Antimicrobial activity of the volatile compound 3,5-dichloro-4-methoxybenzaldehyde, produced by the mushroom Porostereum spadiceum, against plant-pathogenic bacteria and fungi.

AIMS: In this study, volatile compounds released from mycelia of some aromatic mushrooms were investigated for their inhibitory activity against plant-pathogenic bacteria and fungi. METHODS AND RESULTS: A screening revealed that volatile compounds from mycelia of Porostereum spadiceum remarkably inhibited the colony formation of plant-pathogenic bacteria, including Clavibacter michiganensis subsp. michiganensis and Ralstonia solanacearum while also inhibiting the conidial germination of plant-pathogenic fungi including Alternaria brassicicola and Colletotrichum orbiculare. The volatile compounds were isolated from the culture filtrate of P. spadiceum, and 3,4-dichloro-4-methoxybenzaldehyde (DCMB) was identified as a major compound. DCMB significantly inhibited bacterial colonization at 10 µg ml-1 and fungal conidial germination at 0·1-1 µg ml-1 as a vapour. CONCLUSIONS: This is the first report on the production of the volatile compound DCMB by P. spadiceum and on the antimicrobial activity of DCMB against plant-pathogenic bacteria and fungi at low concentrations. It may be possible to use the compound as an agent for protecting crops from bacterial and fungal diseases during cultivation and storage. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides an understanding of antimicrobial activity of the mushroom volatile compound that may be useful as a novel biological control agent for protecting various plant diseases.

Multidimensional standard curve for the development process of human fetuses.

The present paper considers a multidimensional view of the standard for the development process of human fetuses. An efficient method by which to find a multidimensional standard curve for the development process of human fetuses is proposed in which a logistic function with three parameters is utilized as an underlying model and a nonlinear regression method is applied. The proposed method also identifies an approximate prediction region, which can be efficiently applied to diagnose fetal malformation.

Reg I-knockout mice reveal its role in regulation of cell growth that is required in generation and maintenance of the villous structure of small intestine.

Reg I (regenerating gene product I) is a growth factor that plays a central role in the generation and regeneration of the gastric mucosal architecture. On the other hand, mouse Reg I mRNA is expressed at the highest levels in the small intestine among the gastrointestinal tissues. In the current study, with the aim to clarify the role of Reg I protein in the small intestine, the temporal and spatial pattern of Reg I expression and the phenotype of Reg I-knockout mice in the tissue were examined. In the wild-type mice, immunohistochemistry localized Reg I protein expression in absorptive cells located in the lower half of the intestinal villi. Reg I expression was undetectable until embryonic day 13 (E13), when the fetal intestine still lacks villous structure; however, it dramatically increased at E17 along with the formation and maturation of the fetal intestinal villi. In the small intestine of the adult Reg I-knockout mice, less densely packed, round-shaped aberrant morphology of the absorptive cells was observed light microscopically, and electron microscopical examination revealed a strikingly loose connection of these cells to the basement membrane. Antiproliferating cell nuclear antigen staining and anti-Ki67 staining demonstrated the marked decrease in the number of proliferating cells in the small intestinal mucosa of the knockout mice. The cell migration speed visualized by one shot labeling of 5-bromodeoxyuridine was significantly slower in the knockout mice. These phenotypes of Reg I-knockout mice emerged, in accordance with the temporal pattern of Reg I expression described above, from E17. Reg I was considered to be a regulator of cell growth that is required to generate and maintain the villous structure of the small intestine.

Alternaria brassicae produces a host-specific protein toxin from germinating spores on host leaves.

