Expression of RNase Rh from Rhizopus niveus in yeast and characterization of the secreted proteins.

The full-length cDNA encoding RNase Rh, which is secreted extracellularly by Rhizopus niveus, was isolated and its nucleotide sequence was determined. It was placed under control of the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene of Saccharomyces cerevisiae in a high expression vector in yeast. Since yeast cells transformed by this plasmid poorly secreted RNase into the medium, the plasmid pYE RNAP-Rh was constructed, in which the signal sequence of RNase Rh was replaced by the prepro-sequence of aspartic proteinase-I, one of the extracellular enzymes secreted by R. niveus. Yeast cells harboring pYE RNAP-Rh produced RNase efficiently (ca. 40 micrograms/ml) into the medium. The product was a mixture of six enzymes (RNase RNAP-Rhs) having 3, 5, 9, 13, 14, and 16 additional amino acid residues attached to the amino terminus of the mature RNase Rh. The major product was the RNase with three additional amino acids at the amino terminus. Limited digestion of RNase RNAP-Rhs with staphylococcal V8 protease succeeded in shortening the various lengths of extra amino acid residues attached to the amino terminus of RNase Rh, yielding an RNase that has 3 additional amino acids at the amino terminus. It has been named RNase RNAP-Rh. The RNase RNAP-Rh showed the same specific activity and CD spectra as those of RNase Rh, suggesting that the two have similar conformations to each other around aromatic amino acid residues and the peptide backbone.     

Primary structure of a base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi

The complete primary structure of a base non-specific and adenylic acid preferential RNase (RNase M) from Aspergillus saitoi was determined. The sequence was determined by analysis of the peptides generated by digestion of heat-denatured RNase M with lysylendopeptidase, and the peptides generated from RCM RNase M by digestion with staphylococcal V8 protease or chemical cleavage with BrCN. It consisted of 238 amino acid residues and carbohydrate moiety attached to the 74th asparagine residue. The molecular weight of the protein moiety deduced from the sequence was 26,596. The locations of 10 half cystine residues are almost superimposable on those of RNase Rh from Rhizopus niveus and RNase T2 from Aspergillus oryzae which have similar base specificity. The homology between RNase M and RNase Rh and RNase T2 amounted to 97 and 160 amino acid residues, respectively. The amino acid sequences conserved in the three RNases are concentrated around the three histidine residues, which are supposed to form part of the active sites of these RNases.     

The subsite structures of guanine-specific ribonucleases and a guanine-preferential ribonuclease. Cleavage of oligoinosinic acids and poly I

In order to estimate the size of the active site of guanylic acid specific RNases (RNase T1 from Aspergillus oryzae and RNase St from Streptomyces erythreus) and guanine-preferential RNase (RNase Ms from A. saitoi), the depolymerization reaction of oligoinosinic acid, (Ip)nI greater than p, having various chain lengths was studied. The kinetic parameters for depolymerization of oligoinosinic acids, (pKm, log V and log V/Km) by the three RNases increased with increase of the chain length of the substrates, and became almost constant at n = 2 or more. Thus, the size of the active site of RNase T1, RNase St, and RNase Ms was estimated to be three nucleotides in length.     

Modification of an arginine residue of a base-nonspecific ribonuclease from Aspergillus saitoi.

1. A base-nonspecific ribonuclease from Aspergillus saitoi [RNase Ms, EC 3.1.4.23; molecular weight, 12,500] was modified with phenylglyoxal (PG) and 1,2-cyclohexanedione (CHD) in order to determine whether a single arginine residue was involved in the active site of the enzyme. 2. RNase Ms was inactivated by both PG and CHD with concomitant loss of one arginine residue. A competitive inhibitor of RNase Ms, 2',(3')-AMP, protected the enzyme from inactivation by PG. These findings strongly suggest that one arginine residue is involved in the active site of RNase Ms. 3. Difference CD spectra were measured at pH 5.5 for the binding of 2'-AMP and adenosine to native RNase Ms and the CHD- and PG-modified enzyme derivatives to determine the association constants. The arginine modification brought about a marked decrease in the binding affinity of 2'-AMP for the enzyme, but only a slight decrease for adenosine, suggesting that the arginine residue had interacted with the phosphate groups of the substrate.     

Isolation of Q nucleoside precursor present in tRNA of an E. coli mutant and its characterization as 7-(cyano)-7-deazaguanosine.

