Carbohydrate and lipid balances in the positive plant phenotypic response to arbuscular mycorrhiza: increase in sink strength.

The exchange of phosphorus (P) and carbon (C) between plants and arbuscular mycorrhizal fungi (AMF) is a major determinant of their mutualistic symbiosis. We explored the C dynamics in tomato (Solanum lycorpersicum) inoculated or not with Rhizophagus irregularis to study their growth response under different NaH2 PO4 concentrations (Null P, 0 mM; Low P, 0.065 mM; High P, 1.3 mM). The percentage of AMF colonization was similar in plants under Null and Low P, but severely reduced under High P. However, the AMF mass biomarker 16:1ω5 revealed higher fungal accumulation in inoculated roots under Low P, while more AMF spores were produced in the Null P. Under High P, AMF biomass and spores were strongly reduced. Plant growth response to mycorrhiza was negative under Null P, showing reduction in height, biovolume index, and source leaf (SL) area. Under Low P, inoculated plants showed a positive response (e.g., increased SL area), while inoculated plants under High P were similar to non-inoculated plants. AMF promoted the accumulation of soluble sugars in the SL under all fertilization levels, whereas the soluble sugar level decreased in roots under Low P in inoculated plants. Transcriptional upregulation of SlLIN6 and SlSUS1, genes related to carbohydrate metabolism, was observed in inoculated roots under Null P and Low P, respectively. We conclude that P-limiting conditions that increase AMF colonization stimulate plant growth due to an increase in the source and sink strength. Our results suggest that C partitioning and allocation to different catabolic pathways in the host are influenced by AMF performance.

Pathogenesis in Pseudomonas aeruginosa PAO1 Biofilm-Associated Is Dependent on the Pyoverdine and Pyocyanin Siderophores by Quorum Sensing Modulation.

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium for humans, animals, and plants, through producing different molecular factors such as biofilm, siderophores, and other virulence factors which favor bacterial establishment and infection in the host. In P. aeruginosa PAO1, the production of these factors is regulated by the bacterial quorum sensing (QS) mechanisms. From them, siderophores are involved in iron acquisition, transport, and homeostasis. They are also considered some of the main virulence factors in P. aeruginosa; however, detailed mechanisms to induce bacterial pathogenesis are poorly understood. In this work, through reverse genetics, we evaluated the function of bacterial pathogenesis in the pvd cluster genes, which are required for synthesizing the siderophore pyoverdine (PVD). Single pvdI, pvdJ, pvdL, and double mutant strains were analyzed, and contrary to expected, the pvdL and pvdI mutations increased the concentration of PVD and other phenazines, such as pyocyanin (PYO) and phenazine-1-carboxylic acid (PCA) and also an increased biofilm production and morphology depending on the autoinducer 2-alkyl-4-quinolone (PQS) and the QS molecules acyl-homoserine lactones. Consequently, in the in vivo pathogenicity model of Caenorhabditis elegans, the mutations in pvdI, pvdJ, and pvdL increased the survival of the worms exposed to supernatants or biofilms of the bacterial cultures. However, the double mutant pvdI/pvdJ increased its toxicity in agreeing with the biofilm production, PVD, PYO, and PCA. The findings indicate that the mutations in pvd genes encode non-ribosomal peptide synthetases impacted the biofilm's structure, but suppressively also of the phenazines, confirming that the siderophores contribute to the bacterial establishment and pathogenicity of P. aeruginosa PAO1.

An Updated Review on the Modulation of Carbon Partitioning and Allocation in Arbuscular Mycorrhizal Plants.

