Converging evidence for a pseudoautosomal cytokine receptor gene locus in schizophrenia.

Schizophrenia is a strongly heritable disorder, and identification of potential candidate genes has accelerated in recent years. Genomewide scans have identified multiple large linkage regions across the genome, with fine-mapping studies and other investigations of biologically plausible targets demonstrating several promising candidate genes of modest effect. The recent introduction of technological platforms for whole-genome association (WGA) studies can provide an opportunity to rapidly identify novel targets, although no WGA studies have been reported in the psychiatric literature to date. We report results of a case-control WGA study in schizophrenia, examining approximately 500 000 markers, which revealed a strong effect (P=3.7 x 10(-7)) of a novel locus (rs4129148) near the CSF2RA (colony stimulating factor, receptor 2 alpha) gene in the pseudoautosomal region. Sequencing of CSF2RA and its neighbor, IL3RA (interleukin 3 receptor alpha) in an independent case-control cohort revealed both common intronic haplotypes and several novel, rare missense variants associated with schizophrenia. The presence of cytokine receptor abnormalities in schizophrenia may help explain prior epidemiologic data relating the risk for this illness to altered rates of autoimmune disorders, prenatal infection and familial leukemia.

Gene expression changes in peripheral blood cells provide insight into the biological mechanisms associated with regimen-related toxicities in patients being treated for head and neck cancers.

UNLABELLED: Patients treated with radiotherapy are prone to a constellation of local and systemic toxicities including mucositis, xerostomia, fatigue and anorexia. The biological complexities and similarities underlying the development of toxicities have recently been realized. Mucosal barrier injury is one of the best studied, and gene expression patterns, based on animal tissue samples, have added to its understanding. While investigations gene expression based on tissue samples was valuable, its use precludes more generalizable conclusions relative to common pathogenic mechanisms. Additionally, attempting to define the kinetics of changes in gene expression by sequential sampling is pragmatically unrealistic. Our objectives were: 1. to determine if changes in gene expression could be detected during toxicity development using PBM from patients receiving chemoradiation; 2. to characterize the relationship of expressed genes using graph theory and pathway analysis; and 3. to evaluate potential relationships between the expression of particular genes, canonical pathways, and functional networks in explaining the pathogenesis of regimen-related toxicities. DESIGN: Microarray analysis was performed using PBM-derived cRNA obtained before and 2 weeks after the initiation of chemoradiation in five patients with head and neck cancer who developed documented regimen-related toxicities. We created a database of those genes newly expressed at 2 weeks and evaluated their potential significance relative to toxicity, by canonical pathway analysis, compilation of regional networks around focus genes, and development of a model globalizing the individual functional networks. There was strong concordance between known pathogenic mechanisms of toxicity and the genes, pathways, and networks developed by our data. A role was elicited for unsuspected genes in toxicity development. Our results support the concept that radiation induced toxicities have common underlying mechanisms and demonstrate the utility of PBM as an RNA source for genetic studies. This methodology could be broadly applicable to the study of regimen-related toxicities.

nifU gene product from Azotobacter vinelandii is a homodimer that contains two identical [2Fe-2S] clusters.

The nifU gene product is required for the full activation of the metalloenzyme nitrogenase, the catalytic component of biological nitrogen fixation. In the present work, a hybrid plasmid that contains the Azotobacter vinelandii nifU gene was constructed and used to hyperexpress the NIFU protein in Escherichia coli. Recombinant NIFU was purified to homogeneity and was found to be a homodimer of 33-kDa subunits with approximately two Fe atoms per subunit. The combination of UV/visible absorption, variable-temperature magnetic circular dichroism, EPR, and resonance Raman spectroscopies shows the presence of a [2Fe-2S]2+,+ center (Em = -254 mV) with complete cysteinyl coordination in each subunit. The electronic, magnetic, and vibrational properties of the [2Fe-2S]2+,+ center do not conform to those established for any of the spectroscopically distinct types of 2Fe ferredoxins. These distinctive properties appear to be a consequence of a novel arrangement of coordinating cysteinyl residues in NIFU, and the residues likely to be involved in cluster coordination are discussed in light of primary sequence comparisons to other putative [2Fe-2S] proteins. The observed physicochemical properties of NIFU and its constituent [2Fe-2S] cluster also provide insight into the role of this protein in nitrogenase metallocluster biosynthesis.

