Larval species composition and genetic structures of Carposina sasakii, Grapholita dimorpha, and Grapholita molesta from Korea.

Rapid determination of the larval species composition and understanding of their genetic structure is important to establish the appropriate management system for multiple species infesting in fruits. We established accurate and rapid diagnostic methods based on multiplex polymerase chain reaction (PCR) diagnostic techniques to discriminate the three major lepidopteran species in orchard, Carposina sasakii, Grapholita dimorpha, and Grapholita molesta. Each species was identified by amplifying species-specific PCR products (375 bp for C. sasakii, 125 and 234 bp for G. dimorpha, and 125 bp for G. molesta). Based on species composition analysis from six types of infested fruits, G. dimorpha constituted the highest proportion (47.8%), followed by 35.2 and 13.5% for G. molesta and C. sasakii, respectively. Interestingly, high prevalence was found in G. dimorpha and G. molesta for plum and peach, respectively. Based on genetic diversity analysis, the three insect species exhibited moderate or high haplotype diversity and low nucleotide diversity, ranging from 0.319 to 0.699 and 0.0006 to 0.0045, respectively. Demographic expansion was not detected according to either a neutrality test or mismatch distribution analysis. Moreover, no significant genetic structure corresponding to province, host plant, fruit type, or collection period was observed. These results suggest that the population of each species would have high dispersal ability following fruit-generating periods via intrinsic host adaptation ability regardless of the spatial and temporal conditions. Determination of larval composition on fruit is valuable for establishing appropriate management systems that take the species into consideration; additionally, population genetic approaches can be utilized to understand the effects of environmental factors (province, host fruit, fruit type, etc.) on population structures.

An intrauterine catch-up growth regimen increases food intake and post-natal growth in rats.

Nutritional conditions during the intrauterine stage are an important developmental programming factor that can affect the growth and metabolic status during foetal development and permanently alter the phenotypes of newborn offspring and adults. This study was performed to examine the effects of intrauterine catch-up growth (IUCG) on food intake, post-natal body growth and the metabolic status of offspring and growing rats. Control pregnant rats were fed ad libitum during the entire gestation period. For the IUCG regimen, pregnant rats were fed 50% of the food of the controls from pregnancy days 4 through 11 (8 days), followed by ad libitum feeding from pregnancy days 12 through parturition. The birth weight of offspring was not affected by the IUCG regimen. At weaning, offspring from each treatment group were assigned to two groups and given either a normal diet or high-fat diet (HFD) for 12 weeks until 103 days of age. In the normal diet group, the IUCG offspring showed a 9.0% increase (P < 0.05) in total food intake, were 11.2% heavier (p < 0.05) at 103 days of age and had an 11.0% greater (p < 0.05) daily weight gain compared with control offspring. The IUCG regimen did not affect body glucose and lipid metabolism. After exposure to the HFD, the IUCG regimen has not exacerbated metabolic disorders. In conclusion, our findings suggest that the IUCG nutritional regimen during pregnancy can increase the food intake and post-natal body growth of offspring without inducing metabolic disorders such as obesity and insulin resistance. The IUCG nutritional regimen might be used to improve the food intake and post-natal body growth of domestic animals.

Severe dietary lysine restriction affects growth and body composition and hepatic gene expression for nitrogen metabolism in growing rats.

Dietary lysine restriction may differentially affect body growth and lipid and nitrogen metabolism, depending on the degree of lysine restriction. This study was conducted to examine the effect of dietary lysine restriction on growth and lipid and nitrogen metabolism with two different degree of lysine restriction. Isocaloric amino acid-defined diets containing 1.4% lysine (adequate), 0.70% lysine (50% moderate lysine restriction) and 0.35% lysine (75% severe lysine restriction) were fed from the age of 52 to 77 days for 25 days in male Sprague-Dawley rats. The 75% severe lysine restriction increased (p < 0.05) food intake, but retarded (p < 0.05) growth, increased (p < 0.05) liver and muscle lipid contents and abdominal fat accumulation, increased (p < 0.05) blood urea nitrogen levels and mRNA levels of the serine-synthesizing 3-phosphoglycerate dehydrogenase gene, but decreased (p < 0.05) urea cycle arginase gene mRNA levels. In contrast, the 50% lysine restriction did not significantly (p > 0.05) affect body growth and lipid and nitrogen metabolism. Our results demonstrate that severe 75% lysine restriction has detrimental effects on body growth and deregulate lipid and nitrogen metabolism.

