A defined subset of adenylyl cyclases is regulated by bicarbonate ion

The molecular basis by which organisms detect and respond to fluctuations in inorganic carbon is not known. The cyaB1 gene of the cyanobacterium Anabaena sp. PCC7120 codes for a multidomain protein with a C-terminal class III adenylyl cyclase catalyst that was specifically stimulated by bicarbonate ion (EC50 9.6 mM). Bicarbonate lowered substrate affinity but increased reaction velocity. A point mutation in the active site (Lys-646) reduced activity by 95% and was refractory to bicarbonate activation. We propose that Lys-646 specifically coordinates bicarbonate in the active site in conjunction with an aspartate to threonine polymorphism (Thr-721) conserved in class III adenylyl cyclases from diverse eukaryotes and prokaryotes. Using recombinant proteins we demonstrated that adenylyl cyclases that contain the active site threonine (cyaB of Stigmatella aurantiaca and Rv1319c of Mycobacterium tuberculosis) are bicarbonate-responsive, whereas adenylyl cyclases with a corresponding aspartate (Rv1264 of Mycobacterium) are bicarbonate-insensitive. Large numbers of class III adenylyl cyclases may therefore be activated by bicarbonate. This represents a novel mechanism by which diverse organisms can detect bicarbonate ion

Religiöse Sozialisation und Individualisierung : zum religiösen Kultur- und Identitätskonflikt muslimischer Migranten

In der vorliegenden Dissertation wird ein theoretisches Konzept erarbeitet, welches eine wachsende gesellschaftliche Inhomogenität der Wertorientierungen zu erklären sucht. Die Untersuchung beschäftigt sich jedoch nicht mit dem Verhalten der Bundesbürger gegenüber Ausländern, sondern mit potentiellen Kultur- und Identitätskonflikten muslimischer Migranten, welche sich im Zusammenhang ihrer religiösen Sozialisation ergeben können. Dabei ist die Frage vorrangig, ob das Identitätsverständnis des Islam bzw. dessen Individualisierungspotential für mögliche Konflikte verantwortlich ist. Bezogen auf die "zweite und dritte Generation" konzentriert sich die Analyse auf die Frage, ob der Sozialisationsprozeß innerhalb westlicher Werte- und Normensysteme im Bezugsrahmen muslimisch-religiöser Sozialisation durch Familie und subkulturelle Einflußnahme zu Konflikten führen kann. Somit untersucht die vorliegende Arbeit die Auseinandersetzung muslimischer Migranten mit der sie umgebenden säkularen, westlich-modernen Gesellschaft. Dabei gliedert sich die Arbeit in drei Teile, die aufeinander aufbauen: 1.- Darstellung des Problemfeldes anhand sozialisationstheoretischer und religionssoziologischer Betrachtungen. 2.- Entwicklung theoretischer Konzepte zum Identitätsverständnis auf der Basis der dargelegten Sozialisationsbedingungen und soziologischer Theorien. Im Anschluß hieran werden das Christentum und der Islam dargestellt, wobei deren Identitätskonzepte und Individualisierungspotentiale hergeleitet werden. Hieran anknüpfend wird auch das Identitätsverständnis innerhalb der westlichen Moderne dargelegt. In der Konfrontation unterschiedlicher Identitätsverständnisse innerhalb der Moderne wird die von muslimischen Migranten wahrgenommene, Diskrepanz der Werte- und Normensysteme als Identitätsanomie bewertet. 3.- Reaktionsmuster auf die angenommene Identitätsanomie werden benannt und bezüglich des zu betrachtenden Personenkreises muslimischer Migranten untersucht. Der Vergleich mit "modernen Christen" als auch mit Muslimen innerhalb der Heimatländer der Migranten schließt sich hieran an. Dieser Vergleich dient der Veranschaulichung der im Anschluß erläuterten politischen und gesellschaftlichen Änderungen, die für eine erfolgreiche Integration muslimischer Migranten notwendig erscheinen

Genomic Structure of an Economically Important Cyanobacterium, Arthrospira (Spirulina) platensis NIES-39

A filamentous non-N2-fixing cyanobacterium, Arthrospira (Spirulina) platensis, is an important organism for industrial applications and as a food supply. Almost the complete genome of A. platensis NIES-39 was determined in this study. The genome structure of A. platensis is estimated to be a single, circular chromosome of 6.8 Mb, based on optical mapping. Annotation of this 6.7 Mb sequence yielded 6630 protein-coding genes as well as two sets of rRNA genes and 40 tRNA genes. Of the protein-coding genes, 78% are similar to those of other organisms; the remaining 22% are currently unknown. A total 612 kb of the genome comprise group II introns, insertion sequences and some repetitive elements. Group I introns are located in a protein-coding region. Abundant restriction-modification systems were determined. Unique features in the gene composition were noted, particularly in a large number of genes for adenylate cyclase and haemolysin-like Ca2+-binding proteins and in chemotaxis proteins. Filament-specific genes were highlighted by comparative genomic analysis