Resonance Raman studies of iron-only hydrogenases.

The nature of the iron-sulfur clusters in oxidized and reduced forms of Fe-only hydrogenases from Desulfovibrio vulgaris, Thermotoga maritima, and Clostridium pasteurianum has been investigated by resonance Raman spectroscopy. The results indicate the presence of ferredoxin-like [4Fe-4S]2+,+ and [2Fe-2S]2+,+ clusters in both T. maritima hydrogenase and C. pasteurianum hydrogenase I, but only [4Fe-4S]2+,+ clusters in D. vulgaris hydrogenase. This necessitates a reevaluation of the iron-sulfur cluster composition of C. pasteurianum hydrogenase I and indicates that the resonance Raman bands in the oxidized hydrogenase that were previously attributed to the hydrogen activating center [Macor, K. A., Czernuszewicz, R. S., Adams, M. W. W., & Spiro, T. G. (1987) J. Biol. Chem. 262, 9945-9947] arise from an indigenous [2Fe-2S]2+ cluster. No resonance Raman bands that could be uniquely attributed to the oxidized or reduced hydrogen activating center were observed. This suggests that the hydrogen activating center is a novel Fe center that is unrelated to any known type of Fe-S cluster.

The unusual metal clusters of nitrogenase: structural features revealed by x-ray anomalous diffraction studies of the MoFe protein from Clostridium pasteurianum.

Nitrogenase (EC 1.18.6.1) catalyzes the conversion of dinitrogen to ammonia, the central reaction of biological nitrogen fixation. X-ray anomalous diffraction data were analyzed to probe the structures of the metal clusters bound by nitrogenase MoFe protein. In addition to one FeMo cofactor, each half-molecule of MoFe protein binds one large FeS cluster of a type not previously observed in a protein. The FeS cluster contains roughly eight Fe atoms, comprises two subclusters, and is separated from the FeMo cofactor by an edge-to-edge distance of 14 A. The inorganic framework of the FeMo cofactor is not resolved into subclusters, but the Mo atom is located at its periphery. FeMo cofactors in different half-molecules are 70 A apart and cannot promote binuclear activation of dinitrogen by two Mo atoms.

Mapping the site(s) of MgATP and MgADP interaction with the nitrogenase of Azotobacter vinelandii. Lysine 15 of the iron protein plays a major role in MgATP interaction.

Nitrogenase binds and hydrolyzes 2MgATP yielding 2MgADP and 2Pi for each electron that is transferred from the iron protein to the MoFe protein. The iron protein alone binds but does not hydrolyze 2MgATP or 2MgADP and the binding of these nucleotides is competitive. Iron protein amino acid sequences all contain a putatitive mononucleotide-binding region similar to a region found in other mononucleotide-binding proteins. To examine the role of this region in MgATP interaction, we have substituted glutamine and proline for conserved lysine 15. The amino acid substitutions, K15Q and K15P, both yielded a non-N2-fixing phenotype when the genes coding for them were substituted into the Azotobacter vinelandii chromosome in place of the wild-type gene. The iron protein from the K15Q mutant was purified to homogeneity, whereas the protein from the K15P mutant could not be purified in its native form. Unlike wild-type iron protein, the purified K15Q iron protein showed no acetylene reduction, H2 evolution, or ATP hydrolysis activities when complemented with wild-type MoFe protein. The K15Q iron protein and the normal iron protein had a similar total iron content and both proteins showed the characteristic rhombic EPR signal resulting from the reduced state of the single 4Fe-4S cluster bridging the two subunits. Unlike the wild-type iron protein, addition of MgATP to the K15Q iron protein did not result in the perturbation necessary to change the EPR signal of its 4Fe-4S center from a rhombic to an axial line shape. Also unlike the wild-type iron protein, addition of MgATP to K15Q iron protein in the presence of the iron chelator, alpha,alpha'-dipyridyl, did not result in a time-dependent transfer of iron to the chelator. Thus, even though the K15Q iron protein contains a normal 4Fe-4S center, it does not respond to MgATP like the wild-type protein. Examination of the ability of the K15Q iron protein to bind MgADP showed no change from the wild-type iron protein, but its ability to bind MgATP decreased to 35% of the wild-type protein. Thus, in A. vinelandii iron protein, lysine 15 is not needed for interaction with MgADP but is involved in the binding of ATP, presumably through charge-charge interaction with the gamma-phosphate. Based on the above data, this lysine appears to be essential for the MgATP induced conformational change of wild-type iron protein that is required for activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetic and spectroscopic analysis of the inactivating effects of nitric oxide on the individual components of Azotobacter vinelandii nitrogenase.

