Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

Physiological Diversity of Rhizoplane Diazotrophs of the Saltmeadow Cordgrass,Spartina patens: Implications for Host Specific Ecotypes

Diazotrophic bacteria are important contributors to salt marsh productivity, but the biotic and abiotic factors that influence their distributions and function and the extent of their diversity cannot be understood in the absence of physiological information. Here we examine the physiological diversity and distribution patterns of diazotrophic bacteria associated with the rhizoplane of the saltmeadow cordgrass, Spartina patens, in comparison with diazotrophs from other intertidal grasses (tall and short form Spartina alterniflora and Juncus roemerianus) from the same salt marsh. S. patens plants were collected from two distinct habitats, and a total of 115 strains (111 Gram negative and 4 Gram positive strains) were isolated into pure culture by stab inoculating roots and rhizomes into combined nitrogen-free semisolid media. Most strains were microaerophilic and approximately one-half were motile. API test strips were used to eliminate redundancy within the culture collection, resulting in 21 physiologically different API groups (17 Gram negative and 4 Gram positive groups). A representative strain from each API group was selected for dot blot hybridization with a nifH specific probe and 16 strains (13 Gram negative and 3 Gram positive) were scored as positive. The nifH positive API group representative strains were characterized further using BIOLOG test plates. Substrate utilization potentials defined two S. patens strain clusters, and only one S. patens strain was physiologically similar to any other strain from a different host plant origin. No distinctions could be made based on the different S. patens habitats, suggesting that the host plant may have a greater impact than abiotic environmental conditions on the distributions of the rhizoplane diazotrophs recovered.     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

The Genome Sequence of Psychrobacter arcticus 273-4, a Psychroactive Siberian Permafrost Bacterium,Reveals Mechanisms for Adaptation to Low-Temperature Growth

Psychrobacter arcticus strain 273-4, which grows at temperatures as low as −10°C, is the first cold-adapted bacterium from a terrestrial environment whose genome was sequenced. Analysis of the 2.65-Mb genome suggested that some of the strategies employed by P. arcticus 273-4 for survival under cold and stress conditions are changes in membrane composition, synthesis of cold shock proteins, and the use of acetate as an energy source. Comparative genome analysis indicated that in a significant portion of the P. arcticus proteome there is reduced use of the acidic amino acids and proline and arginine, which is consistent with increased protein flexibility at low temperatures. Differential amino acid usage occurred in all gene categories, but it was more common in gene categories essential for cell growth and reproduction, suggesting that P. arcticus evolved to grow at low temperatures. Amino acid adaptations and the gene content likely evolved in response to the long-term freezing temperatures (−10°C to −12°C) of the Kolyma (Siberia) permafrost soil from which this strain was isolated. Intracellular water likely does not freeze at these in situ temperatures, which allows P. arcticus to live at subzero temperatures.Temperature is one of the most important parameters that determine the distribution and extent of life on earth, and it does this by affecting cell structure and function. High temperatures break covalent bonds and ionic interactions between molecules, inactivating proteins and disrupting cell structures. Low temperatures reduce biochemical reaction rates and substrate transport and induce the formation of ice that damages cell structures. Not surprisingly, an organism''s compatibility with the temperature of its habitat is ultimately determined by its underlying genetic architecture.The strong emphasis in research on mesophile biology (temperatures in the 20°C to 37°C range) has given us a misimpression of the importance of cold on earth. However, 70% of the Earth''s surface is covered by oceans with average temperatures between 1°C and 5°C (11), 20% of the Earth''s terrestrial surface is permafrost (47), and a larger portion of the surface undergoes seasonal freezing, making our planet a predominantly cold environment. Hence, cold adaptation in the microbial world should be expected (55).Permafrost is defined as soils or sediments that are continuously exposed to a temperature of 0°C or less for at least 2 years (44). Permafrost temperatures range from −10°C to −20°C in the Arctic and from −10°C to −65°C in the Antarctic, and permafrost has low water activity, often contains small amounts of carbon (0.85 to 1%), and is subjected to prolonged exposure to damaging gamma radiation from ⁴⁰K in soil minerals (49). Liquid water occurs as a very thin, salty layer surrounding the soil particles in the frozen layer. Despite the challenges of the permafrost, a variety of microorganisms successfully colonize this environment, and many microorganisms have been isolated from it (54, 70). The bacterial taxa most frequently isolated from the Kolyma permafrost of northeast Siberia include Arthrobacter, Exiguobacterium, Flavobacterium, Sphingomonas, and Psychrobacter (71). Rhode and Price (56) proposed that microorganisms can survive in frozen ice for very long periods due to the very thin film of water surrounding each cell that serves as a reserve of substrates. Permafrost is a more favorable environment than ice as a result of its heterogeneous soil particles and larger reservoirs of nutrients.The genus Psychrobacter comprises a group of Gram-negative, rod-shaped, heterotrophic bacteria, and many Psychrobacter species are capable of growth at low temperatures. Members of this genus can grow at temperatures between −10°C and 42°C, and they have frequently been isolated from various cold environments, including Antarctic sea ice, ornithogenic soil and sediments, the stomach contents of Antarctic krill (Euphausia), deep seawater, and permafrost (9, 36, 57, 70, 71, 76; http://www.bacterio.cict.fr/p/psychrobacter.html). Psychrobacter arcticus 273-4 is a recently described species (4) that was isolated from a 20,000- to 30,000-year-old continuously frozen permafrost horizon in the Kolyma region in Siberia that was not exposed to temperatures higher than 4°C during isolation (70). This strain, the type strain of the species, grows at temperatures ranging from −10°C to 28°C, has a generation time of 3.5 days at −2.5°C, exhibits excellent long-term survival under freezing conditions, and has temperature-dependent physiological modifications in membrane composition and carbon source utilization (50). The fact that Psychrobacter has been found to be an indicator genus for permafrost and other polar environments (66) suggests that many of its members are adapted to low temperatures and increased levels of osmotica and have evolved molecular-level changes that aid survival at low temperatures.Early studies on cold adaptation in microorganisms revealed physiological strategies to deal with low temperatures, such as changes in membrane saturation, accumulation of compatible solutes, and the presence of cold shock proteins (CSPs) and many other proteins with general functions (62). However, many of the studies were conducted with mesophilic microorganisms, which limits the generality of the conclusions. We addressed the question of cold adaptation by studying microorganisms isolated from subzero environments using physiologic and genomic methods. We chose P. arcticus as our model because of its growth at subzero temperatures and widespread prevalence in permafrost. This paper focuses on the more novel potential adaptations.     

Effect of coculturing canine notochordal,nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration

IntroductionEarly degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs.MethodsCanine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture.ResultsNCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa.ConclusionsNo regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0569-6) contains supplementary material, which is available to authorized users.     

The distribution and evolutionary history of the PRP8 intein

Background  

We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal.