Anatomical network analysis of the musculoskeletal system reveals integration loss and parcellation boost during the fins‐to‐limbs transition

Tetrapods evolved from within the lobe‐finned fishes around 370 Ma. The evolution of limbs from lobe‐fins entailed a major reorganization of the skeletal and muscular anatomy of appendages in early tetrapods. Concurrently, a degree of similarity between pectoral and pelvic appendages also evolved. Here, we compared the anatomy of appendages in extant lobe‐finned fishes (Latimeria and Neoceratodus) and anatomically plesiomorphic amphibians (Ambystoma, Salamandra) and amniotes (Sphenodon) to trace and reconstruct the musculoskeletal changes that took place during the fins‐to‐limbs transition. We quantified the anatomy of appendages using network analysis. First, we built network models—in which nodes represent bones and muscles, and links represent their anatomical connections—and then we measured network parameters related to their anatomical integration, heterogeneity, and modularity. Our results reveal an evolutionary transition toward less integrated, more modular appendages. We interpret this transition as a diversification of muscle functions in tetrapods compared to lobe‐finned fishes. Limbs and lobe‐fins show also a greater similarity between their pectoral and pelvic appendages than ray‐fins do. These findings on extant species provide a basis for future quantitative and comprehensive reconstructions of the anatomy of limbs in early tetrapod fossils, and a way to better understand the fins‐to‐limbs transition.     

The relationship between the chromophore moiety and the cation binding sites in bacteriorhodopsin

Bleaching of the purple membrane strongly reduces the number of divalent cation binding sites as well as their affinities. Conversely, deionization of the bleached membrane drastically inhibits the chromophore regeneration. Proteolysis experiments using bromelain show that the bleached membrane has an additional cleavage site probably located at the fifth loop, whereas in the blue membrane, the C-terminal tail is no longer susceptible to proteolysis. It is suggested that there exists a close relationship between the retinal environment and one or more of the cation binding sites.     

Characterization of pancreatic somatostatin binding sites with a I-somatostatin 28 analog

Somatostatin binding to guinea pig pancreatic acinar cell plasma membranes was characterized with an iodinated stable analog of somatostatin 28 (S₂₈): ¹²⁵I-[Leu⁸, DTrp²²,Tyr²⁵] S₂₈. The binding was highly dependent on calcium ions. In 0.2 mM free Ca²⁺ medium, binding at 37°C was saturable, slowly reversible and exhibited a single class of high affinity binding sites (K_D=0.05±0.01 nM, B_max=157±33 fmol/mg protein). Dissociation of bound radioactivity occurred with biphasic kinetics. Rate of dissociation increased when dissociation was measured at a time before equilibrium binding was reached. In 30 nM free Ca²⁺ medium, binding affinity and maximal binding capacity were decreased by about 4-fold. Decreasing calcium concentrations increased the amount of rapidly dissociating form of the receptor. Somatostatin 14 antagonist, Des AA^1,2[AzaAla^4–5,DTrp⁸,Phe^12–13]-somatostatin was active at the membrane level in inhibiting the binding. We conclude that using ¹²⁵I-[Leu⁸,DTrp²²,Tyr²⁵]S₂₈ as radioligand allows us to characterize a population of specific somatostatin receptors which are not different from those we previously described with the radioligand ¹²⁵I-[Tyr¹¹]-somatostatin. Somatostatin receptors could exist in two interconvertible forms. Calcium ions are an essential component in the regulation of the conformational change of somatostatin receptors.     