Spore suspensions of Alternaria brassicae, the causal agent of gray leaf spot in Brassica plants, were incubated on the leaves of cabbage (B. oleracea) and spore germination fluid (SGF) was collected after 48 h. A high molecular weight (HMW) fraction (>10 kDa) was separated from the SGF by ultrafiltration. In a detached leaf assay, the HMW fraction induced visible symptoms only on host leaves and the toxicity was lost by treatment with proteinase K or heat at 60 degrees C for 15 min, indicating the presence of host-specific protein toxin(s). A protein toxin in the HMW fraction was purified by several chromatography steps. The toxin induced water-soaked symptoms followed by chlorosis at concentrations of 0.5 to 1 microg/ml on host leaves, but not on nonhost leaves even at 50 microg/ml. The toxin also had infection-inducing activity when added to spore suspension of a nonpathogenic isolate of A. alternata, causing symptoms similar to the infection of A. brassicae only on host leaves. These results indicate that a new host-specific protein toxin named ABR-toxin is released from germinating spores of A. brassicae on host leaves. ABR-toxin migrated as a protein of 27.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of ABR-toxin was estimated to be approximately 7.0 and 21 N-terminal amino acid residues were sequenced.

Myocardial ischemia induces differential regulation of KATP channel gene expression in rat hearts.

The cardiac ATP-sensitive potassium (KATP) channel is thought to be a complex composed of an inward rectifier potassium channel (Kir6.1 and/or Kir6.2) subunit and the sulfonylurea receptor (SUR2). This channel is activated during myocardial ischemia and protects the heart from ischemic injury. We examined the transcriptional expression of these genes in rats with myocardial ischemia. 60 min of myocardial regional ischemia followed by 24-72 h, but not 3-6 h, of reperfusion specifically upregulated Kir6.1 mRNA not only in the ischemic (approximately 2.7-3.1-fold) but also in the nonischemic (approximately 2.0-2.6-fold) region of the left ventricle. 24 h of continuous ischemia without reperfusion also induced an increase in Kir6.1 mRNA in both regions, whereas 15-30 min of ischemia followed by 24 h of reperfusion did not induce such expression. In contrast, mRNAs for Kir6.2 and SUR2 remained unchanged under these ischemic procedures. Western blotting demonstrated similar increases in the Kir6.1 protein level both in the ischemic (2.4-fold) and the nonischemic (2.2-fold) region of rat hearts subjected to 60 min of ischemia followed by 24 h of reperfusion. Thus, prolonged myocardial ischemia rather than reperfusion induces delayed and differential regulation of cardiac KATP channel gene expression.

Critical role of Caenorhabditis elegans homologs of Cds1 (Chk2)-related kinases in meiotic recombination.

Although chromosomal segregation at meiosis I is the critical process for genetic reassortment and inheritance, little is known about molecules involved in this process in metazoa. Here we show by utilizing double-stranded RNA (dsRNA)-mediated genetic interference that novel protein kinases (Ce-CDS-1 and Ce-CDS-2) related to Cds1 (Chk2) play an essential role in meiotic recombination in Caenorhabditis elegans. Injection of dsRNA into adult animals resulted in the inhibition of meiotic crossing over and induced the loss of chiasmata at diakinesis in oocytes of F(1) animals. However, electron microscopic analysis revealed that synaptonemal complex formation in pachytene nuclei of the same progeny of injected animals appeared to be normal. Thus, Ce-CDS-1 and Ce-CDS-2 are the first example of Cds1-related kinases that are required for meiotic recombination in multicellular organisms.

Life-style activities in systemic lupus erythematosus.

OBJECTIVE: We evaluated the life-style activities of outpatients with SLE and factors that reduce their social activities. SUBJECTS: SLE group = 60 patients, Control 1 = 30 healthy subjects and Control 2 = 30 patients with other autoimmune diseases. The Frenchay Activity Index (FAI), Zung's self-rating depression scale (SDS), and the Japanese version of the Philadelphia Geriatric Center morale scale-revised (MS) were compared between groups. Relation between FAI and age, disease duration, steroid dose, SDS, and MS were examined in the SLE group, Control 1, and Control 2. RESULTS: Total scores by FAI was 28.1 +/-8.0 points in Control 1, whereas it was 26.5 +/- 5.8 points in Control 2 and 24.5 +/- 7.7 points in the SLE group. While there was no statistical difference between the SLE group and Control 2, the scores were significantly lower in the SLE group than in Control 1 (P < 0.05). In SLE patients, age, the duration of the disease, and the steroid dose had no correlation, but MS had a positive correlation (P < 0.05) and SDS had a negative correlation (P < 0.05). In Control 2, age, the duration of the disease, the steroid dose, MS and SDS had no correlation whereas there was significant negative relation between FAI and SDS in Control 1 (r= -0.516, P<0.005). CONCLUSION: The significant relation between life-style activities and subjective well-being, and depression in SLE suggests that detection and treatment of mental status is important in improving the life-style activities of SLE patients.

