Development of PCR Primers to Identify Fusarium oxysporum f. sp. fragariae.

Fusarium oxysporum f. sp. fragariae is a fungal pathogen causing Fusarium wilt on strawberry. Polymerase chain reaction (PCR) primers that can discriminate F. oxysporum f. sp. fragariae from nonpathogenic F. oxysporum would greatly assist pathogen identification. In order to develop a molecular diagnostic tool for this pathogen, transposable elements in the pathogen were characterized and used for designing a specific set of PCR primers. Portions of the transposable elements Fot3, Han, Hop, Hornet1, and Skippy were detected in all 33 strains of F. oxysporum f. sp. fragariae tested by PCR, whereas Foxy was detected in 32 strains and Impala sequences were detected in 30 strains. Two types of sequences were detected for Hop, two types for Impala, and three types for Skippy. The genomic region between Han and Skippy was amplified by an inter-retrotransposon amplified polymorphism technique, and PCR primers (FofraF and FofraR) to specifically identify F. oxysporum f. sp. fragariae were designed from this region. The developed PCR primers discriminated F. oxysporum f. sp. fragariae strains from nonpathogenic F. oxysporum strains and five other formae speciales. Conidia of F. oxysporum f. sp. fragariae could be detected in brown lowland-type soil by PCR using the primers. After preculturing the soil sample on FoG2 medium, 1 × 102 conidia/g of soil could be detected; without preculturing, 1 × 103 conidia/g of soil were detected.

Differential impacts of CYP2C19 gene polymorphisms on the antiplatelet effects of clopidogrel and ticlopidine.

We examined the influence of CYP2C19 polymorphisms on the antiplatelet effects of clopidogrel and ticlopidine. The platelet aggregation induced by 20 µmol/l adenosine diphosphate (ADP) and CYP2C19 single-nucleotide polymorphisms (*2 and *3) was determined in patients with coronary artery disease (CAD) who were taking aspirin alone (n = 21), aspirin plus clopidogrel (n = 97), or aspirin plus ticlopidine (n = 47). The degree of platelet aggregation in the clopidogrel group, although not in the ticlopidine group, depended on the CYP2C19 polymorphism, and the maximal platelet aggregation in poor metabolizers (PMs) taking clopidogrel was equivalent to that in the group taking aspirin alone. After being switched from clopidogrel to ticlopidine, all seven of the PMs showed markedly lower platelet aggregation. These results suggest that CYP2C19 polymorphisms have a profound impact on the antiplatelet effect of clopidogrel but not on that of ticlopidine. Ticlopidine may be an effective therapeutic option for CYP2C19 PMs.

Bepridil up-regulates cardiac Na+ channels as a long-term effect by blunting proteasome signals through inhibition of calmodulin activity.

BACKGROUND AND PURPOSE: Bepridil is an anti-arrhythmic agent with anti-electrical remodelling effects that target many cardiac ion channels, including the voltage-gated Na+ channel. However, long-term effects of bepridil on the Na+ channel remain unclear. We explored the long-term effect of bepridil on the Na+ channel in isolated neonatal rat cardiomyocytes and in a heterologous expression system of human Na(v)1.5 channel. EXPERIMENTAL APPROACH: Na+ currents were recorded by whole-cell voltage-clamp technique. Na+ channel message and protein were evaluated by real-time RT-PCR and Western blot analysis. KEY RESULTS: Treatment of cardiomyocytes with 10 micromol.L(-1) bepridil for 24 h augmented Na+ channel current (I(Na)) in a dose- and time-dependent manner. This long-term effect of bepridil was mimicked or masked by application of W-7, a calmodulin inhibitor, but not KN93 [2-[N-(2-hydroxyethyl)-N-(4-methoxy benzenesulphonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine], a Ca2+/calmodulin-dependent kinase inhibitor. During inhibition of protein synthesis by cycloheximide, the I(Na) increase due to bepridil was larger than the increase without cycloheximide. Bepridil and W-7 significantly slowed the time course of Na(v)1.5 protein degradation in neonatal cardiomyocytes, although the mRNA levels of Na(v)1.5 were not modified. Bepridil and W-7 did not increase I(Na) any further in the presence of the proteasome inhibitor MG132 [N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide]. Bepridil, W-7 and MG132 but not KN93 significantly decreased 20S proteasome activity in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS: We conclude that long-term exposure of cardiomyocytes to bepridil at therapeutic concentrations inhibits calmodulin action, which decreased degradation of the Na(v)1.5 alpha-subunit, which in turn increased Na+ current.