The adenylyl cyclase cyaB1 from Anabaena sp. PCC 7120 is a cAMP-sensitive protein

Das Gen CyaB1 aus dem Cyanobakterium Anabaena sp. PCC 7120 codiert für eine Adenylatcyclase, die aus zwei N-terminalen GAF-Domänen (GAF-A und GAF-B), einer PAS-Domäne, einer Klasse-IIIb-AC katalytischen Domäne und einer TPR-ähnlichen Domäne besteht. Die katalytische Domäne von CyaB1 wurde kinetisch charakterisiert und mit Punktmutationen wurden die durch Sequenzvergleiche vorhergesagten katalytischen Aminosäuren verifiziert. Mit Rekonstitutionsversuchen von Punktmutanten, die einzeln inaktiv sind, wurde die Bildung katalytisch aktiver Homodimere nachgewiesen. Die spezifische Aktivität des Holoenzyms stieg exponentiell mit der Inkubationszeit an, da das Reaktionsprodukt cAMP dosisabhängig und nukleotidspezifisch als Aktivator der enzymatischen Aktivität (EC50 bei 1 µM) fungiert. Damit wurde erstmalig cAMP als GAF-Domänen Ligand identifiziert. Punktmutationen in der GAF-A oder GAF-B Domäne von CyaB1 zeigten, dass für die Aktivierung eine Bindung von cAMP an die GAF-B Domäne erfolgt. Das cyanobakterielle GAF-Domänen Ensemble konnte gegen das GAF-A/-B-Ensemble der cGMP stimulierten Ratten-PDE-2 ausgetauscht werden und verwandelte CyaB1 in eine cGMP stimulierte Adenylatcyclase. Das demonstriert die funktionelle Konservierung des bewährten Prinzips der Aktivierung durch GAF-Domänen, das vor über 3 Milliarden Jahren entwickelt wurde und sich in alle Phyla ausbreitete.Weitergehende Versuch mit einzeln ausgetauschten oder deletierten GAF-Domänen sowie Chimären mit der mycobakteriellen AC Rv1625c zeigen, dass Tandem-GAF-Domänen nur als fein aufeinander abgestimmtes Ensemble funktionieren. In Cyanobakterien könnte CyaB1 als cAMP gesteuerter Nano-Schalter zur Stabilisierung einmal getroffener Entwicklungsentscheidungen dienen.The gene cyaB1 from cyanobacterium Anabaena sp. PCC 7120 codes for a modular protein consisting of two N-terminal GAF (GAF-A and GAF-B) domains and a PAS and single class III adenylyl cyclase catalytic domain. The kinetics of the catalytic domain were characterised and the catalytic active amino acids were defined using point mutations. The adenylyl cyclase is active as a homodimer as demonstrated by reconstitution from complementary inactive point mutants. The specific activity of the recombinant and purified holoenyzme increased exponentially with time because the reaction product cAMP dose-dependently and nucleotide-specifically activated cyaB1 (half-maximally at 1µM). This established cAMP as a novel GAF domain ligand. Using inactivating point mutants of either GAF-A or GAF-B domain revealed that cAMP activated cyaB1 via the GAF-B domain. We replaced the cyanobacterial GAF domain ensemble in the cyaB1 adenylyl cyclase by the GAF-A/GAF-B assemblage from the rat cGMP-stimulated phosphodiesterase type 2. This way we converted the cyanobacterial adenylyl cyclase to a cGMP-stimulated adenylyl cyclase and established in principle the functional conservation of the GAF domain ensemble over more than 2 billion years since the divergence of bacterial and eukaryotic lineages. Further experiments using single GAF-domain deletionmutants and chimeric proteins with single PDE-2 GAF-domains or the catalytic domain of the mycobacterial adenylyl cyclase Rv1625c revealed that the GAF-domains work as a delicate fine tuned ensemble

A Physiologically-Based Pharmacokinetic (PBPK) Model Network for the Prediction of CYP1A2 and CYP2C19 Drug–Drug–Gene Interactions with Fluvoxamine, Omeprazole, S-mephenytoin, Moclobemide, Tizanidine, Mexiletine, Ethinylestradiol, and Caffeine

Physiologically-based pharmacokinetic (PBPK) modeling is a well-recognized method for quantitatively predicting the effect of intrinsic/extrinsic factors on drug exposure. However, there are only few verified, freely accessible, modifiable, and comprehensive drug–drug interaction (DDI) PBPK models. We developed a qualified whole-body PBPK DDI network for cytochrome P450 (CYP) CYP2C19 and CYP1A2 interactions. Template PBPK models were developed for interactions between fluvoxamine, S-mephenytoin, moclobemide, omeprazole, mexiletine, tizanidine, and ethinylestradiol as the perpetrators or victims. Predicted concentration–time profiles accurately described a validation dataset, including data from patients with genetic polymorphisms, demonstrating that the models characterized the CYP2C19 and CYP1A2 network over the whole range of DDI studies investigated. The models are provided on GitHub (GitHub Inc., San Francisco, CA, USA), expanding the library of publicly available qualified whole-body PBPK models for DDI predictions, and they are thereby available to support potential recommendations for dose adaptations, support labeling, inform the design of clinical DDI trials, and potentially waive those

