Over-production of the D1 protein of photosystem II reaction centre in the cyanobacterium Synechococcus sp. PCC 7942

The unicellular cyanobacterium Synechococcus sp. PCC 7942 has three psbA genes encoding two different forms of the photosystem II reaction centre protein D1 (D1:1 and D1:2). The level of expression of these psbA genes and the synthesis of D1:1 and D1:2 are strongly regulated under varying light conditions. In order to better understand the regulatory mechanisms underlying these processes, we have constructed a strain of Synechococcus sp. PCC 7942 capable of over-producing psbA mRNA and D1 protein. In this study, we describe the over-expression of D1:1 using a tac-hybrid promoter in front of the psbAI gene in combination with lacI ^Q repressor system. Over-production of D1:1 was induced by growing cells for 12 h at 50 mol photons m^-2 s^-1 in the presence of 40 or 80 g/ml IPTG. The amount of psbAI mRNA and that of D1:1 protein in cells grown with IPTG was three times and two times higher, respectively. A higher concentration of IPTG (i.e., 150 g/ml) did not further increase the production of the psbAI message or D1:1. The over-production of D1:1 caused a decrease in the level of D1:2 synthesised, resulting in most PSII reaction centres containing D1:1. However, the over-production of D1:1 had no effect on the pigment composition (chlorophyll a or phycocyanin/number of cells) or the light-saturated rate of photosynthesis. This and the fact that the total amounts of D1 and D2 proteins were not affected by IPTG suggest that the number of PSII centres within the membranes remained unchanged. From these results, we conclude that expression of psbAI can be regulated by using the tac promoter and lacI ^Q system. However, the accumulation of D1:1 protein into the membrane is regulated by the number of PSII centres.     

Orcein and Litmus

Orcein was separated into 14 dyes by partition chromatography. Their constitutions were determined mainly by spectroscopy and led to formulae that are derived from 7-amino-2-phenoxazone, 7-hydroxy-2-phenoxazone, and 7-amino-2-phenoxazime, and that were confirmed by syntheses. The major constituent of litmus is assembled polymerically from 7-hydroxy-2-phenoxazone chromophores. The mechanism of formation is elucidated.     

Force-dependent stepping kinetics of myosin-V

Myosin-V is a processive two-headed actin-based motor protein involved in many intracellular transport processes. A key question for understanding myosin-V function and the communication between its two heads is its behavior under load. Since in vivo myosin-V colocalizes with other much stronger motors like kinesins, its behavior under superstall forces is especially relevant. We used optical tweezers with a long-range force feedback to study myosin-V motion under controlled external forward and backward loads over its full run length. We find the mean step size remains constant at approximately 36 nm over a wide range of forces from 5 pN forward to 1.5 pN backward load. We also find two force-dependent transitions in the chemomechanical cycle. The slower ADP-release is rate limiting at low loads and depends only weakly on force. The faster rate depends more strongly on force. The stronger force dependence suggests this rate represents the diffusive search of the leading head for its binding site. In contrast to kinesin motors, myosin-V's run length is essentially independent of force between 5 pN of forward to 1.5 pN of backward load. At superstall forces of 5 pN, we observe continuous backward stepping of myosin-V, indicating that a force-driven reversal of the power stroke is possible.     

Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes

Oxygenic photosynthesis produces various radicals and activeoxygen species with harmful effects on photosystem II (PSII).Such photodamage occurs at all light intensities. Damaged PSIIcentres, however, do not usually accumulate in the thylakoidmembrane due to a rapid and efficient repair mechanism. Theexcellent design of PSII gives protection to most of the proteincomponents and the damage is most often targeted only to thereaction centre D1 protein. Repair of PSII via turnover of thedamaged protein subunits is a complex process involving (i)highly regulated reversible phosphorylation of several PSIIcore subunits, (ii) monomerization and migration of the PSIIcore from the grana to the stroma lamellae, (iii) partial disassemblyof the PSII core monomer, (iv) highly specific proteolysis ofthe damaged proteins, and finally (v) a multi-step replacementof the damaged proteins with de novo synthesized copies followedby (vi) the reassembly, dimerization, and photoactivation ofthe PSII complexes. These processes will shortly be reviewedpaying particular attention to the damage, turnover, and assemblyof the PSII complex in grana and stroma thylakoids during thephotoinhibition–repair cycle of PSII. Moreover, a two-dimensionalBlue-native gel map of thylakoid membrane protein complexes,and their modification in the grana and stroma lamellae duringa high-light treatment, is presented. Key words: Arabidopsis thylakoid membrane proteome, assembly of photosystem II, D1 protein, light stress, photosystem II photoinhibition, repair of photosystem II     

