An unanticipated role for survivin in organ transplant damage.

Ischemia/reperfusion (I/R) injury is a major determinant of graft survival in kidney transplantation. Survivin, an inhibitor of apoptosis that participates in the control of mitosis and cell cycle progression, has been implicated in renal protection and repair after I/R injury; however, no study has been performed in the transplant setting. We investigated the role of survivin in modulating posttransplant I/R injury in syngeneic and allogeneic kidney grafts, and studied whether protection from I/R injury impacted on the recipient immune system, on chronic allograft nephropathy and rejection. We used genetically engineered mice with survivin haploinsufficiency and WT mice in which survivin over-expression was induced by gene-delivery. Survivin haploinsufficiency in syngeneic grafts was associated with exuberant I/R tissue injury, which triggered inflammation eventually resulting in graft loss. Conversely, survivin over-expression in the grafts minimized I/R injury and dysfunction in syngeneic grafts and in a clinically relevant fully MHC-mismatched allogeneic combination. In the latter, survivin over-expression translated into limited anti-donor adaptive immune response and less long-term allograft injury with protection from renal parenchymal damage. Our data support survivin over-expression in the graft as a novel target for protocols aimed at limiting tissue damage at the time of transplant ultimately modulating the recipient immune system.

The conformational behaviour of phosphatidylinositol in model membranes: 2H-NMR studies.

Dimyristoylphosphatidylinositol (DMPI) has been synthesized with the appropriate natural stereochemistry and labelled with deuterium at specific sites in the D-myo-inositol headgroup. 2H-NMR spectroscopy of DMPI in its lamellar phase at a molar ratio of water-to-lipid RW/L of 129 and at 70 degrees C reveals quadrupolar splittings delta v of 3.83 and 2.17 kHz, respectively, for the five axially oriented C-D bonds and the single equatorially oriented C-D bond of the D-myo-inositol headgroup. Between RW/L ratios of 129 and 210 and between 30 degrees C and 80 degrees C the value of the ratio of these splittings delta nu ax/delta nu eq varies significantly (between 1.17 and 4.38). If it is assumed that, at a particular temperature, there is a single preferred orientation of the inositol headgroup, and that motion of the DPMI molecule establishes axial symmetry with respect to the bilayer normal then the ratio of these quadrupolar splittings can be used to impose constraints on that orientation. For example, the data are inconsistent with a situation in which the inositol ring lies parallel to the membrane surface and are difficult to reconcile with an arrangement where the inositol ring lies perpendicular to the surface. Computational modelling identifies four possible 'tilted' orientations, all of which are consistent with the data, and two of these allow good intramolecular hydrogen bonds to be formed. In one there is hydrogen bonding between the inositol C2-OH and the phosphate pro-R oxygen. This is close to the conformation previously identified as being dominant in DMSO solution (Bushby, R.J., Byard, S.J., Hansbro, P.M. and Reid, D.G. (1990) Biochim. Biophys. Acta 1044, 231-236).

Synthesis and biological activity of the four stereoisomers of 6-[4-[3-[[2-hydroxy-3-[4-[2- (cyclopropylmethoxy)ethyl]phenoxy]propyl]amino]-propionamido] phenyl]-5-methyl-4,5-dihydro-3(2H)-pyridazinone, a combined vasodilator and beta-adrenoceptor antagonist.

6-[4-[3-[[2-Hydroxy-3-[4-[2- (cyclopropylmethoxy)ethyl]phenoxy]propyl]amino]propionamido] phenyl]- 5-methyl-4,5-dihydro-3(2H)-pyridazinone (3) consists of a mixture of four stereoisomers, i.e., two racemates, as a consequence of the two asymmetric centers contained in the structure. An approximately equimolar mixture of these two racemates exhibits a novel combination of vasodilation and beta-adrenergic antagonist activity. This paper describes the synthesis of each of the four possible stereoisomers of 3 and provides clear evidence for the different pharmacological profile of each of the stereoisomers. The RA,SB isomer 3a has an overall profile slightly better than the complete mixture; the other three isomers all show reduced activity as vasodilators and/or beta-adrenergic antagonists.

Selective thyromimetics. Cardiac-sparing thyroid hormone analogues containing 3'-arylmethyl substituents.

