A study of conformational stability of poly(L-alanine), poly(L-valine), and poly(L-alanine)/poly(L-valine) blends in the solid state by (13)C cross-polarization/magic angle spinning NMR

13C cross-polarization/magic angle spinning (CP/MAS) NMR and (1)H T(1rho) experiments of poly(L-alanine) (PLA), poly(L-valine) (PLV), and PLA/PLV blends have been carried out in order to elucidate the conformational stability of the polypeptides in the solid state. These were prepared by adding a trifluoroacetic acid (TFA) solution of the polymer with a 2.0 wt/wt % of sulfuric acid (H(2)SO(4)) to alkaline water. From these experimental results, it is clarified that the conformations of PLA and PLV in their blends are strongly influenced by intermolecular hydrogen-bonding interactions that cause their miscibility at the molecular level.     

Structural analysis of an insect lysozyme exhibiting catalytic efficiency at low temperatures

Bombyx mori lysozyme (BmLZ), from the silkworm, is an insect lysozyme. BmLZ has considerable activity at low temperatures and low activation energies compared with those of hen egg white lysozyme (HEWLZ), according to measurements of the temperature dependencies of relative activity (lytic and glycol chitin) and the estimation of activation energies using the Arrhenius equation. Being so active at low temperatures and low activation energies is characteristic of psychrophilic (cold-adapted) enzymes. The three-dimensional structure of BmLZ has been determined by X-ray crystallography at 2.5 A resolution. The core structure of BmLZ is similar to that of c-type lysozymes. However, BmLZ shows some distinct differences in the two exposed loops and the C-terminal region. A detailed comparison of BmLZ and HEWLZ suggests structural rationalizations for the differences in the catalytic efficiency, stability, and mode of activity between these two lysozymes.     

The effects of aggregation-inducing motifs on amyloid formation of model proteins related to neurodegenerative diseases

To examine the effects of aggregation-inducing motifs related to neurodegenerative diseases on amyloid formation of host protein, we prepared several chimera myoglobins, in which various aggregation-inducing motifs were inserted. The focused aggregation-inducing motifs included five (R5) or two (R2) oligopeptide repeats in yeast Sup35p, five octapeptide repeats (OPR) in the human prion protein, a nonamyloid beta component (NAC) in alpha-synuclein, and tandem repeats of 50 glutamines (Q50). Circular dichroism and infrared spectroscopies suggested that the OPR, R5, and Q50 motifs formed an antiparallel beta sheet as well as a random coil, whereas the R2 and NAC motifs mainly formed random coils. The OPR, R5, and Q50 mutants, but not the R2 and NAC mutants, readily formed the SDS-resistant aggregates under physiological condition, and electron microscopy revealed that the aggregates contained amyloid fibrils. The destabilization and increase in gyration radius of the OPR, R5, and Q50 mutants correlated with the tendency to form amyloid fibrils. A control mutant bearing a nonamyloidgenic sequence was also moderately destabilized but did not form amyloid fibrils. Therefore, we concluded that the OPR, R5, and Q50 motifs, even in a quite stable protein such as myoglobin, led the host protein to formation of amyloid fibrils under physiological condition.     

Phosphorylation of hUPF1 induces formation of mRNA surveillance complexes containing hSMG-5 and hSMG-7

Eukaryotic mRNAs containing premature termination codons (PTCs) are degraded by a process known as nonsense-mediated mRNA decay (NMD). NMD has been suggested to require the recognition of PTC by an mRNA surveillance complex containing UPF1/SMG-2. In multicellular organisms, UPF1/SMG-2 is a phosphoprotein, and its phosphorylation contributes to NMD. Here we show that phosphorylated hUPF1, the human ortholog of UPF1/SMG-2, forms a complex with human orthologs of the C. elegans NMD proteins SMG-5 and SMG-7. The complex also associates with protein phosphatase 2A (PP2A), resulting in dephosphorylation of hUPF1. Overexpression of hSMG-5 mutants that retain interaction with P-hUPF1 but which cannot induce its dephosphorylation impair NMD, suggesting that NMD requires P-hUPF1 dephosphorylation. We also show that P-hUPF1 forms distinct complexes containing different isoforms of hUPF3A. We propose that sequential phosphorylation and dephosphorylation of hUPF1 by hSMG-1 and PP2A, respectively, contribute to the remodeling of the mRNA surveillance complex.     

Thelephantins D-H: five p-terphenyl derivatives from the inedible mushroom Thelephora aurantiotincta

Five p-terphenyl derivatives named thelephantins D-H (1-5) together with nine known compounds, thelephantins A-C (6-8), ganbajunin E (9), p-hydroxylbenzoic acid (10), ganbajunin C (11), thelephorin A (12), 2-O-methylatromentin (13) and atromentin (14), were isolated from the methanolic extract of fruit bodies of the Thelephoraceous Basidiomycete Thelephora aurantiotincta. Their structures were elucidated by high-resolution MS, 2D NMR, IR and UV spectroscopic analysis.     

