Search

This report summarizes the findings presented at the Chromosome 4 Workshop of the Sixth World Congress on Psychiatric Genetics (October 1998, Bonn, Germany). Chromosome 4 linkage and association results for several psychiatric phenotypes including bipolar affective disorder, schizophrenia, alcoholism, and mental retardation are reviewed. In bipolar affective disorder, positive linkage results for markers on 4q35 were reported by three independent groups. In addition, findings in bipolar disorder were reported for markers spanning 4p14–16, and of particular interest are the results that coincide with the original Blackwood et al. [1996: Nat Genet 12:427–430] region at 4p16. For schizophrenia, modest positive results were reported for 4q31, as well as for marker D4S2917 at a region of 4q close to the centromere. Chromosome 4 continues to demonstrate interesting results in alcoholism, particularly in the region of the alcohol dehydrogenase gene cluster; however, it is not clear how to interpret the contrast in the susceptibility versus protective loci that are being reported in this region. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:224–228, 1999.

In a series of sibpairs with late onset Alzheimer's disease, we have examined the segregation of the loci involved in the early onset, autosomal dominant form of the disorder by using flanking microsatellite repeat markers: thus we have used APP-PCR3 and D21S210 to examine the segregation of the amyloid-β precursor protein (APP) gene, the markers Dl 4S77 and D14S284 to examine the segregation of the presenilin 1 (PSI) gene and the markers D1S227, D1S249 and D1S419 to examine the segregation of presenilin 2 (PS2). We carried out our analyses on the whole dataset of 291 affected sibpairs, and on subsets comprising those sibpairs in which neither had an apolipoprotein E4 allele (65 affected sibpairs) and those in which both had an apolipoprotein E4 allele (165 affected sibpairs). We used the programs SPLINK to generate allele frequencies and MAPMAKER/SIBS to analyze our results. We examined the segregation of the markers D19S908 and D19S918 that are close to the apolipoprotein E (ApoE) gene as a positive control to assess whether the methods we are employing have the capability to identify known loci. The sibpair approach to the identification of genetic risk loci is relatively insensitive as indicated by the failure of the ApoE locus to reach statistical significance (P=0.06). Nevertheless, these data suggest that neither the PS1 nor the PS2 gene is a major locus for late-onset AD, but that the APP gene cannot be ruled out as a risk locus in those sibships without an E4 allele (P=0.014). The possibility that APP is indeed a locus for late onset disease will need confirmation in other series of familial cases.

The recombinant fusion protein rF450[mRat4A1/mRatOR]L1, containing the heme domain of P450 4A1 and the flavin domains of NADPH–P450 reductase, when incubated with dilaurylphosphatidylcholine (DLPC), Chaps, cytochromeb5, and a 20-fold excess of purified NADPH–P450 reductase, catalyzes the omega-oxidation of lauric acid at a rate of about 300 nmol/min/nmol P450. This is the first report of a mammalian P450 enzyme with such a high turnover number. The resultant 12-hydroxydodecanoic acid [12-hydroxylauric acid (12-OH LA)] is further oxidized by the P450 oxygenase reaction to dodecanedioic acid (decane-1,10-dicarboxylic acid) via 12,12-dihydroxydodecanoic acid. Spectral binding studies show that 12-OH LA inhibits the binding of lauric acid to the active site of P450 with aKiof about 1.9 μM. The construction and expression of recombinant P450 4A1 containing a six-member polyhistidine domain at the carboxy-terminus of the protein is described. Reconstitution experiments with this purified recombinant P450 4A1, DLPC, Chaps,b5, and purified NADPH–P450 reductase show results similar to those obtained with the purified fusion protein, albeit at lower turnover rates. The requirement for normal-phase HPLC in resolving the metabolites formed during lauric acid metabolism is demonstrated.

