Regeneration of horseradish hairy roots incited by Agrobacterium rhizogenes infection

Surface-sterilized leaf disks of horse-radish (Armoracia lapathifolia) were immersed in a suspension of Agrobacterium rhizogenes harboring the root-inducing plasmid (pRi) and cultured on a solid medium. Within about 10 days after inoculation, adventitious roots (hairy roots) emerged from the leaf disks. No roots emerged from the uninoculated leaf disks. The excised hairy roots grew vigorously in the dark and exhibited extensive lateral branches in the absence of phytohormones. When the hairy roots were moved into the light, numerous adventitious buds thrust out of the roots within about 10 days, and they developed into complete plants (R0 generation). R0 plants revealed leaf wrinkle. Root masses of cultured R0 plants were of two types. One had fibrous roots only and the other had both fibrous and tuberous roots Leaf disks of the R0 plants proliferated adventitious roots (R1 generation) on a solid medium after 1–2 weeks of culture. Phenotypical characters of the R1 roots were the same as those observed with the initial hairy roots. The T-DNA sequences of pRi were detected within DNA isolated from the hairy roots and their regenerants.     

Organ specific expression of ras oncoproteins during growth and development of the rat

Summary Expression of proteins encoded by the ras proto-oncogenes was examined in extracts from normal rat organs using anti-ras p21 antibodies generated against synthetic peptides. The highest level of ras p21 was found in brain (cerebrum) and was predominantly of c-Ha-ras origin. Levels of brain ras p21 did not vary from the newborn period of 3 months of age. Moderate levels of ras p21s were detected in lung, spleen and thymus. In contrast to the p21 in brain, these levels varied with the age of the rats and were encoded by other members of ras proto-oncogene family (Ki-ras or N-ras). This organ specific expression of different ras genes might be related to developmental control of gene expressions.     

Isolation and partial characterization of a compound with siderophore activity from Vibrio parahaemolyticus

A compound with siderophore activity was purified by successive column and thin layer chromatographic procedures from Dowex 1 x 8 extracts of culture supernatants of Vibrio parahaemolyticus AQ 3354. The strain synthesized the compound in culture media containing less than 2 microM added FeCl3. Hydrolysis of the compound yielded alanine, ethanolamine, citric acid and 2-ketoglutaric acid. The 1H-NMR spectrum exhibited the presence of a residue from each of these components in the intact molecule. The fast-atom bombardment mass spectrum of the methyl ester derivative indicated a prominent ion at m/z 477, probably corresponding to [M + 1] ion. Other strains of V. parahaemolyticus were also found to produce this compound when grown in an iron-limited medium.     

Purification and characterization of subtilisin inhibitors 'Marinostatin' produced by marine Alteromonas sp.

The four novel peptide subtilisin inhibitors, which have been named Marinostatin B-1, B-2, C-1 and C-2, were purified from the culture supernatant fluid of a marine bacterium, Alteromonas sp. A combination of Diaion HP-20 and CM-cellulofine ion exchange column chromatographies, Sephadex G-25 gel filtration and preparative high performance liquid chromatography (HPLC) were employed. Final preparations gave a single peak on HPLC. The molecular weights by gel filtration of Marinostatin B-1 and C-1 were estimated to be 1500, and those of B-2 and C-2 were 1700. All the inhibitors were stable in acidic (pH range 4–6) but less stable in alkaline solutions (pH 10). The metal ions Co²⁺ and Fe²⁺ repressed the inhibitory activity by 30 and 20%, respectively. The four inhibitors had inhibitory effects on serine proteases like α-chymotrypsin.     

