Ribonucleotide reductases: divergent evolution of an ancient enzyme

Ribonucleotide reductases (RNRs) are uniquely responsible for converting nucleotides to deoxynucleotides in all dividing cells. The three known classes of RNRs operate through a free radical mechanism but differ in the way in which the protein radical is generated. Class I enzymes depend on oxygen for radical generation, class II uses adenosylcobalamin, and the anaerobic class III requires S-adenosylmethionine and an iron–sulfur cluster. Despite their metabolic prominence, the evolutionary origin and relationships between these enzymes remain elusive. This gap in RNR knowledge can, to a major extent, be attributed to the fact that different RNR classes exhibit greatly diverged polypeptide chains, rendering homology assessments inconclusive. Evolutionary studies of RNRs conducted until now have focused on comparison of the amino acid sequence of the proteins, without considering how they fold into space. The present study is an attempt to understand the evolutionary history of RNRs taking into account their three-dimensional structure. We first infer the structural alignment by superposing the equivalent stretches of the three-dimensional structures of representatives of each family. We then use the structural alignment to guide the alignment of all publicly available RNR sequences. Our results support the hypothesis that the three RNR classes diverged from a common ancestor currently represented by the anaerobic class III. Also, lateral transfer appears to have played a significant role in the evolution of this protein family.     

A cellular tensegrity model to analyse the structural viscoelasticity of the cytoskeleton

This study describes the viscoelastic properties of a refined cellular-tensegrity model composed of six rigid bars connected to a continuous network of 24 viscoelastic pre-stretched cables (Voigt bodies) in order to analyse the role of the cytoskeleton spatial rearrangement on the viscoelastic response of living adherent cells. This structural contribution was determined from the relationships between the global viscoelastic properties of the tensegrity model, i.e., normalized viscosity modulus (eta(*)), normalized elasticity modulus (E(*)), and the physical properties of the constitutive elements, i.e., their normalized length (L(*)) and normalized initial internal tension (T(*)). We used a numerical method to simulate the deformation of the structure in response to different types of loading, while varying by several orders of magnitude L(*) and T(*). The numerical results obtained reveal that eta(*) remains almost independent of changes in T(*) (eta(*) proportional, variant T(*+0.1)), whereas E(*) increases with approximately the square root of the internal tension T(*) (from E(*) proportional, variant T(*+0.3) to E(*) proportional, variant T(*+0.7)). Moreover, structural viscosity eta(*) and elasticity E(*) are both inversely proportional to the square of the size of the structure (eta(*) proportional, variant L(*-2) and E(*) proportional, variant L(*-2)). These structural properties appear consistent with cytoskeleton (CSK) mechanical properties measured experimentally by various methods which are specific to the CSK micromanipulation in living adherent cells. Present results suggest, for the first time, that the effect of structural rearrangement of CSK elements on global CSK behavior is characterized by a faster cellular mechanical response relatively to the CSK element response, which thus contributes to the solidification process observed in adherent cells. In extending to the viscoelastic properties the analysis of the mechanical response of the cellular 30-element tensegrity model, the present study contributes to the understanding of recent results on the cellular-dynamic response and allows to reunify the scattered data reported for the viscoelastic properties of living adherent cells.     

Expression and self-assembly of norwalk virus capsid protein from venezuelan equine encephalitis virus replicons

The Norwalk virus (NV) capsid protein was expressed using Venezuelan equine encephalitis virus replicon particles (VRP-NV1). VRP-NV1 infection resulted in large numbers of recombinant NV-like particles that were primarily cell associated and were indistinguishable from NV particles produced from baculoviruses. Mutations located in the N-terminal and P1 domains of the NV capsid protein ablated capsid self-assembly in mammalian cells.     

New mutations of CIAS1 that are responsible for Muckle-Wells syndrome and familial cold urticaria: a novel mutation underlies both syndromes

Mutations of CIAS1 have recently been shown to underlie familial cold urticaria (FCU) and Muckle-Wells syndrome (MWS), in three families and one family, respectively. These rare autosomal dominant diseases are both characterized by recurrent inflammatory crises that start in childhood and that are generally associated with fever, arthralgia, and urticaria. The presence of sensorineural deafness that occurs later in life is characteristic of MWS. Amyloidosis of the amyloidosis-associated type is the main complication of MWS and is sometimes associated with FCU. In FCU, cold exposure is the triggering factor of the inflammatory crisis. We identified CIAS1 mutations, all located in exon 3, in nine unrelated families with MWS and in three unrelated families with FCU, originating from France, England, and Algeria. Five mutations--namely, R260W, D303N, T348M, A439T, and G569R--were novel. The R260W mutation was identified in two families with MWS and in two families with FCU, of different ethnic origins, thereby demonstrating that a single CIAS1 mutation may cause both syndromes. This result indicates that modifier genes are involved in determining either a MWS or a FCU phenotype. The finding of the G569R mutation in an asymptomatic individual further emphasizes the importance of such modifier a gene (or genes) in determining the disease phenotype. Identification of this gene (or these genes) is likely to have significant therapeutic implications for these severe diseases.     

