Same patient, different advice: a study into why doctors vary.

AIM: To understand why doctors differ in their recommendations in situations where there is little certainty about the long term outcomes of the possible treatment options. METHODS: A correlational design was used to examine the relation between preference for different treatment options and beliefs about likely outcomes for these options. Eighty doctors, with a mean of nine years in paediatric cardiology/surgery, attending a conference on serious congenital heart disease were studied. Main outcome measures were: ratings of the extent to which each of four treatment options were favoured; and subjective probabilities for three outcomes-death, survival with "good heart function" (New York Heart Association functional class (NYHA) I or II), and survival with "poor heart function" (NYHA III or IV)-for different treatment options over a 20 year time frame. RESULTS: Preference for one treatment option over another was most closely associated with the subjective estimate of the additional years with "good heart function" that it offered 10-20 years after surgery (Pearson's r = 0.66, p < 0.001). In influencing a preference, the possibility of early death was subordinate to optimising the late outcome. CONCLUSIONS: Doctors' treatment preferences are consistent with selecting the option that maximises the chance of the best outcome (long term survival with good heart function). Doctors' recommendations imply that they place more value on years of life in the child's far future than on life-years in the immediate future.

Differences in belief about likely outcomes account for differences in doctors' treatment preferences: but what accounts for the differences in belief?

Doctors, patients, and their relatives face a complex decision when there are multiple treatment options that differ in their profiles of risk and benefit over time. Doctors from a single specialist paediatric cardiac unit participated in a correlational study that used a novel tool (subjective multi-state survival graphs) to elicit their beliefs about the likely outcome of different treatments. Doctors' preferences were more closely related to their beliefs about long term, rather than short term, outcomes. This is consistent with placing greater value on far future than on immediate life years, highlighting the importance of incorporating patients' values for these outcomes into decisions of this kind. Beliefs about likely outcomes differed with whether or not doctors encountered former paediatric patients who were now adults, illustrating the difficulty of deciding what risk information should be available when the evidence base on outcomes is limited. Some problems of risk communication are identified, and the value of multi-state survival graphs as an aid to communication is discussed.

Synaptic exocytosis and nervous system development impaired in Caenorhabditis elegans unc-13 mutants.

C. elegans mutants defective in unc-13 exhibited severe behavioral abnormalities including paralyzed locomotion and slow pharyngeal pumping and irregular defecation cycle. Consistent with the phenotypes, the mutants accumulated abnormally high levels of the neurotransmitter acetylcholine and were resistant to acetylcholinesterase inhibitors. The unc-13 gene was expressed in most, if not all, neurons when analyzed by using chimeric constructs consisting of the unc-13 promoter and green fluorescence protein or beta-galactosidase reporter gene. While Ca(2+)-regulated acetylcholine release is lacking, the mutants were still able to release acetylcholine in vivo and in vitro at similar levels to that mediated by the regulated mechanism. Double mutants defective in both unc-13 and other genes involved in synaptic transmission showed the Unc-13 phenotype, rather than other mutant phenotypes, in terms of locomotion as well as of acetylcholine accumulation. Furthermore, electron microscopic reconstruction of the mutant nervous system uncovered that a majority of neurons developed and connected as those in the wild type except for subtle abnormalities including inappropriate connections through gap junctions and morphological alterations of neurons. These results demonstrate that the unc-13 gene product plays an essential role at a late stage in Ca(2+)-regulated synaptic exocytosis. Neurotransmitters released through the Ca(2+)-regulated mechanism are required for, but do not play major roles in the nervous system development. The large amount of Ca(2+)-independent neurotransmitter release observed in the unc-13 mutants suggests that there may be a distinct mechanism from evoked or spontaneous release in neurotransmission.

Expression of multiple UNC-13 proteins in the Caenorhabditis elegans nervous system.

The Caenorhabditis elegans UNC-13 protein and its mammalian homologues are important for normal neurotransmitter release. We have identified a set of transcripts from the unc-13 locus in C. elegans resulting from alternative splicing and apparent alternative promoters. These transcripts encode proteins that are identical in their C-terminal regions but that vary in their N-terminal regions. The most abundant protein form is localized to most or all synapses. We have analyzed the sequence alterations, immunostaining patterns, and behavioral phenotypes of 31 independent unc-13 alleles. Many of these mutations are transcript-specific; their phenotypes suggest that the different UNC-13 forms have different cellular functions. We have also isolated a deletion allele that is predicted to disrupt all UNC-13 protein products; animals homozygous for this null allele are able to complete embryogenesis and hatch, but they die as paralyzed first-stage larvae. Transgenic expression of the entire gene rescues the behavior of mutants fully; transgenic overexpression of one of the transcripts can partially compensate for the genetic loss of another. This finding suggests some degree of functional overlap of the different protein products.

Imaging protein kinase C activation in living sea urchin eggs after fertilization.

