Metabolomic responses of an estuarine benthic amphipod to heavy metals at urban-runoff concentrations.

Heavy metals released from urban areas have toxic effects on aquatic organisms. Heavy metals in aquatic environments exist in various forms and methods designed to assess their effects need to consider their bioavailability. This study aimed to explore biomarkers in an estuarine amphipod, Grandidierella japonica, for exposure to heavy metals using metabolomics. We exposed G. japonica to different heavy metals and analyzed their metabolomes using high-resolution mass spectrometry. Partial least squares discriminant analysis (PLS-DA) was used to extract biomarkers of exposure for each heavy metal. As a result, three models were built to predict discrimination based on metabolomic profiles, and 70, 106, and 168 metabolites were extracted as biomarkers for exposure to Cu, Zn, and Cd, respectively. Our results suggest that PLS-DA was effective in extracting biomarkers, and this study demonstrated the usefulness of metabolomics as biomarkers.

Multilayer coated grazing incidence condenser for large numerical aperture objective at wavelength of 4.5 nm.

A grazing incidence condenser is developed for objectives with large numerical aperture working in Carbon-window wavelength region (λ=4.4-5.0 nm) with the use of a point light source. The condenser is composed of four pieces of toroidal mirrors and a piece of the mirror was fabricated to evaluate the performance of the mirror. The radii of the toroidal mirror are determined by ray-trace calculation, and each radius of the mirror substrate and the roughness of the polished surface were evaluated to satisfy the designed parameter. A Co/C reflection multilayer is also designed to reflect soft x-ray light at 4.5 nm wavelength, and the reflection multilayer was deposited on the mirror surface. Measured reflectance of the toroidal mirror with the reflection multilayer is higher than 0.32 at 4.5 nm wavelength.

Large current MOSFET on photonic silicon-on-insulator wafers and its monolithic integration with a thermo-optic 2 × 2 Mach-Zehnder switch.

n-channel body-tied partially depleted metal-oxide-semiconductor field-effect transistors (MOSFETs) were fabricated for large current applications on a silicon-on-insulator wafer with photonics-oriented specifications. The MOSFET can drive an electrical current as large as 20 mA. We monolithically integrated this MOSFET with a 2 × 2 Mach-Zehnder interferometer optical switch having thermo-optic phase shifters. The static and dynamic performances of the integrated device are experimentally evaluated.

The Wilms' tumor gene WT1-GFP knock-in mouse reveals the dynamic regulation of WT1 expression in normal and leukemic hematopoiesis.

The Wilms' tumor gene WT1 is overexpressed in most of human leukemias regardless of disease subtypes. To characterize the expression pattern of WT1 during normal and neoplastic hematopoiesis, we generated a knock-in reporter green fluorescent protein (GFP) mouse (WT1(GFP/+)) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term (LT) hematopoietic stem cells (HSC) and very few (<1%) of the multipotent progenitor cells. In contrast, in murine leukemias induced by acute myeloid leukemia 1 (AML1)/ETO+TEL/PDGFbetaR or BCR/ABL, WT1 was expressed in 40.5 or 38.9% of immature c-kit(+)lin(-)Sca-1(+) (KLS) cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSCs). WT1 expression was minimal in normal fetal liver HSCs and mobilized HSCs, both of which are stimulated for proliferation. In addition, overexpression of WT1 in HSCs did not result in proliferation or expansion of HSCs and their progeny in vivo. Thus, the mechanism by which expansion of WT1-expressing cells occurs in leukemia remains unclear. Nevertheless, our results demonstrate that the WT1(GFP/+) mouse is a powerful tool for analyzing WT1-expressing cells, and they highlight the potential of WT1, as a specific therapeutic target that is expressed in LSCs but not in normal HSCs.

Identification of a gene element essential for leukemia-specific expression of transgenes.

