The proliferation inhibitory proteins p27(Kip1) and retinoblastoma are involved in the control of equine lymphocyte proliferation

Observations in early equine pregnancy clearly reveal maternal immune recognition of and response to the presence of the conceptus. Nevertheless, both maternal cellular and humoral responses appear ineffective in destroying the developing placenta and fetus in early pregnancy. Our previous studies had shown that the pre-conditioned medium generated from the culture of equine invasive trophoblast inhibited mitogen-induced lymphocyte proliferation and the expression of cytokine messenger RNA in vitro. Those findings also suggested that lymphocytes might have been halted in the G0/G1 phase of the cell cycle. To characterize the cell cycle and the intracellular mechanisms involved in the inhibition of lymphocyte proliferation, equine peripheral blood lymphocytes were cultured in the presence or absence of pokeweed mitogen (PWM) in fresh medium, or in medium pre-conditioned through cell culture of invasive trophoblast cells or fetal fibroblasts. Two-color flow cytometric analysis for bromodeoxyuridine (BrdU) incorporation by stimulated lymphocytes, and concomitant DNA staining with 7-amino-actinomycin D (7-AAD), indicated that a greater proportion of lymphocytes were found in the G0/G1 phase of the cell cycle when cultured in the invasive trophoblast cell pre-conditioned medium compared to controls. Analysis using carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescence intensity demonstrated that lymphocytes cultured in the presence of invasive trophoblast cell pre-conditioned medium had fewer cells going through division, but that those fewer cells sustained similar numbers of cell divisions as in control cultures. Hypophosphorylated retinoblastoma (Rb) protein expression was increased and p27Kip1 expression was maintained at higher levels in lymphocytes cultured in invasive trophoblast pre-conditioned medium compared to fresh medium. In agreement with these data, flow cytometric measurement of the Ki-67 protein expression in lymphocytes cultured in invasive trophoblast pre-conditioned medium was lower in comparison to controls. These findings suggest that the equine lymphocyte proliferation is at least partially regulated by the expression of proliferation inhibitory proteins such as p27Kip1 and hypophosphorylated Rb. These proteins seem to be important regulators of cell cycle transition between G1 and S phase in equine lymphocytes.     

Rural income generation through improving crop-based pig production systems in Vietnam: Diagnostics,interventions, and dissemination

Sweetpotato-pig production is an important system that generates income, utilizes unmarketable crops, and provides manure for soil fertility maintenance. This system is widely practiced from Asia to Africa, with many local variations. Within this system, pigs are generally fed a low nutrient-dense diet, yielding low growth rates and low economic efficiency. Our project in Vietnam went through a process of situation analysis, participatory technology development (PTD), and scaling up over a seven-year period to improve sweetpotato-pig production and to disseminate developed technologies. The situation analysis included a series of pig production assessments in several provinces in northern and southern Vietnam, and pig supply-market chain identification was conducted in 13 provinces. The analysis of these studies informed the project of the following: (1) appropriate locations for our activities; (2) seasonal available feedstuff and farmers feeding practices; (3) market fluctuation and requirements; and (4) feeding and management improvement needs based on which the subsequent phase of PTD was designed. The PTD involved a limited number of farmers participating in sweetpotato varietal selection, sweetpotato root and vine silage processing, seasonal feeding combination, and pig feeding with balanced crop-feed diet and silage. Six years of multi-location and multi-season sweetpotato selection resulted in a few promising varieties that yielded up to 75% more dry matter and have since been formally released. The most significant results of silage processing and feeding trials include improved growth, higher feeding efficiency, increased year-round local feedstuff, and considerable labor reduction from eliminated cooking and vine cutting. Once these technologies were developed, a farmer-to-farmer training model was designed for scaling up the adoption and impact. Farmer trainers from seven communes in seven provinces received training in these technologies. In turn, they undertook the responsibility of training other farmers on sweetpotato selection, processing, and feeding. An impact study was also administered to monitor and evaluate (M&E) the dissemination process and to document the impact of the new technologies and farmer-to-farmer training model on pig growth and farmer income generation. The results showed that both participating and non-participating farmers have taken up the technologies, although the former demonstrates higher rates of adoption than the latter. The participants also generated more income and saved more labor from the adoption of the technologies. While the scaling up and M&E activities are on-going, the project has since broadened from a sweetpotato-pig system perspective to a pig-cropfeed system perspective based on farmers needs. It has included other crop feeds such as cassava and peanut stems in the research portfolio. New technologies based on on-going PTD will continuously be incorporated into the future training curriculum.Dai Peters is currently a senior scientist with the International Center for Tropical Agriculture (CIAT) based in Hanoi, Vietnam. This publication is based on research conducted while she was a research scientist with the International Potato Center (CIP). She holds a PhD from North Carolina State University. Her research interests include on-farm participatory research methodologies, agroenterprise development, and sweetpotato-pig farming systems in Vietnam and Papua, Indonesia. Her recent publications include a manual on crop-based pig feed systems, post-harvest fermentation of sweetpotato roots and vines, and agroenterprise development.Nguyen Thi Tinh is currently a coordinator of the sweetpotato-pig improvement project at the International Potato Center in Vietnam. Ms. Tinh holds a Masters degree in animal nutrition from Wageningen University, Netherlands. She participated in the project on pig feeding trials for five years.Mai Thach Hoanh is a sweetpotato breeder with the Root Crop Research Center of Vietnam Agricultural Science Institute (VASI). He holds a PhD in sweetpotato breeding from the same institute. He participated in the project for seven years on sweetpotato selection. Nguyen The Yen is a crop scientist in the Food Crop Research Institute of Vietnam. He holds a PhD in sweetpotato breeding from VASI. He participated in the project for five years on sweetpotato selection.Pham Ngoc Thach is a lecturer with the Hanoi Agricultural University. He holds a PhD in veterinarian science from the same university. He participated in the project for five years in controlling pig diseases during the feeding trials.Keith Fuglie is a regional coordinator and research scientist in the International Potato Center based at Bogor, Indonesia. He holds a PhD in agriculture economics from the University of Wisconsin. He designed the impact study for the project.     

