Analysis of low concentrations of 5-bromo-2-deoxyuridine on sister chromatid exchanges in human lymphocytes

To determine a concentration of 5-bromo-2-deoxyuridine (BrdU) sufficient for sister chromatid differentiation (SCD), and yet having a minimal effect on the number of sister chromatid exchanges (SCEs), we assessed the effect produced on the number of SCEs by low concentrations (1, 3, and 10 micrograms/mL) of BrdU. SCD was not obtained in 19% of the 31 subjects with 1 microgram/mL of BrdU, while the differentiation was adequate for all samples treated with 3 and 10 micrograms/mL. We statistically analysed the effects of these three different doses and found no significant difference in the number of SCEs obtained with the doses of 1 and 3 micrograms/mL, but a significant difference was observed between these two concentrations and 10 micrograms/mL. We therefore suggest that the dose of 3 micrograms/mL, while sufficient to produce reliable differential staining, still permits an adequate evaluation of the base line of SCEs and appears to enhance the sensitivity of the test to evaluate between-individual variations. Our experiments also underline that SCE counts should include the centromere exchanges.     

Dynamic G- and R-banding of human chromosomes for electron microscopy

Synchronized human lymphocytes were exposed to 5-bromo-2-deoxyuridine (BrdUrd) for incorporation in either G-or R-bands. The substituted bands were revealed by monoclonal anti-BrdUrd antibodies disclosed with either gold-labeled antibodies or with the protein A-gold complex. Sharp G-or R-banding, specific for electron microscopy (EM), was obtained. These banding patterns, referred to as GB-AAu (G-bands by BrdUrd using Antibodies and gold [Au]) and RB-AAu (R-bands by BrdUrd using Antibodies and gold [Au]), resemble dynamic band patterns (GBG and RBG) much more than they do morphologic band patterns (GTG and RHG). The G- and R- band patterns allow accurate chromosome identification and karyotyping. An actual karyotype of human GB-AAu-banded chromosomes at the 750 band level, photographed in the EM, is presented. The method produces excellent band separation and band contrast. Variations in band staining intensities were noted and correlated with BrdUrd enrichment. The C-band regions were positively stained after GB-AAu banding while they were negatively stained after RB-AAu banding. Telomeres appeared heterogeneous after GB-AAu banding suggesting that part of the telomeric bands might be late replicating.     

High-resolution dynamic and morphological G-bandings (GBG and GTG): a comparative study

Summary A high-resolution replication banding technique, dynamic GBG banding (G-bands after 5-bromodeoxyuridine [BrdUrd] and Giemsa), showed that, at a resolution of 850 bands/genome, GBG banding and GTG banding (G-bands after trypsin and Giemsa) produce almost identical patterns. RBG band (R-bands after BrdUrd and Giemsa) and RHG band (R-bands after heat denaturation and Giemsa) patterns were previously shown to be only 75%–85% coincident; thus GTG banding more accurately reflects replication patterns than does RHG banding. BrdUrd synchronization uses high concentrations of BrdUrd both to substitute early replicating DNA and to arrest cells before the late bands replicate. Release from the block is via a low thymidine concentration. The banding is revealed by the fluorochrome-photolysis-Giemsa (FPG) technique and produces the GBG banding that includes concomitant staining of constitutive heterochromatin. As opposed to other replication G-banding procedures, BrdUrd synchronization and GBG banding produces a reproducible replication band pattern. The discordance between homologs after GBG banding is similar to that after GTG banding and no lateral asymmetry of the constitutive heterochromatin has been observed. Also, BrdUrd synchronization neither significantly depresses the mitotic index, nor induces chromosome breaks. Thus, GBG banding seems as clinically useful as GTG banding and provides important information regarding replication time.     

Endocytosis of an antibody ricin A-chain conjugate (immuno-A-toxin) adsorbed on colloidal gold. Effects of ammonium chloride and monensin

An immunotoxin (IT) formed by a specific antibody coupled to the ricin A chain was adsorbed on colloidal gold particles (IT-Au). Binding and internalization of IT-Au in human lymphoblastic CEM cells were studied using electron microscopy. IT-Au showed specific cytotoxic activity toward the target cells. After 1 h at 4 degrees C, IT-Au were linked diffusely to the plasma membrane with 45% of the particles regrouped in clusters. Upon transfer to 37 degrees C, the particles carrying the ligand were regrouped more frequently and internalized into the cell by endocytosis through smooth microinvaginations or coated pits of the plasma membrane. After 15 min, IT-Au was observed in endocytic vacuoles, or receptosomes, in tubular structure near the Golgi apparatus and in lysosomes. Entry of IT-Au into lysosomes was rapid (around 50% of intracellular IT-Au particles after 30 min). NH4Cl or monensin, well-known potentiators of immunotoxin activity, when present in incubation medium, altered neither the processes nor the rate of IT-Au endocytosis. In the presence of either of these substances, IT-Au accumulated in the normal or often enlarged endocytic vacuoles, and entry into the lysosomes was slowed down (50% of particles after 2 h 15 min). We conclude that this intense slowing-down in the speed of IT-Au transportation into lysosomes and the functional modifications of these organelles help to explain the increased efficacy of immunotoxins in the presence of potentiators.     