Arbuscular mycorrhizal fungi (AMF) are obligate biotrophs that supply mineral nutrients to the host plant in exchange for carbon derived from photosynthesis. Sucrose is the end-product of photosynthesis and the main compound used by plants to translocate photosynthates to non-photosynthetic tissues. AMF alter carbon distribution in plants by modifying the expression and activity of key enzymes of sucrose biosynthesis, transport, and/or catabolism. Since sucrose is essential for the maintenance of all metabolic and physiological processes, the modifications addressed by AMF can significantly affect plant development and stress responses. AMF also modulate plant lipid biosynthesis to acquire storage reserves, generate biomass, and fulfill its life cycle. In this review we address the most relevant aspects of the influence of AMF on sucrose and lipid metabolism in plants, including its effects on sucrose biosynthesis both in photosynthetic and heterotrophic tissues, and the influence of sucrose on lipid biosynthesis in the context of the symbiosis. We present a hypothetical model of carbon partitioning between plants and AMF in which the coordinated action of sucrose biosynthesis, transport, and catabolism plays a role in the generation of hexose gradients to supply carbon to AMF, and to control the amount of carbon assigned to the fungus.

Wnt/ß-Catenin Signaling as a Molecular Target by Pathogenic Bacteria.

The Wnt/ß-catenin signaling pathway is crucial to regulate cell proliferation and polarity, cell determination, and tissue homeostasis. The activation of Wnt/ß-catenin signaling is based on the interaction between Wnt glycoproteins and seven transmembrane receptors-Frizzled (Fzd). This binding promotes recruitment of the scaffolding protein Disheveled (Dvl), which results in the phosphorylation of the co-receptor LRP5/6. The resultant molecular complex Wnt-Fzd-LRP5/6-Dvl forms a structural region for Axin interaction that disrupts Axin-mediated phosphorylation/degradation of the transcriptional co-activator ß-catenin, thereby allowing it to stabilize and accumulate in the nucleus where it activates the expression of Wnt-dependent genes. Due to the prominent physiological function, the Wnt/ß-catenin signaling must be strictly controlled because its dysregulation, which is caused by different stimuli, may lead to alterations in cell proliferation, apoptosis, and inflammation-associated cancer. The virulence factors from pathogenic bacteria such as Salmonella enterica sv Typhimurium, Helicobacter pylori, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Citrobacter rodentium, Clostridium difficile, Bacteroides fragilis, Escherichia coli, Haemophilus parasuis, Lawsonia intracellularis, Shigella dysenteriae, and Staphylococcus epidermidis employ a variety of molecular strategies to alter the appropriate functioning of diverse signaling pathways. Among these, Wnt/ß-catenin has recently emerged as an important target of several virulence factors produced by bacteria. The mechanisms used by these factors to interfere with the activity of Wnt/ß-catenin is diverse and include the repression of Wnt inhibitors' expression by the epigenetic modification of histones, blocking Wnt-Fzd ligand binding, activation or inhibition of ß-catenin nuclear translocation, down- or up-regulation of Wnt family members, and inhibition of Axin-1 expression that promotes ß-catenin activity. Such a variety of mechanisms illustrate an evolutionary co-adaptation of eukaryotic molecular signaling to a battery of soluble or structural components synthesized by pathogenic bacteria. This review gathers the recent efforts to elucidate the mechanistic details through which bacterial virulence factors modulate Wnt/ß-catenin signaling and its physiological consequences concerning the inflammatory response and cancer.

Delayed and Abbreviated Environmental Enrichment after Brain Trauma Promotes Motor and Cognitive Recovery That Is Not Contingent on Increased Neurogenesis.

Environmental enrichment (EE) confers motor and cognitive recovery in pre-clinical models of traumatic brain injury (TBI), and neurogenesis has been attributed to mediating the benefits. Whether that ascription is correct has not been fully investigated. Hence, the goal of the current study is to further clarify the possible role of learning-induced hippocampal neurogenesis on functional recovery after cortical impact or sham injury by utilizing two EE paradigms (i.e., early + continuous, initiated immediately after TBI and presented 24 h/day; and delayed + abbreviated, initiated 4 days after TBI for 6 h/day) and comparing them to one another as well as to standard (STD) housed controls. Motor and cognitive performance was assessed on post-operative Days 1-5 and 14-19, respectively, for the STD and early + continuous EE groups and on Days 4-8 and 17-22, for the delayed + abbreviated EE groups. Rats were injected with bromodeoxyuridine (BrdU, 500 mg/ kg; intraperitoneally) for 3 days (12 h apart) before cognitive training and sacrificed 1 week later for quantification of BrdU+ and doublecortin (DCX+) labeled cells. Both early + continuous and delayed + abbreviated EE promoted motor and cognitive recovery after TBI, relative to STD (p < 0.05), and did not differ from one another (p > 0.05). However, only early + continuous EE increased DCX+ cells beyond the level of STD-housed controls (p < 0.05). No effect of EE on non-injured controls was observed. Based on these data, two novel conclusions emerged. First, EE does not need to be provided early and continuously after TBI to confer benefits, which lends credence to the delayed + abbreviated EE paradigm as a relevant pre-clinical model of neurorehabilitation. Second, the functional recovery observed after TBI in the delayed + abbreviated EE paradigm is not contingent on increased hippocampal neurogenesis. Future studies will elucidate alternate viable mechanisms mediating the benefits induced by EE.

