Chinese and Americans agree on what is fair, but disagree on what is best in societal decisions affecting health and safety risks.

Through surveys of students and junior professionals and interviews with business and government executives, we studied Chinese choices and fairness perceptions in risky health and safety decisions. The survey responses were compared with American responses from an earlier study by Keller and Sarin. The survey results show that the American and Chinese respondents had similar fairness perceptions, but the Chinese did not make decisions that were consistent with their fairness perceptions, whereas the Americans did. We found that the middle-age Chinese professionals tended to make choices that were more different from the Americans than were the choices of the young Chinese management students. It is likely that these discrepancies were caused by cultural differences, with the younger Chinese tending to face a stronger Western influence. The insights from the survey results were enriched by interviews that revealed fairness perceptions of Chinese business and government executives. A framework to interpret cultural influences on decision making was also proposed.

DNA elements regulating alpha1-tubulin gene induction during regeneration of eukaryotic flagella.

Eukaryotic flagella are complex organelles composed of more than 200 polypeptides. Little is known about the regulatory mechanisms governing synthesis of the flagellar protein subunits and their assembly into this complex organelle. The unicellular green alga Chlamydomonas reinhardtii is the premier experimental model system for studying such cellular processes. When acid shocked, C. reinhardtii excises its flagella, rapidly and coordinately activates transcription of a set of flagellar genes, and ultimately regenerates a new flagellar pair. To define functionally the regulatory sequences that govern induction of the set of genes after acid shock, we analyzed the alpha1-tubulin gene promoter. To simplify transcriptional analysis in vivo, we inserted the selectable marker gene ARG7 on the same plasmid with a tagged alpha1-tubulin gene and stably introduced it into C. reinhardtii cells. By deletion of various sequences, two promoter regions (-176 to -122 and -85 to -16) were identified as important for induction of the tagged alpha1-tubulin gene. Deleting the region between -176 and -122 from the transcription start site resulted in an induction level which was only 45 to 70% of that of the resident gene. Deleting the region upstream of -56 resulted in a complete loss of inducibility without affecting basal expression. The alpha1-tubulin promoter region from -85 to -16 conferred partial acid shock inducibility to an arylsulfatase (ARS) reporter gene. These results show that induction of the alpha1-tubulin gene after acid shock is a complex response that requires diverse sequence elements.

Calcium influx signals normal flagellar RNA induction following acid shock of Chlamydomonas reinhardtii.

Acid shock of Chlamydomonas results in flagellar excision and induction of flagellar protein RNAs. The magnitude of flagellar RNA accumulations after flagellar excision by mechanical shear depends on the extracellular Ca2+ concentration. In this report, we demonstrate that the magnitude and duration of flagellar RNA accumulations are signaled by an acid shock-induced Ca2+ influx. RNA accumulations were greater in cells acid shocked in 500 microM CaCl2 than in 200 microM CaCl2, although the accumulation durations were similar. RNA accumulations of lower magnitude and shorter duration were observed in cells in Ca2+-containing buffer treated with CdCl2. RNA accumulations were of still lower magnitude and shorter duration in cells shocked in buffer without added CaCl2 than in cells shocked in 200 or 500 microM CaCl2 or in the presence of CdCl2. RNA accumulations similar to those in cells shocked in buffer without added CaCl2 were measured in cells following acid shock in buffer containing 200 microM CaCl2 and supplemented with neomycin, ruthenium red, or LaCl3. Acid shock of the adf-1 mutant resulted in RNA accumulations of shorter duration and lower magnitude than those measured in adf-1 cells stimulated by mechanical shear. These results are consistent with an hypothesis that acid shock generates two genetically and pharmacologically distinct signals governing flagellar RNA induction; the first signal is independent of a Ca2+ influx and flagellar excision and results in low magnitude accumulations of short duration, and the second is a consequence of a Ca2+ influx and results in accumulations of high magnitude and long duration.

Ion selectivity in the Chlamydomonas reinhardtii flagellar regeneration system.

