Transit peptide cleavage sites of integral thylakoid membrane proteins

A set of 58 nuclearly encoded thylakoid-integral membrane proteins from four plant species was identified, and their amino termini were assigned unequivocally based upon mass spectrometry of intact proteins and peptide fragments. The dataset was used to challenge the Web tools ChloroP, TargetP, SignalP, PSORT, Predotar, and MitoProt II for predicting organelle targeting and transit peptide proteolysis sites. ChloroP and TargetP reliably predicted chloroplast targeting but only reliably predicted transit peptide cleavage sites for soluble proteins targeted to the stroma. SignalP (eukaryote settings) accurately predicted the transit peptide cleavage site for soluble proteins targeted to the lumen. SignalP (Gram-negative bacteria settings) reliably predicted peptide cleavage of integral thylakoid proteins inserted into the membrane via the "spontaneous" pathway. The processing sites of more common thylakoid-integral proteins inserted by the signal recognition peptide-dependent pathway were not well predicted by any of the programs. The results suggest the presence of a second thylakoid processing protease that recognizes the transit peptide of integral proteins inserted via the spontaneous mechanism and that this mechanism may be related to the secretory mechanism of Gram-negative bacteria.     

Arthritogenicity ofYersinia enterocolitica O:8 in Hamsters: Analysis of the immune response

An animal model, hamster, was used for the study ofYersinia-induced arthritis. The development of arthritis, estimated by measuring the inflammation on hind paws after infection, was correlated with the kinetics of the immune response. Hostological and immunofluorescence (IFI) studies and serum antibody measurements were performed. Two inflammatory peaks were observed: an acute one on day 11 post-infection (p.i) and a chronic one on days 26–35 p.i. Joint cultures were positive until day 14 p.i. IFI was used to demonstrate the deposit of bacterial antigens in the joint. A persistent response of cellular extract-specific IgG antibodies was observed until day 94. Lipopolysaccharide-specific IgG was statistically significant on day 26 p.i. Antibodies against bands 66 and 54 were observed by immunoblotting. Polyclonal activation was detected during reactive arthritis. It is shown thatY. enterocolitica is arthritogenic in hamsters, immune mechanisms participating in the development of this disease.     

Changes in the photosynthetic light response curve during leaf development of field grown maize with implications for modelling canopy photosynthesis

Changes in the photosynthetic light-response curve during leaf development were determined for the fourth leaf of maize crops sown on 23 April and 10 June. Temperatures were unusually mild during late spring/early summer and neither crop experienced chilling damage. The concept of thermal time was used to take into account the effects of different temperature regimes on developmental stage, thereby enabling photosynthetic light-response data to be combined for both crops to describe the general response. Large variations in the upper asymptote (A_sat) and convexity () of the light-response curve occurred during leaf development, but the maximum quantum yield of CO₂ assimilation remained relatively constant throughout. Dark respiration rates showed a small but significant decrease with leaf age and generally ranged between 5 and 10% of A_sat. A simple mathematical model was developed to assess the sensitivity of daily leaf photosynthesis (A_L) to reductions in the A_sat, and the initial slope () of the light-response curve at different stages of leaf development. On bright sunny days, and at all developmental stages, A_L was ca. twice as sensitive to reductions in A_sat than to reductions in and . In overcast conditions, however, all three parameters contributed significantly to reductions in leaf photosynthesis, although the contribution of was greatest during early leaf growth, while older leaves were most sensitive to depressions in A_sat. The implications of these results for modelling the sensitivity of canopy photosynthesis to chill-induced photoinhibition of the light-response curve are discussed.     

