Chloroform deposition in renal cyst fluid of hemodialysis patients with renal cystic disorders.

Chronic exposure to chloroform (CHCl3) induces renal neoplasms in rodents and may be carcinogenic in humans, but studies on chronic CHCl3 deposition in the human body have not been performed. In this study, we examined 27 hemodialysis patients with renal cystic diseases including acquired cystic disease of the kidney (ACDK) accompanied by renal tumors at high frequency. Intracystic and serum CHCl3 concentrations were determined using a headspace gas chromatography/mass spectrometry analysis. CHCl3 was not detected in the serum in any cases, but levels ranging from <0.1 to 0.659 mg/L were found in the cyst fluid in most cases, including patients with ACDK and autosomal dominant polycystic kidney disease. Because intracystic CHCl3 deposition was not confined to ACDK cases, we were unable to evaluate the relationship between CHCl3 accumulation and carcinogenesis in ACDK. However, our results suggest that compounds such as CHCl3 accumulate in renal cyst fluid in hemodialysis patients with renal cystic diseases.

Significance of chemokine receptor expression in aggressive NK cell leukemia.

Natural killer (NK) cell-type lymphoproliferative diseases of granular lymphocytes can be subdivided into aggressive NK cell leukemia (ANKL) and chronic NK cell lymphocytosis (CNKL). One reason for the poor outcome in ANKL is leukemic infiltration into multiple organs. The mechanisms of cell trafficking associated with the chemokine system have been investigated in NK cells. To clarify the mechanism of systemic migration of leukemic NK cells, we enrolled nine ANKL and six CNKL cases, and analyzed the expression profiles and functions of chemokine receptors by flowcytometry and chemotaxis assay. CXCR1 was detected on NK cells in all groups, and CCR5 was positive in all ANKL cells. Proliferating NK cells were simultaneously positive for CXCR1 and CCR5 in all ANKL patients examined, and NK cells with this phenotype did not expand in CNKL patients or healthy donors. ANKL cells showed enhanced chemotaxis toward the ligands of these receptors. These results indicated that the chemokine system might play an important role in the pathophysiology of ANKL and that chemokine receptor profiling might be a novel tool for discriminating ANKL cells from benign NK cells.

Detection of 1733insC mutations in an Asian family with Birt-Hogg-Dubé syndrome.

BACKGROUND: Birt-Hogg-Dubé syndrome (BHD) is a rare autosomal dominant genodermatosis characterized by skin tumours, including multiple fibrofolliculomas, trichodiscomas and acrochordons. BHD patients also may suffer from associated renal and colonic carcinomas. The defective gene in BHD has been recently identified and is suspected of being a tumour suppressor gene. Several mutations of the BHD gene have been reported only in Caucasian patients. OBJECTIVES: This study reports the first Asian family that has been demonstrated to carry a BHD mutation. PATIENTS/METHODS: The proband was a 26-year-old Japanese man with multiple asymptomatic, soft skin-coloured papules on his face, neck and trunk, which were clinically thought to be acrochordon. His father was also affected. Histopathologically, the papules revealed a fibrofolliculoma that had a circumscribed proliferation of fibroblasts and collagen fibres surrounding an abnormal hair follicle. RESULTS: Mutational analysis of the BHD gene of the proband and the father detected 1733insC, a cytosine insertion mutation in an eight-cytosine tract (nucleotides 1733-1740) in exon 11. Analysis of fibrofolliculoma in the proband showed heterozygous 1733insC mutation, suggesting the absence of loss of heterozygosity. Interestingly, previous mutational analysis in Caucasian patients revealed that both1733insC and 1733delC mutations were hot spots. CONCLUSIONS: This study is the first to find the same hot-spot 1733insC mutation in Asian kindred. The mutations in this polycytosine tract may have a wide, global distribution despite their arising from a different ethnic background.

Brain malformations caused by retroviral vector-mediated gene transfer of hippocampal cholinergic neurostimulating peptide precursor protein into the CNS via embryonic mice ventricles.

