Connective tissue growth factor is a substrate of ADAM28

ADAM28, a member of the ADAM (a disintegrin and metalloproteinase) gene family, is over-expressed by carcinoma cells and the expression correlates with carcinoma cell proliferation and progression in human lung and breast carcinomas. However, information about substrates of ADAM28 is limited. We screened interacting molecules of ADAM28 in human lung cDNA library by yeast two-hybrid system and identified connective tissue growth factor (CTGF). Binding of CTGF to proADAM28 was demonstrated by yeast two-hybrid assay and protein binding assay. ADAM28 cleaved CTGF in dose- and time-dependent manners at the Ala¹⁸¹-Tyr¹⁸² and Asp¹⁹¹-Pro¹⁹² bonds in the hinge region of the molecule. ADAM28 selectively digested CTGF in the complex of CTGF and vascular endothelial growth factor₁₆₅ (VEGF₁₆₅), releasing biologically active VEGF₁₆₅ from the complex. RT-PCR and immunohistochemical analyses demonstrated that ADAM28, CTGF and VEGF are commonly co-expressed in the breast carcinoma tissues. These data provide the first evidence that CTGF is a novel substrate of ADAM28 and suggest that ADAM28 may promote VEGF₁₆₅-induced angiogenesis in the breast carcinomas by the CTGF digestion in the CTGF/VEGF₁₆₅ complex.     

Further studies on thermal treatment of two-cell stage embryos to produce complete embryonic stem-cell-derived mice by cell-aggregation methods

Employing aggregation techniques with two embryonic sources, one from two-cell stage embryos treated by thermal stimulation and the other from mouse embryonic stem (ES) cells that had been obtained from a feeder layer, simple and most effective methods of producing a complete generation of mice from ES cells were explored. Although thermal treatment affected embryos at various developmental stages, the embryos at the two-cell stage of development were selected because of the remarkably reduced number of cells present in the inner cell mass (ICM) at blastocyst stage after thermal conditioning. Under these conditions, a combination of thermally treated host embryos and an aggregated ES cell-clump was found to produce a high rate of live newborns by natural delivery. That the newborns were completely derived from ES cells was checked by two criteria: microsatellite analysis and coat color analysis. Importantly, all of these mice were healthy and fertile. The aggregation techniques reported here might well be applied to other animal species whose ES cells form stable colonies on a feeder layer.     

Involvement of two cytosolic enzymes and a novel intermediate, 5'-oxoaverantin, in the pathway from 5'-hydroxyaverantin to averufin in aflatoxin biosynthesis

During aflatoxin biosynthesis, 5'-hydroxyaverantin (HAVN) is converted to averufin (AVR). Although we had previously suggested that this occurs in one enzymatic step, we demonstrate here that this conversion is composed of two enzymatic steps by showing that the two enzyme activities in the cytosol fraction of Aspergillus parasiticus were clearly separated by Mono Q column chromatography. An enzyme, HAVN dehydrogenase, catalyzes the first reaction from HAVN to a novel intermediate, another new enzyme catalyzes the next reaction from the intermediate to AVR, and the intermediate is a novel substance, 5'-oxoaverantin (OAVN), which was determined by physicochemical methods. We also purified both of the enzymes, HAVN dehydrogenase and OAVN cyclase, from the cytosol fraction of A. parasiticus by using ammonium sulfate fractionation and successive chromatographic steps. The HAVN dehydrogenase is a homodimer composed of 28-kDa subunits, and it requires NAD, but not NADP, as a cofactor for its activity. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of tryptic peptides of the purified HAVN dehydrogenase revealed that this enzyme coincides with a protein deduced from the adhA gene in the aflatoxin gene cluster of A. parasiticus. Also, the OAVN cyclase enzyme is a homodimer composed of 79-kDa subunits which does not require any cofactor for its activity. Further characterizations of both enzymes were performed.     

Quantitative analysis of biological responses to ionizing radiation, including dose, irradiation time, and dose rate

Because biological responses to radiation are complex processes that depend on both irradiation time and total dose, consideration of both dose and dose rate is necessary to predict the risk from long-term irradiations at low dose rates. Here we mathematically and statistically analyzed the quantitative relationships between dose, dose rate and irradiation time using micronucleus formation and inhibition of proliferation of human osteosarcoma cells as indicators of biological response. While the dose-response curves did not change with exposure times of less than 20 h, at a given dose, both biological responses clearly were reduced as exposure time increased to more than 8 days. These responses became dependent on dose rate rather than on total dose when cells were irradiated for 20 to 27 days. Mathematical analysis demonstrates that the relationship between effective dose and dose rate is well described by an exponential function when the logarithm of effective dose is plotted as a function of the logarithm of dose rate. These results suggest that our model, the modified exponential (ME) model, can be applied to predict the risk from exposure to low-dose/low-dose-rate radiation.     

Cyclic GMP-dependent protein kinase EGL-4 controls body size and lifespan in C elegans

We designed an automatic system to measure body length, diameters and volume of a C. elegans worm. By using this system, mutants with an increased body volume exceeding 50% were isolated. Four of them are grossly normal in morphology and development, grow longer to be almost twice as big, and have weak egg-laying defects and extended lifespan. All the four mutants have a mutation in the egl-4 gene. We show that the egl-4 gene encodes cGMP-dependent protein kinases. egl-4 promoter::gfp fusion genes are mainly expressed in head neurons, hypodermis, intestine and body wall muscles. Procedures to analyze morphology and volume of major organs were developed. The results indicate that volumes of intestine, hypodermis and muscle and cell volumes in intestine and muscle are increased in the egl-4 mutants, whereas cell numbers are not. Experiments on genetic interaction suggest that the cGMP-EGL-4 signaling pathway represses body size and lifespan through DBL-1/TGF-beta and insulin pathways, respectively.