Rapid and Scalable DNA Extraction and Real-Time PCR Assay from Boxwood Tissue for the Detection of Calonectria pseudonaviculata, Causal Agent of Boxwood Blight.

Boxwood blight can be challenging to detect in the field, especially when symptoms are mild, thus requiring large numbers of plants to be screened. Therefore, a rapid diagnostic assay that can detect the pathogen from large amounts of plant tissue would be useful. Here, we present a crude DNA extraction protocol that is rapid and scalable. The DNA extraction protocol can process large volumes of tough boxwood tissue rapidly without using cetyltrimethylammonium bromide or phenol-chloroform to remove inhibitors. Additionally, to detect the boxwood blight pathogen Calonectria pseudonaviculata, we developed a TaqMan probe to use with previously described PCR primers for a real-time PCR assay. The assay's limit of detection was determined by diluting symptomatic boxwood leaves in nondiseased leaves and by adding spores to nondiseased leaves to simulate diagnostic scenarios. The assay was able to detect the pathogen in symptomatic leaves diluted up to 1 × 104- to 1 × 105-fold in nondiseased leaves and from as low as 1,000 to 10,000 spores added to 1.2 g of nondiseased leaves. The ability to extract DNA from large volumes of plant tissue facilitates screening more plant tissue using the real-time PCR assay without increasing the number of samples to process in the lab.

JSAP1/JIP3 and JLP regulate kinesin-1-dependent axonal transport to prevent neuronal degeneration.

Axonal transport is critical for neuronal development and function, and defective axonal transport has been implicated in neurodegenerative diseases. However, how axonal transport is regulated, or how defective transport leads to neuronal degeneration, remains unclear. Here, we report that c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1, also known as JNK-interacting protein 3 (JIP3)) and JNK-associated leucine zipper protein (JLP) are essential for postnatal brain development. Mice with a double-knockout (dKO) in Jsap1 and Jlp in the dorsal telencephalon developed progressive neuron loss. Using a primary neuron culture system with induced disruption of targeted genes, combined with gene rescue experiments, we show that JSAP1 and JLP regulate kinesin-1-dependent axonal transport with functional redundancy. We also show that the binding of JSAP1 and JLP to kinesin-1 heavy chain is crucial for interactions between kinesin-1 and microtubules. Furthermore, we describe a molecular mechanism by which defective kinesin-1-dependent axonal transport in Jsap1:Jlp dKO neurons causes axonal degeneration and subsequent neuronal death. JNK hyperactivation because of increased intra-axonal Ca(2+) in the Jsap1:Jlp dKO neurons was found to mediate both the axonal degeneration and neuronal death, in cooperation with the Ca(2+)-dependent protease calpain. Our results indicate that axonal JNK may relocate to the nucleus in a dynein-dependent manner, where it activates the transcription factor c-Jun, resulting in neuronal death. Taken together, our data establish JSAP1 and JLP as positive regulators of kinesin-1-dependent axonal transport, which prevents neuronal degeneration.

Improvement of emotional healthcare system with stress detection from ECG signal.

Our emotional healthcare system is designed to cope with users' negative emotions in daily life. To make the system more intelligent, we integrated emotion recognition by facial expression to provide appropriate services based on user's current emotional state. Our emotion recognition by facial expression has confusion issue to recognize some positive, neutral and negative emotions that make the emotional healthcare system provide a relaxation service even though users don't have negative emotions. Therefore, to increase the effectiveness of the system to provide the relaxation service, we integrate stress detection from ECG signal. The stress detection might be able to address the confusion issue of emotion recognition by facial expression to provide the service. Indeed, our results show that integration of stress detection increases the effectiveness and efficiency of the emotional healthcare system to provide services.

Pretreatment of donor islets with the Na(+) /Ca(2+) exchanger inhibitor improves the efficiency of islet transplantation.