The effects of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4- and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C2H2-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactivate Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by the presence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicated that the effect of NO on Av2 involves oxidation of the [4Fe-4S] center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S = 1/2 and S = 3/2 signals. Appearance of the classical and intense iron-nitrosyl signal (g = 20.3) was only observed when Av2 was incubated with large molar excesses of NO and the appearance of this signal did not correlate with the loss of Av2 activity. The effects of NO on the MoFe protein (Av1) were more complex than for Av2. A time-dependent inactivation of Av1 activity (C2H2 reduction) was observed which required considerably higher concentrations of NO than those required to inactivate Av2 (up to 10 kPa).(ABSTRACT TRUNCATED AT 250 WORDS)

Resonance Raman studies of the [4Fe-4S] to [2Fe-2S] cluster conversion in the iron protein of nitrogenase.

Resonance Raman spectroscopy has been used to investigate the Fe-S stretching modes of the [4Fe-4S]2+ cluster in the oxidized iron protein of Clostridium pasteurianum nitrogenase. The results are consistent with a cubane [4Fe-4S] cluster having effective Td symmetry with cysteinyl coordination for each iron. In accord with previous optical and EPR studies [(1984) Biochemistry 23, 2118-2122], treatment with the iron chelator alpha, alpha'-dipyridyl in the presence of MgATP is shown to effect cluster conversion to a [2Fe-2S]2+ cluster. Resonance Raman data also indicate that partial conversion to a [2Fe-2S]2+ cluster is induced by thionine-oxidation in the presence of MgATP in the absence of an iron chelator. This result suggests new explanations for the dramatic change in the CD spectrum that accompanies MgATP-binding to the oxidized Fe protein and the anomalous resonance Raman spectra of thionine-oxidized Clostridium pasteurianum bidirectional hydrogenase.

Circular dichroism and magnetic circular dichroism of Azotobacter vinelandii ferredoxin I.

Room temperature circular dichroism (CD) and low temperature magnetic circular dichroism (MCD) spectra of air-oxidized and dithionite-reduced Azotobacter vinelandii ferredoxin I (FdI), a [( 4Fe-4S]2+/1+, [3Fe-4S]1+/0) protein, are reported. Unlike the CD of oxidized FdI, the CD of dithionite-reduced FdI exhibits significant pH dependence, consistent with protonation-deprotonation at or near the cluster reduced: the [3Fe-4S] cluster. The MCD of reduced FdI, which originates in the paramagnetic reduced [3Fe-4S]0 cluster, is also pH-dependent. Detailed studies of the field dependence and temperature dependence of the MCD of oxidized and reduced FdI, in the latter case at pH 6.0 and 8.3, are reported. The low-field temperature dependence of the MCD of oxidized FdI, which originates in the paramagnetic oxidized [3Fe-4S]1+ cluster, establishes the absence of a significant population of excited electronic states of this cluster up to 60 K. The low-field temperature dependence of the MCD of reduced FdI establishes that the ground-state manifold of the reduced [3Fe-4S]0 cluster possesses S greater than or equal to 2 at both pH 6.0 and 8.3. Analysis, assuming S = 2 and an axial zero-field splitting Hamiltonian, leads to D = -2.0 and -3.5 cm-1 at pH 6.0 and 8.3, respectively. The site of the (de)protonation affecting the spectroscopic properties of the [3Fe-4S] cluster remains unknown.

