Light-Induced Changes within Photosystem II Protects Microcoleus sp. in Biological Desert Sand Crusts against Excess Light

The filamentous cyanobacterium Microcoleus vaginatus, a major primary producer in desert biological sand crusts, is exposed to frequent hydration (by early morning dew) followed by desiccation during potentially damaging excess light conditions. Nevertheless, its photosynthetic machinery is hardly affected by high light, unlike “model” organisms whereby light-induced oxidative stress leads to photoinactivation of the oxygen-evolving photosystem II (PSII). Field experiments showed a dramatic decline in the fluorescence yield with rising light intensity in both drying and artificially maintained wet plots. Laboratory experiments showed that, contrary to “model” organisms, photosynthesis persists in Microcoleus sp. even at light intensities 2–3 times higher than required to saturate oxygen evolution. This is despite an extensive loss (85–90%) of variable fluorescence and thermoluminescence, representing radiative PSII charge recombination that promotes the generation of damaging singlet oxygen. Light induced loss of variable fluorescence is not inhibited by the electron transfer inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), nor the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), thus indicating that reduction of plastoquinone or O₂, or lumen acidification essential for non-photochemical quenching (NPQ) are not involved. The rate of Q_A ⁻ re-oxidation in the presence of DCMU is enhanced with time and intensity of illumination. The difference in temperatures required for maximal thermoluminescence emissions from S₂/Q_A ⁻ (Q band, 22°C) and S_2,3/Q_B ⁻ (B band, 25°C) charge recombinations is considerably smaller in Microcoleus as compared to “model” photosynthetic organisms, thus indicating a significant alteration of the S₂/Q_A ⁻ redox potential. We propose that enhancement of non-radiative charge recombination with rising light intensity may reduce harmful radiative recombination events thereby lowering ¹O₂ generation and oxidative photodamage under excess illumination. This effective photo-protective mechanism was apparently lost during the evolution from the ancestor cyanobacteria to the higher plant chloroplast.     

Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of the plasma membrane.

BACKGROUND: Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria-dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time-consuming and inaccurate, and the latter is still limited by sample size. METHODS: We developed a rapid and reliable technique to detect cytochrome c release during drug-induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL-60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells. RESULTS: The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4-8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c. CONCLUSIONS: This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose.     

Coincident but distinct messages of midbrain dopamine and striatal tonically active neurons

Midbrain dopamine and striatal tonically active neurons (TANs, presumed acetylcholine interneurons) signal behavioral significance of environmental events. Since striatal dopamine and acetylcholine affect plasticity of cortico-striatal transmission and are both crucial to learning, they may serve as teachers in the basal ganglia circuits. We recorded from both neuronal populations in monkeys performing a probabilistic instrumental conditioning task. Both neuronal types respond robustly to reward-related events. Although different events yielded responses with different latencies, the responses of the two populations coincided, indicating integration at the target level. Yet, while the dopamine neurons' response reflects mismatch between expectation and outcome in the positive domain, the TANs are invariant to reward predictability. Finally, TAN pairs are synchronized, compared to a minority of dopamine neuron pairs. We conclude that the striatal cholinergic and dopaminergic systems carry distinct messages by different means, which can be integrated differently to shape the basal ganglia responses to reward-related events.     

Benzoic acid, a weak organic acid food preservative, exerts specific effects on intracellular membrane trafficking pathways in Saccharomyces cerevisiae

Microbial spoilage of food causes losses of up to 40% of all food grown for human consumption worldwide. Yeast growth is a major factor in the spoilage of foods and beverages that are characterized by a high sugar content, low pH, and low water activity, and it is a significant economic problem. While growth of spoilage yeasts such as Zygosaccharomyces bailii and Saccharomyces cerevisiae can usually be retarded by weak organic acid preservatives, the inhibition often requires levels of preservative that are near or greater than the legal limits. We identified a novel synergistic effect of the chemical preservative benzoic acid and nitrogen starvation: while exposure of S. cerevisiae to either benzoic acid or nitrogen starvation is cytostatic under our conditions, the combination of the two treatments is cytocidal and can therefore be used beneficially in food preservation. In yeast, as in all eukaryotic organisms, survival under nitrogen starvation conditions requires a cellular response called macroautophagy. During macroautophagy, cytosolic material is sequestered by intracellular membranes. This material is then targeted for lysosomal degradation and recycled into molecular building blocks, such as amino acids and nucleotides. Macroautophagy is thought to allow cellular physiology to continue in the absence of external resources. Our analyses of the effects of benzoic acid on intracellular membrane trafficking revealed that there was specific inhibition of macroautophagy. The data suggest that the synergism between nitrogen starvation and benzoic acid is the result of inhibition of macroautophagy by benzoic acid and that a mechanistic understanding of this inhibition should be beneficial in the development of novel food preservation technologies.     

The COOH terminus of GATE-16, an intra-Golgi transport modulator,is cleaved by the human cysteine protease HsApg4A

Docking of a vesicle at the appropriate target membrane involves an interaction between integral membrane proteins located on the vesicle (v-SNAREs) and those located on the target membrane (t-SNAREs). GATE-16 (Golgi-associated ATPase enhancer of 16 kDa) was shown to modulate the activity of SNAREs in the Golgi apparatus and is therefore an essential component of intra-Golgi transport and post-mitotic Golgi re-assembly. GATE-16 contains a ubiquitin fold subdomain, which is terminated at the carboxyl end by an additional amino acid after a conserved glycine residue. In the present study we tested whether the COOH terminus of GATE-16 undergoes post-translational cleavage by a protease which exposes the glycine 116 residue. We describe the isolation and characterization of HsApg4A as a human protease of GATE-16. We show that GATE-16 undergoes COOH-terminal cleavage both in vivo and in vitro, only when the conserved glycine 116 is present. We then utilize an in vitro assay to show that pure HsApg4A is sufficient to cleave GATE-16. The characterization of this protease may give new insights into the mechanism of action of GATE-16 and its other family members.     

Glutathione is involved in the antimalarial action of chloroquine and its modulation affects drug sensitivity of human and murine species of Plasmodium

Ferriprotoporphyrin IX (FP) is released inside the food vacuole of the malaria parasite during the digestion of host cell hemoglobin. FP is detoxified by its biomineralization to hemozoin. This process is effectively inhibited by chloroquine (CQ) and amodiaquine (AQ). Undegraded FP accumulates in the membrane fraction and inhibits enzymes of infected cells in parallel with parasite killing. FP is demonstrably degraded by reduced glutathione (GSH) in a radical-mediated mechanism. This degradation is inhibited by CQ and AQ in a competitive manner, thus explaining the ability of increased GSH levels in Plasmodium falciparum-infected cells to increase resistance to CQ and vice versa, and to render Plasmodium berghei that were selected for CQ resistance in vivo sensitive to the CQ when glutathione synthesis is inhibited. Some over-the-counter drugs that are known to reduce GSH in body tissues when used in excess were found to enhance the antimalarial action of CQ and AQ in mice infected either with P. berghei or Plasmodium vinckei. In contrast, N-acetyl-cysteine which is expected to increase the cellular levels of GSH, antagonized the action of CQ. These results suggest that some over-the-counter drugs can be used in combination with some antimalarials to which the parasite has become resistant.