Pancreatic islet transplantation is an attractive therapy for the treatment of insulin-dependent diabetes mellitus. However, the low efficiency of this procedure necessitating sequential transplantations of islets with the use of 2-3 donors for a single recipient, mainly due to the early loss of transplanted islets, hampers its clinical application. Previously, we have shown in mice that a large amount of HMGB1 is released from islets soon after their transplantation and that this triggers innate immune rejection with activation of DC, NKT cells and neutrophils to produce IFN-γ, ultimately leading to the early loss of transplanted islets. Thus, HMGB1 release plays an initial pivotal role in this process; however, its mechanism remains unclear. Here we demonstrate that release of HMGB1 from transplanted islets is due to hypoxic damage resulting from Ca(2+) influx into ß cells through the Na(+) /Ca(2+) exchanger (NCX). Moreover, the hypoxia-induced ß cell damage was prevented by pretreatment with an NCX-specific inhibitor prior to transplantation, resulting in protection and long-term survival of transplanted mouse and human islets when grafted into mice. These findings suggest a novel strategy with potentially great impact to improve the efficiency of islet transplantation in clinical settings by targeting donor islets rather than recipients.

Relaxant effect of adrenomedullin on bovine isolated iris sphincter muscle under resting conditions.

1. The mechanisms involved in the fine adjustment of iris sphincter muscle tone are largely unknown. The aim of the present study was to clarify the effects of adrenomedullin on the resting tension of the bovine isolated iris sphincter muscle. 2. The motor activity of the bovine isolated iris sphincter muscle was measured isometrically. The effects of adrenomedullin on resting tension were analysed in the presence of indomethacin. The presence of adrenomedullin mRNA in the preparation was determined by reverse transcription-polymerase chain reaction. Immunolabelling for adrenomedullin was also performed. 3. Adrenomedullin significantly decreased the resting tension of the muscle. The relaxant effect of adrenomedullin was significantly inhibited by adrenomedullin (22-52), a putative antagonist for the adrenomedullin receptor, or calcitonin gene-related peptide (CGRP) (8-37), a putative antagonist for the CGRP1 receptor. The relaxant effect was almost completely blocked by a combination of adrenomedullin (22-52) and CGRP (8-37). 4. The relaxant effect of adrenomedullin was also significantly diminished by 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, N(G)-nitro-L-arginine, an inhibitor of nitric oxide synthesis, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. 5. Reverse transcription-polymerase chain reaction analysis showed that adrenomedullin mRNA was expressed in the muscle strip. Immunopositive staining for adrenomedullin was detected in blood vessel cells and in the iris sphincter muscle cells. 6. These results suggest that adrenomedullin may be an autocrine and paracrine regulator of the resting tension of the iris sphincter muscle. Its biological effects may be due to the direct involvement of adrenomedullin receptors and also to the stimulation of CGRP1 receptors. The stimulation of these receptors by the peptide leads to the activation of adenylate cyclase and soluble guanylate cyclase and subsequent relaxation of the muscle strip.

Japanese collaborative study for fibrinogen assay: variability of the fibrinogen assay between different laboratories does not improve when a common calibrator is used.

Several national and local external quality assurance schemes have been developed to improve the plasma fibrinogen assay in Japan over the past 30 years. Now most commercial calibrant plasma may be calibrated against an International Standard preparation, in order to achieve agreement of results obtained by different laboratories. However, we have never achieved satisfactory results, according to an external quality control survey regarding the fibrinogen assay. Therefore, we distributed two kinds of fibrinogen standards to be used as common calibrators, along with three plasma samples, among 183 general laboratories in Japan. The results of this collaborative study showed that the assigned value for the commercially available calibrators remained problematic. Furthermore, it was concluded that the between-laboratory variability could not be improved beyond a certain degree of standardization, even if a common calibrator was used for the Clauss-derived assay carried out by an automatic coagulometer.

Involvement of serotonergic and dopaminergic mechanisms in hyperthermia induced by a serotonin-releasing drug, p-chloroamphetamine in mice.