[Aortic regurgitation caused by prolapsed noncoronary cusp associated with dissecting aortic aneurysm treated by placing a prosthetic graft onto the anulus: report of a case].

A 70-year-old man with severe aortic regurgitation (AR) associated with dissecting aortic aneurysm underwent a radical operation. AR was thought to be due to dilated sinotubular junction and prolapsed noncoronary cusp caused by dissecting flap extended into the sinus of Valsalva. At operation, the noncoronary cusp was slightly prolapsed into the left ventricle, but all cusps were seemed to be thin and pliable. An isolated "tongue shaped" graft was placed onto the anulus of the noncoronary cusp, and a 26 mm Woven Dacron graft was used to replace the ascending aorta. Postoperative angiogram showed mild AR and improved left ventricular (LV) function. This procedure was effective to repair AR caused by prolapsed noncoronary cusp without elongation or thickening of the valve.

[Assessment of blood flow of the internal thoracic artery in patients with aortic stenosis].

The number of patients undergoing combined aortic valve replacement (AVR) for aortic stenosis (AS) and coronary artery bypass grafting (CABG) has been increasing. In CABG, the internal thoracic artery (ITA) is the preferred conduit for its long-term patency. Although Doppler studies on ITA have been widely used, flow characteristics of the vessel in patients with AS have not been reported. To evaluate blood flow pattern of the ITA in AS, duplex scanning was performed in 10 patients before and after AVR. Peak systolic velocity was measured, and blood flow was calculated from mean velocity and cross-sectional area. The mean diameters of the vessels were approximately 1.8 mm on both sides. AVR caused an increase in systolic velocities from 61.2 cm/sec to 85.5 cm/sec in right ITA and from 58.4 cm/sec to 84.7 cm/sec in left ITA. The flow volumes increased from 32.2 ml/min to 46.7 ml/min in right and increased from 31.6 ml/min to 46.3 ml/min in left after AVR. In simultaneous AVR for AS and CABG, suitability of the ITA should be assessed before its use, and concomitant AVR may be quite important to provide adequate flow of the ITA as a conduit.

[Surgical treatment of Budd-Chiari syndrome associated with superior vena cava obstruction; report of a case].

We report a case of a 37-year-old woman with Budd-Chiari syndrome who underwent a radical treatment. She had had ascites, general edema, and liver dysfunction for 5 years. Preoperative cineangiogram showed a membranous stenosis at the retrohepatic inferior vena cava and the catheter could not be advanced into superior vena cava. In the operation, obstructed superior vena cava was identified. Membranous tissue at retrohepatic inferior vena cava was removed, and patch cavoplasty was performed under circulatory arrest through Senning procedure. Postoperative cavogram revealed good patency of inferior vena cava and her symptoms were disappeared.

[Assessment of left internal thoracic artery grafts by atrial pacing].

The flow reactivity of a left internal thoracic artery graft (LITAG) in response to atrial pacing was evaluated in 14 patients who underwent coronary artery bypass grafting (CABG) with LITAG to left anterior descending artery (LAD). Systolic peak velocity and diastolic peak velocity were recorded using a duplex scanner of 7.5 MHz, and flow volumes in each phase and flow ratio were calculated. The external temporary atrial pacing was used to increase heart rates 25 and 50%. Diastolic peak velocity and flow volume increased predominantly on both pacing rates. In contrast, systolic peak velocity decreased when heart rate was raised 50%, and there was no significant difference between the pacing modes in systolic flow volumes. As a result, flow ratio increased predominantly on both pacing rates. Based on the present studies, there may be some advantages with atrial pacing to increase the LITAG flow in response to the myocardial oxygen demand.

[Assessment of saphenous vein grafts flow by Doppler echocardiography].

In an effort to evaluate flow characteristics of the saphenous vein grafts (SVG) after coronary artery bypass grafting, we performed duplex scanning of SVG which were anastomosed to the left anterior descending artery in 12 patients, and compared those indexes with 34 internal thoracic artery grafts (ITAG). The SVG were observed with a 7.5 MHz duplex scanner through the anterior intercostal space. The diameter of the vessel, systolic peak velocity, and diastolic peak velocity were recorded in both groups, and systolic flow volume, diastolic flow volume, and velocity ratio were calculated. The systolic and diastolic peak velocity of SVG were predominantly lower than ITAG. No difference in the diameter and flow ratio could not be demonstrated between 2 groups. The flow volume of SVG were also predominantly lower than that of ITAG throughout cardiac cycle. This study reveals that advanced stenotic change were caused in the SVG group and suggest the occurrence of vein grafts disease long after coronary artery bypass grafting.