Experimental evidence of masculinization by continuous illumination in a temperature sex determination teleost (Atherinopsidae) model: is oxidative stress involved?

The present study evaluates the influence of continuous light on phenotypic sex ratios in Chirostoma estor, a temperature sex determination animal model. Relative gene expression levels of 5 day old larvae were performed on two early gonad differentiation genes (sox9 and foxl2), two stress axis activation genes (gcr1 and crf) and four reactive oxygen species (ROS) antagonist effector genes (sod2, ucp2, gsr and cat). Two light treatments were applied from fertilization; control (12L:12D) simulated natural photoperiod and a continuous illumination photoperiod. By the end of the trial (12 weeks after hatching), differentiated and normal gonads were clearly identifiable in both treatments by histological observations. Regarding sex ratio, 73% of phenotypic males were found in continuous illumination compared with 40% in controls. Consistently, the sox9 gene (involved in early testis differentiation) showed an over expression in 64% of the individual larvae analysed compared with foxl2 (ovarian differentiation) suggesting a masculinization tendency in continuous illumination. On the other hand, only 36% of individuals showed the same tendency in the control treatment consistent with phenotypic sex ratios found under normal culture conditions. Relative gene expression results did not show significant difference in sod2, ucp2 and gcr1 levels, but cat, gsr and crf showed significantly higher expression levels in the continuous illumination treatment suggesting that both, the stress axis and ROS response mechanisms were activated at this time. This study suggests, a link between continuous light, oxidative stress and environmental sex determination in vertebrates. However, further research is necessary to describe this possible upstream mechanism that may drive some aspects of sexual plasticity in vertebrates.

Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus.

Glycogen synthase kinase 3 (GSK3) is a constitutive enzyme implicated in the regulation of cytokine expression and the inflammatory response during bacterial infections. Mammals have two GSK3 isoforms named GSK3α and GSK3ß that plays different but often overlapping functions. Although the role of GSK3ß in cytokine regulation during the inflammatory response caused by bacteria is well described, GSK3α has not been found to participate in this process. Therefore, we tested if GSK3α may act as a regulatory isoform in the cytokine expression by bovine endothelial cells infected with Staphylococcus aureus because this bacterium is one of the major pathogens that cause tissue damage associated with inflammatory dysfunction. Interestingly, although both isoforms were phosphorylated-inactivated, we consistently observed a higher phosphorylation of GSK3α at Ser21 than that of GSK3ß at Ser9 after bacterial challenge. During a temporal course of infection, we characterized a molecular switch from pro-inflammatory cytokine expression (IL-8), promoted by nuclear factor-kappa B (NF-κB), at an early stage (2 h) to an anti-inflammatory cytokine expression (IL-10), promoted by cAMP response element binding (CREB), at a later stage (6 h). We observed an indirect effect of GSK3α activity on NF-κB activation that resulted in a low phosphorylation of CREB at Ser133, a decreased interaction between CREB and the co-activator CREB-binding protein (CBP), and a lower expression level of IL-10. Gene silencing of GSK3α and GSK3ß with siRNA indicated that GSK3α knockout promoted the interaction between CREB and CBP that, in turn, increased the expression of IL-10, reduced the interaction of NF-κB with CBP, and reduced the expression of IL-8. These results indicate that GSK3α functions as the primary isoform that regulates the expression of IL-10 in endothelial cells infected with S. aureus.

