The assignment of carbon monoxide association rate constants to the alpha and beta subunits in native and mutant human deoxyhemoglobin tetramers

The association kinetics of CO binding to site-directed mutants of human deoxyhemoglobin were measured by stopped-flow rapid mixing techniques at pH 7.0, 20 degrees C. Hemoglobin tetramers were constructed from one set of native subunits and one set of mutated partners containing His(E7) to Gly, Val(E11) to Ala, or Val(E11) to Ile substitutions. The reactivity of beta Cys93 with p-hydroxymercuribenzoate was measured to ensure that the mutant deoxyhemoglobins were capable of forming T-state quaternary conformations. Time courses for the complete binding of CO were measured by mixing the deoxygenated proteins with a 5-fold excess of ligand in the absence and presence of inositol hexaphosphate. Association rate constants for the individual alpha and beta subunits in the T-state conformation were assigned by measuring time courses for the reaction of a small, limiting amount of CO with a 20-fold excess of deoxyhemoglobin (i.e. Hb4 + CO----Hb4(CO)). The effects of the E7 and E11 mutations in T-state alpha subunits were qualitatively similar to those observed for the same subunit in the R-state (Mathews, A.J., Rohlfs, R.J., Olson, J.S., Tame, J., Renaud, J-P., and Nagai, K. (1989) J. Biol. Chem. 264, 16573-16583). The alpha His58(E7) to Gly and Val62(E11) to Ala substitutions caused 80- and 3-fold increases, respectively, in k'CO for T-state alpha subunits, and the alpha Val62(E11) to Ile mutation caused a 3-fold decrease. The beta His63(E7) to Gly and Val67(E11) to Ala substitutions produced 70- and 8-fold increases, respectively, in k'CO for T-state beta subunits whereas these same mutations caused little effect on the rate of CO binding to R-state beta subunits. The beta Val67(E11) to Ile mutation produced the same large effect, a 23-fold reduction in k'CO, in both quaternary conformations of beta subunits. These kinetic results can be interpreted qualitatively in terms of differences between the alpha and beta subunits in the deoxy and liganded crystal structures of human hemoglobin (Perutz, M.F. (1990) Annu. Rev. Physiol. 52, 1-25). Both the structural and functional data suggest that the distal portion of the beta heme pocket is tightly packed in deoxyhemoglobin whereas the CO binding site in R-state beta subunits is much more open. In contrast, the distal portion of the alpha heme pocket is restricted sterically in both quaternary states.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.     

Larval Dispersal Modeling of Pearl Oyster Pinctada margaritifera following Realistic Environmental and Biological Forcing in Ahe Atoll Lagoon

Studying the larval dispersal of bottom-dwelling species is necessary to understand their population dynamics and optimize their management. The black-lip pearl oyster (Pinctada margaritifera) is cultured extensively to produce black pearls, especially in French Polynesia''s atoll lagoons. This aquaculture relies on spat collection, a process that can be optimized by understanding which factors influence larval dispersal. Here, we investigate the sensitivity of P. margaritifera larval dispersal kernel to both physical and biological factors in the lagoon of Ahe atoll. Specifically, using a validated 3D larval dispersal model, the variability of lagoon-scale connectivity is investigated against wind forcing, depth and location of larval release, destination location, vertical swimming behavior and pelagic larval duration (PLD) factors. The potential connectivity was spatially weighted according to both the natural and cultivated broodstock densities to provide a realistic view of connectivity. We found that the mean pattern of potential connectivity was driven by the southwest and northeast main barotropic circulation structures, with high retention levels in both. Destination locations, spawning sites and PLD were the main drivers of potential connectivity, explaining respectively 26%, 59% and 5% of the variance. Differences between potential and realistic connectivity showed the significant contribution of the pearl oyster broodstock location to its own dynamics. Realistic connectivity showed larger larval supply in the western destination locations, which are preferentially used by farmers for spat collection. In addition, larval supply in the same sectors was enhanced during summer wind conditions. These results provide new cues to understanding the dynamics of bottom-dwelling populations in atoll lagoons, and show how to take advantage of numerical models for pearl oyster management.     

Phagocytosis in Tetrahymena thermophila: naloxone-reversible inhibition by opiates

1. Nanomolar concentrations of opiates inhibit phagocytosis in the ciliated protozoan Tetrahymena thermophila. 2. Naloxone and naltrexone counteract the effect of the opiate agonists tested. 3. The dose-response curves are U-shaped, with no detectable effect at low or high concentrations. 4. An increase in extracellular calcium and dopamine counteract the inhibition caused by metenkephalin. 5. The recognition mechanism for opiates in Tetrahymena cannot be classified as belonging to any of the mammalian opiate receptor subtypes and is perhaps a primitive receptor.     

Demonstration of prolactin messenger RNA after amplification in the brain in rats]

By means of immunocytochemistry, a central neuronal network containing a prolactin-like substance has been described in the rat. In order to demonstrate the synthesis of this peptide in these cells, we examined the presence of prolactin messenger RNA (PRL mRNA) in several brain samples including the pituitary gland. Amplification of the PRL mRNA was performed by the polymerase chain reaction technique, followed by southern blotting and hybridization with a specific oligonucleotide. Results showed the presence of the expected cDNA (468 bp) in the hypothalamus. Another cDNA with a lower molecular weight was also observed.     

Calcium regulates the regeneration of cilia in Tetrahymena thermophila

A study of calcium metabolism in Tetrahymena during the regeneration of cilia evidenced that the process is inhibited by nifedipine and trifluoperazine. This suggests that calcium ions play an important regulatory role in this process. This was confirmed by studies on calcium uptake and efflux which showed that there was a net increase in calcium uptake prior to the reinitiation of motility. The increase coincided with a period of sensitivity to the calcium antagonist TMB-8 and with an increase in the intracellular level of cGMP. The process was also inhibited by neomycin and stimulated by phorbol esters, which suggests that hydrolysis of phosphatidylinositol phosphates may take place as part of the calcium regulatory network during the regeneration of cilia.