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3D cultured cell aggregates, including spheroids, reflect the gene expression patterns of living tissues/organs. Mass preparation of spheroids enables high-throughput drug screening (HTS). However, conventional optical imaging of spheroids makes it difficult to obtain sufficient resolution of individual living cells in the thick cellular stack. Rapid and accurate assessment of cellular responses in spheroids is required for effective drug screening. Here, we show that negative contrast imaging (NCI) of spheroids overcomes this issue. Hydrophilic fluorescent dye added into the culture medium rapidly diffused into the intercellular space of living spheroids within a few minutes. Confocal microscopy showed the NCI of individual cells as dark and detailed contours clearly separated with fluorescence signals in the intercellular space. NCI enables the visualization of the alteration of cell morphology after anti-tumor drug application to living spheroids and the measurement of the fluorescent dye diffusion rate without any complicated pretreatments. Using this system, we found that the antitumor drug doxorubicin reduced the intercellular space of spheroids consisting of the human hepatocyte carcinoma cell line HepG2, through the activation of TGF-β signaling and upregulation of ECM protein expression, implicating a drug resistance mechanism. Collectively, the combination of NCI of spheroids and HTS may enhance the efficiency of drug discovery. [ABSTRACT FROM AUTHOR]

Background: Altered expression of collapsin response mediator proteins (CRMPs) has been reported in several malignant tumors, including downregulation of CRMP1 in lung cancer and upregulation of CRMP2 in colorectal cancer. This study aimed to investigate the relationship between CRMP expression and clinicopathological characteristics in patients with breast cancer. Methods: Twenty-two breast cancer and four normal breast tissues were used to assess CRMP mRNA expression. The average expression level of each CRMP ( CRMP1- 5) mRNA was analyzed in a subset of breast cancer specimens and compared with that in normal breast tissue by real-time quantitative reverse-transcription polymerase chain reaction. Furthermore, 173 breast cancer specimens and matching normal breast controls were used for immunohistochemistry based on the tissue microarray technique. Levels of CRMP2 and phosphorylated CRMP2 protein were assessed, and possible correlations between the clinicopathological characteristics were evaluated. Results:: The expression of CRMP2 mRNA was significantly decreased in breast cancer tissues, while that of the other CRMPs was similar between normal and breast cancer tissues. Immunohistochemistry revealed that CRMP2 protein expression was also decreased in breast cancer tissues ( P < 0.001). Phosphorylated CRMP2 was observed in the nuclei of breast cancer cells but not in normal mammary cells ( P < 0.001). Furthermore, nuclear phosphorylated CRMP2 expression was increased in proportion to the histological grade and triple-negative subtype. Conclusions: Reduced CRMP2 expression and elevated expression of nuclear phosphorylated CRMP2 may be associated with breast cancer progression. [ABSTRACT FROM AUTHOR]

Background: Pancreatic cancer is an aggressive malignancy with one of the worst mortality rates of all cancers. Recently, collapsin response mediator proteins (CRMPs) were reported to be associated with proliferation, apoptosis, differentiation, and invasion in several cancers. However, CRMP expression and their role in pancreatic cancer have not been investigated. This study aimed to clarify the clinical significance of CRMPs in pancreatic cancer. Methods: Expression of crmp genes in 11 pairs of pancreatic cancer and corresponding noncancerous pancreas tissues were examined by real-time RT-PCR. Knockdown of CRMP4 expression using siRNA was examined in pancreatic cancer cell lines to determine whether CRMP4 regulates cell proliferation and invasion in vitro. Furthermore, CRMP4 protein levels in primary tumors of pancreatic cancer ( n = 53) were examined by immunohistochemistry and compared with the clinicopathological features of the tumors. Results: Of all the CRMPs, only CRMP4 was differentially expressed in pancreatic cancer tissues ( p = 0.008). CRMP4 knockdown using siRNA reduced cellular invasion, but did not affect proliferation. The expression of CRMP4 was detected immunohistochemically in 34 (64.2 %) of the 53 pancreatic cancer samples, and CRMP4 expression was correlated with severe venous invasion ( p = 0.044), stage ( p = 0.019), and liver metastasis ( p = 0.021). Multivariate analyses suggested that venous invasion and CRMP4 overexpression were prognostic factors for survival. Conclusions: Our results suggested that CRMP4 is significantly associated with poor prognosis by promoting liver metastasis and can serve as a novel therapeutic target for pancreatic cancer. [ABSTRACT FROM AUTHOR]