Introduction of specific arylmethyl groups at the 3'-position of the thyroid hormone 3,3',5-triiodo-L-thyronine (T3), and its known hormonally active derivatives, gives liver-selective, cardiac-sparing thyromimetics, with potential utility as plasma cholesterol lowering agents. Selectivity-conferring 3'-substituents include substituted benzyl, e.g. p-hydroxybenzyl, and heterocyclic methyl, e.g. 2-oxo-1,2-dihydropyrid-5-ylmethyl and 6-oxo-1,6-dihydropyridazin-3-ylmethyl. Correlations between in vivo and in vitro receptor binding affinities show that liver/heart selectivity does not depend on receptor recognition but on penetration or access to receptors in vivo. QSAR studies of the binding data of a series of 20 3'-arylmethyl T3 analogues show that electronegative groups at the para position increase both receptor binding and selectivity in vivo. However, increasing 3'-arylmethyl hydrophobicity increases receptor binding but reduces selectivity. Substitution at ortho and meta positions reduces both binding and selectivity. Replacement of the 3,5-iodo groups by halogen or methyl maintains selectivity, with 3,5-dibromo analogues in particular having increased potency combined with oral bioavailability. Diphenyl thioether derivatives also have improved potency but are less orally active. At the 1-position, the D enantiomer retains selectivity, but removal of the alpha-amino group to give a propionic acid results in loss of selective thyromimetic activity.

ATP-citrate lyase as a target for hypolipidemic intervention. Design and synthesis of 2-substituted butanedioic acids as novel, potent inhibitors of the enzyme.

ATP-citrate lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. Inhibitors of the enzyme represent a potentially novel class of hypolipidemic agent, which are anticipated to have combined hypocholesterolemic and hypotriglyceridemic properties. A series of 2-substituted butanedioic acids have been designed and synthesized as inhibitors of the enzyme. The best compounds, 58, 68, 71, 74 have reversible Ki's in the 1-3 microM range against the isolated rat enzyme. As representative of this compound class, 58, has been shown to exert its inhibitory action through a mainly competitive mechanism with respect to citrate and a noncompetitive one with respect to CoA. None of the inhibitors were able to inhibit cholesterol and/or fatty acid synthesis in HepG2 cells. This has been attributed to the adverse physicochemical properties of the molecules leading to a lack of cell penetration. Despite this, a lead structural class of compound has been identified with the potential for modification into potent, cell-penetrant, and efficacious inhibitors of ATP-citrate lyase.

Modified selective and differential isolation medium for Vibrio parahaemolyticus.

The semiselective salt-starch-agar formulation of Baross and Liston was modified as the result of a systematic study of the effect of each constituent on the growth of Vibrio parahaemolyticus and competitive species characteristic of the marine environment. The selection of nutrient constituents depended on an analysis of their effect on generation time. The addition of inhibitors depended on an analysis of minimal inhibitory concentrations. The modified formulation included: peptone, 2.0%; yeast extract, 0.2%; corn starch, 0.5%; NaCl, 3.0%; agar, 1.5% (pH 8.0). Penicillin at 2 to 5 units/ml increased selectivity without significantly inhibiting Vibrio in pure suspensions. Over 62% of the most sensitive strain (YM-K33) was recovered at a concentration of 5 units of penicillin per ml. The per cent recovery of V. parahaemolyticus from fish homogenate compared favorably with other selective formulations. At an initial concentration of 10(5) cells/ml, recovery varied with the strain used from 60 to 119%, whereas at 10(2) cells/ml the range was 36 to 94%. Applications of the medium for Vibrio quantification are discussed.

Serum substitute in epithelial cell culture media: nonfat dry milk filtrate.

