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The phenomenon of “elliptical” expressions in Japanese has been extensively studied in the field of Japanese linguistics. However, this phenomenon has been often treated as a general syntactic feature of Japanese, and the question of how this feature is realized in actual use of the language has been rather neglected. The present paper is intended to analyze how speakers of Japanese actually deal with the task of interpreting unexpressed elements that emerge in their talk in interaction. Using video- and audio-data of naturally occurring conversations in Japanese, it is shown that, in producing and understanding utterances involving unexpressed referents, conversational parties utilize not only their morphological and syntactic knowledge but also various, multilayered resources that are available to them in the immediate context of interaction.

This study investigates, from a conversation-analytic perspective, the phenomenon in which a Japanese language speaker uses the first-person singular pronoun watashi to initiate a “parenthetical insert” in the course of producing telling. Some of them are led by first-person pronouns with the so-called topic marker wa, while others are led by first-person pronouns without wa or any other particle. We explore the differences in the interactional workings between them. It will be established that the parenthetical inserts led by watashi without any particle are used to supplement or qualify the immediately preceding part of the telling that the speaker may take as potentially puzzling or misleading to the recipient, while parenthetical inserts led by watashi with wa parenthetically clarify the position/status from which the speaker is producing the current turn of telling.

This study examines, from a conversation-analytic perspective, the use of [NP+wa?] format turns ([NP+wa?] turns) by young Japanese children aged 23–33 months, who have started using the so-called topic marker wa. Most of their [NP+wa?] turns are used in one of the following environments: 1) the NP in an [NP+wa?] turn refers to a person or object absent from the scene, 2) the NP refers to an object of noticing that is present at the scene, and 3) [NP+wa?] turns are used repeatedly. I will show that each of these situations provides co-participants with guidance to determine how the NP referent is relevant to the here-and-now and what kind of response is expected. I will then claim that [NP+wa?] turns provide these children with a convenient and powerful tool to engage with the everyday social world around them; that is, to explore how the life-world is organized and ordered. Furthermore, I will propose that wa can be seen as marking the speaker's claim that the recipient should be able to detect the preceding NP referent's relevance to the here-and-now. I claim that this is what is fundamentally involved in the “topic-marking” function that is conventionally associated with the particle wa.

This study investigates the Japanese interjections anoo and sonoo, both of which are pragmaticized from adnominal forms of distal demonstratives, and have been often associated with speaker's internal difficulties in searching for appropriate words or expressions. Observing these two tokens in natural conversation, we find that the differentiated interactional work that each interjection performs go far beyond marking a momentary problem with one's own speech production. We demonstrate, instead, that by deploying anoo, speakers foreshadow an emerging shift in the interactional trajectory, i.e., what will follow is not a continuation of the immediately preceding context; while by deploying sonoo, speakers explicitly project that the following talk is in-line with what has been already established in the interaction, and should thus be construed as a non-deviation with reference to the existing or ongoing activity or project. Thus, while each of these two interjections work in halting the progressivity of the turn-at-talk, they are differentially used as resources to help participants orient to the emerging talk in its relation to the present context, and are thus useful linguistic resources that Japanese speakers employ to delicately navigate intersubjective orientation and understanding between participants.

Ectodomain shedding is a post-translational modification mechanism by which the entire extracellular domain of membrane proteins is liberated through juxtamembrane processing. Because shedding rapidly and irreversibly alters the characteristics of cells, this process is properly regulated. However, the molecular mechanisms governing the propensity of membrane proteins to shedding are largely unknown. Here, we present evidence that negatively charged amino acids within the stalk region, an unstructured juxtamembrane region at which shedding occurs, contribute to shedding susceptibility. We show that two activated leukocyte cell adhesion molecule (ALCAM) protein variants produced by alternative splicing have different susceptibilities to ADAM metallopeptidase domain 17 (ADAM17)-mediated shedding. Of note, the inclusion of a stalk region encoded by a 39-bp-long alternative exon conferred shedding resistance. We found that this alternative exon encodes a large proportion of negatively charged amino acids, which we demonstrate are indispensable for conferring the shedding resistance. We also show that the introduction of negatively charged amino acids into the stalk region of shedding-susceptible ALCAM variant protein attenuates its shedding. Furthermore, we observed that negatively charged amino acids residing in the stalk region of Erb-B2 receptor tyrosine kinase 4 (ERBB4) are indispensable for its shedding resistance. Collectively, our results indicate that negatively charged amino acids within the stalk region interfere with the shedding of multiple membrane proteins. We conclude that the composition of the stalk region determines the shedding susceptibility of membrane proteins.

