Cloning and Characterization of Two Novel Human cDNAs (NELL1 and NELL2) Encoding Proteins with Six EGF-like Repeats

From a human fetal-brain cDNA library we isolated two novel genes encoding peptides containing six EGF-like repeats. Both showed significant homologies with nel, a gene strongly expressed in neural tissues of chicken. The cDNAs, designated NELL1 (nel-like, type 1) and NELL2 (nel-like, type 2), contained open reading frames encoding 810 and 816 amino acids, respectively. NELL2 is strongly expressed in brain of adult and fetus but only weakly in fetal kidney. NELL1 and NELL2 were mapped by FISH to chromosomal bands 11p15.1–p15.2 and 12q13.11–q13.12, respectively.     

Lipofection of Synthetic Oligodeoxyribonucleotide Having a Palindromic Sequence of AACGTT to Murine Splenocytes Enhances Interferon Production and Natural Killer Activity

A synthetic 22-mer oligodeoxyribonucleotide having an AACGTT palindrome, AAC-22, induced interferon (IFN) production and augmented the natural killer (NK) activity in murine splenocytes, whereas its analogue, ACC-22, having an ACCGGT palindrome, did not. The binding of AAC-22 to splenocytes was not different from that of ACC-22. Lipofection of AAC-22 to splenocytes remarkably enhanced IFN production and NK cell activity, whereas that of ACC-22 caused little enhancement. These results strongly suggest that the prerequisite for IFN production is not the binding of AAC-22 to the cell surface receptors, but its penetration into the spleen cells.     

Adsorption of BSA on QAE-dextran: equilibria

Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a strong-base (QAE) dextran-type ion exchanger have been determined experimentally. They were not affected by the initial concentration of BSA but were affected by pH considerably. They were correlated by the Langmuir equation when pH >/= 5.05 and by the Freundlich equation of pH 4.8, which is close to pl approximately 4.8 of BSA. The contribution of ion exchange to adsorption of BSA on the ion exchanger was determined experimentally. The maximum amounts of inorganic anion exchanged for BSA were 1% and 0.4% of the exchange capacity of the ion exchanger at pH 6.9, respectively. Since the effect of the ion exchange on the adsorption appeared small, BSA may be adsorbed mainly by electrostatic attraction when pH >/= 5.05 and by hydrophobic interaction or hydrogen bonding at pH 4.8. When NaCl coexisted in the solution, the shape of the isotherm was similar to the Langmuir isotherm, but it is shifted to the right. When the concentration of NaCl was 0.2 mol/dm(3), BsA was not adsorbed on the resin. When BSA was dissolved in pure water, the saturation capacity of BSA on HPO(4) (2-),-orm resin was about 2 times larger than that for adsorption from the solution with buffer (pH 6.9 and 8.79). The saturation capacity for adsorption of BSA in pure water on HPO(4) (2-) + H(2)O(4) (-)-from resin was much smaller than that from the solution with buffer. The isotherms for univalent Cl(-)-and H(2)PO(4) (-)-form resin was peculiar; that is, the amount of BSA adsorbed decreased with increasing the liquid-phase equilibrium concentration of BSA. (c) 1993 John Wiley & Sons, Inc.     

Partitioning behavior and enrichment of proteins with reversed micellar extraction: I. forward extraction of proteins from aqueous to reversed micellar phase

Protein extractions using aerosol OT (AOT)-isooctane reverse micelle solutions have been studied to explore the potential for separating and enriching proteins with the reversed micellar extraction. The effects of pH, ionic strength, and different cations of chlorides in a bulk aqueous phase and of AOT concentration in an organic phase on the partitioning of lysozyme and myoglobin and the solubilization of water are presented in detail. The extraction of lysozyme was affected by the concentration of potassium or barium but was almost independent of that of sodium or calcium, whose ionic diameter is smaller than that of potassium and barium. For the extraction of myoglobin, however, the effect of barium concentration was not appreciable. Lysozyme could be enriched into the reversed micellar phase up to 30 times the aqueous feed concentration. (c) 1993 John Wiley & Sons, Inc.     

Ischemic changes in myocardial ionic contents of the isolated perfused rat hearts as studied by NMR

Using³¹P-,²³Na- and³⁹K-NMR, we assessed ischemic changes in high energy phosphates and ion contents of isolated perfused rat hearts continuously and systematically. To discriminate intra- and extracellular Na⁺, a shift reagent (Dy(TTHA)^3–) was used in²³Na-NMR study. In³⁹K-NMR study, the extracellular K⁺ signal was suppressed by inversion recovery pulse sequence in order to obtain intracellular K⁺ signal without using shift reagnets. During the early period of ischemia, increases in intracellular Na⁺ and inorganic phosphate (Pi) were observed in addition to the well-documented decreases in creatine phosphate and ATP and a fall of intracellular pH, suggesting an augmented operation of Na⁺–H⁺ exchange triggered by a fall of the intracellular pH resulted from breakdown of ATP. At around 15 min of ischemia, a second larger increase in intracellular Na⁺ and a decrease in intracellular K⁺ were observed in association with a second increase in Pi. This was accompnanied by an abrupt rise of the ventricular end-diastolic pressure. As there was a depletion of ATP at this time, the increase in intracellular Na⁺ and associated decrease in intracellular K⁺ may be explained by inhibition of the Na⁺–K⁺ ATPase due to the depletion of ATP. A longer observation with³¹P-NMR revealed a second phosphate peak (at lower magnetic field to ordinary Pi peak) which increased its intensity as ischemic time lengthened. The pH of this 2nd peak changed in parallel with the changes in pH of the bathing solution, indicating the appearance of a compartment whose hydrogen concentration is in equilibrium with that of the external compartment. Thus, the peak could be used as an index of irreversible membrane damage of the myocardium.     

