Isomorphic coalescence of aster cores formed in vitro from microtubules and kinesin motors.

We report fluorescence microscopy studies of the formation of aster-like structures emerging from a cellular element-based active system and a novel analysis of the aster condensation. The system consists of rhodamine labeled microtubules which are dynamically coupled by functionalized kinesin motor proteins cross-linked via streptavidin-coated quantum dots (QDs). The aster-shaped objects contain core structures. The cores are aggregates of the QD-motor protein complexes, and result from the dynamic condensation of sub-clusters that are connected to each other randomly. The structural specificity of the aster core reflects a configuration of the initial connectivity between sub-clusters. Detailed image analysis allows us to extract a novel correlation between the condensation speed and the sub-cluster separation. The size of the core is scaled down during the condensation process, following a power law dependence on the distance between sub-clusters. The exponent of the power law is close to two, as expected from a geometric model. This single exponent common to all the contractile lines implies that there exists a time regime during which an isomorphic contraction of the aster core continues during the condensation process. We analyze the observed contraction by using a model system with potential applicability in a wide range of emergent phenomena in randomly coupled active networks, which are prevalent in the cellular environment.

Impact of immunoreactive substances contained in apheresis platelet concentrate on postoperative respiratory function in surgical patients receiving platelet transfusion: a prospective cohort study.

OBJECTIVES: To construct an alternative policy for the donor selection of platelet concentrate (PC), a clinical study exploring the features of lung injury following PC administration is needed. BACKGROUND: Although a male-donor-only policy for plasma products appears to have efficiently reduced transfusion-related acute lung injury (TRALI), this policy may not be applied to PC because of supply shortages. METHODS AND MATERIALS: We prospectively examined pulmonary function after the transfusion of PC in informed surgical patients treated at a tertiary university hospital in Japan. The contributions of immunoreactive substances contained in the PC to respiratory function after PC transfusion was then statistically examined. RESULTS: Eighty-six patients (56 men, 30 women) were enrolled in the analysis. Fifty-four cases experienced respiratory failure (PaO2 /FiO2 <300 mmHg) after transfusion. Five cases were diagnosed as possible TRALI based on permeability pulmonary oedema, while 23 cases were diagnosed as transfusion-associated circulatory overload (TACO) based on chest radiograph findings. A multivariate logistic regression analysis identified the presence of anti-granulocyte antibody as a significant predictor of possible TRALI [P = 0.023; odds ratio (OR), 13.0; 95% confidence interval (CI), 1.4-118.3]. Meanwhile, anti-leukocyte antibody class II was identified as a significant independent predictor of TACO (P = 0.010; OR, 18.4; 95% CI, 2.0-170.1). CONCLUSIONS: Our data suggest that antibodies contained in PC may contribute to the deterioration of respiratory function after PC transfusion, although the diagnoses of TACO and TRALI may have overlapped among the patients with pulmonary distress in this cohort.

Improvement of the long-term stability for dioxin toxicity evaluation method by enzyme-linked immunosorbent assay.

A dioxin enzyme immunoassay (EIA) is one of the methods that may satisfy the requirements to reduce the cost and turn around time for the dioxin analysis. We developed a dioxin enzyme-linked immunosorbent assay (ELISA) to rapidly analyze for trace levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in human milk. In this study, to aim to supply the stable assay quality, the development of long-term stable coated plates for the ELISA system was reported. To the conventional coated plate (wet plate), the dry plate ELISA indicated the stability to be able to store for 1.5 years at 11 degrees C. The IC(50) of this ELISA was 17 +/- 4 pg/well. The standard curve showed almost the same as that of the wet plate. A fairly good correlation between cross-reactivity of the ELISA and WHO-TEF was achieved for environmental matrices. This ELISA should be more practical for environmental sample monitoring.

Pyrrhotites: synthetics having two new superstructures.

Synthetic pyrrhotites Fe(1-x)S, synthesized at various compositions and temperatures, show the presence of two new superstructures based on the hexagonal subcell of the NiAs type (axes A and C): one, in the range 1-x=0.89 to 0.93, has a = 90A and c = 3C; the other, in the range 1 -x = 0.935 to 0.975, has a = 2A but c irrationally related to C, varying with composition.

Pyrrhotites: Stoichiometric Compounds with Composition Fen--1Sn (nge8).