In vitro antioxidant properties of the iron chelator pyridoxal isonicotinoyl hydrazone and some of its analogs

Since there are several problems with desferrioxamine (DFO) therapy, pyridoxal isonicotinoyl hydrazone (PIH) has been studied for more than 10 years as a promising new candidate for iron chelation therapy in iron-overload diseases. Iron chelation could also be helpful for experimental treatment of several other pathologies including rheumatoid arthritis and heart ischemia/reperfusion, due to the generation of oxyradicals and lipid peroxidation mediated by delocalized iron. We demonstrate here that sub-millimolar levels of PIH can inhibit the Fe(III)-EDTA/ascorbate-mediated formation of hydroxyl-like radicals as tested by the release of ethylene from 2-keto-4-methylthiobutyric acid (KMB assay) and the formation of malonaldehyde from 2-deoxyribose damage. PIH could also decrease the rates of Fe(III)-EDTA-mediated oxidation of ascorbate and block the peroxidation of liposomes of rat brain phospholipids induced by ferrous iron-EDTA. In all cases the in vitro antioxidant effectiveness of PIH was comparable to its analogs—including salicylaldehyde isonicotinoyl hydrazone—and to DFO. We conclude that PIH and its analogs are effective new candidates against iron-mediated oxidative stress for use in experimental medicine.     

Orcein and Litmus

Orcein was separated into 14 dyes by partition chromatography. Their constitutions were determined mainly by spectroscopy and led to formulae that are derived from 7-amino-2-phenoxazone, 7-hydroxy-2-phenoxazone, and 7-amino-2-phenoxazime, and that were confirmed by syntheses. The major constituent of litmus is assembled polymerically from 7-hydroxy-2-phenoxazone chromophores. The mechanism of formation is elucidated.     

Anti-α4 Antibody Treatment Blocks Virus Traffic to the Brain and Gut Early,and Stabilizes CNS Injury Late in Infection

Four SIV-infected monkeys with high plasma virus and CNS injury were treated with an anti-α4 blocking antibody (natalizumab) once a week for three weeks beginning on 28 days post-infection (late). Infection in the brain and gut were quantified, and neuronal injury in the CNS was assessed by MR spectroscopy, and compared to controls with AIDS and SIV encephalitis. Treatment resulted in stabilization of ongoing neuronal injury (NAA/Cr by 1H MRS), and decreased numbers of monocytes/macrophages and productive infection (SIV p28⁺, RNA⁺) in brain and gut. Antibody treatment of six SIV infected monkeys at the time of infection (early) for 3 weeks blocked monocyte/macrophage traffic and infection in the CNS, and significantly decreased leukocyte traffic and infection in the gut. SIV – RNA and p28 was absent in the CNS and the gut. SIV DNA was undetectable in brains of five of six early treated macaques, but proviral DNA in guts of treated and control animals was equivalent. Early treated animals had low-to-no plasma LPS and sCD163. These results support the notion that monocyte/macrophage traffic late in infection drives neuronal injury and maintains CNS viral reservoirs and lesions. Leukocyte traffic early in infection seeds the CNS with virus and contributes to productive infection in the gut. Leukocyte traffic early contributes to gut pathology, bacterial translocation, and activation of innate immunity.     

Phylogeography, genetic structure and population divergence time of cheetahs in Africa and Asia: evidence for long-term geographic isolates