Genetic Transformation in Helicobacter pylori

Genetic transformation in Helicobacter pylori was investigated by using its chromosomal and plasmid DNAs. Six out of the eight strains exhibited the natural competence for incorporation of H. pylori chromosomal DNA, and all the strains incorporated the donor DNA efficiently by washing and concentrating the cells, with a glycerol solution. The much higher frequency of transformation was obtained in each strain by means of electroporation. Electroporation experiments were also conducted by use of the recombinant DNAs consisting of the H. pylori and Escherichia coli plasmids as the donors, and the occurrence of the homologous recombination was demonstrated between the incoming H. pylori plasmid-derived region and the corresponding region of the originally residing plasmid in H. pylori.     

Transport of chimeric proteins that contain a carboxy-terminal targeting signal into plant microbodies

Malate synthase is a glyoxysome-specific enzyme. The carboxy-terminal tripeptide of the enzyme is Ser—Arg—Leu (SRL), which is known to function as a peroxisomal targeting signal in mammalian cells. To analyze the function of the carboxy-terminal amino acids of pumpkin malate synthase in plant cells, a chimeric gene was constructed that encoded a fusion protein which consisted of β-glucuronidase and the carboxyl terminus of the enzyme. The fusion protein was expressed and accumulated in transgenic Arabidopsis that had been transformed with the chimeric gene. Immunocytochemical analysis of the transgenic plants revealed that the carboxy-terminal five amino acids of pumpkin malate synthase were sufficient for transport of the fusion protein into glyoxysomes in etiolated cotyledons, into leaf peroxisomes in green cotyledons and in mature leaves, and into unspecialized microbodies in roots, although the fusion protein was no longer transported into microbodies when SRL at the carboxyl terminus was deleted. Transport of proteins into glyoxysomes and leaf peroxisomes was also observed when the carboxy-terminal amino acids of the fusion protein were changed from SRL to SKL, SRM, ARL or PRL. The results suggest that tripeprides with S, A or P at the −3 position, K or R at the −2 position, and L or M at the carboxyl terminal position can function as a targeting signal for three kinds of plant microbody.     

Reconstitution of a pentameric complex of dimeric transforming growth factor beta ligand and a type I,II, III receptor in baculoviral-infected insect cells

Summary Two transmembrane serine-threonine kinases (type I and II receptors), a membrane-anchored proteoglycan (type III), and a homodimeric ligand participate in the transforming growth factor beta type on (TGFβ1) signal transduction complex. The expression of recombinant receptors in insect cells co-infected with up to three recombinant baculoviruses was employed to study interactions among the ectodomains of the three types of receptors and the TGFβ1 ligand in absence of uncontrollable extrinsic factors in mammalian cells. Multi-subunit complexes were assembled in intact cells and purified on glutathione-conjugated beads for analysis by tagging one of the subunits with glutathione S-transferase (GST). Intrinsic ligand-independent interactions were observed among receptor subunits as follows: type III–III type I–I, type III-I, and type II-I. The homeotypic complex of type II–II receptors and the heterotypic type III-II interaction was ligand dependent. The type I, but not the type III, subunit displaced about 50% of the type II component in either ligand-dependent homomeric type II-type II complexes or heteromeric type III-type II complexes to form type II-I or type III-II-I oligomers, respectively. The type II subunit displaced type I subunits in oligomers of the type I subunit. Specificity of type I receptors may result from differential affinity for the type II receptor rather than specificity for ligand. A monomeric subunit of the TGFβ1 ligand bound concurrently to type III and type II or type III and type I receptors, but failed to concurrently bind to the type II and type I subunits. The binding of TGFβ1 to the type I kinase subunit appears to require an intact disulfide-linked ligand dimer in the absence of a type III subunit. The combined results suggest a pentameric TGFβ signal transduction complex in which one unit each of the type III, type II, and type I components is assembled around the two subunits of the dimeric TGFβ1 ligand. An immobilized GST-tagged subunit of the receptor complex was utilized to assemble multi-subunit complexesin vitro and to study the phosphorylation events among subunits in the absence of extrinsic cell-derived kinases. The results revealed that (a) a low level of ligand-independent autophosphorylation occurs in the type I kinase; (b) a high level of autophosphorylation occurs in the type II kinase; (c) both the type III and type I subunits aretrans-phosphorylated by the type II subunit; and (d) the presence of both type I and II kinases complexed with the type III subunit and dimeric TGFβ1 ligand in a pentameric complex causes maximum phosphorylation of all three receptor subunits.