Rac1 is required for the formation of three germ layers during gastrulation.

The Rac1, a member of the Rho family proteins, regulates actin organization of cytoskeleton and cell adhesion. We used genetic analysis to elucidate the role of Rac1 in mouse embryonic development. The rac1 deficient embryos showed numerous cell deaths in the space between the embryonic ectoderm and endoderm at the primitive streak stage. Investigation of the primary epiblast culture isolated from rac1 deficient embryos indicated that Rac1 is involved in lamellipodia formation, cell adhesion and cell migration in vivo. These results suggest that Rac1-mediated cell adhesion is essential for the formation of three germ layers during gastrulation.

K-ras is essential for the development of the mouse embryo.

ras genes encode members of the small GTP-binding proteins. Ras protein in highly conserved in various species from yeast to humans and plays a key role in signal transduction. Ras is related to cell proliferation and differentiation. While, in addition, mutations in the ras genes are implicated in a variety of tumors. However, the physiological functions and specific roles of each ras gene, H-ras, K-ras and N-ras, are still not fully understood. To clarify the role of the K-Ras in vivo, we generated K-ras mutant mice by gene targeting. In contrast to the findings that H-Ras-deficient mice and N-Ras-deficient mice are born and grow normally, the K-Ras-deficient embryos die progressively between embryonic day 12.5 and term. At embryonic day 15.5, their ventricular walls are extremely thin. Besides, at embryonic day 11.5, they demonstrate increased cell death of motoneurons in the medulla and the cervical spinal cord. Our results thus indicate K-Ras to be essential for normal development in mice and residual Ras composed of H-Ras and N-Ras cannot compensate for the loss of K-Ras function in the mutant mice.

Vacuolar H(+)-ATPase plays a crucial role in growth and phenotypic modulation of myofibroblasts in cultured human saphenous vein.

BACKGROUND: The molecular mechanism of neointimal hyperplasia after vein graft surgery remains elusive. Vacuolar H(+)-ATPase (V-ATPase) is involved in intracellular trafficking and may play a crucial role in neointimal cell growth. METHODS AND RESULTS: Cultured human saphenous vein segments developed neointimal formation within 10 days. Neointimal cells were positive for vimentin and alpha-smooth muscle actin but negative for desmin, which is indicative of myofibroblasts. Those myofibroblasts were found to have originated from periadventitial fibroblasts, which upregulated the expression of 16-kDa proteolipid of V-ATPase before proliferation and phenotypic modulation. Neointimal myofibroblast growth and survival were highly sensitive to inhibition of V-ATPase by bafilomycin A(1) (BA(1)), because the incorporation of [(3)H]thymidine into the myofibroblasts was significantly inhibited by nanomolar concentrations of BA(1) and apoptotic cell death was induced by a similar concentration range of BA(1). In contrast, endothelial cells and differentiated smooth muscle cells were resistant to apoptosis by BA(1). CONCLUSIONS: These results suggest that V-ATPase plays a crucial role in growth and phenotypic modulation of myofibroblasts that contributes to neointimal formation in cultured human saphenous vein.

Myocardial protection by preconditioning of heart with losartan, an angiotensin II type 1-receptor blocker: implication of bradykinin-dependent and bradykinin-independent mechanisms.

