Interactions of Divalent Cations in Their Action on ATPases from Cucumber Roots

A microsomal fraction (10,000–30.000 g) was prepared from roots of Cucumis satirus L. (cv. Bestseller Fl) grown in solution culture. The ATPase activity was stimulated by Mg and Mn with optima between pH 6 and 7. Stimulation by Ca was obtained only above pH 7. Activations by Mg and Mn were inhibited by Ca, Mn and Mg interacted, so that Mn appeared strongly inhibitory for activation by Mg and Mg weakly inhibitory for activation by Mn: the simplest interpretation for this would be two separate enzymes. Cucumber accumulates and deposits Ca contrary to wheat and oat, which contain Ca-activated ATPase in the pH region 6 to 7. The Ca data for cucumber are compared with earlier findings from wheat and oat and are tentatively related to known physiological differences.     

Structural basis for selective inhibition of purine nucleoside phosphorylase from Schistosoma mansoni: Kinetic and structural studies

Selectivity plays a crucial role in the design of enzyme inhibitors as novel antiparasitic agents, particularly in cases where the target enzyme is also present in the human host. Purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive target for the discovery of potential antischistosomal agents. In the present work, kinetic studies were carried out in order to determine the inhibitory potency, mode of action and enzyme selectivity of a series of inhibitors of SmPNP. In addition, crystallographic studies provided important structural insights for rational inhibitor design, revealing consistent structural differences in the binding mode of the inhibitors in the active sites of the SmPNP and human PNP (HsPNP) structures. The molecular information gathered in this work should be useful for future medicinal chemistry efforts in the design of new inhibitors of SmPNP having increased affinity and selectivity.     

Targeted expression of a dominant-negative fibroblast growth factor (FGF) receptor in gonadotropin-releasing hormone (GnRH) neurons reduces FGF responsiveness and the size of GnRH neuronal population

Increasing evidence suggests that fibroblast growth factors (FGFs) are neurotrophic in GnRH neurons. However, the extent to which FGFs are involved in establishing a functional GnRH system in the whole organism has not been investigated. In this study, transgenic mice with the expression of a dominant-negative FGF receptor mutant (FGFRm) targeted to GnRH neurons were generated to examine the consequence of disrupted FGF signaling on the formation of the GnRH system. To first test the effectiveness of this strategy, GT1 cells, a GnRH neuronal cell line, were stably transfected with FGFRm. The transfected cells showed attenuated neurite outgrowth, diminished FGF-2 responsiveness in a cell survival assay, and blunted activation of the signaling pathway in response to FGF-2. Transgenic mice expressing FGFRm in a GnRH neuron-specific manner exhibited a 30% reduction in GnRH neuron number, but the anatomical distribution of GnRH neurons was unaltered. Although these mice were initially fertile, they displayed several reproductive defects, including delayed puberty, reduced litter size, and early reproductive senescence. Overall, our results are the first to show, at the level of the organism, that FGFs are one of the important components involved in the formation and maintenance of the GnRH system.     

Optimization of the measurement of outdoor airborne allergens using a protein microarrays platform

Increased knowledge on allergenic molecules in the environmental air helps in the information on environmental air quality and in the prevention and treatment of allergies. The aim of this study is to develop and validate a new methodology for the simultaneous detection and quantification of several airborne allergens using protein microarray technology, which has been created for the clinical detection of allergens. The immunological method was performed with Immuno Solid-phase Allergen Chip (ISAC) inhibition assay. Reagents for the validation studies include the following: (1) three sera from patients allergic to grass pollen each with different IgE levels as the detection reagents, (2) recombinant Phl p 1 major allergen as the inhibitor for the inhibition assays, (3) “natural” Phl p 1 released by Phleum pratense (timothy grass) pollen grains as the “biologically” relevant aeroallergen and (4) samples of airborne pollens collected by a Multi-vial Cyclone Sampler for comparison of levels of pollen detection versus the protein allergen detection by the microarray assay. The results obtained showed that ISAC inhibition is a sensitive technique able to detect 2.1 pg/mL of Phl p 1 and the allergens released from 1 grain of natural pollen. Also, the airborne allergen samples analyzed showed a good correlation with the concentration of grass pollen in the air. The use of ISAC inhibition will greatly improve future airborne simultaneous allergen quantification, becoming a valuable option in air quality control.     

