Behavioural and molecular endophenotypes in psychotic disorders reveal heritable abnormalities in glutamatergic neurotransmission.

Psychotic disorders such as schizophrenia are biologically complex and carry huge population morbidity due to their prevalence, persistence and associated disability. Defined by features such as delusions and hallucinations, they involve cognitive dysfunction and neurotransmitter dysregulations that appear mostly to involve the dopaminergic and glutamatergic systems. A number of genetic and environmental factors are associated with these disorders but it has been difficult to identify the biological pathways underlying the principal symptoms. The endophenotype concept of stable, heritable traits that form a mechanistic link between genes and an overt expression of the disorder has potential to reduce the complexity of psychiatric phenotypes. In this study, we used a genetically sensitive design with individuals with a first episode of psychosis, their non-affected first-degree relatives and non-related healthy controls. Metabolomic analysis was combined with neurocognitive assessment to identify multilevel endophenotypic patterns: one concerned reaction times during the performance of cognitive and emotional tests that have previously been associated with the glutamate neurotransmission system, the other involved metabolites involved directly and indirectly in the co-activation of the N-methyl-D-aspartate receptor, a major receptor of the glutamate system. These cognitive and metabolic endophenotypes may comprise a single construct, such that genetically mediated dysfunction in the glutamate system may be responsible for delays in response to cognitive and emotional functions in psychotic disorders. This focus on glutamatergic neurotransmission should guide drug discovery and experimental medicine programmes in schizophrenia and related disorders.

Speed of facial affect intensity recognition as an endophenotype of first-episode psychosis and associated limbic-cortical grey matter systems.

BACKGROUND: Psychotic disorders are highly heritable such that the unaffected relatives of patients may manifest characteristics, or endophenotypes, that are more closely related to risk genes than the overt clinical condition. Facial affect processing is dependent on a distributed cortico-limbic network that is disrupted in psychosis. This study assessed facial affect processing and related brain structure as a candidate endophenotype of first-episode psychosis (FEP). METHOD: Three samples comprising 30 FEP patients, 30 of their first-degree relatives and 31 unrelated healthy controls underwent assessment of facial affect processing and structural magnetic resonance imaging (sMRI) data. Multivariate analysis (partial least squares, PLS) was used to identify a grey matter (GM) system in which anatomical variation was associated with variation in facial affect processing speed. RESULTS: The groups did not differ in their accuracy of facial affect intensity rating but differed significantly in speed of response, with controls responding faster than relatives, who responded faster than patients. Within the control group, variation in speed of affect processing was significantly associated with variation of GM density in amygdala, lateral temporal cortex, frontal cortex and cerebellum. However, this association between cortico-limbic GM density and speed of facial affect processing was absent in patients and their relatives. CONCLUSIONS: Speed of facial affect processing presents as a candidate endophenotype of FEP. The normal association between speed of facial affect processing and cortico-limbic GM variation was disrupted in FEP patients and their relatives.

Nucleotide variations in the lxd region of Drosophila melanogaster: characterization of a candidate modifier of lifespan.

We have investigated the structure and function of several proteins that might influence adult lifespans in Drosophila melanogaster. The present report focuses on the gene lxd ('low xanthine dehydrogenase'), which lies in a region of chromosome III identified by QTL-mapping as potentially important for lifespan. DNA sequence of a 3780 bp genomic fragment containing the lxd locus reveals differences between long-lived and control inbred lines. In order to determine the importance of nucleotide replacements, the intron/exon boundaries have been determined, based on peptide alignment and conserved amino acids. We identified four exons in the lxd coding region. The deduced amino acid sequence of exon 4 shows 46.5% identity with Escherichia coli MoaC sequences. There are eight nucleotide substitutions in exons differentiating the inbred lines, three in exon 3 and five in exon 4. One of the exon 4 substitutions has resulted in a Thr-Ile replacement at the protein surface, but not entirely solvent exposed. This substitution is potentially a modifier of lifespan via oxygen defense, but since the activities of three molybdoenzymes are unaffected in inbred lines, this possibility seems remote.

Molecular-functional studies of adaptive genetic variation in prokaryotes and eukaryotes.

