HPLC analysis of plant DNA methylation: a study of critical methodological factors.

HPLC analysis of nucleosides is important for determining total DNA methylation in plants and can be used to help characterise epigenetic changes during stress, growth and development. This is of particular interest for in vitro plant cultures as they are highly susceptible to genetic change. HPLC methodologies have been optimised for mammalian and microbial DNA, but not for plants. This study examines critical methodological factors in the HPLC analysis of plant DNA methylation using in vitro cultures of Ribes ciliatum. HPLC revealed that complete removal of RNA from plant DNA extractions is difficult using RNase (A and T1) digestions and LiCl precipitation. This suggests that base analysis should be avoided when using these RNA removal techniques, as bases from residual RNA fragments will inflate peak areas for DNA-derived bases. Nucleoside or nucleotide analysis is therefore recommended as a more suitable option as RNA and DNA constituents can be readily separated. DNA digestion was also a critical factor as methylation was under-estimated following incomplete nuclease digestion and over-estimated following incomplete phosphatase digestion. The units of enzyme required for complete DNA digestion was optimised and found to be 20-200 times less for nuclease P1 and 15 times less for alkaline phosphatase as compared with previous protocols. Digestion performance was conveniently monitored using marker peaks that indicate incomplete digestion products. This study identifies critical components of HPLC analysis and offers a comprehensive guide for the stringent analysis of DNA methylation in plants.     

Heightened stress response in primary fibroblasts expressing mutant eIF2B genes from CACH/VWM leukodystrophy patients

Childhood ataxia with central nervous system hypomyelination (CACH), also called vanishing white matter (VWM) leukoencephalopathy, is a fatal genetic disease caused by mutations in eukaryotic initiation factor 2B (eIF2B) genes. The five subunits eIF2B factor is critical for translation initiation under normal conditions and regulates protein synthesis in response to cellular stresses. Primary fibroblasts from CACH/VWM patients and normal individuals were used to measure basal eIF2B activity as well as global protein synthesis and ATF4 induction in response to stress in the endoplasmic reticulum. We show that although the cells expressing mutant eIF2B genes respond normally to stress conditions by reduced global translation rates, they exhibit significantly greater increase in ATF4 induction compared to normal controls despite equal levels of stress and activity of the upstream eIF2α kinase. This heightened stress response observed in primary fibroblasts that suffer from minor loss of basal eIF2B activity may be employed as an initial screening tool for CACH/VWM leukodystrophy.     

ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth

Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF-1 in this context, the role of additional mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2alpha or by a dominant-negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities.     

The nematode Panagrellus redivivus is susceptible to killing by human pathogens at 37 degrees C

Caenorhabditis elegans has been used as a host for the study of bacteria that cause disease in mammals. However, a significant limitation of the model is that C. elegans is not viable at 37 degrees C. We report that the gonochoristic nematode Panagrellus redivivus survives at 37 degrees C and maintains its life cycle at temperatures up to and including 31.5 degrees C. The C. elegans pathogens Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, but not Yersinia pseudotuberculosis, reduced P. redivivus lifespan. Of four strains of Burkholderia multivorans tested, one reduced P. redivivus lifespan at both temperatures, one was avirulent at both temperatures and two strains reduced P. redivivus lifespan only at 37 degrees C. The mechanism by which one of these strains killed P. redivivus at 37 degrees C, but not at 25 degrees C, was investigated further. Killing required viable bacteria, did not involve bacterial invasion of tissues, is unlikely to be due to a diffusible, bacterial toxin and was not associated with increased numbers of live bacteria within the intestine of the worm. We believe B. multivorans may kill P. redivivus by a temperature-regulated mechanism similar to B. pseudomallei killing of C. elegans.     

Subcellular localization determines MAP kinase signal output

The Raf-MEK-ERK MAP kinase cascade transmits signals from activated receptors into the cell to regulate proliferation and differentiation. The cascade is controlled by the Ras GTPase, which recruits Raf from the cytosol to the plasma membrane for activation. In turn, MEK, ERK, and scaffold proteins translocate to the plasma membrane for activation. Here, we examine the input-output properties of the Raf-MEK-ERK MAP kinase module in mammalian cells activated in different cellular contexts. We show that the MAP kinase module operates as a molecular switch in vivo but that the input sensitivity of the module is determined by subcellular location. Signal output from the module is sensitive to low-level input only when it is activated at the plasma membrane. This is because the threshold for activation is low at the plasma membrane, whereas the threshold for activation is high in the cytosol. Thus, the circuit configuration of the module at the plasma membrane generates maximal outputs from low-level analog inputs, allowing cells to process and respond appropriately to physiological stimuli. These results reveal the engineering logic behind the recruitment of elements of the module from the cytosol to the membrane for activation.     