Hippocampal cholinergic neurostimulating peptide precursor protein (HCNP-pp) is a unique multifunctional protein, being not only the precursor of HCNP, which promotes the phenotype development of septo-hippocampal cholinergic neurons, but also the binding protein of phosphatidylethanolamine, ATP, Raf-1 kinase (known as "Raf-1 kinase inhibitory factor" in peripheral organs), and serine protease. We obtained a high-titer retroviral vector harboring HCNP-pp cDNA by the use of a modified packaging cell line and centrifugation, and by injecting it into embryonic mouse ventricles, we investigated the function of its gene product within the central nervous system (CNS). We found that efficient transduction into hippocampal pyramidal neurons can be achieved by injecting the vector into embryonic brain ventricles on embryonic day 14 (E14). Three days after receiving the intraventricular injection of the high-titer HCNP-pp retrovirus vector on E14, the tissues around the ventricles showed an overexpression of HCNP-pp. This was accompanied by a reduced amount of activated MEK and Erk (as analyzed by histochemical and Western blot methods), suggesting that HCNP-pp also regulates the MAP-kinase cascade within the CNS. Surprisingly, mouse brains that received the HCNP-pp retroviral vector showed massive malformation of the hippocampus and cerebellum when examined 30 days after birth. This shows that strictly regulated HCNP-pp gene expression is necessary for the normal development of the mouse brain, and that the moderate overexpression achieved by retroviral vector-mediated gene transfer is sufficient to cause severe abnormality of entire brain structures.

Prenatal and neonatal exposure to bisphenol-A enhances the central dopamine D1 receptor-mediated action in mice: enhancement of the methamphetamine-induced abuse state.

Bisphenol-A (BPA), one of the most common environmental endocrine disrupters, has been extensively evaluated for toxicity in a variety of tests in rodents, including developmental and reproductive toxicity, and carcinogenicity. However, little is known about its action on the CNS. In this report, we show that prenatal and neonatal exposure to BPA in mice leads to the enhancement of the dopamine D1 receptor-dependent rewarding effect induced by a psychostimulant methamphetamine. Furthermore, this treatment with BPA markedly enhanced hyperlocomotion and its sensitization induced by methamphetamine, which reflects extensive abuse associated with sociological and psychiatric problems. We also demonstrated that chronic exposure to BPA produced an up-regulation of dopamine D1 receptor function to activate G-protein in the mouse limbic forebrain, which is thought to be a critical site for the expression of rewarding effects by abuse drugs. Additionally, chronic BPA exposure produced a significant increase in levels of the dopamine D1 receptor mRNA in the whole brain. In contrast, no change in protein levels of methamphetamine-targeted proteins, dopamine transporter or the type 2 vesicle monoamine transporter in the brain was observed by prenatal and neonatal exposure to BPA. The present data provide the first evidence that prenatal and neonatal exposure to BPA can potentiate the central dopamine D1 receptor-dependent neurotransmission, resulting in supersensitivity of methamphetamine-induced pharmacological actions related to psychological dependence on psychostimulants.

In vitro assay of hydrolysis and chlorohydroxy derivatives of bisphenol A diglycidyl ether for estrogenic activity.

Bisphenol A diglycidyl ether (BADGE) is an epoxy resin monomer. Epoxy-based solution coatings are used in many applications as additives for a variety of plastic coatings in food packaging. It is well known that unreacted BADGE can migrate from epoxy-based packing materials into foods. Not only BADGE but also its derivatives can easily migrate into foods and it is likely that we intake BADGE and its derivatives through food or drink. Recently, endocrine disrupting chemicals (EDCs) have attracted attention because they have been shown to affect reproduction in wildlife. The estrogenic activity of BADGE derivatives has not previously been investigated. Therefore, we investigated the estrogenic activity of the BADGE derivatives, dihydrolysed BADGE (BADGE-4OH) and chlorohydroxy BADGE (BADGE-2Cl), using breast cancer cell (T47D) proliferation assay and estrogen receptor (ER) (alpha) binding assay. These chemicals exhibited T47D cell proliferation at concentrations of 10(-14)-10(-4) M. However, these chemicals did not bind to ER (alpha) in the binding assay.

Opening of mitochondrial K(ATP) channels attenuates the ouabain-induced calcium overload in mitochondria.