Cardiovascular and biological effects of K+ channel openers, a class of drugs with vasorelaxant and cardioprotective properties

Several new chemical entities (RP 52891, cromakalim and its derivatives) are potent and specific openers of vascular K+ channels. This mechanism is also shared, at least partially, by drugs such as minoxidil, diazoxide, pinacidil and nicorandil. The opening of plasmalemma K+ channels produces loss of cytosolic K+. This effect results in cellular hyperpolarization and functional vasorelaxation. In normotensive or hypertensive rats, K+ channel activators decrease aortic blood pressure (by producing a directly mediated fall in systemic vascular resistance) and reflexly increase heart rate. The former effect is not modified by specific blockers of classical vascular receptors but it is completely antagonized by the hypoglycemic sulphonylurea, glibenclamide, an established blocker of ATP-regulated K+ channels. K+ channel openers produce selective coronary vasodilatation and afford functional and biochemical protection to the ischemic myocardium. This salutary effect is mediated via cardiac K+ channel modulation and may result from an improved myocardial oxygen balance in the ischemic region. K+ channel openers increase plasma renin activity in animals as well as in man. However, only diazoxide, but not cromakalim or RP 52891, lowers plasma insulin concentration. The dose of glibenclamide entirely blocking the latter effect is over 50-fold smaller than that antagonizing the hypotensive and hyper-reninemic responses to diazoxide. In conclusion, K+ channel activators are potent vasorelaxant and cardioprotective agents possessing an original mechanism of action which is the opening of plasmalemma ATP-regulated K+ channels. Their clinical use as antihypertensive agents may be accompanied by undesirable effects (characteristic of peripheral vasodilators) which are likely to be attenuated or avoided by controlled release formulations. However, inasmuch as low doses of K+ channel openers may be sufficient to produce selective coronary artery dilatation and cardioprotection, these compounds could be of particular value in treating patients with coronary artery disease efficaciously and possibly without adverse cardiovascular effects.     

Pan-Arctic soil moisture control on tundra carbon sequestration and plant productivity

Long-term atmospheric CO₂ concentration records have suggested a reduction in the positive effect of warming on high-latitude carbon uptake since the 1990s. A variety of mechanisms have been proposed to explain the reduced net carbon sink of northern ecosystems with increased air temperature, including water stress on vegetation and increased respiration over recent decades. However, the lack of consistent long-term carbon flux and in situ soil moisture data has severely limited our ability to identify the mechanisms responsible for the recent reduced carbon sink strength. In this study, we used a record of nearly 100 site-years of eddy covariance data from 11 continuous permafrost tundra sites distributed across the circumpolar Arctic to test the temperature (expressed as growing degree days, GDD) responses of gross primary production (GPP), net ecosystem exchange (NEE), and ecosystem respiration (ER) at different periods of the summer (early, peak, and late summer) including dominant tundra vegetation classes (graminoids and mosses, and shrubs). We further tested GPP, NEE, and ER relationships with soil moisture and vapor pressure deficit to identify potential moisture limitations on plant productivity and net carbon exchange. Our results show a decrease in GPP with rising GDD during the peak summer (July) for both vegetation classes, and a significant relationship between the peak summer GPP and soil moisture after statistically controlling for GDD in a partial correlation analysis. These results suggest that tundra ecosystems might not benefit from increased temperature as much as suggested by several terrestrial biosphere models, if decreased soil moisture limits the peak summer plant productivity, reducing the ability of these ecosystems to sequester carbon during the summer.     

Solution conformation of Lewis a-derived selectin ligands is unaffected by anionic substituents at the 3'- and 6'- positions

The selectins are a family of proteins that mediate leukocytetethering and rolling along the vascular endothelium. E-, P-,and L-selectin recognize various derivatives of the Lewis^a andLewis^x trisaccharides. The distribution of negative chargeson the Lewis^a and Lewis^x oligosaccharides appears to be an importantfactor in their binding by the selectins. Previous work exploringthis electrostatic dependence found that a series of syntheticanionic trisaccharides, 3'-sulfo, 3'-phospho, 6'-sulfo, and3',6'-disulfo Lewis^a. (Glc), exhibited differing selectin inhibitoryefficacies. To explore the possibility that these differencesarise from conformational differences between the sugars, thesolution structures of these trisaccharides were determinedusing NMR and molecular dynamics simulations. Interproton distancesand interglycosidic torsion angles were determined at 37°Cusing NOESY buildup curves and 1D LRJ experiments, respectively.Data from both experiments agreed well with predictions madefrom 2000 picosecond unrestrained molecular dynamics simulations.We found that 3'-sulfation did not alter the core Lewis^a conformation,a finding that reaffirms the results of previous study. In addition,we found that sulfation at the 6' position also leaves the trisaccharideconformation unperturbed. This is significant because the proximityof the 6'-sulfate group to the fucose ring might have alteredthe canonical Lewis^a structure. The disulfate exhibited greaterflexibility than the other derivatives in dynamics simulations,but not so much as to affect NOE and heteronuclear couplingconstant measurements. Taken together, our findings supportthe use of Lewis^a as a template onto which charged groups maybe added without significantly altering the trisaccharide'sstructure. oligosaccharides molecular dynamics simulations NMR sulfated Lewis^a phosphorylated Lewis^a