In naturally occurring everyday caregiver–child interaction, a major part of what is hearable as storytelling or an incipient form of it is talk about participants’ (mostly children’s) past experiences. Adopting a conversation-analytic approach, this study attempts to show how explicit references to children’s past actions formulated in the form of [(X) did (Y)], where X is the young child interacting with the caregiver, can engender opportunities for participants to develop telling activities. Through the detailed analysis of talk and embodied features of telling sequences in each case, the analysis will reveal how the [(X) did (Y)]-format utterance is utilized for co-constructing the telling, and what social and interactional consequences are accomplished through the telling occasioned by such reference.

Eeto is one of the most frequently occurring Japanese vocal markers. Often characterized as a mere time-buyer, or “filler”, this token has been frequently said to reflect ongoing internal cognitive processing or reflection. Examining naturally occurring instances of eeto by focusing on its occurrences at the beginning of responses to information–seeking questions, however, we found that eeto-prefaced responses all provide a carefully constructed answer in contexts where the responses might otherwise be heard as not aligning in the most straightforward way. We argue that eeto affords Japanese conversationalists a way through which they can project the maximally prosocial stance of interactional commitment to undertaking the task at hand. Rather than a marker of an internal processing state, eeto, we argue, is instead a useful linguistic resource to publically display a respectful stance toward the questioner while the respondent is carefully building an appropriately contextualized response.

An increase of cellular phosphocholine (PC) and total choline-containing compounds (tCho) as well as alterations in lipids have been consistently observed in cancer cells and tissue. These metabolic changes are closely related to malignant transformation, invasion, and metastasis. The study of cancer cells in culture plays an important role in understanding mechanisms leading to altered choline and lipid metabolism in cancer, as it provides a carefully controlled environment. A solid tumor, however, is a complex system with a unique tumor microenvironment frequently containing hypoxic and acidic regions, and areas of nutrient deprivation and necrosis. Cancer cell – stromal cell interactions and the extracellular matrix may also alter choline and lipid metabolism. Human tumor xenograft models in mice are useful to mimic the growth of human cancers and provide insights into the influence of in vivo conditions on metabolism. Here we have compared metabolites, obtained with high resolution 1H MRS of extracts from human breast and prostate cancer cells in a 2-dimensional (2D) monolayer culture, and from solid tumor xenografts derived from these cells, as well as the protein expression of enzymes that regulate choline and lipid metabolism. Our data demonstrate significant differences in choline and lipid metabolism and protein expression patterns between human breast and prostate cancer cells in culture and tumors derived from these cells. These data highlight the influence of the tumor microenvironment on choline and lipid metabolism.

The ability to destroy cancer cells while sparing normal tissue is highly sought after in cancer therapy. Small interfering RNA (siRNA)-mediated silencing of cancer-cell specific targets, or the use of a prodrug enzyme delivered to the tumor to convert a non-toxic prodrug to an active drug are two promising approaches in achieving this goal. Combining both approaches into a single treatment strategy can amplify selective targeting of cancer cells while sparing normal tissue. Noninvasive imaging can assist in optimizing such a strategy, by determining effective tumor delivery of the siRNA and prodrug enzyme to time prodrug administration, and detecting target downregulation by siRNA, and prodrug conversion by the enzyme. In proof-of-principle studies, we synthesized a nanoplex carrying magnetic resonance imaging (MRI) reporters for in vivo detection, and optical reporters for microscopy, to image the delivery of siRNA and a functional prodrug enzyme in breast tumors, and achieve image-guided molecular targeted cancer therapy. siRNA targeting of choline kinase-α (Chk-α), an enzyme significantly up regulated in aggressive breast cancer cells, was combined with the prodrug enzyme bacterial cytosine deaminase (bCD) that converts the non-toxic prodrug 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU). In vivo MRI and optical imaging showed efficient intratumoral nanoplex delivery. siRNA-mediated downregulation of Chk-α and the conversion of 5-FC to 5-FU by bCD were detected noninvasively with 1H MR spectroscopic imaging and 19F MR spectroscopy. Combined siRNA and prodrug enzyme activated treatment achieved higher growth delay than either treatment alone. The strategy can be expanded to target multiple pathways with siRNA.