A new type of natural pyrrhotite, orthorhombic 11C type (a = 6.892, b = 11.952, c = 5.744 x 11 angstroms), and the hexagonal 6C type (a = 6.89, c = 5.76 x 6 angstroms) are described. Their compositions are Fe(10)S(11) and Fe(11)S(12), respectively. Pyrrhotites stable in nature have essentially stoichiometric composition, Fe(n)-(l)S(n) (n>/=8), with the structures of n/2C type for n even and of nC type for n odd. The solid solutions between Fe(11)S(12) and Fe(10)S(11), and between Fe(10)S(11) and Fe(9)S(10) are considered metastable in nature.

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.

Nitric oxide mediates murine cytomegalovirus-associated pneumonitis in lungs that are free of the virus.

4 wk after intraperitoneal inoculation of 0.2 LD50 (50% lethal dose) of murine cytomegalovirus (MCMV) in adult BALB/c mice, MCMV remained detectable in the salivary glands, but not in the lungs or other organs. When the T cells of these mice were activated in vivo by a single injection of anti-CD3 monoclonal antibody, interstitial pneumonitis was induced in the lungs that were free of the virus with an excessive production of the cytokines. In the lungs of such mice persistently infected with MCMV, the mRNA of the cytokines such as IL-2, IL-6, TNF-alpha, and IFN-gamma were abundantly expressed 3 h after the anti-CD3 injection, and the elevated levels continued thereafter. A marked expression of inducible nitric oxide synthetase (iNOS) was then noted in the lungs, suggesting that such cytokines as TNF-alpha and IFN-gamma may have induced iNOS. Although the increase in NO formation was demonstrated by the significant elevation of the serum levels of nitrite and nitrate, the interstitial pneumonitis was not associated with either increased superoxide formation or peroxynitrite-induced tyrosine nitration. Nevertheless, the administration of an NO antagonist also alleviated the interstitial pneumonitis provoked by anti-CD3 mAb. Based on these findings, it was concluded that MCMV-associated pneumonitis is mediated by a molecule of cytokine-induced NO other than peroxynitrite.

Role of nitric oxide and peroxynitrite in the cytokine-induced sustained myocardial dysfunction in dogs in vivo.

Studies in vitro suggested that inflammatory cytokines could cause myocardial dysfunction. However, the detailed mechanism for the cytokine-induced myocardial dysfunction in vivo remains to be examined. We thus examined this point in our new canine model in vivo, in which microspheres with and without IL-1beta were injected into the left main coronary artery. Left ventricular ejection fraction (LVEF) was evaluated by echocardiography for 1 wk. Immediately after the microsphere injection, LVEF decreased to approximately 30% in both groups. While LVEF rapidly normalized in 2 d in the control group, it was markedly impaired in the IL-1beta group even at day 7. Pretreatment with dexamethasone or with aminoguanidine, an inhibitor of inducible nitric oxide synthase, prevented the IL-1beta-induced myocardial dysfunction. Nitrotyrosine concentration, an in vivo marker of the peroxynitrite production by nitric oxide and superoxide anion, was significantly higher in the myocardium of the IL-1beta group than in that of the control group or the group cotreated with dexamethasone or aminoguanidine. There was an inverse linear relationship between myocardial nitrotyrosine concentrations and LVEF. These results indicate that IL-1beta induces sustained myocardial dysfunction in vivo and that nitric oxide produced by inducible nitric oxide synthase and the resultant formation of peroxynitrite are substantially involved in the pathogenesis of the cytokine-induced sustained myocardial dysfunction in vivo.

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.

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.

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.

UV-radiation-specific p53 mutation frequency in normal skin as a predictor of risk of basal cell carcinoma.

BACKGROUND: A strong association has been found between skin cancer and exposure to UV radiation. The p53 tumor suppressor gene (also known as TP53), which is frequently mutated in human cancers, is believed to be an early target in UV radiation-associated skin carcinogenesis. We have previously developed a sensitive, polymerase chain reaction-based method capable of detecting and quantifying a UV radiation-specific mutation in the p53 gene (codons 247 and 248: AAC CGG --> AAT TGG) in normal skin. We have used this method to examine whether UV radiation-specific mutation frequency is associated with risk of basal cell carcinoma (BCC) and with sun exposure. METHODS: This case-control study in Australia involved 53 case subjects with BCC and 75 control subjects. DNA was isolated from normal skin (mirror-image anatomic site to the cancer site for case subjects and a randomly selected site for control subjects) and assayed for p53 mutation. Relationships between p53 mutation frequency and risk of BCC, sun sensitivity, or sun exposure were estimated by use of odds ratios (ORs) and 95% confidence intervals (95% CIs). RESULTS: Case subjects were more likely to have a p53 mutation than control subjects (OR = 3.1; 95% CI = 1.3-7.1). In addition, the odds of BCC increased monotonically with increasing frequency of p53 mutation. No statistically significant associations could be demonstrated between p53 mutation frequency and age, sex, sensitivity to the sun, pigmentary characteristics, total lifetime sun exposure, or sun exposure to the biopsy site. CONCLUSIONS: Our results indicate that tandem CC --> TT mutations involving codons 247 and 248 of the p53 gene are associated with an increased risk of BCC but cannot be used as an accurate measure of total UV-radiation exposure.