The Glycogen Synthase Kinase 3α and ß Isoforms Differentially Regulates Interleukin-12p40 Expression in Endothelial Cells Stimulated with Peptidoglycan from Staphylococcus aureus.

Glycogen synthase kinase 3 (GSK3) is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3ß is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN), one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3ß activity in bovine endothelial cells (BEC) regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3ß at Ser9, and NF-κB p65 subunit (p65) at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3ß. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor). Inhibition of GSK3α and GSK3ß with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3ß. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3ß gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus.

The Wnt/ß-catenin signaling pathway controls the inflammatory response in infections caused by pathogenic bacteria.

Innate immunity against pathogenic bacteria is critical to protect host cells from invasion and infection as well as to develop an appropriate adaptive immune response. During bacterial infection, different signaling transduction pathways control the expression of a wide range of genes that orchestrate a number of molecular and cellular events to eliminate the invading microorganisms and regulate inflammation. The inflammatory response must be tightly regulated because uncontrolled inflammation may lead to tissue injury. Among the many signaling pathways activated, the canonical Wnt/ß-catenin has been recently shown to play an important role in the expression of several inflammatory molecules during bacterial infections. Our main goal in this review is to discuss the mechanism used by several pathogenic bacteria to modulate the inflammatory response through the Wnt/ß-catenin signaling pathway. We think that a deep insight into the role of Wnt/ß-catenin signaling in the inflammation may open new venues for biotechnological approaches designed to control bacterial infectious diseases.

Long-distance abscisic acid signalling under different vertical soil moisture gradients depends on bulk root water potential and average soil water content in the root zone.

To determine how root-to-shoot abscisic acid (ABA) signalling is regulated by vertical soil moisture gradients, root ABA concentration ([ABA](root)), the fraction of root water uptake from, and root water potential of different parts of the root zone, along with bulk root water potential, were measured to test various predictive models of root xylem ABA concentration [RX-ABA](sap). Beans (Phaseolus vulgaris L. cv. Nassau) were grown in soil columns and received different irrigation treatments (top and basal watering, and withholding water for varying lengths of time) to induce different vertical soil moisture gradients. Root water uptake was measured at four positions within the column by continuously recording volumetric soil water content (θv). Average θv was inversely related to bulk root water potential (Ψ(root)). In turn, Ψ(root) was correlated with both average [ABA](root) and [RX-ABA](sap). Despite large gradients in θv, [ABA](root) and root water potential was homogenous within the root zone. Consequently, unlike some split-root studies, root water uptake fraction from layers with different soil moisture did not influence xylem sap (ABA). This suggests two different patterns of ABA signalling, depending on how soil moisture heterogeneity is distributed within the root zone, which might have implications for implementing water-saving irrigation techniques.

Role of glycogen synthase kinase-3 beta in the inflammatory response caused by bacterial pathogens.

Glycogen synthase kinase 3ß (GSK3ß) plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3ß may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3ß and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB) activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection.

The phosphoinositide-3-kinase-Akt signaling pathway is important for Staphylococcus aureus internalization by endothelial cells.

Internalization of Staphylococcus aureus in bovine endothelial cells (BEC) is increased by tumor necrosis factor alpha stimulation and NF-κB activation. Because the phosphoinositide-3-kinase (PI3K)-Akt signaling pathway also modulates NF-κB activity, we considered whether the internalization of S. aureus by BEC is associated with the activity of PI3K and Akt. We found a time- and multiplicity of infection-dependent phosphorylation of Akt on Ser473 in BEC infected with S. aureus. This phosphorylation was inhibited by LY294002 (LY), indicating the participation of PI3K. Inhibition of either PI3K with LY or wortmannin, or Akt with SH-5, strongly reduced the internalization of S. aureus. Transfection of BEC with a dominant-negative form of the Akt gene significantly decreased S. aureus internalization, whereas transfection with the constitutively active mutant increased the number of internalized bacterium. Inhibition of PDK1 activity with OSU-03012 did not affect the level of S. aureus internalization, demonstrating that phosphorylation of Akt on Thr308 is not important for this process. Compared to the untreated control, the adherence of S. aureus to the surface of BEC was unaltered when cells were transfected or incubated with the pharmacological inhibitors. Furthermore, Akt activation by internalized S. aureus triggered a time-dependent phosphorylation of glycogen synthase kinase-3α (GSK-3α) on Ser21 and GSK-3ß on Ser9 that was partially inhibited with SH-5. Finally, treatment of BEC with LY prior to S. aureus infection inhibited the NF-κB p65 subunit phosphorylation on Ser536, indicating the involvement of PI3K. These results suggest that PI3K-Akt activity is important for the internalization of S. aureus and phosphorylation of GSK-3α, GSK-3ß, and NF-κB.

