Melatonin modulates proliferation of pancreatic stellate cells through caspase-3 activation and changes in cyclin A and D expression

Journal of Physiology and Biochemistry - In this study, the effects of melatonin (1 μM–1 mM) on pancreatic stellate cells (PSC) have been examined. Cell viability and...     

Paradigm of Monoterpene (β-phellandrene) Hydrocarbons Production via Photosynthesis in Cyanobacteria

A direct “photosynthesis-to-fuels” approach envisions application of a single organism, absorbing sunlight, photosynthesizing, and converting the primary products of photosynthesis into ready-made fuel. The work reported here applied this concept for the photosynthetic generation of monoterpene (β-phellandrene) hydrocarbons in the unicellular cyanobacteria Synechocystis sp. PCC 6803. Heterologous expression of a codon-optimized Lavandula angustifolia β-phellandrene synthase (β-PHLS) gene in Synechocystis enabled photosynthetic generation of β-phellandrene in these microorganisms. β-phellandrene accumulation occurred constitutively and in tandem with biomass accumulation, generated from sunlight, CO₂, and H₂O. Results showed that β-phellandrene diffused through the plasma membrane and cell wall of the cyanobacteria and accumulated on the surface of the liquid culture. Spontaneous β-phellandrene separation from the biomass and its removal from the liquid phase alleviated product inhibition of cellular metabolism and enabled a continuous production process. The work showed that oxygenic photosynthesis can be directed to generate monoterpene hydrocarbons, while consuming CO₂, without a prior requirement for the harvesting, dewatering, and processing of the respective biomass.     

Warming reduces the growth and diversity of biological soil crusts in a semi-arid environment: implications for ecosystem structure and functioning

Biological soil crusts (BSCs) are key biotic components of dryland ecosystems worldwide that control many functional processes, including carbon and nitrogen cycling, soil stabilization and infiltration. Regardless of their ecological importance and prevalence in drylands, very few studies have explicitly evaluated how climate change will affect the structure and composition of BSCs, and the functioning of their constituents. Using a manipulative experiment conducted over 3 years in a semi-arid site from central Spain, we evaluated how the composition, structure and performance of lichen-dominated BSCs respond to a 2.4°C increase in temperature, and to an approximately 30 per cent reduction of total annual rainfall. In areas with well-developed BSCs, warming promoted a significant decrease in the richness and diversity of the whole BSC community. This was accompanied by important compositional changes, as the cover of lichens suffered a substantial decrease with warming (from 70 to 40% on average), while that of mosses increased slightly (from 0.3 to 7% on average). The physiological performance of the BSC community, evaluated using chlorophyll fluorescence, increased with warming during the first year of the experiment, but did not respond to rainfall reduction. Our results indicate that ongoing climate change will strongly affect the diversity and composition of BSC communities, as well as their recovery after disturbances. The expected changes in richness and composition under warming could reduce or even reverse the positive effects of BSCs on important soil processes. Thus, these changes are likely to promote an overall reduction in ecosystem processes that sustain and control nutrient cycling, soil stabilization and water dynamics.     

Truncated Photosystem Chlorophyll Antenna Size in the Green Microalga Chlamydomonas reinhardtii upon Deletion of the TLA3-CpSRP43 Gene