BACKGROUND: Ischemic preconditioning (PC) represents a state-of-the-art technique for myocardial preservation. Although certain intracellular mediators have been shown to play a role in PC, the exact nature of the trigger for PC is not known. Our previous study demonstrated that intracellular bradykinin released from the heart during ischemia/reperfusion plays a role in myocardial preservation. This study was undertaken to further examine the mechanism of bradykinin-mediated PC. METHODS AND RESULTS: Since the bradykinin B(2) receptor is likely to provide cardioprotection by blocking angiotensin II formation, we determined the effects of an angiotensin II type 1 (AT(1)) receptor blocker, losartan, and a bradykinin B(2) receptor blocker, HOE 140, on myocardial protection. Isolated rat hearts were perfused initially by the Langendorff mode with Krebs-Henseleit buffer (KHB) for 15 minutes in the absence (control) or presence of losartan (4.5 micromol/L) and/or HOE 140 (10 micromol/L). After conversion to the working mode for 10 minutes (baseline), randomly assigned control and experimental hearts were subjected to 30 minutes of normothermic global ischemia followed by 2 hours of reperfusion. Myocardial function, infarct size, cardiomyocyte apoptosis, and amount of bradykinin/angiotensin released from the hearts were measured at baseline and during reperfusion while in the working mode. Significant postischemic ventricular recovery was demonstrated by improved developed pressure and aortic flow and reduced myocardial infarct size and apoptotic cell death with losartan, whereas the reverse was true for HOE 140. The functional recovery and infarct size-lowering ability of losartan were partially blocked and the antiapoptotic function of losartan was completely blocked by HOE 140. CONCLUSIONS: The results document that losartan reduced whereas HOE 140 increased myocardial ischemia/reperfusion injury by blocking AT(1) and bradykinin B(2) receptors, respectively, suggesting a role of the bradykinin B(2) receptor in PC. Losartan provided cardioprotection through both bradykinin-dependent and bradykinin-independent mechanisms.

Long-term right ventricular volume overload increases myocardial fluorodeoxyglucose uptake in the interventricular septum in patients with atrial septal defect.

BACKGROUND: Several studies have shown that long-term right ventricular (RV) overload in animal models alters myocardial energy substrate metabolism. However, whether long-term RV volume overload alters this metabolism in the human is unclear. METHODS AND RESULTS: We performed positron emission tomography with [(18)F]fluorodeoxyglucose (FDG) and single-photon emission tomography (SPECT) with [(201)Tl]TlCl (Tl) and [(123)I]15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid (BMIPP) in 11 patients with atrial septal defect (ASD) and 11 control subjects. In the FDG study, we calculated myocardial metabolic rate of glucose (MMR) in interventricular septum (IVS) and left ventricular (LV) free wall. MMR was significantly increased in IVS compared with LV free wall in the ASD patients (420+/-35 versus 333+/-32 mol x kg(-1) x min(-1); P<0.05) but not in the control group (347+/-27 versus 357+/-25 mol x kg(-1) x min(-1)). In both ASD and control groups, SPECT count was not significantly different between IVS and LV free wall in Tl (ASD, 160+/-11 versus 177+/-12; control, 141+/-12 versus 157+/-14 counts per 15 minutes) and BMIPP studies (ASD, 203+/-14 versus 212+/-18; control, 162+/-16 versus 176+/-16 counts per 15 minutes). MMR in the IVS/LV free wall ratio in the ASD group significantly correlated with indices related to RV volume overload. CONCLUSIONS: Given the assumption that long-term RV volume overload did not affect the lumped constant, the present study suggests that, unlike myocardial perfusion or fatty acid analogue uptake, myocardial glucose utilization in IVS relative to LV free wall is increased in relation to long-term RV volume overload in patients with ASD.

Diabetic environment and genetic predisposition as causes of congenital malformations in NOD mouse embryos.

