Developmental toxicity potential of grayanotoxin I in mice and chicks

Developmental toxicity potential of grayanotoxin I (GTX I), a toxic diterpenoid contained in plants of the family Ericaceae, with sodium ionophore activity, was studied in mice and chicks. In mice, intraperitoneal injections of 1.5 mg GTX I/kg body weight of mouse for three consecutive days during the organogenetic period caused some dams to die, but neither embryotoxicity nor teratogenicity was detected. In chicks, a single injection of 0.1-1.0 microgram GTX I per egg into the extraembryonic coelom on day 1.5 or 2 of incubation, or into the amnionic cavity on day 3 or 4 of incubation induced neither embryotoxic nor teratogenic signs, but 10 micrograms GTX I per egg showed lethal effects when applied on each of those days.     

Effects of iodine-enriched egg (IE-egg) on nasal allergy: basic and clinical investigations

The effect of iodine-enriched egg (IE-egg) on nasal allergy was investigated using an experimental allergic model. In addition, the effect of IE-egg was investigated using patients with yearly nasal allergy. IE-egg could suppress the leakage of pontamine sky blue dye in the experimental allergic model. beta-Glucuronidase activity in the perfusate was suppressed with the ingestion of IE-egg. The symptoms of the patients with yearly nasal allergy were mitigated by the ingestion of IE-egg. beta-Glucuronidase activity in the pituita of nasal allergic patients tended to be decreased.     

Block of sodium channels by tyramine and its analogue (N-feruloyl tyramine) in frog ventricular myocytes.

Pharmacological effects of tyramine and its analogue, N-feruloyl tyramine (NFT), on sodium and calcium currents in frog ventricular myocytes were examined using the whole-cell voltage-clamp technique. To improve the temporal and spatial control of the membrane potential, sodium currents (INa) were recorded in 45.5 mM [Na+]o at 10 degrees C. Both tyramine and NFT (1-100 microM) induced a concentration-dependent decrease in INa evoked from a holding potential of -80 mV without affecting a change in either the time to peak or the time constant for the falling phase of INa. Similarly the reversal potential for INa remained unchanged at a value close to that predicted from the Nernst equation. The finding that both tyramine and NFT decreased INa when activated maximally, from a holding potential of -120 mV, indicates that the amplitude of INa can be reduced independently of a change in the kinetics of the current. In addition, tyramine (100 microM) shifted the membrane potential for half maximal inactivation (Vh) of the steady-state inactivation (h infinity)-curve from -74 to -84 mV without affecting its slope. In contrast, NFT failed to affect the h infinity-curve. The calcium current (ICa) recorded in the presence of 0.3 microM TTX was not affected by either 100 microM tyramine or NFT. We concluded that tyramine directly blocks Na channel by shifting h infinity-curve and by suppressing maximum Na channel conductance, while NFT suppresses only maximum Na channel conductance.     

Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis

Pulmonary lymphangioleiomyomatosis (LAM) is a destructive lung disease characterized by a diffuse hamartomatous proliferation of smooth muscle cells (LAM cells) in the lungs. Pulmonary LAM can occur as an isolated form (sporadic LAM) or in association with tuberous sclerosis complex (TSC) (TSC-LAM), a genetic disorder with autosomal dominant inheritance with various expressivity resulting from mutations of either the TSC1 or TSC2 gene. We examined mutations of both TSC genes in 6 Japanese patients with TSC-LAM and 22 patients with sporadic LAM and identified six unique and novel mutations. TSC2 germline mutations were detected in 2 (33.3%) of 6 patients with TSC-LAM and TSC1 germline mutation in 1 (4.5%) of 22 sporadic LAM patients. In accordance with the tumor-suppressor model, loss of heterozygosity (LOH) was detected in LAM cells from 3 of 4 patients with TSC-LAM and from 4 of 8 patients with sporadic LAM. Furthermore, an identical LOH or two identical somatic mutations were demonstrated in LAM cells microdissected from several tissues, suggesting LAM cells can spread from one lesion to another. Our results from Japanese patients with LAM confirmed the current concept of pathogenesis of LAM: TSC-LAM has a germline mutation but sporadic LAM does not; sporadic LAM is a TSC2 disease with two somatic mutations; and a variety of TSC mutations causes LAM. However, our study indicates that a fraction of sporadic LAM can be a TSC1 disease; therefore, both TSC genes should be examined, even for patients with sporadic LAM. Received: August 30, 2001 / Accepted: November 2, 2001     

Feasibility of using decades-old archival tissues in molecular oncology/epidemiology.

