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PubMed1993

Use of Escherichia coli strains containing fad mutations plus a triple plasmid expression system to study the import of myristate, its activation by Saccharomyces cerevisiae acyl-CoA synthetase, and its utilization by S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase.

A system is described for studying protein N-myristoylation, a eukaryotic protein modification, in Escherichia coli strains containing components of eukaryotic metabolic pathways that regulate metabolism of myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) substrates. Three recombinant plasmids were used to simultaneously direct synthesis of Saccharomyces cerevisiae Nmt1p, a substrate protein (S. cerevisiae ADP-ribosylation factor 1, Arf1p), and one of the acyl-CoA synthetases produced by S. cerevisiae (Faa1p) in isogenic strains of bacteria with wild type or mutant alleles of genes comprising the regulon for fatty acid degradation (FadR, FadE, FadL and FadD). Incorporation of exogenous tritiated myristate into Arf1p and bacterial phospholipid biosynthetic pathways was analyzed. Removal of FadL, a 448-residue protein necessary for efficient transport of fatty acids across the outer membrane, had no detectable effect on Nmt1p-dependent N-myristoylation of Arf1p. This finding is consistent with the notion that permeation of C14:0 across the bacterial inner membrane can occur by simple diffusion. Studies of strains that contain a mutation in FadE which inhibits beta-oxidation of exogenous fatty acids, confirm that Nmt1p retains its specificity for myristoyl-CoA over palmitoyl-CoA in E. coli. A mutation that inactivates FadD, a 580-residue protein which is the only acyl-CoA synthetase produced by this bacterium, completely blocks incorporation of exogenous myristate into Arf1p. This failure to be incorporated indicates that myristoyl-acyl carrier protein, generated by inner membrane acyl-acyl carrier protein synthetase, is not a substrate for Nmt1p. S. cerevisiae Faa1p can partially complement this mutant fadD allele. It can fully "restore" N-myristoylation of Arf1p. Faa1p can also rescue growth at 37 degrees C of fadD- strains on minimal media supplemented with C12:0, although this rescue becomes less efficient as the chain length of the supplemental fatty acid increases. In addition, S. cerevisiae Faa1p is better able to direct myristoyl-CoA to the bacteria's phospholipid biosynthetic pathways than FadD, while FadD is more efficient at directing myristoyl-CoA to the genetically engineered protein N-myristoylation pathway. Since cellular acyl-CoA synthetase activity in S. cerevisiae has been distributed to at least two functionally differentiated proteins, this system should be useful for comparing their structure-activity relationships as well as their interactions with Nmt1p in an organelle-free environment.

Acyltransferases↗
PubMedDate not supplied

[Potential difference across the membrane of subcellular particles. IV. Study of the part of the respiratory chain from FAD to cytochrome c by the ion penetration technic].

Ability of mitochondria and submitochondrial particles (SMP) to generate a membrane potential on the addition of unpenetrating electron donors and acceptors and trimethylhydroquinone was used for specification of organization of the electron transfer chain. Ferri- and ferrocyanide added to mitochondria induce the membrane potential generation exchanging electrons only with cytochromes c or c1. Ferricyanide added to SMP induces the membrane potential taking electrons in three places: from NAD-H dehydrogenase, succinate dehydrogenase and in the presence of antimicyne A from cytochrome beta region. The difference of two sides of mitochondrial membrane is in agreement with the chemielectric hypothesis. According to this hypothesis protons from internal part of mitochondria are taken up to input H+-channel after electrons. That is why electrons come near the inner surface of the membrane and can be transferred from respiratory chain to water soluble acceptors. In output channels protons move under the action of intramembrane electrostatic field and in the direction of rising polarization of the medium. Protons are released to output channels while electrons are transferred to next carrier within the hydrophobic part of the membrane far from external surface of the membrane and cannot be transferred to water soluble acceptors.

Electron Transport↗
PubMedDate not supplied

Adolescent trends in the late 20th century: fad or societal alienation?

Since teens are engaging in a variety of body modifications at increasingly early ages, health care providers should be aware of the extent and the possible harm of these activities. This article reviews the historical context, medical uses, and risks of tattooing and other forms of body modification. There is little historical precedent for the extent of body piercing as carried out by the youth today. Body building is also practiced in excess. There is little legislative control of body modification other than for tattooing. The need for even young adolescents to improve their appearance and self-esteem with such extreme and even risky measures is evidence of a regressive trend in our culture.

Adolescent↗
PubMed1995

Beta APP mRNA transcription is increased in cultured fibroblasts from the familial Alzheimer's disease-1 family.