Identification of an antioxidant,ethyl protocatechuate,in peanut seed testa

The antioxidant activity and identification of the antioxidant component of peanut seed testa were investigated. The antioxidant activity of peanut seed testa was studied in the linoleic acid model system by using the ferric thiocyanate method. Among the five organic solvent extracts, the ethanolic extracts of peanut seed testa (EEPST) produced higher yields and stronger antioxidant activity than other organic solvent extracts. EEPST was separated into 17 fractions on silica gel column chromatography. Fraction 17, which showed the largest yield and significant antioxidant activity, was separated by thin-layer chromatography. Four major antioxidative subfractions were present. Subfraction 17-2 was found to be effective in preventing oxidation of linoleic acid. This subfraction was further fractionated and isolated and characterized by UV, MS, IR, and (1)H NMR techniques. The active compound was identified as ethyl protocatechuate (3,4-dihydroxybenzoic acid ethyl ester).     

Changes in sorption/bioavailability of imidacloprid metabolites in soil with incubation time

Changes in sorption/bioavailability of two metabolites, imidacloprid-urea {1-[(6-chloro-3-pyridinyl)methyl]-2-imidazolidinone} and imidacloprid-guanidine {1-[(6-chloro-3-pyridinyl)methyl]-4,5-dihydro-1H-imidazol-2-amine} of the insecticide imidacloprid {1-[(6-chloro-3-pyridinyl)-methyl]-N-nitro-2-imidazolidinimine} with aging in different soils were determined. Soil moisture was adjusted to -33 kPa and ¹⁴C- and analytical-grade imidacloprid-urea and imidacloprid-guanidine were added to the soil at a rate of 1.0 mg kg^-1. Spiked soils were incubated at 25°C for 8 weeks. Replicate soil samples were periodically extracted successively with 0.01 N CaCl₂, acetonitrile, and 1 N HCl. Imidacloprid-urea sorption, as indicated by sorption coefficient values, was highest in the soil with highest organic C content, and increased by an average factor of 2.6 in three soils during the 8-week incubation period. Imidacloprid-guanidine sorption increased by a factor of 2.3 in the same soils. The increase in sorption was the result of a decrease in the metabolite extractable with CaCl₂ (solution phase); the amount of metabolite extractable with acetonitrile and HCl (sorbed phase) did not significantly change with incubation time. It appears the increase in sorption was because the rate of degradation in solution and on labile sites was faster than the rate of desorption from the soil particles. It may have also been due to metabolite diffusion to less accessible or stronger binding sites with time. Regardless of the mechanism, these results are further evidence that increases in sorption during pesticide aging should be taken into account during characterization of the sorption process for mathematical models of pesticide degradation and transport.     

Induction of apoptosis by the Anthocyanidins through regulation of Bcl-2 gene and activation of c-Jun N-terminal kinase cascade in hepatoma cells

Anthocyanidins that are reddish pigments widely distributed in fruit and vegetables have been reported to possess antioxidant and anticancer activities. To understand the molecular basis of the putative anticancer activity of anthocyanidins, we investigated the antiproliferation effects of anthocyanidins in human hepatoma cell lines. Delphinidin, cyanidin, and malvidin exhibited strong growth inhibitory effects against human hepatoma HepG(2), but were less effective against Hep3B. According to the appearance of the caspase-3 fragments and stimulated proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) in time-dependent studies, delphinidin induced apoptotic cell death characterized by internucleosomal DNA fragmentation and caused a rapid induction of caspase-3 activity. RT-PCR and Western blot data revealed that delphinidin stimulated an increase in the c-Jun and JNK phosphorylation expression at mRNA and protein levels, respectively. Moreover, delphinidin-induced apoptotic cell death was accompanied by up-regulation of Bax and down-regulation of Bcl-2 protein. Dephinidin-induced DNA fragmentation was blocked by N-acetyl-l-cysteine and catalase, suggesting that the death signaling was triggered by oxidative stress. Our experiments provide evidence that delphinidin is an effective apoptosis inducer in HepG(2) cells through regulation of Bcl-2 family moleculars and activation of c-Jun N-terminal kinase cascade. The results suggest that induction of apoptosis by anthocyanidins is a pivotal mechanism of their cancer chemopreventive functions.     

Determination of caffeine, theobromine, and theophylline in standard reference material 2384, baking chocolate, using reversed-phase liquid chromatography

A rapid and selective isocratic reversed-phase liquid chromatographic method has been developed at the National Institute of Standards and Technology to simultaneously measure caffeine, theobromine, and theophylline in a food-matrix standard reference material (SRM) 2384, Baking Chocolate. The method uses isocratic elution with a mobile phase composition (volume fractions) of 10% acetronitrile/90% water (pH adjusted to 2.5 using acetic acid) at a flow rate of 1.5 mL/min with ultraviolet absorbance detection (274 nm). Total elution time for these analytes is less than 15 min. Concentration levels of caffeine, theobromine, and theophylline were measured in single 1-g samples taken from each of eight bars of chocolate over an eight-day period. Samples were defatted with hexane, and beta-hydroxyethyltheophylline was added as the internal standard. The repeatability for the caffeine, theobromine, and theophylline measurements was 5.1, 2.3, and 1.9%, respectively. The limit of quantitation for all analytes was <100 ng/mL. The measurements from this method were used in the value-assignment of caffeine, theobromine, and theophylline in SRM 2384.