Solubilization of nitrate reductase from the blue-green alga Anabaena cylindrica

Nitrate reductase was solubilized and purified from Anabaenacylindrica by Triton X-100 treatment of particulate preparationsfollowed by adsorption on calcium phosphate gel. Reduced methylviologen, FAD or FMN, but not ferredoxin, served as an effectiveelectron donor for the nitrate reduction by solubilized nitratereductase. ¹This work was supported by a grant (4061) from the Ministryof Education (Received June 25, 1970; )     

Homoeotic Gene ETc Controls the Cell Number of Embryonic Ectodermal Layers of Bombyx mori

The formation of segments of Bombyx larvae involves differentiation of legged and legless segments during embryogenesis. Observations by light microscopy of serial sections of developing embryos of Bombyx showed that the cell number of the ectodermal layers increased more rapidly in segments where legs were being extruded than in those where no appendages were formed. In the embryos of a homoeotic mutant for the E -pseudoallelic locus (about 0.0–VI), E^Tc/E^Tc , in which all the abdominal segments were legless, the cell number of the ectodermal layers did not increase as in normal embryos. These findings suggest that the E^Tc gene controls the cell number of the ectodermal layers in relation to the differentiation of abdominal segments.     

Isolation of an Insulin-Responsive Preadipose Cell Line and a Mammary Tumor Virus-Producing, Dome-Forming Epithelial Cell Line from a Mouse Mammary Tumor

Two functional tissue culture cell lines, MTD and MTF cell lines, have been isolated from a mouse mammary tumor. MTD cells are epithelial and retain the ability to transport fluid leading to the formation of three-dimensional fluid-filled multicellular structures called "domes" or "hemicysts". Another property of MTD cells is the production of murine mammary tumor virus (MTV). Release of MTV into the culture medium was verified by immunological, electrophoretic and enzymatic analyses. Addition of dexamethasone in the culture medium enhanced both the formation of domes and the production of MTV. Thus, MTD cells retain the morphological and functional properties of the original mammary tumor cells.
MTF cells show the fibroblastic morphology in subconfluent cultures. After reaching confluence, however, these cells gradually accumulated triglycerides in the cytoplasm and eventually assumed the morphology of fat cells. This adipose conversion was greatly enhanced by the presence of insulin in the culture medium. The morphological resemblance of adipose-converted MTF cells to the mammary fat cells suggests that the MTF cell line was derived from the mammary fat pad stroma. These functional cell lines will be useful to study cell differentiation as well as cell-to-cell interactions in the mammary gland.     

HYDROGENASE ACTIVITY OF CELL-FREE PREPARATION OBTAINED FROM THE BLUE-GREEN ALGA, ANABAENA CYLINDRICA

A cell-free preparation of hydrogenase was obtained from acetone-driedcells of Anabaena cylindrica. This preparation was capable ofcatalyzing the reduction of various redox dyes by molecularhydrogen and evolution of hydrogen from reduced methyl viologen.PMS, methylene blue, DPIP and toluidine blue were effective,in this order, as hydrogen acceptors. Ferricyanide, p-quinone,phenosafranine and neutral red were not reduced by the presentpreparation. PPNR obtained from this alga was effective on neitherreduction of NAD and NADP by hydrogen nor stimulation of hydrogenuptake with methylene blue and PMS. Coupled with hydrogenasereaction, there occurred reduction of nitrate to ammonia whenmethylene blue was added to the reaction mixture. ¹Present address: Department of Zoology, University of Texas,Austin, Texas, U.S.A.     

Purification and properties of nitrite reductase from the blue-green alga Anabaena cylindrical

Nitrite reductase was purified about 40-fold from the blue-greenalga Anabaena cylindrica by acetone precipitation and chromatographyon DEAE-cellulose columns. The nitrite reductase had its pHoptima at about 7.6 with Tris-HCl and at about 7.4 with phosphatewhen reduced methyl viologen was used as an electron donor.The Km's for nitrite, methyl viologen and ferredoxin were 510^–55,210^–4 and 510^–6M, respectively. A stoichiometryof one molecule of ammonia formation per one molecule of nitritedisappearance was confirmed. Ferredoxin which had been reducedeither chemically with dithionite or enzymatically with NADPHin the presence of diaphorase was active as an electron donor.Dithionite-reduced FAD and FMN were inactive. NADPH could notgive electrons directly to nitrite reductase. Hydroxylaminereductase was segregated from nitrite reductase by DEAE-cellulosecolumn chromatography. Purified nitrite reductase showed noactivity for sulfite reduction. A molecular weight of 68,000was estimated for nitrite reductase using a calibrated SephadexG-200 column. ¹This work was supported by grants 4090 and 955008 from theMinistry of Education. ²This work was supported by grants 4090 and 955008 from theMinistry of Education. 2 Present address: Department of Botany,Faculty of Science, University of Tokyo, Tokyo.     