Pulsed electron paramagnetic resonance studies of the interaction of Mg-ATP and D2O with the iron protein of nitrogenase.

Mg-ATP binds to the iron protein component of nitrogenase. The magnetic field dependence of the linear electric field effect (LEFE) in pulsed EPR is consistent with a single 4Fe-4S cluster. The LEFE is virtually unaltered when Mg-ATP is bound. Electron spin echo envelope modulation techniques were employed to evaluate the possibility of a magnetic interaction between 31P of Mg-ATP and the Fe-S center of the iron protein. None was detected. However, weak modulations possibly attributable to peptide 14N were seen, and these were slightly shifted by Mg-ATP addition. Further, protons in the vicinity of the Fe-S cluster of the protein readily exchange with D2O, and this process is unaffected by Mg-ATP.

Comparison of redox and EPR properties of the molybdenum iron proteins of Clostridium pasteurianum and Azotobacter vinelandii nitrogenases.

Both heterologous crosses of the Clostridium pasteurianum and Azotobacter vinelandii nitrogenase components are completely inactive, although the reasons for this incompatibility are not known. We have compared a number of properties of the MoFe proteins from these organisms (Cp1 and Av1, respectively) in an attempt to find differences that could explain this lack of functional activity. Optical and CD spectroscopic titrations are similar for both Av1 and Cp1, but EPR titrations are significantly different, suggesting different chemical reactivity patterns and/or magnetic interaction behavior. Similarly, reduction measurements on the six-electron-oxidized state of Cp1 and Av1 at controlled potentials indicate a difference in both the relative reduction sequence of the redox centers and the numerical values for their measured midpoint potentials. EPR measurements as a function of temperature also demonstrate that the relaxation behavior of the S = 3/2 MoFe centers associated with the proteins differ markedly. The Cp1 EPR signal only begins to undergo broadening above 65 K, whereas the Av1 signal is severely broadened above 25 K. These variations in the EPR properties for the two proteins are not likely to be due to differences in the stoichiometry and/or geometry of the MoFe cluster units themselves since similar EPR studies of the isolated cofactors showed them to be essentially identical. Thus, the different EPR behavior of the two proteins seems to arise either from protein constraints imposed on identical cofactors, and/or from magnetic interactions due to neighboring metal clusters.

Azotobacter vinelandii ferredoxin I: cloning, sequencing, and mutant analysis.

The structure of Azotobacter vinelandii ferredoxin I (AvFdI) has been extensively characterized by a variety of techniques. Although its physiological function is unknown, it has long been implicated as being involved in electron donation to nitrogenase. Here we report that the AvFdI gene (fdxA) has been cloned from an EcoRI digest lambda library using a synthetic oligonucleotide probe and that its sequence has been determined. The amino acid sequence deduced from the DNA sequence is identical to the previously published protein sequence. Analysis of the promoter region indicates that AvFdI is not a nif specific gene product. A mutant of A. vinelandii has been constructed which is identical to the wild-type, at the DNA level, except that the fdxA gene has been interrupted by insertion of a kanamycin cartridge. This mutant, called LM100, does not synthesize AvFdI but does synthesize the Fe and MoFe proteins of nitrogenase and grows at wild-type rates under N2-fixing conditions. This demonstrates that AvFdI is not required for N2 fixation by A. vinelandii. There is a small acidic protein, which is present in wild-type A. vinelandii, whose level is dramatically increased in LM100. The nature of this protein is under further investigation.

[4Fe-4S]-cluster-depleted Azotobacter vinelandii ferredoxin I: a new 3Fe iron-sulfur protein.

Fe(CN)6(-3) oxidation of the aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I, (7Fe)FdI, is a degradative reaction. Destruction of the [4Fe-4S] cluster occurs first, followed by destruction of the [3Fe-3S] cluster. At a Fe(CN)6(-3)/(7Fe)FdI concentration ratio of 20, the product is a mixture of apoprotein and protein containing only a [3Fe-3S] cluster, (3Fe)FdI. This protein mixture, after partial purification, has been characterized by absorption, CD, magnetic CD, and EPR and Fe x-ray absorption spectroscopies. EPR and magnetic CD spectra provide strong evidence that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in (7Fe)FdI. Analysis of the extended x-ray absorption fine structure (EXAFS) of (3Fe)FdI finds Fe scattering at an average Fe...Fe distance of approximately equal to 2.7 A. The structure of the oxidized [3Fe-3S] cluster in solutions of oxidized (3Fe)FdI, and, by extension, of oxidized (7Fe)FdI, is thus different from that obtained by x-ray crystallography on oxidized (7Fe)FdI. Possible interpretations of this result are discussed.

