Dynamic regulation of interregional cortical communication by slow brain oscillations during working memory.

Transiently storing information and mentally manipulating it is known as working memory. These operations are implemented by a distributed, fronto-parietal cognitive control network in the brain. The neural mechanisms controlling interactions within this network are yet to be determined. Here, we show that during a working memory task the brain uses an oscillatory mechanism for regulating access to prefrontal cognitive resources, dynamically controlling interactions between prefrontal cortex and remote neocortical areas. Combining EEG with non-invasive brain stimulation we show that fast rhythmical brain activity at posterior sites are nested into prefrontal slow brain waves. Depending on cognitive demand this high frequency activity is nested into different phases of the slow wave enabling dynamic coupling or de-coupling of the fronto-parietal control network adjusted to cognitive effort. This mechanism constitutes a basic principle of coordinating higher cognitive functions in the human brain.

Nucleotide sequence of the Rhodobacter capsulatus hemH gene.

The last step in heme synthesis is the insertion of iron into the ring of protoporphyrin IX. The enzyme which catalyzes this reaction, ferrochelatase (FC), is encoded by the hemH gene. A clone containing this gene from Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium, has been sequenced. A single open reading frame was found which could encode a protein of 351 amino acids. This putative protein is very similar to other FC and contains the FC signature sequence.

Cloning and overexpression of the Rhodobacter capsulatus hemH gene.

In photosynthetically grown Rhodobacter capsulatus, heme is a qualitatively minor end product of the common tetrapyrrole pathway, but it may play a significant regulatory role. Heme is synthesized from protoporphyrin by the product of the hemH gene, ferrochelatase. We have cloned the R. capsulatus hemH gene by complementation of an Escherichia coli hemH mutant. When a plasmid carrying the hemH gene is returned to R. capsulatus, ferrochelatase activity increases, aminolevulinate synthase activity decreases, and bacteriochlorophyll levels are dramatically lowered. This is the first in vivo evidence to suggest that heme feedback inhibits aminolevulinate synthase in R. capsulatus, thereby reducing porphyrin synthesis.

Risk perception and the location of a repository for spent nuclear fuel.

This study investigates lay people's reactions to a repository for nuclear waste. Risk perception is seen as a complex concept, comprising both affective and cognitive components. Attitude towards nuclear power and trust in experts and authorities had a substantial impact on risk perception, while personal knowledge about nuclear waste disposal had no effect. Thus, the more positive one's attitude towards nuclear power is and the more trust one has in experts and authorities, the lower one's risk perception is. Also, reactions were expected to vary with distance between the home district and the location of a repository. These variations differed in nature for people with alternative levels of risk judgement. The distance between the home and a repository affected approval of the proposed site. Distance between home and repository also had an effect on risk feelings and somewhat less on beliefs about consequences. Estimated total risk was directly mediated by beliefs about consequences, but even more so by risk feelings. With regard to risk, one can conclude that it is important to make a distinction between an emotional and a cognitive component of risk perception.

Oxygen-regulated steps in the Rhodobacter capsulatus tetrapyrrole biosynthetic pathway.

The effect of exogenous aminolevulinate and porphobilinogen on protoporphyrin accumulation in Rhodobacter capsulatus was measured. Oxygen inhibited protoporphyrin accumulation in strain AJB456, a bchH mutant, even in the presence of exogenous aminolevulinate, suggesting that some step in the formation of protoporphyrin from aminolevulinate is regulated by oxygen. In contrast, in the presence of exogenous porphobilinogen, oxygen did not inhibit protoporphyrin accumulation. The results presented in this study indicate that oxygen regulates the formation of porphobilinogen from aminolevulinate.

Characterization of a coproporphyrin-protein complex from Rhodobacter capsulatus.

Rhodobacter capsulatus strain AJB530 excretes large amounts of coproporphyrin into the culture supernatant. The coproporphyrin was precipitable with ammonium sulfate, suggesting that it was part of a macromolecular complex. Analysis of an ammonium sulfate fraction indicated that the coproporphyrin was bound to a 66-kDa protein with a pI' of 4.0. The same protein was found in the culture supernatant of the bch+ strain PAS100.

Use of a lacZ fusion to study transcriptional regulation of the Rhodobacter capsulatus hemA gene.

An EcoRI fragment containing the Rhodobacter capsulatus hemA promoter has been cloned into a lacZ translational fusion vector. The resulting plasmid produced a hemA-lacZ fusion protein with a molecular mass of 147,000. Expression of the hemA-lacZ fusion, as measured by production of beta-galactosidase, was regulated 2- to 3-fold by oxygen tension. The unexpectedly small change in beta-galactosidase levels suggests that transcriptional regulation of the hemA gene is not the major factor in oxygen-mediated control of porphyrin synthesis.

Isolation of a Rhodobacter capsulatus bioB mutant and cloning of the bioB gene.

We isolated a Tn5 insertion mutant of Rhodobacter capsulatus which requires biotin for growth. Crossfeeding studies with Escherichia coli bio mutants indicated that the insertion is in the bioB gene. A cosmid that complements the bioB insertion was isolated, and the bioB gene was localized to a 2.85-kilobase EcoRI-PstI fragment.

Isolation of a Rhodobacter capsulatus mutant that lacks c-type cytochromes and excretes porphyrins.

