Lipoxygenase distribution in coffee (Coffea arabica L.) berries

In this paper lipoxygenase (LOX) presence was investigated in coffee berries to determine its involvement in lipid degradative metabolism of plants grown in organic and conventional cultivations. An immunochemical analysis has evidenced a ca. 80 kDa protein, cross-reacting with an anti-LOX antibody, only in the pulp fraction of berries obtained from plants of both cultivations. LOX activity in this fraction could be monitored either as conjugated diene formation or reaction products (determined by HPLC) and was mainly associated with a heavy membrane fraction (HMF, enriched in tonoplast, endoplasmic reticulum, plasma membrane, and mitochondria) and a light membrane fraction (LMF, enriched in plasma membrane and endoplasmic reticulum, with low levels of tonoplast and mitochondria). The LOX activity of LMF from berries of both cultivations showed an optimum at pH 8.0. The HMF exhibited a different activity peak in samples from conventional (pH 8.0) and organic (pH 5.5) cultures, suggesting the presence of different isoenzymes. These findings were also confirmed by variation of the ratio of 9- and 13-hydroperoxides in organic (1:1) and conventional cultivations (1:10), indicating that the organic one was subjected to an oxidative stress in the coffee pulp fraction leading to the expression of an acidic LOX. Such de novo synthesized LOX activity could be responsible for the production of secondary metabolites, which may interfere with the organoleptic profile of coffee.     

Use of random amplified polymorphic DNA (RAPD) to identify races 1, 2, 4 and 8 of Fusarium oxysporum f. sp. dianthi in Italy

The RAPD fingerprinting procedure was used in combination with pathogenicity assays on differential cultivars to characterize a representative collection of 72 Fusarium spp. isolates of different geographic origin collected from diseased carnation. In F. oxysporum f. sp. dianthi, isolates were grouped according to the physiologic race: group 1 included isolates of race 4; group 2 was formed by isolates of race 2 and single representatives of races 5 and 6; group 3 included isolates of races 1 and 8. No correlation was found between RAPD data and geographic origin of the isolates tested: representatives of race 2 isolated in Italy, Israel and Japan had the same amplification profile. Three isolates which showed a low level of pathogenicity on all carnation cultivars tested shared an identical amplification pattern and are probably saprophytic F. oxysporum. Finally, two F. redolens isolates from Japan and seven non-pathogenic isolates of F. proliferatum collected from diseased carnation in Italy, Israel and The Netherlands were clearly distinguishable according to their RAPD fingerprint. The results are discussed in relation to previous studies on the genetic diversity of F. oxysporum f. sp. dianthi and to the development of forma specialis- and pathotype-specific diagnostic tools.     

Paraneoplastic leukocytosis in a dog with a renal carcinoma

A 7-year-old male German Shepherd dog in poor body condition had a 3-month history of intermittent hematuria. Nonregenerative anemia, mild leukocytosis, marked hypoalbuminemia, and hematuria were observed. Subsequently, marked neutrophilia and moderate monocytosis were noted; anemia, hypoalbuminemia, and hematuria persisted; and the dog developed disseminated intravascular coagulation. Ultrasonographic examination of the abdomen revealed the presence of an enlarged and irregularly shaped right kidney with a large area of cavitation, and a nephrectomy was performed 30 days after initial examination. Cytologic examination of fine-needle aspirates and imprints of the right kidney revealed a neoplastic cell population suggestive of renal carcinoma. The histopathologic diagnosis was chromophobic cystic-papillary renal carcinoma. The tumor cells expressed granulocytic/macrophage-colony-stimulating factor (GM-CSF), detected by immunohistochemical staining, and elaboration of GM-CSF by the tumor may have mediated the leukocytosis in this dog. Following excision of the tumor, neutrophil and monocyte counts were only mildly increased. The dog died 135 days after initial presentation, and a necropsy was not permitted. Paraneoplastic neutrophilic leukocytosis is an uncommon finding and may be caused by elaboration of CSF by neoplastic cells.     