A GAF-domain-regulated adenylyl cyclase from Anabaena is a self-activating cAMP switch

The gene cyaB1 from the cyanobacterium Anabaena sp. PCC 7120 codes for a protein consisting of two N-terminal GAF domains (GAF-A and GAF-B), a PAS domain and a class III adenylyl cyclase catalytic domain. The catalytic domain is active as a homodimer, as demonstrated by reconstitution from complementary inactive point mutants. The specific activity of the holoenyzme increased exponentially with time because the product cAMP activated dose dependently and nucleotide specifically (half-maximally at 1 µM), identifying cAMP as a novel GAF domain ligand. Using point mutants of either the GAF-A or GAF-B domain revealed that cAMP activated via the GAF-B domain. We replaced the cyanobacterial GAF domain ensemble in cyaB1 with the tandem GAF-A/GAF-B assemblage from the rat cGMP-stimulated phosphodiesterase type 2, and converted cyaB1 to a cGMP-stimulated adenylyl cyclase. This demonstrated the functional conservation of the GAF domain ensemble since the divergence of bacterial and eukaryotic lineages >2 billion years ago. In cyanobacteria, cyaB1 may act as a cAMP switch to stabilize committed developmental decisions

Multiple splice variants encode a novel adenylyl cyclase of possible plastid origin expressed in the sexual stage of the malaria parasite Plasmodium falciparum.

We report the characterization of an unusual adenylyl cyclase gene from Plasmodium falciparum, here designated PfACalpha. The level of mRNA expression is maximum during development of gametocytes (the sexual blood stage of the parasite life cycle). The gene is highly interrupted by 22 introns, and reverse transcriptase-PCR analysis revealed that there are multiple mRNA splice variants. One intron has three alternative 3'-splice sites that confer the potential to encode distinct forms of the enzyme using alternative start codons. Deduced amino acid sequences predict membrane-spanning regions, the number of which can vary between two and six depending on the splice variant. Expression of a synthetic form of two of these variants in Xenopus oocytes and in Dictyostelium adenylyl cyclase-deficient mutants, confirms that PfACalpha is a functional adenylyl cyclase. These results identify a novel mechanism in P. falciparum for the generation of multiple isoforms of a key, membrane-bound signaling molecule from a single genomic copy. Comparisons of the catalytic domains of PfACalpha and a second putative P. falciparum adenylyl cyclase (PfACbeta) with those from other species reveal an unexpected similarity with adenylyl cyclases from certain prokaryotes including the cyanobacteria (blue green algae). In addition, the presence of an unusual active site substitution in a position that determines substrate specificity, also characteristic of these prokaryotic forms of the enzyme, further suggests a plastid origin for the Plasmodium cyclases

The cyanobacterial tandem GAF domains from the cyaB2 adenylyl cyclase signal via both cAMP-binding sites

The tandem GAF domains from the cyanobacterium Anabaena PCC7120 cyaB2 adenylyl cyclase form an antiparallel dimer with cAMP bound to all four binding sites. cAMP binding causes highly cooperative allosteric enzyme activation (>500-fold; EC(50) = 1 μM; Hill coefficient >2.0). The cyaB2 GAF domains, like those of the cyclic nucleotide phosphodiesterases (PDEs), contain conserved NKFDE motifs that when mutated in the PDEs abrogate cyclic nucleotide binding. We mutated the aspartic acids within this motif in cyaB2 to determine which domains were required for signaling. Constructs containing an Asp/Ala mutation in either GAF domain still showed positive cooperative cAMP stimulation but with reduced Hill coefficients. The cyaB2 GAF domain NKFDE motifs contain inserts of 14 (GAF-A) and 19 (GAF-B) amino acids not present in PDE2 or cyaB1. Constructs having these inserts deleted could still be activated by cAMP (23- to 100-fold) but lost all positive cooperative activation, suggesting that the inserts play an important role in domain interaction and/or stabilization of the cAMP-binding pockets. In the shortened constructs, even those with a single Asp/Ala mutation in the NKFDE motifs could still be activated by cAMP. However, in a double Asp/Ala mutant of the shortened construct, stimulation by cAMP was almost completely lost, and the EC(50) shifted far to the right. Overall, the data suggest that in GAF domains without these inserts, only the canonical lysine:aspartate salt bridge keeps the α4-helix and the α4-β5 linker that close over the cyclic nucleotide properly oriented, thereby stabilizing the binding pocket. The cyaB2 GAF ensemble appears to be an evolutionary intermediate where both GAF domains still participate in allosteric activation by cAMP