Production of R-(-)-mandelic acid from mandelonitrile by Alcaligenes faecalis ATCC 8750.

R-(-)-Mandelic acid was produced from racemic mandelonitrile by Alcaligenes faecalis ATCC 8750. Ammonium acetate or L-glutamic acid as the carbon source and n-butyronitrile as the inducer in the culture medium were effective for bacterial growth and the induction of R-(-)-mandelic acid-producing activity. The R-(-)-mandelic acid formed from mandelonitrile by resting cells was present in a 100% enantiomeric excess. A. faecalis ATCC 8750 has an R-enantioselective nitrilase for mandelonitrile and an amidase for mandelamide. As R-(-)-mandelic acid was produced from racemic mandelonitrile in a yield of 91%, whereas no S-mandelonitrile was left, the S-mandelonitrile remaining in the reaction is spontaneously racemized because of the chemical equilibrium and is used as the substrate. Consequently, almost all the mandelonitrile is consumed and converted to R-(-)-mandelic acid. R-(-)-Mandelic acid was also produced when benzaldehyde plus HCN was used as the substrate.     

Induction of CAT gene expression on a plasmid vector (L factor) by retinoic acid in mouse embryonal carcinoma (F9) cells

We reported previously that composite DNA constructed from a mammalian plasmid (L factor) and foreign gene can be reestablished as a plasmid in mouse embryonal carcinoma (F9) cells after transfection and the plasmid-bearing F9 cells undergo normal in vitro differentiation in response to retinoic acid, an inducer for F9 cell differentiation. We constructed F9 cells bearing plasmidal L factor DNA in which a reporter (chloramphenicol acetyltransferase; CAT) gene was placed under the control of a differentiation-responsive viral (Moloney murine leukemia virus or simian virus 40) enhancer-promoter. When such plasmid-bearing cells were treated with retinoic acid, the CAT gene was inducibly expressed. These results indicate that mammalian gene expression can be studied with the plasmidal expression vector which is structurally dissociated from complex chromosomes.     

Construction of an excretion vector and extracellular production of human growth hormone from Escherichia coli

A new excretion vector, pEAP8, was constructed to develop an excretion system for Escherichia coli. This plasmid, derived from pEAP37, carried the weakly activated kil gene of plasmid pMB9 [Kobayashi et al., J. Bacteriol. 166 (1986) 728-732] and the penicillinase promoter and signal region of an alkalophilic Bacillus sp. to excrete foreign gene products. A gene for human growth hormone (hGH) was joined to this signal sequence through the HindIII site. The recombinant plasmid p8hGH1 thus constructed, was introduced into E. coli. The hybrid protein which was produced in E. coli carrying p8hGH1 was processed during transport through the inner membrane, with the mature hGH being excreted into the medium through the outer membrane which was made permeable by the action of the kil gene. The N-terminal amino acid sequence and the biological activity of the extracellular hGH were consistent with those of the authentic hGH.     

Cytoplasmic localization of human repetitive DNA revealed by in situ hybridization

Previously, we showed that a human repetitive DNA sequence (Sau3A family) belonging to a satellite DNA is unstable and constantly excised from the chromosomes (R. Kiyama, H. Matsui, and M. Oishi, 1986, Proc. Natl. Acad. Sci. USA 83, 4665). The unusual property of the repetitive DNA, along with another repetitive DNA (Alu sequence), was further investigated by in situ hybridization in several different human cells including HeLa, bone marrow, and peripheral blood cells. We found that the excised repetitive DNA sequences are localized not only in nuclei, but also in cytoplasm. These results have confirmed the instability of these DNA sequences in the chromosomes and further suggest that the alpha satellite DNA and the Alu sequence which were excised from the chromosomes are released from nuclei to cytoplasm.     

Application of Overall Dynamic Body Acceleration as a Proxy for Estimating the Energy Expenditure of Grazing Farm Animals: Relationship with Heart Rate

Estimating the energy expenditure of farm animals at pasture is important for efficient animal management. In recent years, an alternative technique for estimating energy expenditure by measuring body acceleration has been widely performed in wildlife and human studies, but the availability of the technique in farm animals has not yet been examined. In the present study, we tested the potential use of an acceleration index, overall dynamic body acceleration (ODBA), as a new proxy for estimating the energy expenditure of grazing farm animals (cattle, goats and sheep) at pasture with the simultaneous evaluation of a conventional proxy, heart rate. Body accelerations in three axes and heart rate for cows (n = 8, two breeds), goats (n = 6) and sheep (n = 5) were recorded, and the effect of ODBA calculated from the body accelerations on heart rate was analyzed. In addition, the effects of the two other activity indices, the number of steps and vectorial dynamic body acceleration (VeDBA), on heart rate were also investigated. The results of the comparison among three activity indices indicated that ODBA was the best predictor for heart rate. Although the relationship between ODBA and heart rate was different between the groups of species and breeds and between individuals (P<0.01), the difference could be explained by different body weights; a common equation could be established by correcting the body weights (M: kg): heart rate (beats/min) = 147.263∙M ^-0.141 + 889.640∙M ^-0.179∙ODBA (g). Combining this equation with the previously reported energy expenditure per heartbeat, we estimated the energy expenditure of the tested animals, and the results indicated that ODBA is a good proxy for estimating the energy expenditure of grazing farm animals across species and breeds. The utility and simplicity of the procedure with acceleration loggers could make the accelerometry technique a worthwhile option in field research and commercial farm use.