Chemical characterization and antioxidant properties of coffee melanoidins

Melanoidins, the brown polymers formed through Maillard reaction during coffee roasting, constitute up to 25% of the coffee beverages' dry matter. In this study chemical characterization of melanoidins obtained from light-, medium-, and dark-roasted coffee beans, manufactured from the same starting material, was performed. Melanoidins were separated by gel filtration chromatography and studied by MALDI-TOF mass spectrometry. Results showed that the amount of melanoidins present in the brews increased as the intensity of the thermal treatment increased, while their molecular weight decreased. The antioxidant activity of melanoidins isolated from the different brews was studied by using different methodologies. Melanoidins antiradical activity determined by ABTS(*)(+) and DMPD(*)(+) assays decreased as the intensity of roasting increased, but the ability to prevent linoleic acid peroxidation was higher in the dark-roasted samples. Data suggest that melanoidins must be carefully considered when the relevance of coffee intake in human health is studied.     

Use of electrochemical biosensor and gas chromatography for determination of dichlorvos in wheat

Two methods for the determination of dichlorvos in durum wheat by electrochemical assay and gas chromatography, respectively, have been developed. Dichlorvos, an organophosphorus anticholinesterase pesticide, was extracted from wheat with hexane, and the filtered extract was directly analyzed by gas chromatography with nitrogen-phosphorus flame detection (NPD). Recoveries of dichlorvos from milled wheat spiked at 0.25-1.5 microg/g ranged from 96.5 to 100.9%, and the limit of detection was 0.02 microg/g. The electrochemical assay was based on the detection of choline, the acetylcholinesterase product, via a choline oxidase biosensor. An aliquot of the filtered hexane extract was partitioned with phosphate buffer solution, and the organic layer was evaporated prior to electrochemical analysis. A limit of detection of 0.05 microg/g of dichlorvos was obtained with mean recoveries of 97-103% at spiking levels of 0.25-1.5 microg/g. A good correlation (r = 0.9919) was found between the results obtained with the electrochemical and those obtained with the gas chromatographic methods. The electrochemical method was peer-validated in two laboratories that analyzed 10 blind samples (5 duplicates), including a blank and 4 spiked samples with dichlorvos at levels of 0.25, 0.60, 1.00, and 1.50 microg/g. Within-laboratory repeatability (RSDr) and between-laboratory reproducibility (RSDR) ranged from 5.5 to 7.8% and from 9.9 to 17.6%, respectively.     

A New Bioassay Platform Design for the Discovery of Small Molecules with Anticancer Immunotherapeutic Activity

Immunotherapy takes advantage of the immune system to prevent, control, and eliminate neoplastic cells. The research in the field has already led to major breakthroughs to treat cancer. In this work, we describe a platform that integrates in vitro bioassays to test the immune response and direct antitumor effects for the preclinical discovery of anticancer candidates. The platform relies on the use of dendritic cells that are professional antigen-presenting cells (APC) able to activate T cells and trigger a primary adaptive immune response. The experimental procedure is based on two phenotypic assays for the selection of chemical leads by both a panel of nine tumor cell lines and growth factor-dependent immature mouse dendritic cells (D1). The positive hits are then validated by a secondary test on human monocyte-derived dendritic cells (MoDCs). The aim of this approach is the selection of potential immunotherapeutic small molecules from natural extracts or chemical libraries.     

Gastric inflammatory pseudotumour secondary to <Emphasis Type="Italic">Actinomyces hordeovulneris</Emphasis> infection in a cat

Background

The authors report the first case of feline gastric actinomycosis associated with infection by Actinomyces hordeovulneris.

Case presentation

A 4-year-old, neutered male, semi-feral European cat, with a 1 year history of chronic vomiting, was referred to the clinic. Abdominal ultrasound examination identified a hypoechoic focal transmural thickening with loss of normal wall layering and hyperechoic speckles at the gastric body. Initial gastroscopic examination showed a tumour-like gastric mass with an ulcerated depression at the level of the greater curvature. Histologic examination of endoscopic biopsy specimens was consistent with a severe lymphoplasmacytic gastritis. After 2 months, due to persistence of abdominal discomfort, surgical exploration and intraoperative sampling of gross abnormalities was recommended. Full thickness gastric wall biopsies, and fine needle aspiration of the gastric thickening and gastric lymph node, were performed. Histopathological examination identified a transmural pyogranulomatous gastritis. Aspirate samples of the gastric wall cultured positive, with colony morphology, biochemical testing and PCR of the 16 s rRNA gene compatible with Actinomyces hordeovulneris. After 4 months of treatment with cefovecin (8 mg/kg subcutaneously every 14 days), the vomiting completely resolved, as well as the ultrasonographic gastric alteration.