A small number of fishers in Chiba Prefecture of eastern Japan use cotton gill nets to catch Japanese spiny lobster Panulirus japonicus. To examine the advantages of cotton gill nets, we analyzed changes in mesh breaking load of a new cotton gill net used in a fishing operation. A new cotton gill net was also soaked in a seawater tank to simulate ghost fishing conditions. The average mesh breaking load of new cotton mesh was 50.3 N. This value decreased to 19.0 N after 38 days (∼912 h), and after 82 days (∼1968 h) the mesh could be easily torn (breaking load 0.07 N). Under fishing conditions, the cumulative soak time was only 744.4 h over 19 months. The average breaking load at the end of this period was 43.1 N, a strength 86% that of the presoaked mesh. The mesh breaking load of a cotton gill net continuously soaked for 744.4 h was 26.1 N, as estimated from tank experiment data. Thus, a cotton gill net maintains reasonable strength under typical use conditions, but will degrade if lost at sea. [ABSTRACT FROM AUTHOR]

In this short review paper, we summarize some of our ideas to utilize gold nanoparticles for the enhancement of surface plasmon resonance signals on DNA microarray. The hybridization of target-DNA capped gold nanoparticles with probe DNA on surface provides ca. ten times stronger optical contrast compared with that of target-DNA molecules. Our simulation result based on the Maxwell-Garnet theory explains well our experimental data and proves a potential of metallic nanoparticles for the substantial sensitivity enhancements for biosensor application in DNA diagnostics and bio-affinity studies, which leads to the fabrication of high resolution DNA microarrays. [ABSTRACT FROM AUTHOR]

Identifies Nogo as a Reticulon protein. Expression of Nogo by oligodendrocytes but not by Schwann cells; Association of Nogo with the endoplasmic reticulum; Molecular basis of the contribution of Nogo to the failure of axonal regeneration in the adult central nervous system.

Scanning electron microscopy (SEM) observation showed that fully differentiated spherical adipocytes were embraced by a network of collagens and fibroblastic preadipocytes. The properties of both the collagen networks and the preadipocytes allow the adipocytes to be interconnected, forming a fat-cell cluster, which can anchor to the bottom of a culture dish. In this network structure, collagen fibrils and fibrillar bundles were closely arranged and stratified. We found that immunostained collagens appeared to form extracellular network structures, which can be observed by SEM. The extracellular network of fibronectin was the first to develop among the extracellular matrix (ECM) components, though it became degraded with the progress of adipocyte differentiation. The type I collagen network was the last to develop and remained well organized through the late stage of adipocyte differentiation. The extracellular networks of type III, V, and VI collagen developed by the mid-stage and remained in the late stage of adipocyte differentiation. The network structures of type IV collagen and laminin became degraded during the differentiation process and localized at the surface of spherical cells. In addition to these basement membrane components, types III, V, and VI collagens also showed pericellular spherical staining patterns. These results demonstrated that the constitution and distribution of the ECM are altered during adipocyte differentiation, suggesting that the organization of each ECM component into a suitable structure is a requirement for the differentiation and maintenance of unilocular adipocytes. [ABSTRACT FROM AUTHOR]

Presents a study which used an oocyte expression system to identify molecules involved in collapsin signalling. Mechanism of collapsin action poorly understood; Participation of heterotrimeric GTP-binding proteins in collapsin reactions; CRMP-62 as an intracellular component of a signalling cascade initiated by a transmembrane collapsin-binding protein.