Gluconacetobacter diazotrophicus levansucrase is involved in tolerance to NaCl, sucrose and desiccation, and in biofilm formation.

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane, which expresses levansucrase, a fructosyltransferase exoenzyme with sucrose hydrolytic and levan biosynthetic activities. As a result of their physical properties, the levan can provide protection against stress caused by abiotic or biotic factors and participate in the formation of biofilms. In this study, we investigated the construction and function of a levansucrase-defective mutant of G. diazotrophicus. The lsdA mutant showed a decreased tolerance (65.5%) to 50-150 mM NaCl and a decrease of 89% in 876 mM (30%) sucrose, a reduction (99%) in tolerance to desiccation after 18 h, and a decrease (36.9-58.5%) in the ability to form cell aggregates on abiotic surfaces. Complementation of the mutant with the complete lsdA gene leads to a recovery of the ability to grow on sucrose-containing medium and to form slimy colonies, the ability to form the cell aggregates on abiotic surfaces and the tolerance to NaCl. This report demonstrates the importance of levansucrase in environmental adaptation of G. diazotrophicus under high osmotic stress and in biofilm formation.

TNF-alpha reduces the level of Staphylococcus epidermidis internalization by bovine endothelial cells.

Staphylococcus epidermidis is an environmental opportunistic pathogen associated with bovine intramammary infections. In bacterial infections, the endothelial tissue plays an important role during inflammation and it is the target of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha). Therefore, this work was designed to explore the effect of TNF-alpha on the interaction of S. epidermidis with bovine endothelial cells (BEC). We show that cell signaling activated by TNF-alpha caused a marked reduction in the number of intracellular S. epidermidis, suggesting that molecules participating in this pathway were involved in the internalization of this bacterium. We also found that S. epidermidis internalization was not associated with basal levels of nuclear factor kappa B (NF-kappaB) activity because the intracellular number of bacteria recovered after treating BEC with the NF-kappaB inhibitors, SN50 or BAY 11-7083, was similar to that of the untreated control. Interestingly, inhibition of the basal activity of JNK with SP600125 and p38 with SB203580 caused a decrease in the number of intracellular S. epidermidis. These results suggest that activation of the signaling pathway initiated by TNF-alpha could play an important role in the phagocytosis of this bacterium. However, the basal activity of NF-kappaB was shown not to be important for the internalization process of S. epidermidis.

The capacity of bovine endothelial cells to eliminate intracellular Staphylococcus aureus and Staphylococcus epidermidis is increased by the proinflammatory cytokines TNF-alpha and IL-1beta.

Staphylococcus aureus is a pathogenic bacterium causing clinical and subclinical bovine mastitis. Infections of the udder by S. aureus are frequently associated with the presence of Staphylococcus epidermidis, an opportunistic pathogen. We reported previously that the capacity of bovine endothelial cells (BEC) to endocytize S. aureus is associated with the activation of NF-kappaB and modulated by the proinflammatory cytokines TNF-alpha and IL-1beta. In this work, we explore the ability of BEC to eliminate intracellular S. aureus and S. epidermidis and their response to these cytokines. Time-kinetics survival experiments indicated that BEC eliminate intracellular S. epidermidis more efficiently. Replication of S. aureus, but not S. epidermidis, inside BEC was evident by an increase in intracellular bacteria recovered at 2 h postinfection. Afterwards, the intracellular number of staphylococci decreased gradually, reaching the lowest value at 24 h. Treatment of BEC with TNF-alpha or IL-1beta potentiated the capacity of BEC to eliminate both Staphylococcus species at the times tested. These results indicate that activation of the intrinsic antistaphylococcal response in BEC, enhanced by TNF-alpha and IL-1beta, is effective to eliminate S. aureus and S. epidermidis and suggest that endothelial cells may play a prominent role in the defense against infections caused by these bacteria.