Familial (autosomal dominant) Alzheimer's disease (FAD) is a genetically heterogeneous disorder. Mutations in exons 16 and 17 of the amyloid beta-protein precursor (beta PP) gene currently account for less than 2% of FAD kindreds. No known defect in beta PP quantity, structure, or processing accounts for disease-associated beta-amyloid deposition in the majority of early-onset FAD kindreds. Only two out of a sample of 48 pedigrees (particularly the early onset FAD 4 kindred) contributed noticeably to evidence of linkage at the D21S16/13 and S1/S11 loci in the chromosomal region 21q21 [75]. Many early onset FAD pedigrees (including the FAD 1 and FAD 4 kindreds) show strong evidence of linkage to markers in the chromosome 14q24.3 region. Patients with trisomy 21 (Down's syndrome, DS) virtually always develop a histopathological phenotype indistinguishable from FAD, presumably on the basis of increased beta PP gene dosage and transcription. Whereas no beta PP gene duplication has been found in FAD, other mechanisms that augment beta PP production by effects at the transcriptional level could explain some FAD cases. Here, we report that cultured fibroblasts from affected members of the FAD 1 pedigree show a approximately 1.9 fold increase (P = 0.007) in beta PP mRNA levels compared to unaffected members when the cells are grown under stressed conditions in 0.5% serum. The elevated levels of beta PP mRNA in cells cultured in 0.5% serum also cosegregate with haplotypes in the 14q24.3 region when analyzed by linkage methods (LOD score = 3.26 at theta = 0.001). This is the chromosomal region to which FAD in this family has previously been mapped. As expected, fibroblasts from patients with DS used as a control show a similar beta PP mRNA increase. Fibroblasts from the FAD 4 pedigree did not show this defect under the conditions utilized here. beta PP and A beta protein levels were determined quantitatively after metabolic labeling and immunoprecipitation and found to increase 2.0 and 2.5 fold, respectively, in the fibroblasts from affected FAD 1 members. Finally, transient transfections of a beta PP promoter/chloramphenicol acetyl transferase reporter gene construct demonstrated a approximately 3-4 fold increase in beta PP promoter activity in affected fibroblasts from the FAD 1 but not the FAD 4 pedigree. Taken together, these data raise the possibility that an increase in beta PP transcription may underlie the AD phenotype in at least some of the chromosome 14-linked FAD families.

Alzheimer Disease↗
PubMed2006

Absorption and fluorescence spectroscopic characterization of cryptochrome 3 from Arabidopsis thaliana.

The blue light photoreceptor cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized at room temperature in vitro in aqueous solution by optical absorption and emission spectroscopic studies. The protein non-covalently binds the chromophores flavin adenine dinucleotide (FAD) and N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). In the dark-adapted state of cry3, the bound FAD is present in the oxidized form (FAD(ox), ca. 38.5%), in the semiquinone form (FADH., ca. 5%), and in the fully reduced neutral form (FAD(red)H2) or fully reduced anionic form (FAD(red)H-, ca. 55%). Some amount of FAD (ca. 1.5%) in the oxidized state remains unbound probably caused by chromophore release and/or denaturation. Förster-type energy transfer from MTHF to FAD(ox) is observed. Photo-excitation reversibly modifies the protein conformation causing a slight rise of the MTHF absorption strength and an increase of the MTHF fluorescence efficiency (efficient protein conformation photo-cycle). Additionally there occurs reversible reduction of bound FAD(ox) to FAD(red)H2 (or FAD(red)H-, FAD(ox) photo-cycle of moderate efficiency), reversible reduction of FADH. to FAD(red)H2 (or FAD(red)H-, FADH. photo-cycle of high efficiency), and modification of re-oxidable FAD(red)H2 (or FAD(red)H-) to permanent FAD(red)H2 (or FAD(red)H-) with low quantum efficiency. Photo-excitation of MTHF causes the reversible formation of a MTHF species (MTHF', MTHF photo-cycle, moderate quantum efficiency) with slow recovery to the initial dark state, and also the formation of an irreversible photoproduct (MTHF'').

Absorption↗
PubMed1990

Evidence for direct interaction between cysteine 138 and the flavin in thioredoxin reductase. A study using flavin analogs.

