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.     

RP S19 C-terminal peptide trimer acts as a C5a receptor antagonist

We have demonstrated that ribosomal protein S19 (RP S19) polymer, when crosslinked between Lys122 and Gln137 by activated coagulation factor XIII, acts as a C5a receptor (C5aR) antagonist/agonist. Based on experimental data obtained using RP S19 analog peptide and recombinant protein monomer, we suggested that L₁₃₁DR, I₁₃₄AGQVAAAN and K₁₄₃KH moieties in the RP S19 C‐terminus act in, respectively, C5aR binding, penetration of the plasma membrane, and interaction with either an apoptosis-inducing molecule in neutrophils (delta lactoferrin) or a calcium channel-activating molecule (annexin A3) to induce the p38 MAPK pathway in macrophages. Recently, we observed RP S19 trimer in serum. To study the effects of this RP S19 trimer on C5aR, we prepared mutant RP S19 C‐terminal peptide (RP S19^122-145) dimer and trimer, and examined their chemotactic activities and signal transduction pathways in human C5aR-overexpressing squamous cell carcinoma HSC-1 (HSC-1^C5aR) cells using 24 trans-well chamber and western blotting assays, respectively. HSC-1^C5aR cells were attracted by RP S19^122-145 dimer and vice versa by RP S19^122-145 trimer. The RP S19^122-145 dimer-induced attraction was competitively blocked by pre-treatment with RP S19^122-145 trimer. Moreover, RP S19^122-145 trimer-induced p38 MAPK phosphorylation was stronger than RP S19^122-145 dimer-induced p38 MAPK phosphorylation. RP S19^122-145 trimer appeared to act as a C5aR antagonist. The agonistic and antagonistic effects of RP S19^122-145 dimers and trimers were reflected by monocytic, THP-1-derived macrophage-like cells. Unlike the C5aR agonist C5a, which acts at the inflammation phase of acute inflammation, RP S19 trimer might act as a C5aR antagonist at the resolution phase.     

Full-term development of mouse blastomere nuclei transplanted into enucleated two-cell embryos

The nuclei from four- and eight-cell mouse embryos were transplanted into enucleated two-cell embryos. It was found that such embryos not only developed to the blastocyst stage in vitro (72% and 35%), but also developed to full term (22% and 8%) after transfer to recipient mice. However, development of embryos which contained nuclei from the inner cell mass was not observed. Since the development of enucleated zygotes which contain advanced nuclei is limited (the present study; McGrath and Solter: Science, 226:1317-1319, '84; Robl, Gilligan, Critser, and First: Biol. Reprod., 34:733-739, '86), it appears that cytoplasmic factors are important for the development of nuclei from advanced cells.     

Nucleotide sequence of the bovine parainfluenza 3 virus genome: the genes of the F and HN glycoproteins.

By analysing complementary DNA clones constructed from genomic RNA of bovine parainfluenza 3 virus (BPIV3), we determined the nucleotide sequence of the region containing the entire F and HN genes. Their deduced amino acid sequences showed about 80% homologies with those of human parainfluenza 3 virus (HPIV3), about 45% with those of Sendai virus, and about 20% with those of SV5 and Newcastle disease virus (NDV), indicating, together with the results described in the preceding paper on the NP, P, C and M proteins of BPIV3, that BPIV3, HPIV3 and Sendai virus constitute a paramyxovirus subgroup, and that BPIV3 and HPIV3 are very closely related. The F and HN proteins of all these viruses, including SV5 and NDV, however, were shown to have protein-specific structures as well as short but well-conserved amino acid sequences, suggesting that these structures and sequences are related to the activities of these glycoproteins.     

Electrofusion for the pronuclear transplantation of mouse eggs

The present study was undertaken to determine the efficiency of HVJ treatment and electrofusion for pronuclear transplantation in the mouse. The output voltage and duration of the pulses were fixed to 200 μsec at 10 V or to 150 μsec at 15 V for electrofusion, because the maximum rates of blastomere fusion of 2-cell embryos and development of fused embryos in vitro were obtained under these conditions. Although the proportion of eggs with fused karyoplast (78%) and the fused eggs developed to morulae or blastocysts (67%) was significantly lower than those obtained after HVJ treatment (94% and 94%), the proportion of pregnant recipients and young obtained after treatment of fused eggs was not significantly different between these two procedures. It is advised that electrofusion can be used as a fusogenic procedure for pronuclear transplantation in the mouse in some cases where HVJ cannot be applied.     