Leukemia-specific promoters and enhancers for gene therapy had never been reported. Since the Wilms' tumor gene WT1 is overexpressed in almost all types of leukemia, WT1 is an ideal target of leukemia-specific therapy. To explore the possibility of gene therapy for leukemia using WT1 promoter and enhancer, their activities in several kinds of cells were analyzed by using the enhanced green fluorescent protein (EGFP) gene as a reporter. First, we identified the best combination (654P/EGFP/int3- enh/3'-enh vector) of the 654-bp WT1 promoter and the two WT1 enhancers located in intron 3 and at the 3' end of the WT1 gene for inducing EGFP expression in K562 cells, which endogenously expressed WT1. When this was transfected into WT1-expressing leukemia cells (K562, HEL), WT1-nonexpressing hematopoietic cells (Daudi, U937), and WT1-expressing nonhematopoietic cells (TYK-nu-CPr, SW480, 293 T), 19.8, 22.9, 1.47, 1.43, 4.50, 4.16, and 1.09 times EGFP expression was induced, respectively, compared to that by the promoter-less EGFP vector. These results showed that the 654P/EGFP/int3-enh/3'-enh vector specifically induced high levels of EGFP expression in WT1-expressing leukemia cells. 654P/int3- enh/3'-enh vector containing transgenes such as suicide genes might become useful tools for leukemia-specific gene therapy.

Thalamic projections to the hippocampal and entorhinal areas in the cat.

The thalamic projections to the hippocampal formation and to the subicular and entorhinal areas in the cat have been studied with retrograde transport of horseradish peroxidase (HRP) or wheat germ agglutinin conjugated to HRP (WGA-HRP) and anterograde transport of WGA-HRP. Retrograde transport tracers injected in various parts of these cortices resulted in labeled cells in the midline, anterior, and lateral dorsal nuclei. Injections into the hippocampal formation or the subiculum led to retrograde labeling of cells in the reuniens nucleus of the ipsilateral thalamus throughout its rostrocaudal extent, whereas the restricted injections into the dentate gyrus and the inferior region of the hippocampus led to no labeling. Following an injection into the pre- and parasubiculum, a large number of labeled cells were seen not only in the reuniens nucleus but in other midline nuclei. In addition, a substantial number of labeled cells were also detected in the anterior and lateral dorsal nuclei, particularly in the anterodorsal nucleus, which contained densely arranged labeled cells throughout almost the entire rostrocaudal extent. An injection into the medial entorhinal area labeled a number of cells in the anterior nuclei and in the reuniens nucleus, particularly its dorsal part. Injections into various subdivisions of the lateral entorhinal area yielded different patterns of distribution of labeled cells in the thalamic nuclei. An injection into the ventromedial division (VMEA) led to abundant labeling of cells in the paraventricular and reuniens nuclei. After an injection into the ventral division (VLEA), numerous labeled cells were detected in the reuniens nucleus and a lesser number in the paraventricular nucleus at anterior levels. When an injection was made into the dorsal division (DLEA), a large number of labeled cells were detected in the reuniens nucleus, and less numerous labeled cells were found in the central medial nucleus. There appears to be a topographic arrangement of cortical projections of the reuniens nucleus. The pre- and parasubiculum receive projections from the most medial part of the reuniens nucleus near the midline, and the DLEA receives projections from the medial part of the nucleus. The cells projecting to the VLEA and MEA are distributed in the central part of the reuniens nucleus, and those to the VMEA are distributed in the lateral part. Anterograde experiments were also performed; injections of WGA-HRP into the reuniens nucleus resulted in terminal labeling in the superficial layers of the subicular area and the neighboring hippocampus and in the entorhinal area.

Intracellular recordings of pontine medial gigantocellular tegmental field neurons in the naturally sleeping cat: behavioral state-related activity and soma size difference in order of recruitment.

