NMR studies of L-dopa melanin-manganese(II) complex in water solution

The study of water relaxation rates of solutions of melanins with paramagnetic metal ions provides a powerful tool for investigating the binding sites and for obtaining useful structural and dynamic information. The measure of 1H- and 2H-longitudinal and transverse relaxation rates at a single, high-magnetic field for H2O/D2O (80:20) solutions allows the determination of tau c, tau R, tau e, tau M, r, q, and Z (the outer sphere contribution to the overall relaxation rate) for Mn(II)-L-Dopa melanin system.     

A chronobiological study of delta-amino levulinic acid urinary excretion

Determination of urinary delta-amino levulinic acid (ALA) is now systematically used in occupational health to detect an excessive exposure to lead in professionally exposed workers. However, to determine whether circadian changes of the urinary excretion of ALA alter the significance of the test, we quantified the ALA levels in the urine of 19 healthy young adults. Urine samples were taken every 3 hr between 0700 and 2300 hr and ALA levels were determined by a spectrocolorimetric method. The data indicated that the 24-hr mean ALA level was: 1.81 mg/g creatinine. The peak values (2.24 +/- 0.24) were obtained between 1400 and 1700 hr whereas the lowest ALA levels were found between 2200 and 0300 hr. Cosinor analysis revealed a significant circadian rhythm while no significant difference could be found according to sex. Possible explanations of our findings are discussed.     

Comparison of the relative sensitivity of human lymphocytes and mouse splenocytes to two spindle poisons

Aneugenic compounds act on non-DNA targets to exert genotoxicity via an indirect mechanism. In contrast to DNA-binding agents, these compounds are expected to possess threshold levels of activity. Therefore, the risk for adverse effects following human exposure to an aneugen could be minimal, if the threshold of activity has been clearly determined in vivo and in vitro and providing the human exposure level is below this threshold. Thus, the development of a single-cell model to allow comparisons between in vitro and in vivo threshold values for aneugenic compounds is of importance.The in vivo micronucleus test is one of the main assays used in genetic toxicology, and is often performed in the mouse. Thus, an extensive database is available in the literature. However, there are only few data concerning the in vitro micronucleus assay using mouse cells, as the majority of in vitro micronucleus assays have been performed using human lymphocytes. In addition, there is a lack of data concerning thresholds for any compound using this model.First, we evaluated whether the use of mouse splenocytes would be an acceptable alternative to that of human lymphocytes to identify aneugens. To allow valid comparisons, the two protocols were first harmonized. Thus, phytohemagglutinin (PHA) and concanavalin A were used as specific mitogens for human lymphocytes and mouse splenocytes, respectively, in order to achieve similar cell-proliferation rates. To achieve similar and sufficient numbers of binucleated cells, cytochalasin B was added 44 and 56 h after culture initiation of the human and mouse cells, respectively.Second, we compared the sensitivity of the mouse protocol with that of the human protocol by exposing the cells to the aneugens nocodazole and paclitaxel.There was good reproducibility of the cytotoxic/genotoxic responses of the two cell models following exposure to the aneugens. The sensitivity of the mouse splenocytes to paclitaxel was higher than that of the human lymphocytes. The two cell types were equally sensitive to nocodazole.     

De novo root formation in thin cell layers of tobacco: changes in free and bound polyamines

Thin cell layers excised from tobacco ( Nicotiana tabacum L. cv. Samsun) stem internodes, with an appropriate exogenous hormonal balance, were able to form a greater number of roots, and in a larger percentage of the explants (93%) than when they were excised from pedicels (40%). The developmental sequence of root formation and explant growth were followed by histological analysis. Free and bound [trichloroacetic acid (TCA)-soluble and -insoluble] putrescine and spermidine increased in the explants, particularly when root meristemoids appeared. These meristemoids originated in the superficial (day 6 in culture) or deep (days 10–11) layers and inside the newly formed callus (day 25). At those times, TCA-soluble and, to a lesser extent, TCA-insoluble bound putrescine predominated over the other polyamines. Spermine was always present in trace amounts. Polyamines decreased again when root and callus formation was completed (day 30). The involvement of these three classes of polyamines (free, TCA-soluble and -insoluble) in morphogenic processes is discussed.     

