Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris

The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a “closed” conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile¹⁶⁵ and Met³³¹) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala⁴⁷) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme''s k_cat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature.     

Toward deciphering proteomes of formalin-fixed paraffin-embedded (FFPE) tissues

Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for both prospective and retrospective biomarker discovery. Unlocking the proteomic profile of clinicopathological FFPE tissues is a critically essential step for annotating clinical findings and predicting biomarkers for ultimate disease prognosis and therapeutic follow-up.     

Evolutionary game dynamics in a finite asymmetric two-deme population and emergence of cooperation

We consider evolutionary game dynamics in a finite population subdivided into two demes with both unequal deme sizes and different migration rates. Assuming viability differences in the population according to a linear game within each deme as a result of pairwise interactions, we specify conditions for weak selection favoring a mutant strategy to go to fixation, under the structured-coalescent assumptions, and their connections with evolutionary stability concepts. In the framework of the Iterated Prisoner's Dilemma with strategy ‘tit-for-tat’ as mutant strategy and ‘always defect’ as resident strategy, we deduce a condition under which the emergence of cooperation is favored by selection, when the game matrix is the same in both demes. We show how this condition extends the one-third law for a panmictic population and when an asymmetry in the spatial structure of a two-deme population facilitates the emergence of the cooperative tit-for-tat strategy in comparison with both its symmetric and panmictic population structure counterparts. We find that the condition is less stringent in the asymmetric scenario versus the symmetric scenario if both the fraction of the population in the deme where the mutant was initially introduced, and the expected proportion of migrant offspring in this deme among all migrant offspring after population regulation, are smaller than, or equal to, , provided they are not too small. On the other hand, the condition is less stringent than the one-third law, which holds in the panmictic case, if the latter proportion remains not too close to 1.     

Probability of fixation under weak selection: a branching process unifying approach

We link two-allele population models by Haldane and Fisher with Kimura's diffusion approximations of the Wright-Fisher model, by considering continuous-state branching (CB) processes which are either independent (model I) or conditioned to have constant sum (model II). Recent works by the author allow us to further include logistic density-dependence (model III), which is ubiquitous in ecology. In all models, each allele (mutant or resident) is then characterized by a triple demographic trait: intrinsic growth rate r, reproduction variance sigma and competition sensitivity c. Generally, the fixation probability u of the mutant depends on its initial proportion p, the total initial population size z, and the six demographic traits. Under weak selection, we can linearize u in all models thanks to the same master formula u = p + p(1 - p)[g(r)s(r) + g(sigma)s(sigma) + g(c)s(c)] + o(s(r),s(sigma),s(c), where s(r) = r' - r, s(sigma) = sigma-sigma' and s(c) = c - c' are selection coefficients, and g(r), g(sigma), g(c) are invasibility coefficients (' refers to the mutant traits), which are positive and do not depend on p. In particular, increased reproduction variance is always deleterious. We prove that in all three models g(sigma) = 1/sigma and g(r) = z/sigma for small initial population sizes z. In model II, g(r) = z/sigma for all z, and we display invasion isoclines of the 'mean vs variance' type. A slight departure from the isocline is shown to be more beneficial to alleles with low sigma than with high r. In model III, g(c) increases with z like ln(z)/c, and g(r)(z) converges to a finite limit L > K/sigma, where K = r/c is the carrying capacity. For r > 0 the growth invasibility is above z/sigma when z < K, and below z/sigma when z > K, showing that classical models I and II underestimate the fixation probabilities in growing populations, and overestimate them in declining populations.     

Treatment of the Fixation Surface Improves Glenoid Prosthesis Longevity in vitro

Many commercial cemented glenoid components claim superior fixation designs and increased survivability. However, both research and clinical studies have shown conflicting results and it is unclear whether these design variations do improve loosening rates. Part of the difficulty in investigating fixation failure is the inability to directly observe the fixation interface, a problem addressed in this study by using a novel experimental set-up.Cyclic loading-displacement tests were carried out on 60 custom-made glenoid prostheses implanted into a bone substitute. Design parameters investigated included treatment of the fixation surface of the component resulting in different levels of back-surface roughness, flat-back versus curved-back, keel versus peg and more versus less conforming implants. Visually-observed failure and ASTM-recommended rim-displacements were recorded throughout testing to investigate fixation failure and if rim displacement is an appropriate measure of loosening.Roughening the implant back (R_a > 3 µm) improved resistance to failure (P < 0.005) by an order of magnitude with the rough and smooth groups failing at 8712 ± 5584 cycles (mean ± SD) and 1080 ± 1197 cycles, respectively. All other design parameters had no statistically significant effect on the number of cycles to failure. All implants failed inferiorly and 95% (57/60) at the implant/cement interface. Rim-displacement correlated with visually observed failure.The most important effect was that of roughening the implant, which strengthened the polyethylene-cement interface. Rim-displacement can be used as an indicator of fixation failure, but the sensitivity was insufficient to capture subtle effects.Level of Evidence: Basic Science Study, Biomechanical Analysis.     