Congenital malformations such as neural tube defects and a kinky or waved vertebral column were observed at higher incidence in embryos from nonobese diabetic (NOD) female mice with overt diabetes (NOD-D; 40.3%, P less than 0.005) or without overt diabetes (NOD-N; 8.4%, P less than 0.05) than in control Institute of Cancer Research (ICR) mouse embryos (1%) at day 13 of gestation. In vivo and in vitro preimplantation development of NOD-N, NOD-D, and ICR embryos did not differ in rate of development, size, or morphology. Embryos cultured from one-cell to early blastocyst stage were mutually transferred to uterine horns of pseudopregnant females between NOD-D and ICR mice and examined at day 13 of gestation. There were significant decreases in ratios of implantation and of viable embryos in ICR embryos transferred to NOD-D recipients (52%, P less than 0.001 and 14%, P less than 0.001, respectively) compared with those ratios in ICR embryos transferred to ICR uteri (79.2 and 56.2%) or those in NOD-D embryos transferred to ICR uteri (70.3 and 33.1%). Furthermore, 18 of 45 viable ICR embryos transferred to NOD-D dams had malformations, whereas there were no malformations in 73 viable ICR embryos transferred to ICR recipients, suggesting deleterious effects of maternal diabetic environment to embryos. On the other hand, 8 of 58 viable NOD-D embryos that were cultured in vitro and transferred to ICR uteri had malformations such as neural tube defects.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallization and preliminary X-ray diffraction studies of curculin. A new type of sweet protein having taste-modifying action.

A taste-modifying protein, curculin, has been crystallized by the vapor diffusion method using polyethylene glycol 400 as a precipitant. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions: a = 105 A, b = 271 A, c = 48.7 A. The crystals diffract X-rays to at least a resolution of 3.0 A and are suitable for X-ray crystallographic studies.

A novel Drosophila nuclear protein serine/threonine kinase expressed in the germline during its establishment.

Nuclear protein kinases are believed to play important roles in regulating gene expression. We report here the identification and developmental expression of Dmnk (Drosophila maternal nuclear kinase), a Drosophila gene encoding a putative nuclear protein serine/threonine kinase with no apparent homology to previously identified protein kinases and located at 38B on the second chromosome. Dmnk mRNAs are transcribed in nurse cells and are subsequently localized in the anterior of oocytes during oogenesis, in a manner similar to several maternal transcripts regulating oogenesis and early embryogenesis. At early cleavage-stages Dmnk transcripts are transiently present throughout the embryo, but become restricted to the posterior pole and then to the newly-formed primordial germ cells (pole cells) by the blastoderm stage. The transcripts are sustained in the pole cells during gastrulation until they pass through the midgut pocket wall into the body cavity. Immunostaining with specific antibodies revealed that Dmnk proteins are localized to the nuclei in a speckled pattern. Dmnk proteins become detectable in both somatic and germ line cell nuclei upon their arrival at the periplasm of the syncytial embryo, but then disappear from the somatic cell nuclei. Consistent with mRNA expression, Dmnk proteins in pole cell nuclei are sustained during gastrulation. Taken together, Dmnk represents a novel class of nuclear protein kinases and the dynamic expression of Dmnk suggests a role in germ line establishment. The results are discussed in the light of recent findings concerning germ line establishment in Caenorhabditis and Drosophila.

Fetal jaw movement affects condylar cartilage development.

Using a mouse exo utero system to examine the effects of fetal jaw movement on the development of condylar cartilage, we assessed the effects of restraint of the animals' mouths from opening, by suture, at embryonic day (E)15.5. We hypothesized that pre-natal jaw movement is an important mechanical factor in endochondral bone formation of the mandibular condyle. Condylar cartilage was reduced in size, and the bone-cartilage margin was ill-defined in the sutured group at E18.5. Volume, total number of cells, and number of 5-bromo-2'-deoxyuridine-positive cells in the mesenchymal zone were lower in the sutured group than in the non-sutured group at E16.5 and E18.5. Hypertrophic chondrocytes were larger, whereas fewer apoptotic chondrocytes and osteoclasts were observed in the hypertrophic zone in the sutured group at E18.5. Analysis of our data revealed that restricted fetal TMJ movement influences the process of endochondral bone formation of condylar cartilage.

MRI study of atherosclerotic plaque progression using ultrasmall superparamagnetic iron oxide in Watanabe heritable hyperlipidemic rabbits.