Genetic factors are major determinants of phenotypic variability in a mouse model of the DiGeorge/del22q11 syndromes.

The del22q11 syndrome is associated with a highly variable phenotype despite the uniformity of the chromosomal deletion that causes the disease in most patients. Df1/+ mice, which model del22q11, present with reduced penetrance of cardiovascular defects similar to those seen in deleted patients but not with other del22q11-like findings. The reduced penetrance of cardiovascular defects is caused by the ability of mutant embryos to recover from a fourth pharyngeal arch artery growth abnormality that is fully penetrant in early embryos. Here we show that genetic background has a major effect on penetrance of cardiovascular defects by affecting this embryonic recovery process. This effect could not be explained by allelic variation at the haploid locus, and it is likely to be caused by genetic modifiers elsewhere in the genome. We also show that genetic factors control extension of the Df1/+ phenotype to include thymic and parathyroid anomalies, establishing the Df1 mouse as a model for the genetic analysis of three major features of human del22q11 syndrome. We found that in Df1/+ mice, as in human patients, expression of the heart and thymic phenotypes are essentially independent from each other, suggesting that they may be controlled by different genetic modifiers. These data provide a framework for our understanding of phenotypic variability in patients with del22q11 syndrome and the tools for its genetic dissection.

[Assessment of left internal thoracic artery graft by exercise Doppler echocardiography].

Doppler echocardiography has several advantages such as frequent use, noninvasive approach, and physiological evaluation. Supine bicycle exercise testing was conducted for 30 patients undergoing CABG with LITA to LAD. Doppler echocardiography studies were performed before and after exercise to observe the change. On the basis of the angiographic data, patients were divided into two groups: 27 patients with a patent LITA graft, 3 patients with mildly stenosed LITA graft. In the patients who had patent grafts, diastolic flow velocity were increased higher than systolic after exercise. In the stenotic group, the flow pattern was changed to further systolic one. Doppler echocardiography during exercise is thought to be a reliable method to assess the LITA flow.

Establishment of variant PC12 subclones deficient in stimulation-secretion coupling.

Clonal rat pheochromocytoma (PC12) cells have been widely used to study the molecular mechanism of exocytosis. We have isolated variant PC12 subclones with deficiencies in stimulation-secretion coupling, by a single cell recloning, and investigated the defects. PC12-1G2 hardly released dopamine following high-K(+)-induced depolarization, but normal release was evoked by the Ca(2+)-ionophore, ionomycin. Fura-2 fluorometry indicated that a nicardipine-sensitive component of Ca(2+) influx was missing, suggesting that PC12-1G2 has defects in L-type Ca(2+) channel function. PC12-2B3 was not responsive to high-K(+)-induced depolarization and ionomycin, and voltage-dependent Ca(2+) entry was identical to that of the normal clone. Electron microscopy revealed that the number of vesicles adjacent or directly attached to the plasma membrane was decreased in PC12-2B3. The expression of presynaptic proteins was analyzed by immunoblotting using a panel of antibodies. Syntaxin 1, VAMP-2, SNAP-25, Munc18, Rab3C and Sec-6 were decreased compared to the control clone and that of synaptophysin was extremely low. PC12-D60 synthesized and released dopamine normally, but had almost lost its catecholamine-uptake activity. These results show that multiple PC12 cells variants are spontaneously generated, and that recloning can select PC12 subclones useful for the study of the molecular mechanisms of neurotransmitter release.

Activity-dependent transfer of brain-derived neurotrophic factor to postsynaptic neurons.

Neurotrophins such as brain-derived neurotrophic factor (BDNF) are thought to be transferred from post- to presynaptic neurons and to be involved in the formation and plasticity of neural circuits. However, direct evidence for a transneuronal transfer of BDNF and its relation to neuronal activity remains elusive. We simultaneously injected complementary DNAs of green fluorescent protein (GFP)-tagged BDNF and red fluorescence protein into the nucleus of single neurons and visualized expression, localization, and transport of BDNF in living neurons. Fluorescent puncta representing BDNF moved in axons in the anterograde direction, though some moved retrogradely, and transferred to postsynaptic neurons in an activity-dependent manner.

