Curcumin derivatives as new ligands of Aβ peptides

Curcumin derivatives with high chemical stability, improved solubility and carrying a functionalized appendage for the linkage to other entities, have been synthesized in a straightforward manner. All compounds retained Curcumin ability to bind Aβ peptide oligomers without inducing their aggregation. Moreover all Curcumin derivatives were able to stain very efficiently Aβ deposits.     

Host-plant specificity and specialization in eriophyoid mites and their importance for the use of eriophyoid mites as biocontrol agents of weeds

Eriophyoid mites, which are among the smallest plant feeders, are characterized by the intimate relationships they have with their hosts and the restricted range of plants upon which they can reproduce. The knowledge of their true host ranges and mechanisms causing host specificity is fundamental to understanding mite-host interactions, potential mite-host coevolution, and diversity of this group, as well as to apply effective control strategies or to use them as effective biological control agents. The aim of this paper is to review current knowledge on host specificity and specialization in eriophyoid mites, and to point out knowledge gaps and doubts. Using available data on described species and recorded hosts we showed that: (1) 80% of eriophyoids have been reported on only one host species, 95% on one host genus, and 99% on one host family; (2) Diptilomiopidae has the highest proportion of monophagous species and Phytoptidae has the fewest; (3) non-monophagous eriophyoids show the tendency to infest closely related hosts; 4) vagrant eriophyoids have a higher proportion of monophagous species than refuge-seeking and refuge-inducing species; (5) the proportions of monophagous species infesting annual and perennial hosts are similar; however, many species infesting annual hosts have wider host ranges than those infesting perennial hosts; (6) the proportions of species that are monophagous infesting evergreen and deciduous plants are similar; (7) non-monophagous eriophyoid species have wider geographic distribution than monophagous species. Field and laboratory host-specificity tests for several eriophyoid species and their importance for biological control of weeds are described. Testing the actual host range of a given eriophyoid species, searching for ecological data, genetic differentiation analysis, and recognizing factors and mechanisms that contribute to host specificity of eriophyoid mites are suggested as future directions for research.     

Expression,purification, phosphorylation and characterization of recombinant human statherin

This work reports the successful recombinant expression of human statherin in Escherichia coli, its purification and in vitro phosphorylation. Human statherin is a 43-residue peptide, secreted by parotid and submandibular glands and phosphorylated on serine 2 and 3. The codon-optimized statherin gene was synthesized and cloned into commercial pTYB11 plasmid to allow expression of statherin as a fusion protein with intein containing a chitin-binding domain. The plasmid was transformed into E. coli strains and cultured in Luria–Bertani medium, which gave productivity of soluble statherin fusion protein of up to 47 mg per liter of cell culture, while 112 mg of fusion protein were in the form of inclusion bodies. No significant refolded target protein was obtained from inclusion bodies. The amount of r-h-statherin purified by RP–HPLC corresponded to 0.6 mg per liter of cell culture. Attenuated total reflection-Fourier transform infrared spectroscopy experiments performed on human statherin isolated from saliva and r-h-statherin assessed the correct folding of the recombinant peptide. Recombinant statherin was transformed into the diphosphorylated biologically active form by in vitro phosphorylation using the Golgi-enriched fraction of pig parotid gland containing the Golgi-casein kinase.     

Quantitative HIV-1 proviral DNA detection: a multicentre analysis

Despite the widespread use of molecular biology techniques, standardized methods for the measurement of HIV-1 proviral DNA are currently lacking and several discordant results are still present in different studies. To assess the clinical meaning of the proviral DNA load, a study group comprising seven different laboratories was set up to standardize a HIV-1 proviral DNA quantification method able to assess the DNA proviral load of the most relevant circulating HIV-1 subtypes. Reference samples (24 cellular samples infected with HIV-1 clade B, and 40 samples of peripheral blood mononuclear cells containing different concentrations of plasmids expressing different HIV-1 clades) were distributed and tested blindly. All laboratories employed hTERT gene as housekeeping gene and primers within the gag gene to quantify different HIV-1 clades. Inter-laboratory results did not differ statistically but showed only minor variations concerning HIV-1 DNA amounts and different HIV clades, with a good agreement among the laboratories participating in the study. Since test standardization represents a key step for future application in clinical practice, further studies of the patients' samples are in progress to establish the real meaning and utility of the proviral DNA load for clinical management of HIV-1 infected patients.     

