Isolation of Rhodococcus equi from the gastrointestinal contents of earthworms (family Megascolecidae).

Rhodococcus equi was isolated from the gastrointestinal contents of earthworms (family Megascolecidae) and their surrounding soil collected from pastures of two horse-breeding farms in Aomori Prefecture, outdoor pig pens, forest in Towada campus, orange groves and forest where wild boars (Sus scrofa) are established in Tanabe, Wakayama Prefecture. The number of R. equi in the lower gastrointestinal contents of 23 earthworms collected from our campus was significantly larger than that of the upper gastrointestinal content. The mean numbers of R. equi from the gastrointestinal contents of earthworms collected from the various places were 2·3-fold to 39·7-fold more than those of the surrounding soil samples. In all, 1771 isolates from the earthworms and 489 isolates from the soil samples were tested for the presence of vapA and vapB genes using polymerase chain reaction. At the horse-breeding farm N, 9 of the 109 isolates (8·3%) from the earthworms and 7 of the 106 isolates (6·6%) from the soil samples were positive for the vapA gene. At the University's forest, one of the 250 isolates (0·4%) from the gastrointestinal contents of the earthworm was positive for the vapB gene. These results revealed that R. equi can be found in significant quantities in the gastrointestinal contents of earthworms, suggesting that they act as an accumulator of R. equi in the soil environment and as a source or reservoir of animal infection.

Targeting the vulnerability to NAD+ depletion in B-cell acute lymphoblastic leukemia.

Although substantial progress has been made in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), the prognosis of patients with either refractory or relapsed B-ALL remains dismal. Novel therapeutic strategies are needed to improve the outcome of these patients. KPT-9274 is a novel dual inhibitor of p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyltransferase (NAMPT). PAK4 is a serine/threonine kinase that regulates a variety of fundamental cellular processes. NAMPT is a rate-limiting enzyme in the salvage biosynthesis pathway of nicotinamide adenine dinucleotide (NAD) that plays a vital role in energy metabolism. Here, we show that KPT-9274 strongly inhibits B-ALL cell growth regardless of cytogenetic abnormalities. We also demonstrate the potent in vivo efficacy and tolerability of KPT-9274 in a patient-derived xenograft murine model of B-ALL. Interestingly, although KPT-9274 is a dual PAK4/NAMPT inhibitor, B-ALL cell growth inhibition by KPT-9274 was largely abolished with nicotinic acid supplementation, indicating that the inhibitory effects on B-ALL cells are mainly exerted by NAD+ depletion through NAMPT inhibition. Moreover, we have found that the extreme susceptibility of B-ALL cells to NAMPT inhibition is related to the reduced cellular NAD+ reserve. NAD+ depletion may be a promising alternative approach to treating patients with B-ALL.

Joint Effect of Solar UVB and Heat Stress on the Seasonal Change of Egg Hatching Success in the Herbivorous False Spider Mite (Acari: Tenuipalpidae).

Seasonal population dynamics of an herbivorous mite has been documented in terms of the relationship between thermoresponses and temporal biological factors such as resource availability or predation risk. Although recent studies emphasize the deleterious effects of solar ultraviolet-B (UVB; 280-320 nm wavelengths) radiation on plant-dwelling mites, how UVB affects mite population remains largely unknown. On a wild shrub Viburnum erosum var. punctatum in Kyoto, an herbivorous false spider mite, Brevipalpus obovatus Donnadieu, occurs only in autumn. Females of this species lay one-third of their eggs on upper leaf surfaces. Oviposition on upper surfaces is beneficial for avoiding predation by phytoseiids, but exposes eggs to solar UVB and heat stress. To test the hypothesis that the seasonal occurrence of this mite is determined by interactions between solar UVB radiation and temperature, we examined variation in egg hatching success under near-ambient and UV-attenuated sunlight conditions from spring to autumn. The UV-attenuation significantly improved hatching success. However, most eggs died under heat stress regardless of UV treatments in July and August. We established a deterministic heat stress-cumulative UVB dose-egg hatching success response model, which we applied to meteorological data. The model analyses illustrated lower and higher survivability peaks in late May and October, respectively, which partly corresponded to data for annual field occurrence, indicating the importance of solar UVB radiation and heat stress as determinants of the seasonal occurrence of this mite.