Tbx1 haploinsufficieny in the DiGeorge syndrome region causes aortic arch defects in mice.

DiGeorge syndrome is characterized by cardiovascular, thymus and parathyroid defects and craniofacial anomalies, and is usually caused by a heterozygous deletion of chromosomal region 22q11.2 (del22q11) (ref. 1). A targeted, heterozygous deletion, named Df(16)1, encompassing around 1 megabase of the homologous region in mouse causes cardiovascular abnormalities characteristic of the human disease. Here we have used a combination of chromosome engineering and P1 artificial chromosome transgenesis to localize the haploinsufficient gene in the region, Tbx1. We show that Tbx1, a member of the T-box transcription factor family, is required for normal development of the pharyngeal arch arteries in a gene dosage-dependent manner. Deletion of one copy of Tbx1 affects the development of the fourth pharyngeal arch arteries, whereas homozygous mutation severely disrupts the pharyngeal arch artery system. Our data show that haploinsufficiency of Tbx1 is sufficient to generate at least one important component of the DiGeorge syndrome phenotype in mice, and demonstrate the suitability of the mouse for the genetic dissection of microdeletion syndromes.

[A case of lateral origin of the left internal mammary artery].

A 66 years old men, with left anterior descending coronary artery (LAD) stenosis and aortic valve stenosis, underwent coronary artery bypass grafting (CABG) to LAD with left internal mammary artery (LIMA) and aortic valve replacement. His postoperative course was uneventful. But, postoperative angiogram showed that his patent LIMA graft was originated from much lateral side of the left subclavian artery. Internal mammary artery is considered the most ideal graft for CABG, but its' anomality is not well known. We present this rare case with the anomalous origin of LIMA.

Identification of the major Abeta1-42-degrading catabolic pathway in brain parenchyma: suppression leads to biochemical and pathological deposition.

Alzheimer amyloid beta-peptide (Abeta) is a physiological peptide constantly anabolized and catabolized under normal conditions. We investigated the mechanism of catabolism by tracing multiple-radiolabeled synthetic peptide injected into rat hippocampus. The Abeta1-42 peptide underwent full degradation through limited proteolysis conducted by neutral endopeptidase (NEP) similar or identical to neprilysin as biochemically analyzed. Consistently, NEP inhibitor infusion resulted in both biochemical and pathological deposition of endogenous Abeta42 in brain. This NEP-catalyzed proteolysis therefore limits the rate of Abeta42 catabolism, up-regulation of which could reduce the risk of developing Alzheimer's disease by preventing Abeta accumulation.

CROP/Luc7A, a novel serine/arginine-rich nuclear protein, isolated from cisplatin-resistant cell line.

A novel putative SR protein, designated cisplatin resistance-associated overexpressed protein (CROP), has been cloned from cisplatin-resistant cell lines by differential display. The N-half of the deduced amino acid sequence of 432 amino acids of CROP contains cysteine/histidine motifs and leucine zipper-like repeats. The C-half consists mostly of charged and polar amino acids: arginine (58 residues or 25%), glutamate (36 residues or 16%), serine (35 residues or 15%), lysine (30 residues, 13%), and aspartate (20 residues or 9%). The C-half is extremely hydrophilic and comprises domains rich in lysine and glutamate residues, rich in alternating arginine and glutamate residues, and rich in arginine and serine residues. The arginine/serine-rich domain is dominated by a series of 8 amino acid imperfect repetitive motif (consensus sequence, Ser-Arg-Ser-Arg-Asp/Glu-Arg-Arg-Arg), which has been found in RNA splicing factors. The RNase protection assay and Western blotting analysis indicate that the expression of CROP is about 2-3-fold higher in mRNA and protein levels in cisplatin-resistant ACHN/CDDP cells than in host ACHN cells. CROP is the human homologue of yeast Luc7p, which is supposed to be involved in 5'-splice site recognition and is essential for vegetative growth.

The basic diagnostic approaches used in robotic still-image telepathology.

