6-Amino-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonyl derivatives as a new class of potent inhibitors of Interleukin-8-induced neutrophil chemotaxis

A series of 6-amino-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonyl derivatives was synthesized. The compounds demonstrated to be novel, potent and selective inhibitors of Interleukin-8-induced human neutrophil chemotaxis. A SAR study was performed by varying the carbonyl function at position 5 and the chain linked to the amino group at position 6 of the scaffold. All the compounds of the series displayed inhibitory activity at nano- or picomolar concentrations against Interleukin-8-driven migration and no activity against fMLP- and C5a-induced chemotaxis. The binding tests of selected compounds on CXCR1 and CXCR2 receptors were negative. The most potent derivative showed in vivo efficacy in a mouse model of Zymosan-induced peritonitis.     

Mycoplasma and herpesvirus PCR detection in tortoises with rhinitis-stomatitis complex in Spain

Chelonid herpesvirus (ChHV) and mycoplasmal infections cause similar clinical signs in terrestrial tortoises and may be the most important causative agents of rhinitis-stomatitis complex, a common disease in captive tortoises worldwide. Currently, diagnosis of ChHV and Mycoplasma spp. infections is most often based on serologic testing. However, serologic results only detect past exposure, and the specificity of these tests can be reduced due to antigenic cross-reactions with other pathogens. Molecular-based techniques could help to define the causative agent and to better manage infected tortoises. Using polymerase chain reaction, we analyzed 63 tortoises (59 spur-thighed tortoise, Testudo graeca; three Greek tortoise, Testudo ibera; and one Russian tortoise, Agryonemys horsfieldii) with clinical signs of rhinitis-stomatitis complex to identify the causative agent. Molecular evidence of ChHV type I (24%), type II (3%), and Mycoplasma agassizii (6%) infections, as well as coinfection of Mycoplasma-ChHV and both types of ChHV, were detected. Both ChHV and M. agassizii are considered pathogenic in captive tortoises and both are a threat to wild populations. However, neither agent was detected from most of the symptomatic tortoises we evaluated, indicating that other agents could be involved in the rhinitis-stomatitis complex.     

Phosphatidylserine is involved in the ferrichrome-induced plasma membrane trafficking of Arn1 in Saccharomyces cerevisiae

Arn1 is an integral membrane protein that mediates the uptake of ferrichrome, an important nutritional source of iron, in Saccharomyces cerevisiae. In the absence of ferrichrome, Arn1p is sorted directly from the trans-Golgi network to the vacuolar lumen for degradation. In the presence of low levels of ferrichrome, the siderophore binds to a receptor domain on Arn1, triggering the redistribution of Arn1 to the plasma membrane. When extracellular ferrichrome levels are high, Arn1 cycles between the plasma membrane and intracellular vesicles. To further understand the mechanisms of trafficking of Arn1p, we screened 4580 viable yeast deletion mutants for mislocalization of Arn1-GFP using synthetic genetic array technology. We identified over 100 genes required for trans-Golgi network-to-vacuole trafficking of Arn1-GFP and only two genes, SER1 and SER2, required for the ferrichrome-induced plasma membrane trafficking of Arn1-GFP. SER1 and SER2 encode two enzymes of the major serine biosynthetic pathway, and the Arn1 trafficking defect in the ser1Δ strain was corrected with supplemental serine or glycine. Plasma membrane trafficking of Hxt3, a structurally related glucose transporter, was unaffected by SER1 deletion. Serine is required for the synthesis of multiple cellular components, including purines, sphingolipids, and phospholipids, but of these only phosphatidylserine corrected the Arn1 trafficking defects of the ser1Δ strain. Strains with defects in phospholipid synthesis also exhibited alterations in Arn1p trafficking, indicating that the intracellular trafficking of some transporters is dependent on the phospholipid composition of the cellular membranes.     

Analysis of the genus Petagnaea Caruel (Apiaceae), using new molecular and literature data

In Southern Italy, an endemic monotypic genus belonging to family Apiaceae occurs: Petagnaea (P. gussonei), relict of Tertiary flora, belonging to subfamily Saniculoideae. At present, P. gussonei is an endangered species and is included in various lists of species deserving special protection. The genus belongs to scapose hemicryptophytes and shares a sciaphilous habitat (hygrophilous woodland). This study is aimed at doing a complete contribution about the evolutionary history of Petagnaea, using molecular markers as plastidial DNA (cpDNA), nuclear ribosomal DNA (rDNA) and data present in literature. We used nucleotide sequences from four regions of the chloroplast genome (rps16 intron, trnL^(UAA) intron, atpB-rbcL intergenic spacer, and partial matK gene) to investigate possible haplotypes in Petagnaea populations. To have an idea of the molecular relationships of all populations of P. gussonei, the internal transcribed spacer (ITS) sequences, already employed in recent studies, were obtained for 18 populations. These sequences in combination with other Saniculoideae ITS sequences available from GenBank have been used for a further phylogenetic analysis. The results agree with the current classification of Saniculoideae in placing P. gussonei in tribe Saniculeae, since P. gussonei is in basal position to Sanicula. According to intraspecific chloroplast DNA diversity, no different haplotypes were detected. In addition to molecular data, morphology, cytology, phytochemistry and conservation status have been considered in the discussion.     

