Importance of the l-galactonolactone pool for enhancing the ascorbate content revealed by l -galactonolactone dehydrogenase-overexpressing tobacco plants

l-Galactono-1,4-lactone (GalL) dehydrogenase (GLDH) is an enzyme that catalyzes the last step of l-ascorbate (AsA) biosynthesis in plants. To re-evaluate the importance of the enzyme and the possibility of manipulating the AsA content in plants, a cDNA encoding GLDH from sweet potato was introduced into tobacco plants by Agrobacterium-mediated transformation under the control of a CaMV 35S promoter. Protein blot analysis revealed the elevation of GLDH protein contents in three GLDH-transformed lines. Furthermore, these transgenic lines showed 6- to 10-fold higher GLDH activities in the roots than the non-transformed plants, SR1. Despite the elevated GLDH activity, the AsA content in the leaves did not change in all lines; i.e., the AsA content in GLDH-transformed lines was 3–7 μmol g⁻¹ FW, comparable to that in the non-transformed plants. Incubation of leaf discs in a GalL solution led to a rapid 2- to 3-fold increase in the AsA content in both GLDH-transformed and non-transformed plants in the same manner. These results suggest that the supply of GalL is a crucial factor for determining the AsA pool size and that the upstream genes in the AsA biosynthetic pathway are responsible for enhancing the AsA content in plants.     

On the Metabolism of Organic Acids by Clostridium acetobutylicum

Racemiase, an enzyme which catalyzed racemization of lactic acids, was isolated from culture filtrate of Clostridium acetobutylicum by salting-out, and its purification was attained to about twenty-fold by treating with calcium phosphate gel. It was shown that racemiase requires for its full activity cofactors, pyridoxamine phosphate and ferrous ions. These substances were detected in the racemiasc preparation. Several other properties of racemiase were also investigated.     

A simple and quick technique for establishing axenic cultures of the centric diatom Coscinodiscus wailesii Gran

We devised a simple and quick technique for establishing axeniccultures of the centric diatom Coscinodiscus wailesii Gran bywashing the cells with a sieve and soaking them in antibioticmedia.     

Osteoblast Recruitment and Bone Formation Enhanced by Cell Matrix–associated Heparin-binding Growth-associated Molecule (HB-GAM)

Bone has an enormous capacity for growth, regeneration, and remodeling. This capacity is largely due to induction of osteoblasts that are recruited to the site of bone formation. The recruitment of osteoblasts has not been fully elucidated, though the immediate environment of the cells is likely to play a role via cell– matrix interactions. We show here that heparin-binding growth-associated molecule (HB-GAM), an extracellular matrix–associated protein that enhances migratory responses in neurons, is prominently expressed in the cell matrices that act as target substrates for bone formation. Intriguingly, N-syndecan, which acts as a receptor for HB-GAM, is expressed by osteoblasts/osteoblast precursors, whose ultrastructural phenotypes suggest active cell motility. The hypothesis that HB-GAM/N-syndecan interaction mediates osteoblast recruitment, as inferred from developmental studies, was tested using osteoblast-type cells that express N-syndecan abundantly. These cells migrate rapidly to HB-GAM in a haptotactic transfilter assay and in a migration assay where HB-GAM patterns were created on culture wells. The mechanism of migration is similar to that previously described for the HB-GAM–induced migratory response of neurons. Our hypothesis that HB-GAM/N-syndecan interaction participates in regulation of osteoblast recruitment was tested using two different in vivo models: an adjuvant-induced arthritic model and a transgenic model. In the adjuvant-induced injury model, the expression of HB-GAM and of N-syndecan is strongly upregulated in the periosteum accompanying the regenerative response of bone. In the transgenic model, the HB-GAM expression is maintained in mesenchymal tissues with the highest expression in the periosteum. The HB-GAM transgenic mice develop a phenotype characterized by an increased bone thickness. HB-GAM may thus play an important role in bone formation, probably by mediating recruitment and attachment of osteoblasts/osteoblast precursors to the appropriate substrates for deposition of new bone.     

Studies on Storage of Harvested Grapes for Wine Making

When harvested grape clusters were stored at 40°C, the decrement of acidity in grape juice was recognized. The decrement was caused by transformation of malic acid to lactic acid. It is advised to keep the grapes at 40°C as a storing temperature because of drying efficiency, sterile effect on wild microbes and preservation of biological activity of grape clusters. As the period of storage 4 days for Kôshû and 3 days for Merlot were most suitable.     

Inhibitory effect of a self-derived peptide on glucosyltransferase of Streptococcus mutans. Possible novel anticaries measures.

Glucosyltransferase (GTF) plays an important role in the development of dental caries. We examined the possible presence of self-inhibitory segments within the enzyme molecule for the purpose of developing anticaries measures through GTF inhibition. Twenty-two synthetic peptides derived from various regions presumably responsible for insoluble-glucan synthesis were studied with respect to their effects on catalytic activity. One of them, which is identical in amino acid sequence to residues 1176-1194, significantly and specifically inhibited both sucrose hydrolysis and glucosyl transfer to glucan by GTF-I. Double-reciprocal analysis revealed that the inhibition is noncompetitive. Scramble peptides, composed of the identical amino acids in randomized sequence, had no effect on GTF-I activity. Furthermore, the peptide is tightly bound to the enzyme once complexed, even in the presence of sodium dodecyl sulfate (SDS). Kinetic analysis using an optical evanescent resonant mirror cuvette system demonstrated that the enzyme-peptide interaction was biphasic. These results indicate that the peptide directly interacts with the enzyme with high affinity and inhibits its activity in a sequence-specific manner. This peptide itself could possibly be an effective agent for prevention of dental caries, although its effectiveness may be improved by further modification.     

High frequency of tetraploidy in Agrobacterium-mediated transformants regenerated from tuber discs of diploid potato lines

Summary Variations in the ploidy level of 69 transgenic potato (Solanum tuberosum L.) plants regenerated from the tuber discs of 17 diploid lines were studied: 24 plants (35%) were diploid, the other 45 plants (65%) were tetraploid. Seventy-eight control regenerants obtained without Agrobacterium inoculation showed a relatively low tendency to tetraploidization (35%). The results obtained suggested that chromosome doubling occurred frequently in diploid potato lines during the tissue culture process for regeneration. Putative somaclonal changes in in vitro-formed tuber proteins were detected in three out of six transformants by electrophoretic analysis.