Ectopic expression of KNOTTED1-like homeobox protein induces expression of cytokinin biosynthesis genes in rice

Some phytohormones such as gibberellins (GAs) and cytokinins (CKs) are potential targets of the KNOTTED1-like homeobox (KNOX) protein. To enhance our understanding of KNOX protein function in plant development, we identified rice (Oryza sativa) genes for adenosine phosphate isopentenyltransferase (IPT), which catalyzes the rate-limiting step of CK biosynthesis. Molecular and biochemical studies revealed that there are eight IPT genes, OsIPT1 to OsIPT8, in the rice genome, including a pseudogene, OsIPT6. Overexpression of OsIPTs in transgenic rice inhibited root development and promoted axillary bud growth, indicating that OsIPTs are functional in vivo. Phenotypes of OsIPT overexpressers resembled those of KNOX-overproducing transgenic rice, although OsIPT overexpressers did not form roots or ectopic meristems, both of which are observed in KNOX overproducers. Expression of two OsIPT genes, OsIPT2 and OsIPT3, was up-regulated in response to the induction of KNOX protein function with similar kinetics to those of down-regulation of GA 20-oxidase genes, target genes of KNOX proteins in dicots. However, expression of these two OsIPT genes was not regulated in a feedback manner. These results suggest that OsIPT2 and OsIPT3 have unique roles in the developmental process, which is controlled by KNOX proteins, rather than in the maintenance of bioactive CK levels in rice. On the basis of these findings, we concluded that KNOX protein simultaneously decreases GA biosynthesis and increases de novo CK biosynthesis through the induction of OsIPT2 and OsIPT3 expression, and the resulting high-CK and low-GA condition is required for formation and maintenance of the meristem.     

Stabilization of anaphase midzone microtubules is regulated by Rho during cytokinesis in human fibrosarcoma cells

The dynamics of astral and midzone microtubules (MTs) must be separately regulated during cell division, but the mechanism of selective stabilization of midzone MTs is poorly understood. Here we show that, in HT1080 cells, activation of Rho is required to stabilize midzone MTs, and to maintain the midzone structures after anaphase onset or during cytokinesis. Ect2-depleted cells undergoing conventional cytokinesis (cytokinesis A) or contractile ring-independent cytokinesis (cytokinesis B) formed abnormally thin bundles of midzone MTs. C3-loaded mitotic cells with inactivated Rho showed similar but more severe disorganization of midzone MTs. In addition, the bundles of astral MTs were abnormally abundant along the cell periphery in both Ect2-depleted and C3-loaded mitotic cells. Mitotic kinesin-like protein 1 (MKLP1), a component of the spindle midzone required for bundling of MTs, was localized only in the narrower equatorial regions in Ect2-depleted cells, and disappeared from the midzone accompanying the progression of the mitotic phase in C3-loaded cells. Stabilization of MTs by taxol was sufficient to maintain the midzone structures in C3-loaded mitotic cells. These results, when combined with a preceding analysis on another, microtubule-associated Rho GEF (C.J. Bakal, D. Finan, J. LaRose, C.D. Wells, G. Gish, S. Kulkarni, P. DeSepulveda, A. Wilde, R. Rottapel, The Rho GTP exchange factor Lfc promotes spindle assembly in early mitosis, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 9529–9534), suggest that mammalian cells have two potential steps that require active Rho for the stabilization of midzone MTs during mitosis and cytokinesis.     

Different mechanisms of action of poly(ethylene glycol) and arginine on thermal inactivation of lysozyme and ribonuclease A

Proteins tend to undergo irreversible inactivation through several chemical modifications, which is a serious problem in various fields. We have recently found that arginine (Arg) suppresses heat‐induced deamidation and β‐elimination, resulting in the suppression of thermal inactivation of hen egg white lysozyme and bovine pancreas ribonuclease A. Here, we report that poly(ethylene glycol) (PEG) with molecular weight 1,000 acts as a thermoinactivation suppressor for both proteins, especially at higher protein concentrations, while Arg was not effective at higher protein concentrations. This difference suggests that PEG, but not Arg, effectively inhibited intermolecular disulfide exchange among thermally denatured proteins. Investigation of the effects of various polymers including PEG with different molecular weight, poly(vinylpyrolidone) (PVP), and poly(vinyl alchol) on thermoinactivation of proteins, circular dichroism, solution viscosity, and the solubility of reduced and S‐carboxy‐methylated lysozyme indicated that amphiphilic PEG and PVP inhibit intermolecular collision of thermally denatured proteins by preferential interaction with thermally denatured proteins, resulting in the inhibition of intermolecular disulfide exchange. These findings regarding the different mechanisms of the effects of amphiphilic polymers––PEG and PVP––and Arg would expand the capabilities of methods to improve the chemical stability of proteins in solution. Biotechnol. Bioeng. 2012; 109: 2543–2552. © 2012 Wiley Periodicals, Inc.     

