Unusual Branching in the Seedlings of Lotus japonicus--Gibberellins Reveal the Nitrogen-sensitive Cell Divisions within the Pericycle on Roots

We studied the effects of several plant-growth regulators onthe induction of nodule-like structures on roots of Lotus japonicus,which has been proposed as a candidate for a leguminous plantfor molecular genetic analysis. Contrary to our expectations,the addition of gibberellin A₃ (GA₃) at concentrations of 10^-4M to 10^-4 M resulted in the formation of nodule-like structureson roots when seedlings were plated on nitrogen-free Fahraeusagar medium. GA₄ also induced such outgrowths but was less activethan GA₃. Application of an inhibitor of auxin transport, N-(1-naphthyl)-phthalamicacid (NPA) and of kinetin, which have been reported to inducepseudonodules in other legumes, had no effect on L. japonicus.Microscopic observations showed that GA₃-induced nodule-likestructures were caused by cell divisions within the pericycleon the roots. In addition, the outgrowths elicited by GA₃ couldbe completely suppressed by the addition of 15 mM potassiumnitrate or ammonium nitrate. These results show that the pericyclecells of the roots of L. japonicus are specifically sensitiveto gibberellins and that potential for cell division might bemodulated by nitrogen compounds. We also examined the effectsof ancymidol and uniconazole [S-3307; (E)-1-(4-chIorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol],two synthetic plant-growth retardants. Both compounds at 3 x10^-5 M significantly increased the number of stunted lateralroots. The unusual branching could not be counteracted by theexogenous addition of GA₃ but by 10^-6 M brassinolide. We discussthe physiological role of brassinolide in the initiation oflateral roots. (Received August 4, 1995; Accepted March 11, 1996)     

The Excessive Production of Indole-3-Acetic Acid and Its Significance in Studies of the Biosynthesis of This Regulator of Plant Growth and Development

Because of the importance of indole-3-acetic acid (IAA) in thegrowth and development of plants, extensive studies of the biosynthesisof IAA have been performed during the four decades since thediscovery of IAA as a plant hormone. The pathway for the biosynthesisof IAA in plants remains, however, to be unelucidated, eventhough studies within the past decade have revealed unexpectedaspects of such biosynthesis. By contrast, two pathways to IAAhave been characterized in bacteria at the molecular level:the indole-3-acetamide (IAM) pathway (L-tryptophan     

Effect of β-alanyl-L-histidinato zinc on differentiation of osteoblastic MC3T3-El cells: Increases in alkaline phosphatase activity and protein concentration

The effect of -alany-L-histidinato zinc (AHZ) on bone cell function was investigated in osteoblastic MC3T3-E1 cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus AHZ (10^–7–10^–5 M) or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5 M) produced a remarkable increase of alkaline phosphatase activity and protein concentration in osteoblastic cells. Thus increases were seen with the prolonged cultivation (12–21 days). With the culture of 1, 3 and 12 days, the effect of AHZ (10^–6 M) to increase alkaline phosphatase activity and protein concentration was more intensive than the effect of zinc sulfate, (10^–6 M). The AHZ effects were completely abolished by the presence of cycloheximide (10^–6 M), indicating that AHZ stimulates protein synthesis in the cells. The present study suggests that AHZ has a stimulatory effect on cell differentiation, and that this effect is partly involved on protein synthesis in osteoblastic cells.     

Distribution of Vacuolar H+-Pyrophosphatase and a Membrane Integral Protein in a Variety of Green Plants

Vacuolar membranes isolated from several species including fernand moss exhibited pyro-phosphate-dependent H⁺ transport activity.On immunoblot analysis, H⁺ -pyrophosphatase was detected inthe vacuolar membranes. A membrane integral protein of 23,000daltons was not found in the membranes of Chara, Conocephalum,or Kalanchoë. Thus, H⁺-pyrophosphatase may be a universalenzyme among green plants, but the 23-kDa protein is not a commonprotein of central vacuoles. (Received September 10, 1993; Accepted November 29, 1993)     

Stimulatory effect of β-alanyl-L-histidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells

The effect of -alanyl-L-histidinato zinc (AHZ) on bone metabolism was investigated in osteoblastic MC3T3-El cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5M) stimulated the proliferation of cells. AHZ (10^–6 and 10^–5M) increased deoxyribonucleic acid (DNA) content in the cells with 48hr-culture. This increase was completely blocked by the presence of cycloheximide (10^–6M) or hydroxyurea (10^–3M). Also, the presence of cycloheximide (10^–6M) completely inhibited the AHZ (10^–5M)-induced increase in the proliferation of cells. Meanwhile, parathyroid hormone (10^–7M), estrogen (10^–9M) and insulin (10^–M) significantly increased cellular DNA content. However, these hormonal effects clearly lowered in comparison with that of AHZ (10^–5M). Dibutyryl cyclic AMP (10^–4M) and zinc sulfate (10^–5M) did not cause a significant increase in cellular DNA content. The present results support the view that AHZ has a direct specific proliferative effect on osteoblastic cellsin vitro and that this effect is dependent on protein synthesis.     