A Rhodobacter capsulatus mutant lacking cytochrome oxidase activity was isolated by Tn5 mutagenesis. Difference spectroscopy of crude extracts and extracted c-type cytochromes demonstrated that this mutant completely lacked all c-type cytochromes. The strain did, however, synthesize normal amounts of b-type cytochromes and nonheme iron. This mutant also excreted large amounts of coproporphyrin and protoporphyrin and synthesized reduced amounts of bacteriochlorophyll, suggesting a link between the synthesis of c-type cytochromes and the expression of the tetrapyrrole biosynthetic pathway.

Cloning of the Rhodobacter capsulatus hemA gene.

Portions of the Rhodobacter capsulatus hemA gene have been cloned from a hemA::Tn5 insertion strain into the lambda bacteriophage derivative EMBL3. A cosmid containing the wild-type R. capsulatus hemA gene was isolated by complementation of the hemA::Tn5 mutant. The cosmid contains a 1.4-kilobase EcoRI fragment that spans the hemA::Tn5 insertion site. The entire hemA gene is contained in this fragment and the adjacent 0.6-kilobase EcoRI fragment.

Isolation and characterization of an aminolevulinate-requiring Rhodobacter capsulatus mutant.

Using transposon Tn5 mutagenesis, we isolated a mutant strain of Rhodobacter capsulatus that requires aminolevulinate for growth. Southern blot analysis indicated that this strain has a single Tn5 insertion. The addition of 0.1 mM aminolevulinate to the medium allowed the mutant to grow either aerobically or photosynthetically with generation times similar to those of the parental strain. When grown photosynthetically, bacteriochlorophyll accumulation increased with increasing aminolevulinate concentration. The mutant strain had only 10% of the normal aminolevulinate synthase activity, but it had a normal level of porphobilinogen synthase activity. The requirement for aminolevulinate could be satisfied by porphobilinogen, hemin, or protoporphyrin. While the mutant grew well on agar plates containing any of these substrates, growth in liquid media containing hemin or protoporphyrin was poor. Introduction of an R' factor containing all the known R. capsulatus bch genes into the mutant strain did not relieve the requirement for aminolevulinate, suggesting that the Tn5 insertion is not within the bch region.

Control of bacteriochlorophyll accumulation by light in Rhodobacter capsulatus.

The accumulation of bacteriochlorophyll in Rhodobacter capsulatus grown either anaerobically or under low aeration is repressed by bright light. It has been proposed that an intact photosynthetic membrane system is required for light-mediated regulation. This was tested by measuring bacteriochlorophyll accumulation in various mutant strains grown under 3% oxygen. Mutants lacking either the reaction center and B875 complexes or the B800-850 complex exhibited normal regulation of bacteriochlorophyll accumulation by light, suggesting that neither photosynthesis nor the photosynthetic membrane system is involved in light-mediated regulation. Bright light did not reduce transcription from the bchA, bchC, bchE, or bchF gene. Neither a bch+ strain nor bchG or bchH mutants accumulated greater than normal amounts of any bacteriochlorophyll precursor when grown in bright light, indicating that carbon flow over the bacteriochlorophyll biosynthetic pathway was not being regulated by light intensity. When exposed to bright light, R. capsulatus converted aminolevulinate into a colorless compound with Rf values very similar to those of bacteriochlorophyll. These results suggest that in strains grown under low aeration, light intensity controls bacteriochlorophyll accumulation, but does not control bacteriochlorophyll synthesis.

Oxygen does not directly regulate carotenoid biosynthesis in Rhodopseudomonas capsulata.

We examined the role of bacteriochlorophyll synthesis on the regulation of carotenoid synthesis in Rhodopseudomonas capsulata. Strains capable of making bacteriochlorophyll accumulated greater amounts of carotenoids under low oxygen than they did under high oxygen. However, strains unable to produce bacteriochlorophyll did not regulate their carotenoid production in response to changes in oxygen tension. This indicates that oxygen does not directly regulate carotenoid production.

Transcriptional regulation of several genes for bacteriochlorophyll biosynthesis in Rhodopseudomonas capsulata in response to oxygen.

Although it has been shown that bacteriochlorophyll synthesis in Rhodopseudomonas capsulata is repressed by oxygen and high light intensity, few details of regulation by these environmental factors are known, primarily owing to a lack of assays for the biosynthetic enzymes. We have examined regulation at the transcriptional level by isolating and studying fusions between the Mu d1(Apr lac) phage and various bch genes. In these strains, the lacZ gene of the phage is under the control of bch gene promoters. We have found that atmospheric oxygen tension (20% O2) reduces the expression of these fusions at least twofold compared with low oxygen tension (2% O2). Therefore, transcription of the bchA, bchB, bchC, bchG, and bchH genes is regulated in response to oxygen.

Mutations in the ilvY gene of Escherichia coli K-12 that cause constitutive expression of ilvC.

A derivative of Escherichia coli K-12 bearing an ilvC-lac fusion has been studied. beta-Galactosidase formation in this strain is under the control of the ilvC promoter and is therefore induced by the acetohydroxy acids. Derivatives of this fusion strain were isolated that constitutively expressed beta-galactosidase. When an ilvC-containing episome was introduced into these strains, acetohydroxy acid isomeroreductase was also constitutively expressed. The lesions are trans dominant and lie in ilvY, the structural gene specifying a positive control element, v, needed for induction of the isomeroreductase. It was concluded from measurements of beta-galactosidase levels in various diploid strains that, although wild-type v requires inducer to act as a positive control element, it does not act as a repressor in the absence of inducer.