Serotonergic and dopaminergic involvement in hyperthermia induced by a serotonin (5-hydroxytryptamine, 5-HT)-releasing drug, p-chloroamphetamine, was investigated in mice. Neither the 5-HT transporter inhibitor fluoxetine nor the 5-HT depleter p-chlorophenylalanine affected p-chloroamphetamine-induced hyperthermia. The dopamine depleter alpha-methyl-p-tyrosine significantly reduced p-chloroamphetamine-induced hyperthermia. The dopamine D(1) receptor antagonist 7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390) antagonized p-chloroamphetamine-induced hyperthermia, although the dopamine D(2) receptor antagonist sulpiride was without effect. These results indicate that p-chloroamphetamine-induced hyperthermia in mice is mediated by dopamine release followed by activation of the dopamine D(1) receptor.

Establishment of CD7+ human myeloma sister cell lines, KMS-21-PE and KMS-21-BM, carrying t(11;14) and t(8;14).

Two new human myeloma cell lines were established from pleural effusion and bone marrow malignant cells derived from a single patient, who manifested hyperammonemia associated with multiple myeloma, and these were characterized. Both lines possess t(11;14)(q13;q32) and t(8;14)(q24;q32) reciprocal translocations and overexpress cyclin D1, but not c-myc. Human myeloma lines including these new lines produced and secreted excess ammonia into culture medium more than non-myelomatous hematological cell lines. In addition, these two lines were revealed to have high surface CD7 expression correlated with relatively high mRNA expression by MP-RT-PCR. Among 8 human myeloma lines, half of them revealed significant surface expression of CD7 and a positive correlation between expression levels of protein and message. CD7 message was also detected in surface negative lines. Consequently, there may be posttranslational regulation of the CD7 molecule, whose cellular biological role in expressing cells has not been elucidated.

Effects of the 5-HT2 receptor antagonist, ritanserin on hyperthermia and depletion of 5-HT in frontal cortex induced by a 5-HT releasing drug, p-chloroamphetamine (PCA) in mice.

Effects of the 5-HT2 receptor antagonist, ritanserin on hyperthermia and depletion of 5-HT induced by the 5-HT-releasing drug, p-chloroamphetamine (PCA) were investigated. Ritanserin significantly suppressed PCA-induced hyperthermia in mice. PCA elicited decreases in 5-HT levels in the mouse frontal cortex. 5-HT reduction elicited by PCA was also attenuated by pretreatment with ritanserin. Since hyperthermia facilitates neurotoxicity induced by amphetamine analogue, ritanserin may inhibit PCA-induced 5-HT neurotoxicity by inhibiting hyperthermia.

Role of 5-HT(2) receptor subtypes in depletion of 5-HT induced by p-chloroamphetamine in the mouse frontal cortex.

A serotonin (5-hydroxytryptamine, 5-HT)-releasing drug, p-chloroamphetamine elicited decreases in 5-HT levels in the mouse frontal cortex. 5-HT reduction elicited by p-chloroamphetamine was inhibited by the 5-HT(2A/2B/2C) receptor antagonist, LY 53857 and the 5-HT(2A) receptor antagonist, ketanserin. However, the 5-HT(2B/2C) receptor antagonist, SB 206553, enhanced it. LY 53857 and ketanserin can inhibit hyperthermia elicited by p-chloroamphetamine, although SB 206553 enhances it. The effects of the 5-HT(2) receptor antagonists on neurotoxicity are very similar to those on hyperthermia. Since hyperthermia facilitates neurotoxicity induced by amphetamine analogue, these 5-HT(2) receptor antagonists may modify 5-HT depletion induced by p-chloroamphetamine through responses to body temperature.

A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein.