KPT-330 has antitumour activity against non-small cell lung cancer.

BACKGROUND: We investigated the biologic and pharmacologic activities of a chromosome region maintenance 1 (CRM1) inhibitor against human non-small cell lung cancer (NSCLC) cells both in vitro and in vivo. METHODS: The in vitro and in vivo effects of a novel CRM1 inhibitor (KPT-330) for a large number of anticancer parameters were evaluated using a large panel of 11 NSCLC cell lines containing different key driver mutations. Mice bearing human NSCLC xenografts were treated with KPT-330, and tumour growth was assessed. RESULTS: KPT-330 inhibited proliferation and induced cell cycle arrest and apoptosis-related proteins in 11 NSCLC cells lines. Moreover, the combination of KPT-330 with cisplatin synergistically enhanced the cell kill of the NSCLC cells in vitro. Human NSCLC tumours growing in immunodeficient mice were markedly inhibited by KPT-330. Also, KPT-330 was effective even against NSCLC cells with a transforming mutation of either exon 20 of EGFR, TP53, phosphatase and tensin homologue, RAS or PIK3CA, suggesting the drug might be effective against a variety of lung cancers irrespective of their driver mutation. CONCLUSIONS: Our results support clinical testing of KPT-330 as a novel therapeutic strategy for NSCLC.

[Carcinomatous pericarditis successfully treated with thoracoscopic pericardial fenestration; report of a case].

We report a case of malignant pericardial effusion after lung cancer surgery treated with thoracoscopic pericardial fenestration. The patient was admitted to our hospital because of dyspnea. Computed tomography (CT) and ultrasound cardiography (UCG) revealed cardiac tamponade which was diagnosed as carcinomatous pericarditis by cytology. We attempted to inject 25 mg of cisplatin (CDDP) into the pericardial space twice. The response of the treatment was unexpectedly poor for the patient. The thoracoscopic pericardial fenestration was performed and the patient was discharged without a drainage tube on the 17th postoperative day. Malignant pericardial effusion is a common complication of advanced cancers and is often associated with significant morbidity. Thoracoscopic pericardial fenestration appears to be a safe, effective and minimally invasive treatment for patients with malignant pericardial effusion. This surgery might also have a favorable effect on the improvement of the quality of life for patients with malignant pericardial effusion resistant to chemotherapy.

Long-term fluctuation and regional variation of nutrient loads from the atmosphere to lakes.

The atmospheric depositions were collected by ordinary bulk-samplers mounted with a glass or polyethylene funnel of 30 cm orifice. A long-term observation was carried out at a site (35 degrees 01'30"N, 135 degrees 58'07"E) in the urbanized area for 21 years since 1974. The annual volume-weighted average concentrations of total nitrogen (TN) and phosphorus (TP) were 1.02 +/- 0.30 and 0.031 +/- 0.015 mg(-1), respectively, and the loading rates were 14.5 +/- 2.8 and 0.43 +/- 0.16 kg ha(-1) yr(-1). The rates neither had a relationship with the precipitation (1,492 +/- 343 mm yr(-1)) nor showed any diachronic tendencies. In order to obtain the loading rate of TN and TP within 10% uncertainty under the significant level of 0.01, there is no way but to continue the observation for seven and eleven years or more, respectively. In order to clarify the regional variation of the loading amounts of the depositions, the samplers were set at 12 sites distributing throughout the Kinki District for two years. The distance from northernmost (2322 mm yr(-1)) to southernmost (1242 mm yr(-1)) is about 150 km. The average loading rates of the 12 sites were 16.2 +/- 2.5 kg ha(-1) yr(-1) of TN and 0.730 +/- 0.247 kg ha(-1) yr(-1) of TP. The depositions of NO3-N, NH4-N, and TN as well as TP showed tendencies with distances neither from a big city nor from Japan Sea.

Decrease in herbicide concentrations and affected factors in lagoons located around Lake Biwa.

