Ideal Osmotic Spaces for Chlorobionts or Cyanobionts Are Differentially Realized by Lichenized Fungi

Lichens result from symbioses between a fungus and either a green alga or a cyanobacterium. They are known to exhibit extreme desiccation tolerance. We investigated the mechanism that makes photobionts biologically active under severe desiccation using green algal lichens (chlorolichens), cyanobacterial lichens (cyanolichens), a cephalodia-possessing lichen composed of green algal and cyanobacterial parts within the same thallus, a green algal photobiont, an aerial green alga, and a terrestrial cyanobacterium. The photosynthetic response to dehydration by the cyanolichen was almost the same as that of the terrestrial cyanobacterium but was more sensitive than that of the chlorolichen or the chlorobiont. Different responses to dehydration were closely related to cellular osmolarity; osmolarity was comparable between the cyanolichen and a cyanobacterium as well as between a chlorolichen and a green alga. In the cephalodium-possessing lichen, osmolarity and the effect of dehydration on cephalodia were similar to those exhibited by cyanolichens. The green algal part response was similar to those exhibited by chlorolichens. Through the analysis of cellular osmolarity, it was clearly shown that photobionts retain their original properties as free-living organisms even after lichenization.Lichens are ubiquitously found in all terrestrial environments, including those with extreme climates such as Antarctica and deserts; they are pioneer organisms in primary succession (Longton, 1988; Ahmadjian, 1993). Colonization ability is largely owed to lichens’ extreme tolerance for desiccation (Ahmadjian, 1993). Although lichens harbor photosynthetic green algae or cyanobacteria (blue-green algae) within their thalli, they show metabolic activity even when dried at 20°C and under conditions of 54% relative humidity (Cowan et al., 1979). This desiccation tolerance partially results from drought resistance originally exhibited by the photobiont. It is further strengthened by lichen symbiosis (Kosugi et al., 2009). Cyanolichens (symbiosis between a fungus and a cyanobacterium) are desiccation-tolerant organisms that favor humid and shady environments, whereas chlorolichens (symbiosis between a fungus and a green alga) tolerate dry and high-light environments (James and Henssen, 1976; Lange et al., 1988). Chlorolichens can perform photosynthesis when the surrounding humidity is high, but cyanolichens require some water in a liquid state (Lange et al., 1986, 2001; Nash et al., 1990; Ahmadjian, 1993).Most poikilohydric photosynthetic organisms can tolerate rapid drying. Biological activity during desiccation and recovery following drought are scarcely affected by protein synthesis inhibitors (Proctor and Smirnoff, 2000). Moderate drought tolerance is attained by increasing compatible solutes (amino acids, sugars, and sugar alcohols) as protective agents during drought stress (Mazur, 1968; Parker, 1968; Hoekstra et al., 2001). An increase in compatible solutes prevents water loss or increases water uptake from the air when humidity is high (Lange et al., 1988). It has been observed, however, that the intracellular solute concentration is low (corresponding to a sorbitol concentration of approximately 0.22 m) in the desiccation-tolerant terrestrial cyanobacterium Nostoc commune (Satoh et al., 2002; Hirai et al., 2004). N. commune photosynthetic activity is lost when incubated in low sorbitol concentrations (Hirai et al., 2004), whereas a Trebouxia spp. chlorobiont freshly isolated from the desiccation-tolerant chlorolichen Ramalina yasudae remains active under the same conditions (Kosugi et al., 2009).Different solute concentrations in photobionts may dictate habitat preferences for chlorolichens and cyanolichens (James and Henssen, 1976; Lange et al., 1988). One might expect that the ideal cellular osmotic pressure (or cellular solute concentration) of