Binding of flunitrazepam to differentiating neurons cultured in a chemically defined,hormone-supplemented medium

[³H]Flunitrazepam (FNZ) binding to cortical neurons from fetal rat brain was investigated in vitro. The use of a synthetic medium specific for neurons made it possible to plot a developmental curve of³H-FNZ binding in an almost pure neuronal culture. Detectable specific binding was present in vitro at time 0 (that is, the 16th gestational day). A progressive increase of binding, due to an increment in the number of recognition sites, was observed on the subsequent days. The affinity of the specific binding sites to³H-FNZ was enhanced by the addition of exogenous GABA, whereas the density was not affected.     

The complete amino acid sequences of cytosolic and mitochondrial aspartate aminotransferases from horse heart,and inferences on evolution of the isoenzymes

Summary We report here the complete amino acid sequences of the cytosolic and mitochondrial aspartate aminotransferases from horse heart. The two sequences can be aligned so that 48.1% of the amino acid residues are identical. The sequences have been compared with those of the cytosolic isoenzymes from pig and chicken, the mitochondrial isoenzymes from pig, chicken, rat, and human, and the enzyme fromEscherichia coli. The results suggest that the mammalian cytosolic and mitochondrial isoenzymes have evolved at equal and constant rates whereas the isoenzymes from chicken may have evolved somewhat more slowly. Based on the rate of evolution of the mammalian isoenzymes, the geneduplication event that gave rise to cytosolic and mitochondrial aspartate aminotransferases is estimated to have occurred at least 10⁹ years ago. The cytosolic and mitochondrial isoenzymes are equally related to the enzyme fromE. coli; the prokaryotic and eukaryotic enzymes diverged from one another at least 1.3×10⁹ years ago.     

Solubilization of L-triiodothyronine binding site from human erythrocyte membrane

A thyroid binding peripheral membrane protein(s) has been characterized in human red cell. Two classes of affinity sites for triiodothyronine have been demonstrated. The high affinity, low capacity site showed values for dissociation constant of 2 X 10(-10)M. The binding activity depended on the presence of free -SH group and showed a high stereospecificity for L-triiodothyronine, L-thyroxine was less potent (about 1,000-fold) than L-triiodothyronine in competing for this site. The results are discussed with respect to their cellular significance.     

Prenylated bianthrones and vismione F from Psorospermum febrifugum

The roots of Psorospermum febrifugum collected in Malawi contained together with the known vismione D and geranyloxyemodin four new compounds: vismione F and the three bianthrones A₁, A_3a and A_3b. All the isolated compounds contained C- or O-geranyl substituents and showed a close biogenetic relationship.     

Inhibition of lateral wall elongation by mecillinam stimulates cell division in certain cell division conditional mutants of Escherichia coli.

The effect of mecillinam, a beta-lactam antibiotic that specifically binds penicillin-binding protein 2 of Escherichia coli, causes transition from rod to coccal shape, and inhibits cell division in sensitive cells, has been tested on three different E. coli temperature-sensitive cell division mutants. At the nonpermissive temperature, the antibiotic allows an increase in cell number for strains BUG6 and AX655 but not for AX621. In strain AX655, the cell division stimulation was observed only if the antibiotic was added immediately after shifting to the nonpermissive temperature, whereas in BUG6, the rise in cell number was observed also when mecillinam was added after 90 min of incubation at the nonpermissive temperature. In all cases, cell division began occurring 30 min after addition of the antibiotic. Mecillinam had no effect on division of dnaA, dnaB temperature-sensitive mutants or on division of BUG6 derivatives made resistant to this antibiotic. Other beta-lactam antibiotics such as penicillin, ampicillin, cephalexin, and piperacillin and non beta-lactam antibiotics such as fosfomycin, teichomycin, and vancomycin that inhibit cell wall synthesis did not show any effect on cell division for any of the mutants. The response of the three cell division mutants to mecillinam is interpreted in terms of a recently proposed model for shape regulation in bacteria.