Evaluating UV-B effects and EDU protection in cucumber leaves using fluorescence images and fluorescence emission spectra

A newly developed laboratory fluorescence imaging system was used to obtain fluorescence images (FImage) of freshly excised cucumber (Cucumis sativus L.) leaves in spectral bands centered in the blue (F450), green (F550), red (F680), and far-red (F730) spectral regions that resulted from a broad-band (300-400 nm) excitation source centered at 360 nm. Means of relative fluorescence intensities (RFI) from these spectral fluorescence images were compared with spectral fluorescence emission data obtained from excitation wavelengths at 280 nm (280EX, 300-550 nm) and 380 nm (380EX, 400-800 nm) of dimethyl sulfoxide (DMSO) extracts from these leaves. All three fluorescence data types (FImage, 280EX, 380EX) were used to assess ultraviolet-B (UV-B, 280-320 nm) induced physiological changes and the possible use of N-[2-(2-oxo-1-imidazolidinyl) ethyl]-N′-phenylurea (EDU or ethylenediurea) as a chemical protectant against UV-B damage. Plants exhibited well known foliar growth and pigment responses to UV-B exposure (e.g., increased UV-B absorbing compounds and decreased leaf area, chlorophyll a content; and and lower chlorophyll a/b and chlorophyll/carotenoid pigment ratios). Since EDU alone had no effect on foliar variables, there was no evidence that EDU afforded protection against UV-B. Instead, EDU augmented some UV-B effects when provided in conjunction with UV-B irradiation (e.g., reductions in the chlorophyll/carotenoid ratio, total photosynthetic pigments, and chlorophyll b content).Relative fluorescence intensities (RFI) in the longer visible wavelengths (green, red, and far-red) were uncorrelated for comparisons between the FImage and 380EX data sets. However, blue and green RFI were significantly correlated (0.8r0.6; P ≤0.002) for comparisons between FImage and 280EX data sets. UV-B treatment caused an increase in blue RFI (e.g., F450) in both images and 280EX measurements. One explanation is that the UV-B excitation of both 280EX and FImage stimulates processes that produce excess blue fluorescence. The molecules that produce the excess blue fluorescence in both the 280EX and the Fimage data are different electron transfer agents that operate in parallel. For FImage, the UV excitation penetrates leaf surface layers to stimulate fluorescence from compounds in mesophyll and epidermal tissues (as occurs for the extracts of leaf discs), whereas emissions captured at longer, less energetic wavelengths, were primarily from the epidermal layer. UV-B irradiated leaves showed much greater heteorgeneity of RFI in both the green (F550_FImag) and the red (F680_FImag) bands than unirradiated leaves; this was true irrespective of EDU treatment.Although qualitative responses in individual bands differed between FImage and 380EX data, similar results were obtained in the detection of UV-B induced effects when the red/green and blue/far-red fluorescence ratios of these data were compared. The red/green ratio (either F680/F550_FImage or F675/F525_380EX) was lower for UV-B exposed plants in both images and 380EX data. UV-B exposure also significantly enhanced the blue/far-red ratio of images (F450/F740_FImage) and the comparable 380EX ratio (F450/F730_380EX) for the combined UV-B/EDU group. The far-red/red ratios were not useful in separating treatment effects in images or 380EX. Although comparable ratios were not available in 280EX data, the UV/blue ratio (F315/F420_280EX) was substantially reduced by UV-B exposure and was inversely related to total photosynthetic pigment content. These findings suggest that the red/green ratio (FImage, 380EX) and the UV/blue ratio (280EX) may be as useful as the blue/far-red ratio (380EX) reported previously in detection of UV-B stress. Furthermore, the results support the validity of the imaging technique as a non-destructive diagnostic tool for assessing UV-B stress damage in plants.     

Adenylate pool sizes and succinate dehydrogenase activity increase continuously throughout the cell cycle of Escherichia coli

Abstract In synchronous cultures of Escherichia coli , the pool sizes of ATP and ADP increase continuously, probably exponentially, such that the ATP/ADP ratio is invariant during the cell cycle. Resolution of cells from an exponentially growing culture into size, and thus age, classes reveals no significant variation in succinate dehydrogenase activity during the cell cycle. These data reinforce earlier findings that respiratory metabolism in the E. coli cell cycle is not manifestly periodic.     

