Analysis of intergenic spacer regions in the nuclear rDNA of Pharbitis nil.

The nucleotide sequence of a 4.2-kb EcoRI fragment from the intergenic region between the genes for 25S and 18S ribosomal RNA of Pharbitis nil Choisy was determined. The region contained a unique repetitive family of DNA sequences, called the RsaI family, composed of 32-bp units. The 32-bp unit was tandemly repeated in the intergenic region, and four subfamilies of repeating units were clustered as discrete blocks. The RsaI family of repeats was shown to be specific to the genus Pharbitis by Southern blot hybridization.     

Ontogeny of pituitary cell-types and the hypothalamo-hypophysial relationship during early development of chum salmon,Oncorhynchus keta

Tissue non-specific alkaline phosphatase is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic, protein phosphatase, and phosphotransferase activities. This enzyme is distributed virtually in all mammalian tissues, particularly during embryonic development. Its expression is stagespecific and can be demonstrated in the developing embryo as early as the 2-cell stage. It has been suggested that tissue non-specific alkaline phosphatase might play a role in tissue formation. In the study reported here, a genetransfer approach was employed to investigate possible roles for this enzyme by inserting the cDNA for rat tissue non-specific alkaline phosphatase into CHO and LLC-PK1 cells. Permanently transfected cell-lines expressing varying levels of alkaline phosphatase were estblished. The data showed that functional enzyme was expressed in the transfected cells. Cell spreading and attachment were enhanced in transfected CHO cells expressing high levels of tissue non-specific alkaline phosphatase but not in the LLC-PK1 cells. Further, in CHO cells, proliferation was shown to be inversely proportional to the level of the tissue non-specific alkaline phosphatase expression. Homotypic cell association was demonstrated in both alkaline phosphatase-positive and alkaline phosphatase-negative cells in both CHO and LLC-PK1 celllines. Taken together, these findings suggest that in addition to a role in mineralization of bone, tissue nonspecific alkaline phosphatase might also play a role in other cell activities, including those related to differentiation, such as cell-cell or cell-substrate interaction and proliferation.     

Cooperative roles of Bozozok/Dharma and Nodal-related proteins in the formation of the dorsal organizer in zebrafish

In vertebrates, specification of the dorso-ventral axis requires Wnt signaling, which leads to formation of the Nieuwkoop center and the Spemann organizer (dorsal organizer), through the nuclear accumulation of beta-catenin. Zebrafish bozozok/dharma (boz) and squint (sqt), which encode a homeodomain protein and a Nodal-related protein, respectively, are required for the formation of the dorsal organizer. The zygotic expression of boz and sqt in the dorsal blastoderm and dorsal yolk syncytial layer (YSL) was dependent on the maternally derived Wnt signal, and their expression at the late blastula and early gastrula stages was dependent on the zygotic expression of their own genes. The dorsal organizer genes, goosecoid (gsc) and chordin (din), were ectopically expressed in wild-type embryos injected with boz or sqt RNA. The expression of gsc strictly depended on both boz and sqt while the expression of din strongly depended on boz but only partially depended on sqt and cyclops (cyc, another nodal-related gene). Overexpression of boz in embryos defective in Nodal signaling elicited the ectopic expression of din but not gsc and resulted in dorsalization, implying that boz could induce part of the organizer, independent of the Nodal proteins. Furthermore, boz; sqt and boz;cyc double mutants displayed a severely ventralized phenotype with anterior truncation, compared with the single mutants, and boz;sqt;cyc triple mutant embryos exhibited an even more severe phenotype, lacking the anterior neuroectoderm and notochord, suggesting that Boz/Dharma and the Nodal-related proteins cooperatively regulate the formation of the dorsal organizer.     

