Cloning and sequencing of a ligninase gene from a lignindegrading basidiomycete,Phanerochaete chrysosporium

Summary A ligninase gene has been cloned from a Phanerochaete chrysosporium genomic DNA library. Nucleotide sequencing of the gene has revealed that the ligninase structural gene contains 1116 bp of the protein-encoding sequence, of which 84 bp encode the signal peptide. The protein-encoding sequence is interrupted by eight introns which conform to the universal G-T/A-G splicing rule observed for the 3 and 5 intron boundaries. The putative eukaryotic regulatory sequences, i.e. CAAT and TATA box-like sequences, are present in the 5 flanking region.     

Levels of IAA, Cytokinins, ABA and Ethylene in Rice Plants as Affected by a Gibberellin Biosynthesis Inhibitor, Uniconazole-P.

Effects of uniconazole-P, a triazole-type growth retardant,on endogenous levels of IAA, cytokinins, ABA and ethylene inrice seedlings were investigated. Endogenous levels of IAA andABA were similar between control and uniconazole-P-treated riceshoots. Evolution of ethylene was promoted slightly, being 1.8times greater under 0.3 ppm uniconazole-P treatment than thatof control. The most obvious effect was the increase of trans-Zand trans-RZ in shoots. Shoots treated with uniconazole-P (10mg/m² nursery box) contained 3.4 times and 3 times more trans-Zand trans-RZ than control, respectively. No significant differencesof cytokinin levels were recognized in roots except for cis-RZ.The increase of ethylene and active forms of cytokinins, andthe decrease of gibberellin in the shoots may be the basis forphysiological phenomena caused by uniconazole-P, namely thepromotion of flowering in woody plants and the enhancement offemaleness in cucumber. (Received September 9, 1987; Accepted October 20, 1987)     

A Comparative Study of the Structures of {alpha}-Glucans from Suspension-Cultured Nonglutinous and Glutinous Rice Cells

-Glucans (average mol wt, 1.3 ? 10⁴) extracted with perchloricacid from 8-day-old suspension-cultured nonglutinous (var. Sasanishiki)and glutinous rice (var. Miyakogane) cells were compared. Theresults of hydrolysis by alpha;-, ß- and iso-amylasesand methylation analysis of the -glucans suggested that theirbasic structures are almost the same. These -glucans are highly-branchedpolysaccharides with an average chain length of about 9–10,with exterior and interior chain lengths of about 6–7and 2–3, respectively. ¹Current address: Laboratory of Food Science, Faculty of Education,Hirosaki University, Hirosaki, Aomori 036, Japan. (Received April 27, 1987; Accepted March 2, 1988)     

Monoclonal Antibodies Specific for NADH-Ferricyanide Reductase Domain of Spinach Leaf Nitrate Reductase

Two monoclonal antibodies, 17(3)9 and 36(79)4, were preparedagainst nitrate reductase from Spinacia oleracea L. leaves.An enzyme-linked immunosorbent assay showed that 17(3)9, butnot 36(79)4, reacted more strongly to heat-denatured than nativeantigen. These antibodies inhibited NADH-nitrate reductase aswell as its various partial activities including reduced methylvilogen-nitrate reductase, reduced flavin mononucleotide-nitratereductase and NADH-cytochrome c reductase activities, but notNADH-ferricyanide reductase activity. Immunoblotting after electrophoreticseparation of nitrate reductase fragments obtained by Staphyrococcusaureus V8 protease digestion of native enzyme revealed thatthe two monoclonal antibodies bind to different epitopes locatedon the 28 kDa of the NADH-ferricyanide reductase domain. (Received October 2, 1987; Accepted June 9, 1988)     

Morphological differentiation of endothelial cells co-cultured with astrocytes on type-I or type-IV collagen

Summary In this study bovine aortic endothelial cells were co-cultured with astrocytes from fetal Wistar Kyoto rats. Endothelial cells growing on type-I collagen, development. Although some cells appeared to be mature, horseradish peroxidase penetrated within 1 min of incubation through the intercellular junctions of these endothelial elements maintained on type-I collagen. In contrast, endothelial cells on type-IV collagen, co-cultured with astrocytes, were well developed; their intercellular junctions were well established, and plasmalemmal vesicles reduced in number. As a result, horseradish peroxidase was unable to penetrate through the endothelial cells grown on type-IV collagen and co-cultured with astrocytes because of the reduced extent of the junctional and vesicular transport. These findings reveal that (1) type-IV collagen is essential for the differentiation of endothelial cells, (2) endothelial cell-astrocyte interactions occur during co-culture, and (3) endothelial permeability depends on astrocyte-produced factors, in addition to type-IV collagen.     

