Identification of Dynamic Changes in Proteins Associated with the Cellular Cytoskeleton after Exposure to Okadaic Acid

Exposure of cells to the diarrhetic shellfish poison, okadaic acid, leads to a dramatic reorganization of cytoskeletal architecture and loss of cell-cell contact. When cells are exposed to high concentrations of okadaic acid (100–500 nM), the morphological rearrangement is followed by apoptotic cell death. Okadaic acid inhibits the broad acting Ser/Thr protein phosphatases 1 and 2A, which results in hyperphosphorylation of a large number of proteins. Some of these hyperphosphorylated proteins are most likely key players in the reorganization of the cell morphology induced by okadaic acid. We wanted to identify these phosphoproteins and searched for them in the cellular lipid rafts, which have been found to contain proteins that regulate cytoskeletal dynamics and cell adhesion. By using stable isotope labeling by amino acids in cell culture cells treated with okadaic acid (400 nM) could be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton, microtubules and cell adhesion structures. A large number of these okadaic acid-regulated proteins have previously also been shown to be similarly regulated prior to cell proliferation and migration. Our results suggest that okadaic acid activates general cell signaling pathways that induce breakdown of the cortical actin cytoskeleton and cell detachment.     

Preparation and comparative release characteristics of three anthocyanin encapsulation systems

Bilberries (Vaccinium myrtillus L.) and their major polyphenolic constituents, anthocyanins, have preventive activities inter alia against colon cancer and inflammatory bowel diseases. However, anthocyanins are sensitive to environmental conditions; thus their bioavailability in the gastrointestinal tract is an important determinant of their in vivo activity. In the study reported here, the potential benefits of encapsulating an anthocyanin rich bilberry extract (BE) on anthocyanin stability were investigated. Nonencapsulated BE and three different BE loaded microcapsule systems were incubated in simulated gastric fluid (SGF) and fed state simulated intestinal fluid (FeSSIF). After exposure to these media, released anthocyanins were identified and quantified by HPLC with UV/Vis detection. Although a rapid release of anthocyanins was observed within the first 20 min, encapsulation of anthocyanins doubled the amount of available anthocyanins after 150 min of incubation. These results illustrate the ability of encapsulation to inhibit early degradation of anthocyanins in the intestinal system.     

The use of adjuvants to improve spray deposition and Botrytis cinerea control on Chardonnay grapevine leaves

Spray adjuvants have the potential to improve deposition by effecting uniform distribution of the active ingredient on plant surfaces. In order to study whether such a qualitative improvement of spray deposition would lead to improved disease control, a laboratory experiment was conducted on artificially inoculated grapevine (cv. Chardonnay) leaves. Prior to inoculation with Botrytis cinerea conidia in a spore settling tower, leaves were sprayed to pre-runoff with 1 mL of a mixture of fenhexamid, a fluorescent pigment, and one of 15 selected commercial adjuvants to manipulate the deposition quality of a specific quantity of spray. Following an incubation period of 24 h at high relative humidity, leaf discs were plated onto Petri dishes with paraquat-amended water agar and rated for development of B. cinerea from isolated leaf discs 11 d later. Spray deposition on leaves was assessed with a spray assessment protocol using fluorometry, photomicrography and digital image analyses. B. cinerea incidences on the upper and lower surfaces of water-sprayed leaves averaged 90.4% and 95.8%, respectively. Despite full spray cover of leaves, applications with fenhexamid alone did not completely prevent infection and resulted in 34.6% and 40.8% B. cinerea incidences on the upper and lower surfaces of leaves, respectively. Through the addition of certain adjuvants, B. cinerea incidences were significantly reduced (incidences of 2.9–17.1% and 10.0–30.8%, respectively), while some adjuvants did not differ from the fungicide only treatment, even though they might have improved spray deposition. In a histopathology study using epifluorescence microscopy, distinct differences were observed in conidium mortality (20.5% vs. 31.2%), germination (60.4% vs. 51.4%) and germ tube lengths (27.8 μm vs. 19.7 μm) between Hydrosilicote and Solitaire in combination with fenhexamid, even though both adjuvants effected similar quantitative and qualitative spray deposition. The study clearly demonstrated the potential of adjuvants to improve the bio-efficacy of a fungicide directly through improved deposition on grapevine leaf surfaces, although bio-efficacy might be influenced by adjuvant mode of action in some cases.     

Storage and stability of organic matter and fossil carbon in a Luvisol and Phaeozem with continuous maize cropping: A synthesis

Quantitative information about the amount and stability of organic carbon (OC) in different soil organic‐matter (OM) fractions and in specific organic compounds and compound‐classes is needed to improve our understanding of organic‐matter sequestration in soils. In the present paper, we summarize and integrate results performed on two different arable soils with continuous maize cropping (a) Stagnic Luvisol with maize cropping for 24 y, b) Luvic Phaeozem with maize cropping for 39 y) to identify (1) the storage of OC in different soil organic‐matter fractions, (2) the function of these fractions with respect to soil‐OC stabilization, (3) the importance and partitioning of fossil‐C deposits, and (4) the rates of soil‐OC stabilization as assessed by compound‐specific isotope analyses. The fractionation procedures included particle‐size fractionation, density fractionation, aggregate fractionation, acid hydrolysis, different oxidation procedures, isolation of extractable lipids and phospholipid fatty acids, pyrolysis, and the determination of black C. Stability of OC was determined by ¹³C and ¹⁴C analyses. The main inputs of OC were plant litter (both sites) and deposition of fossil C likely from coal combustion and lignite dust (only Phaeozem).     

