Courtship in Drosophila quadrilineata with a unique male behavioral element, abdomen bending

This paper describes courtship of Drosophila quadrilineata of the immigrans group and reports a species-specific element, abdomen bending, that has not been described in Drosophila. The male bends his abdomen right and left as a display to the female. During courtship the male circles around the female, bends his abdomen, and taps her. The male licks the female ovipositor repeatedly, often after the females extrusion. Although the females ovipositor extrusion is thought to indicate rejection of a courting male in many species, the male of D. quadrilineata continues courtship. It is possible that the extrusion is a females mating signal to the courting male. The male of D. quadrilineata mounts the female in a rearward position and the genitalia of both sexes couple after mounting. This mounting position is shared by the species belonging to the immigrans group. We never observed behavior in which it appeared as if the male displayed the black stripes to the female. Males court one another and a courtship chain is frequently formed even in the absence of females. The males abdomen bending and the females extrusion followed by the males licking are discussed.     

Effect of anti-regucalcin antibody on neutral phosphatase activity in rat liver cytosol: Involvement of endogenous regucalcin

The effect of anti-regucalcin monoclonal antibody on neutral phoshatase activity in rat liver cytosol was investigated. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate toward phosphatase asssy. Liver cytosolic phosphatase activity with three phosphoaminoacids was significantly increased in the presence of anti-regucalcin antibody (100 and 200 ng/ml) in the enzyme reaction mixture with calcium chloride (0.1 mM) or EGTA (1.0 mM). The effect of anti-regucalcin antibody was completely abolished in the presence of exogenous regucalcin (1.0 M), indicating the involvement of endogenous regucalcin. The anti-regucalcin anti body- increased phosphatase activity was not significantly altered in the presence of trifluoperazine (20 M), an antagonist of calmodulin, or akadaic acid (10 M), an inhibitor of protein phosphatase, although these inihibitors caused a slight decrease in liver cytosolic phosphatase activity. The effect of endogenous regucalcin might be not related to calmodulin, and it was insensitive to okadaic acid. The present findings suggest that endogenous regucalcin is involved in the regulation of protein phasphatase in rat liver cytoplasm.     

Enzymatic characterization of scytalone dehydratase Val75Met variant found in melanin biosynthesis dehydratase inhibitor (MBI-D) resistant strains of the rice blast fungus

Carpropamid ((1RS,3SR)-2,2-dichloro-N-[(R)-1-(4-chlorophenyl)ethyl]-1-ethyl-3-methylcyclopropanecarboxamide) is a potent chemical against the rice blast fungus, Pyricularia oryzae. In 2001, isolates of the fungus with reduced sensitivity to this fungicide appeared in Saga Prefecture of Japan and were regarded as a potential threat to rice protection by carpropamid. The cause of the resistance has been identified genetically as a point mutation resulting in the Val75Met change in scytalone dehydratase, the primary target of the fungicide. We constructed an overexpression system of the variant enzyme and characterized the kinetics in the catalysis and the inhibition by carpropamid isomers. The variant enzyme retained a significant level of enzymatic activity. Inhibition of the variant enzyme by carpropamid was more than 200-fold reduced in comparison with that of the wild-type. Based on the results, here we propose possible mechanisms of the carpropamid-resistance of the variant enzyme in retaining the normal enzymatic activity.     

Effect of temperature on the floral scent emission and endogenous volatile profile of Petunia axillaris

The floral scent emission and endogenous level of its components in Petunia axillaris under different conditions (20, 25, 30, and 35 degrees C) were investigated under the hypothesis that floral scent emission would be regulated by both metabolic and vaporization processes. The total endogenous amount of scent components decreased as the temperature increased, the total emission showing a peak at 30 degrees C. This decrease in endogenous amount was compensated for by increased vaporization, resulting in an increase of floral scent emission from 20 degrees C to 30 degrees C. The ambient temperature differently and independently influenced the metabolism and vaporization of the scent compounds, and differences in vapor pressure among the scent compounds were reduced as the temperature increased. These characteristics suggest the operation of an unknown regulator to change the vaporization of floral scent.     

The process of displacing the single-stranded DNA-binding protein from single-stranded DNA by RecO and RecR proteins

The regions of single-stranded (ss) DNA that result from DNA damage are immediately coated by the ssDNA-binding protein (SSB). RecF pathway proteins facilitate the displacement of SSB from ssDNA, allowing the RecA protein to form protein filaments on the ssDNA region, which facilitates the process of recombinational DNA repair. In this study, we examined the mechanism of SSB displacement from ssDNA using purified Thermus thermophilus RecF pathway proteins. To date, RecO and RecR are thought to act as the RecOR complex. However, our results indicate that RecO and RecR have distinct functions. We found that RecR binds both RecF and RecO, and that RecO binds RecR, SSB and ssDNA. The electron microscopic studies indicated that SSB is displaced from ssDNA by RecO. In addition, pull-down assays indicated that the displaced SSB still remains indirectly attached to ssDNA through its interaction with RecO in the RecO-ssDNA complex. In the presence of both SSB and RecO, the ssDNA-dependent ATPase activity of RecA was inhibited, but was restored by the addition of RecR. Interestingly, the interaction of RecR with RecO affected the ssDNA-binding properties of RecO. These results suggest a model of SSB displacement from the ssDNA by RecF pathway proteins.     