The flavoenzyme thioredoxin reductase from Escherichia coli contains an oxidation-reduction active disulfide made up of Cys135 and Cys138. Mutations changing each Cys residue to a Ser residue have been effected (Prongay, A. J., engelke, D. R., and Williams, C. H., Jr. (1989) J. Biol. Chem. 264, 2656-2664). The FAD prosthetic group of each altered thioredoxin reductase has been replaced with 1-deaza-FAD (a flavin analog with carbon substituted for nitrogen at position 1), 4-thio-FAD (a flavin analog with sulfur substituted for oxygen at position 4), and 6-thiocyanato-FAD. 1-Deaza-FAD-TRR(Cys135,Ser138) has absorbance and fluorescence spectral properties similar to the oxidized form of wild type apothioredoxin reductase reconstituted with 1-deaza-FAD. The absorbance spectrum of 1-deaza-FAD-TRR(Ser135,Cys138) is similar to the spectrum of the two-electron reduced form of wild type apothioredoxin reductase reconstituted with 1-deaza-FAD, indicating that it is a mixture of two species (O'Donnell, M. E., and Williams, C. H., Jr. (1984) J. Biol. Chem. 259, 2243-2251). The spectrum of one of these species of 1-deaza-FAD-TRR(Ser135,Cys138) resembles the spectrum of oxidized 1-deaza-FAD bound to wild type apothioredoxin reductase. The other species has an absorbance spectrum with a single peak at 400 nm (epsilon 400 = 11,100 M-1 cm-1) and resembles the spectrum of a thiolate adduct at the C4a position of the 1-deaza-FAD. The equilibrium between these species is pH-dependent, with a maximum of 50% C4a-adduct formation at low pH, and is linked to pK alpha values at 8.2 and 9.3. The absorbance spectrum of 4-thio-FAD-TRR(Cys135,Ser138) resembles the spectrum of the unbound 4-thio-FAD, whereas 4-thio-FAD-TRR(Ser135,Cys138) has a spectrum indicative of a mixture of 4-thio-FAD and FAD, suggesting a reaction between the 4-position of the flavin and Cys138. The binding of 6-thiocyanato-FAD to the apoprotein of the mutated enzymes showed no evidence for a reaction between the thiols and the group at the 6-position of the flavin.

Binding Sites↗
PubMed2002

Expression and characterization of ferredoxin and flavin adenine dinucleotide binding domains of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) catalyzes the selective oxidation of methane to methanol, the first step in the primary catabolic pathway of methanotrophic bacteria. A reductase (MMOR) mediates electron transfer from NADH through its FAD and [2Fe-2S] cofactors to the dinuclear non-heme iron sites housed in a hydroxylase (MMOH). The structurally distinct [2Fe-2S], FAD, and NADH binding domains of MMOR facilitated division of the protein into its functional ferredoxin (MMOR-Fd) and FAD/NADH (MMOR-FAD) component domains. The 10.9 kDa MMOR-Fd (MMOR residues 1-98) and 27.6 kDa MMOR-FAD (MMOR residues 99-348) were expressed and purified from recombinant Escherichia coli systems. The Fd and FAD domains have absorbance spectral features identical to those of the [2Fe-2S] and flavin components, respectively, of MMOR. Redox potentials, determined by reductive titrations that included indicator dyes, for the [2Fe-2S] and FAD cofactors in the domains are as follows: -205.2 +/- 1.3 mV for [2Fe-2S](ox/red), -172.4 +/- 2.0 mV for FAD(ox/sq), and -266.4 +/- 3.5 mV for FAD(sq/hq). Kinetic and spectral properties of intermediates observed in the reaction of oxidized MMOR-FAD (FAD(ox)) with NADH at 4 degrees C were established with stopped-flow UV-visible spectroscopy. Analysis of the influence of pH on MMOR-FAD optical spectra, redox potentials, and NADH reaction kinetics afforded pK(a) values for the semiquinone (FAD(sq)) and hydroquinone (FAD(hq)) MMOR-FAD species and two protonatable groups near the flavin cofactor. Electron transfer from MMOR-FAD(hq) to oxidized MMOR-Fd is extremely slow (k = 1500 M(-1) s(-1) at 25 degrees C, compared to 90 s(-1) at 4 degrees C for internal electron transfer between cofactors in MMOR), indicating that cofactor proximity is essential for efficient interdomain electron transfer.

Binding Sites↗
PubMed2004

[Folie à deux in schizophrenia--"psychogenesis" revisited].