Epidermal growth factor (EGF) family members play important roles in the skeletal system. In this study, we examined the role of EGF receptor (EGFR) signaling in osteoblastogenesis in vitro. The expression of HB-EGF and epiregulin (EPR) was transiently induced within 24 h after osteogenic stimulation, but when preosteoblastic MC3T3-E1 cells were incubated with HB-EGF or EPR, osteoblast differentiation was inhibited. These effects were Ras-dependent, and ERK modulated Runx2 activity through the localization of Smad1 and the induction of Twist2. PI3-kinase was also required for the induction of Twist2. However, the inhibition of individual signaling pathways was not sufficient to overcome HB-EGF-mediated inhibition of osteoblast differentiation. Additionally, HB-EGF treatment promoted the proliferation of preosteoblasts, and this was associated with the downregulation of p27 at the protein level. These results suggest that HB-EGF-EGFR signaling inhibits the differentiation of osteoblasts by suppression of Runx2 transcriptional activity and enhances proliferation of preosteoblasts by downregulation of expression of p27.

Elevated phosphocholine (PC) and total choline (tCho) metabolites are widely established characteristics of most cancer cells, including breast cancer. Effective silencing of choline kinase (chk), the enzyme that converts choline to PC, is associated with reduced tumor growth. The functional importance and down-regulation of chk using RNA interference has been previously established. Here, we report on the preclinical evaluation of lentiviral vector–mediated down-regulation of chk using short hairpin RNA (shRNA) in established tumors derived from human breast cancer cells. Concentrated lentivirus expressing shRNA against chk was injected i.v. in the tail vein of MDA-MB-231 tumor-bearing female severe combined immunodeficient mice. Transduction efficiency in cells and tumors in vivo was assessed optically by enhanced green fluorescent protein expression and additionally from chk mRNA and protein levels. An 80% reduction in chk mRNA and protein was achieved following ∼90% transduction efficiency in cells. After transduction with chk-shRNA, 1H magnetic resonance spectroscopy (MRS) of cell and tumor extracts showed decreases in PC and tCho levels (P < 0.01 and 0.05, respectively) in comparison with controls. PC levels were monitored noninvasively by 31P MRS in tumors and by 1H MRS in cell and tumor tissue extracts. Noninvasive 31P MR spectra of chk-shRNA–transduced tumors in vivo showed lower PC and phosphomonoester levels that were associated with reduced tumor growth and proliferation. This study shows the use of lentiviral vectors to target chk in a human breast cancer xenograft and noninvasive MRS detection of this targeting. [Cancer Res 2009;69(8):3464–71]

