Coastal armouring affects intertidal biodiversity across the Alboran Sea (Western Mediterranean Sea).

Intertidal ecosystems are key habitats that are being replaced by artificial hard substrates due to the increment of human activities in coastal areas. These new substrates host generally less biodiversity mainly due to differences in complexity and composition. This is a global phenomenon and has led to the development of strategies in the framework of eco-engineering. However, mitigating measures, such as new eco-designs, must cope with the high spatial variability of the region where they are applied. Therefore, in order to assess if differences in biodiversity detected at local scales in previous studies could be scaled up to predict patterns at a wider scale, we studied taxa richness and taxonomic structure of intertidal communities across the Alboran Sea (western Mediterranean Sea). We compared four different types of artificial substrates (cubes, rip-raps, seawalls and tetrapods) to assess which produces less impact. Overall, artificial substrates host low benthic biodiversity, specially on seawalls, whereas boulder-like artificial structures such as rip-raps were more similar to natural ones. Nevertheless, the effect of a particular type of artificial structure at a regional scale seems unpredictable, highlighting the challenge that eco-engineering measures face in order to establish global protocols for biodiversity enhancement and the importance of local scale in management programmes.

A toddler with phylloid-type pigmentary mosaicism and ambiguous genitalia resulting from trisomy 14 induced by a der(Y)t(Y;14).

A 1-year-old baby with phylloid-type pigmentary mosaicism, hypotonia, ambiguous genitalia, and a positive screening test for congenital adrenal hyperplasia was referred. Previous sonograph, cytogenetics, and metabolic profile were inconclusive, therefore we performed an additional karyotype and a molecular cytogenetics studies. A mosaic karyotype 45,X/46,X,der(Y)t(Y;14) was characterized in peripheral blood. Congenital adrenal hyperplasia genes were sequenced and the results were negative. The ambiguous genitalia was the result of the special gonosomal mosaicism. The low level of trisomy 14 led to minor physical characteristics and mild mental retardation; also, Turner syndrome features can be expected rather than severe trisomy 14 stigmata.

Evaluación clínica y hematológica de pacientes con drepanocitosis esplenectomizados en el Instituto de Hematología e Inmunología de Cuba: 30 años de experiencia / Clinical and hematological evaluation of patients with splenectomized drepanocytosis at the Hematology and Immunology Institute of Cuba: 30 years of experience

Introducción: Desde 1986 se ha introducido en nuestro país la esplenectomía parcial, que logra prevenir la recurrencia de la crisis de secuestro esplénico y disminuir la incidencia de sepsis sobreaguda postesplenectomía. Objetivo: Comparar desde el punto de vista clínico y de laboratorio los pacientes con esplenectomía total y parcial. Pacientes y métodos: Se estudiaron todos los pacientes con drepanocitosis, seguidos en el Instituto de Hematología e Inmunología, que se hubieran sometido a esplenectomía durante la edad pediátrica. Resultados: Se incluyeron 39 pacientes en cada método de esplenectomía; la mayoría eran varones (60,3%) y predominaba la anemia drepanocítica (69,2%). La edad de aparición de la esplenectomía fue menor en la de tipo parcial (4,7 años; p= 0,009) que en la total (6,7 años), y el tiempo de seguimiento fue de 12,5 años. El grupo de esplenectomía total presentó un mayor aumento de hemoglobina, leucocitos y plaquetas (p= 0,039), así como valores elevados de lactato deshidrogenasa (p= 0,015), hemoglobina plasmática (p= 0,001) y velocidad de regurgitación tricuspídea (p= 0,038). La crisis vasooclusiva dolorosa fue más frecuente tras la esplenectomía total (75,8 ± 14,3 frente a 39,8 ± 10,1; p < 0,001), al igual que las úlceras maleolares (p= 0,04). La crisis hepática y la mortalidad fueron también más frecuentes en la esplenectomía total aunque sin significación estadística (p= 007 y p= 0,305, respectivamente). Conclusiones: La esplenectomía parcial presenta menos complicaciones a largo plazo que la total (AU)

