeal carcinoma cells.

In the previous study, we cloned a new gene, named NGX6, related to nasopharyngeal carcinoma (NPC) at 9p. To study its function in the pathogenesis of NPC, we have investigated changes in protein synthesis between NPC cell line HNE1 and that transfected with the gene. Using high-resolution two-dimensional electrophoresis, we found that 22 protein spots showed variations that were significant and reproducible. Analysis of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searches identified seven proteins that were upregulated and seven proteins that were downregulated. These proteins included Fas, zinc-finger protein (ZNF), RAB, and Ah receptor-interacting protein (AIP). The functional implications of the identified proteins are discussed.

Expression of nuclear retinoid receptors in normal, premalignant and malignant gastric tissues determined by in situ hybridization.

Retinoids exhibit multiple functions through interaction with nuclear retinoid receptors and have growth-suppressive activity on gastric cancer cells. To better understand the roles of nuclear retinoid receptors during gastric carcinogenesis, we have used in situ hybridization to investigate expression of retinoic acid receptors (RARs) and retinoid x receptors (RXRs) in premalignant and malignant formalin-fixed paraffin-embedded gastric tissues. Histological sections of eight normal, 17 distal normal and nine gastric cancer tissues were hybridized with non-radioactive RNA probes for subtypes of RAR and RXR. Expression of RAR alpha, RAR beta, RAR gamma, RXR alpha and RXR beta was found in most cell types in gastric mucosa tissues from normal individuals as well as in distal normal tissues from cancer patients. Expression of RAR alpha and RAR beta were found in three and seven cancer tissues, respectively, and levels of RXR alpha mRNA were significantly decreased in poorly differentiated cancer tissues. Among the five investigated nuclear retinoid receptors, only expression of RAR alpha mRNA was significantly decreased in intestinal metaplasia, dysplasia and cancer tissues when compared to adjacent normal tissues. In conclusion, normal gastric mucosa expressed both RARs and RXRs, which supports the physiological role of retinoic acid on normal gastric mucosa. The decrease in RAR alpha expression in premalignant and malignant gastric tissues suggests a significant role of RAR alpha during gastric carcinogenesis.

[Mechanism of scavenging effects of (-)-epigallocatechin gallate on active oxygen free radicals].

The concentration of 50% scavenging ratio (SC50) and the scavenging rate constants (k) and stiochiometric factor (n) were determined when (-)-epigallocatechin gallate [(-)-EGCG] scavenging superoxide anion free radical (O2.-) and hydroxyl radical (.OH). The mechanism of scavenging active free radicals of (-)-EGCG and the promotion of (-)-EGCG free radical and its structure were analyzed in vitro. Our results suggest that the centers of scavenging reaction of (-)-EGCG are B, D, and A cycles, and each (-)-EGCG traps six O2.- or .OH. It is in accord with the value of stiochiometric factor n = 6.

[Effects of epostane on chorio-trophoblast, co-culture of embryos with endometrium and corpora lutea in vitro].

Effects of epostane on human trophoblast cells, co-culture of mouse embryos with endometrium and guinea pig corpora lutea were studied in vitro. Human chorio-trophoblast cells were obtained from induced abortion (2-2.5 months of pregnancy). The tissue was incubated in control medium or in medium containing epostane. The results indicated that epostane at a concentration of 50 micrograms/ml could injure the human trophoblast cells and its function. Obviously damaged cells were observed at the concentration of 100 micrograms/ml. The cells were degenerated with pyknotic nuclei and sometimes only cell fragments were found. Guinea pig corpora lutea produced progesterone in vitro. In the incubation medium of the control group, the total luteal progesterone increased linearly with the prolongation of incubation during the first 30 min of incubation. The addition of hCG at concentration of 10 IU/ml clearly accelerated progesterone production. Epostane at a concentration of 50 micrograms/ml could significantly inhibit both basic and the hCG-stimulated luteal production of progesterone. Epostane at concentrations of 50 and 100 micrograms/ml showed no inhibitory effect on mouse embryos and endometrium in vitro.

Effects of anordrin and its analogue on antifertility.

Anordrin has been used as an effective postcoital contraceptive in China. The mechanism of anordrin and its analogue SIPPR-113 on antifertility has been studied. Anordrin and SIPPR-113 possessed estrogenicities and induced decrease in serum progesterone levels in rats. Their antiprogesterone activities might be mainly caused by their estrogenicities, which were the main but not the only contributors for the antifertility. The direct effects of anordrin and SIPPR-113 on human trophoblast cells were studied. A concentration of 50 micrograms/ml or 100 micrograms/ml of anordrin or SIPPR-113 could injure the human trophoblast cells in vitro. The uterine Pontamine blue reaction of mated rats was inhibited in those treated with anordrin or SIPPR-113 at the dose of 4 mg/kg. Anordrin, SIPPR-113 or AF-45 was given orally, intramuscularly and intravenously. The effects of drugs administered via the three routes were nearly the same. This study further demonstrated that anordrin was hydrolyzed to break its bond of dipropionate and was transformed into its parent steroid AF-45 to exert its antifertility effects in vivo. This study warrants that anordrin should been evaluated further.

The nature of radiation damage of haemopoietic stem cells under continuous irradiation at low dose rate.

Mice irradiated continuously at a low dose rate of 70 rad/day showed not only a drop of CFU-S content in the bone marrow but also an apparent functional disturbance of haemopoietic stem cells. With increase in accumulated dose of radiation, the damage of the haemopoietic microenvironment becomes evident. From the changes caused by chronical irradiation, it is believed that haemopoietic stem cells is more sensitive than haemopoietic microenvironment, and the radiation injury by hemopoiesis involves both damage of haemopoietic stem cells and microenvironment. Furthermore, in the course of recovery of hemopoietic injury, microenvironment plays as important role. In comparison of the growth curves of CFU-S from normal and irradiated bone marrow cells in irradiated recipient spleens or cultured in diffusion chambers, it is suggested that the nature of radiation damage of haemopoietic stem cells in continuous irradiation at low dose rate is mainly attributed to the deterioration of self replicative capacity of CFU-S.

Studies on the property and transplantation of haemopoietic stem cells from peripheral blood.

Comparative studies of the properties of murine haemopoietic stem cells from different sources revealed that the peripheral haemopoietic stem cell is relatively weaker than the haemopoietic stem cell from bone marrow in promoting the recovery of hemopoiesis in the irradiated animals. This is due to the heterogenity of stem cell population in which some aged cellsubpopulations are co-existing. The modified potential in proliferation and differentiation of haemopoietic stem cells in the peripheral blood seems to be irreversible under normal physiological conditions. In a preliminary experiment, the use of an anti-thymocyte immunoglobulin to eliminate immunocompetent cells proved effective in reducing the severity and incidence of secondary diseases and in increasing the number of survivors of lethally irradiated semi-isologous mice after transplantation of parental peripheral mononuclear cells.