One of the E. coli mutants selected for deficiency of modified nucleoside Q was found to contain an analogue of Q and normal guanosine in place of Q. The analogue of Q, designated as preQo, was isolated on a large scale from purified tRNATyr containing preQo. The structure of preQo was determined to be 7-(cyano)-7-deazaguanosine by comparison of its ultraviolet absorption spectra, thin-layer chromatographic mobility and mass spectrum with those of synthetic material.     

Photooxidation and carbethoxylation of a minor ribonuclease from Aspergillus saitoi.

In order to investigate the nature of amino acid residues involved in the active in the active site of a ribonuclease from Aspergillus saitoi, the pH dependence of the rates of inactivation of RNase Ms by photooxidation and modification with diethylpyrocarbonate were studied. (1) RNase Ms was inactivated by illumination in the presence of methylene blue at various pH's. The pH dependence of the rate of photooxidative inactivation of RNase Ms indicated that at least one functional group having pKa 7.2 was involved in the active site. (2) Amino acid analyses of photooxidized RNase Ms at various stages of photooxidative inactivation at pH's 4.0 and 6.0 indicated that one histidine residue was related to the activity of RNase Ms, but that no tryptophan residue was involved in the active site. (3) 2',(3')-AMP prevented the photooxidative inactivation of RNase Ms. The results also indicated the presence of a histidine residue in the active site. (4) Modification of RNase Ms with diethylpyrocarbonate was studied at various pH's. The results indicated that a functional group having pKa 7.1 was involved in the active site of RNase Ms.     

Ca2+ Regulation of Outward Rectifying K+ Channel in the Plasma Membrane of Tobacco Cultured Cells in Suspension: a Role of the K +Channel in Mitigation of Salt-Stress Effects by External Ca2+

The patch-clamp technique was used to study effect of the Ca²⁺on K⁺ channels in the plasma membrane of protoplasts isolatedfrom tobacco (Nicotiana tabacum L., cv. Bright Yellow) culturedcells in suspension. The outward rectifying whole-cell K⁺ currentswere not affected by in-tracellular Ca²⁺, but they were reducedwith increasing extracellular Ca²⁺. Neither extracellular norintracellular Ca²⁺ affected the permeability ratios (p_K+/P_Na+)of the plasma membrane. These results suggest that the inhibitionof outward-rectifying K⁺ channels by extracellular Ca²⁺may partiallycontribute towards the mitigation of detrimental effects ofsalinity on growth by extracellular Ca²⁺. (Received January 19, 1998; Accepted July 30, 1998)     

Distribution of serine proteinase inhibitor, clade B, member 6 (Serpinb6) in the adult mouse brain

In the brain, Serpinb6 was identified as an endogenous inhibitor of neuropsin, a member of the S1 (clan SA) family of serine proteases [J. Biol. Chem. 276 (2001) 14562]. In the present study, we investigated the localization of Serpinb6 in the adult mouse brain using in situ hybridization histochemistry and immunohistochemistry. Region-specific patterns of expression were observed and two characteristics were recognized. First, the forebrain limbic area that expressed neuropsin mRNA contained Serpinb6 mRNA at moderate levels but not the lateral septum. On the other hand, Serpinb6 mRNA was also expressed moderately in the substantia nigra-ventral tegmental area system, whose fibers projected to the lateral septum. Additionally, Serpinb6 protein was detected in the lateral septum. Together, it was suggested that the expression of neuropsin in the brain is regulated entirely by Serpinb6. Second, Serpinb6 mRNA and the protein were strongly expressed in most somatic and visceral motoneurons among cranial nerve nuclei. This suggests that another serine protease is regulated by Serpinb6 in motoneurons and/or fibers.     

Identification of DELE,a novel DAP3-binding protein which is crucial for death receptor-mediated apoptosis induction

Death associated protein 3 (DAP3) is known to be a highly conserved protein, and is responsible for regulating apoptosis induced by various stimuli. To understand the molecular mechanism of how DAP3 induces apoptosis, we performed yeast two-hybrid screening, and identified a novel DAP3-binding protein termed death ligand signal enhancer (DELE). In this report, we show that DELE actually binds to DAP3 in mammalian cells. We found that the cells stably expressing DELE are susceptible to apoptosis induction by the stimulation of TNF-α and TRAIL. In addition, knockdown of DELE expression rescued the HeLa cells from apoptosis induction by these stimuli. Moreover, activation of caspase-3, caspase-8 and caspase-9 induced by stimulation of TNF-α, anti-Fas or TRAIL was significantly inhibited by the knockdown of DELE expression. These results demonstrated the biological significance of DELE for apoptosis signal mediated by death receptors.     

Osteogenic differentiation of human dental papilla mesenchymal cells

We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of beta-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate that mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering.