A simplified and efficient method for in situ hybridization to whole Drosophila embryos, using electrophoresis for removing non-hybridized probes

We report a simplified and reliable method for non-radioactive in situ hybridization to whole Drosophila embryos. In the previous method (Tautz and Pfeifle, 1989) the post-hybridization wash, or the procedure for washing non-hybridized probe away from embryos depends simply on diffusion. We modified the method with application of electrophoresis to the wash. After hybridized with RNA probe, embryos were transferred to a small well where an electric charge was given to drive non-hybridized probe away from the embryos. This procedure enables us to acquire a much higher signal-to-noise ratio than that obtained from a conventional method. Furthermore, this is a time-saving method. We propose a term "electro-wash" for this procedure.     

Suramin interrupts androgen-inducible autocrine loop involving heparin binding growth factor in mouse mammary cancer (Shionogi carcinoma 115) cells

The androgen-dependent clonal cell line SC-3, derived from Shionogi carcinoma 115, secretes a fibroblast growth factor (FGF)-autocrine growth factor in response to androgen, which is able to bind to FGF receptors. In SC-3 cells, FGF receptor expression is upregulated by the SC-3-derived growth factor, providing a means of amplifying an autocrine loop of cell growth. In the present investigations, the effect of the polysulfonated naphthylurea suramin on this autocrine loop and its amplification in SC-3 cells were studied. Suramin inhibited androgen-dependent growth of SC-3 cells in a concentration-dependent fashion: ～50% inhibition was observed at 25 μM. [³H]Thymidine incorporation into the cells stimulated with partially purified SC-3-derived growth factor was inhibited by suramin in a similar way. Additionally, suramin inhibited acidic (a) or basic (b) FGF-induced cell proliferation, though relatively high concentrations were necessary to achieve the maximal inhibition. Pretreatment of SC-3 cells with suramin decreased cell surface ¹²⁵I-bFGF binding without altering dissociation constant (Kd) of the binding sites. When the cells were incubated with 250 μM suramin for 24 h, the maximum binding (Bmax) decreased to almost 50% of the control. Treatment with suramin also decreased the levels of FGF receptor-1 mRNA to a similar extent, whereas it appeared not to affect the levels of β-actin mRNA. Moreover, suramin completely blocked androgen- or bFGF-induced accumulation of FGF receptor-1 mRNA. The inhibitory effects of suramin on FGF receptor expression were reversed by simultaneous addition of high concentrations of bFGF. These results indicate that suramin exerts its potent antiproliferative action on SC-3 cells through inhibition of an androgen-inducible autocrine loop involving SC-3-derived growth factor and FGF receptor. © 1993 Wiley-Liss, Inc.     

Soluble Sema4D cleaved from osteoclast precursors by TACE suppresses osteoblastogenesis

Bone remodelling is mediated by orchestrated communication between osteoclasts and osteoblasts which, in part, is regulated by coupling and anti-coupling factors. Amongst formally known anti-coupling factors, Semaphorin 4D (Sema4D), produced by osteoclasts, plays a key role in downmodulating osteoblastogenesis. Sema4D is produced in both membrane-bound and soluble forms; however, the mechanism responsible for producing sSema4D from osteoclasts is unknown. Sema4D, TACE and MT1-MMP are all expressed on the surface of RANKL-primed osteoclast precursors. However, only Sema4D and TACE were colocalized, not Sema4D and MT1-MMP. When TACE and MT1-MMP were either chemically inhibited or suppressed by siRNA, TACE was found to be more engaged in shedding Sema4D. Anti-TACE-mAb inhibited sSema4D release from osteoclast precursors by ~90%. Supernatant collected from osteoclast precursors (OC-sup) suppressed osteoblastogenesis from MC3T3-E1 cells, as measured by alkaline phosphatase activity, but OC-sup harvested from the osteoclast precursors treated with anti-TACE-mAb restored osteoblastogenesis activity in a manner that compensates for diminished sSema4D. Finally, systemic administration of anti-TACE-mAb downregulated the generation of sSema4D in the mouse model of critical-sized bone defect, whereas local injection of recombinant sSema4D to anti-TACE-mAb-treated defect upregulated local osteoblastogenesis. Therefore, a novel pathway is proposed whereby TACE-mediated shedding of Sema4D expressed on the osteoclast precursors generates functionally active sSema4D to suppress osteoblastogenesis.     