Population ecology, nonlinear dynamics, and social evolution. I. Associations among nonrelatives

Using an individual-based and genetically explicit simulation model, we explore the evolution of sociality within a population-ecology and nonlinear-dynamics framework. Assuming that individual fitness is a unimodal function of group size and that cooperation may carry a relative fitness cost, we consider the evolution of one-generation breeding associations among nonrelatives. We explore how parameters such as the intrinsic rate of growth and group and global carrying capacities may influence social evolution and how social evolution may, in turn, influence and be influenced by emerging group-level and population-wide dynamics. We find that group living and cooperation evolve under a wide range of parameter values, even when cooperation is costly and the interactions can be defined as altruistic. Greater levels of cooperation, however, did evolve when cooperation carried a low or no relative fitness cost. Larger group carrying capacities allowed the evolution of larger groups but also resulted in lower cooperative tendencies. When the intrinsic rate of growth was not too small and control of the global population size was density dependent, the evolution of large cooperative tendencies resulted in dynamically unstable groups and populations. These results are consistent with the existence and typical group sizes of organisms ranging from the pleometrotic ants to the colonial birds and the global population outbreaks and crashes characteristic of organisms such as the migratory locusts and the tree-killing bark beetles.     

The Endoplasmic reticulum-associated maize GL8 protein is a component of the acyl-coenzyme A elongase ivolved in the production of cuticular waxes

The gl8 gene is required for the normal accumulation of cuticular waxes on maize (Zea mays) seedling leaves. The predicted GL8 protein exhibits significant sequence similarity to a class of enzymes that catalyze the reduction of a ketone group to a hydroxyl group. Polyclonal antibodies raised against the recombinant Escherichia coli-expressed GL8 protein were used to investigate the function of this protein in planta. Subcellular fractionation experiments indicate that the GL8 protein is associated with the endoplasmic reticulum membranes. Furthermore, polyclonal antibodies raised against the partially purified leek (Allium porrum) microsomal acyl-coenzyme A (CoA) elongase can react with the E. coli-expressed GL8 protein. In addition, anti-GL8 immunoglobulin G inhibited the in vitro elongation of stearoyl-CoA by leek and maize microsomal acyl-CoA elongase. In combination, these findings indicate that the GL8 protein is a component of the acyl-CoA elongase. In addition, the finding that anti-GL8 immunoglobulin G did not significantly inhibit the 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA dehydrase, and (E) 2,3-enoyl-CoA reductase partial reactions of leek or maize acyl-CoA elongase lends further support to our previous hypothesis that the GL8 protein functions as a beta-ketoacyl reductase during the elongation of very long-chain fatty acids required for the production of cuticular waxes.     

Nonselective currents and channels in plasma membranes of protoplasts from coats of developing seeds of bean

In developing bean (Phaseolus vulgaris) seeds, phloem-imported nutrients move in the symplast from sieve elements to the ground parenchyma cells where they are transported across the plasma membrane into the seed apoplast. To study the mechanisms underlying this transport, channel currents in ground parenchyma protoplasts were characterized using patch clamp. A fast-activating outward current was found in all protoplasts, whereas a slowly activating outward current was observed in approximately 25% of protoplasts. The two currents had low selectivity for univalent cations, but the slow current was more selective for K(+) over Cl(-) (P(K):P(Cl) = 3.6-4.2) than the fast current (P(K):P(Cl) = 1.8-2.5) and also displayed Ca(2+) selectivity. The slow current was blocked by Ba(2+), whereas both currents were blocked by Gd(3+) and La(3+). Efflux of K(+) from seed coat halves was inhibited 25% by Gd(3+) and La(3+) but was stimulated by Ba(2+) and Cs(+), suggesting that only the fast current may be a component in the pathway for K(+) release. An "instantaneous" inward current observed in all protoplasts exhibited similar pharmacology and permeability for univalent cations to the fast outward current. In outside-out patches, two classes of depolarization-activated cation-selective channels were observed: one slowly activating of low conductance (determined from nonstationary noise to be 2.4 pS) and another with conductances 10-fold higher. Both channels occurred at high density. The higher conductance channel in 10 mM KCl had P(K):P(Cl) = 2.8. Such nonselective channels in the seed coat ground parenchyma cell could function to allow some of the efflux of phloem-imported univalent ions into the seed apoplast.     

Radiotherapy: effects on expanded skin

Although the combination of radiation and tissue expansion has been associated with a significant rate of complications, the specific pathophysiology has yet to be clearly elucidated. The objective of this study was to develop a model to identify and examine specific histologic changes associated with tissue expansion and irradiation. Rectangular 50-cc silicone tissue expanders were placed subcutaneously over the midline dorsum of 18 adult New Zealand white rabbits. Preoperative radiographic dosimetry demonstrated that the radiation portal was away from vital intraabdominal structures. The expanders were inflated with 10 cc of saline every other day for a total of 80 cc. Expanders were left in place for 2 to 3 weeks to allow fibrovascular capsule formation. The rabbits were then divided into three groups (six rabbits per group), each receiving one of three nonfractionated doses of radiation (20, 25, or 35 Gy). Half of the expanded skin was irradiated using a single dose, and the other half served as a nonirradiated control. Capsules and skin were harvested 6 weeks after the delivery of radiation, allowing the beginning of chronic radiation changes to occur. Using hematoxylin and eosin staining, histomorphometric analysis was performed. The data were analyzed using Student's test. Although irradiation did not affect dermal thickness, it did cause a statistically significant increase in epidermal thickness. At 20, 25, and 35 Gy the increase in epidermal thickness was 43, 90, and 130 percent, respectively. Although significant epidermal changes could be identified, capsular and dermal alterations were not evident. Further studies evaluating the long-term effects of alterations in capsular formation caused by radiation may be required.