Novel method for detection of butanolides in Streptomyces coelicolor culture broth, using a His-tagged receptor (ScbR) and mass spectrometry

Gamma-butyrolactone derivative molecules in Streptomyces play a crucial role in cell density control, secondary metabolism, and cell differentiation. As their synthesis level in the cell is very low compared to those of similar N-acyl homoserine lactone molecules from gram-negative bacteria, it is very hard to analyze them even with several hundredfold concentration of the culture broth. We have developed a very quick and easy detection method using an affinity capture technique with His-tagged receptor proteins and electrospray tandem mass spectrometry. Using Streptomyces coelicolor as a model system, SCB1 was detected from only 100 ml of the culture broth after solvent extraction. This method can be further applied to detection and quantitative analysis of butanolides and inhibitor screening of the receptor molecules.     

Hypoglycemic effect of crude exopolysaccharides produced by a medicinal mushroom Phellinus baumii in streptozotocin-induced diabetic rats

The antidiabetic effect of the crude exopolysaccharides (EPS) produced from submerged mycelial culture of Phellinus baumii in streptozotocin (STZ)-induced diabetic rats was investigated. The produced EPS consisted of two different heteropolysaccharides and two proteoglycans. The food intake of the diabetic control rats (STZ) was increased by 28.1%, whereas body weight gain was reduced by 44.1% as compared to the nondiabetic animals (NC). The plasma glucose level in the EPS-fed rats (EPS) was substantially reduced by 52.3% as compared to the diabetic rats (STZ), which is the highest hypoglycemic effect among mushroom-derived materials documented in literature. The activities of alanine aminotransferase (ALT) and asparate aminotransferase (AST) were significantly decreased by administration of P. baumii EPS, thereby exhibiting a remedial role in liver function. The significant increase in weights of liver, spleen, and kidney was observed in diabetic groups (both STZ and EPS) compared to NC. The results suggest that orally administrated P. baumii EPS exhibited considerable hypoglycemic effect in STZ-induced diabetic rats and that these EPS may be useful for the management of diabetes mellitus.     

Gamma-radiation-induced follicular degeneration in the prepubertal mouse ovary

Prepubertal mice were whole-body irradiated with a mean lethal dose (LD50) of gamma-radiation using a 60Co source with a total dose of 7.2 Gy and a dose rate of 12.0 cGy/min. At day 0 before the irradiation and at day 1, 2, and 3 after the irradiation, the ovaries were collected and the morphological changes were assessed. The ratios (%) of atretic or polymorphonuclear leukocytes (neutrophil)-infiltrated follicles in the largest cross sections were calculated. In the early atretic follicle of the control mouse ovary, both apoptotic and mitotic cells were observed and occasionally neutrophils were infiltrated into the follicle cavity. However, in the atretic follicles 2 days post-irradiation, numerous cell fragments, apoptotic cells and bodies, and especially, a number of neutrophils were observed. In the non-irradiated control, the ratios of atretic follicles were 58.0+/-8.6 and 27.3+/-11.2 (mean+/-S.E.M.) in antral and preantral follicles, respectively. The ratios of the number of antral and preantral follicles with one or more neutrophils to the total number of atretic follicles were 29.3+/-12.0. At 2 days post-irradiation, the ratios of atretic follicles were increased to 94.0+/-3.4 and 86.9+/-7.6 in antral and preantral follicles, respectively. The ratios of neutrophil-containing follicles among the atretic one were increased to 65.9+/-11.5 and 57.8+/-15.4 at 2 and 3 days after the irradiation, respectively. Taken together, the present results show that gamma-radiation induces apoptotic and inflammatory degeneration of mouse ovarian follicles. Besides, neutrophils may be involved in the acute atretic degeneration in gamma-irradiated mouse ovarian follicles.     

Dissection of CDK4-binding and transactivation activities of p34(SEI-1) and comparison between functions of p34(SEI-1) and p16(INK4A)

Recent studies showed that p34(SEI-1), also known as TRIP-Br1 or SEI-1, plays a dual role in the regulation of cell-cycle progression. It exhibits the transactivation activity and regulates a number of genes required for G1/S transition, while it also binds and activates cyclin-dependent kinase 4 (CDK4) independent of the inhibitory activity of p16. The goals of this paper are to further dissect the two roles and to compare the functions between SEI-1 and p16. (i) Yeast one-hybrid-based random mutagenesis was first used to identify a number of SEI-1 residues important for LexA-mediated transactivation, including residues L51, K52, L53, H54, L57, and L69 located within the heptad repeat (residues 30-88), a domain required for LexA-mediated transactivation, and two residues M219 and L228 at the C-terminal segment that contributes to transactivation through modulating the heptad repeat. (ii) The functional significance of these residues was further confirmed by site-directed mutagenesis. It was also shown that the heptad repeat-involving transactivation is distinct from the well-known acidic region-involving transactivation. (iii) Yeast two-hybrid-based binding analysis was made possible with the transactivation-negative SEI-1 mutants, and the results showed that some of such mutants retain full ability to bind and activate CDK4. (iv) Site-specific mutants of CDK4 were used to show that there are notable differences among SEI-1, p16, and cyclin D2 in binding to CDK4. (v) The expression levels of SEI-1 and p16 were compared in 32 tumor specimens of human squamous cell carcinomas of the head and neck. The results indicate that SEI-1 was consistently overexpressed, while p16 was consistently underexpressed. These results provide important information on the molecular mechanism of the functions of SEI-1 and on the comparison between SEI-1 and p16 at both molecular and cellular levels.     