Characterization of leachianone G 2"-dimethylallyltransferase, a novel prenyl side-chain elongation enzyme for the formation of the lavandulyl group of sophoraflavanone G in Sophora flavescens Ait. cell suspension cultures

Leachianone G (LG) 2"-dimethylallyltransferase, a novel prenyl side-chain elongation enzyme, was identified in Sophora flavescens Ait. cultured cells. The enzyme transfers a dimethylallyl group to the 2" position of another dimethylallyl group attached at position 8 of LG to form sophoraflavanone G, a branched monoterpenoid-conjugated flavanone characteristic to this plant. This membrane-bound dimethylallyltransferase required Mg2+ (optimum concentration was 10 mm) for the reaction and had an optimum pH of 8.8. It utilized dimethylallyl diphosphate as the sole prenyl donor, and the 2'-hydroxy function in LG was indispensable to the activity. The apparent Km values for dimethylallyl diphosphate and LG were 59 and 2.3 microm, respectively. Subcellular localization of three enzymes that participated in the formation of the lavandulyl group was also investigated by sucrose density gradient centrifugation. Two prenyltransferases, naringenin 8-dimethylallyltransferase and LG 2"-dimethylallyltransferase, were localized in the plastids, whereas 8-dimethylallylnaringenin 2'-hydroxylase, which catalyzes the crucial step in the lavandulyl-group formation, was associated with the endoplasmic reticulum. These results suggest the close cooperation between the plastids and the endoplasmic reticulum in the formation of lavandulyl groups.     

Insertional mutagenesis in a homologue of a Pi transporter gene confers arsenate resistance on chlamydomonas

An arsenate-resistant mutant AR3 of Chlamydomonas reinhardtii is a recessive mutant generated by random insertional mutagenesis using the ARG7 gene. AR3 shows about 10-fold resistance against arsenate toxicity compared with the wild type. By using a flanking region of an inserted tag as a probe, we cloned the corresponding wild-type allele (PTB1) of a mutated gene, which could completely complement the arsenate-resistance phenotype of AR3. The size of PTB1 cDNA is about 6.0 kb and it encodes a putative protein comprising 1666 amino acid residues. This protein exhibits significant sequence similarity with the yeast Pho89 protein, which is known to be a Na(+)/Pi co-transporter, although the PTB1 protein carries an additional Gln- and Gly-rich large hydrophilic region in the middle of its primary structure. Analyses of arsenic accumulation and release revealed that PTB1-disrupted cells show arsenate resistance due to low arsenate uptake. These results suggest that the PTB1 protein is a factor involved in arsenate (or Pi) uptake. Kinetics of Pi uptake revealed that the activity of high-affinity Pi transport component in AR3 is more activated than that in the wild type.     

Relative biological effectiveness and tolerance dose of fission neutrons in canine skin for a potential combination of neutron capture therapy and fast-neutron therapy

To investigate the potential efficacy of fission neutrons from a fast-neutron reactor for the treatment of radioresistant tumors, the relative biological effectiveness (RBE) and tolerance dose of fission neutrons in canine skin were determined. The forelimbs of 34 healthy mongrel dogs received a single dose of fission neutrons (5.6, 6.8, 8.2, 9.6 or 11 Gy) or 137Cs gamma rays (10, 15, 20, 25 or 30 Gy). Based on observations of radiodermatitis for each radiation, the single-fraction RBE of fission neutrons in the sixth month was calculated as approximately 3. The tolerance doses of fission neutrons and gamma rays, defined as the highest doses giving no moist desquamation on the irradiated skin in the recovery phase, were estimated as 7.6 Gy and 20 Gy, respectively. The tolerance dose of 7.6 Gy of fission neutrons included 5.0 Gy of fast neutrons possessing high anti-tumor effects and 1.4 x 10(12) n/cm2 of thermal neutrons, which could be applicable to neutron capture therapy (NCT). The combination of fast-neutron therapy and NCT using a fast-neutron reactor might be useful for the treatment of radioresistant tumors.     

Mapping QTLs influencing rice floral morphology using recombinant inbred lines derived from a cross between <Emphasis Type="Italic">Oryza sativa</Emphasis> L. and <Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff

To understand the genetic basis of floral traits associated with the mating system in rice, we analyzed pistil, stamen and glume traits using a recombinant inbred line population, derived from a cross between an Asian cultivated rice ( Oryza sativa L.), Pei-kuh, and a wild rice ( Oryza rufipogon Griff.), W1944. Quantitative trait loci (QTLs) affecting floral morphology were detected by composite interval mapping using a linkage map constructed using 147 markers, mostly RFLPs. A total of 7, 4, 14 and 6 QTLs were detected for traits related to pistil, stamen, and size and shape of the glume, respectively. Comparison of 31 QTLs affecting these organs revealed ten QTLs affecting the different organs in four adjacent regions on chromosomes 2, 4, 5 and 10, but most QTLs (68%) were located separately on the whole chromosomes. Although four QTLs for stigma breadth, anther length and thickness of lemma and palea explained more than 25% of the total phenotypic variance, most QTLs (87%) had smaller effects. These results suggest that quantitative variation observed for pistil, stamen and glume traits is controlled by several distinct genes with small effects.