Health care professionals and industry: reducing conflicts of interest and established best practices.

The relationship between health care providers and pharmaceutical companies and other commercial interests is ethically complex. The common practice of gift giving takes many forms including free samples, sponsorship of medical education, loan of equipment, and gifts ranging from those of nominal value such as pens to more valuable gifts such as golf outings or dinners. Gift giving is a practice that serves both the recipient and the giver, but, in the medical setting, it raises the question of whether this is to the detriment of patient care. Although health care professionals may believe they are able to ignore influence from commercial interests, human judgment research indicates that decision-makers are generally unaware of biases affecting their decisions. This is an issue of organizational ethics as well. Institutions that allow commercial interests to give some form of gift are allowing the appearance of bias as well as placing the burden of avoiding bias on the individual rather than on the institution. Conflict-of-interest analysis indicates that best practice is to limit or eliminate the influence of commercial interests, ensuring that professionals are better able to exercise their independent objective judgment.

The cyanobacterial toxin, microcystin-LR, can induce apoptosis in a variety of cell types.

Cyanobacterial toxins, especially the microcystins (MCYST), are found in eutrophied waters throughout the world. These toxins cause hepatocyte damage by inhibiting protein phosphatases 1 and 2A, resulting in hyperphosphorylation of cytoskeletal proteins. Acute intoxication of animals and humans has been reported following MCYST exposure. Okadaic acid, a marine biotoxin, has a similar mechanism of action to MCYST and has been shown to cause apoptosis, a form of programmed cell death, in a variety of cell types. In this study, primary rat hepatocytes (in suspension and monolayer culture), human fibroblasts, human endothelial cells, human epithelial cells, and rat promyelocytes were observed following treatment with MCYST for morphological and biochemical changes typical of apoptosis. Hepatocytes underwent cell membrane blebbing, cell shrinkage, organelle redistribution, and chromatin condensation as early as 30 min following MCYST application (0.8 microM). Other cell types treated with MCYST (100 microM) also showed these morphological changes, but required a longer period of treatment. DNA fragmentation and "ladder" formation occurred in most cell types exposed to MCYST. These observations demonstrate that MCYST causes apoptosis in a variety of mammalian cells.

Reconstitution of the 2Fe-2S center and g = 1.89 electron paramagnetic resonance signal into overproduced Nostoc sp. PCC 7906 Rieske protein.

The Rieske 2Fe-2S protein is a distinguishing subunit of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overproduction in Escherichia coli of fusion, full-length, and truncated forms of the Rieske (PetC) protein from the cyanobacterium Nostoc sp. PCC 7906. A glutathione S-transferase/Rieske fusion protein was used to prepare specific chicken egg-yolk antibodies against the Rieske protein. Expression of the nonfusion petC gene in a T7 RNA polymerase promoter vector produced copious quantities of the full-length Rieske protein predominantly as inclusion bodies. The highly enriched, Rieske protein from inclusion bodies has been denatured in guanidine hydrochloride and refolded and the characteristic 2Fe-2S cluster reconstituted in vitro by incubation with iron and sulfide under reducing conditions. Purification by chromatography on Whatman DE52 cellulose and ultrafiltration through a 30000 molecular weight cutoff membrane yielded pure and predominantly monomeric Rieske protein. Reconstituted Rieske preparations showed intense and highly characteristic gx = 1.74, gy = 1.89, and gz = 2.03 "Rieske-type" electron paramagnetic resonance signals at 15 K. Two methods of reconstitution yielded Rieske preparations in which 20-60% of the protein contained 2Fe-2S clusters as determined by EPR spin quantitation. The reconstituted Rieske protein was soluble and stable at 4 degrees C in buffers containing nonionic detergents and showed a redox midpoint potential of +321 mV at pH 7.0 as determined by optical circular dichroism (CD) spectroscopy. These data demonstrate the in vitro restoration of a Cys and His liganded 2Fe-2S cluster and provide the basis for mutational and structural analysis of a PetC Rieske protein of oxygenic photosynthesis.

Teloplasm formation in a leech, Helobdella triserialis, is a microtubule-dependent process.

