Timing of the evolutionary history of Corallinaceae (Corallinales,Rhodophyta)

The temporal dimension of the most recent Corallinaceae (order Corallinales) phylogeny was presented here, based on first occurrence time estimates from the fossil record. Calibration of the molecular clock of the genetic marker SSU entailed a separation of Corallinales from Hapalidiales in the Albian (Early Cretaceous ~105 mya). Neither the calibration nor the fossil record resolved the succession of appearance of the first three emerging subfamilies: Mastophoroideae, Corallinoideae, and Neogoniolithoideae. The development of the tetra/bisporangial conceptacle roofs by filaments surrounding and interspersed among the sporangial initials was an evolutionary novelty emerging at the Cretaceous–Paleogene boundary (~66 mya). This novelty was shared by the subfamilies Hydrolithoideae, Metagoniolithoideae, and Lithophylloideae, which diverged in the early Paleogene. Subclades within the Metagoniolithoideae and Lithophylloideae diversified in the late Oligocene–middle Miocene (~28–12 mya). The most common reef corallinaceans (Hydrolithon, Porolithon, Harveylithon, “Pneophyllum” conicum, and subclades within Lithophylloideae) appeared in this interval in the Indo‐Australian Archipelago.     

Inhibition of basal and calmodulin-activated Ca2+-pump ATPase by fractionated compound 48/80

Compound 48/80 (48/80), a mixture of polycationic compounds was fractionated using affinity chromatography on calmodulin-Sepharose. Unfractionated 48/80 and various fractions were tested for their potential inhibitory effects on ATPase activities of isolated human red blood cell membranes. ATPase activities tested included: Mg2+-ATPase, the Na+/K+-pump ATPase, and the Ca2+-pump ATPase in both its basal (calmodulin-independent) and calmodulin-activated state. Neither 48/80 nor its various fractions were very potent or efficacious inhibitors of the Mg2+-ATPase or the Na+/K+-pump ATPase. In agreement with previous reports, 48/80 was found to be an inhibitor of the calmodulin-activated Ca2+-pump ATPase. By contrast, we found that unfractionated, as well as some fractionated, material inhibited both the basal (calmodulin-independent) and calmodulin-activated Ca2+-pump ATPase activity. A fraction designated as Fraction III bound to calmodulin-Sepharose in the presence of Ca2+ and low salt and was eluted in the absence of Ca2+ and 0.15 M NaCl. By gel filtration, Fraction III had an apparent average molecular weight of 2064 (1320 for unfractionated material). Fraction III was the most potent inhibitor of the Ca2+-pump ATPase with IC50 values for the basal and calmodulin-activated forms of the enzyme of 0.6 and 1.2 micrograms/ml, respectively. Inhibition by Fraction III was cooperative with n apparent values of 2.4 and 5.7, respectively, for the basal and calmodulin-activated forms of the enzyme. Thus, binding of 48/80 constituents to calmodulin can not fully account for the observed data. Direct interaction of 48/80 constituent(s) with the enzyme and/or the lipid portion of the membrane is suggested.     

Babesia bigemina sexual stages are induced in vitro and are specifically recognized by antibodies in the midgut of infected Boophilus microplus ticks

Babesia bigemina, a causative agent of bovine babesiosis, is transmitted from one bovine to another only by infected ticks. The life cycle of B. bigemina includes a sexual phase in the tick host; however, molecules from sexual stages of any Babesia species have not been characterized. This is the first report of the induction of sexual stages of any Babesia species in vitro, free of tick antigens. Intraerythrocytic parasites were cultured in vitro for 20h using an induction medium. Extraerythrocytic parasites were first seen 3h post induction; elongated stages with long projections appeared at 6h post induction and by 9h they paired and fused to form larger stages. Round zygotes appeared 20h post induction. Moreover, by using Percoll gradients, sexual stages were purified free of contaminating intraerythrocytic stages. Purified parasites were used to generate polyclonal antibodies, which specifically bound to antigens expressed in sexual stages induced in vitro, but not to antigens expressed in intraerythrocytic stages. Importantly, these antibodies specifically identified sexual stages from midguts of female Boophilus microplus ticks fed on infected cattle.     