Selective oxidative destruction of iron-sulfur clusters. Ferricyanide oxidation of Azotobacter vinelandii ferredoxin I.

The destructive oxidation of aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I [(7Fe)FdI] by Fe(CN)3-6 is examined using low-temperature magnetic circular dichroism (MCD) and EPR. The results demonstrate that oxidation of the [3Fe-3S] cluster occurs only after essentially complete destruction of the [4Fe-4S] cluster. It is therefore feasible by controlled Fe(CN)3-6 oxidation to obtain a partially metallated form of FdI, (3Fe)FdI, containing only a [3Fe-3S] cluster. The MCD and EPR data demonstrate that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in the native protein.

Spectroscopic studies of ferricyanide oxidation of Azotobacter vinelandii ferredoxin I.

The Fe(CN)3-(6) oxidation of the crystallographically characterized [[3Fe-3S], [4Fe-4S]] ferredoxin I of Azotobacter vinelandii has been studied using absorption, circular dichroism, magnetic circular dichroism, and EPR spectroscopies. A paramagnetic intermediate is observed en route to Fe-S cluster-free apoprotein, possessing an anisotropic g approximately equal to 2 EPR signal, surviving to temperatures greater than 77 K. This species is shown to result from 3-electron oxidation of the [4Fe-4S] cluster, without modification of the [3Fe-3S] cluster. However, it does not give rise to observable paramagnetic magnetic circular dichroism in the visible-near UV spectral region and is therefore neither an oxidized HIPIP [4Fe-4S] cluster nor an oxidized [3Fe-3S] cluster. We identify the paramagnetic species as a cysteinyldisulfide radical formed on dissociation of an oxidized cysteinate and an oxidized sulfide ion from the [4Fe-4S] cluster. This conclusion is consistent with the observed reaction stoichiometry, the spectroscopic results obtained, known EPR spectra of disulfide radicals, and the reconstitution of the native [4Fe-4S] cluster by dithiothreitol alone. This reaction, earlier interpreted as a HIPIP-type oxidation, is a previously uncharacterized oxidation reaction of [4Fe-4S] clusters.

Effects of growth substrate and respiratory chain composition on bioenergetics in Acinetobacter sp. strain HO1-N.

The relationship between respiratory chain composition and efficiency of coupling phosphorylation to electron transport was examined in Acinetobacter sp. strain HO1-N. Cells containing only cytochrome o as a terminal oxidase displayed the same stoichiometries of adenosine 5'-triphosphate synthesis and proton extrusion as cells which contained both cytochromes o and d as terminal oxidases. In addition, CO inhibition and photo-relief of cytochromes o or d did not alter the efficiency of energy coupling. These findings indicate that adenosine 5'-triphosphate synthesis is coupled to electron transport through both cytochromes o and d in Acinetobacter.

Respiratory properties of cytochrome-c-deficient mutants of Azotobacter vinelandii.

Cytochrome-c-deficient mutants of Azotobacter vinelandii have been isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. These mutants grow well under nitrogen-fixing conditions and studies of the physiology and energy conservation efficiency show no apparent differences from those of the parent strain. Under oxygen-limited growth conditions, the growth rate of the cytochrome-c-deficient mutant was slightly slower (approx. 15%) than that of the parent strain. Cytochromes of the c-type are required for the oxidation of artificial electron donors such as reduced N,N,N',N'-tetramethyl-p-phenylenediamine [Ph(NMe2)2]. This study could not demonstrate a physiological role for the c-type cytochromes which supports the idea that the minor Ph(NMe2)2-oxidizing pathway of the electron transport chain may be independent of the major pathway terminated by cytochrome d.