Intracellular recordings and neurobiotin labeling of medial pontine gigantocellular tegmental field (m-PFTG) neurons in the undrugged, naturally sleeping cat were performed to establish the relationship between soma size and membrane potential (MP) activity before and during the onset of the rapid eye movement (REM) phase of sleep. Initial recordings without labeling revealed that recorded neurons in the m-PFTG had a tonic, sustained membrane depolarization in REM sleep as compared with more polarized MP levels in slow-wave sleep (S) and phasic depolarizations in wakefulness (W) on a more polarized MP level. In neurobiotin-labeled neurons, there was a strong correlation between the soma size of m-PFTG neurons and the 'lead time', the time of onset relative to the beginning of REM, of a sustained increase in membrane depolarization. Thirty-nine m-PFTG neurons with soma cross-sectional areas ranging from 2098 microm(2) to 5958 microm(2) (mean value 3833.8 microm(2)) were analyzed. A majority of these m-PFTG neurons showed an increase in membrane depolarization associated with depolarizing postsynaptic potentials (PSPs) and spike generation that occurred before electrographic signs of REM sleep onset, while the rest of the neurons depolarized at the beginning of or just after REM sleep onset. Our previous work had suggested that many of these m-PFTG neurons were output neurons to the spinal cord. Analysis of the onset time of sustained membrane depolarization (Leadtime(MP)) revealed that larger cells had a longer lead time, while analysis of the lead times for onset of sustained PSPs and action potentials (Leadtime(AP)) showed this measure not to be dependent on soma size, but to be rather uniform, occurring just before the onset of REM sleep. Hence recruitment time, defined as the difference between Leadtime(AP) and Leadtime(MP), was dependent on cell soma size, implying that larger neurons may take longer to depolarize to an MP level critical for generating sustained action potentials, while smaller neurons may require less time.

The ultrastructural findings of Charcot-Leyden crystals in stroma of mastocytoma.

Charcot-Leyden crystals (CLCs) have been found in many conditions associated with eosinophilia, but their occurrence in skin diseases is very rare. We report ultrastructural observations on the presence of CLCs in the cutaneous lesions of two cases of mastocytoma. Electron microscopy documented CLCs located in phagosomes of morphologically activated macrophages as well as free CLCs in the stromal tissue, close association between CLCs formation and damaged and lysed eosinophils was present. These findings provided evidence that the formation of CLCs in mastocytoma implicated the individual and interrelated biology of mast cells, eosinophils and macrophages. Phagosomes probably acted as the site of CLCs formation. The clinic and pathologic role of CLCs in mastocytoma deserves further investigation.

Antidromic activation of vagal and sympathetic afferents does not produce intestinal contractions in dogs.

The question has been asked whether vagal and sympathetic afferents activated antidromically play a role as motor nerves on the in vivo small intestine in dogs anesthetized with urethane. The vagus nerve of one side was cut above the nodose ganglion and the efferent fibers allowed to degenerate. Peripheral stimulation (5-50 Hz, 0.5-3 ms, 5-25 V) of an intact cervical vagus, being able to excite both efferent and afferent fibers, caused large contractions in the jejunum and stomach, whereas stimulation of the contralateral cut cervical vagus could not produce any response in the jejunum but small contractions in the stomach. Peripheral stimulation of the cut cervical vagus did not produce bradycardia and hypotension. Single- and multi-unit discharges to distension of the jejunal segments could be recorded from the peripheral cut end of the cut cervical vagus. Immunohistochemically, there were many substance P-containing cells in both nodose ganglia. Antidromic stimulation of the dorsal roots (T7-T10) did not induce any response in the jejunum but contractions in the stomach. The results may confirm that vagal and sympathetic afferents have no antidromic motor function at least in the in vivo canine small intestine.

Monoclonal antibody that binds to both the prenatal and postnatal astroglia in rodent cerebellum.

A monoclonal antibody (MAb), generated by immunizing mice with homogenized guinea pig cerebellum, labeled cerebellar astroglia including perikarya, radial fibers and veil-like processes in adult rats, mice and guinea pigs. Cell bodies and processes of the immature radial glia in the ventricular neuroepithelium of fetal mice cerebellum were definitely stained by the MAb on the 14th day of gestation. The astroglial components continued to show selective immunoreactivity to the MAb after the 14th day of gestation.

Staining of amyloid with cotton dyes.