The truncated light-harvesting antenna size3 (tla3) DNA insertional transformant of Chlamydomonas reinhardtii is a chlorophyll-deficient mutant with a lighter green phenotype, a lower chlorophyll (Chl) per cell content, and higher Chl a/b ratio than corresponding wild-type strains. Functional analyses revealed a higher intensity for the saturation of photosynthesis and greater light-saturated photosynthetic activity in the tla3 mutant than in the wild type and a Chl antenna size of the photosystems that was only about 40% of that in the wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western-blot analyses showed that the tla3 strain was deficient in the Chl a/b light-harvesting complex. Molecular and genetic analyses revealed a single plasmid insertion in chromosome 4 of the tla3 nuclear genome, causing deletion of predicted gene g5047 and plasmid insertion within the fourth intron of downstream-predicted gene g5046. Complementation studies defined that gene g5047 alone was necessary and sufficient to rescue the tla3 mutation. Gene g5047 encodes a C. reinhardtii homolog of the chloroplast-localized SRP43 signal recognition particle, whose occurrence and function in green microalgae has not hitherto been investigated. Biochemical analysis showed that the nucleus-encoded and chloroplast-localized CrCpSRP43 protein specifically operates in the assembly of the peripheral components of the Chl a/b light-harvesting antenna. This work demonstrates that cpsrp43 deletion in green microalgae can be employed to generate tla mutants with a substantially diminished Chl antenna size. The latter exhibit improved solar energy conversion efficiency and photosynthetic productivity under mass culture and bright sunlight conditions.There is current interest and ongoing efforts to renewably generate fuel and chemical products for human consumption through the process of microalgal photosynthesis. Such bioproducts include H₂ (Hankamer et al., 2007; Melis, 2007), biofuel and chemical molecules (Hu et al., 2008; Greenwell et al., 2010; Mata et al., 2010; Melis, 2012), antigens (Dauvillée et al., 2010; Michelet et al., 2011), and high-value biopharmaceuticals (Mayfield et al., 2007). For this effort, sunlight energy conversion in photosynthesis must take place with the utmost efficiency, as this would help to make renewable fuel and chemical processes economically feasible. In plants and algae, the solar energy conversion efficiency of photosynthesis is thus a most critical factor for the economic viability of renewable fuel and chemical production (Melis, 2009, 2012).Green microalgae and other photosynthetic systems tend to develop large arrays of light-harvesting complexes, especially when cultivated under high-density mass culture conditions. This physiological response of the cells reflects an effort to absorb as much sunlight as possible as they compete in a light-limited environment (Kirk, 1994). However, in mass culture with cells possessing large chlorophyll (Chl) antennae, cells at the surface of the reactor would absorb incident sunlight (intensity of 2,500 μmol photons m⁻² s⁻¹) with rates that far exceed the capacity of the photosynthetic apparatus to utilize them (light saturation of photosynthesis occurs at less than 500 μmol photons m⁻² s⁻¹). The excess absorbed sunlight energy is dissipated via a process of nonphotochemical quenching to prevent photodamage and photoinhibition phenomena at the thylakoid membrane level (for review, see Müller et al., 2001).It has been shown that high-density cultures of microalgae with a truncated Chl antenna size are photosynthetically more productive under bright sunlight due to the elimination of overabsorption and wasteful dissipation of excess energy (Nakajima and Ueda, 1997, 1999; Melis et al., 1999; Polle et al., 2002, 2003; Melis, 2009). Identification of genes that confer a permanently truncated light-harvesting antenna size phenotype in plants and algae is thus of interest, as they could be applied in efforts to improve solar-to-product conversion efficiencies (Mitra and Melis, 2008; Melis, 2009; Ort et al., 2011). To this end, and to better understand the genetic mechanism that defines the size of the light-harvesting antenna in green microalgae, and also in an effort to generate truncated light-harvesting antenna size (tla) mutants, we generated and screened a library of Chlamydomonas reinhardtii DNA insertional mutagenesis strains. This work presents a molecular, genetic, and physiological analysis of one of these mutants, termed tla3, which exhibited a stably truncated light-harvesting Chl antenna size. The corresponding TLA3 gene was cloned and found to encode a homolog of the chloroplast signal recognition particle protein CpSRP43. Detailed functional analysis revealed that the phenotype of the tla3-ΔCpSRP43 mutant in C. reinhardtii entailed substantial reductions of the light-harvesting Chl antenna size. Accordingly, the cpsrp43 mutant phenotype and the CrCpSRP43 gene can be employed in C. reinhardtii, and possibly other green microalgae and plants, as a tool by which to truncate the Chl antenna size without affecting the function of the photosystems or thylakoid membrane electron transport properties of the chloroplast.     

