Oxaloacetate decarboxylase of <Emphasis Type="Italic">Archaeoglobus fulgidus</Emphasis>: cloning of genes and expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Archaeoglobus fulgidus harbors three consecutive and one distantly located gene with similarity to the oxaloacetate decarboxylase Na⁺ pump of Klebsiella pneumoniae (KpOadGAB). The water-soluble carboxyltransferase (AfOadA) and the biotin protein (AfOadC) were readily synthesized in Escherichia coli, but the membrane-bound subunits AfOadB and AfOadG were not. AfOadA was affinity purified from inclusion bodies after refolding and AfOadC was affinity purified from the cytosol. Isolated AfOadA catalyzed the carboxyltransfer from [4-¹⁴C]-oxaloacetate to the prosthetic biotin group of AfOadC or the corresponding biotin domain of KpOadA. Conversely, the carboxyltransferase domain of KpOadA exhibited catalytic activity not only with its pertinent biotin domain but also with AfOadC.     

Wildlife crime promoted by weak governance

Wildlife crime such as illegal hunting of elephants and rhinos is currently one of the major issues in conservation. In order to combat poaching, law enforcement is essential. Here, we reviewed wildlife cases from the Northern Zone Anti‐Poaching Unit, Arusha, Tanzania (APU‐Arusha), focusing on the types of wildlife offence committed, prosecution outcomes and the sentences awarded to the offenders who were found guilty by the courts. Eighty‐two registered cases were thoroughly reviewed. The majority of wildlife offences committed were unlawful possession of, and failure to report, government trophies. It was found that only 16% of the accused were found guilty, 6% were acquitted, 30% of the charges were withdrawn, and 48% of the cases were discharged by the director of public prosecution (DPP) or magistrate. The poor conviction rate was attributed to weak evidence, failure of the prosecution to prove cases beyond reasonable doubt, lack of cooperation between the zonal game office and the DPP, and in some cases, the accused jumped their bails. Understanding the factors which allow individuals to continue to engage in poaching and the factors that promote the practice of good governance is vital in trying to stop wildlife crimes.     

Insect Stage-Specific Receptor Adenylate Cyclases Are Localized to Distinct Subdomains of the Trypanosoma brucei Flagellar Membrane

Increasing evidence indicates that the Trypanosoma brucei flagellum (synonymous with cilium) plays important roles in host-parasite interactions. Several studies have identified virulence factors and signaling proteins in the flagellar membrane of bloodstream-stage T. brucei, but less is known about flagellar membrane proteins in procyclic, insect-stage parasites. Here we report on the identification of several receptor-type flagellar adenylate cyclases (ACs) that are specifically upregulated in procyclic T. brucei parasites. Identification of insect stage-specific ACs is novel, as previously studied ACs were constitutively expressed or confined to bloodstream-stage parasites. We show that procyclic stage-specific ACs are glycosylated, surface-exposed proteins that dimerize and possess catalytic activity. We used gene-specific tags to examine the distribution of individual AC isoforms. All ACs examined localized to the flagellum. Notably, however, while some ACs were distributed along the length of the flagellum, others specifically localized to the flagellum tip. These are the first transmembrane domain proteins to be localized specifically at the flagellum tip in T. brucei, emphasizing that the flagellum membrane is organized into specific subdomains. Deletion analysis reveals that C-terminal sequences are critical for targeting ACs to the flagellum, and sequence comparisons suggest that differential subflagellar localization might be specified by isoform-specific C termini. Our combined results suggest insect stage-specific roles for a subset of flagellar adenylate cyclases and support a microdomain model for flagellar cyclic AMP (cAMP) signaling in T. brucei. In this model, cAMP production is compartmentalized through differential localization of individual ACs, thereby allowing diverse cellular responses to be controlled by a common signaling molecule.     

Syntheses and pharmacological characterization of novel thiazole derivatives as potential mGluR5 PET ligands

Four novel thiazole containing ABP688 derivatives were synthesized and evaluated for their binding affinity towards the metabotropic glutamate receptor subtype 5 (mGluR5). (E)-3-((2-(Fluoromethyl)thiazol-4-yl)ethynyl)cyclohex-2-enone O-methyl oxime (FTECMO), the ligand with the highest binding affinity (K_i = 5.5 ± 1.1 nM), was labeled with fluorine-18. [¹⁸F]-FTECMO displayed optimal lipophilicity (log D_pH7.4 = 1.6 ± 0.2) and high stability in rat and human plasma as well as sufficient stability in rat liver microsomes. In vitro autoradiography with [¹⁸F]-FTECMO revealed a heterogeneous and displaceable binding in mGluR5-rich brain regions. PET imaging with [¹⁸F]-FTECMO in Wistar rats, however, showed low brain uptake. Uptake of radioactivity into the skull was observed suggesting in vivo defluorination. Thus, although [¹⁸F]-FTECMO is an excellent ligand for the detection of mGluR5 in vitro, its in vivo characteristics are not optimal for the imaging of mGluR5 in rats in vivo.     