Knowledge of both prokaryotic and eukaryotic organisms is essential to the study of molecular evolution. Their common ancestry mandates that their molecular functions share many aspects of adaptation and constraint, yet their differences in size, ploidy, and structural complexity also give rise to divergent evolutionary options. We explore the interplay of adaptation, constraint, and neutrality in their evolution by the use of genetic variants to probe molecular function in context of molecular structure, metabolic organization, and phenotype-environment interactions. Case studies ranging from bacteria to butterflies, flies, and vertebrates emphasize, among other points: the importance of moving from initial recording of evolutionary pattern variation to studying the processes underlying the patterns, by experiment, reconstructive inference, or both; the complementarity, not conflict, of finding different performance and fitness impacts of natural variants in prokaryotes or eukaryotes, depending on the nature and magnitude of the variants, their locations and roles in pathways, the nature of molecular function affected, and the resulting organismal phenotype-environment interactions leading to selection or its absence; the importance of adaptive functional interaction of different kinds of variants, as in gene expression variants versus variants altering polypeptide properties, or interaction of changes in enzymes' active sites with complementary changes elsewhere that adjust catalytic function in different ways, or coadaptation of different steps' properties in pathways; the power afforded by combining structural and functional analyses of variants with study of the variants' phenotype-environment interactions to understand how molecular changes affect (or fail to affect) adaptive mechanisms "in the wild." Comparative study of prokaryotes and eukaryotes in this multifaceted way promises to deliver both new insights into evolution and a host of new and productive questions about it.

Enzyme evolution explained (sort of).

Sites in proteins evolve at markedly different rates; some are highly conserved, others change rapidly. We have developed a maximum likelihood method to identify regions of a protein that evolve rapidly or slowly relative to the remaining structure. We also show that solvent accessibility and distance from the catalytic site are major determinants of evolutionary rate in eubacterial isocitrate dehydrogenases. These two variables account for most of the rate heterogeneity not ascribable to stochastic effects.

Rapid evolution in plant chitinases: molecular targets of selection in plant-pathogen coevolution.

Many pathogen recognition genes, such as plant R-genes, undergo rapid adaptive evolution, providing evidence that these genes play a critical role in plant-pathogen coevolution. Surprisingly, whether rapid adaptive evolution also occurs in genes encoding other kinds of plant defense proteins is unknown. Unlike recognition proteins, plant chitinases attack pathogens directly, conferring disease resistance by degrading chitin, a component of fungal cell walls. Here, we show that nonsynonymous substitution rates in plant class I chitinase often exceed synonymous rates in the plant genus Arabis (Cruciferae) and in other dicots, indicating a succession of adaptively driven amino acid replacements. We identify individual residues that are likely subject to positive selection by using codon substitution models and determine the location of these residues on the three-dimensional structure of class I chitinase. In contrast to primate lysozymes and plant class III chitinases, structural and functional relatives of class I chitinase, the adaptive replacements of class I chitinase occur disproportionately in the active site cleft. This highly unusual pattern of replacements suggests that fungi directly defend against chitinolytic activity through enzymatic inhibition or other forms of chemical resistance and identifies target residues for manipulating chitinolytic activity. These data also provide empirical evidence that plant defense proteins not involved in pathogen recognition also evolve in a manner consistent with rapid coevolutionary interactions.

The yeast mitochondrial citrate transport protein. Probing the secondary structure of transmembrane domain iv and identification of residues that likely comprise a portion of the citrate translocation pathway.

The mitochondrial citrate transport protein (CTP) has been investigated by replacing 22 consecutive residues within transmembrane domain IV, one at a time, with cysteine. A cysteine-less CTP retaining wild-type functional properties served as the starting template. The single Cys CTP variants were overexpressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to three methanethiosulfonate reagents was evaluated by determining the pseudo first order rate constants for inhibition of CTP function. These rate constants varied by seven orders of magnitude. With three independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of four was observed from residues 177-193. Based on the pattern of accessibility we conclude that residues 177-193 exist as an alpha-helix. Furthermore, a water-accessible face of the helix has been defined consisting of Pro-177, Val-178, Arg-181, Gln-182, Asn-185, Gln-186, Arg-189, Leu-190, and Tyr-193, and a water-inaccessible face has been delineated consisting of Ser-179, Met-180, Ala-183, Ala-184, Ala-187, Val-188, Gly-191, and Ser-192. We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer.