Assembly of the light-harvesting chlorophyll antenna in the green alga Chlamydomonas reinhardtii requires expression of the TLA2-CpFTSY gene

The truncated light-harvesting antenna2 (tla2) mutant of Chlamydomonas reinhardtii showed a lighter-green phenotype, had a lower chlorophyll (Chl) per-cell content, and higher Chl a/b ratio than corresponding wild-type strains. Physiological analyses revealed a higher intensity for the saturation of photosynthesis and greater P(max) values in the tla2 mutant than in the wild type. Biochemical analyses showed that the tla2 strain was deficient in the Chl a-b light-harvesting complex, and had a Chl antenna size of the photosystems that was only about 65% of that in the wild type. Molecular and genetic analyses showed a single plasmid insertion in the tla2 strain, causing a chromosomal DNA rearrangement and deletion/disruption of five nuclear genes. The TLA2 gene, causing the tla2 phenotype, was cloned by mapping the insertion site and upon complementation with each of the genes that were deleted. Successful complementation was achieved with the C. reinhardtii TLA2-CpFTSY gene, whose occurrence and function in green microalgae has not hitherto been investigated. Functional analysis showed that the nuclear-encoded and chloroplast-localized CrCpFTSY protein specifically operates in the assembly of the peripheral components of the Chl a-b light-harvesting antenna. In higher plants, a cpftsy null mutation inhibits assembly of both the light-harvesting complex and photosystem complexes, thus resulting in a seedling-lethal phenotype. The work shows that cpftsy deletion in green algae, but not in higher plants, can be employed to generate tla mutants. The latter exhibit improved solar energy conversion efficiency and photosynthetic productivity under mass culture and bright sunlight conditions.     

Adherence of platelets under flow conditions results in specific phosphorylation of proteins at tyrosine residues

Collagen is a powerful platelet activating agent that promotes adhesion and aggregation of platelets. To differentiate the signals generated in these processes we have analyzed the tyrosine phosphorylation occurring in platelets after activation with collagen in suspension or under flow conditions. For the suspension studies, washed platelets were activated with different concentrations of purified type I collagen (ColI). Studies under flow conditions were performed using two different adhesive substrata: ColI and endothelial cells extracellular matrix (ECM). Coverslips coated with ColI or ECM were perfused through a parallel-plate perfusion chamber at 800 s(-1) for 5 min. After activation of platelets either in suspension or by adhesion, samples were solubilized and proteins were resolved by electrophoresis. Tyrosine-phosphorylated proteins were detected in immunoblots by specific antibodies. Activation of platelet suspensions with collagen induced tyrosine phosphorylation before aggregation could be detected. Profiles showing tyrosine-phosphorylated proteins from platelets adhered on ColI or on ECM were almost identical and lacked proteins p95, p80, p66, and p64, which were present in profiles from platelets activated in suspension. The intensity of phosphorylation was quantitatively weaker in those profiles from platelets adhered on ECM. Results from the present work indicate that activation of platelets in suspension or by adhesion induces differential tyrosine phosphorylation patterns. Phosphorylation of proteins p90 and p76 may be related to early activation events occurring during initial contact and spreading of platelets. Considering that adhesion is the first step of platelet activation, studies on signal transduction mechanisms under flow conditions may provide new insights to understand the signaling processes taking place at earliest stages of platelet activation.     

Endothelial Progenitor Cells Predict Cardiovascular Events after Atherothrombotic Stroke and Acute Myocardial Infarction. A PROCELL Substudy

Introduction

The aim of this study was to determine prognostic factors for the risk of new vascular events during the first 6 months after acute myocardial infarction (AMI) or atherothrombotic stroke (AS). We were interested in the prognostic role of endothelial progenitor cells (EPC) and circulating endothelial cells (CEC)

Methods

Between February 2009 and July 2012, 100 AMI and 50 AS patients were consecutively studied in three Spanish centres. Patients with previously documented coronary artery disease or ischemic strokes were excluded. Samples were collected within 24h of onset of symptoms. EPC and CEC were studied using flow cytometry and categorized by quartiles. Patients were followed for up to 6 months. NVE was defined as new acute coronary syndrome, transient ischemic attack (TIA), stroke, or any hospitalization or death from cardiovascular causes. The variables included in the analysis included: vascular risk factors, carotid intima-media thickness (IMT), atherosclerotic burden and basal EPC and CEC count. Multivariate survival analysis was performed using Cox regression analysis.

Results

During follow-up, 19 patients (12.66%) had a new vascular event (5 strokes; 3 TIAs; 4 AMI; 6 hospitalizations; 1 death). Vascular events were associated with age (P = 0.039), carotid IMT≥0.9 (P = 0.044), and EPC count (P = 0.041) in the univariate analysis. Multivariate Cox regression analysis showed an independent association with EPC in the lowest quartile (HR: 10.33, 95%CI (1.22–87.34), P = 0.032] and IMT≥0.9 [HR: 4.12, 95%CI (1.21–13.95), P = 0.023].

Conclusions

Basal EPC and IMT≥0.9 can predict future vascular events in patients with AMI and AS, but CEC count does not affect cardiovascular risk.     

Reproductive performance of the wild collared peccary (Tayassu tajacu) female in the Peruvian Amazon

Based on the gross anatomic examination of 218 adult female reproductive tracts, we characterised the reproductive biology of the wild collared peccary in the Northeastern Peruvian Amazon. Collared peccary apparently breed year round in the Northeastern Peruvian Amazon and the number of pregnant females was 92 (42.2%). Estimations suggest 1.12 births/year and a yearly reproductive production of 1.98 piglets per pregnant female. Collared peccary females presented a mean ovulation rate of 2.25 ± 0.58 CLs, a litter size of 1.77 ± 0.48 embryos or foetuses and a reproductive wastage of 0.45 ± 0.65 (21.3%) oocytes or embryos per pregnant female. A phenomenon of embryo migration was observed in 31.8% of pregnant females. This study provides reproductive information that should be taken into account in management plans.     

Effect of hydrogen peroxide on secretory response, calcium mobilisation and caspase-3 activity in the isolated rat parotid gland

The parotid glands are highly active secretory systems subjected to continuous stress, which in turn, can lead to several pathophysiological conditions. Damage of the parotid glands are caused by radical oxygen species (ROS) as by-products of oxygen metabolism. This study investigated the effect of hydrogen peroxide (H(2)O(2)) on Carbachol (CCh)-evoked secretory responses and caspase-3 activity in the isolated rat parotid gland to understand the role of oxidative stress on the function of the gland. Amylase secretion, cytosolic calcium concentration ([Ca(2+)](i)) and caspase-3 activity in parotid gland tissue were measured using fluorimetric methods. H(2)O(2) had little or no effect on amylase secretion compared to basal level. Combining H(2)O(2) with CCh resulted in an attenuation of the CCh-evoked amylase secretion compared to the effect of CCh alone. CCh can evoke a large increase in [Ca(2+)](i) comprising an initial peak followed by a plateau. In a Ca(2+)-free medium containing 1 mM EGTA, CCh evoked only the initial peak of [Ca(2+)](i). H(2)O(2) alone evoked a gradual and dose-dependent increase in [Ca(2+)](i). Combining H(2)O(2) with CCh resulted in a decrease in [Ca(2+)](i) compared to the effect of CCh alone. In a Ca(2+)-free medium, H(2)O(2) still evoked a small increase in [Ca(2+)](i), but this response was less compared to the results obtained with H(2)O(2) in normal [Ca(2+)](0). Combining H(2)O(2) with CCh resulted in only a small transient increase in [Ca(2+)](i). Following CCh stimulation, H(2)O(2) application resulted in a large increase in [Ca(2+)](i) in normal [Ca(2+)](0). This effect of H(2)O(2) was partially abolished in a nominally free Calcium medium containing EGTA. H(2)O(2) can stimulate caspase-3 activity in parotid gland tissue. Similar response was obtained with betulinic acid and thapsigargin (TPS) on caspase-3 activity compared to basal. The results have demonstrated that like CCh, H(2)O(2) can also mobilise Ca(2+) from intracellular stores and facilitate its influx into the cell from extracellular medium. This effect of H(2)O(2) may be due to its activity to induce apoptosis in the parotid gland, since H(2)O(2) can stimulate the activity of caspase-3, a marker of cellular apoptosis.