Introduction: Since 1986 has been introduced in our country partial splenectomy, which prevent the recurrence of splenic sequestration crisis and possible reduce the number of overhelming septicemia. Objective: Compare clinical and laboratory aspects in patients with total and partial splenectomy. Patients and methods: All patients with sickle cell disease were studied, followed at the Instituto de Hematología e Inmunología, who have had a splenectomy in childhood. Results: 39 patients were included in each method of splenectomy, where a predominance of males (60.3%) and sickle cell anemia (69.2%). Splenectomy age was lower in the partial, 4.7 years than in total splenectomy, 6.7 years (0.009). Follow-up time was 12.5 years. Total splenectomy group had greater increase in hemoglobin, leucocytes and platelets (p= 0.039), elevated LDH levels (p= 0.015), plasma hemoglobin (p= 0.001) and tricuspid regurgitation velocity (p= 0.038). Vaso-occlusive painful crises was more frequent after total splenectomy (75.8 ± 14.3 vs. 39.8 ± 10.1; p <0.001), as leg ulcer (p= 0.04). Hepatic crisis (p <0.07) and mortality were higher in individuals with complete splenectomy (p= 0.305) but withouth significative statistics. Conclusions: Partial splenectomy has fewer long-term complications that total (AU)

Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme.

The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.

High performance liquid chromatographic method for the determination of lobenzarit disodium in a sustained release tablet formulation.

A rapid and simple high performance liquid chromatographic method is described and validated for the determination of lobenzarit disodium (CAS 64808-48-6) in a sustained release tablet formulation. The calibration graph was linear over the range 20-105 micrograms/ml. The sensitivity (discriminator capacity) was 2.079 micrograms/ml. The coefficient of variations for repeatability and reproducibility were less than 1.60% and 1.30%, respectively. The accuracy of the method did not depend on lobenzarit concentration in tablets. The mean recovery was found to be 100.62%. The method was selective, even when degradation products were present.

A sensor protein involved in induction of nitrate assimilation in Azotobacter chroococcum.

Nitrogen-fixing Azotobacter chroococcum cells, but not ammonium- or nitrate-grown cells, exhibited two polypeptide components of 22 and 35 kDa, respectively, that we termed P22 and P35. Bidimensional polyacrylamide gel electrophoresis analysis of preparations from N2-fixing cells that had been transferred to nitrate medium and then incubated for 2 h revealed that P22 had shifted to a more acidic part of the gel while P35 did not change its electrophoretic pattern. Using [32P]orthophosphoric acid it could be demonstrated that the shift in mobility of P22 was due to the phosphorylation of the polypeptide dependent on nitrate (nitrite). The A. chroococcum TR1 strain, which is unable to use nitrate as a nitrogen source and displays activities of nitrogenase, nitrate reductase and nitrite reductase, exhibited both polypeptides. In contrast, P22 and P35 were absent from A. chroococcum MCD1, a mutant strain that cannot assimilate nitrate and lacks the nitrate-reducing enzymatic system. The results suggest that P22 could act as a sensor protein for nitrate in A. chroococcum.

Posttranslational regulation of nitrogenase activity by fixed nitrogen in Azotobacter chroococcum.

Using anti-(Fe protein) antibody raised against the Fe protein of the photosynthetic bacterium Rhodospirillum rubrum, it was found that the Fe protein component of nitrogenase (EC 1.18.2.1) from Azotobacter chroococcum cells subjected to an ammonium shock, and hence with an inactive nitrogenase, appeared as a doublet in Western blot analysis of cell extracts. The Fe protein incorporated [32P]phosphate and [3H]adenine in response to ammonium treatment, and L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase (L-glutamate: ammonia ligase (ADP forming), EC 6.3.1.2), prevented Fe protein from inhibition and radioisotope labelling. These results support that A. chroococcum Fe protein is most likely ADP-ribosylated in response to ammonium. After ammonium treatment, when in vivo activity was completely inhibited, Fe-protein modification was still increasing. This suggests the existence of another mechanism of nitrogenase inhibition faster than Fe-protein modification. When ammonium was intracellularly generated instead of being externally added, as occurs with the short-term nitrate inhibition of nitrogenase activity observed in A. chroococcum cells simultaneously fixing molecular nitrogen and assimilating nitrate, a covalent modification of the Fe protein was likewise demonstrated.

In vivo modification of Azotobacter chroococcum glutamine synthetase.

