Interactions of actin and tubulin with human deoxyhemoglobin. Their possible occurrence within erythrocytes

Short actin filaments are an essential component of the red-cell membrane skeleton, and microtubules are also present in nucleated erythrocytes as a marginal band. Actin and tubulin share the property of possessing a very anionic terminal peptide. Since deoxyhemoglobin (Hb) is known to be a strong polyanion-binding protein, we have considered how it may interact with actin and tubulin within the intact cell. Here we demonstrate that actin and tubulin form in vitro a high-affinity complex with Hb. This is shown by measuring, by stopped-flow experiments, the decrease of the binding rate constant of CO to Hb in the presence of increasing amounts of actin and tubulin. One tetramer of Hb is bound by an actin monomer, and about two tetramers by an alpha, beta-tubulin heterodimer. Binding assays in batch experiments with immobilized tubulin give the same stoichiometry. Formation of the complexes involves the 2,3-bisphosphoglycerate-binding site of Hb and a negatively charged domain, most likely the highly acidic N and C-terminal peptides of actin and tubulin. In addition to providing new opportunities to study the structural and functional properties of actin and tubulin, these results support the idea that in the case of partial metabolic depletion of bisphosphoglycerate and ATP in erythrocytes, Hb may interact with oligomeric actin and tubulin present in the cytoskeleton.     

Caldesmon is present in human and pig erythrocytes

Caldesmon, a major actin- and calcium-dependent calmodulin-binding protein, is now considered as an essential inhibitory factor of the actomyosin machinery in smooth muscle cells as well as in non-muscle cells. Since its structure seems to vary with the cell in a same species and because platelet and erythrocyte have a common embryonic origin, we have used the affinity purified antibody raised against the platelet caldesmon to determine whether this protein is present in erythrocyte. Using the immunoblotting technique, we show here that, in whole erythrocytes, only a single polypeptide crossreacts with this polyclonal antibody. A 71-72 kDa Mr has been calculated from SDS-PAGE. It is therefore different from those of the gizzard (Mr 145-150 kDa) or the platelet (Mr 80 kDa) proteins. Furthermore, we also give evidence that it is not adducin since this newly discovered calcium-dependent calmodulin-binding protein of erythrocyte, does not crossreact with the polyclonal affinity purified antibody raised against platelet caldesmon.     

Kinetics of water transport in sickle cells

This paper reports the results of stopped-flow studies on differences in the kinetics of osmotic water transport of sickle and normal erythrocytes. The kinetics of inward osmotic water permeability are similar in sickle and normal red blood cells. In contrast, the kinetics of outward water flux are significantly (approx. 38%) decreased in sickle cells. Deoxygenation does not modify the water influx kinetics in either type of cells, but accelerates considerably the rate of water efflux in sickle cells. No significant variation of water transport kinetics was observed in density-separated cell fractions of either type. The results suggest that membrane-associated hemoglobin may decrease the outward water permeability and that in deoxygenated sickle cells the fraction of hemoglobin S near the lipid bilayer is lower than in oxygenated conditions.     

Hormone-resistant mutants of Arabidopsis have an attenuated response to agrobacterium strains

We have examined the response of the hormone-resistant mutants axr1 and axr2 of Arabidopsis thaliana to inoculation by Agrobacterium tumefaciens and Agrobacterium rhizogenes. Our results indicate that recessive mutations in the axr1 gene affect the frequency of tumor formation after inoculation with either Agrobacterium strain. In addition, tumors produced on axr1 plants were smaller than those growing on wild-type plants. These results indicate that the product of the AXR1 gene is important for both crown gall and hairy root tumor formation. In contrast, the dominant axr2 mutation has a more severe effect on the development of crown gall tumors than on hairy root tumors. Crown gall tumors produced on axr2 plants had a different morphology than wild-type tumors and did not grow when they were removed from the explant. In contrast, a large number of hairy root tumors were produced on wild-type and axr2 plants, and both types of tumors grew when they were removed from the explant. Like the roots of axr2 plants, roots produced on axr2 explants lacked root hairs.     

Development and validation of a UPLC/MS method for a nutritional metabolomic study of human plasma

In order to study the effect of a diet on metabolites found in body fluids such as plasma, we have developed and validated a UPLC/MS method. While methods using NMR have been well established to analyse different biological tissues, recent studies have described robust untargeted UPLC-MS methods for plasma analysis. One major concern when profiling plasma is the presence of an important quantity of proteins which have to be precipitated without any loss of metabolites prior to LC/MS analysis. The utilization of untargeted approaches in nutritional metabolomics still suffers from the lack of identification of specific biomarkers. We therefore suggest an alternative method still using a global approach but focusing at the same time on metabolites previously described in human plasma in order to detect biomarkers of metabolic dysregulations. Thus, to fulfil our objectives, analytical parameters were tested (i) the anticoagulant type for sample collection, (ii) the protein precipitation method and (iii) UPLC/MS analytical conditions. Three protein precipitation methods and two anticoagulants were tested and compared. The method utilizing blood collection on heparin and methanol precipitation was chosen for giving the most reproducible results while keeping the complexity of the sample. Finally, a validation was proposed to evaluate the stability of this analytical method applied to a large batch of samples for nutritional metabolomic studies.     

Sex and hormonal modification of 6-keto-PGF1α release by rat aorta

The stable breakdown product of prostacyclin, 6-keto-PGF_1α, was estimated in plasma samples after incubation with rat aortic rings. The 6-keto-PGF_1α concentration obtained with the male aortae was two-fold higher than that of the female. Ovariectomy markedly increased 6-keto-PGF_1α six-fold, but castration had no effect. Estradiol and progesterone treatment of the ovariectomized female suppressed (by 50%) and enhanced (two-fold) 6-keto-PGF_1α. Testosterone was without effect in gonadectomized males and females. Castrate males did not respond to gonadal steroid treatment.