Population dynamics of japanese serow in relation to social organization and habitat conditions. I. Stability of Japanese serow density in stable habitat conditions

The density of a Japanese serow (Capricornis crispus) population, which has been in stable habitat conditions and free from hunting and predation, was investigated by direct observation of serows at Kusoudomari (336 ha), Wakinosawa village, Aomori Prefecture, Japan, during 1977–89. The vegetation, composed of mature mixed forest ofThujopsis with deciduous trees (43.2%), secondary forest of deciduous trees (23.4%) and a plantation of coniferous trees (30.0%) showed little change during the study period. The serows were counted 17 times each winter for 14 consecutive years. The serow population maintained a stable density with a mean of 12.5 individuals km⁻² (SD=1.4) ranging from 8.6 to 14.6 km⁻². The ratio of the proportion of kids (<1 year old) to serows more than 1 year old decreased, but density did not decrease during the study period. Though extremely severe winters with heavy snowfall in 1984–86 caused a decline in serow density, the density recovered within a few years. It was considered that the stability of the serow density resulted mainly from the stable food supply and the lack of human disturbance, such as forest cutting in the habitat. Stable population density for a long period is probably related to the social organization of the serow.     

In vivo cross-linking of nucleosomal histones catalyzed by nuclear transglutaminase in starfish sperm and its induction by egg jelly triggering the acrosome reaction.

A histone heterodimer, designated as p28, which contains an Nepsilon(gamma-glutamyl)lysine cross-link between Gln9 of histone H2B and Lys5 or Lys12 of histone H4, is present in starfish (Asterina pectinifera) sperm. Treatment of sperm nuclei with micrococcal nuclease produced soluble chromatin, which was size-fractionated by sucrose-gradient centrifugation to give p28-containing oligonucleosome and p28-free mononucleosome fractions, indicating that the cross-link is internucleosomal. When sperm nuclei were incubated with monodansylcadaverine, a fluorescent amine, in the presence or absence of Ca(2+), histone H2B was modified only in the presence of Ca(2+). Gln9, in the N-terminal region, was modified, but the other Gln residues located in the internal region were not, suggesting that the modification takes place on the surface of the nucleosome core by the in situ action of a Ca(2+)-dependent nuclear transglutaminase. Treatment of sperm with the egg jelly, which activates Ca(2+) influx to induce the acrosome reaction, resulted in a significant elevation of the p28 content in the nucleus. This is the first demonstration of an in vivo activation of transglutaminase leading to the formation of a cross-link in intracellular proteins.     

Carcinogenesis in accessory sex organs of rats induced by 3,2′-dimethyl-4-aminobiphenyl: response to androgen deprivation

 It is generally accepted that early human prostate cancers reveal higher androgen dependency than do advanced ones. In the present study, we examined whether the animal model of prostate cancer has already lost androgen dependency at the early stages of carcinogenesis. At experimental week 46, androgen deprivation was induced in rats and the incidences of atypical hyperplasia and cancer were examined in the ventral, dorsolateral prostate, coagulating glands, and seminal vesicles. Androgen deprivation significantly lowered the incidence of atypical hyperplasia in all four organs. As for the incidence of cancer, no significant differences were observed in the coagulating glands and seminal vesicles. Regarding atypical hyperplasia, androgen deprivation significantly decreased the proliferative cell nuclear antigen labeling index in the coagulating gland and seminal vesicles. The presence of cancer was also decreased in the coagulating gland but not in the seminal vesicles. With control group specimens, more intense staining of androgen receptor was observed in atypical hyperplasias than in cancers. Compared with the atypical hyperplasias, the cancers revealed low androgen dependency at the early stages of carcinogenesis. The cancers in the seminal vesicles also revealed higher androgen independency than did those in the coagulating gland. Accepted: 6 May 1997