Identification and characterization of three cholinesterases from the common bed bug, Cimex lectularius.

We identified and characterized the full-length cDNA sequences encoding two acetylcholinesterases (ClAChE1 and ClAChE2) and a salivary gland-specific cholinesterase-like protein (ClSChE) from the common bed bug, Cimex lectularius. All three cholinesterase genes (Clac1, Clace2 and Clsce) have conserved motifs, including a catalytic triad, a choline-binding site and an acyl pocket. Phylogenetic analysis showed that ClAChE1 belongs to the insect AChE1 clade, whereas ClAChE2 belongs to the insect AChE2 clade. ClSChE was grouped into the clade containing all AChE1s, suggesting a paralogous relationship to ClAChE1. Transcription levels of Clace1 were higher than those of Clace2 in all tissues examined, including the central nervous system (CNS). In contrast, the Clsce transcript was not detected in the CNS but specifically found in the salivary gland at much higher levels (>3000-fold) than those of Clace1 and Clace2. Western blot analysis using anti-ClAChE antibodies, in conjunction with activity staining, revealed that ClAChE1 is more active than ClAChE2, whereas ClSChE has little enzyme activity. Three-dimensional structure modelling suggested that ClAChEs and ClSChE shared structural similarities, but had some differences in the residues forming the acyl pocket and oxyanion hole. The current findings should provide valuable insights into the evolution and functional diversification of insect cholinesterase.

A point mutation in a glutamate-gated chloride channel confers abamectin resistance in the two-spotted spider mite, Tetranychus urticae Koch.

The molecular mechanisms and genetics of abamectin resistance mediated by target site insensitivity in the two-spotted spider mite, Tetranychus urticae, were investigated by comparing two isogenic abamectin-susceptible (AbaS) and abamectin-resistant (AbaR) strains. Cloning and sequencing of full-length cDNA fragments of gamma-amino butyric acid (GABA)-gated chloride channel genes revealed no polymorphisms between the two strains. However, sequence comparison of the full-length cDNA fragment of a T. urticae glutamate-gated chloride channel gene (TuGluCl) identified a G323D point mutation as being tentatively related with abamectin resistance. In individual F(2) progenies obtained by backcrossing, the G323D genotype was confirmed to correlate with abamectin resistance. Bioassays using progeny from reciprocal crossings revealed that the abamectin resistance trait resulting from TuGluCl insensitivity is incompletely recessive.

Inhibitory effects of orobol 7-O-D-glucoside from banaba (Lagerstroemia speciosa L.) on human rhinoviruses replication.

AIMS: The anti-human rhinovirus (HRV) activity of orobol 7-O-d-glucoside (O7G) from Lagerstroemia speciosa L. (Lythraceae) was evaluated in Hela cells. METHODS AND RESULTS: We tested anti-HRV activity of O7G using a cytopathic effect (CPE) reduction method, which exhibited broad-spectrum anti-HRVs activity with a 50% inhibitory concentration (IC(50)) ranging from 0.58 to 8.80 microg ml(-1). The 50% cytotoxicity concentration (CC(50)) of O7G is more than 100 microg ml(-1), and the derived therapeutic indices are more than 12. Ribavirin didn't possess antiviral activity against HRV15, HRV3 and HRV5, but exhibited weak antiviral activity against HRV2 and HRV3, and showed strong anti-HRV6 and -14 activities. CONCLUSIONS: These results suggest that O7G is a novel drug class with broad spectrum antiviral activity against HRV species A (HRV1B, HRV2, HRV15 and HRV40) and species B (HRV3, HRV6 and HRV14), as well as pleconaril-resistant virus (HRV5). SIGNIFICANCE AND IMPACT OF THE STUDY: Therefore, these findings provide important information for the utilization of Q7G promising broad spectrum for human rhinovirus treatment.