Influence of cations on the blue to purple transition of bacteriorhodopsin. Comparison of Ca2+ and Hg2+ binding and their effect on the surface potential

We have investigated the effect of Ca2+ and Hg2+ binding on various properties of the blue membrane prepared by deionization of the Halobacterium halobium purple membrane. Binding of radioactive 45Ca2+ and 203Hg2+ was monitored by a filtration technique. Five high and medium affinity sites for Ca2+ and seven low affinity sites for Hg2+ were found per bacteriorhodopsin. Competitive binding was observed only for three Ca2+ and three Hg2+. Visible absorption studies indicated that Ca2+ binding could restore the purple color of bacteriorhodopsin while Hg2+ was inefficient. Hg2- could partially reverse to blue the Ca2+-regenerated purple membrane in parallel with the displacement of three Ca2+. Effects of cation binding on the surface potential of the membrane were measured by Electron Spin Resonance spectroscopy using a cationic spin-labeled amphiphile. Cations such as La3+, Ca2+, Mg2+, or Na+ strongly increased (i.e. rendered less negative) the surface potential. An univocal correlation was found between the cation-induced variation of surface potential and the extent of regeneration of the purple color. Hg2+ induced a smaller increase in surface potential than that corresponding to the effective divalent cations. This lower effect appears to be due to binding to sites not related to those of other cations.     

Mobilifilum chasei: Morphology and ecology of a spirochete from an intertidal stratified microbial mat community

Spirochetes were found in the lower anoxiphototrophic layer of a stratified microbial mat (North Pond, Laguna Figueroa, Baja California, Mexico). Ultrastructural analysis of thin sections of field samples revealed spirochetes approximately 0.25 m in diameter with 10 or more periplasmic flagella, leading to the interpretation that these spirochetes bear 10 flagellar insertions on each end. Morphometric study showed these free-living spirochetes greatly resemble certain symbiotic ones, i.e., Borrelia and certain termite spirochetes, the transverse sections of which are presented here. The ultrastructure of this spirochete also resembles Hollandina and Diplocalyx (spirochetes symbiotic in arthropods) more than it does Spirochaeta, the well known genus of mud-dwelling spirochetes. The new spirochete was detected in mat material cellected both in 1985 and in 1987. Unique morphology (i.e., conspicuous outer coat of inner membrane, large number of periplasmic flagella) and ecology prompt us to name a new free-living spirochete.This article is dedicated to the memory of our dear friend and colleague David G. Chase (1935–1987)     

A quantitative ultrastructural study of Chromatium minus in the bacterial layer of Lake Cisó (Spain)

A quantitative ultrastructural study was performed with samples taken throughout a layer of the purple sulfur bacterium Chromatium minus in Lake Cisó (Spain). Ultrathin sections of cells were analyzed by transmission electron microscopy, in order to study the size, number and volume of intracytoplasmic membranes (ICM), sulfur globules and poly--hydroxybutyrate (PHB) granules per unit volume of cell. Important differences were seen between cells from the top (receiving 60 E · m^–2 · s^–1 at noon) on the one hand, and cells from the peak and bottom parts of the bacterial layer (receiving less than 1 E · m^–2 · s^–1) on the other hand. The amount of ICM per cell increased as a function of depth being about three times higher in bottom cells than in top cells. Neither statistically significant differences in cell size, nor in numbers of sulfur globules were found, but the ultrastructure changed with depth. Finally, the most important changes throughout depth were detected in PHB granules. Top cells had 0.5% of their volume occupied by PHB granules, whereas in the bottom cells the corresponding value was 12.2%. These changes were due to the number of PHB granules per unit volume of cell since globule size was constant.Non-common abbreviations ECM intracytoplasmic membrane systems - PHB poly--hydroxybutyrate - Bchl bacteriochlorophyll - SED sphere equivalent diameter     

2-Hydroxy-5-nitrobenzyl bromide as a specific reagent for tryptophan residues in membrane proteins: bacteriorhodopsin as an example

The use of 2-hydroxy-5-nitrobenzyl bromide for the modification of tryptophan residues in integral membrane proteins is exemplified by its application to bacteriorhodopsin from Halobacterium halobium. Complete elimination of the unreacted reagent requires delipidation of the sample with detergents and posterior chromatography. This method also allows separation of the modified from the unmodified bacteriorhodopsin molecules. Modified molecules have lost the retinal, and are thus bleached, whereas the unmodified molecules appear to retain all the characteristics of solubilized native bacteriorhodopsin.