Effects of NaCl and iso-osmotic PEG stress on growth,osmolytes accumulation and antioxidant defense in cultured sugarcane cells

In order to discriminate between the ionic and osmotic components of salt stress, sugarcane (Saccharum officinarum L. cv. Co 86032) calli were cultured on media containing NaCl or polyethylene glycol (PEG) 8000 that exerted the same osmotic pressure (−0.7 MPa). PEG stress exposure for 15 days led to significant growth reduction and loss in water content than salt stressed and control tissues. Osmotic adjustment (OA) was observed in callus tissues grown on salt, but was not evident in callus grown on PEG. Oxidative damage to membranes, estimated in terms of accumulation of thiobarbituric acid reactive substances-TBARS and electrolytic leakage was significantly higher in both the stressed calli than the control however, the extent of damage was more in the PEG stressed calli. The stressed callus tissues showed inhibition of ascorbate peroxidase activity, while catalase activity was increased. These results indicate sensitivity of cells to PEG-mediated stress than salt stress and differences in their OA to these two stress conditions. The sensitivity to the osmotic stress indicate that expression of the stress tolerance response requires the coordinated action of different tissues in a plant and hence was not expressed at the cellular level.     

Gene expression changes associated with cytotoxicity identified using cDNA arrays

In order to investigate gene expression changes associated with cytotoxicity, we used cDNA arrays to monitor the expression of over 5,000 genes in response to toxic stress in the HepG2 liver cell line. Cells were treated with cytotoxic doses of acetaminophen, caffeine or thioacetamide for nine time points ranging from 1 to 24 h. Samples of mRNA from each time point were used to prepare radiolabeled cDNA, which was hybridized to nylon-membrane-based cDNA arrays. High-stringency washes were applied to reduce cross-hybridization. Analysis of spot intensities revealed that each compound led to approximately 150-250 gene expression changes that were sustained over at least three adjacent time points. The affected genes could be classified into clusters based on their temporal patterns of differential expression. A common set of 44 genes showed similar expression changes in response to all three compounds. Of these changes, 90% could be confirmed by quantitative RT-PCR analysis. The results indicate that detailed array-based time-course studies, coupled with a sensitive and highly specific confirmation assay, provide a powerful means of identifying cytotoxicity-associated gene expression changes. Electronic Publication     

A study of GDP binding to purified thermogenin protein from brown adipose tissue.

1. The binding of GDP to purified thermogenin protein was studied by using fluorescence spectroscopy and equilibrium dialysis. 2. GDP binding to thermogenin diminished fluorescence emission in a concentration-dependent manner that exhibited saturation. 3. Kd values for binding of nucleoside di- and tri-phosphates were lower than those for nucleoside monophosphates. 4. The GDP-induced fluorescence quenching was decreased by increasing pH, but the apparent Kd was unaltered by pH changes. 5. Equilibrium dialysis showed a Kd change from 3 to 6 microM when the pH was increased from 6.6 to 8.5. 6. The apparent pK of the fluorescence changes induced by pH (8.3) was identical with the apparent pK of the GDP-binding response. 7. The data are consistent with the existence of protonated and non-protonated forms of thermogenin protein that both bind GDP.     

Plasma volume, osmolarity, total protein and electrolytes during treadmill running and cycle ergometer exercise

While haemoconcentration due to loss of plasma volume is well established during cycling, the existence of similar changes during running remains contentious. This study compared the changes in plasma volume and associated blood indices during 60 min of running and cycling at the same relative intensity (approximately 65% VO2max), with all changes referenced to blood indices obtained after 30 min seated at rest on a cycle ergometer. Plasma osmolarity increased similarly with both forms of exercise but was less than predicted for water loss alone, such that there was a net loss of sodium during exercise and of potassium postexercise, with essentially no loss of protein. Plasma volume decreased similarly (approximately 6.5%) in both exercise trials, but while that with cycling was initiated by exercise itself and was essentially maximal within 5 min, the reduction in plasma volume in the running trial was induced by adopting the upright posture and was complete before exercise began. These data would indicate that different mechanisms are responsible for the changes in plasma volume induced by running and cycling, while the similarity of change would suggest that there is a lower limit to any reduction in plasma volume, regardless of mechanism. Furthermore, the observation that the changes in plasma volume were complete before or early in exercise, would imply that oral water ingestion during prolonged exercise, which is essential for thermoregulation, may be more concerned with homeostasis of extravascular water rather than plasma volume.     

Some factors that influence the plasma lipoprotein 1H NMR spectra of normal and cancer patients: an oncolipid test?

