High-level production of recombinant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease.

Infantile Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. We constructed a plasmid containing a 5'-shortened human acid alpha-glucosidase cDNA driven by the cytomegalovirus promoter, as well as the aminoglycoside phosphotransferase and dihydrofolate reductase genes. Following transfection in dihydrofolate reductase-deficient Chinese hamster ovary cells, selection with Geneticin, and amplification with methotrexate, a cell line producing high levels of the alpha-glucosidase was established. In 48 hr, the cells cultured in Iscove's medium with 5 mM butyrate secreted 110-kDa precursor enzyme that accumulated to 91 micrograms.ml-1 in the medium (activity, > 22.6 mumol.hr-1.ml-1). This enzyme has a pH optimum similar to that of the mature form, but a lower Vmax and Km for 4-methylumbelliferyl-alpha-D-glucoside. It is efficiently taken up by fibroblasts from Pompe patients, restoring normal levels of acid alpha-glucosidase and glycogen. The uptake is blocked by mannose 6-phosphate. Following intravenous injection, high enzyme levels are seen in heart and liver. An efficient production system now exists for recombinant human acid alpha-glucosidase targeted to heart and capable of correcting fibroblasts from patients with Pompe disease.

Influence of alkyltransferase activity and chromosomal locus on mutational hotspots in Chinese hamster ovary cells.

High-density mutational spectra have been established for exon 3 of the gene encoding adenine phosphoribosyltransferase (APRT) of the Chinese hamster ovary (CHO) cell line derivative D422 and closely related and/or modified lines by using the mutagen ethyl methanesulfonate (EMS). The total number of selectable sites (GC-->AT transitions yielding a selectable APRT- phenotype) was estimated at 31 based on our own accumulated data base of 136 sequenced exon 3 mutations and on literature reports. D422 and two other APRT hemizygous lines each yielded very similar spectra and showed two populations of mutable sites: (i) 24 "baseline" sites that followed the Poisson distribution and therefore were equally susceptible to mutation and (ii) two hotspots, one comprising a cluster at nucleotides 1293-1309 and the other at nucleotide 1365. Collectively, the latter sites were about 10-fold more frequently mutated than the others. CHO cells are mer- as they lack the repair enzyme O6-methylguanidine methyltransferase (EC 2.1.1.63). In modified repair-proficient CHO cells, the distribution of mutations among all of the 31 sites was random, with only 3 of the 19 GC-->AT transitions in the above hotspots. To determine whether the distribution was locus-dependent, two independent lines carrying single copies of transfected APRT genes were generated from a derivative of D422 carrying a deletion in the endogenous APRT gene. Nucleotides 1293-1309 were again no longer preferentially mutated, but the site at nucleotide 1365 was still a hotspot. We conclude that mutational spectra in mer- cells are at least in part locus dependent and that some sequences are particularly susceptible to EMS mutagenesis and perhaps also to methyltransferase repair.

Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.

Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to the cytotoxic action of mitomycin C under oxygenated and hypoxic conditions. In contrast, porfiromycin was considerably less cytotoxic to wild-type parental cells than was mitomycin C in air and markedly more cytotoxic under hypoxia. Two FpT-transfected clones were selected that expressed 19- and 27-fold more FpT activity than the parental line. Levels of other oxidoreductases implicated in the activation of the mitomycins were unchanged. Significant increases in sensitivity to mitomycin C and porfiromycin in the two FpT-transfected clones were seen under both oxygenated and hypoxic conditions, with the increases in toxicity being greater under hypoxia than in air. These findings demonstrate that FpT can bioreductively activate the mitomycins in living cells and implicate FpT in the differential aerobic/hypoxic toxicity of the mitomycins.

Muscarinic acetylcholine receptor down-regulation limits the extent of inhibition of cell cycle progression in Chinese hamster ovary cells.

Cellular desensitization is believed to be important for growth control but direct evidence is lacking. In the current study we compared effects of wild-type and down-regulation-resistant mutant m3 muscarinic receptors on Chinese hamster ovary (CHO-K1) cell desensitization, proliferation, and transformation. We found that down-regulation of m3 muscarinic acetylcholine receptors was the principal mechanism of desensitization of receptor-activated inositol phosphate phospholipid hydrolysis in these cells. Activation of wild-type and mutant receptors inhibited anchorage-independent growth as assayed by colony formation in agar. However, the potency for inhibition of anchorage-independent growth was greater for cells expressing the mutant receptor. Activation of either receptor also initially inhibited anchorage-dependent cell proliferation in randomly growing populations. Rates of DNA synthesis and cell division were profoundly reduced by carbachol in cells expressing either receptor at early time points. Analysis of cell cycle parameters indicated that cell cycle progression was inhibited at transitions from G1 to S and G2/M to G1 phases. However, mutant receptor effects on anchorage-dependent growth were sustained, whereas wild-type receptor effects were transient. Thus, receptor down-regulation restored cell cycle progression. In contrast, activation of either receptor blocked entry into the cell cycle from quiescence, and this response was not reduced by receptor down-regulation. Therefore, activation of m3 muscarinic acetylcholine receptors inhibited CHO cell anchorage-dependent and -independent growth. In anchored cells carbachol inhibited the cell cycle at three distinct points. Inhibitions at two of these points were eliminated by wild-type receptor down-regulation while the other was not. These results directly demonstrate that desensitization mechanisms can act as principal determinants of cellular growth responses.

