Engineering of novel tamavidin 2 muteins with lowered isoelectric points and lowered non-specific binding properties

The avidin-biotin interaction is widely employed as a universal tool in numerous biotechnological applications. In avidin-biotin technology, non-specific binding to biological macromolecules is a hindrance. The major origin of this non-specific binding is the electrical charge of the surface of biotin-binding proteins. Tamavidin 2, a fungal avidin-like protein that binds biotin with an extremely high affinity, can be produced as a soluble recombinant protein in Escherichia coli. The isoelectric point of tamavidin 2 is 7.4-7.5, lower than avidin (10.0), and slightly higher than that of streptavidin (6.0-7.5). Here, we genetically engineered charge mutants of tamavidin 2 to reduce non-specific binding. By substituting an acidic residue (glutamic acid) for basic residues (arginine and lysine), we constructed three mutant proteins (muteins) and confirmed their high-level production in soluble form in E. coli, as well as that of tamavidin 2.?We then tested these proteins for non-specific binding to salmon sperm DNA, glycoproteins (integrin and fibronectin), and IgG from human sera. The muteins showed lower non-specific binding than tamavidin 2 to these macromolecules. In particular, one mutein, tamavidin-R104EK141E, which had the lowest isoelectric point (5.8-6.2) among avidin, streptavidin and tamavidin 2, displayed the lowest non-specific binding. The affinity of this mutein to biotin was high, comparable with that of tamavidin 2. These findings indicate that tamavidin-R104EK141E has the potential to serve as a robust tool in the numerous applications of biotin-binding proteins.     

Critical bubble radius for expansion in extrusion cooking

Strong wheat flour, weak wheat flour and rice flour were expanded by sudden reduction in pressure at the discharge port of an extruder. Their micropore-volume distributions were measured by nitrogen adsorption isotherms. The results were compared with the theoretical critical radius for expansion calculated from the balance of forces acting inside and outside a bubble. The micropore volume near the theoretical critical radius for expansion of an extrudate decreased remarkably after extrusion cooking, indicating that the theoretical critical radius for expansion almost coincided with the experimental one.     

Ion beam modification of ePTFE for improving the blood compatibility

Expanded polytetrafluoroethylene (ePTFE), which is a durable biomaterial because of its excellent biological inertness, is now widely used for prostheses in clinical medicine. However, conversely, the inert nature of ePTFE results in poor adaptability to the surrounding tissue due to lack of a cell-adhesive property. In this study, the surface of ePTFE was modified with ion beam irradiation to improve the blood compatibility. The surface modification of ePTFE sheets by He⁺, Ne⁺, Ar⁺ and Kr⁺ ion beams was performed at an energy of 150 keV with fluences of 1 × 10¹⁴, 5 × 10¹⁴ and 1 × 10¹⁵ ions/cm². To investigate anti-thrombogenicity, Ca²⁺-replenished platelet-rich plasma (PRP) was placed in contact with the surfaces for 10 min. Compared to the non-modified ePTFE surface, platelet response was inhibited on the surfaces modified with He⁺, Ne⁺ and Ar⁺: 5 × 10¹⁴ and 1 × 10¹⁵ ions/cm², and Kr⁺: 5 × 10¹⁴ ions/cm²; however, platelet response was promoted on the surfaces modified with He⁺, Ne⁺ and Ar⁺: 1 × 10¹⁴ ions/cm², and Kr⁺: 1 × 10¹⁴ and 1 × 10¹⁵ ions/cm². The significant morphological changes in ePTFE surface associated with ion beam modification are thought to be one of the reasons for the inhibition of platelet response. Endothelial cells were cultured on the surfaces for 3 days to evaluate the cellular response. Endothelial cell growth was significantly promoted on all of the surfaces of ion beam-modified ePTFE, although the non-modified ePTFE surface dramatically inhibited this growth. It is concluded that ion beam modification of ePTFE surface can improve the blood compatibility through not only the promotion of endothelial cell growth but also the inhibition of platelet response.     

Effect of hinokitiol impregnated sheets on shelf life and quality of “KEK‐1” tomatoes during storage

Hinokitiol (β‐thujaplicin, volatile oil extracted from the wood of Hiba arborvitae [Thujopsis dolabrata var. hondae], cypress family) is a natural preservative, antimicrobial, and chelating agent, used to prevent decay and extend the shelf life of fruits and vegetables. In the present study, we used nonwoven rayon sheets impregnated with hinokitiol to conduct packaging studies. Mature green tomatoes var. “KEK‐1” were packaged under three packaging conditions, ie, modified atmosphere packaging (MAP), MAP + hinokitiol (MH), and perforated film package (as control), and their quality attributes were compared during storage. The packaging materials used were low‐density polyethylene (LDPE) film (40 μm) and fresh sheets of nonwoven rayon impregnated with hinokitiol. O₂ in the MAP packaged tomatoes were retained throughout the storage period (3% to 5% O₂). Results showed that MH had slightly improved quality attributes throughout the storage period when compared with MAP alone. Expression levels of LeACS, LeADH, and LeTBG4 genes were higher in 5 and 9 days of control when compared with MAP and MH. “KEK‐1” tomatoes had longer shelf life under MAP and MH packaging and maintained the quality at 15°C. Hence, the effect of the MAP with bioactive packaging treatments could be effective in the future application for the extension of shelf life and quality of fruits and vegetables.     

