Evaluation of ammonium and phosphate release from intertidal and subtidal sediments of a shallow coastal lagoon (Ria Formosa – Portugal): a modelling approach

During an annual cycle, overlying water and sediment cores were collected simultaneously at three sites (Tavira, Culatra and Ramalhete) of Ria Formosa’s intertidal muddy and subtidal sandy sediments to determine ammonium, nitrates plus nitrites and phosphate. Organic carbon, nitrogen and phosphorus were also determined in superficial sediments. Ammonium and phosphate dissolved in porewater were positively correlated with temperature (P < 0.01) in muddy and sandy sediments, while the nitrogen-oxidized forms had a negative correlation (P < 0.02) in muddy sediments probably because mineralization and nitrification/denitrification processes vary seasonally. Porewater ammonium profiles evidenced a peak in the top-most muddy sediment (380 μM) suggesting higher mineralization rate when oxygen is more available, while maximum phosphate concentration (113 μM) occurred in the sub-oxic layer probably due to phosphorus desorption under reduced conditions. In organically poor subtidal sandy sediments, nutrient porewater concentrations were always lower than in intertidal muddy sediments, ranging annually from 20 μM to 100 μM for ammonium and from 0.05 μM to 16 μM for phosphate. Nutrient diffusive fluxes predicted by a mathematical model were higher during summer, in both muddy (104 nmol cm⁻² d⁻¹––NH₄⁺; 8 nmol cm⁻² d⁻¹––HPO₄⁻²) and sandy sediments (26 nmol cm⁻² d⁻¹––NH₄⁺; 1 nmol cm⁻² d⁻¹––HPO₄⁻²), while during lower temperature periods these fluxes were 3–4 times lower. Based on simulated nutrient effluxes, the estimated annual amount of ammonium and phosphate exported from intertidal areas was three times higher than that released from subtidal areas (22 ton year⁻¹––NH₄⁺; 2 ton year⁻¹––HPO₄⁻²), emphasizing the importance of tidal flats to maintain the high productivity of the lagoon. Global warming scenarios simulated with the model, revealed that an increase in lagoon water temperature only produces significant variations (P < 0.05) for NH₄⁺ in porewater and consequent diffusive fluxes, what will probably affect the system productivity due to a N/P ratio unbalance.     

In situ measurement of pH in the secreting canaliculus of the gastric parietal cell and adjacent structures

The gastric H⁺/K⁺-ATPase is located within an infolding (secretory canaliculus) of the apical plasma membrane of gastric parietal cells. Our aim was to measure the pH values in the cytosol and canaliculus of the acid-secreting parietal cell and the adjacent gland lumen in situ. We used ultrafine double-barreled tip-sealed microelectrodes at high acceleration rates for intracellular and canalicular measurements. Immunohistochemical staining of the parietal cells was used to identify the track of the electrode and to estimate the position of the electrode tip at the time of the last intracellular measurement. En route to the deepest regions of the mucosa, where the average gland lumen pH was approximately 3, and on advancing in steps of 2 μm, the electrode entered the cytosol of the parietal cells, where the pH value was 7.4. Advancing the electrode further resulted, in several instances, in a sharp decrease in pH to an average value of 1.7 ± 0.2, which we interpreted as the measurement within the canaliculus. When the electrode was advanced even further, the pH reading returned to the cytosolic value. From the difference in pH between the secreting canaliculus and the adjacent gland lumen, we concluded that the released acid was immediately buffered. Thus, the only cellular structure directly exposed to the highly acidic canalicular content is the apical membrane forming the canaliculus in the parietal cell.     

Assessment of three-dimensional biofilm structure using an optical microscope

A method allowing the evaluation of the structure and the calculation of the volume of a biofilm, using an optical microscope, is proposed based on the linear relation between the intensity of a pixel in biofilm images grabbed on the x-y plane and the corresponding number of cells in the z direction, which allows the calculation of the biofilm thickness. The method is intended to overcome the need for expensive microscopes to study biofilms.     

