Inhibition of Nitrogen Fixation in Soybean Plants Supplied with Nitrate I. Nitrite Accumulation and Formation of Nitrosylleghemoglobin in Nodules

The accumulation of nitrite in nodules was investigated to elucidatethe mechanism of inhibition of nitrogen fixation in nodulesof soybean (Glycine max. [L.] Merr.) plants supplied with nitrate.Acetylene-reducing activity (ARA) in nodules fell within 24h as a result of the supply of exogenous nitrate, accompaniedby an increase in the accumulation of nitrite in the cytosolbut not in the bacteroids of nodules. Nitrate reductase (NR)activity in the nodule cytosol remained high, irrespective ofthe supply of nitrate. Nitrosylleghemoglobin (LbNO) was detectedspectrophotometrically in the extract from nodules in whichnitrogen fixation was inhibited by nitrate. In experiments invitro, it was found that LbNO was easily formed from leghemoglobinin the presence of nitrite and dithionite. Thus, it is suggested that nitrogen fixation was inhibited primarilyby a decrease in the function of leghemoglobin, attributableto the formation of LbNO, which was caused by the accumulationof nitrite generated from nitrate by NR in the nodule cytosol. (Received August 22, 1989; Accepted January 24, 1990)     

Selection and reproductive success in males of the dragonfly,Orthetrum japonicum (Odonata: Libellulidae)

Reproductive success, copulation success, and mating success were measured for a population of male dragonflies,Orthetrum japonicum. Copulation success explained the greatest variation in reproductive success. The proportion of copulations followed by oviposition was positively correlated with the number of oviposited eggs per mating. Directional selection on four morphological characters was estimated. The effect of selection on correlated traits was comparable to that of direct selection. Directional selection varied between traits and between episodes in a single trait. The probability that the observed directional selection on the four morphological traits was expected under the condition of the selective neutrality of traits was not smaller than 5%.     

Molecular analysis redefines three human chromosome 14 deletions

We have used a panel of 13 DNA markers in the distal region of chromosome 14q to characterize deletions in three patients determined cytogenetically to have a ring or terminally deleted chromosome 14. We have characterized one patient with a ring chromosome 14 [r (14) (p13q32.33)] and two with terminal deletions [del (14) (pterq32.3:)]. The two patients with cytogenetically identical terminal deletions of chromosome 14 were found to differ markedly when characterized with molecular markers. In one patient, none of the markers tested were deleted, indicating that the apparent terminal deletion is actually due to either an undetected interstitial deletion or a cryptic translocation event. In the other patient, the deletion was consistent with the cytogenetic observations. The deleted chromosome was shown to be of paternal origin. The long-arm breakpoint of the ring chromosome was mapped to within a 350-kb region of the immunoglobulin heavy chain gene cluster (IGH). This breakpoint was used to localize markers D14S20 and D14S23, previously thought to lie distal to IGH, to a more proximal location. The ring chromosome represents the smallest region of distal monosomy 14q yet reported.     

NUCLEAR CHROMATIN PROTEINS FROM RABBIT CEREBRUM, CEREBELLUM AND LIVER: SYNTHESIS AND PHOSPHORYLATION

Abstract— In order to investigate synthesis and phosphorylation of the various fractions of nuclear proteins. [³H]leucine and [³²P] phosphate incorporation were studied with tissue slices in vitro. Cerebral cortex and cerebellum were used to delineate the similarity and dissimilarity within CNS, and liver was taken to compare the extraneural organ. There were significant differences in [³H]leucine incorporation into nuclear proteins among those tissue sources examined, while [³²P]phosphate incorporation showed very similar results among them. Although the acidic chromatin protein demonstrated high activity in each tissue source for both synthesis and phosphorylation, 0.14M-NaCl soluble protein showed the activity as high as or even higher than the acidic chromatin protein. Both [³H]leucine incorporation and [³²P]phosphate incorporation were relatively low in histone. When the acidic chromatin protein was further fractionated with SDS-acrylamide gel electrophoresis, significant difference was found between CNS tissue and liver for synthesis and phosphorylation. However, considerable difference was also observed even between cerebral cortex and cerebellum. The present investigation demonstrated complicity and diversity of nuclear chromatin proteins in different organs, not only for their protein constituents but also for their synthesis and phosphorylation.     

Porcine kidney -amino acid oxidase: the three-dimensional structure and its catalytic mechanism based on the enzyme–substrate complex model

The three-dimensional structure of porcine kidney -amino acid oxidase (DAO), an FAD-dependent oxidase, has been solved by X-ray crystallography. The overall structure is a dimer, subunits of which are correlated by a non-crystallographic two-fold axis. Each subunit comprises two domains, ‘αβ domain’ and ‘pseudo-barrel domain’. The coenzyme FAD is in an elongated conformation and is bound at the N-terminal βαβ dinucleotide binding motif. The active site is located in the boundary region between the two domains. The crystal structure of DAO in complex with a substrate analog, o-aminobenzoate, was also solved and is used for modeling the DAO- -leucine complex, i.e. Michaelis complex, by means of molecular mechanics simulation. The Michaelis-complex model provided structural information leading to two alternative hypothetical mechanisms for the reductive half-reaction of DAO. These two hypotheses characterize themselves by electron transfer from the lone-pair orbital of the substrate amino nitrogen to flavin C(4a) and by proton transfer from the substrate α-position to flavin N(5) which acts as a catalytic base.     