A number of milk types and milk fractions were investigated as possible substitutes for serum in cell culture media. A filtrate of reconstituted nonfat dry milk showed promise. Culture fluids containing 5% of the nonfat dry milk filtrate were used to propagate primary and continuous cell cultures, and the cell growth from these cultures was compared with that of cells grown in a serum-containing medium. The nonfat dry milk filtrate-supplemented medium supported the growth of all epithelial cells tested, but two fibroblast-type cultures failed to replicate. Cells grown in the medium containing the milk filtrate grew slowly for 2 to 3 days and then propagated to confluency in 6 to 8 days. Viable cell counts of 9 days were comparable to those of serum-grown cells that had been propagated for 7 days. Cells grown in the milk filtrate could be split 1 to 4 when subcultures were prepared. Cell growth could be stimulated by refeeding on days 2 to 3 or by the addition of 30 microM 2-mercaptoethanol to the growth medium. Virus susceptibility and titer comparisons with poliovirus 1, coxsackievirus B2, echovirus 7, and herpes simplex virus indicated that approximately the same data were obtained when either the nonfat dry milk filtrate-treated or the serum-treated cells were studied. The nonfat dry milk filtrate is inexpensive, is easily prepared, and is a substitute for serum in epithelial cell culture media.

Comparative Hemolytic Activity of Vibrio parahaemolyticus and Related Vibrios.

The hemolytic activities of 91 strains of Vibrio parahaemolyticus isolated from human diarrheal stools, sea fish, and sea water; 21 suspected V. parahaemolyticus cultures isolated from wound infections; 14 nonpathogenic marine vibrios; and 21 V. parahaemolyticus isolated from moribund blue crabs Callinectes sapidus were compared. Potentially pathogenic V. parahaemolyticus strains could be differentiated from the related nonpathogenic marine vibrios, because the former hemolyzed hamster, sheep, and human blood, whereas the latter were nonhemolytic. In addition, V. parahaemolyticus isolated from tissue infections could be differentiated from those of the first group isolated from sea fish or human stools, because the former exhibited primarily an alpha-hemolytic reaction on chicken blood; the latter exhibited mostly beta. It is suggested that V. parahaemolyticus isolated from blue crabs may be differentiated from the first group on the basis of their hemolysis of human blood. A useful schema of the differential hemolytic reactions, exhibited by V. parahaemolyticus, tissue infection vibrios, and nonpathogens on hamster, sheep, chicken, goose, and human blood is given. The patterns of hemolytic activity of these groups on special human blood-agar plates (Kanagawa hemolysis) resembled that seen on ordinary human blood-agar.

Synthesis and evaluation of (+) and (-)-2,2-difluorocitrate as inhibitors of rat-liver ATP-citrate lyase and porcine-heart aconitase.

The enantiomers (+) and (-)-2,2-difluorocitrate have been synthesized. Both are good inhibitors of ATP-citrate lyase, showing competitive inhibition against citrate, with Kis = 0.7 microM for (+)-2,2-difluorocitrate and 3.2 microM for (-)-2,2-difluorocitrate. The inhibition patterns with either ATP or CoA as the varied substrate were uncompetitive and mixed, respectively, but with much weaker inhibition constants. Neither isomer undergoes carbon-carbon bond cleavage as a substrate and there is no evidence of irreversible time-dependent inactivation. When ATP-citrate lyase is incubated with CoA and difluorocitrate, the maximal intrinsic ATPase rate is 10% of the citrate-induced rate for the (+)-enantiomer and 2% for the (-)-enantiomer. 19F-NMR studies confirm that only the (+)-enantiomer is chemically processed. The effects of the difluorocitrate enantiomers on the reaction catalysed by aconitase were examined. (-)-2,2-Difluorocitrate is a competitive inhibitor against citrate (Kis = 1.5 microM), whereas the (+)-enantiomer is a relatively poor mixed inhibitor (Ki greater than 300 microM). The (-)-enantiomer irreversibly inactivates aconitase at 1.1 min-1.mM-1 at 25 degrees C and pH 7.4, whereas no irreversible inhibition is seen with the (+)-enantiomer. Therefore, it would be expected that the (+)-enantiomer would slow the rate of acetyl-CoA synthesis in vivo, without inhibiting the citric acid cycle.

A thyromimetic that decreases plasma cholesterol levels without increasing cardiac activity.

A number of agents that mimic the ability of the thyroid hormone, T3, to decrease plasma cholesterol levels are described; one is as effective as T3 at reducing cholesterol levels and stimulating liver function, but has very little effect on cardiac function and is thus less likely to be toxic. The agent may be useful in the treatment of atherosclerosis.