Expression of Fas/APO-1 during the progression of astrocytomas.

Fas/APO-1 (CD95) is an apoptosis-signaling receptor molecule on the surface of cells. To investigate the possible role of Fas during malignant transformation of glial cells, we analyzed the expression of Fas mRNA by reverse transcription-PCR in human astrocytic brain tumors. Expression was found in 1 of 4 (25%) juvenile pilocytic astrocytomas (WHO grade I), 1 of 9 (11%) low-grade astrocytomas (WHO grade II), 6 of 12 (50%) anaplastic astrocytomas (WHO grade III), and all of 9 glioblastomas (WHO grade IV). Thus, the frequency of Fas expression appears to correlate with the malignancy grade of astrocytomas. The soluble form of the Fas mRNA lacking the transmembrane domain was detected in one anaplastic astrocytoma and in two glioblastomas.

Cell-type-specific ras mutations but no microsatellite instability in chemically induced mouse skin tumors and transformed 3T3 cells.

In mouse skin, both papillomas/carcinomas or fibrosarcomas can be induced by 7,12-dimethylbenz[alpha]anthracene (DMBA) depending on the mode of administration. Thus, upon DMBA painting (or transplacental exposure by i.p. injection to pregnant mothers) followed by 12-O-tetradecanoylphorbol-13-acetate applications to the skin of CD1 mice, papillomas and carcinomas appeared, whereas fibrosarcomas were induced when DMBA was s.c. injected. Molecular analysis of these tumors revealed that the majority of papillomas (17/20) and carcinomas (9/10) showed DMBA-specific mutations (A to T transversion at the 61st codon) in the Ha-ras gene. On the other hand, many fibrosarcomas (5/9) showed the same mutation only in the Ki-ras gene. When microsatellites were studied in these tumors at nine loci containing CA repeats, none of them showed an instability. In addition, when we analyzed 14 BALB/c 3T3 cell lines transformed by various carcinogens (including 3 clones induced by DMBA which have the A to T mutation in the Ki-ras gene), no changes in CA repeats were observed. These results suggest that DMBA-induced mouse tumors/transformed cells show cell-type-specific ras gene mutations, and these occur independently in the absence of microsatellite instability. While murine cells are considered to be relatively susceptible to cancer induction partially due to genomic instability, our results indicate that microsatellite instability is not induced in these cells by chemical carcinogens.

Evidence for UV-associated activation of telomerase in human skin.

Telomerase activation plays a crucial role in the immortalization of human cells and carcinogenesis; however, the temporal and pathophysiological aspects of the activation in vivo are poorly understood. We found telomerase activity not only in malignant tumors (91%) but also in most benign (60%) and premalignant (89%) skin tumors. This suggests the involvement of telomerase activation in a crucial biological step of human skin carcinogenesis. Because UV light is a major factor in skin carcinogenesis, we further examined telomerase activity in normal skin samples and in normal skin samples adjacent to benign, premalignant, and malignant skin lesions. Data for chronically sun-exposed body sites were compared with those for covered sites. Among normal skin samples, 39% (26 of 67) had telomerase activity, and this activity was unrelated to neighboring lesions but strongly associated with the level of sun exposure. Fifty-four % (21 of 39) of normal skin samples from chronically sun-exposed sites were telomerase-positive, compared with only 12% (3 of 26) of samples from covered sites. When we examined telomerase activity and CC to TT mutations at codons 247/8 of the p53 gene (which are considered to be UV specific) in the same normal skin samples, only 43% (7 of 16) of telomerase-positive normal skin samples at sun-exposed sites contained the p53 mutations, whereas all (7 of 7) of the samples with UV-specific p53 mutations showed telomerase activity (P = 0.019). These data suggest that telomerase activation is involved at an early stage of human skin carcinogenesis and that activation may precede the acquisition of UV-associated p53 mutations in the skin. Telomerase activity was also found in plucked hair follicles and enzymatically separated epidermis, which may be associated with the presence of stem cells in the skin.

Patched (ptch)-associated preferential expression of smoothened (smoh) in human basal cell carcinoma of the skin.