The genetic link between the Chinese bamboo partridge (Bambusicola thoracica) and the chicken and junglefowls of the genus Gallus.

Further comparison of mitochondrial control-region DNA base sequences of 16 avian species belonging to the subfamily Phasianinae revealed the following: (i) Generalized perdicine birds (quails and partridges) are of ancient lineages. Even the closest pair, the common quail (Coturnix coturnix japonica) and the Chinese bamboo partridge (Bambusicola thoracica), maintained only 85.71% identity. (ii) The 12 species of phasianine birds previously and presently studied belonged to three distinct branches. The first branch was made exclusively of members of the genus Gallus, while the second branch was made of pheasants of the genera Phasianus, Chrysolophus, and Syrmaticus. Gallopheasants of the genus Lophura were distant cousins to these pheasants. The great argus (Argusianus argus) and peafowls of the genus Pavo constituted the third branch. The position of peacock-pheasants of the genus Polyplectron in the third branch was similar to that of the genus Lophura in the second branch. Members of the fourth phasianine branch, such as tragopans and monals, were not included in the present study. (iii) The one perdicine species, Bambusicola thoracica, was more closely related to phasianine genera Gallus and Pavo than to members of other perdicine genera. The above might indicate that Bambusicola belong to one-stem perdicine lineage that later splits into two sublineages that yielded phasianine birds, one evolving to Gallus, and the other differentiating toward Pavo and its allies.

Exposure to Chinese imports andlocal labor market outcomes. An Analysis for Spanish provinces

In the period 1999-2007 Spanish imports from China multiplied by six, making that Asian country the fourth largest supplier to the Spanish economy. In this paper, we analyse whether this massive increase in imports impacted on the labour markets of Spanish provinces to differing degrees, due to differences in their initial productive specialization. Our results show that Spanish provinces with a higher exposure to Chinese imports experienced larger drops in manufacturing employment as a share of the working-age population. However, this reduction was compensated for by increases in non-manufacturing employment.

The First Child In A Chinese Family Who Could Read Prior To Entry Into Elementary School: A Qualitative Intrinsic Case Study

ABSTRACT This study is an account of the literacy-related human environment a Chinese girl experienced as the first person in the history of her family who was able to read prior to entry into elementary school. Temporally speaking, the study spanned more than a decade from the initial, tentative research question to the formal, primary research question. Spatially speaking, it crossed three cultures: the Chinese, Korean, and American cultures. The study was inspired by the Zero Project in China, known as the "Project of Quality Education and Implementation for Children Aged Zero (fetus) to Six." The significance of the content issue in a child's literacy curriculum was explored in an interdisciplinary way. Case study served as a holistic research approach and provided the researcher with free temporal and spatial distance to pursue the indefinably multi-dimensional intricacies of a child's early literacy acquisition among generations in the family. Interpretation of the case was based on the relevant concepts within the scope of the researcher's knowledge of Chinese culture. Major findings revealed that the child's literacy acquisition was inseparably related to her parents' background as well as their awareness of and attitudes towards literacy, and that the foundation of all this was the harmony of the family. Through the lens of generational attitudes towards literacy and especially the lens of the researcher's multicultural life experiences, this study contributes to the field of curriculum studies in general and early literacy curriculum in particular by stimulating people to reconsider what to read to children, besides how to read to them. It calls attention once again to the classic curriculum question, "What knowledge is of most worth?" as well as what is the most essential spiritual food human beings need besides physical needs. This study suggests that Chinese philosophy should be included in a child's early literacy curriculum in China and calls for dialogues on the content issue of curriculum to gain a deeper understanding of human nature so that humans might co-live peacefully with all beings in the universe.