Recently, several groups have developed green fluorescent protein (GFP)-based Ca(2+) probes. When applied in cells, however, these probes are difficult to use because of a low signal-to-noise ratio. Here we report the development of a high-affinity Ca(2+) probe composed of a single GFP (named G-CaMP). G-CaMP showed an apparent K(d) for Ca(2+) of 235 nM. Association kinetics of Ca(2+) binding were faster at higher Ca(2+) concentrations, with time constants decreasing from 230 ms at 0.2 microM Ca(2+) to 2.5 ms at 1 microM Ca(2+). Dissociation kinetics (tau approximately 200 ms) are independent of Ca(2+) concentrations. In HEK-293 cells and mouse myotubes expressing G-CaMP, large fluorescent changes were observed in response to application of drugs or electrical stimulations. G-CaMP will be a useful tool for visualizing intracellular Ca2+ in living cells. Mutational analysis, together with previous structural information, suggests the residues that may alter the fluorescence of GFP.

Involvement of the 5-HT(2) receptor in hyperthermia induced by p-chloroamphetamine, a serotonin-releasing drug in mice.

The effects of a serotonin (5-hydroxytryptamine, 5-HT)-releasing drug, p-chloroamphetamine (PCA), on body temperature were investigated in mice. PCA induced hyperthermia in mice. PCA-induced hyperthermia was inhibited by the 5-HT(2A/2B/2C) receptor antagonist, 4-isopropyl-7-methyl-9-(2-hydroxy-1-methyl-propoxycarbonyl)-4,6A,7 , 8,9,10,10A-octahydro-indolo[4,3-FG]quinolone maleate (LY53857). The 5-HT(2A) receptor antagonist, ketanserin, reduced the PCA-induced hyperthermia, while the 5-HT(2B/2C) receptor antagonist, N-3-pyridinyl-3,5-dihydro-5-methyl-benzo[1,2-b:4, 5-b']dipyrrole-1(2H)-carboxamide (SB 206553), enhanced it. LY 53857, ketanserin and SB 206553 did not affect hyperactivity in mice treated with PCA. These results suggest that PCA-induced hyperthermia in mice is mediated by 5-HT(2A) receptors and is not related to changes in locomotor activity.

Cardiac tumor biopsy under the guidance of intracardiac echocardiography.

Transthoracic echocardiography or transesophageal echocardiography is sometimes useful in intracardiac tumor biopsy. Intracardiac echocardiography was used as an alternative to either of these for performing a biopsy of a right cardiac tumor in a 79-year-old woman. The procedure was well tolerated and no complications occurred. Histopathological findings and immunohistological staining were compatible with the diagnosis of neurogenic sarcoma.

Identification of 30 kDa protein for Ca(2+) releasing action of myotoxin a with a mechanism common to DIDS in skeletal muscle sarcoplasmic reticulum.

The molecular mechanism of Ca(2+) release by myotoxin a (MTYX), a polypeptide toxin isolated from the venom of prairie rattlesnakes (Crotalus viridis viridis), was investigated in the heavy fraction of sarcoplasmic reticulum (HSR) of rabbit skeletal muscles. [(125)I]MYTX bound to four HSR proteins (106, 74, 53 and 30 kDa) on polyvinylidene difluoride (PVDF) membrane. DIDS, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, bound predominantly to 30 kDa protein on the PVDF membrane, the molecular weight of which was similar to one of the MYTX binding proteins. The maximum (45)Ca(2+) release induced by caffeine (30 mM) was further increased in the presence of MYTX (10 microM) or DIDS (30 microM), whereas that induced by DIDS (30 microM) was not affected by MYTX (10 microM). MYTX inhibited [(3)H]DIDS binding to HSR in a concentration-dependent manner. Furthermore, [(125)I]MYTX binding to 30 kDa protein was inhibited by DIDS in a concentration-dependent manner. These results suggest that MYTX and DIDS release Ca(2+) from HSR in a common mechanism. The 30 kDa protein may be a target protein for the Ca(2+) releasing action of MYTX and DIDS.

A new scorpion toxin (BmK-PL) stimulates Ca2+-release channel activity of the skeletal-muscle ryanodine receptor by an indirect mechanism.

