pH effects on the hyaluronan hydrolysis catalysed by hyaluronidase in the presence of proteins: Part I. Dual aspect of the pH-dependence

Hyaluronan (HA) hydrolysis catalysed by hyaluronidase (HAase) is strongly inhibited when performed at a low ratio of HAase to HA concentrations and at low ionic strength. This is because long HA chains can form non-active complexes with HAase. Bovine serum albumin (BSA) is able to compete with HAase to form electrostatic complexes with HA so freeing HAase which then recovers its catalytic activity. This BSA-dependence is characterised by two main domains separated by the optimal BSA concentration: below this concentration the HAase activity increases when the BSA concentration is increased, above this concentration the HAase activity decreases. This occurs provided that HA is negatively charged and BSA is positively charged, i.e. in a pH range from 3 to 5.25. The higher the pH value the higher the optimal BSA concentration. Other proteins can also modulate HAase activity. Lysozyme, which has a pI higher than that of BSA, is also able to compete with HAase to form electrostatic complexes with HA and liberate HAase. This occurs over a wider pH range that extends from 3 to 9. These results mean that HAase can form complexes with HA and recover its enzymatic activity at pH as high as 9, consistent with HAase having either a high pI value or positively charged patches on its surface at high pH. Finally, the pH-dependence of HAase activity, which results from the influence of pH on both the intrinsic HAase activity and the formation of complexes between HAase and HA, shows a maximum at pH 4 and a significant activity up to pH 9.     

Proteome analysis of the phenotypic variation process in Photorhabdus luminescens

Photorhabdus luminescens is an insect pathogen associated with specific soil nematodes. The bacterium has a complex life cycle with a symbiotic stage in which bacteria colonize the intestinal tract of the nematodes, and a pathogenic stage against susceptible larval-stage insect. Symbiosis-"deficient" phenotypic variants (known as secondary forms) arise during prolonged incubation. Correspondence analysis of the in silico proteome translated from the genome sequence of strain TT01 identified two major biases in the amino acid composition of the proteins. We analyzed the proteome, separating three classes of extracts: cellular, extracellular, and membrane-associated proteins, resolved by 2-DE. Approximately 450 spots matching the translation products of 231 different coding DNA sequences were identified by PMF. A comparative analysis was performed to characterize the protein content of both variants. Differences were evident during stationary growth phase. Very few proteins were found in variant II supernatants, and numerous proteins were lacking in the membrane-associated fraction. Proteins up-regulated by the phenotypic variation phenomenon were involved in oxidative stress, energy metabolism, and translation. The transport and binding of iron, sugars and amino acids were also affected and molecular chaperones were strongly down-regulated. A potential role for H-NS in phenotypic variation control is discussed.     

MyoD binds to Mos and inhibits the Mos/MAP kinase pathway

When ectopically expressed, the serine/threonine kinase Mos can induce oncogenic transformation of somatic cells by direct phosphorylation of MAP kinase/ERK kinase (MEK1), activating the mitogen-activated protein kinases ERK1 and ERK2. On the other hand, overexpression of Mos in C2C12 myoblasts is not transforming. Mos activates myogenic differentiation by promoting heterodimerization of the MyoD/E12 proteins, increasing the expression of myogenic markers and the positive autoregulatory loop of MyoD. In this study, we show that in myogenic cells, the mitogenic and oncogenic signalling from the Mos/MEK/ERK pathway is suppressed by MyoD through the formation of a heterotrimeric complex.     

Speedy: a novel cell cycle regulator of the G2/M transition

Stage VI Xenopus oocytes are suspended at the G2/M transition of meiosis I, and represent an excellent system for the identification and examination of cell cycle regulatory proteins. Essential cell cycle regulators such as MAPK, cyclins and mos have the ability to induce oocyte maturation, causing the resumption of the cell cycle from its arrested state. We have identified the product of a novel Xenopus gene, Speedy or Spy1, which is able to induce rapid maturation of Xenopus oocytes, resulting in the induction of germinal vesicle breakdown (GVBD) and activation of M-phasepromoting factor (MPF). Spy1 activates the MAPK pathway in oocytes, and its ability to induce maturation is dependent upon this pathway. Spy1-induced maturation occurs much more rapidly than maturation induced by other cell cycle regulators including progesterone, mos or Ras, and does not require any of these proteins or hormones, indicating that Spy1-induced maturation proceeds through a novel regulatory pathway. In addition, we have shown that Spy1 physically interacts with cdk2, and prematurely activates cdk2 kinase activity. Spy1 therefore represents a novel cell cycle regulatory protein, inducing maturation through the activation of MAPK and MPF, and also leading to the premature activation of cdk2.     

Elasticity of the human red blood cell skeleton

We have measured by optical tweezers micromanipulations the area expansion and the shear moduli of spectrin skeletons freshly extracted from human red blood cells, in different controlled salinity conditions. At medium osmolarity (150 mOsm/kg), we measure KC=9.7+/-3.4 microN/m, muC=5.7+/-2.3 microN/m, KC/muC=2.1+/-0.7. When decreasing the osmolarity, both KC and muC decrease, while KC/muC is nearly constant and equal to about 2. This result is consistent with the predictions made when modeling the spectrin skeleton by a two-dimensional triangular lattice of springs. From the measured elastic moduli we estimate the persistence length of a spectrin filament: xi approximately 2.5 nm at 150 mOsm/kg.     