Archival tissues are a bountiful resource for various studies. Polymerase chain reaction permits the use of such tissues for molecular biological analyses of disease causation. However, a comprehensive study using a large number of decades-old samples (20 or more years) for molecular oncology/epidemiology has never been shown to be feasible. We have relied upon the unique tumor registry of atomic bomb survivors to show that such studies are possible using 275 hepatocellular carcinoma and 41 skin cancer cases. We used 23 relatively recent thyroid papillary carcinoma cases from persons living in the vicinity of the Chernobyl nuclear reactor accident for comparison. Degradation of DNA is severe in autopsy hepatocellular carcinoma samples but can be compensated for by decreasing the polymerase chain reaction product size. Increasing the amount of DNA that is used by a factor of 8 improved amplification efficiency from approximately 60 to 80%. Age of the samples was not as great a problem as was the source of procurement. The extracted DNA can be used for all types of assays that require polymerase chain reaction amplification, such as restriction fragment length polymorphism, single-strand conformation polymorphism, and direct sequencing.     

Mechanism of nerve membrane depolarization caused by grayanotoxin I

1. The mechanism of depolarization of squid axon membranes caused by grayanotoxin I has been studied by means of internal perfusion and voltage clamp techniques.2. The depolarization induced by either internal or external application of grayanotoxin I was reversed by decreasing the external sodium concentration from 449 to 1 mm.3. No depolarization was observed when both external and internal media were devoid of sodium ions, indicating that the depolarization by grayanotoxin I in normal media is due to a specific increase in resting sodium permeability.4. The resting sodium permeability as measured by voltage clamp was increased to 1.31 x 10(-6) cm/sec by internal application of 1 x 10(-5)m grayanotoxin I, an increase by a factor of about 90.5. The apparent dissociation constant of internally applied grayanotoxin I in increasing the resting sodium permeability was estimated to be 4.12 x 10(-5)m, and the toxin interacts with the membrane receptor on a one-to-one stoichiometric basis.6. Tetrodotoxin antagonized the action of grayanotoxin I in increasing the resting sodium permeability in a non-competitive manner.     

Establishment of 2 human thyroid-carcinoma cell-lines (8305c, 8505c) bearing p53 gene-mutations

New cell lines, designated 8305C and 8505C, were established from undifferentiated thyroid carcinomas of a 67 year-old-female patient and a 78-year-old-female patient, respectively. Pathologically both these primary undifferentiated carcinoma tissues contained residual well differentiated components, suggesting well differentiated to undifferentiated carcinoma progression. Cell kinetic analysis indicate that the cell population doubling time is 43 h for 8305C and 36 h for 8505C. The saturation density at confluency is 5.7 x 10(4) cells/cm2 for 8305C and 1.1 x 10(5) cells/cm2 for 8505C. To identify genetic changes that may have occurred in these two cell lines, tumor suppressor genes p53, Rb, APC and MCC were analyzed. Sequence analysis confirmed a C:G to T:A transition at the first base of p53 gene codon 273 in 8305C and a C:G to G:C transversion at the first base of p53 codon 248 in 8505C. Polymerase chain reaction-loss of heterozygosity assays confirmed allelic deletion of p53 gene from the 8505C cell line. Loss of heterozygosity of other tumor suppressor genes were not observed. Given that p53 mutations associate with undifferentiated carcinoma but not with well differentiated carcinoma during multistep carcinogenesis of the thyroid, these cell lines should prove useful for research into the role of p53 gene mutations in malignant transformation.     