The intensity of the total choline (tCho) signal in spectroscopic images of tumors is spatially heterogeneous. The likewise heterogeneous physiologic tumor microenvironment may contribute to this heterogeneity. We therefore investigated the relationship between hypoxia, choline metabolites, and choline kinase (Chk) in a human prostate cancer model. Human PC-3 prostate cancer cells were engineered to express enhanced green fluorescent protein (EGFP) under hypoxic conditions. These PC-3-5HRE-EGFP cells were characterized in culture and as tumors transplanted in mice using 1H magnetic resonance spectroscopy (MRS) and MRS imaging (MRSI) combined with EGFP fluorescence microscopy and imaging. Hypoxic EGFP-fluorescing tumor regions colocalized with regions of high tCho in combined MRSI and optical imaging studies. Cellular phosphocholine (PC) and tCho concentrations as well as Chk expression levels significantly increased following exposure of PC-3 cells to hypoxia. A putative promoter region located 5′ of the translation start site of the human chk-α gene was cloned and luciferase (Luc)-based reporter vector constructs were generated. Luc reporter assays provided evidence that some of the putative hypoxia response elements (HRE) within this putative chk-α promoter region functioned in vitro. Chromatin immunoprecipitation assays using an antibody against hypoxia-inducible factor (HIF)-1α showed that HIF-1 can directly bind this region of the endogenous chk-α promoter in hypoxic PC-3-5HRE-EGFP cells. These data suggest that HIF-1 activation of HREs within the putative chk-α promoter region can increase Chk-α expression within hypoxic environments, consequently increasing cellular PC and tCho levels within these environments. [Cancer Res 2008;68(1):172–80]

Lysosomes contain multiple proteases, which play a crucial role in breast cancer invasion and metastasis. Noninvasive labeling of lysosomes in breast cancer cells and solid breast tumor models is therefore useful to study lysosomal trafficking and its role in invasion. We have synthesized a novel compound, 6'-O-lissamine-rhodamine B-glucosamine, to fluorescently label lysosomes, and evaluated the compound in human breast cancer cells in cell culture or in orthotopic human breast cancer models. We demonstrated that this novel compound biosynthetically labeled lysosomal proteins following addition to cell culture medium or following intravenous injection into mouse models of breast cancer. Fluorescence from 6'-O-lissamine-rhodamine B-glucosamine colocalized with several well-established lysosomal markers, such as lysosome-associated proteins 1 and 2 (LAMP-1 and -2) and CD63. We also demonstrated the feasibility of performing in vivo fluorescence imaging of 6'-O-lissamine-rhodamine B-glucosamine to image lysosomes in human breast cancer models.

The present study reports on the use of a linguistic category "interrogative," which has been traditionally associated with the act of questioning, and its use in argument talk in Japanese. Based on the observation that interrogative utterances in argument data are regularly followed by non-answers, it is argued that interrogative utterances in argument sequences may not be designed/interpreted as doing questioning. Such use of interrogatives can become an orderly practice to which participants orient themselves in social activities recognizable as arguments. However, though an answer is not expected, the recipient invariably provides some form of response, or the initial speaker seeks such a response when none is provided. Thus the nature of interrogatives as a grammatical category seems to reside in the basic structural unit of social interaction [recipient-oriented action]-[response]. In general, this study is intended to show the dynamically interlocking relationship between grammar and interaction by exploring the intricate interplay between a local action for which interrogative grammar is employed, and the sequential environment and activity framework in which the action takes place.

This report presents a method for examining grammar as a participants' resource for conduct in interaction. By situating the analysis of grammar in the interactional context of turn-construction and action sequence organization we are able to establish a technical basis for comparing elements of grammatical organization across languages and cultures. By focusing on the co-construction of single turn-constructional units, we are able to describe participants' treatment of sentences-in-progress in terms of a sequentially informed syntax. Through the co-investigation of languages with dissimilar grammatical practices we are able to isolate and describe the use of language-specific structures as constitutive elements of turn-construction.