The cheetah (Acinonyx jubatus) has been described as a species with low levels of genetic variation. This has been suggested to be the consequence of a demographic bottleneck 10 000–12 000 years ago (ya) and also led to the assumption that only small genetic differences exist between the described subspecies. However, analysing mitochondrial DNA and microsatellites in cheetah samples from most of the historic range of the species we found relatively deep phylogeographic breaks between some of the investigated populations, and most of the methods assessed divergence time estimates predating the postulated bottleneck. Mitochondrial DNA monophyly and overall levels of genetic differentiation support the distinctiveness of Northern‐East African cheetahs (Acinonyx jubatus soemmeringii). Moreover, combining archaeozoological and contemporary samples, we show that Asiatic cheetahs (Acinonyx jubatus venaticus) are unambiguously separated from African subspecies. Divergence time estimates from mitochondrial and nuclear data place the split between Asiatic and Southern African cheetahs (Acinonyx jubatus jubatus) at 32 000–67 000 ya using an average mammalian microsatellite mutation rate and at 4700–44 000 ya employing human microsatellite mutation rates. Cheetahs are vulnerable to extinction globally and critically endangered in their Asiatic range, where the last 70–110 individuals survive only in Iran. We demonstrate that these extant Iranian cheetahs are an autochthonous monophyletic population and the last representatives of the Asiatic subspecies A. j. venaticus. We advocate that conservation strategies should consider the uncovered independent evolutionary histories of Asiatic and African cheetahs, as well as among some African subspecies. This would facilitate the dual conservation priorities of maintaining locally adapted ecotypes and genetic diversity.     

Culturing and characterization of astrocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss)

An access to brain cell cultures from fish would enable screening of possible neurotoxic chemicals contaminating the aquatic environment. In the present study, a protocol for a successful routine isolation and culturing of brain cells from juvenile rainbow trout was worked out. The coating material was shown to be of importance for cell proliferation. Cells grow better on a surface coated with laminin than on those coated with poly-L-lysine (PLL), poly-D-lysin (PDL) or poly-L-ornithine (PLO). The best cell growth was obtained on double-coated surfaces (PLL, PDL or PLO plus laminin). On such a culture substrate and with a seeding density of 1 x 10(7) cells/cm(2) confluence was obtained within 3-4 weeks at an incubation temperature of 18 degrees C. Approximately 95% of the cells were identified as astrocytes on the basis of a positive staining with antibodies against the astrocyte specific glial protein (GFAP). No oligodendrocytes or fibroblasts were identified in the cultures, and despite several efforts, neurons did not grow under the culture conditions used. When challenged with ligands known to awake a calcium transient in mammalian astrocytes, 44% of the cells responded to ATP with an increase in [Ca 2+](i), 38% to norepinephrine, 27% to 5-hydroxytryptamine, 7% to histamine and 6% to glutamate. Kainate, quisqualate and gamma-aminobutyric acid did not awake a calcium transient in the cells. Using a proper protocol, it is thus quite easy to get an almost pure culture of astrocyte, whereas neurones proved to very difficult to culture.     

Protection against activated oxygen following re-aeration of hypoxically pretreated wheat roots. The response of the glutathione system

This paper shows the effects of re-aeration on the glutathionepool following a prolonged period of root hypoxia. An increased content of total glutathione has been measuredin roots of wheat seedlings (Triticum aestivum L. cv. Alcedo),grown in a nitrogen-flushed nutrient solution (HI) with theirshoots in air compared with roots of aerobically grown plants(C). Re-aeration of hypoxically pretreated roots causes oxidativeinjury indicated by the oxidation of reduced glutathione (GSH),decrease of total thiol-groups and increased formation of TBAreactive material (lipid peroxidation). Re-admission of oxygen results in a 50% rise in oxygen uptakeover the whole 16 h re-aeration period compared with the control.During this time the overall glutathione pool of HI treatmentincreases to almost double that of the control, essentiallyreflected in the amount of oxidized glutathione (GSSG). Hypoxically pretreated roots showed lower glutathione reductaseactivity (GR) than the control. Immediately following re-aerationthe activity was further decreased to a limiting value whichseems to prevent full reduction of the newly formed glutathione.Therefore, the capacity to reduce the GSSG pool is below thecapacity for net glutathione synthesis. This results in a declineof the GSH/GSSG ratio which reflects oxidative stress. The enzymeactivity recovers slowly after re-aeration exceeding the valuesof aerobically grown roots only after 16 h correlating witha high reduction state of the glutathione pool. Copper, known to induce the formation of reactive oxygen species,strengthened the effect of re-aeration and enhanced the post-anoxicinjury irreversibly. The importance of the glutathione system in roots to cope withvarying oxygen tension is discussed. Key words: Hypoxia, re-aeration, glutathione system, glutathione reductase, wheat