The contamination levels and changes in the concentrations in four lagoons around Lake Biwa of paddy-use herbicide were studied. Four lagoons, Sone-numa (52 days of HRT (hydraulic residence time) estimated from the lagoon volume and the average discharge at the outlet, 21 ha area), Yanagihira-ko (40 days, 5.0 ha), Noda-numa (11 days, 6.0 ha), and Iba-naiko (2 days, 55.5 ha), were selected as monitoring sites. Intensive water sampling was carried out once a week from May to June at the outlet of each lagoon. Although twelve of the monitored herbicides were detected, the maximum concentrations did not exceed the guidelines for water-supply law in Japan. The relation between half-lives in herbicide concentrations and characteristics of a lagoon such as HRT and chlorophyll-a concentrations were examined. The shorter half-lives of herbicide concentrations in lagoons with shorter HRT means that replacement by influent water effectively decreased the pesticide concentrations. Shorter half-lives in lagoons with high chlorophyll-a concentrations between the lagoons with similar HRT suggest that biological degradation during the residence time worked more efficiently in the lagoon with high chlorophyll-a concentrations.

[Thoracoscopic lung biopsy in the diagnosis of diffuse lung disease].

The diagnosis of diffuse lung disease can only be made accurately by performing a thoracoscopic lung biopsy. We evaluated the safety and efficacy of thoracoscopic lung biopsy in diffuse lung disease. Between May 1999 and April 2003, 34 patients were referred to us for thoracoscopic lung biopsy. We compared the clinical characteristics, number of biopsies, operation times, postoperative course, complications, and mortality in 19 patients with dyspnea and 15 patients who were asymptomatic. Although the number of biopsies, operation times, duration of chest drainage, and postoperative stay did not differ significantly between the 2 groups, postoperative oxygen support was needed for significantly longer in the dyspnea group (p =0.038). A pathological diagnosis was established in all patients. Postoperative complications developed in 4 patients. A prolonged air leak in 1 patient and a wound infection in another resolved conservatively, but 2 patients died of acute exacerbation. Thus, we conclude that the indications for surgery must be carefully considered in dyspneic patients with diffuse lung to prevent acute exacerbation.

Unusual hypersensitivity to warfarin in a critically ill patient.

A patient was admitted to the intensive care unit because of respiratory failure, and warfarin therapy was started at 2 mg/day for the treatment of pulmonary embolism, together with other medications. Despite the low dosage of warfarin, international normalized ratio (INR) was markedly elevated from 1.15 to 11.28 for only 4 days, and bleeding symptoms concurrently developed. Vitamin K2 was infused along with discontinuation of warfarin. One day later, the INR was found to have decreased, and bleeding was also improved. An objective causality assessment indicated a probable relationship between clotting abnormality and warfarin administration, although the degree of elevation of the INR was unusual in the light of the daily warfarin dose and duration of its exposure. Based on the clinical status of the patient, it was suspected that several conditions contributed to the abnormal hypersensitivity to warfarin. Contributory factors probably included pharmacokinetic interactions with co-administrated drugs, vitamin K deficiency caused by decreased dietary intake, reduced gut bacterial production, impaired intestinal absorption and hepatic synthetic capacity, and increased consumption of clotting factors. In view of our experience in the present case, it should be stressed that close monitoring of coagulation capacity is necessary in critically ill patients in order to avoid fatal haemorrhage after initiating warfarin therapy regardless of the dosage.

Night-time restricted feeding normalises clock genes and Pai-1 gene expression in the db/db mouse liver.

AIMS/HYPOTHESIS: An increase in PAI-1 activity is thought to be a key factor underlying myocardial infarction. Mouse Pai-1 (mPai-1) activity shows a daily rhythm in vivo, and its transcription seems to be controlled not only by clock genes but also by humoral factors such as insulin and triglycerides. Thus, we investigated daily clock genes and mPai-1 mRNA expression in the liver of db/db mice exhibiting high levels of glucose, insulin and triglycerides. METHODS: Locomotor activity was measured using an infrared detection system. RT-PCR or in situ hybridisation methods were applied to measure gene expression. Humoral factors were measured using measurement kits. RESULTS: The db/ db mice showed attenuated locomotor activity rhythms. The rhythmic expression of mPer2 mRNA was severely diminished and the phase of mBmal1 oscillation was advanced in the db/db mouse liver, whereas mPai-1 mRNA was highly and constitutively expressed. Night-time restricted feeding led to a recovery not only from the diminished locomotor activity, but also from the diminished Per2 and advanced mBmal1 mRNA rhythms. Expression of mPai-1 mRNA in db/db mice was reduced to levels far below normal. Pioglitazone treatment slightly normalised glucose and insulin levels, with a slight reduction in mPai-1 gene expression. CONCLUSIONS/INTERPRETATION: We demonstrated that Type 2 diabetes impairs the oscillation of the peripheral oscillator. Night-time restricted feeding rather than pioglitazone injection led to a recovery from the diminished locomotor activity, and altered oscillation of the peripheral clock and mPai-1 mRNA rhythm. Thus, we conclude that scheduled restricted food intake may be a useful form of treatment for diabetes.