Extracellular Ca2+ mediates the cellular and molecular responses to cell stimulation of Chlamydomonas reinhardtii. Extracellular Ca2+ concentrations ([Ca2+]e) must exceed certain threshold values to support flagellar excision by acid shock and to stimulate flagellar outgrowth following mechanical shear of the flagella. Also, the magnitude and duration of flagellar RNA accumulations following acid shock or mechanical shear increase with increasing [Ca2+]e. To better understand the role of Ca2+ in flagellar excision, RNA induction, and outgrowth, we have performed a survey of the ion selectivity of each of these responses to acid shock. We found that flagellar excision in vivo following acid shock was supported by Sr2+ and Ca2+, but no other ion tested. LaCl3 and neomycin prevented flagellar excision following acid shock of cells in Ca2+- or Sr2+-containing buffer. Sr2+ addition to detergent-permeabilized cell models, however, failed to elicit flagellar excision in vitro. Cells failed to regrow flagella following flagellar excision in Sr2+-containing buffer unless exogenous Ca2+ was added. Flagellar RNA accumulations of lower magnitude and shorter duration were measured in cells acid-shocked in Sr2+-containing buffer than in Ca2+-containing buffer. These results demonstrate that a Sr2+ influx can evoke flagellar excision following acid shock, but cannot directly activate the machinery for flagellar excision, suggesting that a Sr2+ influx induces excision by stimulating an intracellular Ca2+ release. Furthermore, they suggest that flagellar outgrowth and normal flagellar RNA induction have a strict requirement for Ca2+, which is not satisfied by the proposed intracellular Ca2+ release.

Measuring heart patients' willingness to pay for changes in angina symptoms.

Willingness-to-pay (WTP) measures of the effects of changes in health on a person's welfare are more comprehensive than traditional cost-of-illness (COI) measures, but they are sometimes difficult to obtain. The authors investigated two approaches for measuring heart patients' WTP for changes in their angina symptoms. First, actual expenditures and perceived angina episodes avoided were used to infer an averting-behavior measurement of WTP. Second, a contingent-valuation approach was used to ask direct WTP questions regarding a hypothetical medical treatment that could be purchased to avoid additional angina episodes. The results indicated that although negligible COI changes were expected with small changes in angina frequency, the subjects had significant WTP to avoid increases in angina. The average WTP to avoid additional angina episodes revealed by the averting-behavior questions was comparable to the directly-elicited WTP, providing a test of the validity of the contingent-valuation approach.

Ca2+ signaling in the Chlamydomonas flagellar regeneration system: cellular and molecular responses.

In response to certain extracellular stimuli, Chlamydomonas reinhardtii cells excise their flagella, induce expression of more than 200 different flagellar mRNAs, and assemble a new flagellar pair. Normally, flagellar excision, gene induction and outgrowth are tightly coupled temporally. Our previous studies showed that uncoupling the cellular response of flagellar excision from flagellar outgrowth resulted in submaximal flagellar gene induction, and led us to propose that normal flagellar gene induction is a composite response. The present study extends these observations by measuring flagellar gene induction in Chlamydomonas cells stimulated under conditions where both flagellar excision and flagellar outgrowth are blocked. We find that the flagellar genes are induced in a Ca(2+)-dependent manner in response to stimulation in the absence of flagellar excision and outgrowth. Flagellar gene induction is therefore independent of flagellar excision and outgrowth but sensitive to extracellular Ca2+ levels. Thus, flagellar excision, flagellar outgrowth and flagellar gene induction are three responses to a common stimulus that are related by their requirement for extracellular Ca2+.

A multiattribute-utility-function approach to weighing the risks and benefits of pharmaceutical agents.

Both the selection of doses of pharmaceutical agents and comparisons between pharmaceutical agents have long been based on the nonquantified concept of the risk-benefit ratio. Though useful, this concept implies a data comparison that is difficult to make: the toxicity versus the efficacy of a drug compound. This research demonstrates an approach for weighing risks and benefits by combining utility functions for human efficacy and toxicity with animal and laboratory toxicity information to develop an overall multiattribute utility function for an ophthalmic pharmaceutical agent, I-bunolol, intended for the treatment of glaucoma. With this multiattribute function and a small portion of the published data available for this drug, the expected utilities for six doses (including a control) could be compared and the value of this approach in drug-dosage selection demonstrated.

