Relationship between cell density and prolidase activity in human skin fibroblasts: effects of ascorbate and fructose

To evaluate the influence of cell density on the activity of fibroblast prolidase (EC 3.4.13.9), we determined this activity in sparse and dense cultures. We also investigated, the effects of different concentrations of β-d(?) fructose and l(+) ascorbate, which both increased cell density at confluency. For a fructose concentration of 25 mM, we observed that in the absence of glucose, intracellular total proteins increased 1.5-fold and prolidase specific activity, 1.8-fold. For ascorbate, a broad optimum concentration was found (range 0.01 – 0.50 mM). Addition to cultures of 0.1 mM ascorbate increased total proteins 1.4-fold, and doubled prolidase activity. This investigation was prompted by our previous results [J. Metab. Dis. 1983, 6, 27–31], confirmed here, and suggesting that increased prolidase activity at confluency was due to a rise in cell density.     

Structure of Supercritical Fluid Krypton at Small Scattering Angle Using Parallel Molecular Dynamics Simulation

Abstract

We present a parallel algorithm for molecular dynamics involving short-range two- and three-body potentials and the pair-correlation function, g(r). The method is based on a spatial decomposition of the simulation box that takes advantage of a linked-cell list, and allows a load balanced partition of the computations of both the forces and g(r) over the processors. The tests of the program is conducted by evaluating the efficiency for both the thermalization phase and the production phase of the simulation. This method is successfully applied to the calculation of the direct correlation function of fluid krypton at small scattering angle along the T = 297 K supercritical isotherm.     

Recognition of the acetylcholine receptor binding site of a long-chain neurotoxin by toxin-specific monoclonal antibodies.

The present paper reports the preparation and characterization of two neutralizing monoclonal antibodies (Mabs), called MST1 and MST2, which bind at the central loop of a long-chain neurotoxin from cobra venom. The central loop is a critical region for the binding of the toxin to the nicotinic acetylcholine receptor. Some of the residues incorporated in the epitopes recognized by MST1 and MST2 have been identified on the basis of competition experiments using a set of 'chemical mutants' of the toxin. We show that MST1 and MST2 bind at the base and at the tip of the central loop of the toxin, respectively, however, only MST2 actually overlaps the acetylcholine receptor binding site. Accordingly, only MST2 is capable of recognizing all homologous toxins so far examined. MST2, therefore, mimicks, at least partially, the site by which the nicotinic acetylcholine receptor recognizes a long-chain neurotoxin.     

Cenicriviroc,a Novel CCR5 (R5) and CCR2 Antagonist,Shows In Vitro Activity against R5 Tropic HIV-2 Clinical Isolates

Background

Maraviroc activity against HIV-2, a virus naturally resistant to different HIV-1 antiretroviral drugs, has been recently demonstrated. The aim of this study was to assess HIV-2 susceptibility to cenicriviroc, a novel, once-daily, dual CCR5 and CCR2 antagonist that has completed Phase 2b development in HIV-1 infection.

Methods

Cenicriviroc phenotypic activity has been tested using a PBMC phenotypic susceptibility assay against four R5-, one X4- and one dual-tropic HIV-2 clinical primary isolates. All isolates were obtained by co-cultivation of PHA-activated PBMC from distinct HIV-2-infected CCR5-antagonist-naïve patients included in the French HIV-2 cohort and were previously tested for maraviroc susceptibility using the same protocol. HIV-2 tropism was determined by phenotypic assay using Ghost(3) cell lines.

Results

Regarding the 4 R5 HIV-2 clinical isolates tested, effective concentration 50% EC₅₀ for cenicriviroc were 0.03, 0.33, 0.45 and 0.98 nM, similar to those observed with maraviroc: 1.13, 0.58, 0.48 and 0.68 nM, respectively. Maximum percentages of inhibition (MPI) of cenicriviroc were 94, 94, 93 and 98%, similar to those observed with maraviroc (93, 90, 82, 100%, respectively). The dual- and X4-tropic HIV-2 strains were resistant to cenicriviroc with EC50 >1000 nM and MPI at 33% and 4%, respectively.

Conclusions

In this first study assessing HIV-2 susceptibility to cenicriviroc, we observed an in vitro activity against HIV-2 R5-tropic strains similar to that observed with maraviroc. Thus, cenicriviroc may offer a once-daily treatment opportunity in the limited therapeutic arsenal for HIV-2. Clinical studies are warranted.     

The Ca2+-releasing messenger NAADP, a new player in the nervous system.