[Negative pleural cytology after intrapleural perfusion hyperthermo-chemotherapy for malignant pleural effusion].

We gave intrapleural perfusion hyperthermo-chemotherapy to a 72-year-old woman in whom malignant pleural effusion developed after surgery for primary cancer. This procedure involved irrigating the pleural space for 2 hours with a water solution at 42-43 degrees containing 240 mg cisplatin using specially devised extracorporeal circuits. Thoracoscopy was used to examine the intrapleural cavity and to place the catheters for perfusion. The patient had an uneventful postoperative course and was discharged for hospital on the second postoperative day. Thereafter, she experienced good quality of life with negative pleural cytology. Unfortunately, the patient died 3 months after the therapy, but the cause of death was unknown and there was no cancer recurrence. This technique may be safe and feasible for controlling malignant effusion to preserve quality of life, although the survival benefit has not yet been clarified.

Constant light housing attenuates circadian rhythms of mPer2 mRNA and mPER2 protein expression in the suprachiasmatic nucleus of mice.

Constant light (LL) or constant dark (DD) environmental lighting conditions cause a free-running period and activity reduction in the rodent behavioral circadian rhythm. In order to understand the molecular process underlying behavioral rhythms in LL or DD housing conditions, we examined the circadian profile of mPer2 mRNA and mPER2 in the suprachiasmatic nucleus (SCN), a main oscillator, of free-running mice. The circadian expression rhythm of mPer2 in the SCN was dampened under 7-day LL conditions, whereas that of mPER2 protein was moderately attenuated and its expression peak delayed. The circadian expression of mPer2 and its product was slightly attenuated and advanced by 7-day DD conditions. With arrhythmic behavioral activity caused by long-term LL housing, mPER2 protein lost its rhythmicity in the SCN. On the other hand, LL or DD housing did not affect the mPer2 gene and its product in the cerebral cortex. The present results suggest that mPER2 circadian expression in the SCN corresponds well with behavioral circadian oscillation under LL or DD conditions. Thus, the behavioral circadian rhythm seems to correlate with molecular clock works in the SCN.

[Subjective criteria of chest CT findings for predicting pathological features and postoperative outcomes of small peripheral lung cancer (< or = 2 cm)].

One hundred fifty five patients with completely resected peripheral non-small cell lung cancer, clinically diagnosed 2 cm or less in diameter, are retrospectively reviewed on their preoperative chest CT films, clinico-pathological features, and postoperative outcomes. Pathologic type was classified according to Noguchi's classification. 7% and 8% of all the patients had pathologic N 1 and N 2 diseases, respectively. 19% of all the patients undergone limited resection (segmentectomy or partial resection). Maximum area of the tumor/soft tissue density area of the tumor (M/S ratio) was manually measured by chest CT film. According to the logistic regression analysis, M/S ratio was the only predicting factor of regional lymph node metastasis among factors including pleural indentation, spiculation, and maximum area of the tumor. Univariate analysis showed that maximum area of the tumor, pleural indentation, and M/S ratio were the significant factor for postoperative disease free survival. According to multivariate analysis of postoperative disease free survival with adjustment for operative modality, the result was same as that of univariate analysis. In conclusion, our determined criteria of the chest CT accurately predicted pathological status and postoperative outcome of patients with small peripheral lung cancer. These factors would be useful for stratification factor of prospective clinical study.

Surgically treated primary lung cancer associated with Brugada syndrome: report of a case.

