Oms38 is the first identified pore-forming protein in the outer membrane of relapsing fever spirochetes

Relapsing fever is a worldwide, endemic disease caused by several spirochetal species belonging to the genus Borrelia. During the recurring fever peaks, borreliae proliferate remarkably quickly compared to the slow dissemination of Lyme disease Borrelia and therefore require efficient nutrient uptake from the blood of their hosts. This study describes the identification and characterization of the first relapsing fever porin, which is present in the outer membranes of B. duttonii, B. hermsii, B. recurrentis, and B. turicatae. The pore-forming protein was purified by hydroxyapatite chromatography and designated Oms38, for outer membrane-spanning protein of 38 kDa. Biophysical characterization of Oms38 was done by using the black lipid bilayer method, demonstrating that Oms38 forms small, water-filled channels of 80 pS in 1 M KCl that did not exhibit voltage-dependent closure. The Oms38 channel is slightly selective for anions and shows a ratio of permeability for cations over anions of 0.41 in KCl. Analysis of the deduced amino acid sequences demonstrated that Oms38 contains an N-terminal signal sequence which is processed under in vivo conditions. Oms38 is highly conserved within the four studied relapsing fever species, sharing an overall amino acid identity of 58% and with a strong indication for the presence of amphipathic β-sheets.     

A function for dystroglycan in pronephros development in Xenopus laevis

Dystroglycan (Dg) is a laminin receptor that is expressed at the interface between the basement membrane and the cell membrane. Dg has been reported to play a role in skeletal muscle cell stability, morphogenesis of neuroepithelial tissues, and in regulating cytoskeletal organization, cell polarization, and cell signalling. In this study, we have focused our analysis on the expression of Dg-mRNA and protein at different developmental stages in the pronephros of Xenopus laevis. In order to study its role, we performed loss-of-function experiments mediated by Dg antisense morpholinos and dominant negative mutant. We show that Dg expression is first detectable when epithelialization begins in the pronephric anlage and persists later during tubulogenesis. Loss-of-function experiments induced a disorganization of the basement membrane, a drastic reduction of pronephric tubules and duct that can lead to a renal agenesis. A diminished proliferation of pronephric cell progenitors was also observed in Dg depleted embryos. Together, these data indicate that Dg plays a key role for laminin-1 assembly and pronephric cell anchoring to the basement membrane during early development of the pronephros. They also indicate that Dg may induce a signal transduction pathway controlling cell proliferation needed for the formation of tubules and their growth.     

Ordered progression of nematogenesis from stem cells through differentiation stages in the tentacle bulb of Clytia hemisphaerica (Hydrozoa, Cnidaria)

Nematogenesis, the production of stinging cells (nematocytes) in Cnidaria, can be considered as a model neurogenic process. Most molecular data concern the freshwater polyp Hydra, in which nematocyte production is scattered throughout the body column ectoderm, the mature cells then migrating to the tentacles. We have characterized tentacular nematogenesis in the Clytia hemisphaerica hydromedusa and found it to be confined to the ectoderm of the tentacle bulb, a specialized swelling at the tentacle base. Analysis by a variety of light and electron microscope techniques revealed that while cellular aspects of nematogenesis are similar to Hydra, the spatio-temporal characteristics are markedly more ordered. The tentacle bulb nematogenic ectoderm (TBE) was found to be polarized, with a clear progression of successive nematoblast stages from a proximal zone (comprising a majority of undifferentiated cells) to the distal end where the tentacle starts. Pulse-chase labelling experiments demonstrated a continuous displacement of differentiating nematoblasts towards the tentacle tip, and that nematogenesis proceeds more rapidly in Clytia than in Hydra. Compact expression domains of orthologues of known nematogenesis-associated genes (Piwi, dickkopf-3, minicollagens and NOWA) were correspondingly staggered along the TBE. These distinct characteristics make the Clytia TBE a promising experimental system for understanding the mechanisms regulating nematogenesis.     

An interstate highway affects gene flow in a top reptilian predator (<Emphasis Type="Italic">Crotalus atrox</Emphasis>) of the Sonoran Desert

Roads can substantially impact the population connectivity of a wide range of terrestrial vertebrates, often resulting in loss of genetic diversity and an increase of spatial genetic structure. We studied the Western Diamond-backed Rattlesnake (Crotalus atrox), a large and abundant venomous predator, to test the hypothesis that a large and relatively new roadway in Arizona (Interstate Highway I-10) is a barrier that impacts gene flow and population genetics via habitat fragmentation. Based on 72 C. atrox sampled from three specific sampling sites (“subpopulations”) on both the west and east corridors of I-10, we used 30 nuclear microsatellite DNA loci and three mitochondrial DNA genes (2615 bp) to assess genetic diversity and structure, estimate effective population size (N _e ), and describe patterns of gene flow. We found no evidence for loss of genetic diversity or a decrease in N _e between the three subpopulations. Our microsatellite analysis showed that two subpopulations in close proximity (4 km), but separated by I-10, showed greater levels of genetic differentiation than two subpopulations that were separated by a greater distance (7 km) and not by I-10 or any other obvious barriers. Mitochondrial DNA analyses showed no significant genetic differentiation nor any indication of historically impeded gene flow. Tajima’s D and mismatch distribution tests revealed that demographic expansion is occurring in the overall population (all three subpopulations). Bayesian clustering and spatial genetic autocorrelation analyses of microsatellite data showed resistance to gene flow at the approximate location of I-10. Simulations that investigated gene flow between the subpopulations (with and without a highway barrier present) were consistent with our molecular results. We conclude that I-10 has reduced gene flow in a population of an important reptilian predator of the Sonoran Desert in southern Arizona and make conservation recommendations for reversing this trend.     

