Ethical and legal dilemmas arising during predictive testing for adult-onset disease: the experience of Huntington disease.

The goal of predictive testing is to modify the risk for currently healthy individuals to develop a genetic disease in the future. Such testing using polymorphic DNA markers has had major application in Huntington disease. The Canadian Collaborative Study of Predictive Testing for Huntington Disease has been guided by major principles of medical ethics, including autonomy, beneficence, confidentiality, and justice. Numerous ethical and legal dilemmas have arisen in this program, challenging these principles and occasionally casting them into conflict. The present report describes these dilemmas and offers our approach to resolving them. These issues will have relevance to predictive-testing programs for other adult-onset disorders.     

Effects of temperature on the phase behavior and permeability of thylakoid lipid vesicles : relevance to chilling stress

Large unilamellar vesicles composed of thylakoid glycolipids, phosphatidylglycerol, and varying proportions of dipalmitoylphosphatidylglycerol (DPPG) have been examined for the temperature dependence of their permeability to ⁸⁶Rb⁺ and for the occurrence of liquid-crystalline to gel (L_α-to-L_β) phase separations. In vesicles in which the normal 12 mole percent of moderately unsaturated thylakoid phosphatidylglycerol was partially or completely replaced by DPPG, analysis by differential scanning calorimetry indicated that an L_α-to-L_β phase separation did not occur between 0 and 60°C. However, in similar vesicle dispersions that were first subjected to a freeze-thaw cycle, L_α-to-L_β phase separations were observed to occur between 17 and 53°C. The temperature and enthalpy of these phase separations were closely related to the proportion of DPPG in the original lipid mixture. In parallel experiments, large unilamellar vesicles were measured for their permeability to ⁸⁶Rb⁺ between 7 and 30°C. There were no systematic increases in permeability to ⁸⁶Rb⁺ as a function of DPPG content at the temperatures relevant to chilling stress in higher plants. It is concluded that (a) L_α-to-L_β phase separations do not occur in well-defined galactolipid vesicles containing ≤12 mole percent DPPG between 0 and 60°C and (b) these vesicles show no alterations in permeability to ⁸⁶Rb⁺ between 7 and 30°C that are relevant to chilling stress in higher plants.     

Mutations at the dimer, hexamer, and receptor-binding surfaces of insulin independently affect insulin-insulin and insulin-receptor interactions.

Mutagenesis of the dimer- and hexamer-forming surfaces of insulin yields analogues with reduced tendencies to aggregate and dramatically altered pharmacokinetic properties. We recently showed that one such analogue, HisB10----Asp, ProB28----Lys, LysB29----Pro human insulin (DKP-insulin), has enhanced affinity for the insulin receptor and is useful for studying the structure of the insulin monomer under physiologic solvent conditions [Weiss, M. A., Hua, Q. X., Lynch, C. S., Frank, B. H., & Shoelson, S. E. (1991) Biochemistry 30, 7373-7389]. DKP-insulin retains native secondary and tertiary structure in solution and may therefore provide an appropriate baseline for further studies of related analogues containing additional substitutions within the receptor-binding surface of insulin. To test this, we prepared a family of DKP analogues having potency-altering substitutions at the B24 and B25 positions using a streamlined approach to enzymatic semisynthesis which negates the need for amino-group protection. For comparison, similar analogues of native human insulin were prepared by standard semisynthetic methods. The DKP analogues show a reduced tendency to self-associate, as indicated by 1H-NMR resonance line widths. In addition, CD spectra indicate that (with one exception) the native insulin fold is retained in each analogue; the exception, PheB24----Gly, induces similar perturbations in both native insulin and DKP-insulin backgrounds. Notably, analogous substitutions exhibit parallel trends in receptor-binding potency over a wide range of affinities: D-PheB24 greater than unsubstituted greater than GlyB24 greater than SerB24 greater than AlaB25 greater than LeuB25 greater than SerB25, whether the substitution was in a native human or DKP-insulin background. Such "template independence" reflects an absence of functional interactions between the B24 and B25 sites and additional substitutions in DKP-insulin and demonstrates that mutations in discrete surfaces of insulin have independent effects on protein structure and function. In particular, the respective receptor-recognition (PheB24, PheB25), hexamer-forming (HisB10), and dimer-forming (ProB28, LysB29) surfaces of insulin may be regarded as independent targets for protein design. DKP-insulin provides an appropriate biophysical model for defining structure-function relationships in a monomeric template.     