Selected factors have been evaluated in order to determine their influences on the plasma lipoprotein proton NMR spectra of normal and cancer patients. The variables were donor''s diet (fasting/non-fasting), temperature and time of sample storage, processing procedure, centrifugation speed, and water pre-saturation time. Plasma samples from fasting individuals that were placed immediately on ice, spun at 1,000 and 3,000 g for 15 minutes, and the proton NMR spectrum acquired with the Carr-Purcell Meiboom-Gill (CPMG) pulse sequence, using a two-second water pre-saturation time, consistently gave reproducible results. Resonances attributed to lactate were minimized under these processing conditions. Centrifugation speed and pre-saturation time did not affect the average line width; however, donor fasting state, processing temperature, and storage time did alter the line width. Most important, blood chemistry analysis revealed an inverse correlation between triglyceride levels and average methyl and methylene line widths. Thus, these factors alone caution against the indiscriminate use of proton NMR spectra to differentiate plasma from normal and cancer patients.     

The isolation and immunolocalization of iron-binding compounds produced by Gloeophyllum trabeum

Summary Low molecular weight iron-binding compounds are produced by the brown-rot fungus Gloeophyllum trabeum. These chelators may function in scavenging transition metals for fungal metabolism and extracellular enzyme production. Because of the low molecular mass of the chelate-metal complex (below 1000 Da), and the oxidizing potential of the bound transition metals, certain chelating compounds could also play a role in the early stages of cellulose depolymerization by brown-rot fungi. High-affinity iron-binding compounds were isolated and partially purified from both liquid cultures of the brown-rot Gloeophyllum trabeum and from infected wood. Chelating compounds purified by thin-layer chromatography were used to prepare specific antibodies. These antibodies were shown to detect the chelator in infected wood and liquid fungal cultures by enzyme-linked immunosorbent assay and could be used in immunotransmission electron microscopy to visualize the high-affinity iron-binding compounds in situ. Elucidating the physiological roles of fungal chelate-metal complexes and determining their function in lignocellulose depolymerization will help us to better understand the mechanism of wood biodegradation.Publication no. 1549 Maine Agricultural Experiment Station Offprint requests to: J. Jellison     

Fine needle aspiration biopsy of metastatic melanoma. A morphologic analysis of 174 cases

The prognostic and therapeutic decisions in cases of metastatic melanoma depend upon the morphologic documentation of metastatic disease, which may rapidly and accurately be done by fine needle aspiration (FNA) biopsy of clinically suspicious lesions. The tumor cells derived from malignant melanomas demonstrate a wide range of appearances, however, and other neoplasms may be mimicked. Furthermore, additional neoplasms of other types are more frequent in melanoma patients: the possibility of a new primary tumor must be considered if the morphology of the tumor cells is uncharacteristic. Therefore, a study was undertaken to analyze the morphologic changes seen in FNA biopsy specimens from metastatic malignant melanoma and to determine which features could be the most useful in establishing a definitive diagnosis. A total of 174 consecutive cases, comprising 151 malignant aspirates and 23 inconclusive aspirates, were reviewed. The most significant features for identification of melanoma over other tumor types were the cell shape and nuclear position, the presence of numerous isolated neoplastic cells and occasional binucleated or multinucleated cells. Intracellular melanin in neoplastic cells was diagnostic when present, but it was absent in 60% of the cases. Macronucleoli and/or intranuclear cytoplasmic invaginations were characteristic but variable features. Morphology was also found to vary by site and cell type. Lung aspirates were less cellular and more likely to contain melanin. Aspirates of subcutaneous nodules were more often composed of spindle-shaped cells or of other variant cell types. Lymph node aspirates more often yielded epithelioid cells with macronucleoli and/or intranuclear invaginations. Spindle-cell melanomas usually demonstrated inconspicuous nuclei and rarely showed enlarged nucleoli. Epithelioid-cell tumors contained multinucleated cells and areas of cell wrapping more frequently than did spindle-cell tumors. The findings in this study emphasize that a full awareness of the spectrum of morphologic presentations of metastatic melanoma as well as of the clinical history are needed for greater precision in its diagnosis and for avoidance of the pitfall of misdiagnosing nonmelanomas with similar appearances.     

Purification and properties of arylsulfatase B from high- and low-activity mouse strains

Arylsulfatase B (arylsulfate sulfohydrolase; EC 3.1.6.1) activities in C57BL/6J, SWR/J, and A/J mouse liver approximate a 5:3:1 ratio. Each enzyme was purified to apparent homogeneity, and the properties of the three purified enzymes were compared. The purified enzyme behaved as a monomer with an apparent molecular weight of 50,000. The purified enzyme catalyzed the hydrolysis of p-nitrocatechol sulfate (pNCS), 4-methylumbelliferyl sulfate (4MUS), and chondroitin-4-sulfate (C4S) heptasaccharide. Purified SWR/J arylsulfatase B possessed a higher relative electrophoretic mobility at pH 4.0 than the A/J and C57BL/6J isozymes, and the SWR/J enzyme was more thermostable than either the C57BL/6J or the A/J enzyme. No differences were observed among the three enzymes with respect to their Michaelis constants for 4MUS and pNCS, isoelectric points, responses to inhibitors, pH optima, or electrophoretic mobilities at pH 8.3. The relative in vivo rates of synthesis of C57BL/6J, A/J, and SWR/J arylsulfatase B were comparable.