The fluorescent dye NBD-phorbol acetate was used to visualize the activation of protein kinase C (PKC) in living Lytechinus pictus eggs during fertilization. The dye interacts directly with PKC as determined using a competitive binding assay. Quantitative image analysis of sequential images from laser-scanning confocal microscopy showed a significant reorganization of the signal in the vicinity of the cortical granules and the plasma membrane that began immediately following fertilization and persisted up to 1 hr (P<0.0001). At the concentrations employed, the NBD-phorbol dye was not capable of inducing a significant translocation of the fluorescent signal to the membrane, nor did it appear to interfere with the cell cycle. It therefore seems likely that the present in vivo results reflect the previously reported in vitro activation of protein kinase C immediately subsequent to fertilization. Such an interpretation is parsimonious with the results of parallel subcellular fractionation experiments using an N-terminal polyclonal antibody to sea urchin PKC which showed a significant (P<0.037) translocation of the enzyme from the cytosolic fraction to the membrane fraction 40 min subsequent to fertilization. This study supports and extends previous in vitro data suggesting that PKC activation subsequent to fertilization occurs at or near the egg plasma membrane, perhaps in association with arachadonic acid-rich cortical granules.

Synergistic calcium release in the sea urchin egg by ryanodine and cyclic ADP ribose.

A transient rise in cytoplasmic Ca2+ activity in the sea urchin egg occurs during fertilization due to release from an intracellular store. Two intracellular receptor Ca2+ channels for inositol 1,4,5-trisphosphate (IP3) and ryanodine have been identified by physiological and immunological techniques. While IP3 is the endogenous messenger for the IP3 receptor, a corresponding physiological messenger for the ryanodine receptor is unknown. A variety of recent experimental evidences suggest that cyclic ADP ribose (cADPR) may be a possible candidate. In this study using both egg homogenates and intact eggs, we show that subthreshold concentrations of cADPR and ryanodine can act synergistically to potentiate Ca2+ release. Addition of 10-20 nM cADPR, which causes little net increase in Ca2+, generally enhances the action of subthreshold concentrations of ryanodine. Similarly the addition of 60-80 microM ryanodine causes a slight transient increase but potentiates maximal Ca2+ increase by a subsequent subthreshold addition of cADPR. While the target of Ca2+ release by ryanodine and cADPR may be the ryanodine receptor, their actions appear to be different and more complex than simply opening the release mechanism. There are significant differences in the kinetics of release by the two agonists. In addition we used a poorly metabolized analog of IP3 and an inhibitor of endoplasmic reticulum Ca2+ ATPase activity, to show that the unfertilized egg contains a rapidly filled Ca2+ store, which is commonly released by both IP3-mediated and ryanodine-mediated release mechanisms.

Synergistic release of calcium in sea urchin eggs by caffeine and ryanodine.

A transient rise in intracellular Ca2+ during fertilization is necessary for activation of the quiescent sea urchin egg. Several mechanisms contribute to the rise in Ca2+ including influx across the egg plasma membrane and release from intracellular stores. The egg contains both IP3-sensitive and -insensitive Ca2+ release mechanisms and in this study we have used single-cell spectrofluorimetry to examine the effects of caffeine and ryanodine on Ca2+ release in eggs preloaded with fura 2. Caffeine induced a small Ca2+ release that was insensitive to heparin or ruthenium red. Ca2+ liberation by caffeine could be augmented by prior treatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase. Variable Ca2+ releases were observed in response to microinjection of ryanodine. The action of ryanodine appeared to be enhanced by prior injection of heparin and partially inhibited by ruthenium red. The release of Ca2+ by caffeine or ryanodine was generally insufficient to trigger cortical granule exocytosis, thus these eggs could be fertilized and a second Ca2+ release during fertilization was measured. Unlike the caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release mechanism in somatic cells, the graded responses in eggs suggested this caffeine- and ryanodine-sensitive release mechanism is not sensitive to sudden changes in Ca2+. Thus we could examine the combined actions of caffeine and ryanodine on Ca2+ release, which were synergistic. Caffeine treatment of ryanodine-injected eggs or ryanodine injection of caffeine-treated eggs stimulated a Ca2+ release significantly larger than the release by either drug independently. The experiments presented here suggest that sea urchin eggs liberate Ca2+ in response to caffeine and ryanodine; however, the regulation of this release differs from that described for caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release of somatic cells.

Multiple stores of calcium are released in the sea urchin egg during fertilization.

Fertilization initiates a transient increase in intracellular Ca2+ principally by Ca2+ release from intracellular stores. Possible multiple Ca2+ stores and multiple receptor regulation of the same store have been reported. Here we report the presence of at least two independent intracellular Ca2+ stores in the sea urchin egg, which are released during fertilization. Ca2+ release from one store is mediated by inositol 1,4,5-trisphosphate (IP3) and is sensitive to low molecular weight heparin. The other store is heparin insensitive and independent of IP3 regulation, but the regulatory factor remains unidentified. A transient increase in Ca2+ in heparin-loaded eggs is observed during fertilization, which suggests that IP3-independent Ca2+ release mediates the production of IP3 and release of the IP3-dependent store. Experiments presented here do not support the idea of sperm receptor coupling to inositol phospholipid hydrolysis through a GTP-binding protein mediating the fertilization response.