Study of mRNA Expression by Real Time PCR of Cpkk1, Cpkk2 and Cpkk3, three MEKs of Cryphonectria parasitica,in Virus‐free and Virus‐infected Isogenic Isolates

Cpkk1 and Cpkk2 are two previously characterized Mitogen‐activated protein kinase kinases (MEK) from Cryphonectria parasitica. For the characterization of the third MEK, primers designed to a conserved region of the known fungal MEK sequences were used in a PCR reaction to amplify genomic DNA from C. parasitica. The sequence of the resulting amplicon was compared to known sequences in the database using a Blast search. Results of the sequence comparison indicated that the initial fragment obtained encoded for a new MEK from C. parasitica, that had highest homology to Pbs2 from Saccharomyces cerevisiae. By inverse PCR we obtained a genomic fragment spanning the entire coding sequence of this MEK, which was named Cpkk3. The cDNA of Cpkk3 was obtained by compiling the sequences of RT‐PCR products resulting from the amplification of purified mRNA. TaqMan^® Probes were designed to analyse the expression of Cpkk1, Cpkk2 and Cpkk3 mRNA through RT‐Real Time PCR. This protocol allowed the expression of Cpkk3 to be successfully compared to the expression of Cpkk1 and Cpkk2, two previously cloned C. parasitica MEKs. No variation in expression was associated with the presence of a virus after 2 days of growth in standard conditions whereas an increase in the expression level of all the three MEKs was shown after 4 days of growth.     

Extended cardiolipin anchorage to cytochrome c: a model for protein–mitochondrial membrane binding

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c–CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c–CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c–CL complex.     

Expression of pro-inflammatory TACE-TNF-α-amphiregulin axis in Sjögren’s syndrome salivary glands

The tumor-necrosis-factor-converting-enzyme (TACE)-TNF-α-Amphiregulin (AREG) axis plays an important pathogenic role in inflammatory and autoimmune disorders. However, the pathological roles of these proteins in the chronic autoimmune disease Sjögren’s syndrome (SS) remain to be elucidated. It is known that the TACE–AREG axis is clearly part of a larger cascade of signals that starts with the activation of Furin, responsible for maturation of TACE that, in turn, determines the production of active TNF-α, directly involved in the up-regulation of AREG expression. This study showed that Furin, TACE, TNF-α, and AREG proteins, detected in acinar and ductal cells of human salivary glands from SS patients, increased remarkably in comparison with biopsies of labial salivary glands from healthy controls. The changes in Furin, TACE, TNF- α, and AREG proteins’ level detected in salivary glands biopsies of SS patients could be responsible for pro-inflammatory cytokines overexpression characterizing Sjögren’s syndrome.     

pH dependence of the enzymatic processing of collagen I by MMP-1 (fibroblast collagenase), MMP-2 (gelatinase A), and MMP-14 ectodomain

The proteolytic processing of collagen I by three matrix metalloproteinases (MMPs), a collagenase (MMP-1), a gelatinase (MMP-2), and the ectodomain of a membrane-type metalloproteinase (MMP-14), has been investigated at 37 °C between pH 6.0 and 9.2, a pH range reflecting conditions found in different body compartments under various physiopathological processes. In the proteolytic degradation the native collagen triple helix must be partially unwound to allow the binding of α chains to the protease’s active-site cleft. We have found that MMP-1 interacts with the two types of collagen I α chains in a similar fashion, whereas both MMP-2 and MMP-14 bind the two α chains in a different way. The overall enzymatic activity is higher on the α-2 chain for both MMP-1 and MMP-2, whereas the MMP-14 ectodomain preferentially cleaves the α-1 chain. In MMP-2 a marked difference for substrate affinity (higher for the α-1 chain) is overwhelmed by an even more marked propensity to cleave the α-2 chain. As a whole, the three classes of MMPs investigated appear to process collagen I in a significantly different fashion, so various MMPs play different roles in the collagen homeostasis in various compartments (such as bloodstream, synovial fluid, normal and tumoral tissues), where different pH values are observed.     

A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae (green peach aphid)

Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)-mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid salivary proteins that share features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes.