Involvement of GSK-3beta and DYRK1B in differentiation-inducing factor-3-induced phosphorylation of cyclin D1 in HeLa cells

Differentiation-inducing factors (DIFs) are putative morphogens that induce cell differentiation in Dictyostelium discoideum. We previously reported that DIF-3 activates glycogen synthase kinase-3beta (GSK-3beta), resulting in the degradation of cyclin D1 in HeLa cells. In this study, we investigated the effect of DIF-3 on cyclin D1 mutants (R29Q, L32A, T286A, T288A, and T286A/T288A) to clarify the precise mechanisms by which DIF-3 degrades cyclin D1 in HeLa cells. We revealed that T286A, T288A, and T286A/T288A mutants were resistant to DIF-3-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr(286) and Thr(288) were critical for cyclin D1 degradation induced by DIF-3. Indeed, DIF-3 markedly elevated the phosphorylation level of cyclin D1, and mutations introduced to Thr(286) and/or Thr(288) prevented the phosphorylation induced by DIF-3. Depletion of endogenous GSK-3beta and dual-specificity tyrosine phosphorylation regulated kinase 1B (DYRK1B) by RNA interference attenuated the DIF-3-induced cyclin D1 phosphorylation and degradation. The effect of DIF-3 on DYRK1B activity was examined and we found that DIF-3 also activated this kinase. Further, we found that not only GSK-3beta but also DYRK1B modulates cyclin D1 subcellular localization by the phosphorylation of Thr(288). These results suggest that DIF-3 induces degradation of cyclin D1 through the GSK-3beta- and DYRK1B-mediated threonine phosphorylation in HeLa cells.     

Alteration of bond-cleavage pattern in the hydrolysis catalyzed by Saccharomycopsis alpha-amylase altered by site-directed mutagenesis.

The 210th lysine (K) residue in the Saccharomycopsis alpha-amylase (Sfamy) molecule was replaced by arginine (R) and asparagine (N) residues by site-directed mutagenesis. The influences of the replacements on the bond-cleavage pattern for several substrates were analyzed. Both mutant enzymes, K210R and K210N, cleave mainly the first glycosidic bond from the reducing end of maltotetraose (G4), while the native enzyme hydrolyzes mainly the second bond from the reducing end. We changed successfully the major cleavage point in the hydrolysis reaction of G4. The 8th subsite affinities of the K210R and K210N enzymes are calculated to be +2.52 and -0.01 kcal/mol, respectively, whereas that of the native enzyme is +3.32 kcal/mol as reported in the previous paper. These affinity values suggest that the K210 residue composes the 8th subsite, one of major subsites, and that a positively charged amino residue is necessary for the 8th subsite affinity. The K210N enzyme is found to be less active for short substrates like maltotetraose (G4) than for long substrates like amylose A (approximately G18). The reduced catalytic activity specifically for the short substrates is also attributable to the remarkable decrease in the affinity of the 8th subsite.     

Effects of Homologous O-Antibody on Host Responses to Lipopolysaccharide from Yersinia enterocolitica: Neutralization of Its Pyrogenicity

The pyrogenic activity of lipopolysaccharide (LPS) obtained from Yersinia enterocolitica was neutralized by the homologous O-antiserum at the optimal antigen/antibody ratio. The level of neutralization in either antigen excess or extreme antibody excess was significantly lower than that at the optimal ratio. These facts suggested that the structure of the so-called “lattice” of LPS/antibody complex might influence the neutralization of pyrogenic activity. Moreover, when LPS was gradually added to the antiserum to reach the optimal antigen/antibody ratio, the pyrogenic activity of LPS was only slightly neutralized by the antibody. The similar event to Danysz phenomenon that has been known in the diphtheria toxin-antitoxin system was also observed in the LPS-anti-LPS system.     

