Application of molecular techniques to distinguish Liriomyza trifolii from L. sativae (Diptera: Agromyzidae) on tomato cultivation in Japan

A molecular method is applied for differentiating the morphologically related leafminers Liriomyza trifolli and L. sativae on tomato cultivation. The method requires multiplex polymerase chain reaction (PCR) amplification of a region of mitochondrial cytochrome oxidase DNA using multiprimer sets. The method divides the mitochondrial fragment of L. trifolli into two fragments and L. sativae into three fragments. It is faster, less costly, and easier than random amplified polymorphic DNA-PCR, PCR-restriction fragment-length polymorphism, and DNA sequencing and more sensitive than the enzyme electrophoresis method, which are other ways to differentiate these two species. We applied the method to samples from populations of another place, sex, and stage and obtained the same results.     

Synthesis and inhibition mechanism of Delta lac-acetogenins, a novel type of inhibitor of bovine heart mitochondrial complex I

We have synthesized Deltalac-acetogenins that are new acetogenin mimics possessing two n-alkyl tails without an alpha,beta-unsaturated gamma-lactone ring and suggested that their inhibition mechanism may be different from that of common acetogenins [Hamada et al. (2004) Biochemistry 43, 3651-3658]. To elucidate the inhibition mechanism of Deltalac-acetogenins in more detail, we carried out wide structural modifications of original Deltalac-acetogenins and characterized the inhibitory action with bovine heart mitochondrial complex I. In contrast to common acetogenins, both the presence of adjacent bis-THF rings and the stereochemistry around the hydroxylated bis-THF rings are important structural factors required for potent inhibition. The inhibitory potency of a derivative possessing an n-butylphenyl ether structure (compound 7) appeared to be superior to that of the original Deltalac-acetogenins and equivalent to that of bullatacin, one of the most potent natural acetogenins. Double-inhibitor titration of steady-state complex I activity showed that the extent of inhibition of compound 7 and bullatacin is not additive, suggesting that the binding sites of the two inhibitors are not identical. Competition tests using a fluorescent ligand indicated that the binding site of compound 7 does not overlap with that of other complex I inhibitors. The effects of compound 7 on superoxide production from complex I are also different from those of other complex I inhibitors. Our results clearly demonstrate that Deltalac-acetogenins are a novel type of inhibitor acting at the terminal electron-transfer step of bovine complex I.     

Effects of a time-varying strong magnetic field on transient increase in Ca2+ release induced by cytosolic Ca2+ in cultured pheochromocytoma cells

Exposure of pheochromocytoma (PC 12) cells to a time-varying 1.51 T magnetic field inhibited an increase in the intracellular Ca2+ concentration ([Ca2+]i) induced by addition of caffeine to Ca(2+)-free medium. This inhibition occurred after a 15-min exposure and was maintained for at least 2 h. [Ca2+]i sharply increased in cells loaded with cyclic ADP-ribose, and 2-h exposure significantly suppressed the increase. Addition of ATP induced a transient increase in intracellular Ca2+ release mediated by IP3 receptor, and this increase was strongly inhibited by the exposure. Results indicated that the magnetic field exposure strongly inhibited Ca2+ release mediated by both IP3 and ryanodine receptors in PC 12 cells. However, thapsigargin-induced Ca2+ influx (capacitative Ca2+ entry) across the cell membrane was unaffected. The ATP content was maintained at the normal level during the 2-h exposure, suggesting that ATP hydrolysis was unchanged. Therefore, Mg2+ which is known to be released by ATP hydrolysis and inhibit intracellular Ca2+ release may not relate the exposure-caused inhibition. Eddy currents induced in culture medium appear to change cell membrane properties and indirectly inhibit Ca2+ release from endoplasmic reticulum and other Ca2+ stores in PC 12 cells.     

Analysis of the Promoter of the Auxin-Inducible Gene, parC, of Tobacco

The auxin-responsive region (AuxRR) in the promoter of the parCgene was analyzed in transgenic tobacco plants in which the5' flanking region of the parC promoter was placed upstreamof the gene for rß-glucuronidase (GUS). The AuxRRwas located between nucleotides (nt) –226 and –54.Detailed dissection of this segment revealed that the presenceof the non-contiguous sequences from nt –226 to –151and from nt –84 to –54 was required for the expressionof the auxin responsiveness of the parC promoter. The sequencefrom nt –226 to –151 was found to contain a sequencewhich resembles the as-1 element in the 35S promoter of cauliflowermosaic virus (CaMV). Although it has been reported that theas-1 element is involved in auxin responsiveness [Liu and Lam(1994) J. Biol. Chem. 269: 668], we showed that introductionof a point mutation into the as-1-like sequence completely eliminatedauxin responsiveness, a result that suggests that the sequenceis indispensable for auxin responsiveness. However, the presenceof the as-1-like sequence alone was not sufficient for auxinresponsiveness, since the segment (nt –226 to –84)that included the as-1-like sequence failed to confer auxinresponsiveness on the core promoter. It is possible that thetwo separately located sequences play specific roles in interactionswith trans-factors that are required for the expression of theauxin responsiveness of the parC promoter. (Received March 11, 1996; Accepted July 9, 1996)     