Validation of a high throughput method for serum/plasma testosterone using liquid chromatography tandem mass spectrometry (LC-MS/MS)

Testosterone, the major androgenic hormone in humans, is commonly measured to aid in the diagnosis of clinical conditions related to its excess or deficiency. In addition, testosterone measurements are used to monitor testosterone replacement-, or antiandrogen therapy. Most commonly, automated direct immunoassays have been used to measure testosterone in human serum. Their advantage compared with other methodologies, lies in high- and rapid sample throughput with minimal human intervention. However, many automated testosterone immunoassays suffer from poor accuracy at the low concentration levels (<50ng/dL) seen in women and children, or in men undergoing anti-androgen therapy. Our objective was to develop a LC-MS/MS method which measures testosterone in human serum while fulfilling the following criteria: Rapid pre-analytical sample processing with minimal manual sample manipulation; Minimize sample volume requirements; Accurate, precise and unambiguous measurement; Functional sensitivity of 5-10ng/dL; Sample throughput of at least 30 samples per hour. Our validation criteria for precision, accuracy, and linearity was to have accuracy and linearity within mean limits of +/-10%; Intra and inter-assay precision of <15% throughout the reporting range. We also wanted to compare our results to a previously validated LC-MS/MS assay which utilized a manual liquid-liquid extraction and to an automated commercial immunoassay (Bayer ACS:180). We describe here a sensitive and rapid testosterone assay based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilizing on-line sample extraction and multiplexing.     

Apomixis in Eremopogon foveolatus (Gramineae)

The investigation deals with the reproductive behaviour in Eremopogon foveolatus (Del.) Stapf, a member of the tribe Andropogoneae. The magaspore mother cell or the products of its meiotic division degenerate in a majority of ovules and four–nucleate unreduced embryo sacs are formed from the surrounding cells of the nucellus. Eight–nucleate reduced embryo sacs are also formed in some ovules but the percentage of such ovules is very low. The unreduced egg develops parthenogenetically but fertilization of the polar nucleus appears to be essential for the formation of endosperm.     

Differential regulation of Na+/H+ exchange isoform activities by enteropathogenic E. coli in human intestinal epithelial cells

Enteropathogenic Escherichia coli (EPEC) is an important human intestinal foodborne pathogen associated with diarrhea, especially in infants and young children. Although EPEC produces characteristic attaching and effacing lesions and loss of microvilli, the pathophysiology of EPEC-associated diarrhea, particularly during early infection, remains elusive. The present studies were designed to examine the direct effects of EPEC infection on intestinal absorption via Na(+)/H(+) exchanger (NHE) isoforms. Caco-2 cells were infected with EPEC strain E2348/69 or nonpathogenic E. coli HB101 for a period of 60 to 120 min. Total NHE activity was significantly increased at 60 min, reaching approximately threefold increase after 90 min of EPEC infection. Similar findings were seen in HT-29 cells and T84 cells indicating that the response was not cell-line specific. Most surprising was the differential regulation of NHE2 and NHE3 by EPEC. Marked activation of NHE2 (300%) occurred, whereas significant inhibition ( approximately 50%) of NHE3 activity was induced. The activity of basolateral isoform NHE1 was also significantly increased in response to EPEC infection. Mutations that disrupted the type III secretion system (TTSS) ablated the effect of EPEC on the activity of both NHE2 and NHE3. These results suggest that EPEC, through a TTSS-dependent mechanism, exerts differential effects on NHE isoform activity in intestinal epithelial cells. Additionally, NHEs do not appear to play any role in EPEC-mediated inflammation, because the NHE inhibitors amiloride and 5-(N-ethyl-N-isopropyl)amiloride did not prevent EPEC-mediated IkappaBalpha degradation.     

Leucocytozoon (Apicomplexa: Leucocytozoidae) from West African birds, with descriptions of two species

Five species of Leucocytozoon were recovered from 35/828 birds of 95 species examined from 6 sites in West Africa between May 1995 and June 2001. Leucocytozoon pogoniuli n. sp. is described from the tinker barbets Pogoniulus subsulphureus and Pogoniulus atroflavus. Leucocytozoon trachyphoni n. sp. is described from the barbet Trachyphonus purpureus. No leucocytozoids have been reported previously in species of Pogoniulus. Leucocytozoon nectariniae was identified from the sunbird Nectarinia olivacea, and Leucocytozoon brimonti was recovered from 4 species of Pycnonotidae (bulbuls), all of which are new host records. We also report the first Leucocytozoon to be recovered from the phylogenetically isolated bird, Picathartes sp. (Picathartidae). This parasite is similar in appearance to Leucocytozoon sakharoffi, and probably represents a previously undescribed species. In view of the intraspecific variability and, frequently, relatively minor interspecific differences within Leucocytozoidae, we suggest that the development and application of molecular techniques would greatly advance understanding of speciation and relationships within this family.     