Effect of regulated deficit irrigation and crop load on the antioxidant compounds of peaches.

The use of regulated deficit irrigation (RDI) strategies is becoming a common practice in areas with low water availability. Little information is available about the effects of RDI on the antioxidant content of fruits. In this study, the influence of RDI on the content of vitamin C, phenolic compounds and carotenoids was investigated. Two irrigation strategies, fully irrigated (FI) and RDI, were compared at two levels of thinning, commercial and half of the commercial crop load. RDI strategies affected the content of vitamin C, phenolics and carotenoids of Flordastar peaches. RDI caused fruit peel stress lowering the content of vitamin C and carotenoids, while increasing the phenolic content, mainly anthocyanins and procyanidins. Fruit weight was the only quality index influenced by the crop load as it increased in FI fruits at low crop load. In general, fruits from commercial crop load had slightly higher content of antioxidants to fruits from low crop load, although these influences were only observed in the peel. Additionally, the influence of irrigation controlled by two sensors related to plant water level, maximum daily trunk shrinkage (MDS) and sap flow (SF) on the antioxidant constituents of peaches was evaluated. The response of the fruits to SF sensor was similar to that observed for RDI strategy. According to the tested water sensors, SF did not act as a good plant-based water indicator for use in irrigation scheduling, as it caused an increase in the content of phenolics, similar to that observed for fruits subjected to RDI. Therefore, selection of RDI strategies and plant water indicators should be taken into account as they affect the content of antioxidants of peaches.

Innate immune response of bovine mammary gland to pathogenic bacteria responsible for mastitis.

Mastitis (mammary gland inflammation) is one of the most important bovine diseases causing economic losses to dairy producers. Mammary gland inflammation is a consequence of the activity of a number of cell and soluble factors that function together to eliminate invading microorganisms. The factors involved in this inflammatory response differ depending on the infectious agent. This review analyzes the factors involved in the immunologic mechanisms against the main pathogenic bacteria causing mastitis, and emphasizes the innate immune response of the mammary gland. Knowledge, at the molecular level, of the mammary gland immune response during infection by pathogenic bacteria is fundamental to the design of effective therapies to control and eradicate bovine mastitis.

Response of superoxide dismutase isoenzymes in tomato plants (Lycopersicon esculentum) during thermo-acclimation of the photosynthetic apparatus.