Mutations in nucleolar proteins lead to nucleolar accumulation of polyA+ RNA in Saccharomyces cerevisiae

Synthesis of mRNA and rRNA occur in the chromatin-rich nucleoplasm and the nucleolus, respectively. Nevertheless, we here report that a Saccharomyces cerevisiae gene, MTR3, previously implicated in mRNA transport, codes for a novel essential 28-kDa nucleolar protein. Moreover, in mtr3-1 the accumulated polyA+ RNA actually colocalizes with nucleolar antigens, the nucleolus becomes somewhat disorganized, and rRNA synthesis and processing are inhibited. A strain with a ts conditional mutation in RNA polymerase I also shows nucleolar accumulation of polyA+ RNA, whereas strains with mutations in the nucleolar protein Nop1p do not. Thus, in several mutant backgrounds, when mRNA cannot be exported i concentrates in the nucleolus. mRNA may normally encounter nucleolar components before export and proteins such as Mtr3p may be critical for export of both mRNA and ribosomal subunits.     

Kinetic analysis of benzyl alcohol oxidation by alcohol oxidase in aqueous—organic two-phase systems

Alcohol oxidase-catalyzed oxidation of benzyl alcohol to benzaldehyde was studied in three two-liquid phase systems involving butyl acetate, xylene and n-decane. A more highly hydrophobic solvent resulted in more favorable partitioning of the substrate into the aqueous phase and less deleterious effect on the enzyme activity. As a result, the overall production of aldehyde was highest with the n-decane system and lowest with the butyl acetate system. In addition, the organic solvent served as an extractant for the product, which acted both as a competitive inhibitor and as a denaturant of the enzyme. The time courses of benzaldehyde production were simulated by a simple mathematical model assuming Michaelis–Menten kinetics in the aqueous phase, enzyme deactivation by organic solvents and the equilibrium partitioning of the reagents between the two phases.     

Changes in riparian vegetation communities below a large dam in a monsoonal region: Futase Dam,Japan

This paper examines the effects of a dam reservoir flood control on riparian plant communities in an Asian monsoon area subjected to large summer floods under natural conditions. We compared riparian plant communities downstream of the Futase Dam located on the Arakawa River, with communities on the Nakatsu River, a tributary of the Arakawa River with no dam. Historical changes in spatial distribution of riparian vegetation communities were analysed using aerial photographs. Stand ages and flood history were also examined to analyse the likelihood of tree erosion in scenarios with and without flood control. Comparison of the two reaches shows that the reduced flood peaks below the Futase Dam led to the expansion of riparian forest area and an increase in species diversity. Reduction in the flood peaks below the dam means that the toppling moment of the trees is no longer exceeded. The result is that the riparian forest below the dam is able to develop into later successional stages. Copyright © 2004 John Wiley & Sons, Ltd.     

Atomic force microscopy in histology and cytology

This review briefly introduces the principles of the atomic force microscope (AFM) and shows our own results of AFM application to biological samples. The AFM, invented in 1986, is an instrument that traces the surface topography of the sample with a sharp probe while monitoring the interaction forces working between the probe and sample surface. Thus, the AFM provides three-dimensional surface images of the sample with high resolution. The advantage of the AFM for biologists is that AFM can visualize non-conductive materials in a non-vacuous (i.e., air or liquid) environment. AFM images of the plasmid DNA are comparable to those by transmission electron microscopy using a rotary shadowing technique, and have the advantage of examining directly the molecule without staining nor coating. The surface structure of human metaphase chromosomes and mouse collagen fibrils demonstrated in air by the non-contact mode AFM is comparable to that obtained by scanning electron microscopy. Quantitative information on the heights of structures is further obtainable from the AFM images. Embedment-free thin tissue-sections are useful for observing intracellular structures by AFM. The present review also shows AFM images of living cultured cells which have been collected in a contact mode in liquid. This technique afforded us three-dimensional observation of the cellular movement with high resolution. Although there are some innate limitations for AFM imaging, the AFM has great potential for providing valuable new information in histology and cytology.     

Biosynthesis and characterization of novel polyhydroxyalkanoate copolymers consisting of 3-hydroxy-2-methylbutyrate and 3-hydroxyhexanoate

In this study, in order to explore the possibility of biosynthesizing a novel polyhydroxyalkanoate (PHA), copolymerization of 3-hydroxy-2-methylbutyrate (3H2MB) as the α-position methylated monomer and 3-hydroxyhexanoate (3HHx) as the medium-chain-length monomer was performed to obtain P(3H2MB-co-3HHx). The β-oxidation-deficient Escherichia coli LSBJ, harboring the PHA biosynthetic operon from Aeromonas caviae and the propionyl-CoA transferase gene (pct) from Megasphaera elsdenii, was cultured with feeding tiglic and hexanoic acids as the precursors for 3H2MB and 3HHx, respectively. It was observed that pct expression was highly effective to enhance the incorporation of 3H2MB into PHA. The biosynthesized PHA was composed of 3H2MB and 3HHx units only, and the 3H2MB fraction varied in the range of 36–60 mol% depending on the culture conditions. These PHAs exhibited glass transition temperatures between ?11 to ?17 °C; moreover, no melting peak was observed during analysis using differential scanning calorimetry. This study demonstrated the biosynthesis of a hitherto unreported PHA by engineering metabolic pathway in E. coli.