Zinc-phosphorus interactions in two cultivars of tomato (Lycopersicon esculentum L.) grown in chelator-buffered nutrient solutions

Zinc-phosphorus interactions have been frequently studied using a diverse number of crop species, but attainment of reproducible Zn deficiencies, especially severe ones, has been hampered by the use of conventional hydroponic solutions wherein contaminating levels of Zn are often near-adequate for normal growth. We utilized novel, chelator-buffered nutrient solutions for precise imposition of Zn deficiencies. Tomato (Lycopersicon esculentum L. cv. Jackpot or Celebrity) seedlings were grown for 15 to 18 d in nutrient solutions containing 200, 600, or 1200 M P, and 0 to 91 M total Zn. Computed free Zn²⁺ activities were buffered at 10^-10.3 M by inclusion of a 100-M excess (above the sum of the micronutrient metal concentrations) of the chelator DTPA. At total added Zn=0, acute Zn deficiency resulted in zero growth after seedling transfer, and plant death prior to termination. Free Zn²⁺ activities 10^-10.6 M resulted in Zn deficiencies ranging from mild to severe, but activities 10^-11.2 were required to cause hyperaccumulation of shoot P to potentially toxic levels. Despite severe Zn deficiency (i.e. ca. 20% of control growth), tissue Zn levels were usually much higher than the widely reported critical value of 20 mg kg^-1, which may be an artifact of the selection of DTPA for buffering free Zn²⁺. Across Zn treatments, increasing solution P depressed growth slightly, especially in Celebrity, but corresponding increases in tissue P (indicative of enhanced P toxicity) or decreases in tissue Zn (P-induced Zn deficiency) were not observed. The depressive effect of P was also not explained by reductions in the water-soluble Zn fraction. Within 40 h, restoration of Zn supply did not ameliorate high leakage rates (as measured by K⁺ efflux) of Zn-deficient roots. Similarly, transfer of Zn-sufficient plants to deficient solutions did not induce leakiness within 40 h. Foliar sprays of ZnSO₄ almost completely corrected both Zn deficiency and membrane leakiness of plants grown in low-Zn solutions. Hence, maintenance of root membrane integrity appears to depend on the overall Zn nutritional status of the plant, and not on the presence of certain free Zn²⁺ levels in the root apoplasm.     

C-ferroptosis is an iron-dependent form of regulated cell death in cyanobacteria

Ferroptosis is an oxidative and iron-dependent form of regulated cell death (RCD) recently described in eukaryotic organisms like animals, plants, and parasites. Here, we report that a similar process takes place in the photosynthetic prokaryote Synechocystis sp. PCC 6803 in response to heat stress. After a heat shock, Synechocystis sp. PCC 6803 cells undergo a cell death pathway that can be suppressed by the canonical ferroptosis inhibitors, CPX, vitamin E, Fer-1, liproxstatin-1, glutathione (GSH), or ascorbic acid (AsA). Moreover, as described for eukaryotic ferroptosis, this pathway is characterized by an early depletion of the antioxidants GSH and AsA, and by lipid peroxidation. These results indicate that all of the hallmarks described for eukaryotic ferroptosis are conserved in photosynthetic prokaryotes and suggest that ferroptosis might be an ancient cell death program.     

Interspecific Competition for Shelters in Territorial and Gregarious Intertidal Grazers: Consequences for Individual Behaviour

Experiments have shown that interspecific interactions within consumer guilds can alter patterns of distribution, abundance and size of species. Plastic behavioural responses can be modulated by agonistic interactions. In many cases, consumers compete for space and shelters, and these interactions change the manner in which they exploit food. This study investigates the consequences of competition in the spatial and temporal organization of behaviour of intertidal grazers, which share algal resources and the use of rock crevices while resting, but exhibit different body sizes, spatial behaviour and foraging modes. We evaluate interaction strength between small gregarious Siphonaria lessoni and the larger territorial keyhole limpet Fissurella crassa and between S. lessoni and the medium-size gregarious chiton Chiton granosus. Using field manipulations and artificial arenas in the laboratory, we tested whether the use of crevices, micro-spatial distribution and activity are modified by the density of conspecifics and the presence of heterospecifics. Our results show that small-scale spatial segregation observed in the field between S. lessoni and C. granosus result from species-specific differences in habitat use. In turn, we found evidence that spatial segregation between F. crassa and S. lessoni results from highly asymmetric interference competition in the use of shelters. The presence of F. crassa reduced the use of crevices and growth rates of S. lessoni. Effects on growth rates are assumed to result from exposure to harsh environmental conditions rather than food limitation. Thus, neither gregarious behaviour nor differences in activity were sufficient to prevent competition with the larger grazer. Our study illustrates the importance of competition for shelters, which results in behavioural changes of the smaller-sized species, and how these plastic responses can translate into differences in growth rates. Use of shelters can thus be modulated by environmental conditions in a species-specific as well as an interactive manner within consumers’ guilds.     

