A Physical Map of Arabidopsis thaliana Chromosome 3 Represented by Two Contigs of CIC YAC, P1, TAC and BAC Clones

We have constructed a physical map of Arabidopsis thaliana chromosome3 by ordering the clones from CIC YAC, P1, TAC and BAC librariesusing the sequences of a variety of genetic and EST markersand terminal sequences of clones. The markers used were 112DNA markers, 145 YAC end sequences, and 156 end sequences ofP1, TAC and BAC clones. The entire genome of chromosome 3, exceptfor the centromeric and telomeric regions, was covered by twolarge contigs, 13.6 Mb and 9.2 Mb long. This physical map willfacilitate map-based cloning experiments as well as genome sequencingof chromosome 3. The map and end sequence information are availableon the KAOS (Kazusa Arabidopsis data Opening Site) web siteat http://www.kazusa.or.jp/arabi/.     

Thermodynamic parameters of the interaction of Urtica dioica agglutinin with N-acetylglucosamine and its oligomers

The interaction between Urtica dioica agglutinin (UDA) and N-acetylglucosamine (GlcNAc) and its (1-4)-linked oligomers was studied by fluorescence titration and isothermal titration microcalorimetry. UDA possesses one significant binding site that can be measured calorimetrically. This site is composed of three subsites, each subsite accommodating one GlcNAc residue. The interaction is enthalpically driven, and the binding area of UDA is characterized by a H of interaction for a given oligosaccharide considerably smaller than that of wheat germ agglutinin (WGA), despite the fact that they both belong to a family of proteins composed entirely of hevein domains. Relatively high Cp values of the UDA-carbohydrate interactions and more favorable entropy term compared to WGA suggest that binding of the carbohydrate ligands by UDA has a higher hydrophobic component than that of WGA.     

Interaction analysis between tmRNA and SmpB from Thermus thermophilus

Small protein B, SmpB, is a tmRNA-specific binding protein essential for trans-translation. We examined the interaction between SmpB and tmRNA from Thermus thermophilus, using biochemical and NMR methods. Chemical footprinting analyses using full-length tmRNA demonstrated that the sites protected upon SmpB binding are located exclusively in the tRNA-like domain (TLD) of tmRNA. To clarify the SmpB binding sites, we constructed several segments derived from TLD. Optical biosensor interaction analyses and melting profile analyses with mutational studies showed that SmpB efficiently binds to only a 30-nt segment that forms a stem and loop, with the 5' and 3' extensions composed of the D-loop and variable-loop analogues. The conserved sequences, 16UCGA and 319GAC, in the extensions are responsible for the SmpB binding. These results agree with the those visualized by the cocrystal structure of TLD and SmpB from Aquifex aeolicus. In addition, NMR chemical shift mapping analyses, using the 30-nt segment and (15)N-labeled SmpB, revealed the characteristic RNA binding mode. The hydrogen bond pattern around beta2 changes, with the Gly in beta2, which acts as a hinge, showing the largest chemical shift change. It appears that SmpB undergoes structural changes indicating an induced fit upon binding to the specific region of TLD.     

Free dorsoulnar perforator flap transfers for the reconstruction of severely injured digits

The aim of this study was to investigate the feasibility of transferring the free dorsoulnar perforator flap nourished by the cutaneous perforator branched dorsoulnar artery to reconstruct severely injured fingers under upper arm anesthesia. Between April of 2001 and April of 2002, 13 free dorsoulnar perforator flaps were used in 13 patients. There were 11 men and two women ranging in age from 18 to 64 years, with an average age of 38 years. The affected fingers were one thumb, four index fingers, five middle fingers, two ring fingers, and one little finger. All cases were performed under upper arm anesthesia combined with intravenous local anesthesia. The operative time ranged from 103 to 140 minutes, with an average time of 120 minutes. The flap size ranged from 1 x 3 to 3 x 4 cm, and was transferred from the same forearm of the injured finger. All donor sites were closed primarily without a skin graft. The aim of reconstruction for fingers was to repair a traumatic defect (five cases), partial necrosis following replantation (two cases), and soft-tissue defects resulting from resection of a scar (three cases) and to revascularize ischemic fingers (three cases). All flaps survived completely. After repair of the flow-through circulation of the common digital artery and ischemic finger, a postoperative angiogram showed the vascular patency and hypervascularity of the reconstructed fingers, and the patients' complaints were reduced. The free dorsoulnar perforator flap under regional anesthesia is first reported; it may become one valuable option as a very small flap for the treatment of repairing intercalated or segmental defects as a flow-through flap for soft-tissue defects and ischemic fingers.     