We have utilized three cotton dyes, Pagoda Red, RIT Scarlet No. 5, and RIT Cardinal Red No. 9, for the microscopic demonstration of amyloid in cutaneous histopathology. All three dyes stained amyloid specifically, yielding a bright orange color. The preparation of staining solutions and the staining procedure are simple, practical, and can be applied routinely in a dermatopathology laboratory.

Thalamic projections to the hippocampal formation in the cat.

The thalamo-hippocampal projections in the cat were studied by anterograde and retrograde tracing methods. Injections of horseradish peroxidase (HRP) into the hippocampus and dentate gyrus led to retrograde labeling of cells in the nucleus reuniens of the ipsilateral thalamus throughout almost its entire anteroposterior extent. The labeled cells were found mostly in the parvocellular ventral part, but a few cells were labeled in the dorsal part at anterior levels. Anterograde transport following injections of wheat germ agglutinin conjugated to HRP into the nucleus reuniens resulted in terminal labeling in the subicular area, the neighboring parts of the hippocampus (CA1) and the border zone of the dentate gyrus with the hippocampus.

The laminar arrangement of limbic thalamocortical neurons in the lateropulvinar nuclei of the cat thalamus.

The topical organization of limbic cortical projections of the lateropulvinar nuclei of the thalamus in the cat was studied with the horseradish peroxidase (HRP) technique. The dorsal margins of the dorsal lateral, medial pulvinar and lateral pulvinar nuclei project to the postsubicular and presubicular areas (presubiculum in a wide sense), the most dorsal parts of these nuclei projecting to the retrosplenial area, and the dorsal parts to the cingular area. These three zones of limbic thalamocortical neurons in the lateropulvinar nuclei are arranged in lamination from the surface inward, and may be called presubicular, retrosplenial and cingular zones.

Substance P-like immunoreactive projection to the hippocampal formation from the posterior hypothalamus in the cat.

Following the injection of the fluorescent tracer, 4',6-diamidino-2-phenylindol-2HCl (DAPI) or Fluoro-Gold (FG), into the hippocampal formation of the cat, retrogradely-labeled cells were seen mainly in the supramammillary nucleus and the posterior hypothalamic area. Some of these labeled cells contain substance P-like immunoreactivity (SP-LI). When wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) was injected into the supramammillary nucleus and its adjacent regions, anterogradely-labeled terminals were detected, for the most part, in the granular layer and the supragranular molecular layer of the dentate gyrus as well as in the pyramidal layers of the hippocampus and subiculum. All of these layers were included within the terminal areas containing the SP-like immunoreactivity.

Heterogeneity and multiple expression of intermediate filament proteins, S-100 protein and neuron specific enolase in skin mixed tumor.

Intermediate filament proteins, keratin (KL1, PKK1, K8.12) and vimentin, S-100 protein alpha and beta subunits and neuron specific enolase were evaluated immunohistochemically to determine their distribution patterns in the tumor components of mixed tumor of skin. Keratin proteins were distributed widely in tumor epithelial cells or modified myoepithelial (MME) or neoplastic myoepithelial (NME) cells. Luminal cells of the tubulo-ductal structure of the tumor mass showed positive staining of KL1 and PKK1 keratins and an infrequently positive reaction of MoAb K8.12. The outer or basal tumor cells were characterized by coexpression of K8.12 keratin, vimentin, S-100 protein and infrequently neuron specific enolase reactivity. Heterogeneity of keratin distribution was seen in tumor epithelial cells. MME cells or NME cells of skin mixed tumor showed coexpression of keratin and vimentin, and multiple expression of intermediate filament proteins, S-100 protein and neuron specific enolase. Hyaline and chondroid changed cells stained intensely to vimentin and S-100 proteins, as well as to neuron specific enolase. The authors evaluate the histogenesis of skin mixed tumor in relation to epithelial and myoepithelial cells of the sweat gland and their immunohistochemical findings.

Vimentin expression in sweat gland tumours.