In vitro and in vivo activation of murine gamma/delta T cells induces the expression of IgA, IgM, and IgG Fc receptors.

The present studies examined resting and activated murine gamma/delta T lymphocytes, in vitro and in vivo, for surface expression of FcR. Polyclonal gamma/delta TCR+ lymphocytes selectively grown from the spleen and intestine of normal mice did not express FcR when the cells were in a resting state, but when cells were activated with anti-CD3 antibody virtually all of the splenic gamma/delta lymphocytes and a large subpopulation of the intestinal gamma/delta lymphocytes expressed IgA and IgM FcR. This was confirmed by using transgenic mice. Resting gamma/delta TCR+ lymphocytes from the spleen, thymus, lymph node, and blood of gamma/delta TCR transgenic mice did not express FcR for any of the five major classes of Ig H chains. Activation of the gamma/delta TCR+ cells via the CD3/TCR complex induced high levels of IgM and IgA FcR and low levels of IgG FcR. Finally, in hepatic granulomas of schistosome-infected mice, activated gamma/delta TCR+ cells are present and express high levels of IgA and IgM FcR and low levels of IgG FcR. These investigations establish that transition of gamma/delta TCR+ lymphocytes from a resting to an activated state (triggered via the T3Ti TCR complex) is accompanied by the induction of surface membrane receptors specific for Ig H chain isotypes. The activation-linked expression of FcR on gamma/delta TCR+ lymphocytes provides potential mechanisms for coupling the functional activities of gamma/delta T lymphocytes with immune mechanisms that involve Ig molecules, such as antibody-dependent cellular cytotoxicity.     

The actin cytoskeleton mediates the hormonally regulated translocation of type II iodothyronine 5'-deiodinase in astrocytes

Thyroid hormone, specifically thyroxine, alters cytoskeletal organization in astrocytes by modulating actin polymerization and, in turn, regulates the turnover of the short-lived membrane protein, type II iodothyronine 5'-deiodinase. In the absence of thyroxine, approximately 35% of the total cellular actin is depolymerized, and greater than 90% of the deiodinase is found in the plasma membrane and not associated with the cytoskeleton. Addition of thyroxine promotes actin polymerization and decreases the depolymerized actin to approximately 10% of the total actin pool, induces binding of the deiodinase to F-actin, and promotes rapid internalization of the enzyme. These data provide direct evidence that the actin cytoskeleton participates in the inactivation pathway of the deiodinase by translocating this short-lived plasma membrane protein to an internal membrane pool.     

Substrate specificity of Acanthamoeba myosin I heavy chain kinase as determined with synthetic peptides

Phosphorylation of a single threonine (myosin IA) or serine (myosins IB and IC) in the heavy chains of the Acanthamoeba myosin I isozymes is required for expression of their actin-activated Mg2(+)-ATPase activities. We now report that the synthetic peptide Gly-Arg-Gly-Arg-Ser-Ser-Val-Tyr-Ser, which corresponds to the phosphorylated region of Acanthamoeba myosin IC, is a good substrate for myosin I heavy chain kinase: Km = 54 microM, and Vmax = 15 mumols/min.mg. The same serine is phosphorylated as in the native substrate (residue 6 in the above sequence), and kinase activity with the synthetic peptide as substrate is also stimulated by phosphatidylserine-enhanced autophosphorylation of the kinase. These results indicate that all of the essential sequence determinants of kinase specificity are contained within this 9-residue peptide. With the peptide as substrate, we found that another acidic phospholipid, phosphatidylinositol, also enhances autophosphorylation of the kinase whereas the neutral phospholipids phosphatidylcholine and phosphatidylethanolamine do not. By comparing the Km and Vmax values for a series of synthetic peptide substrates, we established that 1 basic amino acid is essential on the NH2-terminal side of the phosphorylation site, and two are preferable, and that a tyrosine is essential 2 residues away on the COOH-terminal side. There is a slight preference for arginines over lysines. All of these local sequence specificity determinants are present in the three native substrates, Acanthamoeba myosins IA, IB, and IC, and in two Dictyostelium myosin I isozymes that are putative substrates for the kinase. Similar sequences do not occur in the myosins I from intestinal brush border, which is not a substrate for the Acanthamoeba kinase.     

