Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis

The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu515) to the beginning of S6 (Ala560). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro527 and Ile541 was compatible with the presence of a alpha-helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions -1, 0, +1, +2 around Asp542 (high Ca2+ affinity site) were non-functional. Whole cell currents of cysteine mutants at +4 and +5 positions were however covalently inhibited by external MTSET and MTSES. Altogether, the pattern of covalent modification by MTS reagents globally supports a KcsA homology-based three-dimensional model whereby the external vestibule in ECaC-TRPV5 encompasses three structural domains consisting of a coiled structure (Glu515 to Tyr526) connected to a small helical segment of 15 amino acids (527PTALFSTFELFLT539) followed by two distinct coiled structures Ile540-Pro544 (selectivity filter) and Ala545-Ile557 before the beginning of S6.     

Sulfate is transported at significant rates through the symbiosome membrane and is crucial for nitrogenase biosynthesis

Legume–rhizobia symbioses play a major role in food production for an ever growing human population. In this symbiosis, dinitrogen is reduced (“fixed”) to ammonia by the rhizobial nitrogenase enzyme complex and is secreted to the plant host cells, whereas dicarboxylic acids derived from photosynthetically produced sucrose are transported into the symbiosomes and serve as respiratory substrates for the bacteroids. The symbiosome membrane contains high levels of SST1 protein, a sulfate transporter. Sulfate is an essential nutrient for all living organisms, but its importance for symbiotic nitrogen fixation and nodule metabolism has long been underestimated. Using chemical imaging, we demonstrate that the bacteroids take up 20‐fold more sulfate than the nodule host cells. Furthermore, we show that nitrogenase biosynthesis relies on high levels of imported sulfate, making sulfur as essential as carbon for the regulation and functioning of symbiotic nitrogen fixation. Our findings thus establish the importance of sulfate and its active transport for the plant–microbe interaction that is most relevant for agriculture and soil fertility.     

El carcinoma diferenciado incidental de tiroides es menos prevalente en la enfermedad de Graves que en el bocio multinodular

ObjectiveRisk factors for differentiated thyroid carcinoma (DTC) are poorly understood, but serum TSH levels, thyroid nodularity, and presence of autoimmunity are well-recognized factors that modulate DTC prevalence. TSH stimulates proliferation of both normal and neoplastic follicular cells. Consequently, thyroid-stimulating immunoglobulins (TSI), because of its TSH-like action, should induce DTC progression in patients with Graves’ disease (GD). The study objective was to compare the prevalence of incidental DTC in patients undergoing thyroidectomy for benign thyroid disease.MethodsThe pathology reports of 372 patients with preoperative diagnosis of euthyroid multinodular goiter (EMG) or hyperthyroidism were reviewed. Scintigraphy results and serum TSI levels were used to diagnosed either GD or hyperactive MG (HMG) to hyperthyroid subjects. Prevalence of DTC in each category was calculated using a Chi-square test.ResultsEMG, GD, and HMG were diagnosed in 221, 125, and 26 patients. There were 58 DTCs, distributed as follows [n (%)]: EMG, 49 (22.2%); GD, 8 (6.4%), and HMG, 1 (3.8%). Difference in prevalence of incidental DTC between the groups was statistically significant (p < 0.001). After adjustment for age, patients with EMG had a greater DTC prevalence than GD patients, with an OR of 4.17 (p < 0.001). Tumor size (mm, mean ± SD) was 6.92 ± 11.26, 1.97 ± 1.85, and 9.0 for EMG, GD and HMG respectively (p = 0.017).ConclusionsIncidental DTC was less prevalent in GD as compared to EMG irrespective of age. This finding may suggest a predisposition to develop DTC in patients with thyroid nodular disease and/or a potential effect of autoimmunity to protect against development of neoplastic disease.     

Effect of lead stress on mineral content and growth of wheat (Triticum aestivum) and spinach (Spinacia oleracea) seedlings

Lead (Pb) is the most common heavy metal contaminant in the environment. Pb is not an essential element for plants, but they absorb it when it is present in their environment, especially in rural areas when the soil is polluted by automotive exhaust and in fields contaminated with fertilizers containing heavy metal impurities. To investigate lead effects on nutrient uptake and metabolism, two plant species, spinach (Spinacia oleracea) and wheat (Triticum aestivum), were grown under hydroponic conditions and stressed with lead nitrate, Pb(NO₃)₂, at three concentrations (1.5, 3, and 15 mM).Lead is accumulated in a dose-dependent manner in both plant species, which results in reduced growth and lower uptake of all mineral ions tested. Total amounts and concentrations of most mineral ions (Na, K, Ca, P, Mg, Fe, Cu and Zn) are reduced, although Mn concentrations are increased, as its uptake is reduced less relative to the whole plant’s growth. The deficiency of mineral nutrients correlates in a strong decrease in the contents of chlorophylls a and b and proline in both species, but these effects are less pronounced in spinach than in wheat. By contrast, the effects of lead on soluble proteins differ between species; they are reduced in wheat at all lead concentrations, whereas they are increased in spinach, where their value peaks at 3 mM Pb.The relative lead uptake by spinach and wheat, and the different susceptibility of these two species to lead treatment are discussed.     