Salivary and serum procalcitonin and C-reactive protein as biomarkers of periodontitis in United States veterans with osteoarthritis or rheumatoid arthritis

Serum procalcitonin (ProCT) is elevated in response to bacterial infections, whereas high sensitivity C-reactive protein (hsCRP) is a nonspecific inflammatory marker that is increased by excess adipose tissue. We examined the efficacy of ProCT and hsCRP as biomarkers of periodontitis in the saliva and serum of patients with arthritis, which is characterized by variable levels of systemic inflammation that potentially can confound the interpretation of inflammatory biomarkers. Blood and unstimulated whole saliva were collected from 33 patients with rheumatoid arthritis (RA) and 50 with osteoarthritis (OA). Periodontal status was assessed by full mouth examination and patients were categorized as having no/mild, moderate or severe periodontitis by standard parameters. Salivary and serum ProCT and hsCRP concentrations were compared. BMI, diabetes, anti-inflammatory medications and smoking status were ascertained from the patient records. Differences between OA and RA in proportionate numbers of patients were compared for race, gender, diabetes, adiposity and smoking status. Serum ProCT was significantly higher in arthritis patients with moderate to severe and severe periodontitis compared with no/mild periodontitis patients. There were no significant differences in salivary ProCT or salivary or serum hsCRP in RA patients related to periodontitis category. Most of the OA and RA patients were middle aged or older, 28.9% were diabetic, 78.3% were overweight or obese, and slightly more than half were either current or past smokers. The OA and RA groups differed by race, but not gender; blacks and males were predominant in both groups. The OA and RA groups did not differ in terms of controlled or uncontrolled diabetes, smoking status or BMI. The RA patients had been prescribed more anti-inflammatory medication than the OA patients. Our results demonstrate that circulating ProCT is a more discriminative biomarker for periodontitis than serum hsCRP in patients with underlying arthritis. Any elevation in salivary and serum hsCRP due to periodontitis apparently was overshadowed by differences among these patients in factors that influence CRP, such as the extent of inflammation between RA and OA, the extent of adipose tissue, the use of anti- inflammatory medications and smoking status. Although our study showed no differences in salivary ProCT related to severity of periodontitis, this biomarker also may be useful with further refinement.     

Interaction of F1L with the BH3 domain of Bak is responsible for inhibiting vaccinia-induced apoptosis

Apoptosis represents an important cellular defence mechanism against viral pathogens by virtue of its ability to remove infected cells. Consequently, many viruses have developed numerous strategies to prevent or delay host cell apoptosis in order to achieve productive replication. Here we report that deletion of the F1L gene from the vaccinia genome results in increased apoptosis during infection. We demonstrate that F1L, which has no sequence homology to Bcl-2 family members, inhibits apoptosis at the level of mitochondria by binding to Bak. As a consequence, F1L prevents Bak activation, oligomerization and interaction with active Bax, all critical steps in the induction of apoptosis. We demonstrate that residues 64-84 of F1L interact directly with the Bcl-2 homology domain 3 (BH3) domain of Bak. This region of F1L has limited sequence similarity to known Bak-interacting BH3 domains. We also find that such additional BH3-like domains exist in the vaccinia genome. We conclude that F1L uses this specific, BH3-like domain to bind and inhibit Bak at the mitochondria.     

Solvent effects on the reactivity of fluorenyl nitrenium ion with DNA-like probes

Laser Flash Photolysis (LFP) experiments carried out on 2-azidofluorene in aqueous systems generate 2-fluorenyl nitrenium ion (lambda(max) = 450 nm) which decays with first order rate constant and is quenched with 2'-deoxyguanosine originating an intermediate, namely the C8 adduct of 2-fluorenyl nitrenium ion, with bimolecular rate constant in the order of 1.3 x 10(9) M(-1) s(-1) in pure water. This intermediate very likely mimics the intermediate formed from carcinogens (i.e.: arylnitrenium ions formed through metabolic activation pathways from aminoaromatic substrates) and DNA rests in vivo. Solvent effects demonstrate and support the further stabilization of this intermediate (with respect to fluorenyl nitrenium ion) through hydrogen bonding as compared to other probe systems, and accounts for the enhanced metabolic carcinogenecity observed for this type of compounds. Diverse solvent systems, such as mixtures of water with acetonitrile, 1,1,1-trifluoroethanol, and 1,1,1,3,3,3-hexafluoroisopropanol, are used to interpret solvent-complex interactions.