OBJECTIVE AND METHOD: Since the 19th century, many studies have reported on folie à deux (FAD) in subjects with endogenous psychosis. However, the etiology of FAD in patients with schizophrenia has remained unclear, primarily because the possible psychogenic or reactive mechanisms of delusional contagion supposed in FAD were not compatible with the classical concept of endogenous psychosis. However, possible psychogenic mechanisms of FAD in schizophrenia were argued by Bleuler, E and Bleuler, M. The problem of how to distinguish the endogenesis from the psychogenesis (reaction) in FAD results in an aporia well-discussed in the German-speaking psychiatry: how induction in endogeneous psychosis might be possible? In the present study, first, a case of FAD in a husband and wife both with schizophrenia was reported. Three case reports of FAD in schizophrenic married couples from Japanese literature were also examined and the concept of the mind which lies in the background of psychogenesis/endogenesis dicotomy was discussed. CASE REPORT: A 34-year-old housewife had constructed a persecutory delusion shortly after the death of her father. She became convinced that her relative who was a Yakuza (Japanese Mafia) member was planning to kill her. She also reported auditory hallucinations. Her 41-year-old husband, who had been diagnosed with schizophrenia six years before the incident, first denied the claim of his wife that she had become a target of the Yakuza. However, shortly after that, he came to be convinced that he had become a target of another Yakuza. The couple took refuge in the husband's parents' home. Nevertheless, each of them thought that only he or she would be assassinated and not his or her spouse. After hospitalization, the husband was separated from his wife and soon recovered from his delusional state. However, the wife partially kept her persecutory delusion and full insight into her mental disorder could not be obtained after treatment. According to Gralnick's definition and sub-classifications of FAD, this case was diagnosed as FAD, subtype of "folie induite". DISCUSSIONS: Observed delusions of folie à deux in schizophrenic couples were classified into the following two categories, based on the subjective pronoun of the delusional statement: 1) We-type: "We are persecuted", generally observed in cases of paranoid type schizophrenia, paranoid disorder, and shared paranoid disorder; 2) I-type: "I am persecuted", observed in cases of non-paranoid schizophrenia in particular. It could be argued that the We-type suggests the establishment of a delusional community within participants not seen in the I-type delusion. However, according to clinical observations, even in We-type delusion, subjects ignored the wills and thoughts of their partners and somehow constructed the delusional community in a somewhat one-sided and egocentric way. In both We-type and I-type delusional formation, true communication was not established because of the limitation to the solipsistic view point inherent to the subjects involved. Therefore, we designated the We-type delusional view point as Paranoid Solipsism in FAD and the I-type as Schizophrenic Solipsism in FAD. According to German traditional psychiatry, We-type Paranoid Solipsism may correspond to "psychogenic" delusional formation mechanism and I-type Schizophrenic Solipsism to "endogenous" mechanism. The structural similarity between Paranoid Solipsism and the Husserlian concept of transcendental ego was also discussed. The author suggested that classical "psychogenesis" theory in psychiatry was founded on the modern concept of mind, which is inseparable from the European philosophical tradition of self-concept by Descartes and Husserl. From the Japanese literature, the author pointed out that the majority of FAD cases in Japan took the form of possession psychosis until the 1970's, and that We-type and I-type delusional formation in FAD appeared only after the late 70's. The implication of these transformations of FAD phenomenology in accordance with social structure change was briefly discussed regarding the relationship between the modernity and FAD delusion.

Adult↗
PubMed1992

Assessment of amyloid beta-protein precursor gene mutations in a large set of familial and sporadic Alzheimer disease cases.

A genetic locus associated with familial Alzheimer disease (FAD) and a candidate gene, APP, encoding the amyloid protein precursor have both been assigned previously to chromosome 21, and, in a few FAD families, mutations of APP have been detected. However, obligate crossovers between APP and FAD have also been reported in several FAD pedigrees, including FAD4, a large kindred showing highly suggestive evidence for linkage of the disorder to chromosome 21. In case the apparent APP crossover in FAD4 actually represented an intragenic recombination event or segregation of different mutations in different family branches, we have performed a more detailed assessment of APP as a candidate gene in this family. The entire coding region of the APP gene was sequenced for FAD4 and for FAD1, a second large kindred. No mutations were found, indicating that, in at least one chromosome 21-linked FAD pedigree, the gene defect is not accounted for by a mutation in the known coding region of the APP gene. A total of 25 well-characterized early- and late-onset FAD pedigrees were typed for genetic linkage to APP, to assess the percentage of FAD families predicted to carry mutations in the APP gene. None of the FAD families yielded positive lod scores at a recombination fraction of 0.0. To estimate the overall prevalence of FAD-associated mutations in the beta A4 domain of APP, we sequenced exons 16 and 17 in 30 (20 early- and 10 late-onset) FAD kindreds and in 11 sporadic AD cases, and we screened 56 FAD kindreds and 81 cases of sporadic AD for the presence of the originally reported FAD-associated mutation, APP717 Val----Ile (by BclI digestion). No APP gene mutations were found in any of the FAD families or sporadic-AD samples examined in this study, suggesting that the mutations in exons 16 and 17 are a rare cause of FAD. Overall, these data suggest that APP gene mutations account for a very small portion of FAD.

Alzheimer Disease↗