Linfoma pulmonar primario no Hodgkin con infiltración de miocardio

A case of primary pulmonary non-Hodgkin's lymphoma is presented. On this occasion, the lymphoma invaded the myocardium, an event which has not previously been reported in the literature. These neoplasms spread by proximity, and invasion of the pericardium, thoracic wall and oesophagus have been described. Our patient died from heart failure. Tumour myocardial infiltration may well have been the determinant cause through various mechanisms, including a decrease in myocardial contractility. Spread into the myocardium may be facilitated by bulky tumour infiltrates in the pleural space.     

Iron and vitamin status biomarkers and its association with physical fitness in adolescents: the HELENA study

There is a lack of studies that analyze the association between micronutrient-related biomarker status and physical fitness in adolescents. In the present study, biochemical parameters for iron and vitamin status were studied, along with objective measures of physical fitness in healthy male and female European adolescents. One thousand eighty-nine adolescents (580 girls, 12.5-17.5 yr) from the Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) cross-sectional study were included. Hierarchical linear models were performed to determine the associations between micronutrient biomarkers and physical fitness. Age, seasonality, latitude, body mass index, menarche (in girls), and physical activity were used as covariates. For cardiorespiratory fitness, concentrations of hemoglobin, retinol, and vitamin C in male adolescents and β-carotene and 25(OH)D in female adolescents were associated with maximal oxygen consumption. For muscular fitness, concentrations of hemoglobin, β-carotene, retinol, and α-tocopherol in male adolescents and β-carotene and 25(OH)D in female adolescents were associated with better performance of the standing long jump test. In summary, concentrations of hemoglobin and most antioxidant vitamins in male adolescents and β-carotene and 25(OH)D in female adolescents were positively associated with cardiorespiratory and muscular fitness, after controlling for relevant confounders. The associations between physical fitness and iron or vitamin status observed in this cross-sectional study in adolescents should be followed up by a study specifically designed to evaluate causal relationships.     

Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

Summary The 10-kb chromosomal fragment of Streptococus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome.A spontaneous deletion beginning 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (10^-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed.Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanil-amide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene. Transfer of the plasmids to B. subtilis gave rise to large deletions to the left of the sul-d marker, but these deletions did not interfere with the sul-d gene function. Restriction maps of pLS80 and its variously deleted derivatives are presented.     

Fitness of the pMV158 replicon in Streptococcus pneumoniae

Promiscuous, rolling-circle replication plasmid pMV158 determines tetracycline resistance to its host and can be mobilized by conjugation. Plasmid pLS1 is a deletion derivative of pMV158 that has lost its conjugative mobilization ability. Both plasmids replicate efficiently and are stably inherited in Streptococcus pneumoniae. We have analyzed the effect of pMV158 and pLS1 carriage on the bacterial growth rate. Whereas the parental plasmid does not significantly modify the cell doubling time, pLS1 slows down the growth of the bacterial host by 8-9%. The bases of the differential burden caused by pMV158 and pLS1 carriage are not yet understood. The negligible cost of the pMV158 parental natural plasmid on the host might explain the prevalence of small, multicopy, rolling-circle replication plasmids, even though they lack any selectable trait.     

Integrin-linked kinase (ILK) modulates wound healing through regulation of hepatocyte growth factor (HGF)

Integrin-linked kinase (ILK) is an intracellular effector of cell–matrix interactions and regulates many cellular processes, including growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. The present work analyzes the role of ILK in wound healing in adult animals using a conditional knock-out of the ILK gene generated with the tamoxifen-inducible Cre-lox system (CRE-LOX mice). Results show that ILK deficiency leads to retarded wound closure in skin. Intracellular mechanisms involved in this process were analyzed in cultured mouse embryonic fibroblast (MEF) isolated from CRE-LOX mice and revealed that wounding promotes rapid activation of phosphatidylinositol 3-kinase (PI3K) and ILK. Knockdown of ILK resulted in a retarded wound closure due to a decrease in cellular proliferation and loss of HGF protein expression during the healing process, in vitro and in vivo. Alterations in cell proliferation and wound closure in ILK-deficient MEF or mice could be rescued by exogenous administration of human HGF. These data demonstrate, for the first time, that the activation of PI3K and ILK after skin wounding are critical for HGF-dependent tissue repair and wound healing.