Neuropilin-1 (NP-1) has been identified as a necessary component of a semaphorin D (SemD) receptor that repulses dorsal root ganglion (DRG) axons during development. SemA and SemE are related to SemD and bind to NP-1, but do not repulse DRG axons. By expressing NP-1 in retinal neurons and NP-2 in DRG neurons, we demonstrate that neuropilins are sufficient to determine the functional specificity of semaphorin responsiveness. SemA and SemE block SemD binding to NP-1 and abolish SemD repulsion in axons expressing NP-1. SemA and SemE seem to have a newly discovered protein antagonist capacity at NP-1 receptors, whereas they act as agonists at receptors containing NP-2. [ABSTRACT FROM AUTHOR]

The article presents a study on the swimming activity and avoidance behavior of fish in detecting the development of toxic condition according to potential of hydrogen (pH) effects. It states that Japanese fat minnow was used as test fish in which D2-value was applied to show avoidance behavior of fish while R-value for swimming activity. Results show increase in R-value after acidification of the water within 30 minutes while no deviation was found in D2-value.

A certain kind of cell in the pituitary gland exhibited immunoreactive keratin and dome formations in vitro. We obtained epithelial cells, which were able to subculture, from the outgrowth of anterior pituitary organ cultures. These cells lacked hormone secretory granules and exhibited immunoreactive keratin. Furthermore, they produced dome formations or cystic structures in monolayer culture and under three-dimensional culture condition using type I collagen gel. Dome formation was stimulated by dibutyryl cyclic AMP (dbcAMP, 10 to 10 M). Their responsiveness to dbcAMP is similar to that of several other epithelial cells that possess transport functions in vivo and in vitro. Although the origin of our cultured cells is unknown, these cells formed dome formations that possessed transport function and were related to cystic structures in the pituitary gland in vivo. [ABSTRACT FROM AUTHOR]

Hemipituitary glands from 30 rats were isotransplanted under renal capsules. At 1, 2, 4, 7 and 20 days after transplantation, the grafts were examined by light and electron microscopy. Two days after transplantation, the central area of graft showed necrosis; however, the peripheral area, where marginal cells of Rathke's cleft were ingesting the remnants of necrotic glandular cells, survived. Four days after transplantation, mitotic figures of marginal cells were observed. Seven days after transplantation, the damaged area of the graft disappeared, and Rathke's cleft was completely lined by marginal cells. Remnants of necrotic glandular cells were not seen in intercellular spaces or in the cytoplasm of marginal cells. Cystlike structures formed in the grafts; some were connected to Rathke's cleft by narrow cavities. The cavity-lining cells of the cysts were agranular and similar to those that lined Rathke's cleft. At 20 days after transplantation, granular cavity-lining cells appeared. It is suggested that marginal cells of immature rats can differentiate into granular cells. [ABSTRACT FROM AUTHOR]

An immunohistochemical study of the anterior pituitary gland of the female Afghan pika was carried out to distinguish the ultrastructural features of GH, PRL, ACTH, TSH and LH cells. The histochemically identified GH cells resembled ultrastructurally oval or round GH cells of the rat laden with large, dense secretory granules. PRL cells were divided into three subtypes based on differences in the diameter of their spherical secretory granules. They lacked polymorphic or irregularly shaped secretory granules. ACTH cells resembled ultrastructurally, in some respects, Siperstein's 'corticotrophs' of the rat with peripheral arrangement of secretory granules. However, they were not always stellate, but elongate or angular in shape. The dense secretory granules were concentrated in the peripheral area of cytoplasm. TSH cells were non-stellate, but usually oval in shape, containing the smallest spherical secretory granules (100-200 nm in diameter). Almost all LH cells reacted also with FSH antiserum. They were irregular in shape, sometimes in contact with or surrounded the GH cells. They contained an abundance of medium-sized secretory granules (140-260 nm in diameter) which were larger than those in the LH cells of the female rat throughout the estrous cycle. Large secretory granules in the LH cells of the female pika seemed to be related to the endocrine state of persistent estrus. [ABSTRACT FROM AUTHOR]