Kazutaka Kano

ER-dependent estrogenic activity of parabens assessed by proliferation of human breast cancer MCF-7 cells and expression of ERα and PR

Estrogenic activities of the phenolic preservatives methylparaben, ethylparaben, propylparaben, butylparaben, isopropylparaben and isobutylparaben were examined by assaying estrogen-receptor (ER)-dependent proliferation of MCF-7 cells. All the compounds stimulated the proliferation to about the same level as the maximal cell yield attained with 3x10(-11) M 17beta-estradiol, but at a concentration in the order of 10(5) to 10(7) higher than 17beta-estradiol. The cell-proliferative effects of parabens were completely suppressed by anti-estrogen ICI 182,780. MCF-7 cells treated with butylparaben and isobutylparaben exhibited a decrease in gene expression of ERalpha and an increase in that of progesterone-receptor (PR), but the effects of these parabens were not as prominent as those of 17beta-estradiol. Western blot analysis indicated that these parabens caused a slight decrease in expression of ERalpha protein. Competitive binding to human ERalpha and ERbeta in vitro revealed that the parabens with longer side-chains showed greater affinity for estrogen receptors, and that they had similar relative binding affinity (RBA) values to both ERalpha and ERbeta. RBA values were much smaller than that of diethylstilbestrol. In conclusion, parabens have ER-dependent estrogenic activities, and their effects on the intracellular signaling pathway might be different from that of 17beta-estradiol.

Enhancement of Apoptosis in Developing Chick Neural Retina Cells by Basic Fibroblast Growth Factor

Abstract: To evaluate the role of various growth factors in naturally occurring cell death during development of the neural retina, we examined the effects of such factors on the nuclear morphology and the size of DNA in cultured chick embryonic neural retina cells. Basic fibroblast growth factor (bFGF) increased internucleosomal cleavage of DNA and nuclear fragmentation in a time‐ and dose‐dependent manner. The effect was inhibited by anti‐bFGF antibody, suramin, and cycloheximide. Epidermal growth factor, platelet‐derived growth factor, nerve growth factor, tumor necrosis factor‐α, and dexamethasone had no effect. These results provide evidence that bFGF may eventually act as a lethal factor inducing apoptotic cell death during the development of the neural retina in chick embryo.

Lipids in Harderian Glands and Their Significance

The Harderian gland, discovered by Harder (1694), is well developed in rodents (rat, mouse, golden hamster, Mongolian gerbil, guinea pig, etc.), lagomorphs (rabbit, pika) and cetaceans, and may have been lost in primates, terrestrial carnivores, and some other species. The Harderian gland occupies a considerable part of the orbit and is located around the posterior half of the eyeball. Histologically, a single layer of columnar epithelial cells surrounds the lumen, with the single excretory duct directly opening onto the eyelid. The surface of the gland is smooth and is covered with endothelium of the orbital venous sinus (Sakai and Yohro 1981).

Structure of the α1 subunit of horse Na, K-ATPase gene

Genomic DNA for Na,K‐ATPase α1 subunit was obtained from libraries of horse kidney genomic DNA in Charon 4A and in EMBL3 bacteriophages by screening with the full sized cDNA probe of the α1 subunit of rat Na,K‐ATPase as probe. The gene spans 30 kb and consists of 23 exons and 22 intervening sequences. Intron‐exon boundaries were analyzed. The protein‐coding nucleotide sequence encodes 1016 amino acids with an M r of 112 264. The putative amino acid sequence of horse α1 is 96–97% homologous to those of other mammalian species.

Effects of cholestanol feeding on corneal dystrophy in mice

A cholestanol-enriched diet administered for 8 months to BALB/c mice produced in 20% two kinds of corneal opacities resembling calcific band keratopathy and Schnyders crystalline dystrophy in humans. The concentrations of cholestanol in serum, liver and cornea of the corneal opacity bearing mice were 30-40-times higher than those of normal mice. On the other hand, brain cholestanol level increased only 7-times in the opacity group as compared with that of control group. There was no significant difference in the cholesterol concentrations of serum and several tissues among opacity, non-opacity and the control group. The crystal particles were observed between epithelial basement membrane and superficial stroma by the electron microscopy. Energy dispersive analysis of the particles revealed that the deposits were composed principally of calcium and phosphorus with other crystalline materials, which was presumed to be cholestanol. These results suggest that the cholestanol may deposit in the cornea from elevated serum levels. Deposition of cholestanol in cornea and related area may be a cause of corneal dystrophy in CTX.

