Improving male mating competitiveness and survival in the field for medfly,Ceratitis capitata (Diptera: Tephritidae) SIT programs

The success of the sterile insect technique (SIT) depends critically upon mating between released sterilized males and wild females. In Hawaii, improvements in the efficiency of sterile males were attempted on two separate fronts – mating enhancement and survival improvement. In the former, two methods have been investigated – selective breeding and aromatherapy. In the latter, flies which survived in field cages for several days were selected and bred to produce progeny with enhanced survival ability compared to control flies. Regarding mating selection, standard laboratory-reared males that successfully mated with wild females in field cages were allowed to breed. F1 offspring were inbred, then the selection procedure was repeated for four additional cycles. In the aromatherapy procedure, laboratory-reared males were exposed to ginger root oil for several hours 1 day prior to testing in field cages. Compared to controls, the selected flies improved the mating competitiveness of male flies ca. 3-fold, irradiation reduced this increase to ca. 2.5-fold. Exposing the selected, hybrid strain raised the fitness of the lab males to ca. 9-fold that of wild males. In the ongoing survival selection study, we have obtained lines in which the selected males survived ca. 2-fold better than laboratory control males over several days in an outdoor field cage, with food and water provided. The goal is to combine the traits of higher survival and mating ability into a single strain for SIT release.     

Targeted disruption of the Tab1 gene causes embryonic lethality and defects in cardiovascular and lung morphogenesis

The transforming growth factor-beta (TGF-beta) superfamily consists of a group of secreted signaling molecules that perform important roles in the regulation of cell growth and differentiation. TGF-beta activated kinase-1 binding protein-1 (TAB1) was identified as a molecule that activates TGF-beta activated kinase-1 (TAK1). Recent studies have revealed that the TAB1-TAK1 interaction plays an important role in signal transduction in vitro, but little is known about the role of these molecules in vivo. To investigate the role of TAB1 during development, we cloned the murine Tab1 gene and disrupted it by homologous recombination. Homozygous Tab1 mutant mice died, exhibiting a bloated appearance with extensive edema and hemorrhage at the late stages of gestation. By histological examinations, it was revealed that mutant embryos exhibited cardiovascular and lung dysmorphogenesis. Tab1 mutant embryonic fibroblast cells displayed drastically reduced TAK1 kinase activities and decreased sensitivity to TGF-beta stimulation. These results indicate a possibility that TAB1 plays an important role in mammalian embryogenesis and is required for TAK1 activation in TGF-beta signaling.     

Insulin-like growth factor I receptor is expressed at normal levels in Nijmegen breakage syndrome cells

Curcumin (diferuloylmethane) is a major component of food flavoring turmeric (Curcuma longa), and has been reported to be anticarcinogenic and anti-inflammatory. Although curcumin was shown to have antioxidant properties, its exact antioxidant nature has not been fully investigated. In this report we have investigated the possible antioxidant properties of curcumin using EPR spectroscopic techniques. Curcumin was found to inhibit the (1)O(2)-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation in a dose-dependent manner. (1)O(2) was produced in a photosensitizing system using rose bengal as sensitizer, and was detected as TEMP-(1)O(2) adducts by electron paramagnetic resonance (EPR) spectroscopic techniques using TEMP as a spin-trap. Curcumin at 2.75 microM caused 50% inhibition of TEMP-(1)O(2) adduct formation. However, curcumin only marginally inhibited (24% maximum at 80 microM) reduction of ferricytochrome c in a xanthine-xanthine oxidase system demonstrating that it is not an effective superoxide radical scavenger. Additionally, there was minor inhibition of DMPO-OH adduct formation by curcumin (solubilized in ethanol) when an ethanol control was included in the EPR spin-trapping study, suggesting that curcumin may not be an effective hydroxyl radical scavenger. Together these data demonstrate that curcumin is able only to effectively quench singlet oxygen at very low concentration in aqueous systems.     

Phosphorylation of Fanconi anemia protein, FANCA, is regulated by Akt kinase

Phosphorylation of the Fanconi anemia complementation group A (FANCA) protein is thought to be important for the function of the FA pathway. However, the kinase for FANCA (so-called FANCA-PK) remains to be identified. FANCA has a consensus sequence for Akt kinase near serine 1149 (Ser1149), suggesting that Akt can phosphorylate FANCA. We performed in vitro kinase assays using as substrate either a GST-fusion wild-type (WT) FANCA fragment or a GST-fusion FANCA fragment containing a mutation from serine to alanine at 1149 (FANCA-S1149A). These experiments confirmed that FANCA is phosphorylated at Ser 1149, in vitro. However, (32)P-orthophosphate labeling experiments revealed that FANCA-S1149A was more efficiently phosphorylated than WT-FANCA. Furthermore, phosphorylation of wild-type FANCA was blocked by coexpression of a constitutively active (CA)-Akt and enhanced by a dominant-negative (DN) Akt. Our results suggest that Akt is a negative regulator of FANCA phosphorylation.     