Immunocytochemical localization of serotonin-like immunoreactivities in the ciliated epithelium of the frog palatine mucosa

Immunocytochemical localization of serotonin (5-HT)-like immunoreactivities was studies in the ciliated epithelium of the frog palatine mucosa by the peroxidase anti-peroxidase (PAP) method. 5-HT-like immunoreactivity was found only in the small granular vesicles (100-150 nm in diameter) and not in any mature large secretory granules or in other cell organellae in the goblet cells. No 5-HT-like immunoreactivities were found in any other epithelial and secretory cells in the palatine epithelium. It appears therefore that 5-HT-like immunoreactive granular vesicles have certain physiological effects on the ciliary movement of the ciliated cells or in the goblet cells.     

Stimulatory effect of n-octanoylated ghrelin on locomotor activity in the goldfish, Carassius auratus

Ghrelin is implicated in growth and feeding regulation in fish. The influence of ghrelin on behavior has not been well studied and the physiological role of des-fatty acid modification of this peptide is unclear. Therefore, the effects of intracerebroventricular (ICV) and intraperitoneal (IP) administration of synthetic n-octanoylated (acyl) goldfish ghrelin and des-n-octanoylated (des-acyl) ghrelin on locomotor and orexigenic activity in the goldfish were examined. ICV administration of acyl ghrelin at doses of 1 and 2 pmol/g body weight (BW) and IP administration at 16 pmol/g BW both induced significant increases in locomotor activity during for 45-60 min after treatment. Cumulative food intake was significantly increased by ICV injection of acyl ghrelin at doses of 1 and 2 pmol/g BW and IP injection at 8 and 16 pmol/g BW during the 60-min post-treatment observation period. In contrast, ICV and IP administration of des-acyl ghrelin produced no changes in locomotor and orexigenic activity. We also analyzed fasting-induced changes in the expression of ghrelin mRNA in the brain and intestine using a real-time PCR method. The level of ghrelin mRNA in the intestine, but not in the brain, obtained from fish fasted for 7 days was significantly higher than that in fish that had been fed normally. These results suggest that, in the goldfish, acyl ghrelin, but not des-acyl ghrelin, stimulates locomotor activity and enhances food intake via central and peripheral pathways.     

Pituitary adenylate cyclase-activating peptide (PACAP) stimulates production of interleukin-6 in rat Müller cells

Pituitary adenylate cyclase-activating peptide (PACAP) is known to regulate not only neurons but also astrocytes. Here, we investigated, both in vitro and in vivo, the effects of PACAP38 on rat Müller cells, which are the predominant glial element in the retina. Müller cells isolated from juvenile Wistar rats were treated with PACAP38 or PACAP6-38, a PACAP selective antagonist. Cell proliferation was determined by measuring the incorporation of bromodeoxyuridine with ELISA. Interleukin-6 (IL-6) levels in the culture medium were determined by a bioassay using B9 cells, IL-6 dependent hybridoma. In adult Wistar rats, the expression of IL-6 in the retina after intravitreal injection of PACAP38 (10 pmol) was assessed by immunohistochemistry. PACAP38 stimulated IL-6 production in Müller cells at a concentration as low as 10(-12) M, which did not induce cell proliferation. This elevation of IL-6 production was inhibited by PACAP6-38. Radial IL-6 expression was observed throughout the retina at 2 and 3 days after PACAP38 injection. These data demonstrate that Müller cells are one of the target cells for PACAP. IL-6, which is released from Müller cells with stimulation by PACAP, may play a significant role in the retina.     

Structural basis for induced-fit binding of Rho-kinase to the inhibitor Y-27632

Rho-kinase is a main player in the regulation of cytoskeletal events and a promising drug target in the treatment of both vascular and neurological disorders. Here we report the crystal structure of the Rho-kinase catalytic domain in complex with the specific inhibitor Y-27632. Comparison with the structure of PKA bound to this inhibitor revealed a potential induced-fit binding mode that can be accommodated by the phosphate binding loop. This binding mode resembles to that observed in the Rho-kinase-fasudil complex. A structural database search indicated that a pocket underneath the phosphate-binding loop is present that favors binding to a small aromatic ring. Introduction of such a ring group might spawn a new modification scheme of pre-existing protein kinase inhibitors for improved binding capability.