3-Methylene-substituted androgens as novel aromatization inhibitors. Evidence of a requirement for C-3 oxygen in C-19 hydroxylations

Substitution of a methylene group for the C-3 oxygen in androstenedione, testosterone, and the corresponding 19-hydroxy and 19-oxo derivatives results in a new category of inhibitors of estrogen biosynthesis by human placental microsomes. The inhibition is of the competitive type with the most effective inhibitors being the 17-ketonic compounds, 3-methyleneandrost-4-en-17-one, 19-hydroxy-3-methyleneandrost-4-en-17-one, and 3-methylene-19-oxoandrost-4-en-17-one with apparent Ki values of 4.7, 13, and 24 nM, respectively. The 3-methylene derivatives of androstenedione and 19-hydroxyandrostenedione were effective substrates for the placental microsomal 17 beta-hydroxy-steroid oxidoreductase but were only marginally hydroxylated at the C-19 position to the respective 19-hydroxy and 19-oxo derivatives. The 3-methylene analogs are thus competitive inhibitors of aromatization but are not substrates for this enzyme complex. Time-dependent inhibition of aromatization by 10 beta-difluoromethylestr-4-ene-3,17-dione and 10 beta-(2-propynyl)estr-4-ene,3,17-dione was abolished by substitution of a methylene function for the C-3 oxygen, suggesting that the presence of an oxygen at C-3 is required for an oxidative transformation at C-19, an initial step in aromatization. The essential role of the C-19 hydroxylation in aromatization is supported by the observation that the 3-methylene derivatives of 19-hydroxy- and 19-oxoandrostenedione showed time-dependent inhibition, but the corresponding 19-methyl compound did not. The 3-methylene androgens are potent inhibitors of placental aromatization but are themselves only marginal substrates for the enzyme. Their high affinity for and inertness to the placental aromatase complex makes them valuable probes of the aromatization process.     

Use of In Ovo Chorioallantoic Membrane Engraftment to Culture Testes from Neonatal Mice

Many attempts have been made to culture germ cells in vitro by mimicking their development in vivo. The objective of this study was to establish an alternative method of xenotransplantation by developing a new approach for the rapid induction of spermatogenesis by using the chorioallantoic membrane of developing chicken embryos. Fertilized chicken eggs were incubated for 7 d, after which a small window was cut into the shell of the egg. We then transplanted testes from 7- to 8-d-old B6D2F1 mice onto the vessels of the chorioallantoic membrane and incubated them at 35.0 °C for 14 d or 37.5 °C for 12 d. After this in ovo CAM (iCAM) culture, the survival rates of the eggs and testes were assessed histologically and immunohistologically. The transplanted testes in the chicken embryos that survived were supported by the CAM, with an associated chronic vascularization response. The testes cultured at 35.0 °C had lower rates of generation and higher rates of death than did those cultured at 37.5 °C. Histologic examination of the testes cultured at 37.5 °C revealed the presence of spermatogonia and primary spermatocyte-like germ cells in the seminiferous tubules. The number of cells positive for synaptonemal complex protein 3 in the seminiferous tubules was significantly higher than that in the noniCAM-cultured testes from control mice. These results suggest that iCAM culturing of neonatal donor testis induces androcyte development. This method could be the foundation for a method that would enable in vitro spermatogenesis.Abbreviations: CAM, chorioallantoic membrane; iCAM, in ovo chorioallantoic membrane; SCP3, synaptonemal complex protein 3In recent studies of in vitro spermatogenesis, many attempts have been made to culture germ cells in vitro by mimicking their development in vivo. However, the complexity of germ cell development has hampered the induction of immature germ cells into functional gametes under culture conditions. It is well known that spermatogenesis is one of the longest processes of sequential cell proliferation and differentiation, taking more than a month for progression from spermatogonial stem cells, through meiosis, to sperm formation. Male germline cells in mice can only be cultured from pachytene spermatocytes to round spermatids to produce the next generation.¹² Until recently, researchers succeeded only in inducing the early steps of spermatogenesis in vitro.¹⁶In the first study to overcome this difficulty, neonatal mice testis, containing only gonocytes or primitive spermatogonia as germ cells, produced spermatids and sperm in vitro after tissue culture for 2 mo.¹⁵ The embryos derived from these cultured spermatozoa yielded live and normal offspring. In the approach, the culture system widely preserved the cytoarchitecture of the gonad, many endogenous factors were produced and released by the mostly intact seminiferous epithelium, and associated somatic cells regulated the germ cells in a manner similar to the in vivo condition.¹⁵ Other investigators showed that in dissected tissue, the supply of oxygen and nutrients is disturbed and that the spermatogenic process consequently is often disrupted and continues, at best, at low efficiency.¹ Therefore, as a valuable alternative approach, a culture system should be established for the development of spermatogenesis in vitro.The avian chorioallantoic membrane (CAM) is the outermost extraembryonic membrane that lines the noncellular eggshell membrane. The CAM is formed by the fusion of the splanchnic mesoderm of the allantois to the somatic mesoderm of the chorion. CAM vessels grow rapidly until day 11, and the vascular system attains its complete structure on day 18 of incubation, just before hatching.¹³ The CAM serves as a support for the extraembryonic respiratory capillaries and actively transports sodium and chloride from the allantois sac and calcium from the eggshell into the embryonic vasculature.¹⁷ The CAM plays a role in the neovascularization of developing tissues and provides an immunologically protected site in which it is possible to observe the development of blood vessels in the living egg. The extraembryonic vessel system of CAM is naturally immunodeficient.⁹ In addition, the degree of neovascularization of the donor testis by host blood vessels can be determined histologically by the presence of nucleated avian erythrocytes in blood vessels.⁵ For these reasons, the CAM system has been applied to implantation studies with tumors, adipocytes, and cartilages as well as ovarian tissues, but no approach to in ovo spermatogenesis has been reported previously.^4,6-8,10,18 Our current study aimed to establish a novel in ovo system for culturing the testes of mice on the CAM and to investigate whether in ovo CAM (iCAM) culture supports the induction of spermatogenesis and meiosis of the cultured germ cells.     

