Translation and functional expression of cell-cell channel mRNA inXenopus oocytes

Summary mRNA from estrogen-stimulated rat myometrium, a tissue known to upregulate cell-cell channels in response to this hormone, was microinjected intoXenopus laevis oocytes. The oocytes had been freed from covering layers of follicle cells and vitelline to allow direct cell membrane interactions when paired. About 4 hours after the mRNA injection, paired oocytes become electrically coupled. This coupling was due to the presence of typical cell-cell channels characterized by size-limited intercellular tracer flux, the presence of gap junctions at the oocyte-oocyte interface, and the reversible uncoupling that occurred in the presence of carbon dioxide. The induction of new cell-cell channels in the oocyte membrane was observed against a zero background or a low level of endogenous coupling, depending on the maturation stage of the oocytes. The time course of development of cell-cell coupling after the microinjection of mRNA was determined. The mRNA capable of inducing cell-cell coupling was confined to an intermediate size class when fractionated on a sucrose gradient.     

A proteomics approach to cloning fenestrin from the nuclear exchange junction of tetrahymena

ABSTRACT. We set out to find the " fenestrin " gene, a gene whose protein is associated with numerous cellular apertures, including the nuclear exchange junction in mating Tetrahymena thermophila . First we developed protocols for imaging and isolating intact nuclear exchange junctions from conjugating cells. Proteins from these junctions were purified using SDS-PAGE, subjected to limited proteolysis, and precise molecular weights were determined by mass spectrometry. Using Protein Prospector^® software and the published Tetrahymena Genome Database, genes for 15 of the most abundant proteins found in our extracts were identified. The most promising candidate was cloned by PCR, fused to yellow fluorescent protein (YFP), and placed under the control of an inducible metallothionein promoter. YFP-localization within live Tetrahymena transformants strongly suggested that one of these genes encoded the fenestrin protein, a result that was subsequently confirmed by Western blotting.     

CD47 Promotes Protective Innate and Adaptive Immunity in a Mouse Model of Disseminated Candidiasis

CD47 is a widely expressed receptor that regulates immunity by engaging its counter-receptor SIRPα on phagocytes and its secreted ligand thrombospondin-1. Mice lacking CD47 can exhibit enhanced or impaired host responses to bacterial pathogens, but its role in fungal immunity has not been examined. cd47^-/- mice on a C57BL/6 background showed significantly increased morbidity and mortality following Candida albicans infection when compared with wild-type mice. Despite normal fungal colonization at earlier times, cd47^-/- mice at four days post-infection had increased colonization of brain and kidneys accompanied by stronger inflammatory reactions. Neutrophil and macrophage numbers were significantly elevated in kidneys and neutrophils in the brains of infected cd47^-/- mice. However, no defect in phagocytic activity towards C. albicans was observed in cd47^-/- bone-marrow-derived macrophages, and neutrophil and macrophage killing of C. albicans was not impaired. CD47-deficiency did not alter the early humoral immune response to C. albicans. Th1, Th2, and Th17 population of CD4⁺ T cells were expanded in the spleen, and gene expression profiles of spleen and kidney showed stronger pro-inflammatory signaling in infected cd47^-/- mice. The chemoattractant chemokines MIP-2α and MIP-2β were highly expressed in infected spleens of cd47^-/- mice. G-CSF, GM-CSF, and the inflammasome component NLRP3 were more highly expressed in infected cd47^-/- kidneys than in infected wild-type controls. Circulating pro- (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10) were significantly elevated, but IL-17 was decreased. These data indicate that CD47 plays protective roles against disseminated candidiasis and alters pro-inflammatory and immunosuppressive pathways known to regulate innate and T cell immunity.     

Random amplified polymorphic DNA markers reveal genetic variation in the symbiotic fungus of leaf-cutting ants

RAPD markers were used to examine the degree of genetic variation within the putatively asexual basidiomycete fungus (Lepiotaceae: provisionally named Leucoagaricus gongylophorus) associated with the leaf-cutting ant species Atta cephalotes. We analyzed fungal isolates from ant nests in two geographically distant sites, two isolates from Panama and five isolates from Trinidad. Ten decamer primers were used to amplify total DNA from these seven fungal isolates, and RAPD banding patterns were compared. Genetic similarity among isolates was determined by pair-wise comparisons of the shared number of DNA bands on an agarose gel. There was considerable genetic variation among isolates of the symbiotic fungus even within sites. Pairs of fungal isolates from the two different sites shared an average of only 36% of the bands in their RAPD profiles, while pairs from the within sites shared an average of 72% of the bands. RAPD markers may be useful for further investigation of the genetic structure of the fungal symbiont within species of leaf-cutting ants.     