We tested whether opening of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels depolarizes mitochondrial membrane potential (DeltaPsi(m)) and thereby prevents the mitochondrial Ca(2+) overload. With the use of a Nipkow disk confocal system, the mitochondrial Ca(2+) concentration ([Ca(2+)](m)) and DeltaPsi(m) in rat ventricular myocytes were measured by loading cells with Rhod-2 and JC-1, respectively. Exposure to ouabain (1 mmol/L) for 30 minutes produced mitochondrial Ca(2+) overload, and the intensity of Rhod-2 fluorescence significantly increased to 173+/-16% of baseline (P<0.001). Treatment of myocytes with the mitoK(ATP) channel opener diazoxide (100 micromol/L) blunted the ouabain-induced mitochondrial Ca(2+) overload (131+/-10% of baseline; P<0.001 versus ouabain). Moreover, diazoxide significantly depolarized the DeltaPsi(m) and reduced the intensity of JC-1 fluorescence during application of ouabain to 89+/-2% of baseline (P<0.05). These effects of diazoxide were blocked by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 micromol/L). These results indicate that opening of mitoK(ATP) channels prevents a mitochondrial Ca(2+) overload in association with DeltaPsi(m) depolarization and thereby protects myocardium against ischemic damage.

Physiological concentration of nitric oxide induces positive inotropic effects through cGMP pathway in isolated rat ventricular myocytes.

We used authentic NO or NO from NO donors to show that the physiological levels of NO (<1 microM) induce a positive inotropic effect and demonstrated that the effect is evoked through a cGMP-dependent pathway. In isolated rat ventricular myocytes, authentic NO at 588 nM increased both cell shortening and the intracellular Ca(2+) ([Ca(2+)]i) transient (133 and 117%, respectively; p < 0.05 vs. baseline), and 0.16-1.7 microM NO elicited reproducible dose-dependent increases in cell shortening. NOC18 (0.1 mM: actual NO concentration 673 nM) or SNAP (0.1 mM: actual NO concentration 285 nM) showed similar effects (shortening 215% and [Ca(2+)]i transient 160% increases, and shortening 148% and [Ca(2+)]i transient 117% increases, respectively). The NO-induced increases in cell shortening and the [Ca(2+)]i transient were inhibited by an inhibitor of soluble guanylate cyclase (ODQ, 30 microM) or by an inhibitor of cAMP-dependent protein kinase (KT5720, 0.1 microM). In the presence of an inhibitor of cGMP-inhibited cAMP-phosphodiesterase (milrinone, 10 microM), NO failed to increase both cell shortening and the [Ca(2+)]i transient. These results suggest that physiological levels of NO induce positive inotropy through a cGMP-dependent pathway.

Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites.

Streptomyces avermitilis is a soil bacterium that carries out not only a complex morphological differentiation but also the production of secondary metabolites, one of which, avermectin, is commercially important in human and veterinary medicine. The major interest in this genus Streptomyces is the diversity of its production of secondary metabolites as an industrial microorganism. A major factor in its prominence as a producer of the variety of secondary metabolites is its possession of several metabolic pathways for biosynthesis. Here we report sequence analysis of S. avermitilis, covering 99% of its genome. At least 8.7 million base pairs exist in the linear chromosome; this is the largest bacterial genome sequence, and it provides insights into the intrinsic diversity of the production of the secondary metabolites of Streptomyces. Twenty-five kinds of secondary metabolite gene clusters were found in the genome of S. avermitilis. Four of them are concerned with the biosyntheses of melanin pigments, in which two clusters encode tyrosinase and its cofactor, another two encode an ochronotic pigment derived from homogentiginic acid, and another polyketide-derived melanin. The gene clusters for carotenoid and siderophore biosyntheses are composed of seven and five genes, respectively. There are eight kinds of gene clusters for type-I polyketide compound biosyntheses, and two clusters are involved in the biosyntheses of type-II polyketide-derived compounds. Furthermore, a polyketide synthase that resembles phloroglucinol synthase was detected. Eight clusters are involved in the biosyntheses of peptide compounds that are synthesized by nonribosomal peptide synthetases. These secondary metabolite clusters are widely located in the genome but half of them are near both ends of the genome. The total length of these clusters occupies about 6.4% of the genome.

A defect in the cytochrome b large subunit in complex II causes both superoxide anion overproduction and abnormal energy metabolism in Caenorhabditis elegans.

