c-Jun NH2-terminal kinase decreases ubiquitination and promotes stabilization of p21(WAF1/CIP1) in K562 cell

Proteasome-dependent degradation of regulatory proteins is a known mechanism of cell cycle control. p21(WAF1/CIP1) (p21), a negative regulator of the cell division cycle, exhibits proteasome-sensitive turnover and ubiquitination. In the present study, we analyzed the regulatory effects of JNK1 on p21 protein accumulation in p53 null K562 cells. We found that JNK1 (wild type, WT) mediated H(2)O(2)-induced p21 protein up-regulation. Over-expression of JNK1 (WT) could elevate endogenous p21 protein level but did not affect p21 mRNA level and also prolong the p21 half-life as well as inhibited the p21 ubiquitination. These findings indicated that JNK1 could regulate cellular p21 level via inhibiting ubiquitination of p21, which provided a new insight for analyzing the regulatory effect of JNK after stress.     

Production of biodiesel fuel from soybean oil catalyzed by fungus whole-cell biocatalysts in ionic liquids

The methanolysis of soybean oil to produce a fatty acid methyl ester (ME, i.e., biodiesel fuel) was catalyzed by lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst in the presence of ionic liquids. We used four types of whole-cell biocatalysts: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL), recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL), Candida antarctica lipase B (r-CALB), and mono- and diacylglycerol lipase from A. oryzae (r-mdlB). w-ROL gave the high yield of fatty acid methyl ester (ME) in ionic liquid [Emim][BF₄] or [Bmim][BF₄] biphasic systems following a 24 h reaction. While lipases are known to be severely deactivated by an excess amount of methanol (e.g. 1.5 Mequiv. of methanol against oil) in a conventional system, methanolysis successfully proceeded even with a methanol/oil ratio of 4 in the ionic liquid biphasic system, where the ionic liquids would work as a reservoir of methanol to suppress the enzyme deactivation. When only w-ROL was used as a biocatalyst for methanolysis, unreacted mono-glyceride remained due to the 1,3-positional specificity of R. oryzae lipase. High ME conversion was attained by the combined use of two types of whole-cell biocatalysts, w-ROL and r-mdlB. In a stability test, the activity of w-ROL was reduced to one-third of its original value after incubation in [Bmim][BF₄] for 72 h. The stability of w-ROL in [Bmim][BF₄] was greatly enhanced by cross-linking the biocatalyst with glutaraldehyde. The present study demonstrated that ionic liquids are promising candidates for use as the second solvent in biodiesel fuel production by whole-cell biocatalysts.     

A Simple Method for Stabilizing and Granulating Fungi

A simple method, 'Stabileze', is described for granulating fungi with water-absorbent starch, sucrose, corn oil and silica. The potential biological control fungi, Colletotrichum gloeosporioides and Fusarium oxysporum , were stabilized using this protocol. Bioassays were performed on C. gloeosporioides and showed retention of pathogenicity. Sucrose and oil were tested for their effects on the viability of F. oxysporum over time, and sucrose appears to be the most beneficial factor.     

Lysozyme-lysozyme self-interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilization

The purpose of the presented study is to understand the physicochemical properties of proteins in aqueous solutions in order to identify solution conditions with reduced attractive protein-protein interactions, to avoid the formation of protein aggregates and to increase protein solubility. This is assessed by measuring the osmotic second virial coefficient (B²²), a parameter of solution non-ideality, which is obtained using self-interaction chromatography. The model protein is lysozyme. The influence of various solution conditions on B²² was investigated: protonation degree, ionic strength, pharmaceutical relevant excipients and combinations thereof. Under acidic solution conditions B²² is positive, favoring protein repulsion. A similar trend is observed for the variation of the NaCl concentration, showing that with increasing the ionic strength protein attraction is more likely. B²² decreases and becomes negative. Thus, solution conditions are obtained favoring attractive protein-protein interactions. The B²² parameter also reflects, in general, the influence of the salts of the Hofmeister series with regard to their salting-in/salting-out effect. It is also shown that B²² correlates with protein solubility as well as physical protein stability.     