Extensive gene duplication of acetylcholinesterase associated with organophosphate resistance in the two-spotted spider mite.

Monocrotophos-resistant two-spotted spider mites (TSSMs), Tetranychus urticae, are known to possess three mutations on the acetylcholinesterase (AChE) gene (Tuace) that are involved in target site insensitivity. Cross-strain comparison of three strains (highly resistant AD, moderately resistant PyriF and susceptible UD strains) revealed that resistant strains have relatively more Tuace copies than the UD strain and that the levels of transcript were directly proportional to copy numbers. AChEs from the AD and PyriF strains had similar V(max) values to those of AChE from the UD strain but increased K(m) and reduced k(cat) constants, suggesting that the mutated, resistant form of AChE may carry a fitness cost. Relative copy numbers of Tuace in field populations varied from 2.4 to 6.1, correlating well with their levels of resistance (r(2)= 0.895). These results are suggestive of the involvement of Tuace gene duplication in resistance. Thus, monocrotophos resistance in TSSMs appears to have evolved through a combination of mutation accumulation and extensive gene duplication.

Steep left ventricle to aortic root angle and hypertrophic obstructive cardiomyopathy: study of a novel association using three-dimensional multimodality imaging.

BACKGROUND: Patients with hypertrophic cardiomyopathy (HCM) exhibit a difference in left ventricular outflow tract (LVOT) obstruction, independently of basal septal thickness (BST). Some patients with HCM have a steeper left ventricle to aortic root angle than controls. OBJECTIVE: To test the predictors of the LV-aortic root angle and the association between LV-aortic root angle and LVOT obstruction using three-dimensional imaging. PATIENTS: 153 consecutive patients with HCM (mean (SD) age 46 (14) years, 68% men) and 62 patients with hypertensive heart disease of the elderly (all >65 years of age, 73 (6) years, 34% men) who underwent whole-heart three-dimensional cardiac magnetic resonance (CMR) angiography (1.5 T) and Doppler echocardiography. Forty-two controls (age 43 (11) years, 38% men) who underwent contrast-enhanced multidetector computed tomography and were free of cardiovascular pathology were also studied. MAIN OUTCOMES: LV-aortic root angle, BST and maximal non-exercise LVOT gradient were measured in patients with HCM and in hypertensive-elderly patients. Additionally, LV-aortic root angle and BST were measured in controls. RESULTS: The mean (SD) LV-aortic root angle was significantly different (p<0.001) in the three groups: HCM (134 (10) degrees ), hypertensive-elderly (128 (10) degrees ), control (140 (7) degrees ). There was an inverse correlation between age and LV-aortic root angle in the three groups (all p<0.001): HCM (r = -0.56), hypertensive-elderly (r = -0.35), control (r = -0.48). On univariate analysis, in the HCM group, LV-aortic root angle (beta = -0.34, p<0.001), age (beta = 0.23, p = 0.01) and end-systolic volume index (beta = -0.20, p = 0.02), but not BST (beta = 0.02, p = 0.8), were associated with LVOT gradient. On multivariate analysis, only LV-aortic root angle was associated with LVOT gradient. CONCLUSIONS: Patients with HCM have a steeper LV-aortic root angle than controls. In patients with HCM, a steeper LV-aortic root angle predicts dynamic LVOT obstruction, independently of BST.

Endovascular strategies for vertebrobasilar dissecting aneurysms.