Functional genetic variation in aminopeptidase A (ENPEP): lack of clear association with focal and segmental glomerulosclerosis (FSGS)

The aminopeptidase A (APA) ectopeptidase is an integral membrane-bound zinc metalloprotease that cleaves aspartic and glutamic acidic residues from the N-terminus of a number of protein substrates that includes angiotensin II. Angiotensin II, the most vasoactive component of the renin-angiotensin-aldosterone (RAAS) pathway, can contribute to renal disease by causing an increase in arterial blood pressure leading to glomerular injury and fibrosis. APA is expressed in many organs, including the kidney where it localizes mainly to the podocyte cell membrane and brush borders of the proximal tubule cells. Antibodies directed to the APA peptide can induce an acute massive albuminuria in wild-type BALB/c mice after intravenous injection. We examined whether variants in the APA encoding gene (ENPEP) are more frequent in individuals with the proteinuric disease focal and segmental glomerulosclerosis (FSGS) compared to control individuals. The ENPEP coding sequence was re-sequenced in 188 FSGS patients and 48 controls. Genetic variants were further genotyped in 181 individuals without any known kidney disease. We then examined the effect of the non-synonymous coding variants identified on their cell surface APA activity after transfection in COS-1 cells. Several of these ENPEP variants lead to reproducibly altered APA activity. However, we did not see a clear correlation between the presence of a functional ENPEP variant and FSGS. However, the existence of these variants with marked effect on APA activity suggests that both rare and common variation in ENPEP may contribute to the development of renal and hypertensive disorders and warrants further study.     

Effect of salinity on composition, viability and germination of seeds of Chenopodium quinoa Willd

Salinity influences plant growth, seed yield and seed quality even of halophytic crops such as Chenopodium quinoa. Plant growth, total seed yield, number of seeds, fresh weight and dry weight of seeds, were all significantly reduced in the presence of salinity. Only at high salinity did the content of proteins (as well as total N) increase significantly in the seeds whereas the content of total carbohydrates (as well as total C) decrease. Aside from that the capacity for germination was diminished by a reduced seed size and a disproportionate reduction of the volume of the perisperm. However, the reduced capacity seemed to be compensated by an accelerated germination owing to high Na and Cl concentrations leading to a low water potential in the walls of the plant ovary. At high salinity the passage of NaCl to the seed interior was hindered by the seed cover. There was an obvious gradient between potentially toxic (Na and Cl) and essentially needed elements (K, Mg, Ca, P and S) across the seed coat of salt treated plants and also a significant change of the distribution of elements in the embryo. The results indicate a highly protected seed interior leading to a high salinity resistance of quinoa seeds.     

Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: I. Stimulation by bone morphogenetic protein-2 in high-density micromass cultures

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. In this study, we have used multipotential murine C3H10T1/2 cells to analyze the effect and mechanism of action of bone morphogenetic protein 2 (BMP-2) on chondrogenesis. C3H10T1/2 cells have been previously shown to undergo multiple differentiation pathways. While chondrogenesis, osteogenesis, myogenesis and adipogenesis have been observed, chondrocytes appear significantly less frequently than the other cell types, and the appearance of chondrocytes exclusive of the other cell types has not been observed. We report here that the appearance of chondrocytes in C3H10T1/2 cells is markedly enhanced as a result of culture under conditions favorable for chondrogenesis, i.e. plating as high-density micromass and treatment with BMP-2. Such cultures contain chondrocyte-like cells, elaborate an Alcian blue stained cartilage-like matrix, express link protein and type II collagen, both cartilage matrix markers, and show increased [35S]sulfate incorporation. The appearance of Alcian blue positive material and increased sulfate incorporation are dependent on the dose of BMP-2, culture time, and cell plating density of the micromass cultures. Differentiation of cells within the micromass was specific to the chondrogenic lineage, as alkaline phosphatase staining revealed only faint staining in the micromass at the highest BMP-2 concentration. The importance of enhanced cell-cell interaction in the chondroinductive effects of BMP-2 on high-density C3H10T1/2 cultures was further implicated by the additional promotion of chondrogenesis in the presence of the polycationic compound, poly-L-lysine, which has been previously reported to enhance cellular interactions and chondrogenesis in embryonic limb mesenchymal cells. Taken together, these findings suggest that chondrogenesis in C3H10T1/2 cells is inducible by BMP-2 and requires cell-cell interaction.