A 71-year-old man with primary lung cancer associated with Brugada syndrome was safely operated on following the placement of an implantable cardioverter defibrillator (ICD). During examinations for Brugada syndrome, a tumor in the apicoposterior segment of the left lung was incidentally detected by chest computed tomography. Following the implantation of an ICD, surgical treatment of the left lung tumor was scheduled. A lung biopsy was thoracoscopically performed and adenocarcinoma was diagnosed based on a frozen section analysis. A left upper lobectomy with lymph node dissection was performed through a standard posterolateral thoracotomy. Ventricular fibrillation, which occurred during the night of the first day following surgery, was successfully managed by the ICD.

Comparison of nutrient budgets between three forested mountain watersheds on granite bedrock.

The budgets of nutrients and major ions between the input flux from atmospheric deposition and the output flux to streams were measured on three forested small mountain watersheds, Aburahi-N (23.8 ha), Aburahi-S (3.8 ha, 2 km south from Aburahi-N) and Myokoji (1.77 ha, 28 km northwest from Aburahi-N), located at 34-35 N and 136 E around the central part of Japan. The bedrock of the watersheds is granite. The main vegetation of Aburahi-N and Aburahi-S was planted with Japanese cypress (Chamaecyparis obtusa Sieb. et Zucc), and Myokoji was a secondary forest growing deciduous broadleaf trees and Japanese red pine. The chemical analysis of the stream waters was carried out once a week at Aburahi-N from 1987 to 1991, at Aburahi-S from 1995 to 1998, and at Myokoji from 1991 to 1993. Wet and dry depositions were collected every month at Aburahi-N. The annual mean concentrations of total nitrogen (TN) of the stream water of Aburahi-N, Aburahi-S and Myokoji were 0.408, 0.614 and 0.349 mg/l, respectively, total phosphorus (TP) contents were 0.0074, 0.0046 and 0.0096 mg/l, and the potassium ion (K+) contents were 0.83, 0.49 and 0.77 mg/l. The average annual output fluxes of TN from the watersheds of Aburahi-N, Aburahi-S and Myokoji were calculated to be 5.85, 4.10 and 12.7 kg/ha/y, TP 0.131, 0.045 and 0.280 kg/ha/y, K+ 9.63, 3.23 and 9.70 kg/ha/y, and discharge 1062, 508 and 1265 mm/y, respectively. The annual outputs tended to increase as the annual precipitation increased. The average annual input fluxes of TN, TP and K+ for six years from 1990-1998 were 12.5, 1.31 and 7.64 kg/ha/y, respectively, and the precipitation averaged 1449 (1019-1947) mm/y.

Propofol activates vanilloid receptor channels expressed in human embryonic kidney 293 cells.

Propofol (2,6-diisopropylphenol) is an intravenous anesthetic agent structurally unrelated to any other intravenous anesthetics. We examined the effect of propofol on a rat vanilloid receptor that was expressed in the human embryonic kidney (HEK) 293 cells by using calcium imaging method. Propofol caused a concentration-dependent increase in [Ca(2+)](i) in the HEK293 cells with the receptor. These responses were inhibited by removing extracellular calcium ions. The propofol-evoked increase in [Ca(2+)](i) in the HEK293 cells with the receptor was partially inhibited by capsazepine, a competitive antagonist of capsaicin. We conclude that propofol acts as an agonist for the receptor.

Variable effects of nitrous oxide at multiple levels of the central nervous system in goats.

The direct and indirect effects of nitrous oxide (N2O) on the nociceptive responses of lumbar dorsal horn neurons, and the indirect effects on midbrain reticular formation (MRF) neurons and thalamic neurons were determined in goats anaesthetized with isoflurane. The technique used enabled the differential delivery of N2O to either the torso or the cerebral circulation, thus allowing assessment of the direct spinal and indirect brain effects of N2O. Systemic delivery of N2O appeared to have divergent effects, facilitating (4/11) or depressing (7/11) the responses of dorsal horn neurons. Such divergent effects were also observed when N2O was differentially delivered to the circulation in the torso (i.e. the spinal cord). Likewise, MRF and thalamic responses to noxious stimulation were variably affected by administration of N2O to the torso, with some cells facilitated (7/13 MRF neurons, 3/8 thalamic neurons) and others depressed (6/13 MRF neurons, 5/8 thalamic neurons). It appears that N2O has variable effects on the caprine CNS. The facilitatory action of N2O might partially explain why it is a relatively weak anaesthetic.