Effector molecules from antitumor macrophages induced with OK-432 and cyclophosphamide

Summary The present study was designed to determine whether antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent on their ability to produce a soluble factor, that is,l-arginine-dependent nitric oxide as measured by nitrite concentration. Nitrite production by peritoneal macrophages from NIH Swiss mice pretreated with OK-432 (125 KE/kg) i.p. twice at 1-week intervals and with cyclophosphamide (200 mg/kg) i.p. 2 days before the second OK-432 treatment, increased with time for 24 h, and proportionally depended on macrophage numbers. Nitrite production was inhibited by actinomycin D and puromycin but not by mitomycin C.N ^G-Monomethyl-l-arginine, a specific competitive inhibitor ofl-arginine-dependent nitric oxide synthesis, also inhibited production. There was a close correlation between nitrite production and antitumor activity in macrophages from mice pretreated with either OK-432 and cyclophosphamide, OK-432, or thioglycolate broth. OK-432 increased both nitrite production and antitumor activities when added to the macrophage from mice pretreated with OK-432 but not with thioglycolate broth. Both activities of macrophages from mice pretreated with OK-432 and cyclophosphamide were enhanced with increasing concentrations ofl-arginine (0.125–1 mM) in the culture medium.d-Arginine, however, did not substitute forl-arginine. Neither activity was affected by contact between the macrophage and the EL4 cell. The macrophage showed antitumor activity through a membrane filter though the activity was greatly reduced. This antitumor activity of macrophages through a membrane was also inhibited byN ^G-Monomethyl-l-arginine, and increased by OK-432. However, conditioned media, obtained by culturing macrophages induced with OK-432 and cyclophosphamide, inhibited growth of EL4 cells. This activity was carried out by dialysable and non-dialysable factors. One of the dialysable factors was nitrite, an oxidized product of nitric oxide. The antitumor activity of non-dialysable factors was heat-stable and production of factors was increased byN ^G-Monomethyl-l-arginine and OK-432. Also, non-dialysable factors increased both antitumor and nitrite production activities of OK-432-elicited macrophages, when incubated with factors. Such activity of factors was also heat-stable. The production of factors increased with incubation time of macrophages, and was not inhibited byN ^G-Monomethyl-l-arginine. These results indicate that in vitro antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent onl-arginine-dependent nitric oxide, and that macrophageassociated soluble factors other than nitric oxide were also needed to inhibit fully tumor growth in vitro.     

Analysis of intergenic spacer regions in the nuclear rDNA of Pharbitis nil.

The nucleotide sequence of a 4.2-kb EcoRI fragment from the intergenic region between the genes for 25S and 18S ribosomal RNA of Pharbitis nil Choisy was determined. The region contained a unique repetitive family of DNA sequences, called the RsaI family, composed of 32-bp units. The 32-bp unit was tandemly repeated in the intergenic region, and four subfamilies of repeating units were clustered as discrete blocks. The RsaI family of repeats was shown to be specific to the genus Pharbitis by Southern blot hybridization.     

Canopy photosynthetic production in a Japanese larch stand. I. Seasonal and vertical changes of leaf characteristics along the light gradient in a canopy

In an 18 year old Japanese larch stand, leaf characteristics such as area, weight, gross photosynthetic rate and respiration rate were studied in order to obtain basic information on estimating canopy photosynthesis and respiration. The leaf growth courses in area and weight from bud opening were approximated by simple logistic curves. The growth coefficient for the area growth curve was 0.155–0.175 day⁻¹, while that for the weight growth was 0.112–0.117 day⁻¹. The larger growth coefficient in area growth caused the seasonal change in specific leaf area (SLA) that increased after bud opening to its peak early in May at almost 300 cm² g⁻¹ and then decreased until it leveled off at about 140 cm²g⁻¹. The change inSLA indicates the possibility that leaf area growth precedes leaf thickness growth. The relationship between the coefficientsa andb of the gross photosynthetic rate (p)-light flux density (1) curve (p=bI/(1+aI)) and the mean relative light flux density (I′/I ₀) at each canopy height were approximated by hyperbolic formulae:a=A/(I′/I ₀)+B andb=C/(I′/I ₀)+D. Leaf respiration rate was also increased with increasingI′/I ₀. Seasonal change of gross photosynthetic rate and leaf respiration rate were related to mean air temperature through linear regression on semilogarithmic co-ordinates.     

Changes of fluorescence lifetime and rotational correlation time of bovine serum albumin labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride in guanidine and thermal denaturations

The nanosecond fluorescence depolarization method was applied to measure the fluorescence lifetime () and the rotational correlation time () of bovine serum albumin (BSA) labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl-Cl). Changes of and of dansyl BSA in the guanidine denaturation and in the thermal denaturation were examined. In parallel, the secondary structural change of dansyl BSA was followed by circular dichroism measurements. The magnitude of was almost unchanged between 1 and 2 M guanidine, where the secondary structure of the protein was predominantly disrupted; whereas that of began to increase before the disruption of secondary structure in the guanidine denaturation. In the thermal denaturation, in contrast, changes of both and occurred in a temperature range where the secondary structure was predominantly disrupted. The volume of equivalent sphere (V _e ) and the axial ratio () for the BSA were 3.6–3.8×10^–19 cm³ and 3.6 at 2M guanidine as against 2.1×10^–19 cm³ and 2.2 in the absence of guanidine (25°C), respectively. The magnitudes ofV _e and were 4.9×10^–19 cm³ and 4.5 at 65°C, respectively. Although the secondary structural change of dansyl BSA was irreversible in the thermal denaturation,V _e and were reversible.