An Epidermal Growth Factor Motif from Del1 Protein Increases the Efficiency of In Vivo Gene Transfer with a Non-Viral Vector

Increasing the efficiency of gene transfer using non-viral vectors, which have the potential to be safe and economical, would improve upon available options for gene therapy. We previously reported that the third EGF motif of the extracellular matrix protein Del1 (E3) increases the transfection efficiency of non-viral vector methods. Here, we asked if E3 could increase the in vivo transfection efficiency of a polyplex-based approach. To test this, cDNA encoding a heat-stable alkaline phosphatase (AP) was first injected intravenously into mice along with recombinant E3. After 24 h, exogenous AP activity in serum was measured. We found that the introduction of E3 resulted in 50 % more AP activity as compared to the control. We next tested transfection into a tumour explant of SCCKN cells, an oral carcinoma-derived cell line. To do this, a cDNA encoding yellow fluorescent protein was locally injected into a tumour explant, followed by local injection of recombinant E3. Use of E3 increased the number of transfected cells to 2.5 times that of the control. Histochemical staining revealed that E3-induced apoptosis in a tumour explant. The data suggest that E3 might be a useful tool for cancer gene therapy using non-viral vectors.     

The narQP genes for a two-component regulatory system from the deep-sea bacterium Shewanella violacea DSS12.

Shewanella violacea DSS12 is facultative piezophile isolated from the deep-sea. The expression of cydDC genes (required for d-type cytochrome maturation) of the organism is regulated by hydrostatic pressure. In this study, we analyzed the nucleotide sequence upstream of cydDC in detail and found that there are putative binding sites for the NarL protein which is part of a two-component regulatory system also containing the sensor protein NarX. Furthermore, we identified the narQP genes (homologues of narXL) from S. violacea DSS12 and demonstrated the heterologous expression of narP in Escherichia coli. These results will be helpful in examining pressure regulation of gene expression in S. violacea at the molecular level.     

Transport-coupled ubiquitination of the borate transporter BOR1 for its boron-dependent degradation

Plants take up and translocate nutrients through transporters. In Arabidopsis thaliana, the borate exporter BOR1 acts as a key transporter under boron (B) limitation in the soil. Upon sufficient-B supply, BOR1 undergoes ubiquitination and is transported to the vacuole for degradation, to avoid overaccumulation of B. However, the mechanisms underlying B-sensing and ubiquitination of BOR1 are unknown. In this study, we confirmed the lysine-590 residue in the C-terminal cytosolic region of BOR1 as the direct ubiquitination site and showed that BOR1 undergoes K63-linked polyubiquitination. A forward genetic screen identified that amino acid residues located in vicinity of the substrate-binding pocket of BOR1 are essential for the vacuolar sorting. BOR1 variants that lack B-transport activity showed a significant reduction of polyubiquitination and subsequent vacuolar sorting. Coexpression of wild-type (WT) and a transport-defective variant of BOR1 in the same cells showed degradation of the WT but not the variant upon sufficient-B supply. These findings suggest that polyubiquitination of BOR1 relies on its conformational transition during the transport cycle. We propose a model in which BOR1, as a B transceptor, directly senses the B concentration and promotes its own polyubiquitination and vacuolar sorting for quick and precise maintenance of B homeostasis.

The borate transporter BOR1 senses the boron concentration through its borate transport activity for K63-linked polyubiquitination and subsequent degradation.     

Efficient solid phase peptide synthesis. Use of methanesulfonic acid alpha-amino deprotecting procedure and new coupling reagent, 2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidinium hexafluorophosphate (BOI).

An efficient method for solid phase peptide synthesis was developed, which consists of N alpha-selective deprotection by dilute methanesulfonic acid, in situ neutralization and rapid coupling reaction using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or 2-(benzotriazol-1-yl)oxy-1,3- dimethylimidazolidinium hexafluorophosphate (BOI) reagent. Selective removal of the N alpha-Boc group by dilute methanesulfonic acid was of more advantage than removal by TFA in terms of stability of semipermanent protecting groups and suppression of undesired side reactions. The use of in situ neutralization and rapid coupling method reduced intramolecular aminolytic cyclization by shortening exposure of the deprotected nucleophilic amino group. A successful synthesis of porcine brain natriuretic peptide (pBNP) has been achieved using this efficient solid phase peptide synthesis scheme.