Subunit gamma of the oxaloacetate decarboxylase Na(+) pump: interaction with other subunits/domains of the complex and binding site for the Zn(2+) metal ion.

The oxaloacetate decarboxylase Na(+) pump of Klebsiella pneumoniae is an enzyme complex composed of the peripheral alpha subunit and the two integral membrane-bound subunits beta and gamma. The alpha subunit consists of the N-terminal carboxyltransferase domain and the C-terminal biotin domain, which are connected by a flexible proline/alanine-rich linker peptide. To probe interactions between the two domains of the alpha subunit and between alpha-subunit domains and the gamma subunit, the relevant polypeptides were synthesized in Escherichia coli and subjected to copurification studies. The two alpha-subunit domains had no distinct affinity toward each other and could, therefore, not be purified as a unit on avidin-sepharose. The two domains reacted together catalytically, however, performing the carboxyl transfer from oxaloacetate to protein-bound biotin. This reaction was enhanced up to 6-fold in the presence of the Zn(2+)-containing gamma subunit. On the basis of copurification with different tagged proteins, the C-terminal biotin domain but not the N-terminal carboxyltransferase domain of the alpha subunit formed a strong complex with the gamma subunit. Upon the mutation of gamma H78 to alanine, the binding affinity to subunit alpha was lost, indicating that this amino acid may be essential for formation of the oxaloacetate decarboxylase enzyme complex. The binding residues for the Zn(2+) metal ion were identified by site-directed and deletion mutagenesis. In the gamma D62A or gamma H77A mutant, the Zn(2+) content of the decarboxylase decreased to 35% or 10% of the wild-type enzyme, respectively. Less than 5% of the Zn(2+) present in the wild-type enzyme was found if the two C-terminal gamma-subunit residues H82 and P83 were deleted. Corresponding with the reduced Zn(2+) contents in these mutants, the oxaloacetate decarboxylase activities were diminished. These results indicate that aspartate 62, histidine 77, and histidine 82 of the gamma subunit are ligands for the catalytically important Zn(2+) metal ion.     

Effect of structural modification at carbon atom 1 of leukotriene B4 on the chemotactic and metabolic response of human neutrophils

Human neutrophils biosynthesize the chemoattractant leukotriene B4 (LTB4) and metabolize LTB4 to omega oxidative products 20-hydroxy-LTB4 (20-OH-LTB4) and 20-carboxy-LTB4 (20-COOH-LTB4). In this study, we prepared the C-1 methyl ester and N-methyl amide of LTB4 and then examined neutrophil chemotaxis and metabolism of these derivatives of LTB4. The results show that chemical modification of LTB4 at carbon atom 1 dramatically affects metabolism of the lipid molecule. The free acid form of LTB4 was taken up and metabolized by human neutrophils, while the methyl ester and N-methyl amide derivatives were poor substrates for omega oxidation. Although human neutrophils were poorly attracted to the methyl ester of LTB4, the amide derivative was a complete agonist of the neutrophil chemotactic response and displayed an ED50 for chemotaxis identical to that of LTB4. Therefore, we concluded that omega oxidation is not a requirement for the neutrophil chemotactic response induced by LTB4. These results also indicate that the N-methyl amide of LTB4 may be a useful ligand for the elucidation of molecular mechanisms operative in neutrophil chemotaxis to LTB4, since the C-1 derivative is not further metabolized. Two separate responses of human neutrophils are elicited by LTB4, resulting in both cellular activation and generation of omega oxidation products. It appears that putative receptors on the neutrophils can distinguish between LTB4 and certain derivatives that are structurally identical except for modification at the C-1 position (i.e., the methyl ester). LTB4 derivatives modified at the C-1 position do not undergo conversion to omega oxidation products by the neutrophil.     

Homing chemokines in rheumatoid arthritis

In about 20% of patients with rheumatoid arthritis, B and T lymphocytes recruited into the inflamed synovium are organized into complex microstructures, which resemble secondary lymphoid organs. The development of such lymphoid aggregates with germinal centers appears to contribute to the pathogenesis of the disease. Growing evidence indicates that chemokines and their receptors control the recruitment and positioning of leukocytes as well as their organization into node-like lymphoid structures. Here, we comment on recent studies highlighting the importance of chemokines in rheumatoid arthritis, in particular of B-cell-activating chemokine-1 in lymphoid neogenesis in the inflamed synovium.     

Vitalfärbestudien mit Methylrot

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