Inositol phospholipid metabolism may trigger flagellar excision in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii cells shed their flagella in response to environmental stress. Under favorable conditions, flagella are quickly regrown. To learn more about the signals that trigger flagellar excision and regrowth we have investigated inositol phospholipid metabolites, molecules implicated in signal transduction in several other systems. After deflagellation by low pH or mastoparan, a potent activator of G proteins, there was a rapid increase in levels of inositol 1,4,5-trisphosphate measured by use of receptor-binding assays and HPLC. This increase was concomitant with a decrease in levels of phosphatidylinositol 4,5-bisphosphate and was followed by an increase in phosphatidic acid, results consistent with activation of phospholipase C and diacylglycerol kinase. Additional experiments suggest that this activated phospholipase C is not important for flagellar regrowth but plays a role in informing the excision apparatus of the environmental stress. Addition of neomycin (an inhibitor of phospholipase C) before exposure of cells to low pH or mastoparan prevented the increase in inositol 1,4,5-trisphosphate and also prevented deflagellation. Addition of neomycin after deflagellation blocked increases in inositol 1,4,5-trisphosphate that normally followed deflagellation, but did not block flagellar assembly. Furthermore, a flagellar excision-defective mutant, fa-1, did not shed its flagella in response to low pH or mastoparan, yet both of these agents activated phospholipase C in these cells. The results suggest that activation of phospholipase C, possibly via a G protein, is a proximal step in the signal transduction pathway inducing deflagellation in Chlamydomonas.

Uncoupling of Chlamydomonas flagellar gene expression and outgrowth from flagellar excision by manipulation of Ca2+.

Chlamydomonas cells respond to certain environmental stimuli by shedding their flagella. Flagellar loss induces a rapid, transient increase in expression of a specific set of genes encoding flagellar proteins, and assembly of a new flagellar pair. While flagellar gene expression and initiation of flagellar outgrowth are normally tightly coupled to flagellar excision, our results demonstrate that these processes can be uncoupled by manipulating Ca2+ levels or calmodulin activity. In our experiments, wild-type cells were stimulated to excise their flagella using mechanical shearing, and at times after deflagellation, flagellar lengths were measured and flagellar mRNA abundance changes were determined by S1 nuclease protection analysis. When extracellular Ca2+ was lowered by addition of EGTA to cultures before excision, flagellar mRNA abundance changes and flagellar outgrowth were temporally uncoupled from flagellar excision. When extracellular Ca2+ was lowered immediately after excision or when calmodulin activity was inhibited with W-7, flagellar outgrowth was uncoupled from flagellar excision and flagellar mRNA abundance changes. Whenever events in the process of flagellar regeneration were temporally uncoupled, the magnitude of the flagellar mRNA abundance change was reduced. These results suggest that flagellar gene expression may be regulated by multiple signals generated from these events, and implicate Ca2+ as a factor in the mechanisms controlling flagellar regeneration.

Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation.

The fate of exogenous DNA introduced into Chlamydomonas reinhardtii by electroporation was analyzed. With single and double electrical pulses, plasmids as large as 14 kb were introduced into cells with and without intact cell walls. Within hours after introduction, exogenous plasmid DNA was associated with nuclei isolated from cells; several weeks after introduction, exogenous DNA was stably integrated into the Chlamydomonas genome. These studies establish electroporation as a method for introducing DNA, and potentially other molecules, into C. reinhardtii.

Analysis of Chlamydomonas reinhardtii histones and chromatin.

Chromatin spreads made from isolated nuclei of the unicellular green alga Chlamydomonas reinhardtii show the beaded fibers typical of eukaryotic polynucleosomes. Micrococcal nuclease digestions confirmed the presence of nucleosomes with a repeat length of 189 base pairs, essentially the same as typical mammalian cells. Basic nuclear proteins extracted from isolated nuclei or chromatin with 1 M calcium chloride and 0.3 M hydrochloric acid are resolved into seven major components by electrophoresis in the presence of sodium dodecyl sulfate (SDS). These seven components were subjected to qualitative peptide mapping with V8 protease on SDS gels for comparison with the major histone components of calf thymus. Finally, the C. reinhardtii basic nuclear proteins were fractionated by reversed phase high performance liquid chromatography and their amino acid composition determined. From these studies, we conclude that C. reinhardtii has a full complement of the five histones with properties very similar to those of both higher animals and higher plants.

Protein synthesis is required for rapid degradation of tubulin mRNA and other deflagellation-induced RNAs in Chlamydomonas reinhardi.

After flagellar detachment in Chlamydomonas reinhardi, there is a rapid synthesis and accumulation of mRNAs for tubulin and other flagellar proteins. Maximum levels of these mRNAs (flagellar RNAs) are reached within 1 h after deflagellation, after which they are rapidly degraded to their predeflagellation levels. The degradation of alpha- and beta-tubulin RNAs was shown to be due to the shortening of their half-lives after accumulation (Baker et al., J. Cell Biol. 99:2074-2081, 1984). Deflagellation in the presence of protein synthesis inhibitors results in the accumulation of tubulin and other flagellar mRNAs by kinetics similar to those of controls. However, unlike controls, in which the accumulated mRNAs are rapidly degraded, these mRNAs are stabilized in cycloheximide. The stabilization by cycloheximide is specific for the flagellar mRNAs accumulated after deflagellation, since there is no change in the levels of flagellar mRNAs in nondeflagellated (uninduced) cells in the presence of cycloheximide. The kinetics of flagellar mRNA synthesis after deflagellation are shown to be the same in cycloheximide-treated and control cells by in vivo labeling and in vitro nuclear runoff experiments. These results show that protein synthesis is not required for the induced synthesis of flagellar mRNAs, and that all necessary transcriptional control factors are present in the cell before deflagellation, but that protein synthesis is required for the accelerated degradation of the accumulated flagellar mRNAs. Since cycloheximide prevents the induced synthesis and accumulation of flagellar proteins, it is possible that the product(s) of protein synthesis required for the accelerated decay of these mRNAs is a flagellar protein(s). The possibility that one or more flagellar proteins autoregulate the stability of the flagellar mRNAs is discussed.

Transcription of alpha- and beta-tubulin genes in vitro in isolated Chlamydomonas reinhardi nuclei.

Removal of the flagella of Chlamydomonas results in increases in both flagellar protein synthesis and tubulin messenger RNA accumulation. These observations led us to examine whether flagellar protein gene sequences are transcribed differentially in nuclei isolated before and after deflagellation. A nuclear isolation protocol was developed using the cell wall-less strain of Chlamydomonas, CW 15, after cell lysis with 0.5% Nonidet P-40. Transcriptional activity of isolated nuclei was determined by incorporating [32P]UTP into TCA-precipitable and phenol-extractable RNA, and by hybridizing newly transcribed RNA to complementary DNA clones containing alpha- and beta-tubulin sequences. Nuclei from deflagellated cells are more active in transcribing sequences that hybridize with alpha- and beta-tubulin complementary DNA probes than are nuclei from nondeflagellated cells. In addition, while total [32P]UTP incorporation is inhibited 45% by alpha-amanitin concentrations of 1.0 micrograms/ml, tubulin RNA synthesis in this system is completely inhibited by this concentration of alpha-amanitin. This demonstration of differential transcription in nuclei before and after cell deflagellation provides the means to study in vitro the mechanisms that signal and regulate flagellar protein gene activity.

Synthesis of adult myosin light chains by embryonic muscle cultures.

Myosin light chain synthesis has been analyzed in cultures of fast and slow muscles from chicken and quail embryos. Synthesis was assayed by [35S]methionine incorporation and two-dimensional electrophoresis of total cell extracts. Our results show that differentiated cultures of embryonic anterior latissimus dorsi and pectoral muscles synthesize proteins that comigrate on two-dimensional gels with the five myosin light chains of adult fast (pectoral) and slow (anterior latissimus dorsi) muscle. Partial proteolytic digestion and peptide analyses further confirm the identity of these proteins as adult light chains. Cultures of dividing myoblasts do not synthesize any of these fiber type isozymes, and synthesis of the isozymes is initiated at myoblast fusion. Also, myogenic clones drived from single myoblasts differentiate to synthesize these five myosin light chains, indicating that individual myoblasts have the potential to express the synthesis of all fiber type light chain isozymes. We conclude that the primary events in muscle differentiation include the initiation of synthesis of the entire set of adult fast and slow myosin light chain isozymes. The developmental and physiological implications of these results for the establishment of fiber type specificity are discussed.