EVOLUTION OF RESISTANCE IN CULEX PIPIENS: ALLELE REPLACEMENT AND CHANGING ENVIRONMENT

Fixation of adaptive mutations in populations is often constrained by pleiotropic fitness costs. The evolutionary pathways that compensate such fitness disadvantages are either the occurrence of modifier genes or replacement of the adaptive allele by less costly ones. In this context, 23 years of evolution of insecticide resistance genes in the mosquito Culex pipiens from southern France are analyzed. The aim of this study is to answer the following points. Is there a fitness cost associated with these resistance genes in natural populations? Does evolution proceed through allele replacement or through selection of modifiers? And finally, how do environmental changes affect the evolution of resistance genes? Samples from the same transect, crossing the boundary between an insecticide-treated and a nontreated area, are analyzed. Clinal analyses indicate a variable fitness cost among the resistance genes and show that allele replacement has been the primary mechanism of resistance evolution in this area. It is also shown that replacement was probably due to environmental changes corresponding to modification in pesticide-treatment intensity.     

APPEARANCE AND SWEEP OF A GENE DUPLICATION: ADAPTIVE RESPONSE AND POTENTIAL FOR NEW FUNCTIONS IN THE MOSQUITO CULEX PIPIENS

Evolution of a new gene function is a fundamental process of adaptation. Gene duplication followed by divergence due to relaxed selection on redundant copies has been viewed as the predominant mechanism involved in this process. At a macroevolutionary scale, evidence for this scenario came from the analysis of sequences of genes families. However, even if several genetic models have described the different potential microevolutionary scenario for a new function to evolve, little is really known about the initial evolutionary dynamics of such processes. We analyze such early dynamics in natural populations of the mosquito Culex pipiens polymorphic for a duplication at Ace.1, a locus involved in insecticide resistance. The date of occurrence and the selective advantages of the duplication were estimated using frequency data. We propose a scenario where the spread of a duplication is driven, from the very beginning, by selection due to insecticide treatment.     

An overview of the evolution of overproduced esterases in the mosquito Culex pipiens

Insecticide resistance genes have developed in a wide variety of insects in response to heavy chemical application. Few of these examples of adaptation in response to rapid environmental change have been studied both at the population level and at the gene level. One of these is the evolution of the overproduced esterases that are involved in resistance to organophosphate insecticides in the mosquito Culex pipiens. At the gene level, two genetic mechanisms are involved in esterase overproduction, namely gene amplification and gene regulation. At the population level, the co-occurrence of the same amplified allele in distinct geographic areas is best explained by the importance of passive transportation at the worldwide scale. The long-term monitoring of a population of mosquitoes in southern France has enabled a detailed study to be made of the evolution of resistance genes on a local scale, and has shown that a resistance gene with a lower cost has replaced a former resistance allele with a higher cost.     

High level production of secreted proteins: example of the human tissue inhibitor of metalloproteinases 1

The major difficulty for high-throughput screening of therapeutic protein candidates in experimental animal models of pathologies or for structural studies is their fast and efficient production. The tissue inhibitors of metalloproteinases (TIMPs) considered to play a role in many physiological and pathological processes, such as arthritis or cancer, by inhibiting matrix metalloproteinases or acting as signalling molecules, have always been produced with huge difficulties. We hereby propose a new method to overproduce human recombinant TIMP-1 by transient expression in HEK293E cells, followed by a one-step chromatography purification, yielding in only 2 weeks, dozens of milligrams of pure, stable, glycosylated and active protein for in vitro and in vivo studies. This easy to set up, rapid, and efficient method could be applied for any naturally secreted mammalian protein.     

The evolution of recombination in a heterogeneous environment

Most models describing the evolution of recombination have focused on the case of a single population, implicitly assuming that all individuals are equally likely to mate and that spatial heterogeneity in selection is absent. In these models, the evolution of recombination is driven by linkage disequilibria generated either by epistatic selection or drift. Models based on epistatic selection show that recombination can be favored if epistasis is negative and weak compared to directional selection and if the recombination modifier locus is tightly linked to the selected loci. In this article, we examine the joint effects of spatial heterogeneity in selection and epistasis on the evolution of recombination. In a model with two patches, each subject to different selection regimes, we consider the cases of mutation-selection and migration-selection balance as well as the spread of beneficial alleles. We find that including spatial heterogeneity extends the range of epistasis over which recombination can be favored. Indeed, recombination can be favored without epistasis, with negative and even with positive epistasis depending on environmental circumstances. The selection pressure acting on recombination-modifier loci is often much stronger with spatial heterogeneity, and even loosely linked modifiers and free linkage may evolve. In each case, predicting whether recombination is favored requires knowledge of both the type of environmental heterogeneity and epistasis, as none of these factors alone is sufficient to predict the outcome.