The systems approach to the prespore-specific activation of sigma factor SigF in Bacillus subtilis

The prespore-specific activation of sigma factor SigF (σ^F) in Bacillus subtilis has been explained mainly by two factors, i.e., the transient genetic asymmetry and the volume difference between the mother cell and the prespore. Here, we systematically surveyed the effect of these two factors on sporulation using a quantitative modeling and simulation architecture named hybrid functional Petri net with extension (HFPNe). Considering the fact that the transient genetic asymmetry and the volume difference in sporulation of B. subtilis finally bring about the concentration difference in two proteins SpoIIAB (AB) and SpoIIAA (AA) between the mother cell and the prespore, we have surveyed the effect of AB and AA concentration on the prespore-specific activation of σ^F occurring in the early stage of sporulation. Our results show that the prespore-specific activation of σ^F could be governed by the ratio of AA to AB rather than their concentrations themselves. Our model also suggests that B. subtilis could maximize the ratio of AA to AB in the prespore and minimize it in the mother cell by employing both the transient genetic asymmetry and the volume difference simultaneously. This might give a good explanation to the co-occurrence of the transient asymmetry and the volume difference during sporulation of B. subtilis. In addition, we suggest for the first time that the σ^F activation in the prespore might be switched off by the decrease in the ratio of AA to AB after the transient genetic asymmetry is to an end by completion of DNA translocation into the prespore.     

Rapid Nucleotide Exchange Renders Asp-11 Mutant Actins Resistant to Depolymerizing Activity of Cofilin,Leading to Dominant Toxicity in Vivo

Conserved Asp-11 of actin is a part of the nucleotide binding pocket, and its mutation to Gln is dominant lethal in yeast, whereas the mutation to Asn in human α-actin dominantly causes congenital myopathy. To elucidate the molecular mechanism of those dominant negative effects, we prepared Dictyostelium versions of D11N and D11Q mutant actins and characterized them in vitro. D11N and D11Q actins underwent salt-dependent reversible polymerization, although the resultant polymerization products contained small anomalous structures in addition to filaments of normal appearance. Both monomeric and polymeric D11Q actin released bound nucleotides more rapidly than the wild type, and intriguingly, both monomeric and polymeric D11Q actins hardly bound cofilin. The deficiency in cofilin binding can be explained by rapid exchange of bound nucleotide with ATP in solution, because cofilin does not bind ATP-bound actin. Copolymers of D11Q and wild type actins bound cofilin, but cofilin-induced depolymerization of the copolymers was slower than that of wild type filaments, which may presumably be the primary reason why this mutant actin is dominantly toxic in vivo. Purified D11N actin was unstable, which made its quantitative biochemical characterization difficult. However, monomeric D11N actin released nucleotides even faster than D11Q, and we speculate that D11N actin also exerts its toxic effects in vivo through a defective interaction with cofilin. We have recently found that two other dominant negative actin mutants are also defective in cofilin binding, and we propose that the defective cofilin binder is a major class of dominant negative actin mutants.     

Kinematic study of root elongation in Arabidopsis thaliana with a novel image-analysis program

The measurement of the spatial profile of root elongation needs to determine matching points between time-lapse images and calculate their displacement. These data have been obtained by laborious manual methods in the past. Some computer-based programs have been developed to improve the measurement, but they require many time-series digital images or sprinkling graphite particles on the root prior to image capture. Here, we have developed GrowthTracer, a new image-analysis program for the kinematic study of root elongation. GrowthTracer employs a multiresolution image matching method with a nonlinear filter called the critical point filter (CPF), which extracts critical points from images at each resolution and can determine precise matching points by analysis of only two intact images, without pre-marking by graphite particles. This program calculates the displacement of each matching point and determines the displacement velocity profile along the medial axis of the root. In addition, a manual input of distinct matching points increases the matching accuracy. We show a successful application of this novel program for the kinematic analysis of root growth in Arabidopsis thaliana.     

Screening of 3,4-Disubstituted Phenyl-L-alanine-producible Microorganisms

Microbial transaminase which catalyzes the reaction between 3,4-disubstituted phenyl pyruvate and certain amino acids to produce 3,4-disubstituted phenyl-L-alanine was investigated. Wide distribution of this enzyme among the various kinds of microorganisms was confirmed.

3,4-Dihydroxyphenyl-L-alanine (L-Dopa) or 3,4-dimethoxyphenyl-L-alanine was isolated from corresponding keto acids using three strains of selected microorganisms.     

Purification and Properties of an Endo β-1,6-Glucanase from Rhizopus chinensis R-69

An endo β-1,6-glucanase (β-1,6-glucan glucanohydrolase, E, C. 3. 2. 1.) has been purified from the culture filtrate of a strain resembling Rhizopus chinensis in homogeneous form. The procedures involved ammonium sulfate fractionation followed by column chromatography of DEAE-cellulose, CM-Sephadex C–50 and BioGel P–60.

Various physicochemical and chemical characteristics of the enzyme have been made clear, including complete amino acid composition. Optimum pH, optimum temperature, apparent activation energy for activity, Km and V_max are 5.5~6.0, 60°C, 4.39 Cal per mole, 9.39×10^?3m glucose equivalents (0.169%) and 43.13 International Units, respectively. The enzyme required no metal ions for its activity, and it hydrolyzed β-1,6-glucan larger than gentiotetraose, forming gentiobiose and gentiotriose as main products.     

Studies on the Reaction Conditions of DOPA Production with Alcaligenes faecalis.

The effects of several factors on the enzymatic production of 3,4-dihydroxyphenyl-l-alanine (DOPA) and 3,4-dimethoxyphenyl-l-alanine (DMPA) by transamination reaction were investigated using wet cells of Alcaligenes faecalis IAM 1015. In addition, some experiments for the cultural conditions for transaminase production were performed. DOPA and DMPA were obtained in 80 and 90% yields, respectively, using the mixture of l-glutamate and l-aspartate as amino donors. Accumulation of DMPA in the culture under the growing state of the bacteria was also confirmed.