REDUCTION OF NITRATE AND NITRITE BY SUBCELLULAR PREPARATIONS OF ANABAENA CYLINDRICA II. REDUCTION OF NITRATE TO NITRITE

Reduction of nitrate to nitrite by particulate preparationsof Anabaena cylindrica was investigated. Preparations whichshowed high activity of nitrate reductase were obtained by sonication(preparation A) or acetone treatment (preparation B). The preparationA also showed a high activity of DPIP-ascorbate photooxidation.The nitrate reductase system accepted electrons from eitherreduced ferredoxin (preparation A & B) or NADH (preparationB), but not directly from NADPH. Ferredoxin was active whenreduced either by action of photochemical system I or by NADPHand NADP-reductase, but dithionite-reduced ferredoxin was completelyinactive. Ferredoxin could be replaced with methyl viologen,benzyl viologen and diquat. Reduced FMN and FAD could serveas electron donors, but the affinity of the reductase towardthese flavin compounds was very low. ¹ This work was supported by grants from the Ministry of Education(4093 and 95612) and from the National Institutes of Health,U.S. (GM-11300).     

Albinism Due to Transposable Element Insertion in Fish

The i locus of the medaka fish, Oryzias latipes, is responsible for tyrosinase expression, and several mutant alleles have been identified. The genotype i¹/i¹ exhibits a complete albino phenotype, having pale orange-red skin and red eyes. This mutant lacks in vivo tyrosinase activity. The genotype i⁴/i⁴, on the other hand, shows a quasi-albino phenotype with skin as bright as that of i¹/i¹ but with red-wine-colored eyes. At the light microscope level, reduced pigmentation is observed both in the skin and eyes of this mutant. The tyrosinase genes for the i¹ and the i⁴ alleles were cloned and sequenced, and compared with that of the wild-type tyrosinase gene. The i¹ allele was found to contain a 1.9-kb transposable element in the 1st exon, and the i⁴ allele was found to contain a 4.7-kb transposable element in the 5th exon. Both i¹ and i⁴ are alleles that were found in a commercial breeding population. The insertion of a transposable element thus appears to constitute a natural cause of mutations that cause albinism in this organism.     

Potential agronomic options for energy-efficient sugar beet-based bioethanol production in northern Japan

Sugar beet (Beta vulgaris L. subsp. vulgaris) is deemed to be one of the most promising bioethanol feedstock crops in northern Japan. To establish viable sugar beet‐based bioethanol production systems, energy‐efficient protocols in sugar beet cultivation are being intensively sought. On this basis, the effects of alternative agronomic practices for sugar beet production on total energy inputs (from fuels and agricultural materials during cultivation and transportation) and ethanol yields (estimated from sugar yields) were assessed in terms of (i) direct drilling, (ii) reduced tillage (no moldboard plowing), (iii) no‐fungicide application, (iv) using a high‐yielding beet genotype, (v) delayed harvesting and (vi) root+crown harvesting. Compared with the conventional sugar beet production system used in the Tokachi region of Hokkaido, northern Japan, which makes use of transplants, direct drilling and no‐fungicide application contributed to reduced energy inputs from raising seedlings and fungicides, respectively, but sugar (or ethanol) yields were also reduced by these practices, to a greater equivalent extent than the reductions in energy inputs. Consequently, direct drilling (6.84 MJ L^?1) and no‐fungicide application (7.78 MJ L^?1) worsened the energy efficiency (total energy inputs to produce 1 L of ethanol), compared with conventional sugar beet production practices (5.82 MJ L^?1). Sugar yields under conventional plow‐based tillage and reduced tillage practices were similar, but total energy inputs were reduced as a result of reduced fuel consumption from not plowing. Hence, reduced tillage showed improved energy efficiency (5.36 MJ L^?1). The energy efficiency was also improved by using a high‐yielding genotype (5.23 MJ L^?1) and root+crown harvesting (5.21 MJ L^?1). For these practices, no major changes in total energy inputs were noted, but sugar yields were consistently increased. Neither total energy inputs nor ethanol yields were affected by extending the vegetative growing period by delaying harvesting.