Studies on iodinated compounds. X. Isolation and purification of iodoglycyltyrosines]

The methods for the isolation and purification of iodoglycyltyrosines [glycyl-3-iodotyrosine (Gly-MIT) and glycyl-3,5-diiodotyrosine (Gly-DIT)] from a reaction mixture were examined by the use of high-performance liquid chromatography (HPLC). Glycyltyrosine (Gly-Tyr) was iodinated with iodine monochloride (ICl) (the molar ratio of Gly-Tyr to ICl was 1:1.5) in 0.1 M NaOH. The synthesized Gly-MIT and Gly-DIT were separated on a mu Bondapak C18 column employing stepwise gradient systems of a 0.1% trifluoroacetic acid/acetonitrile mixture and a water/acetonitrile mixture. Chemically pure Gly-MIT and Gly-DIT were obtained in 30.2% and 28.2% yields, respectively.     

Survey of implementation of the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines in nine Asian countries

After the release of the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines in 2001 and update in 2003, its implementation in nine Asian countries was investigated. Questionnaire surveys involving thoraco-pulmonary physicians or internists investigated the awareness and consensus of the GOLD guidelines including the care and management of chronic obstructive pulmonary disease (COPD) patients in Asian. Two surveys were conducted, in June 2002 and March 2004, through questionnaires by direct mail in Japan and face-to-face interviews in the other countries. Approximately 600 questionnaires were returned with approximately 84% awareness of the publication and its update and nearly 90% appreciated the globalization efforts. The survey revealed great variances concerning the definition of COPD, its diagnosis, and use of computed tomography. As for the implementation, the majority answered the use of the combined local and GOLD guidelines in five of nine countries surveyed, while the GOLD guidelines were implemented mainly in Korea, suggesting the influence in daily practice for care and management of COPD patients. Implication of rehabilitation in clinical practice has not been standardized despite high evidence of its advantages. Most respondents stated the necessity of developing a local or regional guideline for best practice. Our survey revealed: (i) awareness of the GOLD guidelines was high and well accepted; (ii) the possibility of developing a uniform or standard guideline in Asia is low due to local characteristics; (iii) modifications of the GOLD guidelines may be more practical; and (iv) that the multidisciplinary pulmonary rehabilitation program needs to be further activated in GOLD implementation for the Asia–Pacific region.     

Hypothermia attenuates apoptosis and protects contact between myelin basic protein‐expressing oligodendroglial‐lineage cells and neurons against hypoxia–Ischemia

Periventricular leukomalacia (PVL) is a major form of brain injury among preterm infants, which is characterized by extensive loss and dysfunction of premyelinating oligodendrocytes (pre‐OLs) induced by hypoxia–ischemia (HI). Therapeutic hypothermia, which is a standard treatment for term infants with HI encephalopathy, is not indicated for preterm infants because its safety and effect have not been established. Here we investigate the effectiveness and mechanism of hypothermia for the inhibition of pre‐OLs damage in PVL. For in vivo studies, 6‐day‐old rats underwent left carotid artery ligation, followed by exposure to 6% oxygen for 1 hr under hypothermic or normothermic conditions. The loss of myelin basic protein (MBP) was inhibited by hypothermia. For in vitro studies, primary pre‐OLs cultures were subjected to oxygen–glucose deprivation (OGD) under normothermic or hypothermic conditions, and dorsal root ganglion neurons were subsequently added. Hypothermia inhibited apoptosis of pre‐OLs, and, despite specific downregulation of 21.5‐ and 17‐kDa MBP mRNA expression during hypothermia, recovery of the expression after OGD was superior compared with normothermia. OGD caused disarrangement of MBP distribution, decreased the levels of phosphorylated 21.5‐kDa MBP, and disturbed the capacity to contact with neurons, all of which were restored by hypothermia. Pharmacological inhibition of ERK1/2 phosphorylation with U0126 during and after OGD significantly reduced the protective effects of hypothermia on apoptosis and myelination, respectively. These data suggest that phosphorylated exon 2‐containing (21.5‐ and possibly 17‐kDa) MBP isoforms may play critical roles in myelination and that hypothermia attenuates apoptosis and preserves the contact between OLs and neurons via ERK1/2 phosphorylation. © 2014 Wiley Periodicals, Inc.