Prodynorphin mutations cause the neurodegenerative disorder spinocerebellar ataxia type 23

Spinocerebellar ataxias (SCAs) are dominantly inherited neurodegenerative disorders characterized by progressive cerebellar ataxia and dysarthria. We have identified missense mutations in prodynorphin (PDYN) that cause SCA23 in four Dutch families displaying progressive gait and limb ataxia. PDYN is the precursor protein for the opioid neuropeptides, α-neoendorphin, and dynorphins A and B (Dyn A and B). Dynorphins regulate pain processing and modulate the rewarding effects of addictive substances. Three mutations were located in Dyn A, a peptide with both opioid activities and nonopioid neurodegenerative actions. Two of these mutations resulted in excessive generation of Dyn A in a cellular model system. In addition, two of the mutant Dyn A peptides induced toxicity above that of wild-type Dyn A in cultured striatal neurons. The fourth mutation was located in the nonopioid PDYN domain and was associated with altered expression of components of the opioid and glutamate system, as evident from analysis of SCA23 autopsy tissue. Thus, alterations in Dyn A activities and/or impairment of secretory pathways by mutant PDYN may lead to glutamate neurotoxicity, which underlies Purkinje cell degeneration and ataxia. PDYN mutations are identified in a small subset of ataxia families, indicating that SCA23 is an infrequent SCA type (～0.5%) in the Netherlands and suggesting further genetic SCA heterogeneity.     

Calculated pH-dependent population and protonation of carbon-monoxy-myoglobin conformers

X-ray structures of carbonmonoxymyoglobin (MbCO) are available for different pH values. We used conventional electrostatic continuum methods to calculate the titration behavior of MbCO in the pH range from 3 to 7. For our calculations, we considered five different x-ray structures determined at pH values of 4, 5, and 6. We developed a Monte Carlo method to sample protonation states and conformations at the same time so that we could calculate the population of the considered MbCO structures at different pH values and the titration behavior of MbCO for an ensemble of conformers. To increase the sampling efficiency, we introduced parallel tempering in our Monte Carlo method. The calculated population probabilities show, as expected, that the x-ray structures determined at pH 4 are most populated at low pH, whereas the x-ray structure determined at pH 6 is most populated at high pH, and the population of the x-ray structures determined at pH 5 possesses a maximum at intermediate pH. The calculated titration behavior is in better agreement with experimental results compared to calculations using only a single conformation. The most striking feature of pH-dependent conformational changes in MbCO-the rotation of His-64 out of the CO binding pocket-is reproduced by our calculations and is correlated with a protonation of His-64, as proposed earlier.     

Additive effects of intra-accumbens infusion of the cAMP-specific phosphodiesterase inhibitor, rolipram and cocaine on brain stimulation reward

Evidence from cocaine self-administration studies suggests that increasing the activity of cyclic AMP (cAMP) pathways within the nucleus accumbens may produce a reduction in cocaine's reinforcing effects. Rolipram may increase intra-cellular levels of cAMP by selectively inhibiting Type IV phosphodiesterases, enzymes that catalyze cAMP breakdown. The present study was undertaken to test the hypothesis that infusion of rolipram into the nucleus accumbens would decrease cocaine-induced enhancement of the sensitivity of brain stimulation reward (BSR) pathways. BSR thresholds were determined in rats after the systemic administration of cocaine (4 mg/kg IP) and the infusion of rolipram (0.2 microg/side) into the nucleus accumbens both alone and in combination. Thresholds also were determined after the systemic administration of rolipram alone and, as a positive control, for amphetamine (10 microg/side) infused into the nucleus accumbens. BSR thresholds were significantly lowered below baseline levels following d-amphetamine administration suggesting that cannulae were in place to allow perfusion of reward pathways. Compared to values for saline alone, thresholds were lower after the injection of cocaine (4 mg/kg IP) or the infusion of rolipram (0.2 microg/side) into the nucleus accumbens. Treatment with the combination of cocaine and intra-nucleus accumbens rolipram produced a greater lowering of the BSR threshold than did administration of either rolipram or cocaine alone. Systemic administration of rolipram (0.5 mg/kg IP) either blocked the effects of BSR or raised BSR thresholds and produced stimulation-induced head jerking in most of the test animals. These results suggest that infusion into the nucleus accumbens of rolipram, an agent that putatively elevates cAMP levels in this structure, can enhance the sensitivity of reward pathways to BSR and can augment cocaine's actions on these pathways.     

Reconstructing parental genotypes when testing for linkage in the presence of association

Various family-based association methods have recently been proposed that allow testing for linkage in the presence of linkage disequilibrium between a marker and a disease even if there is only incomplete parental-genotype information. For some families, it may be possible to reconstruct missing parental genotypes from the genotypes of their offspring. Treating such a reconstructed family as if parental genotypes have been typed, however, can introduce bias. The reconstruction-combined transmission/disequilibrium test (RC-TDT) and its X-chromosomal counterpart, XRC-TDT, employ parental-genotype reconstruction and correct for the biases involved in this reconstruction without relying on population marker allele frequencies. For the two tests, exact P values can be obtained by numerically calculating the convolution of the null distributions corresponding to the families in the sample.     

