Large-scale production of a therapeutic protein in transgenic tobacco plants: effect of subcellular targeting on quality of a recombinant dog gastric lipase

A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants.     

Activation of the 12-lipoxygenase and signal transducer and activator of transcription pathway during neointima formation in a model of the metabolic syndrome

Insulin resistance (IR) is associated with an increased risk of cardiovascular diseases. The obese Zucker rat (ZR) is a model of IR that shows markedly increased insulin and triglyceride concentrations without major changes in glucose. In this study, we evaluated the response of obese and lean ZR to carotid balloon injury and determined potential mechanisms and treatments. The neointima-to-media ratio of obese ZR was greater than that of lean ZR, starting at 14 days after injury, and persisted until at least day 30. An enhanced inflammatory response to balloon injury in the obese ZR was reflected by significantly higher ED1-positive macrophage cells in the injured vessel wall compared with that in lean ZR at 3, 7, and 14 days after balloon injury. Inflammatory mediators 12-lipoxygenase (12-LO) and STAT4 were studied in neointimal lesions. Expression of 12-LO RNA was increased beginning at day 7 and showed increases of 4.3-fold on day 14 and 7-fold on day 30 in obese ZR compared with lean animals. Staining of phosphorylated STAT4 (PSTAT4), the activated form of STAT4, in lesions from obese ZR was also increased compared with that in leans. We tested the effects of a novel anti-inflammatory agent, lisofylline (LSF), in the obese ZR. LSF markedly reduced neointimal formation in the obese ZR. LSF also reduced monocyte/macrophage infiltration into the vessel wall and the activation of PSTAT4. These studies suggest both the presence of an exaggerated injury response in the insulin-resistant obese ZR model and that inflammation plays a major role in mediating neointimal growth.     

Attitudes of cancer patients, family caregivers, oncologists and members of the general public toward critical interventions at the end of life of terminally ill patients

Background:

Whereas most studies have focused on euthanasia and physician-assisted suicide, few have dealt comprehensively with other critical interventions administered at the end of life. We surveyed cancer patients, family caregivers, oncologists and members of the general public to determine their attitudes toward such interventions.

Methods:

We administered a questionnaire to four groups about their attitudes toward five end-of-life interventions — withdrawal of futile life-sustaining treatment, active pain control, withholding of life-sustaining measures, active euthanasia and physician-assisted suicide. We performed multivariable analyses to compare attitudes and to identify sociodemographic characteristics associated with the attitudes.

Results:

A total of 3840 individuals — 1242 cancer patients, 1289 family caregivers and 303 oncologists from 17 hospitals, as well as 1006 members of the general Korean population — participated in the survey. A large majority in each of the groups supported withdrawal of futile life-sustaining treatment (87.1%–94.0%) and use of active pain control (89.0%–98.4%). A smaller majority (60.8%–76.0%) supported withholding of life-sustaining treatment. About 50% of those in the patient and general population groups supported active euthanasia or physician-assisted suicide, as compared with less than 40% of the family caregivers and less than 10% of the oncologists. Higher income was significantly associated with approval of the withdrawal of futile life-sustaining treatment and the practice of active pain control. Older age, male sex and having no religion were significantly associated with approval of withholding of life-sustaining measures. Older age, male sex, having no religion and lower education level were significantly associated with approval of active euthanasia and physician-assisted suicide.

Interpretation:

Although the various participant groups shared the same attitude toward futile and ameliorative end-of-life care (the withdrawal of futile life-sustaining treatment and the use of active pain control), oncologists had a more negative attitude than those in the other groups toward the active ending of life (euthanasia and physician-assisted suicide).As more attention turns to when and how the lives of terminally ill patients end in the clinical setting, debate about the issues of euthanasia and physician-assisted suicide grows.^1–5 Euthanasia has been discussed in Europe and the United States for more than a century, and the public has become more accepting of it.^4–10 Announcing its first-ever ruling in favour of an unconscious patient’s right to die with dignity, the Korean Supreme Court recently ruled that doctors of an elderly woman in a persistent vegetative state remove the artificial respirator from her on the basis of her presumed wishes.¹¹ A public debate aimed at legalizing withdrawal of futile life-sustaining treatment, exposure to stories of dying patients in the mass media, and the court’s decision may have led to a greater awareness of, and sensibility toward, the rights of terminally ill patients. In 2000, only 16.5% of 535 Korean oncologists surveyed said that they would prescribe morphine for severe cancer pain, and more than half of 655 patients who had pain said they had inadequate pain management.¹²Although much has been written about attitudes toward how the general public would choose to die in the clinical setting,^4,13 most studies have focused on only euthanasia and physician-assisted suicide.^14–18 We conducted a large survey to examine attitudes among cancer patients, family caregivers, oncologists and members of the general public toward critical interventions at the end of life of terminally ill patients.