Characterization of β-amylase and its deficiency in various rice cultivars

 β-Amylase deficiency in various cultivars of rice was examined at the molecular level. Using an antibody against β-amylase purified from germinating seeds of rice, we were able to demonstrate the expression and organization of the β-amylase gene in normal and deficient cultivars. Although β-amylase is a starch-hydrolyzing enzyme, as is α-amylase, the β-amylase protein/gene is expressed differently from the α-amylase protein/gene; i.e. (1) β-amylase is synthesized only in aleurone cells, (2) the enzyme production in the embryo-less half-seeds is not under hormonal control. We identified some cultivars of rice that are deficient for β-amylase activity. We present new evidence that synthesis is blocked at the level of mRNA synthesis in the deficient cultivars. The usefulness of β-amylase as a crop trait is also discussed. Received: 8 May 1998 / Accepted: 5 June 1998     

Isobutanol production from cellobiose in Escherichia coli

Converting lignocellulosics into biofuels remains a promising route for biofuel production. To facilitate strain development for specificity and productivity of cellulosic biofuel production, a user friendly Escherichia coli host was engineered to produce isobutanol, a drop-in biofuel candidate, from cellobiose. A beta-glucosidase was expressed extracellularly by either excretion into the media, or anchoring to the cell membrane. The excretion system allowed for E. coli to grow with cellobiose as a sole carbon source at rates comparable to those with glucose. The system was then combined with isobutanol production genes in three different configurations to determine whether gene arrangement affected isobutanol production. The most productive strain converted cellobiose to isobutanol in titers of 7.64?±?0.19 g/L with a productivity of 0.16 g/L/h. These results demonstrate that efficient cellobiose degradation and isobutanol production can be achieved by a single organism, and provide insight for optimization of strains for future use in a consolidated bioprocessing system for renewable production of isobutanol.     

Changes in chromatin structure during aging of human skin fibroblasts

Human skin fibroblasts from embryo, 16-, 30- and 60-year-old adults were cultivated and passaged in vitro. Their chromatin structures were examined by the sensitivity to micrococcal nuclease and by electron microscopy. When the mode of DNA degradation by the nuclease was analysed during in vitro aging of the embryo skin fibroblasts, the discrete ladder of nucleosomal DNA became obscure in old cells. Analogous change of chromatin structure was also observed even in young cells as their donor ages increased. From the observation with electron microscopy, it became clear that chromatin of fibroblasts from 30-year-old adults does not have regularly spaced nucleosomes, compared with chromatin from embryo. These results suggest that the length of the linker DNA which connects core particles becomes to be heterogeneous by aging, both in vivo and in vitro in human skin fibroblasts.     

Effect of retinoic acid on in vitro proliferation activity and glycosaminoglycan synthesis of mesenchymal cells from palatal shelves of mouse fetuses

To clarify the mechanism by which retinoid causes cleft palate, we investigated the effect of retinoic acid (RA) on proliferation activity and glycosaminoglycan (GAG) synthesis in mouse fetuses palatal mesenchymal (MFPM) cells. MFPM cells were incubated for 1-11 days with various concentrations of RA to examine its effect on growth rate. Also, confluent cultures were incubated with [3H]glucosamine or [35S]sulfate in the presence of various concentrations of RA to investigate the effect of RA on GAG synthesis. RA remarkably inhibited the growth of MFPM cells in a dose-dependent manner. RA also inhibited the synthesis of GAGs, with sulfated GAGs being more severely affected than hyaluronic acid. These data suggest that the inhibition of proliferation activity and GAG synthesis of palatal mesenchymal cells might be involved in the induction of cleft palate by retinoic acid.     

