Alpha(1)-acid glycoprotein is contained in bovine neutrophil granules and released after activation

The present study was designed to investigate the capability of bovine neutrophil granulocytes to produce the minor acute phase protein alpha(1)-acid glycoprotein (AGP, Orososmucoid). Bovine neutrophils contain a high MW (50-60kDa) AGP isoform (PMN-AGP), as determined by Western blotting and confirmed by fluorescence microscopy. The presence of AGP in bovine neutrophils has been confirmed by fluorescence immunocytometry. In addition, bovine neutrophils contain also a 42-45kDa isoform, which has the same MW as plasma-, liver-delivered, AGP. cDNA sequence of plasma- and PMN-AGP revealed that (i) the two proteins are products of the same gene; (ii) the differences in molecular weight are due do different post-translational modifications. This result was confirmed by deglycosylation of the two glycoforms. Exocytosis studies showed that isolated neutrophils exposed to several challengers, including Zymosan activated serum (ZAS) and phorbol 12-myristate 13-acetate (PMA), which mimic the inflammatory activation, released PMN-AGP as early as 15min. AGP's mRNA is physiologically expressed by mature resting neutrophils. Real-time PCR on LPS, ZAS and PMA challenged cells revealed that the level of expression apparently does not increase after inflammatory activation. Collectively, the findings reported in this paper proved that PMN-AGP: (i) is a hyperglycosylated glycoform of plasma AGP, (ii) is stored in granules, and (iii) is released by neutrophils in response to activation. Due to its anti-inflammatory activity, PMN-AGP may work as a fine tuning of the neutrophils functions in the inflammatory focus, i.e. it can reduce the damages caused by an excess of inflammatory response.     

Bovine alpha-1 acid glycoprotein can reduce the chemotaxis of bovine monocytes and modulate CD18 expression

The acute phase protein alpha(1)-acid glycoprotein (AGP--Orosomucoid) is a lipocalin with immunomodulatory functions. The present study provides evidence that the plasma glycoforms of AGP inhibit the migration of bovine monocytes in response to classical chemoattractants. The inhibition is specific, since neutrophils are apparently not affected. To investigate the molecular basis of this finding, the expression of the molecules mostly involved in chemotaxis, including CD18, CD11b and CD47 was studied. It was found that the incubation of activated monocytes with acute phase concentration of AGP (0.9 mg/mL) induces a down-regulation of CD18, and has no apparent influence on CD11b and CD47. RT-PCR expression studies on CD18, CD11b and CD47 mRNA revealed that AGP treatment does not modify the expression rate of these genes. Since AGP treatment is related to a down-regulation of CD18 on the surface of the monocytes, the authors suggest that one of its possible functions consists in specifically reducing the firm adhesion phase of bovine monocytes to the endothelium.     

Sequence finishing and mapping of Drosophila melanogaster heterochromatin

Genome sequences for most metazoans and plants are incomplete because of the presence of repeated DNA in the heterochromatin. The heterochromatic regions of Drosophila melanogaster contain 20 million bases (Mb) of sequence amenable to mapping, sequence assembly, and finishing. We describe the generation of 15 Mb of finished or improved heterochromatic sequence with the use of available clone resources and assembly methods. We also constructed a bacterial artificial chromosome-based physical map that spans 13 Mb of the pericentromeric heterochromatin and a cytogenetic map that positions 11 Mb in specific chromosomal locations. We have approached a complete assembly and mapping of the nonsatellite component of Drosophila heterochromatin. The strategy we describe is also applicable to generating substantially more information about heterochromatin in other species, including humans.     

Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs

The factors responsible for the clinical progress of visceral leishmaniasis (VL) in dogs have not been yet established. The starting hypothesis was the possibility of associating the changing level of a specific type of cytokines with the evolution of the infection towards infection-manifested disease or resistant behaviour. For this purpose the authors have established a connection between Leishmania load, cytokine mRNA accumulation, and the progression of the disease in naturally infected asymptomatic dogs. We made use of real-time (RT) PCR system to detect the expression of cytokine mRNA levels during all the phases of the infection. In particular, we measured the amount of parasites in samples such as blood, lymph nodes and skin, and the expression levels of IFN-gamma, IL-2, IL-4, IL-10, IL-12 and IL-18 cytokines in the blood. We employed different targeted real-time PCR assay on 40 naturally infected dogs, initially asymptomatic; 20 of these progressed to overt disease, and the 20 remaining dogs remained asymptomatic throughout the period of study (2 years). Two other groups included: 20 naturally infected dogs with clinical signs of VL, and 20 healthy dogs living in a non-endemic area. All these animals were employed as positive and negative controls, respectively. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels represents a reliable tool for predicting disease development, and thus for choosing the best treatment for the asymptomatic form of the disease.     

