International perceptions and approval of gene therapy.

Gene therapy is in clinical trials in a number of countries, raising the question of whether different ethical standards can be justified in different countries. One key issue is how divergent are the perceptions and bioethical reasoning of peoples around the world. An International Bioethics Survey with 150 questions, including 35 open ones, was developed to look at how people think about diseases, life, nature, and selected issues of science and technology, biotechnology, genetic engineering, genetic screening, and gene therapy. The mail response survey was conducted in 1993 among the public in Australia, India, Israel, Japan, New Zealand, Russia, and Thailand, and the same written survey was conducted among university students in Australia, Hong Kong, India, Japan, New Zealand, The Philippines, Russia, Singapore, and Thailand. Similar questions were included in an international high school education bioethics survey among high school teachers in Australia, Japan, and New Zealand. Further international comparisons to the United States and Europe are made. About three-quarters of all samples supported personal use of gene therapy, with higher support for children's use of gene therapy. The diversity of views was generally similar within each country. The major reasons given were to save life and increase the quality of life. About 5-7% rejected gene therapy, considering it to be playing God, or unnatural. There was very little concern about eugenics (0.5-2%), and more respondents gave supportive reasons like "improving genes," especially in Thailand and India. Support for specific applications was significantly less for "improving physical characters," "improving intelligence," or "making people more ethical" than for curing diseases like cancer or diabetes, but there was little difference between inheritable or noninheritable gene therapy.

Perception of risks and benefits of in vitro fertilization, genetic engineering and biotechnology.

The use of new biotechnology in medicine has become an everyday experience, but many people still express concern about biotechnology. Concerns are evoked particularly by the phrases genetic engineering and in vitro fertilization (IVF), and these concerns persist despite more than a decade of their use in medicine. Mailed nationwide opinion surveys on attitudes to biotechnology were conducted in Japan, among samples of the public (N = 551), high school biology teachers (N = 228), scientists (N = 555) and nurses (N = 301). People do see more benefits coming from science than harm when balanced against the risks. There were especially mixed perceptions of benefit and risk about IVF and genetic engineering, and a relatively high degree of worry compared to other developments of science and technology. A discussion of assisted reproductive technologies and surrogacy in Japan is also made. The opinions of people in Japan were compared to the results of previous surveys conducted in Japan, and international surveys conducted in Australia, China, Europe, New Zealand, U.K. and U.S.A. Japanese have a very high awareness of biotechnology, 97% saying that they had heard of the word. They also have a high level of awareness of IVF and genetic engineering. Genetic engineering was said to be a worthwhile research area for Japan by 76%, while 58% perceived research on IVF as being worthwhile, however 61% were worried about research on IVF or genetic engineering. Japanese expressed more concern about IVF and genetic engineering than New Zealanders. The major reason cited for rejection of genetic manipulation research in Japan and New Zealand was that it was seen as interfering with nature, playing God or as unethical. The emotions concerning these technologies are complex, and we should avoid using simplistic public opinion data as measures of public perceptions. The level of concern expressed by scientists and teachers in Japan suggest that public education "technology promotion campaigns" will not reduce concern about science and technology. Such concern should be valued as discretion that is basic to increasing the bioethical maturity of a society, rather than being feared.

Public acceptance of human gene therapy and perceptions of human genetic manipulation.

Clinical trials of gene therapy are underway in different countries, and further countries can be expected to use gene therapy soon. Little remains known, however, about public perceptions of gene therapy. Nationwide mail response opinion surveys were conducted in Japan in August-October, 1991. A total of 54% of the public, 65% of the high school biology teachers, and 54% of the scientists who responded said that they would be willing to use gene therapy, and 66%, 73%, and 62%, respectively, said that they would be willing to use gene therapy on their children. There appears to be growing acceptance of gene therapy in Japan, although about one-quarter of the population are against it. The underlying reasoning behind the acceptability of human genetic manipulation and perceived benefits and risks are presented, and these were found to be generally similar to reasoning expressed in a similar survey conducted in New Zealand in May, 1990. Public perceptions are also compared to those in Europe and the United States. People perceive both benefits and risks from genetic manipulation. There appears to be more teaching of ethical, social, and environmental issues associated with genetic engineering in senior high school biology classes in New Zealand than in Japan. In Japan and New Zealand, about 90% of the public would support including discussion of social issues associated with science and technology in the curriculum.

