Determination of total metals in cultivated oysters (Crassostrea gigas) from the northwest coast of Mexico by microwave digestion and atomic absorption spectrometry.

Bivalves such as oysters often accumulate heavy metals, and therefore can be used to monitor changes of pollutant concentrations in the environment. Cultivated oysters from the northwest coast of Mexico are widely used for human consumption and thus have an important commercial value. Information was gathered on the concentration of these elements in oysters (Crassostrea gigas) cultivated on the coast of Sonora. Oysters were randomly collected from April to October 1997, from 6 different locations (65 individuals per site) in 4 different months. Metals were determined by microwave digestion followed by atomic absorption spectroscopy. The mean values (microg/g fresh weight) for each metal were: Cd, 0.76; Cu, 3.64; Zn, 17.71; Pb, 0.50; As, 0.05; Hg, 0.03; and Se, 0.21. The results show that, except for Cd, concentrations of regulated metals were under the maximum permitted values specified by regulatory agencies of Mexico and were comparable to those reported from other areas.

Construction and characterization of a Mycobacterium tuberculosis mutant lacking the alternate sigma factor gene, sigF.

The alternate RNA polymerase sigma factor gene, sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain of Mycobacterium tuberculosis. The growth rate of the DeltasigF mutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type. The mutant showed increased susceptibility to rifampin and rifapentine. Additionally, the DeltasigF mutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-type sigF gene. No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed. Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth. However, mice infected with the DeltasigF mutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P < 0.001). These data indicate that M. tuberculosis sigF is a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro. While the DeltasigF mutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.

Use of microwave digestion and atomic absorption spectrophotometry to determine chromic oxide as a digestibility marker in feed, feces, and ileal content.

Most conventional digestion procedures, such as dry ashing and wet ashing, are tedious and labor intensive. Microwave digestion is a good alternative, because microwave dissolution is faster, safer, and simpler, and provides more controlled reproducible conditions than conventional methods. The purpose of this study was to develop a microwave digestion method for mineralizing meat and bone meal diets, feces, and ileal contents. Each sample was heated on a hot plate for 10 min, dry ashed at 65 degrees C for 4 h, and transferred into microwave vessels. Then, 10 mL 70% HNO3 was added. Samples were digested for 7, 10, and 20 min at 95, 90, and 85% power, respectively. After the heating cycle, 6 mL 30% H2O2 was added, and samples were returned to the microwave for a second heating cycle of 1 and 7 min at 95% and 90% power, respectively. Finally, chromium concentration was determined by flame atomic absorption spectrophotometry. The digestion method was validated by using a standard reference material, SRM domestic sludge 2781, with a certified chromium value of 195 +/- 9 micrograms/g. The value obtained in this study was 178 +/- 11 micrograms/g, for a difference of 17 micrograms/g. Spike recovery experiments resulted in 103.16 and 100.35% recoveries of chromium from diet and feces samples, respectively. Coefficients of variation were 10.8 and 7.8%, respectively.

In vitro antifungal evaluation and studies on mode of action of eight selected species from the Argentine flora.

Twenty nine extracts belonging to eight species of the Argentine flora reported as antifungal in folk medicine, were assayed for antifungal properties by using the agar dilution method, against a panel of yeasts, filamentous fungi as well as dermatophytes. Nine extracts belonging to six species, exhibited a broad spectrum of activity against Microsporum cants, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum and Epidermophyton floccosum, with MICs ranging from 25 to 900 µg/ml. A dichloromethanis extract of Polygonum ferrugineum was the most active extract with MICs from 25-50 µg/ml. To gain an insight into the mode of action of the active extracts they were evaluated for their inhibitory activities toward the fungal cell wall, using the whole cell Neurospora crassa hyphal growth inhibition agar diffusion assay. A hazy zone around the paper disk strongly suggested that the dichloromethane extracts from aerial parts of Polygonum punctatum, Polygonum ferrugineum and the bark of Luehea divaricata acted by inhibiting polymer synthesis or assembly of the cell wall. The clear zone of inhibition produced by the dichloromethane and methanol antifungal extracts of Xanthium spinosum could be ascribed to the fact that these extracts have another effect on fungal cells in addition to inhibition of cell walls.

Allelotype mapping of unstable microsatellites establishes direct lineage continuity between endometrial precancers and cancer.

