Cytosolic thymidine kinase activity in cultured human fibroblasts from individuals with galactokinase deficiency

The structural genes for human galactokinase (GALK) and the human cytosolic form of thymidine kinase (TK1) are located on 17q21–q22. These two loci are tightly linked, and studies on Chinese hamster cell lines have shown that the expression of TK1 and GALK genes may alter simultaneously. We investigated the possibility of a dependent mutation of TK1 and GALK genes in cultured fibroblasts obtained from two patients homozygous for the GALK^G-deficient gene. Since we showed that the TK1 level varies as a function of the passage and the growth rate of a given strain, our experiments were performed on nonstored skin fibroblasts, between the third and the fifth passage for both controls and patients. We found that TK1 levels in GALK-deficient cells were almost 75% of those observed in control strains with a similar growth rate. Previous results in the literature have shown a pronounced decrease in TK1 activity in three GALK-deficient fibroblastic strains. We suggest that these disparities of TK1 levels in GALK-deficient fibroblasts may be related either to genetic heterogeneity of GALK deficiency or to differences in culture conditions. This work was supported in part by grants from La CNAMTS and l’Université de Paris-Sud (AI 86 10).     

Influence of water chemistry and environmental degradation on macroinvertebrate assemblages in a river basin in south-east Brazil

Benthic macroinvertebrate assemblages, water chemistry variables and environmental degradation were investigated in an Atlantic Forest region in Brazil. Seven sites of the Guapimirim river basin were studied during three sampling periods based on the rain regime: end of wet season (May 1998), dry season (August 1998), and wet season (January 1999). Four substrates were collected at each site: sand, stony substrates, litter in pool areas and litter in riffle areas. Relationships between macroinvertebrate assemblages, water chemistry variables and environmental degradation were examined using canonical correspondence analysis (CCA). According to CCA, concentrations of dissolved oxygen and chloride, and the environmental degradation, measured by the Riparian Channel Environment index, exhibited the strongest relationship to macroinvertebrate assemblages. Overall, the loss of community diversity measured by the Shannon Index along the degradation gradient was observed. Some taxa were shown to be sensitive to water pollution, especially among Plecoptera, Trichoptera, Coleoptera and some Ephemeroptera, while others such as Simuliidae, Odonata and molluscs were tolerant to moderate levels of pollutants. The Chironomidae were the only group tolerant to a high level of pollutants and degradation.     

Does the flatfish community of the Mondego estuary (Portugal) reflect environmental changes?

Temporal and spatial patterns of abundance of the flatfish community in the Mondego estuary were investigated from 2003 to 2007, based on monthly beam trawl samples. During the study period there was a severe drought, with consequential reductions in river runoff. A total of eight flatfish species occurred within the estuary, with seasonal specific richness and abundance varying considerably. It was possible to identify three main groups: one composed of species present throughout the study period and in high densities: Platichthys flesus and Solea solea (maximum densities 3.7 and 4.1 Ind 1000 m^?2, respectively); a second group of less abundant but regularly present species: Scophthalmus rhombus and Solea senegalensis (maximum densities 0.2 and 0.3 Ind 1000 m^?2, respectively); and a third group of occasional species that occurred only during the drought period: Arnoglossus laterna, Buglossidium luteum, Dicologlossa hexophthalma and Pegusa lascaris. Flatfish distribution patterns varied according to the estuarine use guild: marine‐estuarine dependent fish occurred mainly in the upper reaches, while marine stragglers and marine‐estuarine opportunists occurred mostly in the downstream areas. Species with more northern latitudinal affinities were the most affected by the drought, related with a lesser extent of river plumes to the coastal area, resulting in a reduction in half of the abundance levels. However, flatfish species with more southern affinities increased in abundance during the drought, benefiting also from an increase in estuarine water temperature. Early summer salinity and precipitation values were good proxies for estimating abundance levels of P. flesus and S. solea, respectively, emphasizing the importance of hydrodynamics for the recruitment and abundance of these commercially important species.     

Calcium transport and homeostasis in gill cells of a freshwater crab Dilocarcinus pagei

Crustaceans present a very interesting model system to study the process of calcification and calcium (Ca²⁺) transport because of molting-related events and the deposition of CaCO₃ in the new exoskeleton. Dilocarcinus pagei, a freshwater crab endemic to Brazil, was studied to understand Ca²⁺ transport in whole gill cells using a fluorescent probe. Cells were dissociated, all of the gill cell types were loaded with fluo-3 and intracellular Ca²⁺ change was monitored by adding Ca as CaCl₂ (0, 0.1, 0.25, 0.50, 1.0 and 5 mM), with a series of different inhibitors. For control gill cells, Ca²⁺ transport followed Michaelis–Menten kinetics with K _m = 0.42 ± 0.04 mM and V _max = 0.50 ± 0.02 μM (Ca²⁺ change × initial intracellular Ca⁻¹ × 180 s⁻¹; N = 14, r ² = 0.99). Verapamil (a Ca²⁺ channel inhibitor) and amiloride (a Na⁺/Ca²⁺ exchanger [NCX] inhibitor) completely reduced intracellular Ca²⁺ transport, while nifedipine, another Ca²⁺ channel inhibitor, did not. Vanadate, a plasma membrane Ca²⁺-ATPase inhibitor (PMCA), increased intracellular Ca²⁺ in gill cells through a decrease in the efflux of Ca²⁺. Ouabain increased intracellular Ca²⁺, similar to the effect of KB-R, a specific NCX inhibitor for Ca²⁺ in the influx mode. Alterations in extracellular [Na] in the saline did not affect intracellular Ca²⁺ transport. Caffeine, responsible for inducing Ca release from sarcoplasmic reticulum in vertebrate muscle, increased intracellular Ca²⁺ compared to control, suggesting an effect of this inhibitor in gill epithelial cells of Dilocarcinus pagei, probably through release of intracellular stores. We also demonstrate here that intracellular Ca²⁺ in gill cells of Dilocarcinus pagei was kept relatively constant in face of an extracellular Ca concentration of 50-fold, suggesting that crustaceans are able to display Ca²⁺ homeostasis through various Ca²⁺ intracellular sequestration mechanisms and/or plasma membrane Ca²⁺ influx and outflux that are highly regulatory. In summary, studies using whole gill cells are an interesting approach for working with real regulatory Ca²⁺ mechanisms in intact cells under physiological Ca levels (mM range), compared to earlier work using isolated vesicles of various epithelial cells.