Fertilized eggs of the leech Helobdella triserialis undergo a cytoplasmic reorganization which generates domains of nonyolky cytoplasm, called teloplasm, at the animal and vegetal poles. The segregation of teloplasm to one cell of the eight-cell embryo is responsible for a unique developmental fate of that cell, i.e., to give rise to segmental ectoderm and mesoderm. We have studied the cytoplasmic movements that generate teloplasm using time-lapse video microscopy; the formation and migration of rings of nonyolky cytoplasm were visualized using transmitted light, while the movements of mitochondria into these rings were monitored with epifluorescence after labeling embryos with rhodamine 123, a fluorescent mitochondrial dye. To examine the likelihood that cytoskeletal elements play a role in the mechanism of teloplasm formation in Helobdella, we examined the distribution of microtubules and microfilaments during the first cell cycle by indirect immunofluorescence and rhodamine-phalloidin labeling, respectively. The cortex of the early embryo contained a network of microtubules many of which were oriented parallel to the cell surface. As teloplasm formation ensued, microtubule networks became concentrated in the animal and the vegetal cortex relative to the equatorial cortex. More extensive microtubule arrays were found within the rings of teloplasm. Actin filaments appeared in the form of narrow rings in the cortex, but these varied apparently randomly from embryo to embryo in terms of number, size, and position. The role of microtubules and microfilaments in teloplasm formation was tested using depolymerizing agents. Teloplasm formation was blocked by microtubule inhibitors, but not by microfilament inhibitors. These results differ significantly from those obtained in embryos of the oligochaete Tubifex hattai, suggesting that the presumably homologous cytoplasmic reorganizations seen in these two annelids have different cytoskeletal dependencies.

Animal and vegetal teloplasms mix in the early embryo of the leech, Helobdella triserialis.

In embryos of the glossiphoniid leech, Helobdella triserialis, as in many annelids, cytoplasmic reorganization prior to first cleavage generates distinct animal and vegetal domains of yolk-deficient cytoplasm, called teloplasm. Both domains are sequestered to the D' macromere, progenitor of the definitive segmental tissues, during the first three rounds of cell division. And it has been believed that during the fourth round of cell division, the obliquely equatorial cleavage of macromere D' cleanly segregates animal teloplasm into an ectodermal precursor, cell DNOPQ, and vegetal teloplasm into a mesodermal precursor, cell DM. But here we report a hitherto unobserved cytoplasmic rearrangement between the second and the fourth divisions that seems to mix the animal and vegetal domains of teloplasm. The newly observed rearrangement consists of the movement of vegetal teloplasm toward the animal pole of cell D' between the second and the fourth cell divisions. Animal and vegetal teloplasms form a single pool of teloplasm in cell D' which is then divided between DM and DNOPQ at the fourth division. The movement of teloplasm was inferred by examination of embryos fixed and sectioned between the second and the fourth rounds of cleavage and was confirmed in living embryos microinjected with rhodamine 123, a fluorescent mitochondrial stain.

Centrifugation redistributes factors determining cleavage patterns in leech embryos.

In the normal development of glossiphoniid leech embryos, cytoplasmic reorganization prior to the first cleavage generates visibly distinct domains of yolk-deficient cytoplasm, called teloplasm. During an ensuing series of stereotyped and unequal cell divisions, teloplasm is segregated primarily into cell CD of the two-cell stage and then into cell D of the four-cell and eight-cell stages. The subsequent fate of cell D is also unique in that it alone undergoes further cleavages which generate five bilateral pairs of embryonic stem cells, the mesodermal (M) and ectodermal (N, O/P, O/P, and Q) teloblasts. Here we report studies on the effects of centrifugation on cleavage pattern and protein composition of individual blastomeres of the leech Helobdella triserialis. Centrifugation partially stratifies the cytoplasm of each cell, generating a layer of clear cytoplasm in cell CD derived largely from teloplasm. After centrifuging embryos at the two-cell stage, clear cytoplasm present in cell CD and normally inherited by cell D is redistributed and can be inherited by both cells C and D at the second cleavage. The developmental fates of cells C and D in centrifuged embryos correlate with the amount of clear cytoplasm they receive. In particular, when clear cytoplasm has been distributed roughly equally between the two cells, both cell C and cell D undergo further cleavages resembling the pattern of divisions normally associated with cell D. Likewise, non-yolk-associated proteins, normally found in higher quantities in cell D than in cell C, appear evenly disbursed between the two cells under conditions which induce this fate change. These results are consistent with the idea that the fates of cells C and D are influenced by the distribution or cellular localization of cytoplasmic components.

Comparison of embryonic and adult torpedo acetylcholine receptor by sedimentation characteristics and antigenicity.

The acetylcholine receptor (AChR) from Torpedo marmorata electric organ exists in a light form (α2ßγδ) of apparent molecular weight 250,000. The association of two light forms via an intermolecular δ-δ disulfide bridge results in the AChR heavy form. In adult Torpedo electric organ extracts the heavy form constitutes about 70% of the AChR. We report that, in contrast, embryonic electric organ extracts contain only about 30% of the heavy form, the rest being light form. In addition, amongst a library of 38 monoclonal antibodies (mAbs), all of those that distinguished between embryonic and adult AChR did so only because they precipitated the heavy form of the AChR better than the light form.