The Hybrid Spirit Animating Chinese Pentecostals in Malaysia

This dissertation interrogates the assertion in postcolonial scholarship, especially from the work of Homi Bhabha that the construction and performance of hybrid identities act as a form of resistance for marginalized communities against structures of oppression. While this study supports this assertion, it also critiques how hybridity fails to address issues of unequal power relations. This has led to an uncritical use of hybridity that reproduces the very idea of static identity which its proponents claim to transcend. Through qualitative study of Chinese members of a Pentecostal church in Malaysia, this study argues that church members engage in "unequal belonging" where they privilege certain elements of their identities over others. In concert with Pierre Bourdieu's conceptions of habitus, field, and capital, unequal belonging highlights how hybridity fails to capture the intersecting and competing loyalties, strategies, and complexities of identity formation on a contextual level. Unequal belonging challenges postcolonial scholars to locate the subtle workings of power and privilege that manifest even among marginalized communities. The study first situates the Chinese through an analysis of the historical legacy of British colonialism that has structured the country's current socio-political configuration along bounded categories of identification. The habitus constrains church members to accept certain Chinese ethnic markers as "givens." Although they face continuous marginalization, interviewee data demonstrates that church members negotiate their Chineseness and construct a "Modern Chinese" ethnic identity as a strategic move away from Chinese stereotypes. Moreover, conversion to Christianity affords church members access to cultural capital. Yet, it is limited and unequal capital. In particular, the "Chinese Chinese," who church members have demarcated as backward and traditional, are unable to gain access to this capital because they lack fluency in English and knowledge in modern, westernized worldviews. Unequal belonging nuances monolithic conceptions of hybridity. It demonstrates how church members' privilege of Christianity over Chineseness exposes the complex processes of power and privilege that makes westernized-English-speaking Chinese Christians culturally "higher" than non-English-speaking, non-Christian, Chinese. This study provides significant contribution to the complex aspect of hybridity where it is both a site of resistance and oppression.

Chinese Intellectuals and China`s Policy toward Japan

This dissertation aims at integrating two scholarships: state-society relation studies and Chinese foreign policy analysis. I created Two-level Perception Gap Model to analyze different intellectual groups' relations with party-state by confirming Chinese intellectuals play a role in CFP making in general, China's Japan policy in particular. This model is an alternative approach, instead of conventional wisdom patron-client approach, to explain and analyze the pluralized intellectual-state relations in China. This model first analyzed the role of two intellectual groups, namely think tank scholars and popular nationalist, in China's Japan policy making, and then based on these analyses it explains the interactional patterns between these two intellectual groups and party-state. I used three case studies, which represented different types of issue, Chinese attitude toward the U.S.-Japan alliance and the Japanese defense policy, the controversy over the Yasukuni Shrine Visit, and the territorial dispute over the Diaoyu/Senkaku Islands, to examine this model. First, I examined think tank scholar groups and the extent they influenced "core interest issue and sensitive issue (Issue 1)," Chinese attitude toward the U.S.-Japan alliance and the Japanese defense policy, and their international patterns with party-state. Chapter 3 compares the responses of Chinese officials to the changes in the defense policy of Japan to the analyses from the think tank scholars. As the model assumes, results show that think tank scholars' analyses are consistent with China's policy position; nevertheless, it is difficult to confirm their analyses have influence on Chinese attitude toward the U.S.-Japan alliance and the Japanese defense policy. Based on the analysis of journal articles, most articles do not provide policy suggestions or simply provide suggestions that do not deviate from the policy. As Gu's theory of pluralist institutionalism and my hypothesis points out, most think tank scholars are establishment intellectuals so they tend to be self-disciplined. Second, this model provide a new concept "patriotic dilemma" for analyzing the challenge and constraints brought by popular nationalist discourses and public mobilization to Chinese foreign policy decision makers. Chapter 4 investigated the cases study of the controversy over the Yasukuni Shrine Visit, defined as "major/minor interest issue/ sensitive issue (Issue 3)," and the discourses from the popular nationalist, mainly focusing on anti-Japanese activists. The chapter also observes their influence on nationalist public opinions and analyzes how the nationalist public opinions constrain the policy choices among decision makers. Results strongly supported the hypothesis of patriotic dilemma that, although the popular nationalist group and public opinions constrained the policy choices of Chinese decision makers in the short term, they were unable to change the fundamental policy direction. Third, chapter 5 also focuses on anti-Japanese activists and examines the model with the case of the territorial dispute over the Diaoyu/Senkaku Islands. The result supported that hypothesis that China's policy change was not because of the influence from popular nationalist's discourses or public opinions but because of the change of priority of this issue, from major/minor interest issue to core interest issue. These two chapters also indicate that the patron-client model is unable to describe the popular nationalist. An alternative approach, such as the concept "patriotic dilemma" is needed to describe the relations between the popular nationalist and the government.

Providing Bereavement Counseling to Chinese Americans: A Cultural Adaptation of a Western Grief Counseling Model.

The aim of this paper is to extend the existing literature and propose an alternative perspective on bereavement counseling with Chinese Americans. This aim is achieved by integrating William Worden's (2009) grief counseling model with several cultural components that are relevant to counseling with Chinese Americans, including: (a) the barriers to seeking counseling, (b) the clinical presentations of Asian Americans, (c) the common coping styles among Asian Americans, (d) the major Chinese religions and philosophies, and (e) the bereavement-related cultural practices. The corresponding treatment recommendations will be explored following the discussion of each cultural element. Finally, a culturally responsive grief counseling model for Chinese Americans will be proposed in the last section, along with a discussion of important caveats.