The applicability of SERVQUAL in different health care environments.

This paper reports on a study that investigates the applicability of a modified SERVQUAL instrument as a means of measuring service quality in two types of health service environments; medical care and health care (incorporating medical, social, cognitive and emotional elements). The research confirms a four factor structure that is stable for both environments, and similar to the service quality dimensions recognised in the literature. However, the relative importance of the dimensions of quality is inconsistent for the two types of health services. These results confirm the suggestion that importance values should be part of the measurement tool. Finally, the extra diagnostic advantage achieved by the use of gap scores to measure service quality, when compared to perception only scores is demonstrated.

Carbocations in the synthesis of prostaglandins by the cyclooxygenase of PGH synthase? A radical departure!

Evidence already available is used to demonstrate that although prostaglandin G/H synthase hydroxylates arachidonic acid through radical intermediates, it effects cyclizations through a carbocation center at C-10. This is produced following migration of H to the initial radical at C-13 and a 1epsilon oxidation. Under orbital symmetry control, the cyclizations can give only the ring size and trans stereochemistry actually observed. After cyclization, the H-shift reverses to take the sequence back into current radical theory for hydroxylation at C-15. Thus 10,10-difluoroarachidonic acid cannot be cyclized, although it can be hydroxylated. Acetylation of Ser516 in the isoform synthase-2 is considered to oppose carbocation formation and/or H-migration and so prevent cyclizations while permitting hydroxylations; the associated inversion of chirality at C-15 can then readily be accommodated without the change in conformation required by other schemes. Suicide inhibition occurs when carbocations form stable bonds upon (thermal) contact with adjacent heteroatoms, etc. Because the cyclooxygenase and peroxidase functions operate simultaneously through the same heme, phenol acts as reducing cosubstrate for the cyclooxygenase, thus enabling it to promote PGG2 production and protect the enzyme from oxidative destruction.

The structural basis of molecular adaptation.

The study of molecular adaptation has long been fraught with difficulties, not the least of which is identifying out of hundreds of amino acid replacements those few directly responsible for major adaptations. Six studies are used to illustrate how phylogenies, site-directed mutagenesis, and a knowledge of protein structure combine to provide much deeper insights into the adaptive process than has hitherto been possible. Ancient genes can be reconstructed, and the phenotypes can be compared to modern proteins. Out of hundreds of amino acid replacements accumulated over billions of years those few responsible for discriminating between alternative substrates are identified. An amino acid replacement of modest effect at the molecular level causes a dramatic expansion in an ecological niche. These and other topics are creating the emerging field of "paleomolecular biochemistry."

Synthesis and antimicrobial activity of pyrazolo[3',4':4,3]pyrido[6,5-c]pyridazine and thieno[2,3-c]pyridazine derivatives.

4-Acetyl-5,6-diphenyl-2(H)pyridazine-3-one (1) was allowed to react with phenyl hydrazine to afford the corresponding hydrazone 2. Hydrazone 2 upon treatment with Vilsmeier's reagent gave pyrazolylpyridazine derivative 3, which was allowed to react with thiosemicarbazide and hydroxyl amine to give the corresponding thiosemicarbazone and oxime 4 and 5, respectively. Treatment of oxime 5 with Ac2O gave the pyrazolylpyridazine carbonitrile derivative 6. Compound 5 reacts with POCl3 to give the corresponding chloro compound 7. The chloro compound 7 was reacted with hydrazine hydrate or aniline to afford pyrazolopyridazodiazepine 9 or pyrazolopyridazopyridazine 10. When compound 1 was allowed to react with POCl3 the chloro derivative 11 resulted. This compound reacts with thiourea, piperidine or hydrazine hydrate to give compounds 12, 14 and 15, respectively. Compound 12 reacted with alpha-haloester or alpha-haloketone to give the thienopyridazines 13a and b, respectively. Most of the newly synthesized compounds were screened for fungicidal and bactericidal activity.

The evolution of bacterial transformation: sex with poor relations.