Can temporal fluctuation in spatial wall shear stress gradient initiate a cerebral aneurysm? A proposed novel hemodynamic index,the gradient oscillatory number (GON)

We propose a new hemodynamic index for the initiation of a cerebral aneurysm, defined by the temporal fluctuations of tension/compression forces acting on endothelial cells. We employed a patient-specific geometry of a human internal carotid artery (ICA) with an aneurysm, and reconstructed the geometry of the ICA before aneurysm formation by artificially removing the aneurysm. We calculated the proposed hemodynamic index and five other hemodynamic indices (wall shear stress (WSS) at peak systole, time-averaged WSS, time-averaged spatial WSS gradient, oscillatory shear index (OSI), and potential aneurysm formation indicator (AFI)) for the geometry before aneurysm formation using a computational fluid dynamics technique. By comparing the distribution of each index at the location of aneurysm formation, we discussed the validity of each. The results showed that only the proposed hemodynamic index had a significant correlation with the location of aneurysm formation. Our findings suggest that the proposed index may be useful as a hemodynamic index for the initiation of cerebral aneurysms.     

Comprehensive analysis of distinctive polyketide and nonribosomal peptide structural motifs encoded in microbial genomes

We developed a highly accurate method to predict polyketide (PK) and nonribosomal peptide (NRP) structures encoded in microbial genomes. PKs/NRPs are polymers of carbonyl/peptidyl chains synthesized by polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). We analyzed domain sequences corresponding to specific substrates and physical interactions between PKSs/NRPSs in order to predict which substrates (carbonyl/peptidyl units) are selected and assembled into highly ordered chemical structures. The predicted PKs/NRPs were represented as the sequences of carbonyl/peptidyl units to extract the structural motifs efficiently. We applied our method to 4529 PKSs/NRPSs and found 619 PKs/NRPs. We also collected 1449 PKs/NRPs whose chemical structures have been determined experimentally. The structural sequences were compared using the Smith-Waterman algorithm, and clustered into 271 clusters. From the compound clusters, we extracted 33 structural motifs that are significantly related with their bioactivities. We used the structural motifs to infer functions of 13 novel PKs/NRPs clusters produced by Pseudomonas spp. and Burkholderia spp. and found a putative virulence factor. The integrative analysis of genomic and chemical information given here will provide a strategy to predict the chemical structures, the biosynthetic pathways, and the biological activities of PKs/NRPs, which is useful for the rational design of novel PKs/NRPs.     

Loss of μ-crystallin causes PPARγ activation and obesity in high-fat diet-fed mice

The thyroid hormone-binding protein μ-crystallin (CRYM) mediates thyroid hormone action by sequestering triiodothyronine in the cytoplasm and regulating the intracellular concentration of thyroid hormone. As thyroid hormone action is closely associated with glycolipid metabolism, it has been proposed that CRYM may contribute to this process by reserving or releasing triiodothyronine in the cytoplasm. We aimed to clarify the relationship between CRYM and glycolipid metabolism by comparing wild-type and CRYM knockout mice fed a high-fat diet. Each group was provided a high-fat diet for 10 weeks, and then their body weight and fasting blood glucose levels were measured. Although no difference in body weight was observed between the two groups with normal diet, the treatment with a high-fat diet was found to induce obesity in the knockout mice. The knockout group displayed increased dietary intake, white adipose tissue, fat cell hypertrophy, and hyperglycemia in the intraperitoneal glucose tolerance test. In CRYM knockout mice, liver fat deposits were more pronounced than in the control group. Enhanced levels of PPARγ, which is known to cause fatty liver, and ACC1, which is a target gene for thyroid hormone and is involved in the fat synthesis, were also detected in the livers of CRYM knockout mice. These observations suggest that CRYM deficiency leads to obesity and lipogenesis, possibly in part through increasing the food intake of mice fed a high-fat diet.