Adaptation to warming but not cooling at slow rates of stimulus change in thermal threshold measurements

The relationship between thermal detection threshold and rate of temperature change of the thermal stimulus when slow (<1 degrees C s(-1)) rates of change are employed was investigated. Using both the reaction time (RT) inclusive Method of Limits and RT exclusive Method of Levels healthy volunteers had warming (WDT) and cooling detection thresholds (CDT) measured at four different rates of temperature change (0.3, 0.5, 0.7 and 1.0 degrees C s(-1)) from the thenar and/or mental regions using a contact thermode. With the Method of Limits, CDT increased linearly with rate of temperature change suggesting increments were due to RT artefacts. This was further supported by threshold assessment with the Method of Levels which showed CDT were unaffected by the rate of change in the RT exclusive method (P > 0.1). In contrast, WDT did not increase linearly with rate of stimulus temperature change when the Method of Limits was used and threshold assessment with the Method of Levels showed WDT assessed using a 0.3 degrees C s(-1) ramp rate were significantly higher than those measured with a 1 degrees C s(-1) rate of change (P < 0.05). This study indicates that adaptation to a warming stimulus can occur at faster rates of stimulus change than previously anticipated and identifies differences in warming and cooling pathways in sensitivity to adaptation.     

Rapid B cell receptor-induced unfolded protein response in nonsecretory B cells correlates with pro- versus antiapoptotic cell fate

The adaptive unfolded protein response (UPR) is essential for the development of antibody-secreting plasma cells. B cells induced by lipopolysaccharide (LPS) to differentiate into plasma cells exhibit a nonclassical UPR reported to anticipate endoplasmic reticulum stress prior to immunoglobulin production. Here we demonstrate that activation of a physiologic UPR is not limited to cells undergoing secretory cell differentiation. We identify B cell receptor (BCR) signaling as an unexpected physiologic UPR trigger and demonstrate that in mature B cells, BCR stimulation induces a short lived UPR similar to the LPS-triggered nonclassical UPR. However, unlike LPS, BCR stimulation does not induce plasma cell differentiation. Furthermore, the BCR-induced UPR is not limited to cells in which BCR induces activation, since a UPR is also induced in transitional immature B cells that respond to BCR stimulation with a rapid apoptotic fate. This response involves sustained up-regulation of Chop mRNA indicative of a terminal UPR. Whereas sustained Chop expression correlates with the ultimate fate of the BCR-triggered B cell and not its developmental stage, Chop-/- B cells undergo apoptosis, indicating that CHOP is not required for this process. These studies establish a system whereby a terminal or adaptive UPR can be alternatively triggered by physiologic stimuli.     

Structural and immunological studies of a pectin and a pectic arabinogalactan from Vernonia kotschyana Sch. Bip. ex Walp. (Asteraceae)

Two polysaccharides, a pectin (Vk100A2b) and a pectic arabinogalactan (Vk100A2a) with mean Mw 2 x 10(4) and 1.15 x 10(6)Da, respectively, were isolated from the dried powdered roots of Vernonia kotschyana Sch. Bip. ex Walp. by hot water extraction followed by fractionation on DEAE-Sepharose fast flow and Sephacryl S-400 HR. The pectin showed low-complement fixation activity and no influence on proliferation of B or T cells, while the pectic arabinogalactan showed a potent, dose-dependent complement fixation activity and a T cell independent induction of B-cell proliferation. Both polysaccharides induced chemotaxis of human macrophages, T cells and NK cells. exo-alpha-L-arabinofuranosidase and exo-beta-D-galactosidase digestion followed by component sugar and methylation analysis indicated that Vk100A2a consisted of a highly branched rhamnogalacturonan core with approximately 50% of the rhamnose 1,2,4-substituted, side chains rich in terminal-, 1,5-linked and 1,3,5-branched arabinose and terminal-, 1,4-, 1,6-linked and 1,3,6-branched galactose. The enzyme resistant part of Vk100A2a still showed strong complement fixating activity, suggesting that this activity may at least in part be expressed by carbohydrate structures present in the enzyme resistant, inner portion of the polymer.