Analysis of genetic diversity in a sweet potato (Ipomoea batatas L.) germplasm collection from Tanzania as revealed by AFLP

Sweet potato (Ipomoea batatas L.) is the fifth most important crop in the developing countries after rice, wheat, maize and cassava. The amplified fragment length polymorphism (AFLP) method was used to study the genetic diversity and relationships of sweet potato accessions in the germplasm collection of Sokoine University of Agriculture, Morogoro and Sugarcane Research Institute, Kibaha, Tanzania. AFLP analysis of 97 sweet potato accessions using ten primer combinations gave a total of 202 clear polymorphic bands. Each one of the 97 sweet potato accessions could be distinguished based on these primer combinations. Estimates of genetic similarities were obtained by the Dice coefficient, and a final dendrogram was constructed with the un-weight pair-group method using arithmetic average. AFLP-based genetic similarity varied from 0.388 to 0.941, with a mean of 0.709. Cluster analysis using genetic similarity divided the accessions into two main groups suggesting that there are genetic relationships among the accessions. Principal Coordinate analysis confirmed the pattern of the cluster analysis. Analysis of molecular variance revealed greater variation within regions (96.19%) than among regions (3.81%). The results from the AFLP analysis revealed a relatively low genetic diversity among the germplasm accessions and the genetic distances between regions were low. A maximally diverse subset of 13 accessions capturing 97% of the molecular markers diversity was identified. We were able to detect duplicates accessions in the germplasm collection using the highly polymorphic markers obtained by AFLP, which were found to be an efficient tool to characterize the genetic diversity and relationships of sweet potato accessions in the germplasm collection in Tanzania.     

Selection of cowpea genotypes based on grain mineral and total protein content

Grain legumes, including cowpea, are the cheapest sources of minerals and protein to enhance human nutrition. Cultivar development and deployment of cowpea with increased grain mineral content and protein composition rely on selection of genetically unique and complementary breeding lines. The objective of this study was to assess the grain minerals and protein composition of diverse cowpea collections of eastern, southern and western Africa to select promising parents to develop a breeding population. Twenty-two genetically diverse cowpea genotypes were field evaluated using a randomised complete block design with three replications in two locations in South Africa. The dried grain mineral contents were determined using an Atomic Absorption Spectrophotometre, while the protein content was determined by the combustion method. Analyses of variance showed significant (P?<?0.05) effects of genotypes, locations and their interactions. The test genotypes showed considerable variation for the following nine mineral contents: calcium (Ca), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), sodium (Na), phosphorus (P), and zinc (Zn) varying from 0.07 to 0.12?mg?kg^?1; 4.46 to 7.81?mg?kg^?1; 0.06 to 0.11?g; 11.82 to 14.45?g; 1.86 to 2.27?g; 9.24 to 13.30?mg?kg^?1; 0.26 to 0.80?g; 4.63 to 5.92?g and 0.03.00 to 0.05?g, respectively. Further, the total protein content varied from 23.16 to 28.13%. Significant correlations were detected among some mineral elements and total protein content, suggesting the possibility of simultaneous selection for these traits. The principal component analysis (PCA) identified four principal components (PCs) contributing to 70.93% of the total explained variation amongst genotypes. Overall the following genotypes with desirable grain mineral and protein attributes were selected: IT90K-59 (Ca), 98K-5301 (Ca and protein), ITOOK-1060 (Cu), ITOOK-1217 and IT845-2246 (Fe), Bensogla (K, Na, and P), TVU11424 and ITOOK-1217 (Mg), CH14 (Mn and Na), TVU12637 (Mn) and Glenda and Vuli (Zn). The selected cowpea genotypes are useful genetic resources for population and cultivar development for grain nutrients composition.     

Parasites of cultured and wild brown‐marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) in Lampung Bay,Indonesia

A total of 210 Epinephelus fuscoguttatus (brown‐marbled grouper) was examined for parasites. During three consecutive seasons (two rainy and one dry season from 2002 to 2004), 35 specimens each taken from floating net cages of the National Sea Farming Development Centre (Balai Budidaya Laut) and from wild catches in Lampung Bay, South Sumatra, Indonesia were studied. Twenty‐five (cultured grouper) and 30 (wild grouper) parasite species/taxa were identified, with an infracommunity ranging from one to nine (cultured) and three to 14 parasite species (wild), demonstrating a species‐rich parasite fauna even in the cultured fish. Protozoans (1 species), microsporeans (1), myxozoans (1), digeneans (8), monogeneans (5), cestodes (3), nematodes (8), acanthocephalans (2) and crustaceans (6) were found. The most abundant parasites were the monogeneans Pseudorhabdosynochus epinepheli and Pseudorhabdosynochus lantauensis for both, cultured and wild grouper during all seasons. For the cultured fish, the prevalence of monoxenous ectoparasites (e.g. P. epinepheli, P. lantauensis, Capsalidae gen. et sp. indet., Benedenia epinepheli) was in most cases higher than that of heteroxenous endoparasites. This contrasts the wild grouper, where heteroxenous parasites such as Allopodocotyle epinepheli and Raphidascaris sp. occurred at a similar prevalence compared with the fairly abundant Pseudorhabdosynochus spp. No seasonality of infestation was observed for both cultured and wild fish. The high levels of infestation of potentially pathogenic monogeneans throughout the year could result in significant parasite outbreaks at the locality studied.