Protein Expression Analysis in Reversible Photobleached Cells of Scenedesmus vacuolatus after High Temperature Stress.

We have analyzed protein expression in the bleached small vegetative cells of synchronous Scenedesmus vacuolatus to investigate how unicellular algae lived through stress. These cells were subjected to heat treatment (46.5 °C for 1h in dark condition) and then cultured under continuous illumination for 24 h. Flow cytometry analysis of the chlorophyll autofluorescence intensity of S. vacuolatus cells indicated that heat-treated cells were completely bleached within 24 h of light cultivation. Transmission electron microscopy (TEM) images showed that bleached cells maintained thylakoid membrane structure, but with lower contrast. The bleached cells regained green color after 72 h, along with a recovery in contrast, which indicated a return of photosynthetic ability. Two-dimensional gel electrophoresis (2DE) showed that the protein expression patterns were very difference between control and bleached cells. ATP synthase subunits and glutamine synthetase were down-regulated among the many differences, while some of phototransduction, stress response proteins were up-regulated in bleached cells, elucidating bleached cells can undergo changes in their biochemical activity, and activate some stress response proteins to survive the heat stress and then revive. In addition, small heat shock proteins (HSPs), but not HSP40 and HSP70 family proteins, protected the bleaching cells.

Effects of combination treatment with dexamethasone and mannitol on neuronal damage and survival in experimental heat stroke.

There is evidence that increased plasma cytokines, elevated brain levels of monoamines and hydroxyl radical production may be implicated in pathogenesis during heat stroke in rats. Acute treatment with a combined therapeutic approach has been repeatedly advocated in cerebral ischemia experiments. The aim of this study was to investigate whether the combined agent (mannitol and dexamethasone) has beneficial efficacy to improve the survival time (ST) and heat stroke-induced damage in experimental heat stroke. Urethane-anesthetized rats underwent instrumentation for the measurement of colonic temperature, mean arterial pressure (MAP), striatal cerebral blood flow (CBF), heart rate, and neuronal damage score. The rats were exposed to an ambient temperature (43 degrees C) to induce heat stroke. Concentrations of the ischemic and damage markers, dopamine, serotonin, and hydroxyl radical production in corpus striatum, and the plasma levels of tumor necrosis factor-alpha (TNF-alpha) were observed during heat stroke. After the onset of heat stroke, the heat stroke rats display decreased MAP, decreased CBF, increased the plasma levels of TNF-alpha, increased cerebral striatal monoamines and hydroxyl radical production release, and severe cerebral ischemia and neuronal damage compared with those of normothermic control rats. However, immediate treatment with the combined agent confers significant protection against heat stroke-induced arterial hypotension, systemic inflammation, cerebral ischemia, cerebral monoamines and hydroxyl radical production overloads, and improves neuronal damage and the ST in heat stroke rats. Our data suggest that administration of this combined agent seems to have more effective to ameliorate the heat stroke-induced neuronal damage and prolong the ST.

Effects of S-adenosylhomocysteine and homocysteine on DNA damage and cell cytotoxicity in murine hepatic and microglia cell lines.

Limited research has been performed on S-adenosylhomocysteine (SAH) or homocysteine (Hcy)-evoked cell damage in hepatic and neuronal cells. In this study, we assessed effects of SAH or Hcy on cell cytotoxicity and DNA damage in hepatic and neuronal cells and attempted to find the underlying mechanism. Cell cytotoxicity and DNA damage were evaluated in murine hepatic cells (BNL CL.2 cell line) and microglia cells (BV-2 cell line) with SAH or Hcy treatment for 48 h. The influences of SAH or Hcy on lipid peroxidation and DNA methylation were also measured in both cell lines. SAH (5-20 microM) or Hcy (1-5 mM) dose dependently inhibited cell cytotoxicity and enhanced DNA damage in both types of cells. Furthermore, SAH treatment markedly increased intracellular SAH levels and DNA hypomethylation, whereas Hcy caused minimal effects on these two parameters at much higher concentrations. Hcy significantly induced lipid peroxidation, but not SAH. The present results show that SAH might cause cellular DNA damage in hepatic and microglia cells by DNA hypomethylation, resulting in irreversible DNA damage and increased cell cytotoxicity. In addition, higher Hcy could induce cellular DNA damage through increased lipid peroxidation and DNA hypomethylation. We suggest that SAH is a better marker of cell damage than Hcy in hepatic and microglia cells.

Cloning, expression, and characterization of thermostable region of amylopullulanase gene from Thermoanaerobacter ethanolicus 39E.