Purification and regiospecificity of multiple enzyme activities of phospholipase A(1) from bonito muscle

Phospholipase A(1) (PLA(1)), which catalyzes the hydrolysis of the sn-1 ester bond of diacyl phospholipids, was purified from 100,000 x g supernatant of bonito muscle to homogeneity by ammonium-sulfate precipitation and four consecutive column chromatographies (DEAE anion-exchange, ether-Toyopeal, hydroxylapatite and Toyopeal HW 50S columns). The final preparation showed a single band above the 67-kDa molecular marker on SDS-PAGE, and the molecular mass was determined to be 71.5 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using bovine serum albumin as a standard for calibration. The N-terminal 8 amino residues were determined to be Ala-Pro-Ala-Glu-Lys-Val-Lys-Try. Regiospecificity of multiple enzyme activities of the PLA(1) was examined using positionally defined synthetic phosphatidylcholine (PC) and lysophosphatidylcholines (LPC). An acyl ester bond at the sn-1 position of PC was exclusively hydrolyzed by phospholipase activity, and 1-acyl LPC was cleaved to fatty acid and glycerophosphocholine by lysophospholipase (LPL) activity. However, the positional isomer, 2-acyl LPC was a poor substrate for LPL activity. PC/transacylation activity was also observed when excess 2-acyl LPC was supplied in the reaction mixture, and fatty acid at the sn-1 position of donor PC was transferred to the sn-1 position of acceptor LPC. These results demonstrate that the multiple enzyme activities of PLA(1), this is lysophospholipase, transacylase as well as phospholipase, have a strict regiospecificity at the sn-1 position of substrates.     

Minimal requirements for the nuclear localization of p27(Kip1), a cyclin-dependent kinase inhibitor

p27(Kip1) is a cyclin-dependent kinase inhibitor, and its nuclear localization is a prerequisite for it to function as a cell cycle regulator. In the present study, the minimal requirement for the nuclear localization signal (NLS) of p27(Kip1) was determined by analyzing the localization of various mutants of p27(Kip1) tagged with green fluorescent protein (GFP) in HeLa cells and porcine aortic endothelial cells. Wild-type p27(Kip1) exclusively localized into nucleus, while GFP alone localized in both cytosol and nucleus. A comparison of various truncation mutants revealed residues 153-166 to be the minimal region necessary for nuclear localization. However, a fusion of this region to GFP showed cytoplasmic retention in addition to nuclear localization, thus suggesting that some extension flanking this region is required to achieve a full function of NLS. The site-directed mutation of the full-length p27(Kip1) therefore showed that four basic residues (K153, R154, K165, R166), especially R166, play a critical role in the nuclear localization of p27(Kip1).     

Influence of a Small Number of Mature T Cells in Donor Bone Marrow Inocula on Reconstitution of Lymphoid Tissues and Negative Selection of a T Cell Repertoire in the Recipient

Allo-chimerism and clonal elimination of self antigen (Ag) (Ia + Mls-1^a) reactive Vβ6+ T cells were analyzed and compared between allogeneic bone marrow (BM) chimeras reconstituted with BM cells which had been treated with anti-Thy-1 monoclonal antibody (mAb) plus complement (C) (T^– chimeras) and BM chimeras which had been reconstituted with BM cells pretreated with anti-Thy-1 mAb alone (T⁺ chimeras). When lethally irradiated AKR (Mls-1^a) mice were reconstituted with BM cells from B10 or B10 H-2 congenic mice, both T⁺ and T^– chimeras were entirely free of signs of graft-versus-host reaction (GVHR). However, complete replacement of the AKR lymphoid tissues by donor BM cells was accomplished at an early stage in T⁺ chimeras but not in T^– chimeras. On the other hand, clonal elimination of Vβ6⁺ T cells reactive to the recipient Ag (Mls-1^a) was abolished in T⁺ chimeras but successfully induced in T^– chimeras. The Vβ6⁺ T cells not eliminated in T⁺ chimeras showed depressed responses against Mls-1^a antigens. The findings herein demonstrate that T cells which contaminate a BM inoculum survive in recipient mice after treatment with anti-Thy-1 mAb without C in vitro followed by BMT. The surviving T cells have been estimated to represent fewer than 0.5% of the BM cells inoculated. These cells appear to accelerate the full replacement of recipient lymphoid tissues by donor cells. Furthermore, the T cells which survive in the marrow inoculum influence eventually the development of a tolerant state in the T cell repertoire of the donor.     