Genetic Design of Stable Metal-Binding Biomolecules,Oligomeric Metallothioneins

Metallothionein (MT) is a suitable model for investigating molecular interactions relating to the handling of metals in cells. However, the production of functional MT proteins in microorganisms has been limited because of the instability of MT—the thiol group of cysteine is easily oxidized and proteolysis occurs. To increase the binding ability and to stabilize MT, we designed genes for dimeric and tetrameric MT and the genes were successfully overexpressed in Escherichia coli to generate functional oligomeric MTs. A human MT-II (hMT-II) synthesized with prokaryotic codons, a linker encoding a glycine tripeptide, and Met-deficient hMT-II was ligated to create a dimeric MT, from which a tetrameric MT was then constructed. The increased molecular size of the constructs resulted in improved stability and productivity in E. coli. Cells of E. coli carrying the oligomeric MT genes showed resistance toward Zn and Cd toxicity. The oligomeric proteins formed inclusion bodies, which were dissolved with dithiothreitol, and the purified apo-MTs were reconstituted with Cd or Zn ions under reducing conditions. The dimeric and tetrameric MT proteins exhibited both Cd and Zn binding activities that were, respectively, two and four times higher than those of the hMT-II monomer protein. These stable oligomeric MTs have potential as a biomaterial for uses such as detoxification and bioremediation for heavy metals.     

Fission yeast Swi1-Swi3 complex facilitates DNA binding of Mrc1

Replication fork protection complex Swi1-Swi3 and replication checkpoint mediator Mrc1 are required for maintenance of replication fork integrity during the course of DNA replication in the fission yeast Schizosaccharomyces pombe. These proteins play crucial roles in stabilizing stalled forks and activating replication checkpoint signaling pathways. Although they are conserved replication fork components, precise biochemical roles of these proteins are not known. Here we purified Mrc1 and Swi1-Swi3 proteins and show that these proteins bind to DNA independently but synergistically in vitro. Mrc1 binds preferentially to arrested fork or D-loop-like structures, although the affinity is relatively low, whereas the Swi1-Swi3 complex binds to double-stranded DNA with higher affinity. In the presence of a low concentration of Swi1-Swi3, Mrc1 generates a novel ternary complex and binds to various types of DNA with higher affinity. Moreover, purified Mrc1 and Swi1-Swi3 physically interact with each other, and this interaction is lost by mutations in the known DNA binding domain of Mrc1 (K235E,K236E). The interaction is also lost in a mutant form of Swi1 (E662K) that is specifically defective in polar fork arrest at a site called RTS1 and causes sensitivity to genotoxic agents, although the DNA binding affinity of Swi1-Swi3 is not affected by this mutation. As expected, the synergistic effect of the Swi1-Swi3 on DNA binding of Mrc1 is also lost by these mutations affecting the interaction between Mrc1 and Swi1-Swi3. Our results reveal an aspect of molecular interactions that may play an important role in replication pausing and fork stabilization.     

Simple procedures for purification and stabilization of human serum paraoxonase-1

Human paraoxonases-1 is one of the most important detoxifying enzymes. In this study using simple chromatographic procedures human paraoxonases-1 was purified from human pooled plasma. The enzyme was purified using DEAE Sephadex an anion exchanger and G-200 a gel filtration chromatographic media. Results showed a single band of approximately 43 KD proteins in SDS–PAGE, corresponding to the human PON1. Using paraoxon as the substrate the activity was related to the concentration of calcium and sodium ions (K_m = 1.2 ± 0.2 mM). Phenyl acetate hydrolyzing activity was independent of sodium and calcium ions (K_m = 0.78 ± 0.08 mM). Keeping at 25 °C for 20 days 75% of the enzyme original activity was restored in 20% (v/v) glycerol. EDTA and zinc chloride both inhibited the enzyme activity. In conclusion the applied procedures can be used for large scale purification. It would greatly facilitate their structural and functional characterization and permit examination of their weak, yet potentially most biologically relevant activities, in the complete absence of other serum proteins.     

Stabilization of the hexameric glutamate dehydrogenase from Escherichia coli by cations and polyethyleneimine

The enzyme glutamate dehydrogenase (GDH) from Escherichia coli is a hexameric protein. The stability of this enzyme was increased in the presence of Li⁺ in concentrations ranging from 1 to 10 mM, 1 M of sodium phosphate, or 1 M ammonium sulfate. A very significant dependence of the enzyme stability on protein concentration was found, suggesting that subunit dissociation could be the first step of GDH inactivation. This effect of enzyme concentration on its stability was not significantly decreased by the presence of 10 mM Li⁺. Subunit crosslinking could not be performed using neither dextran nor glutaraldehyde because both reagents readily inactivated GDH. Thus, they were discarded as crosslinking reagents and GDH was incubated in the presence of polyethyleneimine (PEI) with the aim of physically crosslinking the enzyme subunits. This incubation does not have a significant effect on enzyme activity. However, after optimization, the PEI-GDH was found to almost maintain the full initial activity after 2 h under conditions where the untreated enzyme retained only 20% of the initial activity, and the effect of the enzyme concentration on enzyme stability almost disappeared. This stabilization was maintained in the pH range 5–9, but it was lost at high ionic strength. This PEI-GDH composite was also much more stable than the unmodified enzyme in stirred systems. The results suggested that a real adsorption of the PEI on the GDH surface was required to obtain this stabilizing effect. A positive effect of Li⁺ on enzyme stability was maintained after enzyme surface coating with PEI, suggesting that the effects of both stabilizing agents could not be exactly based on the same mechanism. Thus, the coating of GDH surface with PEI seems to be a good alternative to have a stabilized and soluble composite of the enzyme.     