A monospecific anti-(glutamine synthetase) antibody raised against glutamine synthetase of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 immunoreacted with glutamine synthetase from the N2-fixing heterotrophic bacterium Azotobacter chroococcum. In Western-blotting experiments this antibody recognized a single protein of a molecular mass of 59 kDa corresponding to glutamine synthetase subunit. This protein was in vivo-labelled in response to addition of ammonium, both [3H]adenine and H(3)32PO4 preincubation of the cells being equally effective. Nevertheless, the amount of glutamine synthetase present in A. chroococcum was independent of the available nitrogen source. Modified, inactive glutamine synthetase was re-activated by treatment with snake-venom phosphodiesterase but not by alkaline phosphatase. L-Methionine-DL-sulphoximine, an inhibitor of glutamine synthetase, prevented the enzyme from being covalently modified. We conclude that, in A. chroococcum, glutamine synthetase is adenylylated in response to ammonium and that for the modification to take place ammonium must be metabolized.

Detection of alginate lyase by activity staining after sodium dodecil sulfate-polyacrylamide gel electrophoresis and subsequent renaturation.

A slab gel electrophoretic method for the study of bacterial alginate lyase has been developed. By incorporating alginate in acrylamide gels, the method is based on renaturation of the enzyme after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent staining with cetylpyridinium chloride, and quantification of the spot by densitometric scanning. The molecular mass of alginate lyase can be determined from its position in the gel.

Production of exocellular polysaccharide by Azotobacter chroococcum.

Environmental conditions affect the production of extracellular polysaccharide by Azotobacter chroococcum ATCC 4412. Production of exocellular polymer from a variety of carbon sources depended on the air flow rate. A high sucrose concentration in medium (8%) markedly favored expopolysaccharide production, which reached 14 g/L in about 72 h. In cell suspensions incubated in the presence of 8% sucrose in a nitrogen-free medium, biopolymer final concentration of 9 g/L corresponds to 68 g/g biomass. Maximum efficiency of sucrose conversion into exopolysaccharide peaked at 70% for initial disaccharide concentration of 6%. High performance liquid chromatography and gas liquid chromatography of acid hydrolysates of the exopolymer revealed the presence of mannuronosyl, guluronosyl, and acetyl residues, but not neutral sugars. The infrared spectrum corroborated the presence of carboxylate anions and O-acetyl groups in the exopolymer. Though the presence of more than one kind of polysaccharide cannot be ruled out, these data suggest that, under the experimental conditions used in this work, only a type of alginate-like exopolysaccharide is produced by A. chroococcum ATCC 4412.

Short-term nitrate (nitrite) inhibition of nitrogen fixation in Azotobacter chroococcum.

Nitrate-grown Azotobacter chroococcum ATCC 4412 cells lack the ability to fix N2. Nitrogenase activity developed after the cells were suspended in a combined nitrogen-free medium and was paralleled by a concomitant decrease in nitrate assimilation capacity. In such treated cells exhibiting transitory nitrate assimilation and N2-fixation capacity, nitrate or nitrite caused a short-term inhibitory effect on nitrogenase activity which ceased once the anion was exhausted from the medium. The analog L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase, prevented inhibition of nitrogenase activity by nitrate or nitrite without affecting the uptake of these antions, which were reduced and stoichiometrically released into the external medium as ammonium. Inhibition of nitrogenase by nitrate (nitrite) did not take place in A. chroococcum MCD1, which is unable to assimilate either. We conclude that the short-term inhibitory effect of nitrate (nitrite) on nitrogenase activity is due to some organic product(s) formed during the assimilation of the ammonium resulting from nitrate (nitrite) reduction.

Short-term ammonium inhibition of nitrogen fixation in Azotobacter.

Addition of NH4Cl at low concentrations to Azotobacter chroococcum cells caused an immediate cessation of nitrogenase activity, which was recovered once the added NH+4 was exhausted from the medium. In the presence of inhibitors of ammonium assimilation, such as L-methionine-DL-sulfoximine, L-methionine sulfone or 6-diazo-5-oxo-L-norleucine, externally added NH+4 had no effect on nitrogenase activity and the newly-fixed nitrogen was excreted into the medium as NH+4. It is concluded that, in A. chroococcum, NH+4 must be assimilated to exert its short-term inhibitory effect on nitrogen fixation.