Integrin-linked kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition

The role of integrin-linked kinase (ILK) in transforming growth factor beta (TGFbeta)-mediated epithelial to mesenchymal transition was investigated. A stable transfection of dominant-negative ILK results in the prevention of TGFbeta-mediated E-cadherin delocalization. TGFbeta-mediated phosphorylation of Akt at Ser-473 was inhibited by dominant-negative ILK and PI3K inhibitors, LY294002 and wortmannin. Treatment with TGFbeta stimulated induction of Akt and ILK kinase activity in HaCat control cells. This increased ILK activity by TGFbeta was lowered by PI3K inhibitor, LY294002. In addition, PI3K inhibitor, dominant-negative Akt, and dominant-negative ILK could not block TGFbeta-mediated C-terminal phosphorylation of Smad2. Taken together, these data suggest that PI3K-ILK-Akt pathway that is independent of the TGFbeta-induced Smad pathway is required for TGFbeta-mediated epithelial to mesenchymal transition.     

MAP, a protein interacting with a tumor suppressor, merlin, through the run domain

Merlin (or schwannomin) is a tumor suppressor encoded by the neurofibromatosis type 2 gene. Many studies have suggested that merlin is involved in the regulation of cell growth and proliferation through interactions with various cellular proteins. To better understand the function of merlin, we tried to identify the proteins that bind to merlin using the yeast two-hybrid screening. Characterization of the positive clones revealed a protein of 749 amino acids named merlin-associated protein (MAP), which showed wide tissue distribution in Northern blot analysis. Sequence analysis revealed that MAP is a potential homologue of a yeast check-point protein, BUB2, and contains TBC, SH3, and RUN domains, thereby implicating its role in the Ras-like GTPase signal pathways. MAP and merlin were directly associated in vitro and in vivo, and colocalized in NIH3T3 cells. The RUN domain of MAP and the C-terminus of merlin appeared to be responsible for their interaction. MAP decreased the AP-1-dependent promoter activity additively with merlin in NIH3T3 cells. In addition, merlin and MAP synergistically reduced the colony formation of NIH3T3 cells. These results suggest that MAP may play a cooperative role in the merlin-mediated growth suppression of cells.     

Role of calcium in pancreatic islet cell death by IFN-gamma/TNF-alpha

We studied the intracellular events associated with pancreatic beta cell apoptosis by IFN-gamma/TNF-alpha synergism. IFN-gamma/TNF-alpha treatment of MIN6N8 insulinoma cells increased the amplitude of high voltage-activated Ca(2+) currents, while treatment with IFN-gamma or TNF-alpha alone did not. Cytosolic Ca(2+) concentration ([Ca(2+)](c)) was also increased by IFN-gamma/TNF-alpha treatment. Blockade of L-type Ca(2+) channel by nifedipine abrogated death of insulinoma cells by IFN-gamma/TNF-alpha. Diazoxide that attenuates voltage-activated Ca(2+) currents inhibited MIN6N8 cell death by IFN-gamma/TNF-alpha, while glibenclamide that accentuates voltage-activated Ca(2+) currents augmented insulinoma cell death. A protein kinase C inhibitor attenuated MIN6N8 cell death and the increase in [Ca(2+)](c) by IFN-gamma/TNF-alpha. Following the increase in [Ca(2+)](c), calpain was activated, and calpain inhibitors decreased insulinoma cell death by IFN-gamma/TNF-alpha. As a downstream of calpain, calcineurin was activated and the inhibition of calcineurin activation by FK506 diminished insulinoma cell death by IFN-gamma/TNF-alpha. BAD phosphorylation was decreased by IFN-gamma/TNF-alpha because of the increased calcineurin activity, which was reversed by FK506. IFN-gamma/TNF-alpha induced cytochrome c translocation from mitochondria to cytoplasm and activation of caspase-9. Effector caspases such as caspase-3 or -7 were also activated by IFN-gamma/TNF-alpha treatment. These results indicate that IFN-gamma/TNF-alpha synergism induces pancreatic beta cell apoptosis by Ca(2+) channel activation followed by downstream intracellular events such as mitochondrial events and caspase activation and also suggest the therapeutic potential of Ca(2+) modulation in type 1 diabetes.