Spacing and kinship in the Formosan squirrel living in different habitats

Summary Spacing and kinship of the Formosan squirrel, Callosciurus erythraeus thaiwanensis, were studied in two different habitats. One, native habitat in the woods of Kenting, southern Formosa, was rich in available food throughout the year and had several species of predators. The other, a site in Kamakura, central Japan where squirrels had been introduced, had relatively scanty food and few potential predators. 1. Home ranges among males and between sexes overlapped extensively in both habitats. 2. Females occupied exclusive home ranges in Kamakura but had small overlapping home ranges in Ken-ting. 3. Most males disappeared from their natal areas at 1 year old in both habitats (86% in Kamakura and 93% in Ken-ting), but less females disappeared (36% in Kamakura and 35% in Ken-ting). 4. In Kamakura, daughters settled adjacent to the mother or inherited the home range of the mother, but never shared the mother's home range. In Ken-ting, 35% of daughters shared the home range with their mothers. 5. Tolerance among female kin in Ken-ting was probably facilitated by the richness of available food throughout the year, and functioned to reduce predation risk via alarm calling and mobbing.     

Primary structure of rat liver alkaline phosphatase deduced from its cDNA.

Rat liver alkaline phosphatase (ALP) was markedly induced by treatment of rats by bile-duct ligation and colchicine injection. Taking this advantage for enrichment of ALP mRNA, we constructed a lambda gt11 liver cDNA library using polyadenylated RNA prepared from the treated rat liver, and isolated an ALP cDNA clone. The 2165 bp cDNA contained an open reading frame that encodes a 524-amino-acid-residue polypeptide with a predicted molecular mass of 57737 Da. The precursor protein contained a presumed signal peptide of 17 amino acid residues followed by 28 amino acid residues identical with the N-terminal sequence determined from the purified rat liver ALP. It was also confirmed that amino acid sequences of two CNBr-cleavage peptides obtained from liver ALP were contained within the cDNA-encoded protein. Five possible N-linked glycosylation sites were found in the molecule and a highly hydrophobic amino acid sequence at the C-terminus. The deduced polypeptide of rat liver ALP showed 88% homology to that of the human liver-type enzyme in osteosarcoma cells. RNA blot hybridization analysis identified a single species of ALP mRNA with 2.7 kb in both the control and the treated rat livers. An approx. 20-fold increase of the mRNA was detected in the treated liver at 12 h after the onset of stimulation, compared with that in the control liver.     

Participation of hup gene product in ori2-dependent replication of fertility plasmid F

The fertility plasmid F'gal was not stably maintained in a hupA-hupB double mutant of Escherichia coli. Moreover, mini-F plasmids pFZY1, pFTC1 and pFTC2 were unable to transform the double mutant, though these plasmids efficiently transformed cells harboring a hupA or hupB single mutation. The composite plasmid pFHS1, which consists of the f5 DNA fragment of F plasmid and the whole DNA of a pSC101 derivative that carries a temperature-sensitive mutation for DNA replication, was not stably maintained in the hup double mutant at 42°C. These findings strongly suggest that HU protein is required for ori2-dependent replication of the F plasmid.     

Immunocytochemical identification of axons containing coexistent serotonin and substance P in the cat lumbar spinal cord

Axons containing both serotonin-like (5-HT)-LI and substance P-like (SP)-LI immunoreactivity were identified in all laminae of the cat spinal cord at the level of the lumbar enlargement. Using an immunologically-specific, double immunofluorescence method, coexistent 5-HT-LI and SP-LI immunoreactivity could be visualized in the same tissue section with appropriate FITC and rhodamine fluorescent filter sets. The fewest number of coexistent axons were observed in the superficial laminae of the dorsal horn, while their number increased in the more ventral dorsal horn laminae. Numerous coexistent axons were observed in the area adjacent to the central canal. The greatest number of coexistent axons was found in the ventral horn, especially in the motoneuronal cell groups. This study demonstrates that axons containing coexistent 5-HT-LI and SP-LI immunoreactivity are found in all laminae of the cat lumbar spinal cord and are thus involved in both sensory and motor functions. Their more frequent occurrence in the ventral horn suggests a greater role for coexistent 5-HT and SP in motor function. Since axons containing coexistent 5-HT and SP, and those containing only 5-HT, likely originate from different populations of neurons, our observations provide evidence for a diverse origin of descending 5-HT afferents to the different spinal laminae.