Degradation of sphingoid long-chain base 1-phosphates (LCB-1Ps): functional characterization and expression of AtDPL1 encoding LCB-1P lyase involved in the dehydration stress response in Arabidopsis

Sphingoid long-chain base (LCB) 1-phosphates are degradated by LCB 1-phosphate lyase to C(16) fatty aldehydes and phosphoethanolamine. Here, we confirmed that the At1g27980 gene product, AtDPL1, is a functional LCB-1-phosphate lyase. Expression of green fluorescent protein fusion products in suspension-cultured Arabidopsis cells showed that AtDPL1 is located to the endoplasmic reticulum. The rates of fresh weight decreases of dpl1-1 and dpl1-2 mutants were significantly slower than those of the wild-type plants. This ability to limit their transpiration reflected the leaf temperature of the mutant plants more than that of wild-type plants, suggesting that AtDPL1 plays a role in dehydration stress.     

Localization of myo-inositol-1-phosphate synthase to the endosperm in developing seeds of Arabidopsis

Expression and localization of myo-inositol-1-phosphate synthase (MIPS) in developing seeds of Arabidopsis thaliana was investigated. MIPS is an essential enzyme for production of inositol and inositol phosphates via its circularization of glucose-6-phosphate as the initial step. myo-inositol-6-phosphate (InsP(6) or phytic acid) is the predominant form of phosphorus found in seeds and accumulates as a consequence of MIPS action. Three MIPS genes have been identified in Arabidopsis, all of which were expressed not only in siliques but in both leaves and roots. Immunoelectron microscopy using a MIPS antibody showed that MIPS localizes to the cytosol primarily in the endosperm during seed development and not in the embryo. This is consistent with results obtained using fluorescent microscopy and western blot analysis that showed a similar pattern of localization. However, InsP(6), which is the final product of inositol phosphate metabolism, was present mainly in the embryo. This suggests that a complex interaction between the endosperm and embryo occurs during the synthesis and subsequent accumulation of InsP(6) in developing seeds of Arabidopsis.     

Phosphorylation in the C-terminal domain of Aquaporin-4 is required for Golgi transition in primary cultured astrocytes

Aquaporin-4 (AQP4) is expressed in the perivascular and subpial astrocytes end-feet in mammalian brain, and plays a critical component of an integrated water and potassium homeostasis. Here we examine whether AQP4 is phosphorylated in primary cultured mouse astrocytes. Astrocytes were metabolically labeled with [³²P]phosphoric acid, then AQP4 was immunoprecipitated with anti-AQP4 antibody. We observed that AQP4 was constitutively phosphorylated, which is reduced by treatment with protein kinase CK2 inhibitors. To elucidate the phosphorylation of AQP4 by CK2, myc-tagged wild-type or mutant AQP4 was transiently transfected in primary cultured astrocytes. Substitution of Ala residues for four putative CK2 phosphorylation sites in the C terminus abolished the phosphorylation of AQP4. Immunofluorescent microscopy revealed that the quadruple mutant was localized in the Golgi apparatus. These observations indicate that the C-terminal domain of AQP4 is constitutively phosphorylated at least in part by protein kinase CK2 and it is required for Golgi transition.     

Population structure ofSclerotium rolfsii in peanut fields

Sclerotium rolfsii isolates from peanut fields in Ibaraki were classified into mycelial compatibility groups (MCGs) based on the barrage zone formation. A total of 132 isolates collected from four fields within a 120 m radius in 1994 comprised four MCGs; MCG A (71 isolates), B (34 isolates), C (26 isolates) and D (one isolate). Fields 1 and 2 were occupied exclusively by MCG A. MCG A also predominated in field 3. In field 4, MCGs A, B and C were dominant. Population structure in 3 additional fields was determined in 1997. All 11 isolates from Field 5, which was 400 m distant from field 1, belonged to MCG C. A total of 42 isolates from fields 6 and 7, 2.5 km distant from other fields and 100 m distant from each other, were all MCG A. These results suggested that the population structure ofS. rolfsii was simple. RAPD fingerprintings showed that most isolates of the same MCG were clonal.     

Larval arm resorption proceeds concomitantly with programmed cell death during metamorphosis of the sea urchin Hemicentrotus pulcherrimus

Sea urchins are excellent models to elucidate metamorphic phenomena of echinoderms. However, little attention has been paid to the way that their organ resorption is accomplished by programmed cell death (PCD) and related cellular processes. We have used cytohistochemistry and transmission electron microscopy to study arm resorption in competent larvae of metamorphosing sea urchins, Hemicentrotus pulcherrimus, induced to metamorphose by L-glutamine treatment. The results show that: (1) columnar epithelial cells, which are constituents of the ciliary band, undergo PCD in an overlapping fashion with apoptosis and autophagic cell death; (2) squamous epithelial cells, which are distributed between the two arrays of the ciliary band, display a type of PCD distinct from that of columnar epithelial cells, i.e., a cytoplasmic type of non-lysosomal vacuolated cell death; (3) epithelial integrity is preserved even when PCD occurs in constituent cells of the epithelium; (4) secondary mesenchyme cells, probably blastocoelar cells, contribute to the elimination of dying epithelial cells; (5) nerve cells have a delayed initiation of PCD. Taken together, our data indicate that arm resorption in sea urchins proceeds concomitantly with various types of PCD followed by heterophagic elimination, but that epithelial organization is preserved during metamorphosis.This investigation was supported in part by a Keio University special grant-in-aid for innovative collaborative research projects.