A peptide toxin isolated from the Chinese scorpion Buthus martensi Karsch (BmK-PL) stimulated Ca2+-release channel activity in both triad membranes and reconstituted ryanodine receptors partially purified from rabbit skeletal muscle. In [3H]ryanodine binding experiments, the toxin increased the affinity of ryanodine for the receptor, from a Kd of 24.3 nM to 2.9 nM, which is an enhancement similar to that seen with known receptor activators, such as ATP and high concentrations of KCl. In contrast, toxin enhancement was not observed with purified receptors, although intrinsic binding activity and stimulation by the conventional receptor activators were retained. In single channel recordings of Ca2+-release activity, the toxin increased the open channel probability (Po) from 0.019 to 0.043 (226% of control) in triad preparations. Further toxin enhancement of Po from 0.07 to 0.37 (529% of control) was observed using partially-purified receptors in the presence of ATP. When purified receptors were assayed in the presence of ATP, however, they showed a high value of Po (0.33) and no further increase was observed following application of the toxin. Results derived from two different experimental methods consistently suggest that a molecule(s) required for toxin-induced enhancement is absent from the purified receptor preparation. Western blot analysis of receptors prepared using three different protocols showed that triadin was missing from the purified receptor preparation. The scorpion toxin minimally enhanced Ca2+-release channel activity of cardiac preparations. From these results, we conclude that the toxin preferentially increases the activity of skeletal-muscle ryanodine receptors by an indirect mechanism, possibly binding to associated protein molecule(s). Triadin is a strong candidate for such a molecule.

Anthophyllite exposure and endemic pleural plaques in Kumamoto, Japan.

OBJECTIVES: This study explored the high prevalence of pleural plaques in the town of Matsubase in Kumamoto, Japan. METHODS: Small-size chest X-ray film was used for screening, and all persons with pleural plaques were confirmed by computed tomography (CT). The prevalence rate of pleural plaques in the 4 districts of Matsubase and its surrounding towns and cities were also examined. The age-adjusted mortality rate for lung cancer in this town was compared with that of its surrounding towns and cities. RESULTS: Pleural plaques were found in 1357 persons (724 men and 633 women) among the inhabitants who were more than 20 years of age in Matsubase between 1988 and 1993. CT scans ascertained 938 cases with pleural plaques among the 11 14 persons who participated. Thus at least 9.5% of the inhabitants over 20 years of age in this town had pleural plaques. The neighboring towns had a higher rate than the more distant towns. A large-scale open-cast asbestos mine and mill had been in operation in Matsubase between 1883 and 1970. Mineral analysis revealed anthophyllite fibers. Most of the plaques were found in persons who had never worked in the mine or mill. CONCLUSIONS: The high prevalence of pleural plaques in Matsubase was due to anthophyllite exposure, mainly environmental. No mesotheliomas were found, however. These findings agree with those from an earlier study from Finland.

Properties of specific binding site of myotoxin a, a powerful convulsant, in brain microsomes.

Myotoxin a, a small basic polypeptide from prairie rattlesnakes (Crotalus viridis viridis), induces myonecrosis and binds to a single class of binding sites in skeletal muscle sarcoplasmic reticulum. In the present study, [125I]myotoxin a with a high specific activity was prepared and it was shown to bind mainly to microsomes in rat whole brain. [125I]Myotoxin a was further shown to bind to microsomes prepared from all regions tested in brain. Its specific binding to whole brain microsomes was of approximately 1.9 times lower affinity (KD = 0.76 microM; Bmax = 13.1 nmol/mg) than that to skeletal muscle sarcoplasmic reticulum. [125I]Myotoxin a binding to brain microsomes was displaced by unlabeled myotoxin a with an IC50 value of 4.5 microM. [125I]Myotoxin a binding was markedly reduced by treatment of microsomes with trypsin, suggesting that the binding site of [125I]myotoxin a is partially proteins. The binding was significantly inhibited by Mg2+ at concentrations above 1 mM. Having looked at several drugs, we noted that [125I]myotoxin a binding was noncompetitively inhibited by spermine, whereas it was enhanced by heparin. On the other hand, the i.c.v. injection of myotoxin a in mice induced potent convulsive effects at 0.05 nmol/mouse or more. This paper is the first to show that the specific binding site of myotoxin a is present in mouse brain and that myotoxin a is a novel peptidic convulsant in mice.