Performance of a new family of modular,bed-supported,chromatography devices

Prepacked chromatography columns and cassette filtration units offer many advantages in bioprocessing. These include reduced labor costs and processing times, ease of storage, and enhanced process flexibility. Rectangular formats are particularly attractive as they can be easily stacked and multiplexed together for continuous processing. Cylindrical chromatography beds have dominated bioprocessing even though their bed support and pressure-flow performance vary with bed dimensions. This work presents the performance of novel, rhombohedral chromatography devices with internally supported beds. They are compatible with existing chromatography workstations and can be packed with any standard commercial resin. The devices offer pressure-flow characteristics independent of container-volume, simple multiplexing, and separation performance comparable to cylindrical columns. Their bi-planar, internal bed support allows mechanically less-rigid resins to be used at up to four times higher maximal linear velocities, and productivities approaching 200 g/L/h for affinity resins, compared to the 20 g/L/h typical of many column-based devices. Three 5 L devices should allow processing of up to 3 kg of monoclonal antibody per hour.     

Tissue-Specific Isoforms of Catalase Subunits in Castor Bean Seedlings

Catalases purified from endosperm glyoxysomes and non-specializedmicrobodies from hypocotyls of castor bean seedlings differedin their specific activity [90–164 and 0.89–4.9kunits (mg protein)^–1, respectively] and in their constituentsubunits [two subunits of 54 and 56 kDa for the endosperm enzymeand only one of 56 kDa for the hypocotyl enzyme]. Immunoblotanalysis also showed that particulate fractions from the endospermsand from etiolated and green cotyledons contained two catalasesubunits of 54 and 56 kDa, whereas such fractions from the hypocotylsand roots contained only the 56-kDa subunit. Leaf peroxisomesfrom green leaves had two catalase subunits of around 55 kDaeach. Results of translation in vitro indicated that the 54-and 56-kDa subunits were translated from distinct mRNAs andlevels of both mRNAs increased in the endosperms during germination,prior to increases in levels of catalase proteins. In the hypocotyls,the 56-kDa subunit seemed to be synthesized constitutively. ¹Present addresses: YO, Toyota Central Institute, 31-9 Musashizuka,Nagabuchi, Nagakute, Aichi 480-11, Japan     

Molecular phylogeny based on the κ-casein and cytochrome b sequences in the mammalian suborder Ruminantia

Nucleotide sequences for the -casein precursor proteins have been determined from the genomic DNAs or hair roots of the Ruminantia. The coding regions, exons 2, 3, and 4, were amplified separately via the three kinds of PCRs and then directly sequenced. The primers were designed from the sequence of bovine -casein gene; they were applicable for the amplification of the -casein genes from the 13 species in the Ruminantia except exon 2 of the lesser mouse deer. These results permitted an easy phylogenetic analysis based on the sequences of an autosomal gene. A phylogenetic tree was constructed from the mature K-casein sequences and compared with the tree of the cytochrome b genes which were sequenced from the same individuals. The Cervidae (sika deer, Cervus nippon) were separated from the branch of the Bovidae on the tree of -casein genes with a relatively high confidence level of the bootstrap analysis, but included in the branch of the Bovidae on the tree of cytochrome b genes. The -casein tree indicated a monophyly of the subfamily Caprinae, although the internal branches were uncertain in the Caprinae. The tree based on the nucleotide sequences of cytochrome b genes clearly showed the relationships of the closely related species in the genus Capricornis consisting of serow (C. smatorensis), Japanese serow (C. crispus), and Formosan serow (C. swinhoei). These results would be explained by the difference of resolving power between the -casein and the cytochrome b sequences. Correspondence to: K. Chikuni     

Expression of hepatic calcium-binding protein regucalcin mRNA is elevated by refeeding of fasted rats: Involvement of glucose,insulin and calcium as stimulating factors

The effect of refeeding on the expression of Ca²⁺-binding protein regucalcin mRNA in the liver of fasted rats was investigated. When rats were fasted overnight, the hepatic regucalcin mRNA level was reduced about 70% of that in feeding rats. Refeeding produced a remarkable elevation of hepatic regucalcin mRNA level (about 150–170% of fasted rats). Liver regucalcin concentration was appreciably increased by refeeding, although it was not altered by fasting. The oral administration of glucose (2 g/kg body weight) to fasted rats caused a significant increase in hepatic regucalcin mRNA level. Moreover, hepatic regucalcin mRNA level was clearly elevated by a single subcutaneous administration of insulin (10 and 100 U/kg) to fasted rats. The hormonal effect was not further enhanced by the simultaneous administration of calcium chloride (250 mg Ca/kg) to fasted rats, although calcium administration stimulated regucalcin mRNA expression in the liver. The present study suggests that the expression of hepatic regucalcin mRNA stimulated by refeeding is significantly involved in the action of insulin and/or calcium as stimulating factors.