BACKGROUND AND PURPOSE: Dissecting vertebrobasilar aneurysms are challenging to treat, and standard treatment modalities remain controversial. We retrospectively evaluated our experience using endovascular techniques to treat these aneurysms. MATERIALS AND METHODS: From February 1997 to December 2007, 42 patients with intradural vertebrobasilar dissecting aneurysms underwent endovascular treatment. Twenty-nine patients had ruptured aneurysms, and 13 patients had unruptured dissecting aneurysms. The endovascular modalities for vertebrobasilar dissecting aneurysms were the following: 1) trapping (n = 30), 2) proximal occlusion (n = 3), 3) stent with coil (n = 6), and 4) stent alone (n = 3). RESULTS: Seventeen of the 29 patients with ruptured vertebrobasilar dissecting aneurysms had successful outcomes without procedural complications following endovascular treatment. Procedure-related complications were the following: 1) rebleeding (n = 3), 2) posterior inferior cerebellar artery (PICA) territory infarction (n = 6), 3) brain stem infarction (n = 2), and 4) thromboembolism-related multiple infarctions (n = 1). Clinical outcomes were favorable in 32 patients (76.1%). There were 3 (7.1%) procedure-related mortalities due to rebleeding, and 1 (2.4%) non-procedure-related mortality due to pneumonia sepsis. All 13 patients with unruptured vertebrobasilar dissecting aneurysms had favorable clinical and radiologic outcomes without procedure-related complications. CONCLUSIONS: Endovascular procedures for treatment of unruptured symptomatic dissecting aneurysms resulted in favorable outcomes. Ruptured vertebrobasilar dissecting aneurysms are associated with a high risk of periprocedural complications. Risks can be managed by using appropriate endovascular techniques according to aneurysm location, configuration, and relationship with the PICA.

Antiviral activity of raoulic acid from Raoulia australis against Picornaviruses.

RNA viruses are a major source of respiratory diseases worldwide. The lack of effective therapeutical treatment underlines the importance of research for new antiviral compounds. Raoulic acid is a principal ingredient of the plant Raoulia australis Hook. F. Antiviral assay using cytopathic effect (CPE) reduction method showed that raoulic acid possessed strong antiviral activity against human rhinovirus 2 (HRV2) with a 50% inhibition concentration (IC(50)) value of less than 0.1mug/ml, human rhinovirus 3 (HRV3) with a IC(50) value of 0.19 microg/ml, coxsackie B3 (CB3) virus with IC(50) values of 0.33 microg/ml, coxsackie B4 (CB4) virus with IC(50) values of 0.40 microg/ml, and enterovirus 71 (EV71) virus with IC(50) values of less than 0.1 microg/ml. However, the compound did not possess antiviral activity against influenza A (Flu A/PR, Flu A/WS, H1N1) and B viruses at four concentrations ranging from 0.1 to 100 microg/ml.

Abnormal papillary muscle morphology is independently associated with increased left ventricular outflow tract obstruction in hypertrophic cardiomyopathy.

BACKGROUND: Abnormal papillary muscles (PM) are often found in hypertrophic cardiomyopathy (HCM). OBJECTIVE: To assess the relationship between morphological alterations of PM in patients with HCM and left ventricular outflow tract (LVOT) obstruction, using magnetic resonance imaging (MRI) and echocardiography. METHODS: Fifty-six patients with HCM (mean age 42 years (interquartile range 27, 51), 70% male) and 30 controls (mean age (42 (30, 53) years, 80% male) underwent MRI on a 1.5 T scanner (Siemens, Erlangen, Germany). Standard cine images were obtained in short-axis (base to apex), along with two-, three- and four-chamber views. The presence of bifid PM (none, one or both) and anteroapical displacement of anterolateral PM was recorded by MRI and correlated with resting LVOT gradients obtained by echocardiography. RESULTS: Double bifid PM (70% vs 17%) and anteroapical displacement of anterolateral PM (77% vs 17%) were more prevalent in patients with HCM than in controls (p<0.001). Subjects with anteroapically displaced PM and double bifid PM had higher resting LVOT gradients than controls (45 (6, 81) vs 12 (0, 12) mm Hg (p<0.01) and 42 (6, 64) vs 11 (0, 17) mm Hg (p = 0.02), respectively. In patients with HCM, the odds ratio of having significant (>or=30 mm Hg) peak resting gradient was 7.1 (95% CI 1.4 to 36.7) for anteroapically displaced anterolateral PM and 10.4 (95% CI 1.2 to 91.2) for double bifid PM (both p = 0.005), independent of septal thickness, use of beta-blockers and/or calcium blockers and resting heart rate. CONCLUSIONS: Patients with HCM with abnormal PM have a higher degree of resting LVOT gradient, which is independent of septal thickness.