Transformation experiments showed that spontaneous deletions which result in loss of streptomycin resistance and an increase in conjugal transfer efficiency are present at a frequency of about 10(-4) in plasmid molecules of R6K. Similar deletions were thus readily selected by conjugal transfer of R6K, and their appearance was dependent upon recA+ activity in either donor or recipient host. The deoxyribonucleic acid segment deleted in four mutants examined was concluded to extend from the same terminus of the transposon, TnA, in the same direction, but to different extents, and to retain the TnA region intact. Insertions of a duplicate TnA element were found in R6K plasmids isolated from strains selected for increased ampicillin resistance, which were unstable in recA+ strains. In four plasmids examined after transfer to a recA host, an inverted repeat of the preexisting TnA element was shown to have been inserted at a similar location and was in two instances associated with deletions which extended from the same direction as those described above. The deletions are ascribed to the result of recA+-dependent recombination between direct repeats of TnA.

Wide ranging reviews, of housing policy are a significant feature of policy making in many countries. In England a major review carried out in 1977 was intended to provide a comprehensive basis for the future. In this article the 1977 national housing policy review is considered and conclusions are offered about the lessons to be learned from it on the form of such reviews and the importance of monitoring.

Previous work suggests an association between allele 1 and the 1–1 genotype of an intronic polymorphism in the presenilin-1 (PS-1) gene and late onset Alzheimer's disease. We found an excess of the 1–1 genotype in our late onset clinical sample (p= 0.006, one-tailed) but not in our postmortem confirmed sample, which instead exhibited an excess of allele 1 (p= 0.02, one-tailed). No interaction between PS-1 and ApoE genotype was detected and the findings remained significant when the effects of ApoE were taken into account (p= 0.03, one-tailed). These results suggest that the PS-1 polymorphism, or a locus in linkage disequilibrium with it, acts as a risk factor for late onset AD.

Recombinant DNA technology was used to clone a gene coding for a surface protein of Haemophilus influenzae type b (Hib) into Escherichia coli. Chromosomal DNA from a clinical isolate of Hib was cleaved with EcoRI and ligated into plasmid vectors containing three different translational reading frames. E. coli carrying recombinant plasmids were screened in a colony blot-radioimmunoassay system by using murine monoclonal antibodies (mabs) directed against cell surface-exposed proteins of Hib. mab 7B2, which is specific for a Hib surface protein with an apparent molecular weight of 27,000 (27K), reacted with several recombinant strains of E. coli. Restriction analysis revealed the presence of a 9.1-kilobase DNA insert in each of these recombinant plasmids and also determined that both transcription and translation of the Hib gene(s) coding for the 7B2-reactive antigen were not dependent on the lac operator and promoter of the vectors. Radioimmunoprecipitation and Western blot analyses showed that the antigenic determinant recognized by mab 7B2 in these recombinant E. coli was present in a 27K protein. In addition, this 27K protein was shown to be both localized on the surface of these E. coli cells and accessible to antibody.

Murine monoclonal antibodies directed against a 47,000-dalton immunodominant surface-exposed antigen of Treponema pallidum subsp. pallidum (Nichols) were isolated. These monoclonal antibodies cross-reacted with analogous 47,000-dalton antigens of two other virulent treponemes, T. pallidum subsp. pertenue and T. pallidum subsp. endemicum (Bosnia A), as determined by radioimmunoassay and immunoblot analyses. Immunoelectron microscopy confirmed that the 47,000-dalton antigen of T. pallidum subsp. pallidum was a surface-associated cellular component. Surface binding assays and immunoelectron microscopic studies also suggested that the analogous 47,000-dalton antigenic component of T. pallidum subsp. pertenue may not have been oriented toward the bacterial surface in the same way as the T. pallidum subsp. pallidum antigen or that the relevant antigenic determinant(s) may not have been exposed to the outer surface in the same way. The significance of this antigen relative to its apparent conservation among pathogenic treponemes and its possible diagnostic and vaccinogenic potentials are discussed.