Development of a multi-step leukemogenesis model of MLL-rearranged leukemia using humanized mice

Mixed-lineage-leukemia (MLL) fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ(-/-) (NOG) mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia.     

TRIC-A channels in vascular smooth muscle contribute to blood pressure maintenance

TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation channels postulated to mediate counter-ion movements facilitating physiological Ca(2+) release from internal stores. Tric-a-knockout mice developed hypertension during the daytime due to enhanced myogenic tone in resistance arteries. There are two Ca(2+) release mechanisms in vascular smooth muscle cells (VSMCs); incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization, while agonist-induced activation of inositol trisphosphate receptors (IP(3)Rs) evokes global Ca(2+) transients causing contraction. Tric-a gene ablation inhibited RyR-mediated hyperpolarization signaling to stimulate voltage-dependent Ca(2+) influx, and adversely enhanced IP(3)R-mediated Ca(2+) transients by overloading Ca(2+) stores in VSMCs. Moreover, association analysis identified single-nucleotide polymorphisms (SNPs) around the human TRIC-A gene that increase hypertension risk and restrict the efficiency of antihypertensive drugs. Therefore, TRIC-A channels contribute to maintaining blood pressure, while TRIC-A SNPs could provide biomarkers for constitutional diagnosis and personalized medical treatment of essential hypertension.     

Pyramiding of male-sterile genes in <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> D. Don with the aid of closely linked markers

Gene pyramiding is a breeding method used to combine multiple useful genes. Although several genes have been pyramided in certain crops, gene pyramiding has not previously been applied to forest trees. In this study, we used the markers closely linked to the two male-sterile genes MS1 and MS2 for the effective development of individuals doubly heterozygous for these two genes. This is the first example of gene pyramiding through marker-assisted selection (MAS) in forest trees. The markers gSNP06239, which is closely linked to the MS1 gene, and estSNP00695, which is closely linked to MS2, were used in MAS. On the basis of the linkage phase between the markers and male-sterile loci, we selected five F₁ individuals (S3-64 from Shindai-3 × Kamikiri-31, S3-70 from Shindai-3 × Kamikiri-38, S3-77 from Shindai-3 × Kamikiri-47, S1-22 from Shindai-1 × Nakakubiki-4, and S1-56 from Shindai-1 × Setsugai-20) as parents for artificial crossing. The 268 seedlings obtained from six artificial cross combinations were used in this study. Chi-squared tests showed no significant deviation from the expected Mendelian ratios of genotypes, indicating that MAS using markers closely linked to the male-sterile genes worked very well. Fifteen individuals that showed unexpected genotypes were probably recombinants, because the map distances between the male-sterile locus and the DNA markers were 4.1 cM (gSNP06239 to MS1) and 6.9 cM (estSNP00695 to MS2). Development of markers more closely linked to the male-sterile loci will facilitate precise gene pyramiding in the future.     

Modification of a novel angiogenic peptide, AG30, for the development of novel therapeutic agents

We previously identified a novel angiogenic peptide, AG30, with antibacterial effects that could serve as a foundation molecule for the design of wound-healing drugs. Toward clinical application, in this study we have developed a modified version of the AG30 peptide characterized by improved antibacterial and angiogenic action, thus establishing a lead compound for a feasibility study. Because AG30 has an α-helix structure with a number of hydrophobic and cationic amino acids, we designed a modified AG30 peptide by replacing several of the amino acids. The replacement of cationic amino acids (yielding a new molecule, AG30/5C), but not hydrophobic amino acids, increased both the angiogenic and the antimicrobial properties of the peptide. AG30/5C was also effective against methicillin-resistant Staphylococcus aureus (MRSA) and antibiotic-resistant Pseudomonas aeruginosa. In a diabetic mouse wound-healing model, the topical application of AG30/5C accelerated wound healing with increased angiogenesis and attenuated MRSA infection. To facilitate the eventual clinical investigation/application of these compounds, we developed a large-scale procedure for the synthesis of AG30/5C that employed the conventional solution method and met Good Manufacturing Practice guidelines. In the evaluation of stability of this peptide in saline solution, RP-HPLC analysis revealed that AG30/5C was fairly stable under 5°C for 12 months. Therefore, we propose the use of AG30/5C as a wound-healing drug with antibacterial and angiogenic actions.