Biodegradation of the fungicide iprodione by Zygosaccharomyces rouxii strain DBVPG 6399

Iprodione is a contact fungicide used to control several pathogens such as Botrytis cinerea, Monilia, and Sclerotinia. This paper reports the ability of an iprodione-resistant strain of Zygosaccharomyces rouxii to degrade iprodione at a concentration of 1 mg L(-1). The yeast Z. rouxii was chosen also for its ability to grow at high osmolarity. Also of note is that in bioremediation situations and in the food industry such resistance could be important. The kinetic and metabolic behaviors of the fungicide in the media are described. The results show a new transformation pathway of iprodione by the yeast leading to the formation of N-(3,5-dichlorophenyl)-2,4-dioxoimidazoline, 3-isopropylhydantoin, and 3,5-dichloroaniline. These compounds were identified by 1H NMR, 13C NMR, and GC-MS analyses. This study provides a basis to employ yeast strains in biodegradation studies in relation to their ability in the disappearance and degradation of xenobiotics into simpler molecules.     

Study of efficacy of miltefosine and allopurinol in dogs with leishmaniosis

Visceral leishmaniosis is a life-threatening disease of medical, social and economic importance in endemic areas. It is an opportunistic infection in immunocompromised patients, including human immunodeficiency virus-positive subjects. Dogs are the main reservoir of Leishmania infantum. The aim of this study was to evaluate the efficacy of miltefosine and allopurinol for the control of human leishmaniosis using the dog as a model. The study included 28 sick dogs treated with miltefosine (2 mg/kg/day PO) administered concurrently with allopurinol (10 mg/kg/day, PO) for 30 days, and then with allopurinol alone, at the same dosage, for 1 year. Eight dogs (four of which relapsed) received a second cycle of miltefosine within 6 months of the first cycle. Efficacy was measured by real-time polymerase chain reaction assay on whole blood samples and lymph node aspirates, collected at baseline and every 3 months for 12 months. Of the total number of animals (28), two showed renal insufficiency and died after the start of therapy with miltefosine. Two other dogs presented some side effects to treatment, such as nausea, vomiting and reduction in white and red blood cell counts, and these animals were excluded from the follow-up. The results showed that the first cycle of therapy with miltefosine and allopurinol induced a drastic and progressive reduction of L. infantum load in lymph node aspirates but the second cycle did not eliminate the parasite.     

Characterization of a new potential functional ingredient: coffee silverskin

Dietary fiber (DF) is one of the main dietary factors contributing to consumers' well-being. In this work the possibility of using the roasted coffee silverskin (CS), a byproduct of roasted coffee beans, as a DF-rich ingredient has been evaluated. The results of our investigation showed that this material has 60% total DF, with a relevant component (14%) of soluble DF. Although a small amount of free phenol compounds is present in CS, it has a marked antioxidative activity, which can be attributed to the huge amount of Maillard reaction products, the melanoidins. Static batch culture fermentation experiments showed that CS induces preferential growth of bifidobacteria rather than clostridia and Bacteroides spp. CS can be proposed as a new potential functional ingredient in consideration of the high content of soluble DF, the marked antioxidant activity, and the potential prebiotic activity.     

Flavonoid and carbohydrate contents in Tropea red onions: effects of homelike peeling and storage

The content of anthocyanins, flavonols, and carbohydrates of Tropea red onions (Allium cepa L.) was determined by HPLC and HPLC-MS. Cyanidin derivatives constitute >50% of total anthocyanins, but delphinidin and petunidin derivatives, which have not been reported in red onions thus far, were also detected. The flavonoid distribution in the different layers of the bulbs indicates that, after homelike peeling, the edible portion contains 79% of the total content of quercetin 4'-glucoside but only 27% of the anthocyanins. Storage of onions for 6 weeks in different conditions, all of them mimicking home storage habits, resulted in a decrease to 64-73% of total anthocyanins. The same trend was verified for the total antioxidant activity, which was reduced to 29-36%. A decrease in glucose and fructose content correlated with anthocyanin degradation was also observed. Storage at low temperature seems to better preserve the onion anthocyanins.     

Metal ions restore the proteolytic resistance of denatured conglutin gamma, a lupin seed glycoprotein, by promoting its refolding

The susceptibility to trypsin of conglutin gamma, a lupin seed glycoprotein affected by this enzyme only when in a non-native conformation, was used to study the effect of Zn(2+) and other metal ions on the structural dynamics of the protein. When acid-treated trypsin-susceptible conglutin gamma was incubated at neutral pH in the presence of Zn(2+), it became resistant to tryptic attack, contrary to the protein treated in the absence of Zn(2+). The time course of this refolding event has been quantitatively evaluated by SDS-PAGE. Amino acid sequencing of the major polypeptide fragments, produced by trypsin before completion of the refolding process, indicated that only a few cleavable bonds were accessible to the enzyme. This suggested that the presence of metal ions affected the pathway of degradation of the protein, by inducing its folding. Among the other metal ions tested, Ni(2+) also promoted the adoption of a trypsin-resistant conformation of conglutin gamma, whereas Mn(2+) and Ca(2+) had only much lower effects. The relevance of these findings for a deeper understanding of the in vivo degradation of plant food proteins and how it is affected by metal ions are discussed.