Bacteria are the only organisms known to actively take up DNA and recombine it into their genomes. While such natural transformation systems may provide many of the same benefits that sexual reproduction provides eukaryotes, there are important differences that critically alter the consequences, especially when recombination's main benefit is reducing the mutation load. Here, analytical and numerical methods are used to study the selection of transformation genes in populations undergoing deleterious mutation. Selection for transformability depends on the shape of the fitness function against mutation. If the fitness function is linear, then transformation would be selectively neutral were it not for the possibility that transforming cells may take up DNA that converts them into nontransformable cells. If the selection includes strong positive (synergistic) epistasis, then transformation can be advantageous in spite of this risk. The effect of low quality DNA (from selectively killed cells) on selection is then studied analytically and found to impose an additional cost. The limited data available for real bacterial populations suggest that the conditions necessary for the evolution of transformation are unlikely to be met, and thus that DNA uptake may have some function other than recombination of deleterious mutations.

Protein engineering reveals ancient adaptive replacements in isocitrate dehydrogenase.

Evolutionary analysis indicates that eubacterial NADP-dependent isocitrate dehydrogenases (EC 1.1.1.42) first evolved from an NAD-dependent precursor about 3.5 billion years ago. Selection in favor of utilizing NADP was probably a result of niche expansion during growth on acetate, where isocitrate dehydrogenase provides 90% of the NADPH necessary for biosynthesis. Amino acids responsible for differing coenzyme specificities were identified from x-ray crystallographic structures of Escherichia coli isocitrate dehydrogenase and the distantly related Thermus thermophilus NAD-dependent isopropylmalate dehydrogenase. Site-directed mutagenesis at sites lining the coenzyme binding pockets has been used to invert the coenzyme specificities of both enzymes. Reconstructed ancestral sequences indicate that these replacements are ancestral. Hence the adaptive history of molecular evolution is amenable to experimental investigation.

Structural constraints in protein engineering--the coenzyme specificity of Escherichia coli isocitrate dehydrogenase.

In a previous study we reported on the successful inversion of coenzyme specificity in isocitrate dehydrogenase (IDH) from NADP to NAD [Chen, R., Greer, A. & Dean, A. M. (1995) A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity, Proc. Natl Acad. Sci. USA 92, 11666-11670]. Here, we explore alternative means to generate NAD dependence in the NADP-dependent scaffold of Escherichia coli IDH. The results reveal that engineering a preference for NAD is constrained by the architecture of the IDH coenzyme binding pocket and confirms that the substituted Asp344 in the engineered enzyme is the major determinant of coenzyme specificity. Mutations in the 316-325 loop, which forms part of the coenzyme binding site, reduce activity through transmission of long-range conformational changes into the active site some 14 A distant. Conformational changes seen upon substituting Cys332-->Tyr are not directly involved with improving activity. Replacements at Cys201 reveal that subtle changes in the packing of hydrophobic residues (Met and Ile versus Leu) can elicit markedly different responses. We caution against using sequence alignments as the sole guide for mutagenesis and show how a combination of rational design of active-site residues based on X-ray structures and random substitutions at surrounding residues provides an efficient means to improve enzyme preference and catalytic efficiency towards novel substrates.

Redesigning secondary structure to invert coenzyme specificity in isopropylmalate dehydrogenase.

Rational engineering of enzymes involves introducing key amino acids guided by a knowledge of protein structure to effect a desirable change in function. To date, all successful attempts to change specificity have been limited to substituting individual amino acids within a protein fold. However, the infant field of protein engineering will only reach maturity when changes in function can be generated by rationally engineering secondary structures. Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to systematically invert the coenzyme specificity of Thermus thermophilus isopropylmalate dehydrogenase from a 100-fold preference for NAD to a 1000-fold preference for NADP. The engineered mutant, which is twice as active as wild type, contains four amino acid substitutions and an alpha-helix and loop that replaces the original beta-turn. These results demonstrate that rational engineering of secondary structures to produce enzymes with novel properties is feasible.

Determinants of cofactor specificity in isocitrate dehydrogenase: structure of an engineered NADP+ --> NAD+ specificity-reversal mutant.