Live-cell imaging of viral RNA genomes using a Pumilio-based reporter

We describe a method for localizing plant viral RNAs in vivo using Pumilio, an RNA-binding protein, coupled to bimolecular fluorescence complementation (BiFC). Two Pumilio homology domain (PUMHD) polypeptides, fused to either the N- or C-terminal halves of split mCitrine, were engineered to recognize two closely adjacent eight-nucleotide sequences in the genomic RNA of tobacco mosaic virus (TMV). Binding of the PUMHDs to their target sites brought the split mCitrine halves into close proximity, allowing BiFC to occur and revealing the localization of viral RNA within infected cells. The bulk of the RNA was sequestered in characteristic inclusion bodies known as viral replication complexes (VRCs), with a second population of RNA localized in discrete particles distributed throughout the peripheral cytoplasm. Transfer of the TMV Pumilio recognition sequences into the genome of potato virus X (PVX) allowed the PVX RNA to be localized. Unlike TMV, the PVX RNA was concentrated in distinctive 'whorls' within the VRC. Optical sectioning of the PVX VRCs revealed that one of the viral movement proteins was localized to the centres of the RNA whorls, demonstrating significant partitioning of viral RNA and proteins within the VRC. The utility of Pumilio as a fluorescence-based reporter for viral RNA is discussed.     

New triphosphate conjugates bearing reporter groups: labeling of DNA fragments for microarray analysis

A simple and convenient method for incorporation of fluorescent or ligand groups into 3'-termini of DNA fragments is proposed. A set of triphosphoric acid monoesters bearing fluorescent groups or biotin attached to the triphosphate fragment through linkers of different lengths and structures was synthesized. All the compounds were substrates for calf thymus terminal deoxynucleotidyltransferase and were used for incorporation of marker groups into 3'-termini of DNA fragments. The compounds were successfully applied for DNA labeling during post-PCR target preparation for microarray analysis.     

Preparation of a monolithic capillary column with immobilized alpha-mannose for affinity chromatography of lectins

A simple method for the preparation of an affinity monolithic (also called continuous bed) capillary column for alpha-mannose-specific lectins is described. 2-Hydroxyethyl methacrylate in combination with (+)-N,N -diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) as crosslinkers, were used as monomers for the monolith. After oxidation of DATD with periodate, alpha-mannose with spacer was bound to the aldehyde groups of the polymeric skeleton via reductive amination to form an affinity column for the separation, enrichment or binding studies of mannose-specific lectins. The permeability of the column was excellent. The porosity of the monolith was investigated by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC). The affinity of the monolith was evaluated by frontal analysis (FA) and fluorescence microscopy (FM) using fluorescently labeled concanavalin (Con A). Frontal affinity chromatography showed a specific interaction of two different lectins with the alpha-mannose-modified monolith. According to FM the affinity sites were evenly distributed over the monolithic bed.     

The effect of pH on the production of cellulases in Aspergillus terreus

Summary The optimal pH for the production of extracellular cellulolytic enzymes in the wild strain of Aspergillus terreus was shown to be pH 5.0. After 160 h of cultivation, carboxymethyl cellulase reached 9.0 IU/ml, filterpaper, cellulase 0.5 IU/ml and -glucosidase 0.9 IU/ml. The rate of synthesis of CM- and FP-cellulases decreased after 90 h of cultivation but -glucosidase was produced linearly for 160 h. Some of the enzymes produced were released into the medium during the fungal growth while others remained bound. The binding of enzymes to cells and residual crystalline cellulose was strongly affected by the pH of the medium. FP-cellulase and particularly -glucosidase were bound more effectively, at lower pHs. Cold shock treatment of the cell suspension increased the activities of FP- and CM-cellulases but -glucosidase activity was not affected.     

QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling

The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state "catch or present" model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS-Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.     

The American brine shrimp as an exotic invasive species in the western Mediterranean

The hypersaline environments and salterns present in the western Mediterranean region (including Italy, southern France, the Iberian Peninsula and Morocco) contain autochthonous forms of the brine shrimp Artemia, with parthenogenetic diploid and tetraploid strains coexisting with the bisexual species A. salina. Introduced populations of the American brine shrimp A. franciscana have also been recorded in these Mediterranean environments since the 1980s. Based on brine shrimp cyst samples collected in these countries from 1980 until 2002, we were able to establish the present distribution of autochthonous brine shrimps and of A. franciscana, which is shown to be an expanding invasive species. The results obtained show that A. franciscana is now the dominant Artemia species in Portuguese salterns, along the French Mediterranean coast and in Cadiz bay (Spain). Co-occurrence of autochthonous (parthenogenetic) and American brine shrimp populations was observed in Morocco (Mar Chica) and France (Aigues Mortes), whereas A. franciscana was not found in Italian cyst samples. The results suggest these exotic A. franciscana populations originate as intentional or non-intentional inoculations through aquacultural (hatchery effluents) or pet market activities, and suggest that the native species can be rapidly replaced by the exotic species. This revised version was published online in July 2006 with corrections to the Cover Date.