a lichenized fungus is problematic, as both the fungus and the photobiont are closely associated in the thallus (Kranner et al., 2005). Thus, we may be able to further hypothesize that the solute concentration itself in original photobionts determines the nature of desiccation tolerance in chlorolichens and cyanolichens.To better understand symbiosis in lichens, it is important to examine how the cellular osmotic pressures of both symbionts contribute to lichen photosynthesis. In this study, cellular osmotic pressures of lichens and photobionts were determined by assessing water potential. The cephalodia-possessing lichen Stereocaulon sorediiferum was chosen as a desiccation-tolerant model organism because it separately harbors a green alga and a cyanobacterium in different compartments of the lichen body. The green algal photobiont is contained in the stem- and branch-like structures, whereas the cyanobacterial photobiont (cyanobiont) is contained in the organism’s cephalodia. For comparison, several chlorolichens (R. yasudae, Parmotrema tinctorum, and Graphis spp.), cyanolichens (Collema subflaccidum and Peltigera degenii), green algae (Prasiola crispa, Trebouxia spp., and Trentepohlia aurea), and cyanobacteria (N. commune, Scytonema spp., and Stigonema spp.) were also analyzed (Fig. 1). The cyanobiont of C. subflaccidum is closely related to N. commune (Ahmadjian, 1993), and the cyanobiont of S. sorediiferum belongs to the genus Stigonema (Kurina and Vitousek, 1999). Green algal photobionts of R. yasudae and S. sorediiferum are Trebouxia spp. (Bergman and Huss-Danell, 1983). For the measurements of water potential, we had to use specimens larger than 0.1 g dry weight for one measurement. Furthermore, the specimens should cover approximately 70% of the surface area of a sample cup with 4 cm diameter that was equipped in our dewpoint potentiometer. Considering the statistical analyses, we needed large amounts of lichen and algal samples for the measurement of water potential. To conduct this study, we wanted to use free-living green algae and cyanobacteria, not the photobionts isolated from a lichen body. This is because inconsistent results were reported previously for chlorobionts liberated from lichens (Brock, 1975; Lange et al., 1990). Three major photobionts of lichens, Trebouxia, Trentepohlia, and Nostoc spp., were considered for inclusion. Until now, free-living Trebouxia spp. were not observed convincingly in nature. Therefore, cultivated Trebouxia spp. were used. Other green algae and cyanobacteria were chosen from among free-living species that (1) are closely related to some photobionts, (2) form large communities sufficient to cover the required quantity that will not destroy the local ecosystem by our sampling, (3) are easy to remove from other attached algae/microorganisms, and (4) are tolerant to desiccation. P. crispa forms large communities in nature, and the closely related species Prasiola borealis is known to be a photobiont of Mastodia tessellata. Only two freshwater species of genus Prasiola are found in Japan; P. crispa inhabits a limited area of Hokkaido Island, and Prasiola japonica is a rare species. P. crispa harvested in Antarctica and shown to be desiccation tolerant in our previous work (Kosugi et al., 2010b) was used in this study.Open in a separate windowFigure 1.Lichens analyzed in this study. A, Cyanolichen C. subflaccidum on a rock. B, Wet (left) and dry (right) thalli of cyanolichen Peltigera degenii with green moss. C, Chlorolichen R. yasudae on a rock. D, Chlorolichen Graphis spp. on a Zelkova serrata tree trunk. The grayish basal part of Graphis spp. is the site where the photobiont resides, and the dark-colored streaks are the apothecia. E, Chlorolichen Parmotrema tinctorum on a Z. serrata tree trunk. F, Cephalodia-possessing lichen S. sorediiferum. Some cephalodia are indicated by arrows. The stem- and branch-like structures are the green algae-containing compartments.     