Identification of a set of calcium-binding proteins in reticuloplasm, the luminal content of the endoplasmic reticulum.

A procedure was developed for the isolation of reticuloplasm, the luminal material of the endoplasmic reticulum (ER). A reticuloplasm-rich extract was prepared from a murine plasmacytoma cell line that contains large amounts of ER, by first extracting the cytoplasmic contents using hypotonic lysis to yield ER-rich 'shells' followed by mechanical lysis to release the ER contents. The extract contains five major proteins with apparent molecular weights of 100, 75, 60, 58 and 55 (X 10(3] Mr by SDS-polyacrylamide gel electrophoresis. The 100, 75 and 58 (X 10(3] Mr species were identified as the known ER proteins endoplasmin, BiP and PD1, respectively. The ER association of the 60 and 55 (X 10(3] Mr proteins was confirmed by confocal fluorescence microscopy with affinity-purified antibodies. Equilibrium dialysis with isolated reticuloplasm gave a calcium-binding capacity of 300 nmoles calcium per mg protein with half-maximal binding at 3 mM-Ca2+. Purified endoplasmin bound 280 nmoles calcium per mg protein at a calcium concentration of 5 mM-Ca2+. A calcium overlay test revealed that, in addition to endoplasmin, reticuloplasm contained at least three other calcium-binding proteins: i.e. BiP, PDI and the 55 X 10(3) Mr protein, respectively, with endoplasmin and the 55 X 10(3) Mr protein (CRP55) accounting for the major proportion of the calcium-binding activity. Treatment of cells with calcium ionophore led to the specific over-expression of the major calcium-binding reticuloplasmins endoplasmin, BiP and CRP55. These studies show that the lumen of the ER contains a family of proteins with the capacity to bind significant amounts of calcium in the millimolar range and thereby to confer upon the ER the ability to perform a calcium storage function analogous to that of the sarcoplasmic reticulum in muscle cells.

Endoplasmin is a reticuloplasmin.

The location of endoplasmin in the endoplasmic reticulum was investigated by biochemical and immunoelectron microscopic analyses. The protein could be obtained in a soluble form by procedures that do not involve the use of any detergents. The soluble protein has the amino- and carboxy-terminal sequences of the intact molecule, showing that it has not been proteolysed. Application of the Triton X-114 phase-separation test does not reveal significant hydrophobicity in the molecule. Immunogold labelling studies on cells with a dilated endoplasmic reticulum (ER) lumen show that endoplasmin is uniformly distributed throughout the lumen, with no evidence of a preferential association with the membrane. These studies clearly demonstrate that endoplasmin is a luminal protein of the ER, i.e. a reticuloplasmin, and not an integral membrane protein.

Visualization of the intact endoplasmic reticulum by immunofluorescence with antibodies to the major ER glycoprotein, endoplasmin.

Antibodies to endoplasmin were used to examine the morphology of the endoplasmic reticulum (ER) by immunofluorescence on permeabilized plasmacytoma and fibroblastoid cells. In unfixed cells, permeabilization led to a pronounced vesiculation of the ER. Therefore cells were first fixed lightly prior to permeabilization with detergent. Fibroblastoid cells gave a characteristic reticular pattern surrounding the nucleus with clear staining of the nuclear membrane. Plasmacytoma cells, in the conventional fluorescence microscope, gave a cisternae-like pattern. Optical sectioning with a confocal scanning laser microscope gave a distinct pattern of concentric cisternae similar to those obtained with transmission electron microscopy on cell sections. The overall morphology of the ER in such cells could be revealed by serial optical sectioning. Evidence was obtained that the ER does not undergo extensive vesiculation during mitosis in plasmacytoma cells. Using anti-endoplasmin immunofluorescence monitoring, conditions were developed for the retention of ER morphology in unfixed, permeabilized cells. These studies illustrate the value of endoplasmin as a general marker for the analysis of ER morphology in different types of cells by immunofluorescence microscopy.