The discovery of specific overexpression of a gatekeeper gene, ptch, in basal cell carcinoma (BCC) led to a hypothesis that the human homologue of patched (PTCH) normally functions as a negative regulator of the signaling pathway that is initiated by hedgehogs (HHs) and activated by the human homologue of smoothened (SMOH); however, no evidence for the involvement of smoh and hhs has been provided. Here, we show novel evidence that smoh is also preferentially overexpressed in BCC, together with ptch (P < 0.002), and that Sonic hh was expressed in only some BCCs. Our data, therefore, indicate that such overexpression of smoh may be associated with overexpression or mutation of PTCH and that this overexpression subsequently stimulates the PTCH/SMOH signaling pathway. In an investigation of a possible regulation of ptch and smoh, we demonstrated that expression of exogenous p21WAF1 in immortalized keratinocytes down-regulates both ptch and smoh and that the down-regulation is accompanied by growth arrest, which suggests the involvement of p21WAF1 in regulation of the PTCH/SMOH signaling pathway.

Modulating the microtubule-tau interactions in biomotility systems by altering the chemical environment.

Obstacles in microtubule mediated neuronal transport can trigger dementia. We use bio-motility assays, that simulate the neuron chemistry in axonopathy, to screen chemicals, that retain the microtubule dynamics in healthy neuronal activity. Tau protein inhibits microtubule activity and leads to oligomerization. Iron(iii) untangles, whereas mono-sodium-glutamate destabilizes the microtubule oligomer.

Experimental and theoretical studies of Si-Cl and Ge-Cl σ-bond activation reactions by iridium-hydride.

We report the iridium hydride-mediated Si-Cl and Ge-Cl σ-bond activation in a low-polarity toluene solution owing to diphosphine-chelation, in which the Si-Cl and Ge-Cl σ-bonds are readily cleaved through an SN2-type pathway via the formation of a free chloride anion.

Identification and quantification of a carcinogen-induced molecular initiation event in cell transformation.

All transformed foci of Balb/c 3T3 clone A31-1-1 cells induced by 7,12-dimethylbenz[a]anthracene (DMBA) (42 out of 42 examined) contained an A to T transversion at codon 61 (A182 to T) of the Ki-ras gene. The transformants induced by other carcinogens tested did not contain such a mutation, except one out of nine 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced transformed foci. Thus, we hypothesized that this mutation is a specific DMBA-induced initiating event in Balb/c 3T3 cell transformation and we have measured its frequency of induction before transformation occurs, employing our recently developed method. Such mutations can be detected in the cell population as early as 3 days after exposure to DMBA. The same mutation was also detected in the Ha-ras gene. No detectable level (< 10(-6) of these mutations was induced by other carcinogens tested. The mutation frequency of the Ha-ras gene reached a plateau after 1 week's exposure, but that of the Ki-ras gene continued to increase. These results suggest that the A182 to T mutation of the Ki-ras gene, but not that of the Ha-ras gene, contributes to morphological transformation of Balb/c 3T3 cells. We have demonstrated that the level of expression of ras genes determines the rate of recruitment of cells into transformation. Quantitative analysis of the frequencies of ras gene mutations (initiation) and of transformation suggests that about 25% of those cells with the Ki-ras mutation were recruited into the full transformation process and that, in the presence of the tumor promoter TPA, about 56% of them completed morphological cell transformation.

Effect of sub-skinning concentrations of saponin on intracellular Ca2+ and plasma membrane fluidity in cultured cardiac cells.

To determine the underlying mechanisms of the positive inotropic effect of sub-skinning concentrations of saponin, we studied changes in the intracellular Ca2+ ([Ca2+]i) and plasma membrane fluidity after exposure to digitonin (a representative saponin) in cultured cardiac cells. [Ca2+]i was measured by use of the fluorescent calcium indicator Calcium Green-1. The membrane fluidity was evaluated by measuring the diffusion coefficient using the method of fluorescence recovery after photobleaching. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate was used as the fluorescent probe. Digitonin at a sub-skinning concentration (0.1 to 1 microM) produced an increase in cell motion and an augmentation of [Ca2+]i. Membrane fluidity which is evaluated by the diffusion coefficient (from 0.34.10(-8) to 0.28.10(-8) cm2/s; P < 0.05), decreased in the presence of 0.2 microM digitonin while the cell maintained an augmented motion and an increased [Ca2+]i. The skinning concentration of digitonin (5 microM) produced a rapid contracture with a marked increase in [Ca2+]i. The membrane fluidity was further reduced (diffusion coefficient: 0.24.10(-8) cm2/s; P < 0.001). These results suggest that saponin at the sub-skinning concentration also causes holes in the plasma membrane by interaction with cholesterol, as was shown with the skinning concentration, and it increases [Ca2+]i, which thereby induces a positive inotropic effect.