Cadmium removal by living cells of the marine microalga Tetraselmis suecica

Cadmium removal by living cells of the marine microalga Tetraselmis suecica was tested in cultures exposed to different cadmium concentrations (0.6, 3, 6, 15, 30 and 45 mg/l). The EC50 for growth was 7.9 mg Cd/l after six days of exposure. The cadmium removed was proportional to the concentration of this metal in the medium and it was dependent on the time of exposure; cultures with higher cadmium concentration removed a higher amount of this metal. In cultures exposed to 0.6 mg/l, T suecica cells removed 98.1% of added cadmium with 0.392 x 10(-6) microg Cd/cell, whereas in cultures with 45 mg/l only 7.7% was removed with 16.052 x 10(-6) microg Cd/cell. The highest amount of cadmium removed per liter of culture was observed in cultures exposed to 6 mg/l, with 3.577 mg/l of cadmium. After six days of incubation, the higher proportion of cadmium was bioaccumulated intracellularly in all cultures except in 45 mg/l cultures, the percentage of intracellular cadmium being always more than 50%. The highest percentage of bioadsorbed cadmium (60.1%) was found in cells of cultures with the highest cadmium concentration (45 mg/l). Furthermore, a relation between intracellular cadmium and the concentration of sulfhydryl groups was observed.     

Fine-tuning of the binding and dissociation of CO by the amino acids of the heme pocket of Coprinus cinereus peroxidase

Resonance Raman and infrared spectra and the CO dissociation rates (k(off)) were measured in Coprinus cinereus peroxidase (CIP) and several mutants in the heme binding pocket. These mutants included the Asp245Asn, Arg51Leu, Arg51Gln, Arg51Asn, Arg51Lys, Phe54Trp, and Phe54Val mutants. Binding of CO to CIP produced different CO adducts at pH 6 and 10. At pH 6, the bound CO is H-bonded to the protonated distal His55 residue, whereas at alkaline pH, the vibrational signatures and the rate of CO dissociation indicate a distal side which is more open or flexible than in other plant peroxidases. The distal Arg51 residue is important in determining the rate of dissociation in the acid form, increasing by 8-17-fold in the Arg51 mutants compared to that for the wild-type protein. Replacement of the distal Phe with Trp created a new acid form characterized by vibrational frequencies and k(off) values very similar to those of cytochrome c peroxidase.     

Simultaneous determination of eight beta-blockers by gradient high-performance liquid chromatography with combined ultraviolet and fluorescence detection in corneal permeability studies in vitro

A gradient HPLC method with combined ultraviolet and fluorescence detection was developed for the simultaneous determination of eight beta-blockers (alprenolol, atenolol, metoprolol, nadolol, pindolol, propranolol, sotalol and timolol) in corneal permeability studies in vitro. Fluorescence detection with excitation wavelength at 230 nm and emission at 302 nm was selective for six of the compounds, whereas UV detection at 205 nm was able to detect all the compounds. Calibration was performed with fluorescence detection for six compounds from 50 or 200 nM to 3 microM, and with UV detection for all the eight compounds from 100 or 200 nM to 30 microM. With optimized fluorescence detection, detection limits between 0.7 and 1.3 nM (0.035-0.065 pmol per 50 microl injection) were obtained for atenolol, metoprolol, nadolol and sotalol. A mixture of eight beta-blockers was used in cassette dosing permeability studies with a cultured corneal epithelium. The HPLC method revealed marked differences in the permeation between hydrophilic and lipophilic beta-blockers through the corneal epithelial cell culture model.     

A novel missense mutation (S18N) in the 5′ non-HMG box region of the SRY gene in a patient with partial gonadal dysgenesis and his normal male relatives

Mutations in the sex-determining region of the Y chromosome (the SRY gene) have been reported in low frequency in patients with 46,XY gonadal dysgenesis. We investigated 21 Brazilian 46,XY sex-reversed patients, who presented either complete or partial gonadal dysgenesis or embryonic testicular regression syndrome. Using Southern blotting, polymerase chain reaction, denaturing gradient gel electrophoresis and direct sequencing, we analyzed deletions and point mutations in the SRY gene. We found a missense mutation at codon 18 upstream of the 5′ border of the HMG box of the SRY gene in one patient with partial gonadal dysgenesis. This variant sequence was also found in DNA obtained from blood and sperm cells of his father and in blood cells of his normal brother. The S18N mutation was not found in 50 normal males, ruling out the possibility of a common polymorphism. We identified a novel familial missense mutation (S18N) in the 5’ non-HMG box of the SRY gene in 1 of 21 patients with 46,XY sex reversal. Received: 6 May 1997 / Accepted: 2 October 1997     

Rapid Selection in Modified BHK-21 Cells of a Foot-and-Mouth Disease Virus Variant Showing Alterations in Cell Tropism