(1)H nuclear magnetic resonance spectroscopic analysis for determination of glucuronic and iduronic acids in dermatan sulfate, heparin, and heparan sulfate.

(1)H NMR spectroscopy has been established for the determination of uronate residues in glycosaminoglycans (GAGs) such as dermatan sulfate (DS), heparin (HP), and heparan sulfate (HS). Because of variation in the sulfonation positions in DS, HP, or HS, interpretation of spectra is difficult. Solvolysis was applied to remove O-sulfo groups from these GAG chains in dimethyl sulfoxide containing 10% methanol at 80 degrees C for 5 h. In the cases of HP and HS, N-sulfo groups on glucosamine residues were also removed under the same conditions. The resulting unsubstituted amino groups in HP and HS chains were re-N-acetylated using acetic anhydride to obtain homogeneous core structure with the exception of the variation of uronate residues. The contents of glucuronate and iduronate residues in the chemically modified DS, HP, and HS samples were analyzed by 600-MHz (1)H NMR spectroscopy. These methods were applied to compositional analysis of uronate residues in GAGs isolated from various sources.

Involvement of thioredoxin in the regulation of growth hormone secretion in rat pituitary cell cultures.

We report here an examination of the effect of thioredoxin (TRX) on the secretion of growth hormone (GH) from rat anterior pituitary cells in vitro. Treatment of rat pituitary cells with growth hormone-releasing factor (GRF), but not GH, led to a significant increase in intracellular TRX protein levels. GRF, recombinant human TRX (rhTRX), and a combination thereof were all shown to induce immediate GH secretion from pituitary cells, as evidenced by perifusion experiments. RhTRX, but not other reducing agents such as beta-mercaptoethanol and N-acetyl-L-cysteine, augmented GRF-stimulated and -unstimulated GH secretion from rat pituitary cells in a dose-dependent manner. RhTRX did not significantly affect the GH mRNA expression of pituitary cells stimulated in the presence or absence of GRF. In addition, rhTRX-augmented GH secretion was not significantly affected by the presence of cycloheximide. Collectively, these findings suggest that TRX is induced by stimulation with GRF and plays a regulatory role in GH secretion from rat anterior pituitary cells by enhancing the secretion of stored GH, rather than by the synthesis of GH.

Thiopental directly depresses lumbar dorsal horn neuronal responses to noxious mechanical stimulation in goats.

BACKGROUND: Thiopental has hypnotic actions in the brain, but it also depresses nociceptive transmission. In this study we examined whether thiopental had direct (spinal) and/or indirect (supraspinal) effects on the responses of single lumbar dorsal horn neurons to noxious mechanical stimulation, using a method to deliver thiopental differentially to either the torso or cranial circulation in goats. METHODS: Goats (n=10) were anesthetized with isoflurane and neck dissections performed to permit cranial bypass. A lumbar laminectomy was made to permit single-unit recording of lumbar dorsal horn neuronal activity (1-2 neurons/animal). Isoflurane was maintained at 0.8+/-0.1% to both head and torso throughout the study. During cranial bypass, thiopental was separately administered to the torso (low dose, 1.5+/-0.5 mg/kg; high dose, 3.7+/-0.5 mg/kg) or cranial (low dose, 0.12+/-0.03 mg/kg; high dose, 0.2 mg/kg) circulation. RESULTS: Thiopental administered to the torso significantly depressed dorsal horn neuronal responses to noxious stimulation at the high dose: 757+/-471 to 392+/-305 impulses/min at 1 min post-injection, P<0.006 (n=14); evoked responses recovered at 5 min post-injection. At the low dose, there was a similar numerical decrease, but this did not reach significance: 876+/-780 to 407+/-499 impulses/min at 1 min post-injection, P>0.05 (n=6). No significant change was observed when thiopental was administered to the cranial circulation: low dose, 1061+/-1167 to 965+/-874 impulses/min at 1 min post-injection, P>0.05 (n=10); high dose, 864+/-331 to 917+/-525 impulses/min at 1 min post-injection, P>0.05 (n=8). CONCLUSION: Thiopental has a direct (spinal) depressant effect on dorsal neuronal responses to noxious stimulus, but no significant supraspinal effect.