Dual regulation of the skeletal muscle ryanodine receptor by triadin and calsequestrin.

Triadin, a calsequestrin-anchoring transmembrane protein of the sarcoplasmic reticulum (SR), was successfully purified from the heavy fraction of SR (HSR) of rabbit skeletal muscle with an anti-triadin immunoaffinity column. Since depletion of triadin from solubilized HSR with the column increased the [3H]ryanodine binding activity, we tested a possibility of triadin for a negative regulator of the ryanodine receptor/Ca2+ release channel (RyR). Purified triadin not only inhibited [3H]ryanodine binding to the solubilized HSR but also reduced openings of purified RyR incorporated into the planar lipid bilayers. On the other hand, calsequestrin, an endogenous activator of RyR [Kawasaki and Kasai (1994) Biochem. Biophys. Res. Commun. 199, 1120-1127; Ohkura et al. (1995) Can. J. Physiol. Pharmacol. 73, 1181-1185] potentiated [3H]ryanodine binding to the solubilized HSR. Ca2+ dependency of [3H]ryanodine binding to the solubilized HSR was reduced by triadin, whereas that was enhanced by calsequestrin. Interestingly, [3H]ryanodine binding to the solubilized HSR potentiated by calsequestrin was reduced by triadin. Immunostaining with anti-triadin antibody proved that calsequestrin inhibited the formation of oligomeric structure of triadin. These results suggest that triadin inhibits the RyR activity and that RyR is regulated by both triadin and calsequestrin, probably through an interaction between them. In this paper, triadin has been first demonstrated to have an inhibitory role in the regulatory mechanism of the RyR.

[Werdnig-Hoffmann disease type I with progressive ophthalmoplegia and ptosis].

Although pathological changes are observed in both the oculomotor nucleus and abducens nucleus in autopsied cases of infantile progressive spinal muscular atrophy, external and internal ocular palsy and ptosis have not been previously reported clinically. We presented here two long-surviving cases on respirators which gradually developed ophthalmoplegia and ptosis were presented. From our observation of these cases, it was suggested that there are certain periods of latencies between the occurrence of pathological changes and their clinical manifestation and that the lack of clinical signs of upper cranial nerve involvement in cases with Werdnig-Hoffmann type I is due to their short survival length.

Properties of Ca++ release induced by puff adder lectin, a novel lectin from the snake Bitis arietans, in sarcoplasmic reticulum.

Puff adder lectin (PAL), a novel lectin venom purified from Bitis arietans, induced Ca++ release from the heavy fraction (HSR) but not from the light fraction of skeletal muscle sarcoplasmic reticulum with EC50 congruent to 10 microM. The potency of PAL was approximately 200-fold higher than that of caffeine. The bell-shaped profile of Ca++ dependence for PAL was almost the same as that for myotoxin a (MYTX), a peptide Ca++ releaser, but was different from that for caffeine. Typical blockers of Ca++ release channels, such as Mg+2, procaine, ruthenium red and ryanodine, markedly reduced PAL-induced Ca++ release from HSR. Interestingly, PAL inhibited 125I-MYTX binding to HSR with IC50 approximately equal to 20 microM. Scatchard analysis revealed that the mode of inhibition by PAL was noncompetitive, which suggests that PAL binds to a different site from that of MYTX. PAL did not affect 3H-ryanodine binding to HSR. These results suggest that PAL binds to a different site from that of MYTX to cause Ca++ release from HSR with novel properties.