利用微藻固定烟道气中CO2的实验研究

为获得能适合于烟道气条件下生长的微藻 ,找到一种高效的温室气体固定的方法 ,利用配置烟道气 (CO2 和O2 的浓度分别为 15 %和 2 %)驯化稻田微藻混合试样 ,分离出对高浓度CO2 条件有很强适应力的微藻ZY 1,并研究了在不同培养条件下微藻ZY 1的生长情况 .微藻ZY 1在CO2 浓度从 10 %～ 15 %的范围内有较高生长力 ,在CO2 浓度为 10 %时 ,生长最好 .微藻ZY 1对温度、气体流速、pH值等物理条件也有很宽的适应范围 ,在温度为 2 5～ 30 .C、流速为 0 2 5～ 0 75L·min-1、pH4～ 6范围内 ,生长基本稳定 .在培养条件为 10 %CO2 、2 5 .C、pH5 0时 ,微藻ZY 1的生长率最高 ,CO2 的固定率平均值为 0 397%.可以认为 ,利用该藻固定烟道气温室气体具有一定的可行性 .     

Alcoholic fixation over formalin fixation: A new,safer option for morphologic and molecular analysis of tissues

Formalin is a widely used fixative but there is potential public health risks to exposure. Besides, alcoholic fixation is advantageous over formalin fixation because of faster fixation, optimal preservation and safer workplace environment. Following fixation by EMA and 10% neutral buffered formalin (NBF), we analyzed the tissue morphology, antigenic stability, DNA and RNA quantity with quality (OD value). The findings of EMA fixing on both the tissue morphology and molecular characterization, were satisfactory. Specially, EMA was faster in penetration of tissues than NBF, fixed ideally as early as 8 h of fixation whereas improper fixation was evident for NBF. In Hematoxylin and Eosin (H & E) staining, better cellular details with stronger affinity for staining were observed. In immunohistochemistry, better antigenic stability was reported for EMA-fixed tissues. The nucleic acid analysis revealed that total genomic DNA and RNA yield from EMA fixed tissues were significantly higher (P < 0.05) with superior quality than NBF fixed tissues. Our results suggest that EMA could be a potential alternative to NBF for fixation and preservation of tissues. These data provide new insights into an option for a safer working environment to support study and research.     

Fixation for the 1990's: a Review of Needs and Accomplishments

Neutral buffered formalin (NBF) has been the standard fixative in histopathology for many decades: however, new technologies and increasing time constraints have made this common fixative less widely applicable. The attributes of universal and ideal fixatives are reviewed and compared with those of NBF. On the strength of a growing body of literature and experience in the histopathology field, zinc formalin solutions are recommended as a functional replacement for NBF. Zinc formalin permits better morphological preservation and preserves immunoreactivity even after prolonged fixation times. Coordinated zinc ions are postulated to maintain macromolecules in their original three-dimensional conformation as formaldehyde forms addition products with them. Significant crosslinking and gross distortion of tertiary structure are thereby avoided.     

Rearrangements of F-actin during Stomatogenesis Visualised by Confocal Microscopy in Fixed and Permeabilised Tradescantia Leaf Epidermis

Abstract: New details of F-actin organisation in leaf epidermal and stomatal cells were revealed by rhodamine — and fluorescein — phalloidin staining of fixed epidermal peels of Tradescantia virginiana and visualisation by confocal microscopy. Non-specialised epidermal cells contain highly organised arrays of fine cortical actin filaments aligned in transverse or oblique orientations. In interphase guard mother cells (GMCs), the arrangement of cortical F-actin changes on the periclinal and anticlinal cell walls at different times during differentiation. Initially, cortical F-actin on the periclinal surfaces is oriented transversely and F-actin is evenly distributed around the anticlinal walls. Following polarisation of the adjacent subsidiary mother cells (SMCs), actin in GMCs concentrates on the lateral anticlinal walls, but not on the transverse walls. Subsequently, F-actin on the periclinal walls reorients to radial and then longitudinal. Organisation of F-actin in SMCs appears to be influenced by the adjacent GMCs and co-ordination in F-actin arrangements in cells of the stomatal complex continues through to the formation of the guard cell pair. Our studies indicate that actin bands marking the division site in prophase cells, and detected in microinjected living material, are a particularly labile subset of F-actin. Actin bands were difficult to preserve, even when aldehyde fixation was avoided, in contrast to all interphase and mitotic F-actin.     

Photosynthesis and respiration of Griffithsia monilis (Rhodophyceae): Effect of light,salinity, and oxygen

The giant-celled alga Griffithsia monilis has a low light compensation point and saturates photosynthesis at 60–90 mol photons m^-2s^-1 (oxygen evolution and CO₂ fixation). Under dark and low light intensities ¹⁴C is preferentially incorporated into amino acids (mainly aspartate and alanine). With increasing light a gradual change was observed and, under light saturation, compounds of the anionic fraction (digeneaside and hexosephosphates) were the most strongly labeled compounds, together with the amino acids glycine and serine. To a large extent (30–40% of the total) ¹⁴C was fixed into EtOH-insoluble products, the hydrolysates of which consisted mainly of glucose and mannose. In the steady state the rates of photosynthesis and respiration decreased with increasing salinity. Changes in the rates after hyperosmotic shocks were less severe in cells adapted to high salinities. Photorespiration exists in Griffithsia: Glycine and serine are the major labeled compounds in O₂-saturated media.