Inhibition of Cyclic Nucleotide Phosphodiesterases by Reticulol

Inhibition mechanism of rat cerebral cortex cyclic nucleotide phosphodiesterases (PDE) by reticulol was investigated. The inhibition of PDE by reticulol was not reduced in the presence of excess PDE activating factor (PAF) or/and Ca²⁺ ion. Reticulol showed lower Ki values for Ca²⁺-PAF dependent PDE than for Ca²⁺ independent PDE.     

Effects of yokukansan on behavioral and psychological symptoms of vascular dementia: an open-label trial

Previous clinical trials suggest that the traditional Japanese medicine yokukansan has beneficial effects on the behavioral and psychological symptoms of dementia (BPSD). The present study was conducted to elucidate the efficacy of yokukansan on BPSD in patients with vascular dementia. Thirteen Japanese patients (9 men and 4 women) who were diagnosed as having vascular dementia (VaD) according to the diagnostic criteria of NINDS-AIREN were subjected to the open-label clinical trial in which yokukansan (7.5g/day) has been given for 4 weeks. Their mean age was 71.2±6.5 years. The BPSD was evaluated using the Neuropsychiatric Inventory (NPI), cognitive function was evaluated by the Mini-Mental State Examination (MMSE), the activities of daily living was evaluated by Barthel index (BI) and Disability Assessment for Dementia (DAD), and the extrapyramidal signs were evaluated by United Parkinson's Disease Rating Scale (UPDRS). The mean NPI was 33.0±17.3 and 23.6±13.9 for the baseline and after treatment, respectively. It was significantly improved after treatment (p<0.05). In the NPI-subcategories, there was a significant improvement in agitation and disinhibition after the treatment. There was no significant change in MMSE, BI, DAD or UPDRS before and after the treatment. There was no adverse effect during the treatment period. The present results suggest that yokukansan is beneficial for the treatment of BPSD in VaD patients.     

The origin of an unusually large 19F chemical shift difference between the diastereomeric alpha-cyano-alpha-fluoro-p-tolylacetic acid (CFTA) esters of 3',4',5,7-tetra-O-methylepicatechin

During a study on the relationship between the (19)F chemical shift difference for the diastereomeric alpha-cyano-alpha-fluoro-p-tolylacetic acid (CFTA) esters of chiral secondary alcohols and the absolute configurations of the alcohols, an unusually large 19F chemical shift difference has been observed for the CFTA esters of 3',4',5,7-tetra-O-methylepicatechin. This large chemical shift difference has been reproduced by ab initio calculations of molecular geometry and GIAO calculations of 19F chemical shifts on the stable conformations of the diastereomeric alpha-cyano-alpha-fluorophenylacetic acid (CFPA) esters of cis-flavan-3-ol as model systems. The origin of the large chemical shift difference has been further investigated using NBO analysis. This revealed that the interaction between pi(C=C) of the aryl group in the chiral auxiliary and sigma*(C--F) is a very important determinant of the 19F chemical shifts and this interaction depends on the torsion angle between the plane of aryl group and the C--F bond.     

Possible involvement of radical intermediates in the inhibition of cysteine proteases by allenyl esters and amides

In order to investigate crystallographically the mechanism of inhibition of cysteine protease by alpha-methyl-gamma,gamma-diphenylallenecarboxylic acid ethyl ester 3, a cysteine protease inhibitor having in vivo stability, we synthesized N-(alpha-methyl-gamma,gamma-diphenylallenecarbonyl)-L-phenylalanine ethyl ester 4. Reaction of 4 with thiophenol, the SH group of which has similar pK(a) value to that of cysteine protease, produced oxygen-mediated radical adducts 6 and 7 in ambient air but did not proceed under oxygen-free conditions. Catalytic activities of two thiol enzymes including cathepsin B were also lowered in the absence of oxygen. These results suggest that cysteine protease can act through an oxygen-dependent radical mechanism.     

Metabolism of N-(3′,5′-Dichlorophenyl)succinimide in Rats and Dogs

When N-(3′,5′-dichlorophenyl)succinimide (DSI)-carbonyI-¹⁴C and –pheny-³H were each orally administered to rats, regardless of the label site, most of the dose was readily eliminated. There was no difference in the excretion rate between male and female rats. No radioactive residues were detected in tissues and organs 24 hr after dosing. Urinary metabolites consisted of N-(3′,5′-dichlorophenyl) succinamic acid (DSA), N-(3′,5′-dichlorophe-nyl) malonamic acid (DMA), N-(3′5’-dichlorophenyl)-2-hydroxysuccinamic acid (2-OH-DSA) and 2-OH-DSA derivatives. In dogs, most of the administered dose was excreted in equal amounts in urine and feces. 2-OH-DSA derivatives were main urinary metabolites and most of fecal radiocarbon was due to intact DSL. The results of this study indicate that DSI is a biodegradable compound which is unlikely to leave any persistent residues in animals.

The degradation of DSI to DSA was mediated by an arylamidase-type hydrolase, which was present in the microsomal fraction of rabbit liver. The enzyme activity was found in livers and kidneys of four animal species tested. Depending on the animal species, the enzyme appears to be important for the metabolism of DSI.     