Progressive microsatellite changes in replication error positive (RER+) endometrium were used to reconstruct evolutionary stages of nonfamilial adenocarcinoma. RER+ putative endometrial precancers (atypical endometrial hyperplasias) progress to RER+ carcinomas, which retain some of the altered microsatellites acquired in earlier precursor stages. The RER+ phenotype may provide a specific marker for early-stage endometrial neoplasms that cannot be resolved by routine histopathology and may be a useful tool to stratify stages in the evolution of RER+ tumors.

Baboon and cotton-top tamarin B2m cDNA sequences and the evolution of primate beta 2-microglobulin.

Nonhuman primates represent phylogenetic intermediates for studying the divergence of human and murine beta 2Ms. We report the nucleotide sequences of B2m cDNA clones from a baboon cell line, 26CB-1 (Papio hamadryas; primates: Cercopithecoidea), and a cotton-top tamarin cell line, 1605L (Saguinus oedipus; primates: Ceboidea). The baboon and tamarin B2m sequences indicate a very slow rate of B2m evolution in primates relative to that in murid rodents. Phenotypic evolution of beta 2M has also been very conservative in primates, with only 9-14 substitutions separating baboon or tamarin beta 2Ms from those of humans or orangutans. Analyses of silent and amino-acid-altering nucleotide substitutions provide evidence that negative selection has acted to limit variability in beta strands of primate beta 2Ms, while positive selection has promoted diversity in non-beta-strand regions of murine beta 2Ms. No evidence for the action of selection upon beta 2M residues that contact the class I heavy chain was found in primates or mice. The finding that different selective forces have operated upon primate and murine beta 2Ms suggests that beta 2M may have evolved to serve distinct functions in primates and mice.

Class I-induced resistance to natural killing: identification of nonpermissive residues in HLA-A2.

Structural characteristics of major histocompatibility complex class I antigens associated with natural killer (NK)-resistance phenomena were examined. Previous research has shown that transfection of class I genomic DNA clones into class I-deficient, NK-sensitive target cell lines results in transfectants exhibiting class I+, NK-resistant phenotypes. In contrast to the HLA-A3, -B7, -B27, and -Bw58 class I molecules, the HLA-A2 class I molecules were shown not to protect target cells from NK activity. Here we show that this nonprotective phenotype maps to the alpha 1 domain of the HLA-A2 molecule by examining the NK-protective capacity of the natural interdomain recombinant HLA-Aw69 molecule. HLA-Aw69, which consists of an alpha 1 domain exhibiting homology with HLA-Aw68, and alpha 2/alpha 3/transmembrane-cytoplasmic domains, exhibiting homologies with HLA-A2, mimics HLA-Aw68 and provides HLA-A,B null target cell (C1R) transfectants with increased resistance to NK. Further, the inability of transfected HLA-A2 to confer protection against NK activity can be completely attributed to the expression of a "nonpermissive" residue at position 74 in the alpha 1 domain. Site-directed mutation of the His-74 residue in HLA-A2 to the Asp-74 (HLA-A3, -Aw68, -Aw69, -B7) residue generates a mutant that provides C1R cell line transfectants an NK-resistant phenotype. As His-74 blocks access to a side pocket in the HLA-A2 antigen-binding cleft, these results support the critical involvement of residues within the peptide-binding groove of class I molecules in determining the NK susceptibility phenotype of class I+ target cells.

Epitopes within the HLA-B5,B35 cross-reacting group.

The HLA-B5,B35 cross-reacting group is a large and serologically complex antigen family which includes the World Health Organization-recognized specificities HLA-B5,B51,Bw52,B35,Bw53,B18,Bw70,Bw71, and Bw72. In addition, several variants of antigens in this cross-reacting group have been described in the past but have not yet gained official recognition. A genetic basis for the complexity and the protein and molecular bases of this highly cross-reactive and polymorphic cross-reacting group have yet to be established. The potential contributions of shared amino acid sequences, the occurrence of multiple epitopes on a single HLA-B molecule, and the presence of new HLA-C antigens have been difficult to resolve. To address this issue, we have carefully examined the serologic reactions of more than 900 allo- and monoclonal antibodies (Tenth International Workshop, Third Asia-Oceanic Workshop, and local reagents) versus lymphocytes from 92 individuals of diverse ethnic origin (North American Caucasians, North American blacks, Amerindians, Middle Eastern Caucasians), 84 of whom were informative for the HLA-B5,B35 cross-reacting group and related antigens. Our results demonstrate that the HLA-B5,B35 gene products share different combinations of distinct epitopes. We have constructed a model for the evolution of this cross-reacting group by assigning polarity to distinct diversification steps utilizing principles of maximum parsimony.