S-Allylcysteine, a garlic-derived antioxidant, ameliorates quinolinic acid-induced neurotoxicity and oxidative damage in rats

Excitotoxicity elicited by overactivation of N-methyl-D-aspartate receptors is a well-known characteristic of quinolinic acid-induced neurotoxicity. However, since many experimental evidences suggest that the actions of quinolinic acid also involve reactive oxygen species formation and oxidative stress as major features of its pattern of toxicity, the use of antioxidants as experimental tools against the deleterious effects evoked by this neurotoxin becomes more relevant. In this work, we investigated the effect of a garlic-derived compound and well-characterized free radical scavenger, S-allylcysteine, on quinolinic acid-induced striatal neurotoxicity and oxidative damage. For this purpose, rats were administered S-allylcysteine (150, 300 or 450 mg/kg, i.p.) 30 min before a single striatal infusion of 1 microl of quinolinic acid (240 nmol). The lower dose (150 mg/kg) of S-allylcysteine resulted effective to prevent only the quinolinate-induced lipid peroxidation (P < 0.05), whereas the systemic administration of 300 mg/kg of this compound to rats decreased effectively the quinolinic acid-induced oxidative injury measured as striatal reactive oxygen species formation (P < 0.01) and lipid peroxidation (P < 0.05). S-Allylcysteine (300 mg/kg) also prevented the striatal decrease of copper/zinc-superoxide dismutase activity (P < 0.05) produced by quinolinate. In addition, S-allylcysteine, at the same dose tested, was able to reduce the quinolinic acid-induced neurotoxicity evaluated as circling behavior (P < 0.01) and striatal morphologic alterations. In summary, S-allylcysteine ameliorates the in vivo quinolinate striatal toxicity by a mechanism related to its ability to: (a) scavenge free radicals; (b) decrease oxidative stress; and (c) preserve the striatal activity of Cu,Zn-superoxide dismutase (Cu,Zn-SOD). This antioxidant effect seems to be responsible for the preservation of the morphological and functional integrity of the striatum.     

Construction and characterization of a minimized version of the HIV-1 pNL4-3 plasmid and its application for pseudotyping HIV-1 vectors

The pUC-based pNL4-3 plasmid is the most widely used vector for in vitro manipulations of the HIV-1 proviral sequences. We have developed a minimal plasmid (pCHUS) based on pNL4-3, which may be useful to facilitate the design of HIV-based constructions. The strategy that has allowed us to construct pCHUS includes the following steps: (1) pNL-3 digestion by using restriction sites contained within the long terminal repeats (LTRs), (2) recircularization of the fragment containing the pUC18 sequence, (3) amplification of the LTR region restored in the previous step, (4) double digestion of the products obtained in steps 2 and 3, (5) ligation of the fragment containing ColE1+Amp^R with the LTR fragment, (6) linearization of the intermediate plasmid obtained, and (7) insertion of the fragment containing the proviral genome into the linearized vector. The pCHUS plasmid includes essential information for its replication and antibiotic selection in bacteria, but it lacks all the unnecessary sequences. Our results suggest that pCHUS may be more advantageous than pNL4-3 for in vitro manipulation of the HIV-1 proviral genome. In addition, we describe a potential application of this new vector for pseudotyping HIV-1 particles, using a single plasmid transfection, as a more helpful alternative to the traditionally used cotransfection method.