Immunohistochemical localization of vimentin was studied in 93 cases of sweat gland tumours using a monoclonal anti-vimentin antibody. A strong immunoreactivity of vimentin was observed in modified myoepithelial or neoplastic myoepithelial cells of mixed tumour of the skin, syringoma, and sweat gland adenoma. Tumour cells in outer layers of tubular, ductal, and duct-like structures usually showed positive staining for vimentin, which coincided with modified myoepithelial cells. All tumour cells of clear cell hydroadenoma showed positive vimentin staining. Tumour cells of the luminal border of tubulo-ductal structures of mixed tumours were rarely immunoreactive for vimentin. Positive vimentin staining of tumour cells in the outer zone of tubulo-ductal structures in sweat gland tumours may be related to reactive proliferation of modified myoepithelial cells and simultaneous growth of luminal tumour cells.

Comparative histochemical study of Bowen's disease and actinic keratosis: preserved normal basal cells in Bowen's disease.

The degree of DNA-instability as revealed by immunohistochemical staining with anti-cytidine antibody after acid hydrolysis (DNA-instability test) has been recently used as a marker of malignancy. This technique was applied to examine 17 skin tissue samples of Bowen's disease, 47 of actinic keratosis, 15 of squamous cell carcinoma, 5 of seborrheic keratosis, and 10 of normal skin. All benign neoplastic cells of seborrheic keratosis and normal epidermal cells were negative. On the other hand, all cancer cells were positive with the DNA-instability test, indicating their malignancy, but all basal cells in Bowen's disease were completely negative. Compatible with this result, the basal cells in Bowen's disease were characteristically normal as evident in other histochemical examinations. Thus, they were negative with p53 immunohistochemistry, with normal signals of chromosome 17 in situ hybridisation and argyrophilic nucleolar organiser region, and showed slightly enhanced proliferative activity as revealed by proliferating cell nuclear antigen immunohistochemistry. Immunohistochemical staining with 34 beta E12 (monoclonal antibody against cytokeratins 1, 5, 10, and 14), which stains all normal epidermal keratinocytes including basal cells, showed that only the basal cells of Bowen's disease stained strongly and homogeneously, while all cancer cells in the upper layers of Bowen's disease and all layers of actinic keratosis were only sporadically or weakly stained. Staining with 34 beta B4 (monoclonal antibody against cytokeratin 1), which recognises the whole epidermis except for the basal layer in the normal epidermis, showed that the basal cells in the Bowen's disease were completely negative, and lower layer cells in the actinic keratosis and upper layer cells in Bowen's disease were only sporadically stained positive, although the superficial layer cells in actinic keratosis stained strongly and homogeneously. Our findings clearly indicate that the basal cells in Bowen's disease are normal. In support of this conclusion, the same cells showed normal morphology on electron microscopy with preserved basement membrane, although the latter was often damaged in actinic keratosis.

Epithelioid hemangioendothelioma of the liver associated with thrombocytopenia and coagulopathy.

Epithelioid hemangioendothelioma of the liver is a rare vascular neoplasm with intermediate malignant potential. The prognosis is highly unpredictable. We report the case of a 59-year-old woman who had the tumor radically resected, but multiple metastases of the liver developed associated with thrombocytopenia and consumption coagulopathy, as observed in Kasabach-Merritt syndrome. The patient did not respond to any treatment and the behavior of the tumor was very aggressive. The patient died 15 months after radical resection of the tumor.

The organization of thalamic neurons projecting to the premotor cortex and the caudate nucleus in the cat studied by a fluorescent retrograde double labeling technique.

Thalamic neurons projecting to both the head of the caudate nucleus and the premotor cortex in the cat were studied by the retrograde fluorescent double labeling technique. After injections of Evans blue into the caudate nucleus, and diamidino-phenylindol into the premotor cortex, a small number of double labeled neurons appeared in the ventral anterior, ventral lateral, anteromedial, rhomboid, central dorsal, central lateral, central medial, paracentral and parafascicular nuclei, in addition to numerous single-labeled neurons. This indicates that some neurons in the thalamic nuclei send bifurcating axons to both the head of the caudate nucleus and the premotor cortex. The caudatal projections of these thalamic neurons are organized in a topical manner.