Protein isolates from jumbo squid (Dosidicus gigas) by pH-shift processing

There is a growing interest in adding value to the jumbo squid Dosidicus gigas fishery. This work describes two extraction procedures for processing muscle to obtain protein isolates with suitable functional properties. The effect on muscle protein solubility and protein recovery of combining freezing and grinding raw materials during storage was evaluated. Processes are based on extraction of protein at acid or alkaline pH and subsequent iso-electric precipitation. About 85% of the initial muscle protein was solubilized at pH 3 and 11. Regardless of the pH used for extraction, about 90% of the protein was obtained after precipitation at pH 5.5. The total yield from both procedures was 75%. Treatments during storage did not significantly affect solubility and yield of protein. Wastewater contained negligible amounts of protein and may be reused. Processing by acid and alkaline extraction are feasible alternatives for obtaining protein isolates either from fresh or frozen squid muscle, which is an important consideration when choosing the most appropriate and inexpensive method to scale up this technology.     

Temperature-dependent development,cold tolerance,and potential distribution of Gratiana boliviana (Coleoptera: Chrysomelidae), a biological control agent of tropical soda apple,Solanum viarum (Solanaceae)

The leaf beetle Gratiana boliviana Spaeth (Coleoptera: Chrysomelidae) was introduced from South America into the southeastern United States in 2003 as a classical biological control agent of tropical soda apple, Solanum viarum Dunal (Solanaceae). Temperature-dependent development and survival studies revealed that development was completed at temperatures >16°C and ≤34°C. The number of degree-days required to complete one generation was 341 and the estimated lower developmental threshold was 13.37°C. Using nonlinear regression, the upper lethal threshold was estimated to be 34–35°C. Cold tolerance studies revealed that the lethal time for 90% of adults (LT₉₀) was 12.6 days at 5°C and 8.68 days at 0°C. Based on the developmental and cold tolerance data, a map predicting the areas of establishment and number of generations per year was generated, which suggests that the northern extent of the G. boliviana range in the USA will be near 32–33° north latitude. Fewer generations per year in more northern areas of the southeastern USA may decrease the effectiveness of this biological control agent.     

Biochemical variation to determine phylogenetic relationships betweenHordeum chilense and other American species of the genusHordeum (Poaceae)

Slab gel electrophoresis techniques have been applied to the study of isozyme and kernel protein patterns in 20 accessions ofHordeum chilense and related species in order to elucidate their phylogenetic relationships. On the basis of our results we can conclude that: (1) Conventional classification based on morphological characters does not totally agree with biochemical data. (2) Sectt.Anisolepis andCritesion seem to be clearly differentiated. (3) The accessions classified asH. compressum present biochemical phenotypes quite different from the rest of the species. (4)H. stenostachys, H. muticum andH. chilense constitute a group of variable species with many biochemical similarities and close phylogenetic relationships. (5) The evolutionary pattern of these American species seems to follow a model of reticulate evolution.     

A new karyotype of an endangered primate species (Callicebus personatus) from the Brazilian Atlantic forests

The genus Callicebus has 28 species arranged in five groups: donacophilus, torquatus, moloch, cupreus and personatus. The personatus group occurs in the Brazilian Atlantic forests where it is isolated from the other Callicebus groups. The present paper is the first report on the karyotype of Callicebuspersonatus. We compared the karyotypes of C. personatus and C. nigrifrons utilizing the following classic banding techniques: G-, C-, NOR banding, G/C- and G/NOR sequential banding. The karyotype of C. personatus has 2n=44, while C. nigrifrons presented 2n=42. Both the species diverge in the diploid number in consequence of a rearrangement type centric fusion/fission. Our results support personatus as a valid group and the specific status for C. personatus and C. nigrifrons.     

Computational prediction of atomic structures of helical membrane proteins aided by EM maps

Integral membrane proteins pose a major challenge for protein-structure prediction because only approximately 100 high-resolution structures are available currently, thereby impeding the development of rules or empirical potentials to predict the packing of transmembrane alpha-helices. However, when an intermediate-resolution electron microscopy (EM) map is available, it can be used to provide restraints which, in combination with a suitable computational protocol, make structure prediction feasible. In this work we present such a protocol, which proceeds in three stages: 1), generation of an ensemble of alpha-helices by flexible fitting into each of the density rods in the low-resolution EM map, spanning a range of rotational angles around the main helical axes and translational shifts along the density rods; 2), fast optimization of side chains and scoring of the resulting conformations; and 3), refinement of the lowest-scoring conformations with internal coordinate mechanics, by optimizing the van der Waals, electrostatics, hydrogen bonding, torsional, and solvation energy contributions. In addition, our method implements a penalty term through a so-called tethering map, derived from the EM map, which restrains the positions of the alpha-helices. The protocol was validated on three test cases: GpA, KcsA, and MscL.