A mev-1(kn1) mutant of the nematode Caenorhabditis elegans is defective in the cytochrome b large subunit (Cyt-1/ceSDHC) in complex II of the mitochondrial electron transport chain. We have previously shown that a mutation in mev-1 causes shortened life span and rapid accumulation of aging markers such as fluorescent materials and protein carbonyls in an oxygen-dependent fashion. However, it remains unclear as to whether this hypersensitivity is caused by direct toxicity of the exogenous oxygen or by the damage of endogenous reactive oxygen species derived from mitochondria. Here we report important biochemical changes in mev-1 animals that serve to explain their abnormalities under normoxic conditions: (i) an overproduction of superoxide anion from mitochondria; and (ii) a reciprocal reduction in glutathione content even under atmospheric oxygen. In addition, unlike wild type, the levels of superoxide anion production from mev-1 mitochondria were significantly elevated under hyperoxia. Under normal circumstances, it is well known that superoxide anion is produced at complexes I and III in the electron transport system. Our data suggest that the mev-1(kn1) mutation increases superoxide anion production at complex II itself rather than at complexes I and III. The mev-1 mutant also had a lactate level 2-fold higher than wild type, indicative of lactic acidosis, a hallmark of human mitochondrial diseases. These data indicate that Cyt-1/ceSDHC plays an important role not only in energy metabolism but also in superoxide anion production that is critically involved in sensitivity to atmospheric oxygen.

Peroxynitrite formation from human myocardium after ischemia-reperfusion during open heart operation.

BACKGROUND: Current experimental studies have demonstrated that peroxynitrite (ONOO-) has both cytotoxic and cytoprotective effects on myocardial ischemia-reperfusion injury. However, even myocardial ONOO- formation has not yet been investigated in humans undergoing open heart operation. We measured plasma nitrotyrosine as an indicator of ONOO- formation during open heart operation and examined its association with myocardial ischemia-reperfusion injury. METHODS: Twenty adult patients undergoing mitral valve replacement under cardiopulmonary bypass between 1997 and 1998 were enrolled in this study (6 men and 14 women). Arterial blood (Ao) and coronary sinus effluent (CS) were obtained: (1) before the initiation of cardiopulmonary bypass, (2) just after aortic unclamping, (3) at 5 minutes, (4) at 10 minutes, (5) at 15 minutes, and (6) at 20 minutes after aortic unclamping. RESULTS: At every sampling point after reperfusion, plasma nitrate and nitrite was significantly lower in CS than in Ao, and the percentage ratio of nitrotyrosine to tyrosine (%NO2-Tyr; an index of ONOO- formation) was significantly higher in CS than in Ao. The CS-Ao difference in %NO2-Tyr, myocardium-derived ONOO-, reached its peak at 5 minutes after reperfusion (2.17+/-0.74%), which was significantly correlated with the peak CS-Ao difference in plasma malondialdehyde, and with postoperative maximum creatine kinase-MB. CONCLUSIONS: These results first demonstrate that ONOO- is produced from human myocardium after ischemia-reperfusion during open heart operation, and myocardium-derived ONOO- can be determined by the CS-Ao difference in %NO2-Tyr.

Inducible nitric oxide production is an adaptation to cardiopulmonary bypass-induced inflammatory response.

BACKGROUND: Cardiopulmonary bypass (CPB) increases nitric oxide (NO) production by the activation of NO synthases (NOS). However, the role of NO from inducible NOS (iNOS) in CPB-induced inflammatory response remains unclear. We examined the effect of a selective iNOS inhibitor, aminoguanidine, on CPB-induced inflammatory response in a rat-CPB model. METHODS: Adult Sprague-Dawley rats underwent 60 minutes of CPB (100 mL x kg(-1) x min(-1), 34 degrees C). Group A (n = 10) received 100 mg/kg of aminoguanidine intraperitoneally 30 minutes before the initiation of CPB, and group B (n = 10) served as controls. RESULTS: There were significant time-dependent changes in plasma interleukin (IL)-6, IL-8, nitrate + nitrite, the percentage ratio of nitrotyrosine to tyrosine (%NO2-Tyr, an indicator of peroxynitrite formation), and respiratory index (RI). Three hours after CPB termination, IL-6, IL-8, and RI were significantly higher in group A (IL-6, 397.5+/-80.6 pg/mL; IL-8, 26.99+/-6.57 ng/mL; RI, 1.87+/-0.31) than in group B (IL-6, 316.5+/-73.9 pg/mL, p <0.05; IL-8, 17.21+/-3.12 ng/mL, p < 0.01; RI, 1.57+/-0.24, p < 0.05) although nitrate + nitrite (31.8+/-4.1 micromol/L) and %NO2-Tyr (1.15%+/-0.20%) were significantly lower in group A than in group B (nitrate + nitrite, 50.2+/-5.0 micromol/L, p < 0.01; %NO2-Tyr, 1.46%+/-0.21%, p < 0.01). Western immunoblot analysis from lung tissue of group A identified marked iNOS inhibition without inhibiting endothelial-constitutive NOS, and neutrophil accumulation in the lung specimens was significantly greater in group A (6.5+/-0.7/alveoli) than in group B (4.4+/-0.4/alveoli, p < 0.01). CONCLUSIONS: These results suggest that NO production from iNOS may be an adaptive response for attenuating the CPB-induced inflammatory response.