A fusion protein containing the heme domain of bovine cytochrome P450 17A and the flavin domains of rat NADPH-cytochrome P450 reductase has been genetically engineered by linking the modified cDNAs for each gene with the codons for serine and threonine. Transformation of Escherichia coli(DH5α) and growth under defined conditions permits expression of 600-700 nmol of membrane-bound fusion protein per liter of growth medium (approximately 4% of cellular protein). A method has been developed for the solubilization, isolation, and purification to homogeneity of this protein. In the presence of NADPH the purified fusion protein catalyzes the 17α-hydroxylation of progesterone and pregnenolone as well as the conversion of 17α-hydroxypregnenolone to dehydroepiandrosterone. The 17,20-lyase activity is enhanced sixfold by the addition of purified rat liver cytochrome b5. Further, dehydroepiandrosterone is slowly metabolized to a number of additional more polar metabolites while 17α-hydroxy-pro-gesterone is slowly converted to dihydroxy-progesterone metabolites as well as a small amount of androstenedione in a reaction not influenced by cytochrome b5. Use of 5α-pregnan steroids as substrates show the importance of the 3β-hydroxyl group for cytochrome b5stimulated 17,20-lyase activity. Studies investigating the factors affecting electron transport between the flavin and heme domains suggest that the protein exists as a tight complex functioning as a self-contained biocatalytic unit.

Cell-free culture supernatant (CFCS) prepared from Haemophilus influenzae type b (Hib) was examined for the presence of soluble Hib proteins. Two proteins with apparent molecular weights of 100,000 (100K) and 116K were predominant in the CFCS, and antibodies directed against these proteins could be detected by radioimmunoprecipitation or Western blot analyses of serum from adult rats immunized with Hib. Radioimmunoprecipitation analyses and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these two proteins demonstrated that the 100K CFCS protein was also present on the cell surface of Hib, whereas the 116K CFCS protein was only detectable in culture supernatants. Both the 100K and 116K CFCS proteins were immunogenic in human infants with Hib meningitis and in infant rats systemically infected with Hib. In addition, the first detectable antibodies produced in these Hib-infected rats against Hib proteins were specific for the 100K protein in both its CFCS and cell-associated forms. These two CFCS proteins were also immunogenic in rats immunized with CFCS in the absence of Hib infection. Monoclonal antibody directed against the 100K protein reacted with 34 of 55 Hib strains examined by using a colony blot radioimmunoassay. The immunogenicity of the 100K and 116K CFCS proteins suggests that one or both of these proteins may have potential for vaccine development, either by themselves or covalently coupled to Hib capsular polysaccharide.

We have determined the nucleotide sequence of the URF A6L and ATPase 6 genes of the mitochondrial DNA of wild-type Chinese hamster ovary (CHO) cells and of two independently isolated, cytoplasmically inherited CHO mutant cell lines that are resistant to oligomycin, an inhibitor of the mitochondrial ATP synthase (ATPase) complex. Comparison of the nucleotide sequences of the mutants with that of their parental cell line revealed a single nucleotide difference, a G-to-A transition at nucleotide 433 of the ATPase 6 gene. This single base pair change predicts a nonconservative amino acid change, with a glutamic acid residue being replaced by a lysine residue at amino acid 145 of the ATPase 6 gene product in the mutants. This glutamic acid residue and several others in the surrounding amino acid sequence are conserved among all species examined to date. Analyses of several of the biochemical properties of the oligomycin-resistant CHO mutants indicate that the glutamic acid residue at position 145 of subunit 6 of the mitochondrial ATP synthase complex is important for the binding of oligomycin to the enzyme complex, but is not essential for proton translocation.