Escherichia coli engineered to produce eicosapentaenoic acid becomes resistant against oxidative damages

The colony-forming ability of Escherichia coli genetically engineered to produce eicosapentaenoic acid (EPA) grown in 3mM hydrogen peroxide (H(2)O(2)) was similar to that of untreated cells. It was rapidly lost in the absence of EPA. H(2)O(2)-induced protein carbonylation was enhanced in cells lacking EPA. The fatty acid composition of the transformants was unaffected by H(2)O(2) treatment, but the amount of fatty acids decreased in cultures of cells lacking EPA and increased in cultures of cells producing EPA, suggesting that cellular EPA is stable in the presence of H(2)O(2) in vivo and may protect cells directly against oxidative damage. We discuss the possible role of EPA in partially blocking the penetration of H(2)O(2) into cells through membranes containing EPA.     

Decreased Expression of Matrix Metalloproteinase-9 and Increased Expression of Tissue Inhibitors of Matrix Metalloproteinase-1 in Paratuberculosis-Infected Cattle in the ELISA-Negative Subclinical Stage

We investigated the gene expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitors of matrix metalloproteinases-1 (TIMP-1) in peripheral blood cells from infected cattle with Mycobacterium avium subsp. paratuberculosis (Map) in the ELISA-negative subclinical stage compared with uninfected control cattle. Significant decreased MMP-9 expression and increased TIMP-1 expression were found in peripheral blood cells from Map-infected cattle after stimulation with Map lysate and Map purified protein derivative (PPD) than in control cattle by real-time RT-PCR analysis. In contrast to the uninfected controls, the activity of MMP-9 was also decreased in peripheral blood cell culture supernatants from Map-infected cattle at 24 hr after Map lysate and MapPPD stimulation by gelatin zymography analysis. As a result, the MMP-9 may play an important role in the development of Mycobacterium avium subsp. paratuberculosis disease.     

A single circularly permuted GFP sensor for inositol-1,3,4,5-tetrakisphosphate based on a split PH domain

A fluorescent sensor for the detection of inositol-1,3,4,5-tetrakisphosphate, Ins(1,3,4,5)P₄, was constructed from a split PH domain and a single circularly permuted GFP. A structure-based design was conducted to transduce a ligand-induced subtle structural perturbation of the split PH domain to an alteration in the population of the protonated and the deprotonated states of the GFP chromophore. Excitation of each distinct absorption band corresponding to the protonated or the deprotonated state of GFP resulted an increase and a decrease, respectively, in the intensity of emission spectra upon addition of Ins(1,3,4,5)P₄ to the split PH domain-based sensor. The Ins(1,3,4,5)P₄ sensor retained the ligand affinity and the selectivity of the parent PH domain, and realized the ratiometric fluorescence detection of Ins(1,3,4,5)P₄.     

Dictyostelium myosin II mutations that uncouple the converter swing and ATP hydrolysis cycle

During the ATP hydrolysis cycle of the Dictyostelium myosin II motor domain, two conserved alpha-helices, the SH1/SH2 helix and the relay helix, rotate in a coordinated way to induce the swing motion of the converter domain. A network of hydrophobic and ionic interactions in these two helices and the converter may ensure that the motions of these helices are effectively transmitted to the converter. To examine the roles of these interactions in the ATPase-dependent converter swing, we disrupted two conserved hydrophobic linkages among them by means of a point mutation (I499A or F692A). The resulting mutations induced only limited changes in the kinetic parameters of ATP hydrolysis, except for a marked increase of basal MgATPase activity. However, the mutant myosins completely lost their in vitro and in vivo motor functions. Measurements of the intrinsic tryptophan fluorescence and the GFP-based FRET revealed that the converter domain of these mutants did not swing during steady-state ATP hydrolysis or in the presence of tightly trapped Mg.ADP.V(i), which shows that the point mutations induced the uncoupling of the converter swing and ATP hydrolysis cycle. These results highlight the importance of these hydrophobic linkages for transmitting the coordinated twist motions of the helices to the converter as well as the requirement of this converter swing for force generation.     

Characterization of Rat8 localization and mRNA export in Saccharomyces cerevisiae during the brewing of Japanese sake

Ethanol affects the nuclear export of mRNA in a similar way to heat shock in Saccharomyces cerevisiae. We recently reported that the nuclear accumulation of Rat8 caused by ethanol stress correlates well with blocking of the export of bulk poly(A)⁺ mRNA. Here, we characterize the localization of Rat8 and bulk poly(A)⁺ mRNA in sake (Japanese rice wine) yeast during the brewing of sake. In wine must and synthetic dextrose medium, sake yeast showed the same responses to ethanol regarding changes in the localization of Rat8 as wine yeast and a laboratory strain: i.e., cells began the nuclear accumulation of Rat8 at an ethanol concentration of 6% and completed it at 9%. In contrast, during the sake-brewing process, sake yeast showed unique phenomena: i.e., cells did not start the nuclear accumulation of Rat8 until the ethanol concentration of the sake mash reached around 12% and they showed a normal localization of Rat8 around the nuclear envelope at the late stage of fermentation. These results provide new information about the transport of mRNA in yeast cells during actual alcoholic fermentation.     

Role of the Rab GTP-binding protein Ypt3 in the fission yeast exocytic pathway and its connection to calcineurin function

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.