Genomic checkpoints for exon 10 usage in the luteinizing hormone receptor type 1 and type 2

Alternative splicing is a hallmark of glycoprotein hormone receptor gene regulation, but its molecular mechanism is unknown. The LH receptor (LHR) gene possesses 11 exons, but exon 10 is constitutively skipped in the New World monkey lineage (LHR type 2), whereas it is constitutively spliced in the human (LHR type 1). This study identifies the regulatory elements of exon 10 usage. Sequencing of genomic marmoset DNA revealed that the cryptic LHR exon 10 is highly homologous to exon 10 from other species and displays intact splice sites. Functional studies using a minigene approach excluded the contribution of intronic, marmoset-specific long interspersed nucleotide-1 elements to exon 10 skipping. Sequencing of the genomic regions surrounding exon 10 from several primate lineages, sequence comparisons including the human and mouse LHR gene, revealed the presence of unique nucleotides at 3'-intronic position -19 and -10 and at position +26 within exon 10 of the marmoset LHR. Exon trap experiments and in vitro mutagenesis of these nucleotides resulted in the identification of a composite regulatory element of splicing consisting of cis-acting elements represented by two polypyrimidine tracts and a trans-acting element within exon 10, which affect the secondary RNA structure. Changes within this complex resulted either in constitutive exon inclusion, constitutive skipping, or alternative splicing of exon 10. This work delineates the molecular pathway leading to intronization of exon 10 in the LHR type 2 and reveals, for the first time, the essential function of regulatory and structural elements involved in glycoprotein hormone receptor splicing.     

Discovery of a regular nesting area of loggerhead turtle <Emphasis Type="Italic">Caretta caretta</Emphasis> in southern Italy: a new perspective for national conservation

Loggerhead turtle Caretta caretta nesting in Italy had been reported to be limited to the Pelagian Islands and only sporadically elsewhere. As presence of loggerhead turtle nests had occasionally been reported (1988–1999) along about 200 km of the Ionian coast of Calabria, we carried out a project to assess the actual state of the nesting population between 2000 and 2004. We divided the coastline in two sectors (A: 52 km, and B: 146 km) that were monitored from mid-June to end of July for a total of n = 174 monitoring days and 1,813.6 km patrolled on foot with different intensities (extensive versus intensive). In sector B, through extensive monitoring we did not find any emergence tracks, but in sector A by intensive survey (2002–2004: one survey/3.64 days) we detected 3–8 nests/year. In total, 25 nests (both observed and reported), were recorded in our study area, and an assessment of a total of 15–16 nests/year was suggested. These figures, within the national scenario depicted from the review of known nesting events in the last 40 years (88 records concerning more than 143–144 nests), show that loggerhead turtle nesting has been underestimated in Italy, due to inadequate monitoring protocols, and that nesting is more frequent than expected (at least 30–40 nests/year). Conservation strategies in Italy should then focus not only on the reduction of mortality at sea, but also include large-scale actions to preserve scattered (but regular) nesting events.     

Mycobacterium parascrofulaceum in Acidic Hot Springs in Yellowstone National Park

Mycobacterium parascrofulaceum was found in Norris Geyser Basin, Yellowstone National Park, in a system composed of two acidic (pH 3.0) springs with temperatures between 56°C at the source and 40°C at the confluence of both springs. Growth and survival assays at 56°C for 60 days were performed, confirming the origin of the strain.     

SIMPLE Approach for Isolating Mutants Expressing Fimbriae

Genomes of members of the family Enterobacteriaceae contain large repertoires of putative fimbrial operons. Since many of these operons are poorly expressed in vitro, a convenient method for inducing elaboration of the encoded fimbriae would greatly facilitate their functional characterization. Here we describe a new technique for identifying fimbriated bacteria from a library of transposon mutants by screening with immunomagnetic particles for ligand expression (SIMPLE). The SIMPLE method was applied to identify the T-POP mutants of Salmonella enterica serotype Typhimurium carrying on their surfaces filaments composed of PefA, the major subunit product of a fimbrial operon (pef) that is not expressed during growth in Luria-Bertani broth. Four such mutants were identified from a library of 24,000 mutants, each of which carried a T-POP insertion within the hns gene, which encodes a global silencer of horizontally acquired genes. Our data suggest that the SIMPLE method is an effective approach for isolating fimbriated bacteria, which can be readily applied to fimbrial operons identified by whole-genome sequencing.