The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation

The severe acute respiratory syndrome coronavirus(SARS-CoV) nucleocapsid (N) protein is one of the four structural proteins of the virus and is predicted to be a 46-kDa phosphoprotein. Our in silico analysis predicted N to be heavily phosphorylated at multiple residues. Experimentally, we have shown in this report that the N protein of the SARS-CoV gets serine-phosphorylated by multiple kinases, in both the cytoplasm and the nucleus. The phosphoprotein is stable and localizes in the cytoplasm and coprecipitates with the membrane fraction. Also, using specific inhibitors of phosphorylation and an in vitro phosphorylation assay, we show that the nucleocapsid protein is a substrate of cyclin-dependent kinase (CDK), glycogen synthase kinase, mitogen-activated protein kinase, and casein kinase II. Further, we show that the phosphorylated protein is translocated to the cytoplasm by binding to 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein). 14-3-3 proteins are a family of highly conserved, ubiquitously expressed eukaryotic proteins that function primarily as adapters that modulate interactions between components of various cellular signaling and cell cycle regulatory pathways through phosphorylation-dependent protein-protein interactions. Coincidentally, the N protein was also found to downregulate the expression of the theta isoform of 14-3-3 (14-3-3theta), leading to the accumulation of phosphorylated N protein in the nucleus, in the absence of growth factors. Using short interfering RNA specific to 14-3-3theta we have inhibited its expression to show accumulation of phosphorylated N protein in the nucleus. Thus, the data presented here provide a possible mechanism for phosphorylation-dependent nucleocytoplasmic shuttling of the N protein. This 14-3-3-mediated transport of the phosphorylated N protein and its possible implications in interfering with the cellular machinery are discussed.     

A novel role of lactosylceramide in the regulation of tumor necrosis factor alpha-mediated proliferation of rat primary astrocytes. Implications for astrogliosis following neurotrauma

The present study describes the role of glycosphingolipids in neuroinflammatory disease and investigates tumor necrosis factor alpha (TNFalpha)-induced astrogliosis following spinal cord injury. Astrogliosis is the hallmark of neuroinflammation and is characterized by proliferation of astrocytes and increased glial fibrillary acidic protein (GFAP) gene expression. In primary astrocytes, TNFalpha stimulation increased the intracellular levels of lactosylceramide (LacCer) and induced GFAP expression and astrocyte proliferation. D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCl (PDMP), a glucosylceramide synthase and LacCer synthase (GalT-2) inhibitor, inhibited astrocyte proliferation and GFAP expression, which were reversed by exogenous supplementation of LacCer but not by other glycosphingolipids. TNFalpha caused a rapid increase in the activity of GalT-2 and synthesis of LacCer. Silencing of GalT-2 gene using antisense oligonucleotides also attenuated the proliferation of astrocytes and GFAP expression. The PDMP and antisense-mediated inhibition of proliferation and GFAP expression was well correlated with decreased Ras/ERK1/2 pathway activation. Furthermore, TNFalpha-mediated astrocyte proliferation and GFAP expression was also inhibited by LY294002, a phosphatidylinositol 3-kinase inhibitor, which was reversed by exogenous LacCer. LY294002 also inhibited TNFalpha-induced GalT-2 activation and LacCer synthesis, suggesting a phosphatidylinositol 3-kinase-mediated regulation of GalT-2. In vivo, PDMP treatment attenuated chronic ERK1/2 activation and spinal cord injury (SCI)-induced astrocyte proliferation with improved functional recovery post-SCI. Therefore, the in vivo studies support the conclusions drawn from cell culture studies and provide evidence for the role of LacCer in TNFalpha-induced astrogliosis in a rat model of SCI. To our knowledge, this is the first report demonstrating the role of LacCer in the regulation of TNFalpha-induced proliferation and reactivity of primary astrocytes.     

Phytoremediation of lead by a wild,non-edible Pb accumulator Coronopus didymus (L.) Brassicaceae

Coronopus didymus was examined in terms of its ability to remediate Pb-contaminated soils. Pot experiments were conducted for 4 and 6 weeks to compare the growth, biomass, photosynthetic efficiency, lead (Pb) uptake, and accumulation by C. didymus plants. The plants grew well having no visible toxic symptoms and 100% survivability, exposed to different Pb-spiked soils 100, 350, 1500, and 2500 mg kg^?1, supplied as lead nitrate. After 4 weeks, root and shoot concentrations reached 1652 and 502 mg Pb kg^?1 DW, while after 6 weeks they increased up to 3091 and 527 mg Pb kg^?1 DW, respectively, at highest Pb concentration. As compared to the 4 week experiments, the plant growth and biomass yield were higher after 6 weeks of Pb exposure. However, the chlorophyll content of leaves decreased but only a slight decline in photosynthetic efficiency was observed on exposure to Pb at both 4 and 6 weeks. The Pb accumulation was higher in roots than in the shoots. The bioconcentration factor of Pb was > 1 in all the plant samples, but the translocation factor was < 1. This suggested C. didymus as a good candidate for phytoremediation of Pb-contaminated soils and can be used for future remediation purposes.