How to guarantee optimal stability for most representative structures in the Protein Data Bank

We proposed recently an optimization method to derive energy parameters for simplified models of protein folding. The method is based on the maximization of the thermodynamic average of the overlap between protein native structures and a Boltzmann ensemble of alternative structures. Such a condition enforces protein models whose ground states are most similar to the corresponding native states. We present here an extensive testing of the method for a simple residue-residue contact energy function and for alternative structures generated by threading. The optimized energy function guarantees high stability and a well-correlated energy landscape to most representative structures in the PDB database. Failures in the recognition of the native structure can be attributed to the neglect of interactions between different chains in oligomeric proteins or with cofactors. When these are taken into account, only very few X-ray structures are not recognized. Most of them are short inhibitors or fragments and one is a structure that presents serious inconsistencies. Finally, we discuss the reasons that make NMR structures more difficult to recognizeCopyright 2001 Wiley-Liss, Inc.     

Defective fatty acid uptake in the spontaneously hypertensive rat is a primary determinant of altered glucose metabolism, hyperinsulinemia, and myocardial hypertrophy

Genetic linkage studies implicated deficiency of CD36, a membrane fatty acid (FA) transporter, in the hypertriglyceridemia and hyperinsulinemia of the spontaneously hypertensive rat (SHR). In this study we determined whether loss of CD36 function in FA uptake is a primary determinant of the SHR phenotype. In vivo, tissue distribution of iodinated, poorly oxidized beta-methyliodophenyl pentadecanoic acid (BMIPP) was examined 2 h after its intravenous injection. Fatty acid transport was also measured in vitro over 20 to 120 s in isolated adipocytes and cardiomyocytes obtained from SHR and from a congenic line (SHRchr4) that incorporates a piece of chromosome 4 containing wild-type CD36. SHR heart and adipose tissue exhibited defects in FA uptake and in conversion of diglycerides to triglycerides that are similar to those observed in the CD36 null mouse. However, a key difference in SHR tissues is that fatty acid oxidation is much more severely impaired than fatty acid esterification, which may underlie the 4-5-fold accumulation of free BMIPP measured in SHR muscle. Studies with isolated adipocytes and cardiomyocytes directly confirmed both the defect in FA transport and the fact that it is underestimated by BMIPP. Heart, oxidative muscle, and adipose tissue in the SHR exhibited a large increase in glucose uptake measured in vivo using [(18)F]fluorodeoxyglucose. Supplementation of the diet with short-chain fatty acids, which do not require CD36-facilitated transport, eliminated the increase in glucose uptake, the hyperinsulinemia, and the heart hypertrophy in the SHR. This indicated that lack of metabolic energy consequent to deficient FA uptake is the primary defect responsible for these abnormalities. Hypertension was not alleviated by the supplemented diet suggesting it is unrelated to fuel supply and any contribution of CD36 deficiency to this trait may be more complex to determine. It may be worth exploring whether short-chain FA supplementation can reverse some of the deleterious effects of CD36 deficiency in humans, which may include hypertrophic cardiomyopathy.     

Crystallographic evidence for substrate-assisted catalysis in a bacterial beta-hexosaminidase

beta-Hexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of beta-1,4-linked N-acetylhexosamine residues from oligosaccharides and their conjugates. Heritable deficiency of this enzyme results in various forms of GalNAc-beta(1,4)-[N-acetylneuraminic acid (2,3)]-Gal-beta(1,4)-Glc-ceramide gangliosidosis, including Tay-Sachs disease. We have determined the x-ray crystal structure of a beta-hexosaminidase from Streptomyces plicatus to 2.2 A resolution (Protein Data Bank code ). beta-Hexosaminidases are believed to use a substrate-assisted catalytic mechanism that generates a cyclic oxazolinium ion intermediate. We have solved and refined a complex between the cyclic intermediate analogue N-acetylglucosamine-thiazoline and beta-hexosaminidase from S. plicatus to 2.1 A resolution (Protein Data Bank code ). Difference Fourier analysis revealed the pyranose ring of N-acetylglucosamine-thiazoline bound in the enzyme active site with a conformation close to that of a (4)C(1) chair. A tryptophan-lined hydrophobic pocket envelopes the thiazoline ring, protecting it from solvolysis at the iminium ion carbon. Within this pocket, Tyr(393) and Asp(313) appear important for positioning the 2-acetamido group of the substrate for nucleophilic attack at the anomeric center and for dispersing the positive charge distributed into the oxazolinium ring upon cyclization. This complex provides decisive structural evidence for substrate-assisted catalysis and the formation of a covalent, cyclic intermediate in family 20 beta-hexosaminidases.     

Monomorphic molecules function as additional recognition structures on haptenated target cells for HLA-A1-restricted, hapten-specific CTL

Hapten-specific T cells have been shown to recognize haptenated peptides with high avidity and, in some instances, with promiscuous MHC restriction. In this study, the impact of Ag density on MHC restriction of a CTL response specific to the trinitrophenyl (TNP) hapten was investigated. In this study, we demonstrate a novel recognition mechanism used by TNP-specific CD8(+) CTL in the presence of high Ag doses. Although low levels of TNP epitopes on target cells allowed for HLA-A1-restricted CTL activity only, entirely MHC-independent target cell recognition became operative at high TNP loading. In both cases, recognition was mediated by the TCR. This MHC-independent recognition is target cell type restricted and critically involves in our model direct recognition of the ectonucleotidase family surface molecule CD39 by the CTL.