With persistent foot-and-mouth disease virus (FMDV) in BHK-21 cells, there is coevolution of the cells and the resident virus; the virulence of the virus for the parental BHK-21 cells is gradually increased, and the cells become partially resistant to FMDV. Here we report that variants of FMDV C₃Arg/85 were selected in a single infection of partially resistant BHK-21 cells (termed BHK-Rb cells). Indirect immunofluorescence showed that the BHK-Rb cell population was heterogeneous with regard to susceptibility to C₃Arg/85 infection. Infection of BHK-Rb cells with C₃Arg/85 resulted in an early phase of partial cytopathology which was followed at 6 to 10 days postinfection by the shedding of mutant FMDVs, termed C₃-Rb. The selected C₃-Rb variants showed increased virulence for BHK-21 cells, were able to overcome the resistance of modified BHK-21 cells to infection, and had acquired the ability to bind heparin and to infect wild-type Chinese hamster ovary (CHO) cells. A comparison of the genomic sequences of the parental and modified viruses revealed only two amino acid differences, located at the surface of the particle, at the fivefold axis of the viral capsid (Asp-9→Ala in VP3 and either Gly-110→Arg or His-108→Arg in VP1). The same phenotypic and genotypic modifications occurred in a highly reproducible manner; they were seen in a number of independent infections of BHK-Rb cells with viral preparation C₃Arg/85 or with clones derived from it. Neither amino acid substitutions in other structural or nonstructural proteins nor nucleotide substitutions in regulatory regions were found. These results prove that infection of partially permissive cells can promote the rapid selection of virus variants that show alterations in cell tropism and are highly virulent for the same cells.     

DETECTION OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM FUNDYENSE (DINOPHYCEAE) WITH OLIGONUCLEOTIDE AND ANTIBODY PROBES: VARIABILITY IN LABELING INTENSITY WITH PHYSIOLOGICAL CONDITION

The toxic dinoflagellate Alexandrium fundyense Balech was grown under temperature- and nutrient-limited conditions, and changes in labeling intensity on intact cells were determined for two probe types: an oligonucleotide probe targeting rRNA and a monoclonal antibody (MAb) targeting a cell surface protein. In nutrient-replete batch culture, labeling with the rRNA probe was up to 400% brighter during exponential phase than during stationary phase, whereas MAb labeling did not change significantly with growth stage at the optimal growth temperature. In cultures grown at suboptimal, low temperatures, there was a significant difference between labeling intensity in stationary versus exponential phase for both probe types, with exponential cells labeling brighter with the rRNA probe and slightly weaker with the MAb. The decrease in rRNA probe labeling with increasing culture age was likely due to lower abundance of the target nucleic acid, as extracted RNA varied in a similar manner. With the MAb and the rRNA probes, slower growing cultures at low, nonoptimal temperature labeled 35% and 50% brighter than cells growing faster at warmer temperatures. Some differences in labeling intensity per cell disappeared when the data were normalized to surface area or volume, which indicated that the number of target antigens or rRNA molecules was relatively constant per unit area or volume, respectively. Slow growth accompanying phosphorus and nitrogen limitation resulted in up to a 400% decrease in labeling intensity with the rRNA probe compared to nutrient-replete levels, whereas the MAb labeling intensity increased by a maximum of 60%. With both probes, labeling was more intense under phosphorus limitation than under nitrogen limitation, and for all conditions tested, labeling intensity was from 600% to 3600% brighter with the MAb than with the rRNA probe. Thus, it is clear that significant levels of variability in labeling intensity can be expected with both probe types because of the influence of environmental conditions and growth stage on cellular biochemistry, cell size,rRNA levels, and the number or accessibility of cell surface proteins. Of the two probes tested, the rRNA probe was the most variable, suggesting that in automated, whole-cell assays, it can be used only in a semiquantitative manner. For manual counts, the human eye will likely accommodate the labeling differences. The MAb probe was less variable, and thus should be amenable to both manual and automated counts.     

VARIATION IN NUMBER OF APICAL RAMIFICATIONS AND VEGETATIVE CELL LENGTH IN FRESHWATER POPULATIONS OF CLADOPHORA (ULVOPHYCEAE,CHLOROPHYTA)1

Populations of Cladophora with two different levels of ploidy, n = 18/2n = 36 (18/36) and n = 24/2n = 48 (24/48), are present in creeks in the southern part of the province of Buenos Aires, Argentina. The goals of our study were to 1) relate the number of apical branches · mm^?2 in 18/36 and 24/48 populations with the water velocity at the collection site; 2) correlate the number of apical ramifications · mm^?2 in plants of the same population (24/48) growing in sectors with distinct water velocities; 3) compare cell length among populations with different ploidy levels, analyzing the sources of variation in different sectors of the same creek and in different plants of the same sector; and 4) analyze the sources of variation in cell length in 24/48 populations, including variations among different creeks. Our results suggest that 1) the number of branches · mm^?2 tends to increase with higher water velocity; 2) the 24/48 populations have more ramifications · mm^?2 than the 18/36 ones; 3) the length of vegetative cells is not an adequate criterion for differentiating between 18/36 and 24/48 populations; and 4) variations in vegetative cell length in 24/48 populations are highly significant among plants from different sectors of the same creek.