Development of a simple and rapid immunochromatographic strip test for diarrhea-causative porcine rotavirus in swine stool.

A rapid and simple immunochromatography (IC) strip test, for specific detection of porcine rotavirus (PRV) in stool specimen, was developed. Monoclonal antibodies (mAbs) to the OSU strain of PRV have been produced in mice. Among them, two hybridoma clones that generate mAb-1 and mAb-2, respectively, specific for VP6 protein of PRV, have been selected. In the IC configuration, mAb-1, one of the selected mAbs was used to the designed coat microparticles (MP), while another mAb-2 was used to fix it on the nitrocellulose membrane strip to form a result line. The control line was formed on the same membrane strip past the result line by fixing anti-mouse IgG antibody. The IC test was capable of detecting 1000 plaque-forming units of PRV/ml in less than 5min, and the binding capacity was demonstrated by specific recognition of PRV only, but not other porcine diarrhea viruses, transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). The IC test produced positive results with all the nine PRV-positive stool specimens and negative results with five different non-PRV specimens, which were identified previously by the polymerase chain reaction (PCR) test, respectively. The results indicate an excellent concordance between the two methods, suggesting a potential application of the three combinated IC tests (PRV, TGEV and PEDV) for the on-site, rapid screening of porcine diarrhea cases.

DNA sequence analysis of rdxA and frxA from 12 pairs of metronidazole-sensitive and -resistant clinical Helicobacter pylori isolates.

We previously reported that inactivation of rdxA and/or frxA converted Helicobacter pylori from metronidazole sensitive to metronidazole resistant. To examine the individual roles of rdxA and frxA in the development of metronidazole resistance in H. pylori, we examined the status of rdxA and frxA from 12 pairs of metronidazole-sensitive and -resistant H. pylori isolates obtained following unsuccessful therapy containing metronidazole. Arbitrary primed fingerprinting analyses revealed that the genotypes of 11 sensitive and resistant pairs of strains were essentially identical. Amino acid sequence identities of RdxA and FrxA from the 14 metronidazole-sensitive isolates ranged from 92 to 98% and 95 to 98%, respectively, compared to that of H. pylori J99 (MIC, 1 microg/ml). All strains with high-level metronidazole resistance (MICs, 128 microg/ml) contained premature truncation of both RdxA and FrxA caused by nonsense and/or frameshift mutations. Strains with intermediate resistance to metronidazole (MICs, 32 to 64 microg/ml) contained a single premature truncation and/or altered RdxA and FrxA caused by nonsense, frameshift, and unique missense mutations. The low-level metronidazole-resistant strains (MICs, 8 microg/ml) contained unique missense mutations in FrxA but no specific changes in RdxA. The results demonstrate that alterations in both the rdxA and frxA genes are required for moderate and high-level metronidazole resistance and that metronidazole resistance that develops during anti-H. pylori therapy containing metronidazole is most likely to involve a single sensitive strain infection rather than a coinfection with a metronidazole-resistant strain.

Assembly, annotation, and integration of UNIGENE clusters into the human genome draft.

