Ontogenetic appearance of somatostatin-containing nerve terminals in the median eminence of rats

Summary The development of immunoreactive (ir) somatostatin-containing nerve terminals in the rat median eminence (ME) has been examined electron-microscopically. Nerve fibers containing ir particles scattered throughout the axoplasm are first seen in the external layer of the ME on day 18.5 of gestation, and, on day 21.5 appear to terminate on the basement membrane of the perivascular space of the portal vessels. After birth, the fiber terminals contain several membrane-limited granules, which are labeled with ir PAP particles. Ultrathin, Epon-embedded sections of ME, treated by the protein A gold-labeling method for somatostatin, demonstrate positively labeled granules in the nerve fibers in the postnatal ME, but in the prenatal tissue, no specific gold-labeling is found. These findings show that, in the external layer of the ME, somatostatin storing occurs in the granules in the axonal terminals after birth.     

Induction of angiogenesis by smooth muscle cell-derived factor: Possible role in neovascularization in atherosclerotic plaque

The development of atherosclerotic plaque is associated with neovascularization in the thickened intima and media of vascular walls. Neovascularization may have a role in the progression of atherosclerotic plaque as well as in the development of intraplaque hemorrhage. However, the mechanism and stimulus for neovascularization in atherosclerotic plaque are unknown. We postulated that smooth muscle cells (SMCs), a major cellular component in the vascular wall, might contribute to the induction of neovascularization in atherosclerotic plaque through the secretion of an angiogenic factor. We observed that endothelial cells (ECs) cultured on collagen gel with SMC-conditioned medium became spindle shaped, invaded the underlying collagen gel, and organized a capillary-like branching cord structure in the collagen gel. The conditioned medium also stimulated EC proliferation and increased the EC-associated plasminogen activator activity. The angiogenic factor in SMC-conditioned medium was retained in a heparin-Sepharose column and eluted with 0.9 M NaCl. Neutralizing anti-vascullar endothelial growth factor (VEGF) antibody attenuated the angiogenic activity in the conditioned medium, including the induction of morphologic changes in ECs, mitogenic activity, and increased plasminogen activator activity associated with ECs. Immunoblotting analysis confirmed the secretion of VEGF from SMCs. These observations indicate that SMC may be responsible for the neovascularization in atherosclerotic plaque through the secretion of VEGF. © 1995 Wiley-Liss, Inc.     

Solid-Phase Nested Deletion: A New Subcloning-less Method for Generating Nested Deletions

We have developed a new subcloning-less method for generatingnested deletions which we have termed Solid-Phase Nested Deletion.The basic procedure for this method is as follows. The targetDNA fragment is cloned in the multiple cloning site of a cloningvector, pUC or its derivatives, and amplified by PCR using aset of primers, one of which is 5'-biotinylated. The amplifiedDNA is partially digested by a restriction enzyme with a 4-baserecognition sequence. The digested DNA is ligated with a syntheticadapter DNA. Monodiverse beads coupled with streptavidin (Dynabeads^TMM-280 streptavidin) are added to the mixture and the biotinylatedDNA fragments are separated by applying magnetic field. Theunidirectionally deleted DNA fragments are recovered by PCRfrom the magnetic beads, and size-fractionated by agarose gelelectrophoresis. The DNA fragments are amplified by PCR andused for sequencing. We demonstrate the potential of this methodusinga 4878-bp EcoRI fragment of phage DNA.     

Chemiluminescence sandwich enzyme immunoassay for determination of human granulocyte colony stimulating factor (G-CSF)

A chemiluminescence sandwich enzyme immunoassay, using a glucose oxidase (GO) label, was developed for detecting attomole amounts of human granulocyte colony stimulating factor (G-CSF). Purified goat F(ab′)₂ immobilized on a bead and purified goat Fab′ labelled with GO were selected in combination with a chemiluminescent detection system comprising luminol and ferricyanide. The detection limits for G-CSF were 4amol/assay (1 pg/mL) in buffer solution and 10 amol/assay (2.5 pg/mL) in human serum. Coefficients of variation within assay and between assay ranged from 5.5% to 7.8% and from 3.4% to 16.0%, respectively. The G-CSF content of serum from normal healthy individuals was measurable using this method. G-CSF in 24 normal human sera showed a mean value of 19.3 pg/mL and ranged from 3.6 to 83.0 pg/mL.     

Phylogenetic relationships of the Chinese brown frogs (genus Rana) inferred from partial mitochondrial 12S and 16S rRNA gene sequences

Based on partial sequences of the 12S and 16S ribosomal RNA genes, we estimated phylogenetic relationships among brown frogs of the Rana temporaria group from China. From the phylogenetic trees obtained, we propose to include Rana zhengi in the brown frogs. Monophyly of the brown frogs was not unambiguously supported, but four well-supported clades (A, B, C, and D) always emerged, although relationships among them remained unresolved. Clade A contained brown frogs with 24 chromosomes and was split into two distinct subclades (Subclade A-1: R. chensinensis and R. huanrenensis; Subclade A-2: R. dybowskii). Polytomous relationships among populations of R. chensinensis and R. huanrenensis suggested the necessity of further taxonomic assessment. Rana kunyuensis proved to be the sister group to R. amurensis, and these two species formed Clade B. Clade C was composed of R. omeimontis and R. chaochiaoensis, and Clade D included R. sauteri, which has been placed in other ranid genera. These relationships did not change after adding published data, and monophyly of Subclade A-1, A-2, and other East Asian brown frogs with 24 chromosomes (R. pirica and R. ornativentris) was ascertained, though their relationships were unresolved. Clade C, together with R. japonica and R. longicrus, also formed a monophyletic group. Brown frogs related to Clades A and C were estimated to have dispersed from continental Asia to adjacent regions through multiple events.     