Identification of neurite outgrowth-promoting domains of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, and involvement of phosphatidylinositol 3-kinase and protein kinase C signaling pathways in neuritogenesis

Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. We report that the recombinant ectodomain of NGC core protein enhances neurite outgrowth from rat neocortical neurons in culture. Both protein kinase C (PKC) inhibitors and phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated the NGC-mediated neurite outgrowth in a dose-dependent manner, suggesting that NGC promotes neurite outgrowth via PI3K and PKC pathways. The active sites of NGC for neurite outgrowth existed in the epidermal growth factor (EGF)-like domain and acidic amino acid (AA)-domain of the NGC ectodomain. The EGF-domain caused cells to extend preferentially one neurite from a soma, whereas the AA-domain caused several neurites to develop. The EGF-domain also enhanced neurite outgrowth from GABA-positive neurons, but the AA-domain did not. These results suggest that the EGF-domain and AA-domain have distinct functions in terms of neuritogenesis. From these findings, NGC can be considered to be involved in neuritogenesis in the developing central nervous system.     

A novel and simple method for quantification of small,dense LDL

A preponderance of small, dense (sd) LDL is strongly associated with the development of coronary heart disease, but the method for the measurement of sd LDL is too laborious for clinical use. We report a simple method for the quantification of sd LDL that is applicable to an autoanalyzer. This method consists of two steps: first, to precipitate the lipoprotein of density (d) <1.044 g/ml using heparin-magnesium; and second, to measure LDL-cholesterol in the supernatant by the homogeneous method or apolipoprotein B (apoB) by an immunoturbidometric assay. The cholesterol and apoB values obtained by the precipitation method (45 +/- 26 and 33 +/- 20 mg/dl, respectively) were similar to those obtained in the lipoprotein (d = 1.044-1.063) separated by ultracentrifugation (42 +/- 22 and 31 +/- 17 mg/dl, respectively), and there was an excellent correlation between the two methods for sd LDL-cholesterol (y = 1.05X + 1, r = 0.88, n = 69) and apoB (y = 1.07X, r = 0.90). Sd LDL values had a significant inverse correlation with LDL size. A high correlation was found between sd LDL-cholesterol and apoB values (r = 0.94). Sd LDL value was related to triglyceride, apoB, and LDL-cholesterol, but not to the buoyant LDL level. These results suggest that this precipitation method is a simple and rapid method for the measurement of sd LDL concentration.     

Interrelation between HeLa-S3 cell transfection and hemolysis in red blood cell suspension using pulsed ultrasound of various duty cycles

We have studied the in vitro transfection of a plasmid DNA with the lacZ gene to HeLa-S3 cells and hemolysis in a red blood cell (RBC) suspension under pulsed ultrasound with duty cycles of 10, 20 and 30% using a digital sonifier at a frequency of 20 kHz and an intensity of 6.2 W/cm² on the surface of a horn tip. Cultured HeLa-S3 cells in suspension were exposed to pulsed ultrasound for an apparent exposure time t from 0 to 60 s. HeLa-S3 viability decreased as a single exponential function of the total exposure time t=t with a common time constant =3.8 s for three duty cycles. Transfection was evaluated by counting the number of -galactosidase(-Gal)-positive cells relative to the total number of cells. Pulsed ultrasound provided an enhanced transfer of the -Gal plasmid to HeLa-S3 cells, 3.4-fold as compared with that in the case of the control. The optimal transfection efficiencies were 0.75, 0.80 and 0.74% near t= with =10, 20 and 30%, respectively. The number ratio of -Gal-positive cells to the surviving cells after exposure increased with t according to a modified logistic equation. The degree of hemolysis also increased exponentially with t at a time constant =₀/ for the RBC suspension in physiological saline at a hematocrit concentration of 0.5% with ₀=0.9 s. Thus the total exposure time for the optimal transfection efficiency was , that is, nearly four times of ₀. Hemolysis in the RBC suspension may be a useful model for determining optimal transfection by pulsed ultrasound of various duty cycles.