The 7-fold mutation Cys201Met/Cys332Tyr/Lys344Asp/Tyr345Ile/Val35 1Ala/Tyr391Lys/Arg395Ser converts the cofactor specificity of Escherichia coli isocitrate dehydrogenase from a 7000-fold preference for NADP+ to a 200-fold preference for NAD+, with overall activity comparable to that of wild-type NAD+-dependent isocitrate dehydrogenases. The structure of the NAD+-dependent mutant has been determined and refined to a working R-factor of 0.186 at 1.9 A resolution. The structure shows that NADP+ affinity is destroyed by removing favorable interactions between the 2'-phosphate and Tyr345, Tyr391, and Arg395 and by adding a repulsive interaction with Asp344. NAD+ affinity is enhanced by adding hydrogen bonds between Asp344 and the free 2'-hydroxyl. The favorable Asp344-2'-OH interaction requires a change in the pucker of the ribose to C2' endo and a shift in the adenine ring. The ring shift is made possible by a series of changes in steric packing interactions. The linchpin for repacking in the adenosine binding site is residue 351. The side chain of this "second layer" residue dictates packing of the surrounding "first layer" residues which interact with the 2' moiety and, in turn, directly determine specificity.

Second-site suppression of regulatory phosphorylation in Escherichia coli isocitrate dehydrogenase.

Inactivation of Escherichia coli isocitrate dehydrogenase upon phosphorylation at S113 depends upon the direct electrostatic repulsion of the negatively charged gamma-carboxylate of isocitrate by the negatively charged phosphoserine. The effect is mimicked by replacing S113 with aspartate or glutamate, which reduce performance (kcat/K(i).isocitrat/ Km.NADP) by a factor of 10(7). Here, we demonstrate that the inactivating effects of the electrostatic repulsion are completely eliminated by a second-site mutation, and provide the structural basis for this striking example of intragenic suppression. N115 is adjacent to S113 on one face of the D-helix, interacts with isocitrate and NADP+, and has been postulated to serve in both substrate binding and in catalysis. The single N115L substitution reduces affinity for isocitrate by a factor of 50 and performance by a factor of 500. However, the N115L substitution completely suppresses the inactivating electrostatic effects of S113D or S113E: the performance of the double mutants is 10(5) higher than the S113D and S113E single mutants. These mutations have little effect on the kinetics of alternative substrates, which lack the charged gamma-carboxylate of isocitrate. Both glutamate and aspartate at site 113 remain fully ionized in the presence of leucine. In the crystal structure of the N115L mutant, the leucine adopts a different conformer from the wild-type asparagine. Repacking around the leucine forces the amino-terminus of the D-helix away from the rest of the active site. The hydrogen bond between E113 and N115 in the S113E single mutant is broken in the S113E/N115L mutant, allowing the glutamate side chain to move away from the gamma-carboxylate of isocitrate. These movements increase the distance between the carboxylates, diminish the electrostatic repulsion, and lead to the remarkably high activity of the S113E/N115L mutant.

Determinants of performance in the isocitrate dehydrogenase of Escherichia coli.

The substrate specificity of the NADP-dependent isocitrate dehydrogenase of Escherichia coli was investigated by combining site-directed mutagenesis and utilization of alternative substrates. A comparison of the kinetics of the wild-type enzyme with 2R-malate reveals that the gamma-carboxylate of 2R,3S-isocitrate contributes a factor of 12,000,000 to enzyme performance. Analysis of kinetic data compiled for 10 enzymes and nine different substrates reveals that a factor of 1,650 can be ascribed to the hydrogen bond formed between S113 and the gamma-carboxylate of bound isocitrate, a factor of 150 to the negative charge of the gamma-carboxylate, and a factor of 50 for the gamma-methyl. These results are entirely consistent with X-ray structures of Michaelis complexes that show a hydrogen bond positions the gamma-carboxylate of isocitrate so that a salt bridge can form to the nicotinamide ring of NADP.

A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity.

The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD (calculated as the ratio of kcat/Km). Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to introduce six substitutions in the adenosine binding pocket that systematically shift coenzyme preference toward NAD. The engineered enzyme displays an 850-fold preference for NAD over NADP, which exceeds the 140-fold preference displayed by a homologous NAD-dependent enzyme. Of the six mutations introduced, only one is identical in all related NAD-dependent enzyme sequences--strict adherence to homology as a criterion for replacing these amino acids impairs function. Two additional mutations at remote sites improve performance further, resulting in a final mutant enzyme with kinetic characteristics and coenzyme preference comparable to naturally occurring homologous NAD-dependent enzymes.