Partial purification of cytochrome P450 from rat brain and demonstration of estradiol hydroxylation

Cytochrome P450 was partially purified from brain microsomes of untreated rats. A difference spectrum of the dithionite-reduced CO-complex of the purified P450 showed essentially the hemeprotein absorbing exclusively at 449 nm. The purified brain P450 was able to catalyze estradiol (E2) hydroxylation leading to the formation of 6 alpha- and 6 beta-hydroxy(OH)E2, 4-OHE2, estrone, 6-oxoE2, 2-OHE2, 15 alpha-OHE2 and estriol. These results demonstrate that rat brain P450 is active in estradiol hydroxylation.     

Synthesis of Pseudomonas quorum-sensing autoinducer analogs and structural entities required for induction of apoptosis in macrophages

The synthesis of the analogs of N-3-oxododecanoyl-L-homoserine lactone (1) and their structure-activity relationship for the apoptotic induction in macrophages, P388D1 cells, are described. It was revealed that the position of the oxo group in the acyl side chain in addition to the presence of the L-homoserine lactone unit is crucial for the apoptosis-inducing activity. Furthermore, the long acyl side chains with hydrophobic distal ends are preferable for the activity.     

Verification of Emmert's law in actual and virtual environments

We examined Emmert's law by measuring the perceived size of an afterimage and the perceived distance of the surface on which the afterimage was projected in actual and virtual environments. The actual environment consisted of a corridor with ample cues as to distance and depth. The virtual environment was made from the CAVE of a virtual reality system. The afterimage, disc-shaped and one degree in diameter, was produced by flashing with an electric photoflash. The observers were asked to estimate the perceived distance to surfaces located at various physical distances (1 to 24 m) by the magnitude estimation method and to estimate the perceived size of the afterimage projected on the surfaces by a matching method. The results show that the perceived size of the afterimage was directly proportional to the perceived distance in both environments; thus, Emmert's law holds in virtual as well as actual environments. We suggest that Emmert's law is a specific case of a functional principle of distance scaling by the visual system.