The recombinant fusion protein containing the heme domain of human P450 3A4 and the flavin domains of rat NADPH-cytochrome P450 (P450) reductase (rF450[mHum3A4/mRat0R]L1) requires both phospholipid and detergent as well as cytochrome b₅ (b₅) for the NADPH-dependent catalysis of the 6β-hydroxylation of testosterone. NADPH oxidation results in the formation of hydrogen peroxide in the presence or absence of phospholipid and detergent. NADPH oxidation and hydrogen peroxide formation are inhibited by the addition of b₅ and stimulated greater than 3-fold by the addition of testosterone. Marked differences in the ability of various phospholipids to support the P450-dependent 6β-hydroxylation of testosterone by the fusion protein were seen. Addition of a 4-fold excess of purified NADPH-P450 reductase, in the presence of phospholipid, detergent, and b₅, stimulates the rate of testosterone 6β-hydroxylation approximately 10-fold, providing turnover rates as high as 80 min−1 for P450 3A4. Approximately 30% of the rate bf hydrogen peroxide formation is not sensitive to inhibition by the P450 inhibitor ketoconazole, suggesting hydrogen peroxide (or superoxide anion) formation/directly from the reduced flavin domains of the fusion protein. it is proposed that the stimulation of NADPH oxidation observed following the addition of testosterone to the fusion protein may serve as a useful means of monitoring the interaction of other substrates with this P450 and thereby permit the rapid screening of chemicals to evaluate their potential metabolism by a human P450.Copyright 1995, 1999 Academic Press, Inc.

A T7 expression system is described for the high-level production in Escherichia coli of the membrane-bound form of human and rat cytochrome b₅. The cDNAs of b₅ have been engineered to contain a coding sequence for a four-member histidine domain at the amino-terminus of the recombinant protein permitting the use of a nickel-chelate affinity column for rapid purification of the detergent-solubilized hemoprotein. Results are presented demonstrating the ability of the purified recombinant b₅ proteins to stimulate the rate of oxidation of 17α-hydroxypregnenolone to dehydroepiandrosterone, catalyzed by bovine P450 17A, and to stimulate the 6β-hydroxylation of testosterone, catalyzed by human P450 3A4. These P450-catalyzed reactions have been used to compare the properties of different forms of b₅. Purified b₅ can serve as a "coupling protein" as illustrated by its inhibition of NADPH oxidation, catalyzed by a fusion protein containing the heme domain of P450 3A4 linked to rat NADPH-P450 reductase, and the associated inhibition of hydrogen peroxide formation. Kinetic studies show the formation of a complex of the flavoprotein, NADPH-P450 reductase, with b₅ for the rapid transfer of electrons from NADPH.Copyright 1994, 1999 Academic Press, Inc.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The article reflects on the Great Britain's Department for Communities and Local Government demographically based estimates of housing demand and needs projected for the year 2008. It highlights increment in the number of lone parent households which suggests the future need for social sector housing. It reports that future housing demand and need for second homes and vacant dwellings will increase.

We have constructed a pBR322 plasmid derivative which expresses dnaA protein under the control of the E. coli lac UV5 promotor. Expression of the dnaA protein from the plasmid is inducible by isopropyl-ß-D-thiogalactoside. In a dnaA+ strain induction has no effect on the accumulation of DNA. In contrast, in a thermosensitive dnaA46 strain, induction, at either the permissive or the nonpermissive temperature, results in an immediate stimulation of DNA accumulation. We conclude that, while in a dnaA46 strain dnaA protein limits DNA replication, in a dnaA+ strain dnaA protein activity does not control the timing of replication initiation.

Regenerating the Inner City; Glasgow's Experience. edited by David Donnison and Alan Middleton, London: Routledge and Kegan Paul, 1987. pp. 322. £12.95.Family Demography: Methods and their Application. edited by J. Bongaarts, T. K. Burch and K. W. Wachter. Oxford: Clarendon Press, 1987 pp. 372. £35 cloth.

Correction to: Molecular Psychiatry (2017) 22: 1502-1508; advance online publication, 12 July 2017; doi: 10.1038/mp.2016.97 In the first paragraph of the Results section and Figure 1, the authors incorrectly referred to the finding of SNP rs77897117. The correct SNP is rs77897177. The corrected figure appears in previous page.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.