The prevalence of renal cell carcinoma: a nation-wide survey in Japan in 1997.

BACKGROUND: The present study was conducted to investigate the incidence of renal cell carcinoma by sex, age group and different regions in Japan. METHODS: The survey was conducted from the beginning of January 1997 to the end of December 1997. A total of 1306 Institutions in all 47 prefectures throughout Japan were requested to register cases. RESULTS: There were 6358 persons with renal cell carcinoma, consisting of 4372 men and 1986 women. The age-specific incidence rates showed a peak in the age group of 65-70 years in both men and women. The crude incidence rates per 100 000 population for men and women were 7.1 and 3.1, respectively, and age-standardized incidence rates per 100 000 population for men and women were 4.9 and 1.8, respectively. The incidence rates in the Hokkaido region were significantly higher than in other regions (P < 0.05), among which there was no significant difference in incidence rates. CONCLUSIONS: The present study showed that the incidence rates of renal cell carcinoma in Japan were approximately the same as among Japanese in Los Angeles. The rates were, however, lower than North American and European countries, but higher than China, Central or South American countries and African countries. The reasons for the high incidence of renal cancer in the Hokkaido region are not entirely clear. Further epidemiologic research is required.

Calmodulin kinases II and IV and calcineurin are involved in leukemia inhibitory factor-induced cardiac hypertrophy in rats.

We recently reported that leukemia inhibitory factor (LIF) enhances Ca(2+)](i) through an increase in L-type Ca(2+) current (I(Ca,L)) in adult cardiomyocytes. The aim of this study was to investigate whether LIF activates Ca(2+)-dependent signaling molecules, such as calcineurin and calmodulin kinases II and IV (CaMKII and CaMKIV), and, if so, whether these Ca(2+)-mediated signaling events contribute to LIF-mediated cardiac hypertrophy. We first confirmed that LIF increased I(Ca,L) and [Ca(2+)](i) in primary cultured rat neonatal cardiomyocytes. Calcineurin, CaMKII, and CaMKIV activities increased at 2 minutes and peaked by 1.6-, 2.2-, and 2.2-fold, respectively, at 15 minutes. Nicardipine or verapamil fully inhibited these activities. Autophosphorylation of CaMKII was also observed to parallel the timing of CaMKII activity, and this phosphorylation was blocked by nicardipine, verapamil, or EGTA. LIF treatment led to a 3-fold increase in nuclear factor of activated T cell-luciferase activity. To confirm that inositol triphosphate (IP(3))-induced Ca(2+) release from sarcoplasmic reticulum was not involved in this process, IP(3) content and phosphorylation of phospholipase Cgamma were investigated. LIF did not increase IP(3) content or phosphorylate phospholipase Cgamma. KN62 (an inhibitor of CaMKII and CaMKIV) attenuated c-fos, brain natriuretic peptide, alpha-skeletal actin, and atrial natriuretic peptide expression. KN62 suppressed the LIF-induced increase in [(3)H]phenylalanine uptake and cell size. Cyclosporin A and FK506 slightly attenuated brain natriuretic peptide but did not affect c-fos or atrial natriuretic peptide expression. Cyclosporin A significantly reduced the LIF-induced increase in [(3)H]phenylalanine uptake. These findings indicated that LIF activated CaMKII, CaMKIV, and calcineurin through an increase in I:(Ca,L) and [Ca(2+)](i) and that CaMKII, CaMKIV, and calcineurin are critically involved in LIF-induced cardiac hypertrophy.