Additive effect of TAp63 deficiency on the adrenocortical dysplasia (acd) phenotype

Adrenocortical dysplasia (acd) is a spontaneous autosomal recessive mouse mutation exhibiting caudal truncation, vertebral segmentation defects, hydronephrosis, limb hypoplasia, and perinatal lethality. Acd encodes TPP1, a component of the shelterin complex that maintains telomere integrity, and consequently acd mutant mice have telomere dysfunction and genomic instability. We previously showed that apoptosis is the primary mechanism causing the acd skeletal phenotype, and that p53 deficiency rescues the skeletal defects of the acd phenotype but has no effect on the perinatal lethality. The Trp63 gene encodes multiple isoforms, which play a role in proliferation, apoptosis, and stem/progenitor cell maintenance. Different p63 isoforms exhibit both proapoptotic (TAp63) and antiapoptotic (ΔNp63) functions. We hypothesized that deficiency of proapoptotic TAp63 isoforms might rescue the acd skeletal phenotype, similar to our previous observations with deficiency of p53. Mice heterozygous for a null allele of TAp63 were crossed to heterozygous acd mice to determine the effect of TAp63 deficiency on the acd mutant phenotype. In contrast to our results with the acd?×?p53 cross, skeletal anomalies were not rescued by deficiency of TAp63. In fact, the limb and vertebral anomalies observed in double-mutant embryos were more severe than those of embryos with the acd mutation alone, demonstrating a dose-dependent effect. These studies suggest that TAp63 isoforms do not facilitate p53-like apoptosis during development in response to acd-mediated telomere dysfunction and are consistent with the proposed roles of TAp63 in maintaining genomic stability.     

Phosphate binding to liver alcohol dehydrogenase studied by the rate of alkylation with affinity labels

In this work we report that phosphate anions interact with the anion binding site of alcohol dehydrogenase from horse liver. In protection experiments against the two affinity labels, iodoacetic acid and bromo-imidazolylpropionic acid, the dissociation constant for the enzyme-phosphate complex at pH 7.0 is, based on total phosphate, found to be 20 +/- 5 mM. The 1,4-piperazinediethanesulfonate anion has a lower affinity for the anion binding site, the dissociation at pH 7.0 being 130 +/- 20 mM. The anion-independent dissociation constants for the reversible enzyme-affinity label complexes are at pH 7.0, 1.35 +/- 0.2 mM for iodoacetic acid and 0.39 +/- 0.05 mM for bromo-imidazolylpropionic acid. These findings have important implications with respect to past and future work on this well known enzyme.     

Single‐cell and population level viral infection dynamics revealed by phageFISH,a method to visualize intracellular and free viruses

Microbes drive the biogeochemical cycles that fuel planet Earth, and their viruses (phages) alter microbial population structure, genome repertoire, and metabolic capacity. However, our ability to understand and quantify phage–host interactions is technique‐limited. Here, we introduce phageFISH – a markedly improved geneFISH protocol that increases gene detection efficiency from 40% to > 92% and is optimized for detection and visualization of intra‐ and extracellular phage DNA. The application of phageFISH to characterize infection dynamics in a marine podovirus–gammaproteobacterial host model system corroborated classical metrics (qPCR, plaque assay, FVIC, DAPI) and outperformed most of them to reveal new biology. PhageFISH detected both replicating and encapsidated (intracellular and extracellular) phage DNA, while simultaneously identifying and quantifying host cells during all stages of infection. Additionally, phageFISH allowed per‐cell relative measurements of phage DNA, enabling single‐cell documentation of infection status (e.g. early vs late stage infections). Further, it discriminated between two waves of infection, which no other measurement could due to population‐averaged signals. Together, these findings richly characterize the infection dynamics of a novel model phage–host system, and debut phageFISH as a much‐needed tool for studying phage–host interactions in the laboratory, with great promise for environmental surveys and lineage‐specific population ecology of free phages.