Activation of insulin-secreting cells by pyruvate and halogenated derivatives.

Addition of pyruvate to rat islets perifused in the presence of 5 mM-glucose elicited an immediate pronounced biphasic stimulation of insulin secretion. At lower concentrations of glucose (2.5 mM), only the initial, transient, phase of secretion was observed. Pyruvate inhibited 45Ca2+ efflux from islets at 2.5 mM-glucose and stimulated efflux at 5 mM-glucose. Pyruvate also decreased the rate of efflux of 86Rb+ from perifused islets. A marked stimulation of insulin secretion and 45Ca2+ efflux rate was observed in response to 3-fluoropyruvate and 3-bromopyruvate, compounds which inhibited oxidative metabolism of [14C]glucose and [14C]pyruvate in islets. The stimulatory effects of 3-fluoro- and 3-bromo-pyruvate were associated with enhanced 86Rb+ efflux. Withdrawal of pyruvate or halogenated analogues from the perfusate resulted in a secondary stimulation of insulin release, 45Ca2+ efflux and, to some extent, 86Rb+ efflux rates. Pyruvate, 3-fluoropyruvate and 3-bromopyruvate were all effective in promoting intracellular acidification and a rise in cytosolic Ca2+ concentration, as judged from fluorescence measurements in HIT-T15 cells loaded with 2',7'-biscarboxyethyl-5'(6')-carboxyfluorescein and Quin 2 respectively. It is proposed that oxidative metabolism of pyruvate is not a prerequisite for its stimulatory actions on pancreatic beta-cells. An alternative mechanism of activation by pyruvate and its halogenated derivatives is proposed, based on the possible electrogenic flux of these anions across the cell membrane.     

Effect of Alternaria solani culture filtrate on adventitious shoot regeneration in potato

Summary The effect of Alternaria solani culture filtrate on adventitious shoot regeneration from tuber discs was evaluated using five potato cultivars, which were selected based on their field reaction to Alternaria solani and which represented a range of disease reactions. The culture filtrate stimulated regeneration, a response that could prove to be very useful in the wider utilization of transformation and in vitro selection technology.Research conducted at the Scottish Crop Research Institute during a transfer of work of the senior author     

Inward rectification in rat olfactory receptor neurons.

Inwardly rectifying currents in enzymically dissociated olfactory receptor neurons of rat were studied by using patch-clamp techniques. Upon hyperpolarization to membrane potentials more negative than -100 mV, small inward-current relaxations were observed. Activation was described by a single exponential with a time constant that decreased e-fold for a 21 mV hyperpolarization. The current was not reduced by the external application of 5 mM Ba2+, but was abolished by the addition of 5 mM Cs+ to the bath solution. Increasing the external K+ concentration ([K+]o) to 25 mM dramatically enhanced the current without affecting the voltage range or the kinetics of activation. In 25 mM [K+]o, tail currents reversed at -26 mV, significantly more positive than the K+ equilibrium potential of -44 mV. These characteristics are consistent with those of a mixed Na+/K+ inward rectification that has been reported in several types of neuronal, cardiac and smooth muscle cells. The current may contribute to controlling cell excitability during the response to some odorants.     

Brevibacterium liquefaciens adenylate cyclase and its in vivo stimulation by pyruvate.

Adenylate cyclase of Brevibacterium liquefaciens depends on pyruvate for activity. Growing in a simple medium containing glucose and DL-alanine, the microorganism excreted pyruvate, which reached 20 mM in the medium at stationary phase. Using [3H]adenosine to label the adenosine 5'-triphosphate pool, we showed that pyruvate in the medium stimulated adenylate cyclase of B. liquefaciens in vivo, in a manner similar to the stimulation observed in vitro. Adenylate cyclase in cells harvested at different phases of growth was equally responsive to exogenous pyruvate, indicating that the allosteric site for pyruvate was present in the enzyme throughout the various phases of cell growth. The specific activity of adenylate cyclase was highest in cells harvested at early log phase; thereafter it declined and was substantially lower at stationary phase. Although adenylate cyclase appears to be activated by pyruvate throughout the life span of the cell, the activity appears not to be critical to cell growth, which was comparable whether the medium contained high or low pyruvate.