Single-molecule PCR using water-in-oil emulsion

Polymerase chain reaction (PCR) using a single molecule of DNA is very useful for analysis, detection and cloning of the desired DNA fragment. We developed a simple PCR method utilizing a water-in-oil (W/O) emulsion that included numerous droplets of reaction mixture in bulk oil phase. These droplets, which were stable even at high temperatures, functioned as micro-reactors. This allows the effective concentration of template DNA to be increased, even for low concentrations of template DNA. The present method consists of a two-step thermal cycle. The first step was carried out using the W/O emulsion. During this step, the template DNA was amplified in the limited volume of the droplets in the W/O emulsion. The W/O emulsion was broken and the second PCR step was carried out. This method can be easily applied to amplify a single DNA molecule.     

Determination of the torsion angles of alanine and glycine residues of model compounds of spider silk (AGG)(10) using solid-state NMR methods

Spiders synthesize several kinds of silk fibers. In the primary structure of spider silk, one of the major ampullate (dragline, frame) silks, spidroin 1, and flagelliform silk (core fibers of adhesive spiral), there are common repeated X-Gly-Gly (X = Ala, Leu, Pro, Tyr, Glu, and Arg) sequences, which are considered to be related to the elastic character of these fibers. In this paper, two dimensional spin diffusion solid-state NMR under off magic angle spinning (OMAS), ¹³C chemical shift contour plots, and Rotational Echo DOuble Resonance (REDOR) were applied to determine the torsion angles of one Ala and two kinds of Gly residues in the Ala-Gly-Gly sequence of ¹³C=O isotope-labeled (Ala-Gly-Gly)₁₀. The torsion angles were determined to be (, ) = (–90°, 150° ) within an experimental error of ±10° for each residue. This conformation is characterized as 3₁ helix which is in agreement with the structure proposed from the X-ray powder diffraction pattern of poly(Ala-Gly-Gly). The 3₁ helix of (Ala-Gly-Gly)₁₀ does not change by formic acid treatment although (Ala-Gly)₁₅ easily changes from the silk I conformation (the structure of Bombyx mori silk fibroin before spinning in the solid state) to silk II conformation (the structure of the silk fiber after spinning) by such treatment. Thus, the 3₁ helix conformation of (Ala-Gly-Gly)₁₀ is considered very stable. Furthermore, the torsion angles of the 16th Leu residue of (Leu-Gly-Gly)₁₀ were also determined as (, ) = (–90°, 150° ) and this peptide is also considered to take 3₁ helix conformation.     

Real-time observation of a single DNA digestion by lambda exonuclease under a fluorescence microscope field

A fluorescence microscopy technique has been developed to visualize the behavior of individual DNA and protein molecules. Real-time direct observation of a single DNA molecule can be used to investigate the dynamics of DNA-protein interactions, such as the DNA digestion reaction by lambda exonuclease. In conventional methods it is impossible to analyze the dynamics of an individual lambda exonuclease molecule on a DNA because they can only observe the average behavior of a number of exonuclease molecules. Observation of a single molecule, on the other hand, can reveal processivity and binding rate of an individual exonuclease molecule. To evaluate the dynamics of lambda exonuclease, a stained lambda DNA molecule with one biotinylated terminal was fixed on an avidin-coated coverslip and straightened using a d.c. electric field. Microscopic observation of digestion of a straightened DNA molecule by lambda exonuclease revealed that the DNA digestion rate was approximately 1000 bases/s and also demonstrated high processivity.     

Enteric microflora contribute to constitutive ICAM-1 expression on vascular endothelial cells

Quantitative estimates of endothelial cell adhesion molecule expression have revealed that some adhesion molecules [e.g., intercellular adhesion molecule-1 (ICAM-1)] are abundantly expressed in different vascular beds under normal conditions. The objective of this study was to determine whether the enteric microflora contribute to the constitutive expression of ICAM-1 and other endothelial cell adhesion molecules in the gastrointestinal tract and other regional vascular beds. The dual radiolabeled monoclonal antibody technique was used to measure endothelial expression of ICAM-1, ICAM-2, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in conventional, germ-free mice and germ-free mice receiving the cecal contents of conventional mice to reestablish the enteric microflora (total association). Constitutive ICAM-1 expression was significantly lower in the splanchnic organs (pancreas, stomach, small and large intestine, mesentery, and liver), kidneys, skeletal muscle, and skin of germ-free mice compared with their conventional counterparts. These differences were abolished after total association of germ-free mice with the indigenous gastrointestinal flora. The expression of ICAM-2, VCAM-1, and E-selectin in the various tissues studied did not differ between conventional and germ-free mice. These findings indicate that the indigenous gastrointestinal microflora are responsible for a significant proportion of the basal ICAM-1 expression detected in both intestinal and extraintestinal tissues.