The bifunctional activities of alpha-amylase and pullulanase are found in the cloned recombinant amylopullulanase. It was encoded in a 2.9-kb DNA fragment that was amplified using polymerase chain reaction from the chromosomal DNA of Thermoanaerobacter ethanolicus 39E. An estimated 109-kDa recombinant protein was obtained from the cloned gene under the prokaryotic expression system. The optimum pH of the recombinant amylopullulanase was 6.0. The most stable pH for the alpha-amylase and pullulanase activity was 5.5 and 5.0, respectively. The optimum temperature for the alpha-amylase activity was 90 degrees C, while its most stable temperature was 80 degrees C. Regarding pullulanase activity, the optimum temperature and its most stable temperature were found to be 80 and 75 degrees C, respectively. Pullulan was found to be the best substrate for the enzyme. The enzyme was activated and stabilized by the presence of Ca2+, whereas EDTA, N-bromosuccinimide, and alpha-cyclodextrin inhibited its bifunctional activities. A malto-2-4-oligosaccharide was the major product obtained from the enzymatic reaction on soluble starch, amylose, amylopectin, and glycogen. A single maltotriose product was found in the pullulan hydrolysis reaction using this recombinant amylopullulanase. Kinetic analysis of the enzyme indicated that the Km values of alpha-amylase and pullulanase were 1.38 and 3.79 mg/mL, respectively, while the Vmax values were 39 and 98 micromol/(min x mg of protein), respectively.

Attenuation of circulatory shock and cerebral ischemia injury in heat stroke by combination treatment with dexamethasone and hydroxyethyl starch.

BACKGROUND: Increased systemic cytokines and elevated brain levels of monoamines, and hydroxyl radical productions are thought to aggravate the conditions of cerebral ischemia and neuronal damage during heat stroke. Dexamethasone (DXM) is a known immunosuppressive drug used in controlling inflammation, and hydroxyethyl starch (HES) is used as a volume-expanding drug in cerebral ischemia and/or cerebral injury. Acute treatment with a combined therapeutic approach has been repeatedly advocated in cerebral ischemia experiments. The aim of this study is to investigate whether the combined agent (HES and DXM) has beneficial efficacy to improve the survival time (ST) and heat stroke-induced cerebral ischemia and neuronal damage in experimental heat stroke. METHODS: Urethane-anesthetized rats underwent instrumentation for the measurement of colonic temperature, mean arterial pressure (MAP), local striatal cerebral blood flow (CBF), heart rate, and neuronal damage score. The rats were exposed to an ambient temperature (43 degrees centigrade) to induce heat stroke. Concentrations of the ischemic and damage markers, dopamine, serotonin, and hydroxyl radical productions in corpus striatum, and the serum levels of interleukin-1 beta, tumor necrosis factor-alpha and malondialdehyde (MDA) were observed during heat stroke. RESULTS: After heat stroke, the rats displayed circulatory shock (arterial hypotension), decreased CBF, increased the serum levels of cytokines and MDA, increased cerebral striatal monoamines and hydroxyl radical productions release, and severe cerebral ischemia and neuronal damage compared with those of normothermic control rats. However, immediate treatment with the combined agent at the onset of heat stroke confers significant protection against heat stroke-induced circulatory shock, systemic inflammation; cerebral ischemia, cerebral monoamines and hydroxyl radical production overload, and improves neuronal damage and the ST in rats. CONCLUSIONS: Our results suggest that the combination of a colloid substance with a volume-expanding effect and an anti-inflammatory agent may provide a better resuscitation solution for victims with heat stroke.

A possible origin of the middle phase of polyphasic chlorophyll fluorescence transient.

The rise of the chlorophyll fluorescence of a whole leaf as induced by high-intensity actinic light comprises three distinct phases, and is termed the O-J-I-P polyphasic rise. The initial rise (the O-J phase) was found to be the most sensitive to light intensity, being slower and smaller with decreasing irradiation. The leaf was also found to be transparent for chlorophyll fluorescence to a considerable extent, so that the fluorescence originating from deep inside the sample could still be detected. In contrast, the actinic light used to induce fluorescence was strongly absorbed by chlorophylls, so that a steep light gradient was created along the light path. The fluorescence transient of a leaf, thus, was always a mixture of the fluorescence from the surface of the sample as well as that from the inside of the sample, whose O-J phase is slower as it is induced by a weaker actinic light. We have provided evidence suggesting that, in an intact leaf, the middle phase of the measured polyphasic fluorescence transient (the J-I phase) might actually reflect the initial rise of the transient coming from the abaxial layer of the leaf. Moreover, if the polyphasic fluorescence transient is used as an analytical tool for accessing information on the photosynthetic activities of leaves, the factors of concentration and thickness of the sample must be taken into account. To obtain the 'true' fluorescence transient of a sample, both the chlorophyll concentration and thickness of the sample must be kept as low as possible.