OBJECTIVE: The purpose of this study was to evaluate plaque progression by using MRI with ultrasmall superparamagnetic iron oxide (USPIO) and by histopathological studies. METHODS: We divided 12 Watanabe heritable hyperlipidemic (WHHL) rabbits into 4 groups based on their age (3, 9, 14 and 26 months) and injected them intravenously with 0.8 mmol (Fe) kg(-1) of USPIO (size, 32 nm; concentration, 15 mg dl(-1)). On the fifth post-injection day, they were again given an intravenous injection with 40 µmol kg(-1) of the same USPIO, and MR angiography (MRA) was performed. The signal-to-noise ratio (SNR) in regions of interest in the wall of the upper abdominal aorta was calculated on coronal images. Specimens from the same level of the aorta were subjected to iron staining and RAM-11 immunostaining and used for histopathological study. For statistical analysis of the MRA and histopathological findings, we used analysis of variance [Tukey's honest significant difference (HSD) test]. RESULTS: In 9-month-old rabbits, the SNR was significantly lower than in rabbits of the other ages (p < 0.01), and the area of RAM-11 (DAKO Corporation, Glostrup, Denmark) and iron uptake in the aortic wall was significantly larger (RAM-11, p < 0.01; iron, p < 0.05). These areas were the smallest in 3-month-old rabbits. CONCLUSION: Histopathologically, the number of macrophages was the greatest in 9-month-old rabbits. Our findings indicate that the SNR on MRI scans reflects the number of macrophages in the aortic wall of WHHL rabbits. ADVANCES IN KNOWLEDGE: USPIO-enhanced MRI visualized the accumulation of macrophages in early atherosclerotic plaques of WHHL rabbits in the course of natural progression.

Deuterium oxide (heavy water) accelerates actin assembly in vitro and changes microfilament distribution in cultured cells.

While deuterium oxide (D2O) is known to produce various biological effects in living animals and cultured cells, the detailed mechanisms by which it does so remain unclear. The present study was designed to assess the effects of D2O on microfilaments (MFs) via fluorescence staining of BALB 3T3 cells and in vitro actin polymerization studies. After BALB 3T3 cells had been exposed to a concentration of more than 30% D2O for several hours, stress fibers in the peripheral region became thick and distinct, while the quantity of perinuclear MFs was drastically reduced. This effect was transient and returned to the original distribution within 12 h. Cytoplasmic F-actin (FA) also increased transiently coincident with the enhancement of stress fibers. The pattern of cell locomotion became simpler, and total locomotor activity was suppressed in a D2O concentration-dependent manner. Analysis of in vitro studies demonstrated that, when purified G-actin was polymerized in D2O at a concentration greater than 10%, the rate of actin polymerization was accelerated, whereas the total amount of polymerized actin at the steady state in D2O was the same as that in H2O controls. A gelation assay and transmission electron microscopy (TEM) showed that the network of crosslinked FA with alpha-actinin became denser in 30% D2O than in H2O. These findings concerning actin polymerization and FA gelation suggest that the alteration of stress fibers in cultured cells is caused by a direct effect of D2O on cellular MF dynamics.

Cloning and characterization of a cyclic peptide synthetase gene from Alternaria alternata apple pathotype whose product is involved in AM-toxin synthesis and pathogenicity.

Afternaria afternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide host-specific toxin, AM-toxin. PCR (polymerase chain reaction), with primers designed to conserved domains of peptide synthetase genes, amplified several products from A. alternata apple pathotype that showed high similarity to other fungal peptide synthetases and were specific to the apple pathotype. Screening of a Lambda Zap genomic library with these PCR-generated probes identified overlapping clones containing a complete cyclic peptide synthetase gene of 13.1 kb in length with no introns. Disruption of this gene, designated AM-toxin synthetase (AMT), by transformation of wild-type A. afternata apple pathotype with disruption vectors resulted in toxin-minus mutants, which were also unable to cause disease symptoms on susceptible apple cultivars. AM-toxin synthetase is therefore a primary determinant of virulence and specificity in the A. alternata apple pathotype/apple interaction.