Phosphorylation and inactivation of myeloid cell leukemia 1 by JNK in response to oxidative stress

Oxidative stress induces JNK activation, which leads to apoptosis through mitochondria-dependent caspase activation. However, little is known about the mechanism by which JNK alters mitochondrial function. In this study, we investigated the role of phosphorylation of myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the Bcl-2 family, in oxidative stress-induced apoptosis. We found that JNK phosphorylated Ser-121 and Thr-163 of Mcl-1 in response to stimulation with H(2)O(2) and that transfection of unphosphorylatable Mcl-1 resulted in an enhanced anti-apoptotic activity in response to stimulation with H(2)O(2). JNK-dependent phosphorylation and thus inactivation of Mcl-1 may be one of the mechanisms through which oxidative stress induces cellular damage.     

RINGs hold the key to ubiquitin transfer

Ubiquitylation, the covalent modification of proteins by the addition of ubiquitin, relies on a cascade of enzymes that culminates in an E3 ligase that promotes the transfer of ubiquitin from an E2 enzyme to the target protein. The most prevalent E3 ligases contain a type of zinc-finger domain called RING, and although an essential role for the RING domain in ubiquitin transfer is widely accepted, the molecular mechanism by which this is achieved remains uncertain. In this review, we highlight recent studies that have suggested that the RING domain modulates the stability of the E2-ubiquitin conjugate so that catalysis is promoted. We also review the role of RING dimerisation and emphasise the importance of studying RING domains in the context of the full-length protein.     

Higher inductions of twin and single sister chromatid exchanges by cross-linking agents in Fanconi's anemia cells

Summary Twin and single sister chromatid exchanges (SCEs) induced by short treatments with mitomycin C (MC) and 4,5,8-trimethylpsoralen (TMP)-plus-near ultraviolet light (NUV) were analyzed in colcemid-induced endoreduplicated normal human and typical Fanconi's anemia (FA) fibroblasts with diplochromosomes. The induction rate of twin SCEs that had occurred in the first cycle (S1) after the treatment was 1.7–2.4 times higher in FA cells than in normal cells. The induction rate of single SCEs that had arisen during the second cycle (S2) long after the treatment was also much higher, though less than the twin SCE rate, in FA cells than the almost neglible rate after repair of cross-links and monoadducts in normal cells. These results in FA cells, which specifically lack the first half-excision step of the two-step cross-link repair but retain the normal monoadduct repair, indicate that MC or TMP cross-links remaining unrepaired are indeed responsible for higher inductions of twin (S1 exchange) and single SCEs (S2 exchange). Thus, these findings indicate that Shafer's model of replication bypass for cross-link-induced SCE, which predicts greatly reduced twin SCE formation in FA cells due to half cancellation, is apparently inadequate as such. We present three plausible models, incorporating the ordinary replication model, random unilateral cross-link transfer, and chromatid breakage/reunion, that can account for the probabilistic inductions of single and twin SCEs and even for no SCE formation.This work was supported in part by a grant-in-aid for cancer research from the Ministry of Education, Science and Culture, Japan     

Manganese content, fluorescence yields and the effect of chloride ion on Hill reaction activity of Tris-washed and reactivated chloroplasts

Reversible Tris-inhibition and reductive reactivation of oxygenevolution activity, observed previously in spinach, were studiedin chloroplasts from Japanese-radish, pokeweed (Phytolacca americana),oats and Easter-lily (Lilium longiflorum). Mn content of Tris-washed and reactivated chloroplasts onlymoderately decreased. Inhibition caused by Tris treatment wasalways greater than the decrease in Mn content, suggesting thatinhibition might not be principally due to Mn decrease. Reactivation enhanced the depressed chlorophyll fluorescencein Tris-inhibited chloroplasts to original levels. Chloride ion promoted oxygen evolution in reactivated, as wellas intact, chloroplasts. Reactivated chloroplasts could perform photophosphorylation,but ammonium ion, which promotes oxygen evolution by uncoupling,did not seem to affect them. (Received November 1, 1971; )