NAD(P)+-independent aldehyde dehydrogenase from Pseudomonas testosteroni. A novel type of molybdenum-containing hydroxylase

Aldehyde dehydrogenase from Pseudomonas testosteroni was purified to homogeneity. The enzyme has a pH optimum of 8.2, uses a wide range of aldehydes as substrates and cationic dyes (Wurster's blue, phenazine methosulphate and thionine), but not anionic dyes (ferricyanide and 2.6-dichloroindophenol), NAD(P)+ or O2, as electron acceptors. Haem c and pyrroloquinoline quinone appeared to be absent but the common cofactors of molybdenum hydroxylases were present. Xanthine was not a substrate and allopurinol was not an inhibitor. Alcohols were inhibitors only when turnover of the enzyme occurred in aldehyde conversion. The enzyme has a relative molecular mass of 186,000, consists of two subunits of equal size (Mr 92,000), and 1 enzyme molecule contains 1 FAD, 1 molybdopterin cofactor, 4 Fe and 4 S. It is a novel type of NAD(P)+-independent aldehyde dehydrogenase since its catalytic and physicochemical properties are quite different from those reported for already known aldehyde-converting enzymes like haemoprotein aldehyde dehydrogenase (EC 1.2.99.3), quino-protein alcohol dehydrogenases (EC 1.1.99.8) and molybdenum hydroxylases.     

Citrullination of TNF-α by peptidylarginine deiminases reduces its capacity to stimulate the production of inflammatory chemokines

Citrullination, a posttranslational modification (PTM) recently discovered on inflammatory chemokines such as interleukin-8 (IL-8/CXCL8) and interferon-γ-inducible protein-10 (IP-10/CXCL10), seriously influences their biological activity. Citrullination or the deimination of arginine to citrulline is dependent on peptidylarginine deiminases (PADs) and has been linked to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Chemokines are to date the first identified PAD substrates with receptor-mediated biological activity. We investigated whether cytokines that play a crucial role in RA, like interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), may be citrullinated by PAD and whether such a PTM influences the biological activity of these cytokines. IL-1β and TNF-α were first incubated with PAD in vitro and the occurrence of citrullination was examined by Edman degradation and a recently developed detection method for citrullinated proteins. Both techniques confirmed that human TNF-α, but not IL-1β, was citrullinated by PAD. Citrullination of TNF-α reduced its potency to stimulate chemokine production in vitro on human primary fibroblasts. Concentrations of the inflammatory chemokines CXCL8, CXCL10 and monocyte chemotactic protein-1 (MCP-1/CCL2) were significantly lower in supernatants of fibroblasts induced with citrullinated TNF-α compared to unmodified TNF-α. However, upon citrullination TNF-α retained its capacity to induce apoptosis/necrosis of mononuclear cells, its binding potency to Infliximab and its ability to recruit neutrophils to the peritoneal cavity of mice.     

Knowledge,attitudes, and practices regarding schistosomiasis infection and prevention: A mixed-methods study among endemic communities of western Uganda

IntroductionIn Uganda, schistosomiasis (re)infections have continued to remain high despite the implementation of mass drug administration and sensitization campaigns aimed at controlling the disease. This could imply that there are some barriers to the implemented preventive measures. We conducted a mixed-methods study in Kagadi and Ntoroko districts around Lake Albert to assess knowledge, attitudes, and practices regarding schistosomiasis and to explore and understand perspectives regarding the disease.Materials and methodsSemi-structured survey questionnaires were administered to 337 household adults selected through systematic random sampling. We also interviewed 12 participants and held 28 focus-group discussion sessions with 251 individuals respectively. Quantitative data was analysed using frequencies, percentages, and chi-square tests for associations, while themes and sub-themes were used to analyse qualitative data respectively.FindingsA total of 98.5%, 81.3%, and 78.5% had heard about schistosomiasis, and knew the main transmission modes and symptoms, respectively. The majority (75.8%) said avoiding contact with water was a preventative way, while 67.5% said observing signs and symptoms was a form of diagnosis. Furthermore, 98.4% and 73.4% said it was important to defecate in latrines and to avoid contact with contaminated water respectively. However, it is difficult to avoid contact with lake water because it is the only source of livelihood, especially for fisher communities. Open defecation is commonly practiced along the lake due to insufficient space and difficulties in the construction of latrines. Myths and misconceptions reported include; lake water is safe, gassing in water causes transmission, fetching water early in the morning and from deep water is safe, and feces in the lake water act as a bait for catching fish.Conclusions and recommendationsDespite adequate knowledge of schistosomiasis and a positive attitude towards its prevention, existing myths and misconceptions, coupled with persistent risky water, sanitation, and hygiene practices still pose a challenge. A more robust community-based awareness intervention using bottom-up participatory approaches, accompanied by the provision of clean and safe water sources and increasing latrine coverage, could provide lasting solutions to these barriers.     