Many physiological processes are controlled by a great diversity of Ca2+ signals. Within cell, Ca2+ signals depend upon Ca2+ entry and/or Ca2+ release from internal Ca2+ stores. The control of Ca2+-store mobilization is ensured by a family of messengers comprising inositol 1,4,5 trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). From recent works, new concepts have emerged where activation of the cells by outside stimuli, acting at the plasma membrane, results in the synthesis of multiple Ca2+-releasing messengers which may interact and shape complex Ca2+ signals in the cytosol as well as in the nucleus. This contribution will cover the most recent advances on NAADP signalling with some emphasis on neurons.     

An Investigation of Freezing of Supercooled Water on Anti-Freeze Protein Modified Surfaces

This work investigates how functionalization of aluminium surfaces with natural type III Anti-Freeze Protein (AFP) affects the mechanism of heterogeneous ice nucleation. First the bulk ice nucleation properties of distilled water and aqueous solution of AFP were evaluated by differential scanning calorimetry. Then the modified surface was characterized by Secondary Ions Mass Spectroscopy (SIMS), Fourier Transform InfraRed (FTIR) spectroscopy and contact angle measurement. Freezing experiments were then conducted in which water droplets underwent a slow controlled cooling. This study shows that compared to uncoated aluminium, the anti-freeze proteins functionalized surfaces exhibit a higher and narrower range of freezing temperature. It was found that these proteins that keep living organisms from freezing in cold environment act in the opposite way once immobilized on surfaces by promoting ice nucleation. Some suggestions regarding the mechanism of action of the observed phenomena were proposed based on the Classical Nucleation Theory (CNT).     

Constraints on control: factors influencing reproductive success in male mandrills (Mandrillus sphinx)

Over the last decade, paternity analysis using molecular markershas revealed that observed mating systems do not necessarilycorrelate with reproductive systems and thus cannot providereliable information about male reproductive success (RS). Thisis especially true for primate species with a complex multimale-multifemalesocial organization, such as mandrills (Mandrillus sphinx).Using molecular markers for the measurement of individual RSand a comprehensive data set comprising 193 offspring from 27birth cohorts over a 20-year period of sampling, we investigatedthe social, genetic, and demographic factors that may influencethe probability of paternity by dominant male mandrills, livingin a semi–free-ranging colony. We observed a significantskew in RS towards dominant males, with their probability ofpaternity increasing as the number of adult males in the groupincreased, and when they were closely related to subordinateadolescent males. Conversely, the probability of dominant malessiring infants decreased when the number of simultaneously tumescentfemales increased. Fewer offspring were sired by dominant maleswhen female partners were closely related to them and when therelatedness between dominant and subordinate adult males increased.These two last points suggests particularly that mechanismsof kin recognition are operating to avoid the costs of inbreedingand may also reflect the lower costs to dominant males of losingconception opportunities to more closely related subordinateadult males. This study is, to our knowledge, one of the firstin primates to use an integrative approach and multivariateanalysis to show that multiple factors are involved in determiningthe probability of paternity by dominant males.     

Characterization of the interaction between protein Snu13p/15.5K and the Rsa1p/NUFIP factor and demonstration of its functional importance for snoRNP assembly

The yeast Snu13p protein and its 15.5K human homolog both bind U4 snRNA and box C/D snoRNAs. They also bind the Rsa1p/NUFIP assembly factor, proposed to scaffold immature snoRNPs and to recruit the Hsp90-R2TP chaperone complex. However, the nature of the Snu13p/15.5K–Rsa1p/NUFIP interaction and its exact role in snoRNP assembly remained to be elucidated. By using biophysical, molecular and imaging approaches, here, we identify residues needed for Snu13p/15.5K–Rsa1p/NUFIP interaction. By NMR structure determination and docking approaches, we built a 3D model of the Snup13p–Rsa1p interface, suggesting that residues R₂₄₉, R₂₄₆ and K₂₅₀ in Rsa1p and E₇₂ and D₇₃ in Snu13p form a network of electrostatic interactions shielded from the solvent by hydrophobic residues from both proteins and that residue W₂₅₃ of Rsa1p is inserted in a hydrophobic cavity of Snu13p. Individual mutations of residues in yeast demonstrate the functional importance of the predicted interactions for both cell growth and snoRNP formation. Using archaeal box C/D sRNP 3D structures as templates, the association of Snu13p with Rsa1p is predicted to be exclusive of interactions in active snoRNPs. Rsa1p and NUFIP may thus prevent premature activity of pre-snoRNPs, and their removal may be a key step for active snoRNP production.