Identifying strategies to increase cancer cell kill while sparing normal tissue is critically important in cancer chemotherapy. Choline kinase (Chk), the enzyme that converts choline to phosphocholine (PC), is elevated in cancer cells and presents a novel target for increasing cell kill. Here, we have examined the effects of transiently down-regulating Chk by small interfering RNA against Chk (siRNA-chk) on PC and total choline-containing compound (tCho) levels and on the viability/proliferation of estrogen receptor–negative and estrogen receptor–positive breast cancer cell lines and a nonmalignant mammary epithelial cell line. We investigated the effects of combination treatment with transient siRNA-chk transfection and the anticancer drug 5-fluorouracil (5-FU) in those cell lines. Microarray analysis of the invasive estrogen receptor–negative MDA-MB-231 cell line was done to characterize molecular changes associated with Chk down-regulation. Chk down-regulation decreased PC and tCho levels in the malignant cell lines, whereas the cell viability/proliferation assays detected a decrease in proliferation in these cells. In contrast, Chk down-regulation had an almost negligible effect on PC and tCho levels as well as cell viability/proliferation in the nonmalignant cell line. A combination of siRNA-chk with 5-FU treatment resulted in a larger reduction of cell viability/proliferation in the breast cancer cell lines; this reduction was evident to a much lesser degree in the nonmalignant cells. Microarray analysis showed that Chk down-regulation affected 33 proliferation-related genes and 9 DNA repair–related genes. Chk down-regulation with siRNA-chk may provide a novel alternative to enhance the effect of anticancer drugs in malignant cells. [Cancer Res 2007;67(23):11284–90]

The conjugate of bacterial cytosine deaminase (bCD) and poly-l-lysine (PLL) that was functionalized with biotin, rhodamine, and Gd3+−DOTA was synthesized and characterized. It demonstrated high relaxivity, improved enzymatic specificity to prodrug 5-fluorocytosine, low cytotoxicity, efficient cell uptake, and high enzymatic stability in fresh mouse serum and human breast cancer cell culture.

Elevated lipogenesis is a characteristic feature of cancer. Fatty acid synthase (FASN) overexpression has been found in many human cancers. Tumor cells in culture and solid tumor models are essential tools to study cancer biology. A comparison of the models can reveal tumor microenvironmental influences on lipogenesis. Here we have characterized differences in lipid components such as total fatty acid (tFA) and phosphatidylcholine (PtCho) levels between tumor cells in culture and solid tumors, with 1H MR spectroscopy (MRS) of lipid-soluble cell or tumor extracts derived from breast and prostate cancer cell lines. MCF-12A, nonmalignant human mammalian epithelial cells, MDA-MB-231, an estrogen receptor (ER)/progesterone receptor (PR)-negative metastatic human breast cancer cell line and MCF-7, an ER/PR-positive poorly metastatic human breast cancer cell line, as well as PC-3 and DU-145, which are both androgen independent malignant human prostate cell lines were used in this study. Fully relaxed 1H MR spectra of lipid-soluble extracts from cells in culture and tumors were acquired on a Bruker Avance 500 MR spectrometer using tetramethylsilane (TMS) as an internal concentration standard. Total FA and PtCho levels were quantified as A.U./cell or A.U./g using integrals of the 1.3 ppm and 3.2 ppm signals in the 1H NMR spectra, respectively, normalized to the cell number or tumor weight (g). Patterns of PtCho and tFA levels were similar in cells, but different in tumors. Both PtCho and tFA levels were significantly higher in MCF-7 cells compared to MCF-12A and MDA-MB-231 cells. Interestingly, the levels of PtCho and tFA in nonmalignant MCF-12A cells and malignant MDA-MB-231 cells were comparable. However, the level of tFA in MDA-MB-231 tumors was significantly higher than in MCF-7 tumors. It has been known that hypoxia and low pH conditions activate FASN activity. FASN or/and other lipogenic enzymes in MDA-MB-231 tumors may be more sensitive to those microenvironmental factors. The tFA level of DU-145 cells was significantly higher compared with PC-3 cells. However, PtCho and tFA levels in PC-3 and DU-145 tumors were not significantly different. FASN protein levels in MCF-12A and MCF-7 cells were not significantly different. Since MCF-12A medium contains epidermal growth factor, which activates FASN, we need to further investigate the involvement of FASN without its activators and with only de novo fatty acid. Our results revealed that tFA levels of cells and tumors do not show similar patterns. The differences between cells and tumors can arise from environmental factors found in solid tumors such as depletion of nutrients and oxygen, changes in pH, as well as cancer cell and stromal/endothelial cell interactions. Additional studies are necessary to unravel the tumor microenvironmental factors responsible for the differences in lipid metabolism observed between cells grown in culture as compared to the same cells grown as solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 64.