The recent release of the first draft of the human genome provides an unprecedented opportunity to integrate human genes and their functions in a complete positional context. However, at least three significant technical hurdles remain: first, to assemble a complete and nonredundant human transcript index; second, to accurately place the individual transcript indices on the human genome; and third, to functionally annotate all human genes. Here, we report the extension of the UNIGENE database through the assembly of its sequence clusters into nonredundant sequence contigs. Each resulting consensus was aligned to the human genome draft. A unique location for each transcript within the human genome was determined by the integration of the restriction fingerprint, assembled genomic contig, and radiation hybrid (RH) maps. A total of 59,500 UNIGENE clusters were mapped on the basis of at least three independent criteria as compared with the 30,000 human genes/ESTs currently mapped in Genemap'99. Finally, the extension of the human transcript consensus in this study enabled a greater number of putative functional assignments than the 11,000 annotated entries in UNIGENE. This study reports a draft physical map with annotations for a majority of the human transcripts, called the Human Index of Nonredundant Transcripts (HINT). Such information can be immediately applied to the discovery of new genes and the identification of candidate genes for positional cloning.

Nitrofurantoin quadruple therapy for Helicobacter pylori infection: effect of metronidazole resistance.

BACKGROUND: Antibiotic resistance has increasingly been recognized as the major cause of treatment failure for Helicobacter pylori infection. New therapies for patients with metronidazole- or clarithromycin-resistant H. pylori are needed. AIM: To investigate the role of nitrofurantoin quadruple therapy for the treatment of H. pylori. METHODS: Patients with confirmed H. pylori infection received nitrofurantoin (100 mg t.d.s.), omeprazole (20 mg b.d.), Pepto-Bismol (two tablets t.d.s.), and tetracycline (500 mg t.d.s.) for 14 days. Four or more weeks after the end of therapy, outcome was assessed by repeat endoscopy with histology and culture or urea breath testing. RESULTS: Thirty patients were entered, including 25 men and five women; the mean age was 54.9 years. The most common diagnoses were duodenal ulcer (23%) and GERD (18%). The intention-to-treat cure rate was 70% (95% CI: 50.6-85%). Nitrofurantoin quadruple therapy was more effective with metronidazole-sensitive strains (88%; 15 out of 17) than with metronidazole-resistant strains (33%; three out of nine; P=0.008). Two of the treatment failures had pre-treatment isolates susceptible to metronidazole, which were resistant after therapy. CONCLUSIONS: Because nitrofurantoin quadruple therapy performed inadequately in the presence of metronidazole resistance, we conclude that nitrofurantoin is unlikely to find clinical utility for the eradication of H. pylori.

Analysis of metronidazole, clarithromycin and tetracycline resistance of Helicobacter pylori isolates from Korea.

Antibiotic resistance in Helicobacter pylori varies according to geographical region. We studied the primary resistance rates among 652 H. pylori isolated from Korea in relation to collection date, disease presentation, age and gender. Resistance rates were 40.6% (metronidazole), 5.9% (clarithromycin), 5.3% (tetracycline), 0% (amoxycillin), 1.5% (furazolidone) and 1.5% (nitrofurantoin). Resistance to metronidazole and clarithromycin increased from 1994 to 1999 (from 33.3 to 47.7% and 4.8 to 7.7%, respectively), but the differences only reached significance when rates of metronidazole resistance in women were compared with those in men (48.6 versus 36.9%).

Furazolidone- and nitrofurantoin-resistant Helicobacter pylori: prevalence and role of genes involved in metronidazole resistance.

The prevalence of furazolidone, nitrofurantoin, and metronidazole resistance among Helicobacter pylori strains was assessed with 431 clinical isolates. Fifty-two percent were metronidazole resistant, compared to 2% (7 of 431) with resistance to furazolidone and nitrofurantoin. All seven furazolidone- and nitrofurantoin-resistant isolates were also metronidazole resistant. rdxA, frxA, and fdxB knockouts did not result in furazolidone or nitrofurantoin resistance. These data suggest that furazolidone and nitrofurantoin may be good alternatives to metronidazole for treating H. pylori infection.