Dihydroheptaprenyl and dihydrodecaprenyl monophosphates induce apoptosis mediated by activation of caspase-3-like protease

Dolichyl phosphate, an essential carrier lipid in the biosynthesis of N-linked glycoprotein, has been found to induce apoptosis in rat glioma C6 cells and human monoblastic leukemia U937 cells. In the present study, dolichyl phosphate and structurally related compounds were examined regarding their apoptosis-inducing activities in U937 cells. Dihydroheptaprenyl and dihydrodecaprenyl phosphates, of which isoprene units are shorter than that of dolichyl phosphate, induced apoptosis in U937 cells. This phenomenon occurred in a dose- and time-dependent manner, as seen with dolichyl phosphate-induced apoptosis. Derivatives of the same isoprene units of dolichyl phosphate, such as dolichol, dolichal or dolichoic acid, did not induce DNA fragmentation. Farnesyl phosphate and geranylgeranyl phosphate also failed to induce apoptosis. During apoptosis, the caspase family of cysteine proteases play important roles. We observed that apoptosis induced by dihydroprenyl phosphate was mediated by caspase-3-like (CPP32-like) activation but not by caspase-1-like (ICE-like) activation. This caspase-3-like activation was inhibited by a specific inhibitor of caspase-3, DEVD-CHO, but not by an caspase-1 inhibitor YVAD-CHO. We interpret these results to mean that dihydroprenyl phosphates with more than seven isoprene units have apoptosis-inducing activity and that their signal is mediated by caspase-3-like activation.

Novel regulation of δ-aminolevulinate synthase in the rat harderian gland

The mode of expression of δ-aminolevulinate synthase (ALAS), as well as that of mRNAs for other heme pathway enzymes, was examined in the rat Harderian gland. Northern blot and in situ hybridization analyses demonstrated that the non-specific ALAS (ALAS-N) mRNA is highly expressed in this tissue, whereas the erythroid-specific ALAS (ALAS-E) mRNA is not. Immunoblot analysis of ALAS also confirmed this finding at the protein level. ALAS-N mRNA was maximally induced in the Harderian gland and was not increased further by treatment of animals with 2-allyl-2-isopropylacetamide (AIA). The levels of mRNAs for other heme pathway enzymes, i.e., δ-aminolevulinate dehydratase, porphobilinogen deaminase, uroporphyrinogen decarboxylase, and coproporphyrinogen oxidase, also were increased markedly in the Harderian gland and not influenced by AIA treatment. The level of ferrochelatase (FeC) mRNA in the gland was, however, lower than that in the liver. The gland contained an extremely high level of protoporphyrin, while heme was undetectable. Microsomal heme oxygenase-1 (HO-1) mRNA levels were significantly higher in the Harderian gland than in the liver. When isolated glands were incubated with hemin in vitro in organ cultures, the level of HO-1 mRNA was increased, whereas the ALAS-N mRNA level was not. These findings indicate that markedly elevated levels of protoporphyrin and extremely low levels of heme in the Harderian gland are the results of both decreased expression of FeC and markedly increased expression of ALAS-N and HO-1. The constitutive expression of the ALAS-N gene in the Harderian gland suggests a novel transcriptional control mechanism of this gene.

CPP32 ACTIVATION DURING DOLICHYL PHOSPHATE-INDUCED APOPTOSIS IN U937 LEUKEMIA CELLS

Treatment of U937 cells with dolichyl phosphate led to an increase in the activity of the ICE family protease CPP32, accompanied with cleavage of pre‐CPP32 to generate p17. Peptide inhibitors YVAD‐cmk and Z‐Asp‐CH2‐DCB (specific to ICE) and DEVD‐cho (specific to CPP32) blocked the dolichyl phosphate‐induced apoptosis. The dolichyl phosphate‐induced increase of CPP32 activity was inhibited by adenylate cyclase inhibitors, SQ 22536 and 2′,5′‐dideoxyadenosine. Dolichyl phosphate caused a transient increase of intracellular cAMP concentration. The results suggest that modulation of cAMP synthesis due to the stimulation of adenylate cyclase by dolichyl phosphate plays a critical role in CPP32 activation and apoptosis.

Inhibition of DNA synthesis in the adenovirus DNA replication complex by aphidicolin and 2′,3′-dideoxythymidine triphosphate

Abstract DNA synthesis in the adenovirus DNA replication complex, containing host DNA polymerases-α and -γ, was inhibited completely by aphidicolin and by 2′,3′-dideoxythymidine triphosphate (ddTTP). Double reciprocal plots of DNA polymerase activity in the replication complex against each dNTP gave a straight line although the complex contained two species of DNA polymerase. Inhibition by aphidicolin of DNA polymerase activity was competitive with dTTP but that of purified DNA polymerase-α isolated from adenovirus infected KB cells was competitive with dCTP. The above results suggest that DNA polymerases-α and -γ are integrated in the replication complex to behave as a single enzyme.

Effect of Harderian gland-derived growth factor on the growth of cornea stromal cells.

To investigate the physiological role of Harderian glandderived growth factor (HGDGF), the effects of HGDGF and various other growth factors on the growth of cultured guinea pig cornea stromal cells were examined. HGDGF increased the incorporation of [3H]thymidine to 150% of the control (5% fetal calf serum). The combination of HGDGF with fibroblast growth factors (FGFs) or platelet-derived growth factor enhanced the cell growth over that of either growth factor alone, increasing the incorporation of [3H]thymidine to 180 and 190% of the control, respectively. The combination of HGDGF with transforming growth factor (TGF-(3) decreased the growth to 60% of the control, and epidermal growth factor had no effect on the activity of HGDGF. The growthstimulating activity of HGDGF was inhibited by suramin in a different manner from that of FGFs. These findings suggest that HGDGF binds a specific cell-surface receptor and plays a role in the repair of injured parts of the cornea and in the maintenance of the cornea stromal cells.