Constitutive stabilization of ß-catenin in the dental mesenchyme leads to excessive dentin and cementum formation

Wnt/ß-catenin signaling plays an important role in morphogenesis and cellular differentiation during development. Essential roles of Wnt/ß-catenin signaling in tooth morphogenesis have been well known, but the involvement of Wnt/ß-catenin signaling in the dental hard tissue formation remains undefined. To understand roles of Wnt/ß-catenin signaling in dentin and cementum formation, we generated and analyzed the conditional ß-catenin stabilized mice in the dental mesenchyme. The OC-Cre;Catnb^lox(ex3)/+ mice exhibited malformed teeth characterized by aberrantly formed dentin and excessively deposited cementum. Large amount of dentin was rapidly formed with widened predentin and numerous globular calcifications in the crown. Whereas roots of molars were short and covered with the excessively formed cellular cementum. With age, the coronal pulp chamber and periodontal space were narrowed by the excessively formed dentin and cementum, respectively. To compare the changes of gene expression in the mutant mice, Col1a1 expression was increased but that of Dspp was decreased in the odontoblasts. However, both of Col1a1 and Bsp expression was increased in the cementoblasts. The gene expression changes were consistent with the localization of matrix proteins. Biglycan and PC-1 was increased but Phex was decreased in the odontoblasts and dentin matrix, respectively. TNAP was increased but Dmp1 and FGF23 was decreased in the cementoblasts and cementum matrix, respectively. Our results indicate that persistent stabilization of ß-catenin in the dental mesenchyme leads to premature differentiation of odontoblasts and differentiation of cementoblasts, and induces excessive dentin and cementum formation in vivo. These results suggest that temporospatial regulation of Wnt/ß-catenin signaling plays critical roles in the differentiation of odontoblasts and cementoblasts, and that inhibition of Wnt/ß-catenin signaling may be important for the formation of dentin and cementum during tooth development. Local modulation of Wnt/ß-catenin signaling has therapeutic potential to improve the regeneration of dentin and periodontium.     

NdhP Is an Exclusive Subunit of Large Complex of NADPH Dehydrogenase Essential to Stabilize the Complex in Synechocystis sp. Strain PCC 6803

Two major complexes of NADPH dehydrogenase (NDH-1) have been identified in cyanobacteria. A large complex (NDH-1L) contains NdhD1 and NdhF1, which are absent in a medium size complex (NDH-1M). They play important roles in respiration, cyclic electron transport around photosystem I, and CO₂ acquisition. Two mutants sensitive to high light for growth and impaired in NDH-1-mediated cyclic electron transfer were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in sml0013 encoding NdhP, a single transmembrane small subunit of the NDH-1 complex. During prolonged incubation of the wild type thylakoid membrane with n-dodecyl β-d-maltoside (DM), about half of the NDH-1L was disassembled to NDH-1M and the rest decomposed completely without forming NDH-1M. In the ndhP deletion mutant (ΔndhP), disassembling of NDH-1L to NDH-1M occurred even on ice, and decomposition to a small piece occurred at room temperature much faster than in the wild type. Deletion of the C-terminal tail of NdhP gave the same result. The C terminus of NdhP was tagged by YFP-His₆. Blue native gel electrophoresis of the DM-treated thylakoid membrane of this strain and Western analysis using the antibody against GFP revealed that NdhP-YFP-His₆ was exclusively confined to NDH-1L. During prolonged incubation of the thylakoid membrane of the tagged strain with DM at room temperature, NDH-1L was partially disassembled to NDH-1M and the 160-kDa band containing NdhP-YFP-His₆ and possibly NdhD1 and NdhF1. We therefore conclude that NdhP, especially its C-terminal tail, is essential to assemble NdhD1 and NdhF1 and stabilize the NDH-1L complex.