Genetic relationships among salamanders of the genus Hynobius (Amphibia, Caudata) from Korea and southwestern Japan

We performed allozyme analysis for three Korean (Hynobius leechii, H. quelpaertensis, and H. yangi) and three Japanese (H. nebulosus, H. tsuensis, and H. dunni) salamanders to clarify their interspecific relationships using H. naevius as an outgroup. The genetic distances (Nei's D) within ingroup species ranged from 0.11 to 0.78 with a mean of 0.33. In the NJ and CONTML trees, monophyly of the ingroup was not supported and Korean H. quelpaertensis and H. leechii diverged first from the remaining species, which together formed a weakly supported clade. Korean H. yangi, long identified as H. leechii, was closer to Japanese H. nebulosus (D=0.108) and H. tsuensis (D=0.138) than to Korean H. leechii (D=0.197) and H. quelpaertensis (D=0.305). Hynobius tsuensis and H. nebulosus were very close (D=0.108) despite their different breeding habits. A geohistorical hypothesis is proposed to explain the divergence of the six species.     

Solubilization of the ecdysone binding protein from anterior silk gland cell membranes of the silkworm, Bombyx mori

We previously provided preliminary evidence for the presence of a putative membrane ecdysone receptor (mEcR) anchored in the plasma membranes of anterior silk glands (ASGs) in Bombyx mori. This receptor may act in concert with the conventional EcR in 20E-dependent programmed cell death of these glands. We report here, for the first time, the solubilization of mEcR from ASG membranes using the zwitterionic detergent CHAPS in the presence of NaCl. Our results show by ligand binding assay that mEcR solubilized this way is functionally active and retains 75% of its native binding activity. We also defined experimental conditions that yielded protein/detergent complexes with partial binding activity, which makes it possible to purify the membrane-bound ecdysone binding protein.     

Parkin interacts with LIM Kinase 1 and reduces its cofilin-phosphorylation activity via ubiquitination

Mutations in the PARKIN (PARK2) gene have been found in the majority of early-onset familial Parkinson's disease (PD) patients with autosomal recessive juvenile parkinsonism (ARJP). Parkin protein functions as an ubiquitin (E3) ligase that targets specific proteins for degradation in the 26S proteasome. Here, based on a mass spectrometry analysis of the human dopaminergic neuroblastoma-derived cell line SH-SY5Y that over-expresses parkin, we found that parkin may suppress cofilin phosphorylation. LIM Kinase 1 (LIMK1) is the upstream protein that phosphorylates cofilin, an actin depolymerizing protein. Thus, we postulated a possible connection between parkin and LIMK1. Our studies in other cell lines, using co-transfection assays, demonstrated that LIMK1 and parkin bind each other. LIMK1 also interacted with previously known parkin interactors Hsp70 and CHIP. Parkin enhanced LIMK1-ubiquitination in the human neuroblastoma-derived BE(2)-M17 cell line, but not in the human embryonic kidney-derived HEK293 cell line. In fact, parkin-over-expression reduced the level of LIMK1-induced phosphocofilin in the BE(2)-M17 cells but not in the HEK293 cells. Additionally, in simian kidney-derived COS-7 cells, parkin-over-expression reduced LIMK1-induced actin filament accumulation. LIMK1 in cultured cells regulates parkin reversibly: LIMK1 did not phosphorylate parkin but LIMK1 overexpression reduced parkin self-ubiquitination in vitro and in HEK293 cells. Furthermore, in the cells co-transfected with parkin and p38, LIMK1 significantly decreased p38-ubiquitination by parkin. These findings demonstrate a cell-type dependent functional interaction between parkin and LIMK1 and provide new evidence that links parkin and LIMK1 in the pathogenesis of familial PD.     

Isolated lymphoid follicles are not IgA inductive sites for recombinant Salmonella

In this study, we investigated whether isolated lymphoid follicles (ILF) play a role in the regulation of intestinal IgA antibody (Ab) responses. The transfer of wild type (WT) bone marrow (BM) to lymphotoxin-alpha-deficient (LTalpha(-/-)) mice resulted in the formation of mature ILF containing T cells, B cells, and FDC clusters in the absence of mesenteric lymph nodes and Peyer's patches. Although the ILF restored total IgA Abs in the intestine, antigen (Ag)-specific IgA responses were not induced after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin. Moreover, Ag-specific cell proliferation was not detected in the ILF. Interestingly, no IgA anti-LPS Abs were detected in the fecal extracts of LTalpha(-/-) mice reconstituted with WT BM. On the basis of these findings, ILF can be presumed to play a role in the production of IgA Abs, but lymphoid nodules are not inductive sites for the regulation of Ag-specific intestinal IgA responses to recombinant Salmonella.     

Angiogenic activity of bFGF and VEGF suppressed by proteolytic cleavage by neutrophil elastase

Neutrophil elastase (NE), a serine protease released from the azurophil granules of activated neutrophil, proteolytically cleaves multiple cytokines, and cell surface proteins. In the present study, we examined whether NE affects the biological abilities of angiogenic growth factors such as basic-fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). NE degraded bFGF and VEGF in a time- and concentration-dependent manner, and these degradations were suppressed by sivelestat, a synthetic inhibitor of NE. The bFGF- or VEGF-mediated proliferative activity of human umbilical vein endothelial cells was inhibited by NE, and the activity was recovered by sivelestat. Furthermore, NE reduced the bFGF- or VEGF-induced tubulogenic response of the mice aortas, ex vivo angiogenesis assay, and these effects were also recovered by sivelestat. Neutrophil-derived NE degraded potent angiogenic factors, resulting in loss of their angiogenic activity. These findings provide additional insight into the role played by neutrophils in the angiogenesis process at sites of inflammation.