Conclusion

This case report of feline gastric actinomycosis, caused by Actinomyces hordeovulneris, suggests that gastric bacterial infection should be considered in cases of focal gastric wall thickening associated with chronic vomiting in the cat, which may otherwise closely resemble neoplastic disease. Once a diagnosis of actinomycosis was obtained, a correct treatment with antibiotic therapy can resolve it.

     

Orexin and orexin receptor like peptides in the gastroenteric tract of Gallus domesticus: An immunohistochemical survey on presence and distribution

This study reports the immunohistochemical localization and distribution of orexin A and B-like and their receptors-like peptides in the gastroenteric tract of chicken. The immunoreactivity is distributed in endocrine cells, nerve fibers and neurons, both in the stomach and intestine, and shows a discrete conformity with the data till now reported for Mammals. Our study suggests a possible participation of orexin-like peptides in the modulation of chicken gastroenteric activities and the preservation of their main distribution compared to Mammals. Western blot analysis has confirmed the presence of prepro-orexin and both receptors in the examined tissues.     

Evidence for an expanded host range ofFusarium oxysporum f.sp.raphani

The pathogenicity of four isolates ofFusarium oxysporum obtained from infected cultivated rocket (Eruca vesicaria) and wild (sand) rocket (Diplotaxis tenuifolia) was tested on the following cruciferous hosts: stock, radish, wild and cultivated rockets, and various species in the cabbage tribe: cabbage (Brassica oleracea var.sabauda), cauliflower (Brassica oleracea var.botrytis), Brussels sprouts (Brassica oleracea var.gemmifera), broccoli (Brassica oleracea var.italica), turnip (Brassica rapa var.rapa). The results indicated that isolates ofF. oxysporum from cultivated and wild rocket belong to theforma specialis raphani. The isolates from rocket were pathogenic on cabbage, Brussels sprouts, broccoli, turnip, radish and stock; isolates ofF. oxysporum conglutinans from cabbage and radish, and the isolate ofF. oxysporum f.sp.raphani from rape obtained from the ATCC collection, were pathogenic on both cultivated and wild rocket.     

Spatial evaluation of soil salinity using the WET sensor in the irrigated area of the Segura river lowland

The electrical conductivity of the water within the soil pores (EC_p) measured with the WET sensor, appears to be a reliable estimate of soil salinity. A methodology combining the use of the WET sensor along with geostatistics was developed to delimit and evaluate soil salinity within an irrigated area under arid to semiarid Mediterranean climate in SE Spain. A systematic random sampling of 104 points was carried out. The association between EC_p and the saturation‐extract electrical conductivity (EC_se) was assessed by means of correlation analysis. The semivariograms for EC_p were obtained at three different soil depths. Interpolation techniques, such as ordinary kriging and cokriging, were applied to obtain EC_p levels in the unknown places. For each one of the soil depths, a model able to predict EC_se from EC_p was developed by means of ordinary least squares regression analysis. A good correlation (r = 0.818, p < 0.001) between EC_p and EC_se was found. Spherical spatial distribution was the best model to fit to experimental semivariograms of EC_p at 10, 30, and 50 cm soil depths. Nevertheless, cokriging using the EC_p of an adjacent soil depth as an auxiliary variable provided the best results, compared to ordinary kriging. An analytical propagation‐error methodology was found to be useful to ascertain the contribution of the spatial interpolation and ordinary least squares analysis to the uncertainty of the EC_se mapping. This methodology allowed us to identify 98% of the study area as affected by salinity problems within a rooting depth of 50 cm, with the threshold of EC_se value at 2 dS m^–1. However, considering the crops actually grown and 10% potential reduction yield, the soil‐salinity‐affected area decreased to 83%. The use of sensors to measure soil salinity in combination with geostatistics is a cost‐effective way to draw maps of soil salinity at regional scale. This methodology is applicable to other agricultural irrigated areas under risk of salinization.