Phenolamine-dependent adenylyl cyclase activation in Drosophila Schneider 2 cells

Drosophila Schneider 2 (S2) cells are often employed as host cells for non-lytic, stable expression and functional characterization of mammalian and insect G-protein-coupled receptors (GPCRs), such as biogenic amine receptors. In order to avoid cross-reactions, it is extremely important to know which endogenous receptors are already present in the non-transfected S2 cells. Therefore, we analyzed cellular levels of cyclic AMP and Ca2+, important second messengers for intracellular signal transduction via GPCRs, in response to a variety of naturally occurring biogenic amines, such as octopamine, tyramine, serotonin, histamine, dopamine and melatonin. None of these amines (up to 0.1 mM) was able to reduce forskolin-stimulated cyclic AMP production in S2 cells. Furthermore, no agonist-induced calcium responses were observed. Nevertheless, the phenolamines octopamine (OA) and tyramine (TA) induced a dose-dependent increase of cyclic adenosine monophosphate (AMP) production in S2 cells, while serotonin, histamine, dopamine and melatonin (up to 0.1 mM) did not. The pharmacology of this response was similar to that of the octopamine-2 (OA2) receptor type. In addition, this paper provides evidence for the presence of an endogenous mRNA encoding an octopamine receptor type in these cells, which is identical or very similar to OAMB. This receptor was previously shown to be positively coupled to adenylyl cyclase.     

Five disulfide bridges stabilize a hevein-type antimicrobial peptide from the bark of spindle tree (Euonymus europaeus L.)

A small 45 amino acid residue antifungal polypeptide was isolated from the bark of spindle tree (Euonymus europaeus L.). Though the primary structure of this so-called E. europaeus chitin-binding protein or Ee-CBP is highly similar to the hevein domain, it distinguishes itself from most previously identified hevein-type antimicrobial peptides (AMP) by the presence of two extra cysteine residues that form an extra disulfide bond. Due to these five disulfide bonds Ee-CBP is a remarkably stable protein. Agar diffusion and microtiterplate assays demonstrated that Ee-CBP is a potent antimicrobial protein. IC₅₀-values as low as 1 μg/ml were observed for the fungus Botrytis cinerea. Comparative assays further demonstrated that Ee-CBP is a stronger inhibitor of fungal growth than Ac-AMP2 from Amaranthus caudatus seeds, which is considered one of the most potent antifungal hevein-type plant proteins.     

Short-term effects of biological and physical forces on aggregate formation in soils with different clay mineralogy

The mechanisms resulting in the binding of primary soil particles into stable aggregates vary with soil parent material, climate, vegetation, and management practices. In this study, we investigated short-term effects of: (i) nutrient addition (Hoagland's solution), (ii) organic carbon (OC) input (wheat residue), (iii) drying and wetting action, and (iv) root growth, with or without dry–wet cycles, on aggregate formation and stabilization in three soils differing in weathering status and clay mineralogy. These soils included a young, slightly weathered temperate soil dominated by 2:1 (illite and chlorite) clay minerals; a moderately weathered soil with mixed [2:1 (vermiculite) and 1:1 (kaolinite)] clay mineralogy and oxides; and a highly weathered tropical soil dominated by 1:1 (kaolinite) clay minerals and oxides. Air-dried soil was dry sieved through a 250 m sieve to break up all macroaggregates and 100 g-subsamples were brought to field capacity and incubated for 42 days. After 14 and 42 days, aggregate stability was measured on field moist and air-dried soil, to determine unstable and stable aggregation respectively. In control treatments (i.e., without nutrient or organic matter addition, without roots and at constant moisture), the formation of unstable and stable macroaggregates (> 250 m) increased in the order: 2:1 clay soil < mixed clay soil < 1:1 clay soil. After 42 days of incubation, nutrient addition significantly increased both unstable and stable macroaggregates in the 2:1 and 1:1 clay soils. In all soils, additional OC input increased both unstable and stable macroaggregate formation. The increase in macroaggregation with OC input was highest for the mixed clay soil and lowest for the 1:1 clay soil. In general, drying and wetting cycles had a positive effect on the formation of macroaggregates. Root growth caused a decrease in unstable macroaggregates in all soils. Larger amounts of macroaggregates were found in the mixed clay and oxides soil when plants were grown under 50% compared to 100% field capacity conditions. We concluded that soils dominated by variable charge clay minerals (1:1 clays and oxides) have higher potential to form stable aggregates when OC concentrations are low. With additional OC inputs, the greatest response in stable macroaggregate formation occurred in soils with mixed mineralogy, which is probably a result of different binding mechanisms occurring: i.e., electrostatic bindings between 2:1 clays, 1:1 clays and oxides (i.e. mineral-mineral bindings), in addition to OM functioning as a binding agent between 2:1 and 1:1 clays.     

Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+ -levels in Drosophila S2 cells

The bioluminescent Ca²⁺-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca^{2 +}-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC₅₀ value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca^{2 +}-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I. Revised version received: 12 September 2001 Electronic Publication     

Expanding roles for AMP‐activated protein kinase in neuronal survival and autophagy

AMP‐activated protein kinase (AMPK) is an evolutionarily conserved cellular switch that activates catabolic pathways and turns off anabolic processes. In this way, AMPK activation can restore the perturbation of cellular energy levels. In physiological situations, AMPK senses energy deficiency (in the form of an increased AMP/ATP ratio), but it is also activated by metabolic insults, such as glucose or oxygen deprivation. Metformin, one of the most widely prescribed anti‐diabetic drugs, exerts its actions by AMPK activation. However, while the functions of AMPK as a metabolic regulator are fairly well understood, its actions in neuronal cells only recently gained attention. This review will discuss newly emerged functions of AMPK in neuroprotection and neurodegeneration. Additionally, recent views on the role of AMPK in autophagy, an important catabolic process that is also involved in neurodegeneration and cancer, will be highlighted.