Seedlings of Lycopersicon esculentum Mill. var. Amalia were grown in a growth chamber under a photoperiod of 16 h light at 25 degrees C and 8 h dark at 20 degrees C. Five different treatments were applied to 30-day-old plants: Control treatment (plants maintained in the normal growth conditions throughout the experimental time), heat acclimation (plants exposed to 35 degrees C for 4 h in dark for 3 days), dark treatment (plants exposed to 25 degrees C for 4 h in dark for 3 days), heat acclimation plus heat shock (plants that previously received the heat acclimation treatment were exposed to 45 degrees C air temperature for 3 h in the light) and dark treatment plus heat shock (plants that previously received the dark treatment were exposed to 45 degrees C air temperature for 3 h in the light). Only the heat acclimation treatment increased the thermotolerance of the photosynthesis apparatus when the heat shock (45 degrees C) was imposed. In these plants, the CO(2) assimilation rate was not affected by heat shock and there was a slight and non-significant reduction in maximum carboxylation velocity of Rubisco (V(cmax)) and maximum electron transport rate contributing to Rubisco regeneration (J(max)). However, the plants exposed to dark treatment plus heat shock showed a significant reduction in the CO(2) assimilation rate and also in the values of V(cmax) and J(max). Chlorophyll fluorescence measurements showed increased thermotolerance in heat-acclimated plants. The values of maximum chlorophyll fluorescence (F(m)) were not modified by heat shock in these plants, while in the dark-treated plants that received the heat shock, the F(m) values were reduced, which provoked a significant reduction in the efficiency of photosystem II. A slight rise in the total superoxide dismutase (SOD) activity was found in the plants that had been subjected to both heat acclimation and heat shock, and this SOD activity was significantly higher than that found in the plants subjected to dark treatment plus heat shock. The activity of Fe-SOD isoenzymes was most enhanced in heat-acclimated plants but was unaltered in the plants that received the dark treatment. Total CuZn-SOD activity was reduced in all treatments. Darkness had an inhibitory effect on the Mn-SOD isoenzyme activity, which was compensated by the effect of a rise in air temperature to 35 degrees C. These results show that the heat tolerance of tomatoplants may be increased by the previous imposition of a moderately high temperature and could be related with the thermal stability in the photochemical reactions and a readjustment of V(cmax) and J(max). Some isoenzymes, such as the Fe-SODs, may also play a role in the development of heat-shock tolerance through heat acclimation. In fact, the pattern found for these isoenzymes in heat-acclimated Amalia plants was similar to that previously described in other heat-tolerant tomato genotypes.

Invasive potential of bacterial isolates associated with subclinical bovine mastitis.

This work describes differences in the invasive ability of bacterial isolates associated with mastitis. Invasion ability was determined by the uptake and survival in a primary culture of bovine mammary epithelial cells (BMEC). BMEC were isolated from a healthy lactating cow and characterized by their morphology, immunostaining for cytokeratin and the detection of beta- and kappa-casein mRNAs. Ten bacterial isolates comprising the staphylococcal species Staphylococcus aureus (3), Staphylococcus epidermidis (1), Staphylococcus haemolyticus (1), Staphylococcus equorum (2), Staphylococcus xylosus (1) and Brevibacterium stationis (2) obtained from raw milk of cows with mastitis from backyard farms were assayed for their ability to invade BMEC. Only two S. aureus and one S. epidermidis isolates were able to invade BMEC, at similar levels to the S. aureus control strain ATCC 27543. In conclusion, using the in vitro model of infection used in this study, differences in bacterial invasion capability may be detected.

The plasmid pBMBt1 from Bacillus thuringiensis subsp. darmstadiensis (INTA Mo14-4) replicates by the rolling-circle mechanism and encodes a novel insecticidal crystal protein-like gene.

This work describes a novel rolling-circle replicating (RCR) plasmid pBMBt1 from Bacillus thuringiensis subsp. darmstadiensis (INTA Mo14-4) encoding an insecticidal crystal protein-like gene. pBMBt1 (6700 bp) contains three ORFs and their putative transcription initiation sites and Shine-Dalgarno sequences were localized. ORF1 encodes a 34.6 kDa protein which showed identity with the protein CryC53 from B. thuringiensis subsp. cameroun (24.6%), the Cry15Aa insecticidal crystal protein from B. thuringiensis subsp. thompsoni (21.9%) and the Mtx3 protein from Bacillus sphaericus (27.8%). The ORF2 (52.3 kDa) showed a 74% identity with the Mob protein coded by pUIBI-1 from B. thuringiensis subsp. entomocidus and 64% identity with the Mob protein of pBMY1 from Bacillus mycoides; both Mob proteins belong to the pMV158 superfamily. To evaluate the Mob protein, the plasmid pHTMob14-4 was constructed. This plasmid shows transfer frequencies of 9.1x10(-6) in B. thuringiensis subsp. israelensis (4Q7Gm(R)). The ORF3 (23.6 kDa) gene product is homologous to the Rep protein from the plasmid pBMYdx of B. mycoides (37.6%). A putative double-strand origin with significant homology to that of B. thuringiensis plasmids, and an ssoA-type single-strand origin were also identified. Detection of single-stranded pBMBt1 DNA replicating intermediaries suggests that replication occurs via the rolling-circle mechanism.