We investigated the basic diagnostic processes used in telepathology with our robotic still-image system, OLMICOS, by analysing the steps and patterns used in 20 consecutive tissue section diagnoses. Three basic approaches were recognized. One was magnifying a suspect finding in a low-powered microscopic image. This approach was used mostly for confirming or characterizing a tumour. The second approach was scanning over a low-powered image by magnifying square images to form a mesh. This was found to be useful to confirm the presence or absence of signs and was mostly used as the initial step in judging the surgical margin of malignant cases or diagnosing lesions. The third approach was a combination of these two and was used for delineating the surgical margin, confirming the absence of metastases or diagnosing difficult lesions. Recognition of these three basic diagnostic approaches is important in making a rapid and correct remote diagnosis.

Elevated body fat in rats by the dietary nitric oxide synthase inhibitor, L-N omega nitroarginine.

The influence of the dietary nitric oxide (NO) synthase inhibitor, L-N omega nitroarginine (L-NNA) on body fat was examined in rats. In experiment 1, all rats were fed with the same amount of diet with or without 0.02% L-NNA for 8 wk. L-NNA intake caused elevations in serum triglyceride and body fat, and reduction in serum nitrate (a metabolite of nitric oxide). The activity of hepatic carnitine palmitoyltransferase was reduced by L-NNA. In experiment 2, rats were fed for 8 wk with the same amount of diets with or without 0.02% L-NNA supplemented or not with 4% L-arginine. The elevation in body fat, and the reductions in serum nitrate and in the activity of hepatic carnitine palmitoyltransferase by L-NNA were all suppressed by supplemental L-arginine. The results suggest that lower NO generation elevated not only serum triglyceride, but also body fat by reduced fatty acid oxidation.

Developmental spectrum of cardiac outflow tract anomalies encompassing transposition of the great arteries and dextroposition of the aorta: pathogenic effect of extrinsic retinoic acid in the mouse embryo.

We previously reported that retinoic acid shows a dose-dependent differential induction of various cardiac outflow anomalies: transposition of the great arteries is induced mainly by a high dose (70 mg/kg) and dextroposition of the aorta by a low dose (40-60 mg/kg; Yasui et al., 1995). We subsequently delineated the aberrant outflow tract septation process leading to the transposition (Yasui et al., 1997). The aim of the present study was to illustrate a spectrum of developmental abnormalities by examining mouse embryos treated with a low dose of retinoic acid and comparing them with embryos administered a high dose. We employed in situ observation on live embryos to discern the blood flow streams and scanning electron microscopy to clarify the internal structure. The embryos treated with a low dose of retinoic acid showed several basic phenotypes common to the high dose retinoic acid group, although variable and relatively mild, such as hypoplasia and dysplasia in the proximal outflow cushions, decreased counter-clockwise rotation in the distal outflow tract, and deviation of the edges of the developing outflow septum. In typical cases, the right-sided edge of the developing outflow septum shifted ventrally by various degrees, allowing for the right ventricle-to-aorta pathway, whereas the left-sided edge preserved the continuity with the interventricular septum, as in the normal embryo. These findings indicate that morphogenesis of dextroposition of the aorta and transposition of the great arteries are not only distinct but also show some basic pathways in common.

Situs variation and cardiovascular anomalies in the transgenic mouse insertional mutation, inv.

The inv mouse was reported as a novel strain with situs inversus Yokoyama et al., '93), and a few cases with heterotaxy were found in homozygotes. The original report by Yokoyama et al. described the location of the heart and the stomach using the index of arrangement of body structure. We newly examined 40 homozygous offspring for phenotypes of visceroatrial situs and the incidence of cardiovascular anomalies making use of morphological details defined in each organ structure. According to the arrangement of each organ, which ranged from the almost complete form of situs inversus to left isomerism, visceroatrial situs was classified into four categories: Situs inversus (4 cases), "variation type" of situs (17 cases), "abdominal heterotaxy" (15 cases), and visceroatrial heterotaxy (4 cases). In offspring with situs inversus, only one had aortic stenosis (25%). Seven with the "variation type" of situs had cardiovascular anomalies, such as aortic stenosis, endocardial cushion defect, and posterior vena cava interruption (41%). All 15 offspring with "abdominal heterotaxy" had anomalies of the posterior vena cava, and three of them also had tetralogy of Fallot. The remaining four with visceroatrial heterotaxy had endocardial cushion defect, which was associated with outflow tract anomaly in two cases (i.e. tetralogy of Fallot in one case and transportation of the great arteries in the other). These results revealed that visceroatrial heterotaxy frequently occurred in the inv homozygotes, especially in the abdomen, and often showed a propensity to left isomerism with posterior vena cava interruption.