Antibodies to the rat substance P receptor: production and characterization

1. A protein A-rat substance P receptor (SPR) fusion protein was genetically engineered and used as an immunogen to raise a polyclonal antiserum to the SPR. The fusion protein was expressed in Escherichia coli driven by the heat-inducible lambda promoter (lambda Pr). 2. The fusion protein was purified using an IgG-Sepharose column, which specifically binds proteins containing the protein A moiety. The IgG fraction obtained after the immunization was cleaved to produce Fab fragments, which were subsequently purified using a fusion protein affinity column. The serum (anti-SPR Fab serum) was analyzed by fluorescence-activated cell sorting (FACS) and immunohistochemistry on both a constitutive cell line for the SPR (AR42J) and a cell line transfected with the SPR (KNRKSPR). 3. Specificity of the antiserum for SPR was confirmed by immunohistochemistry on cells using antiserum that had been preincubated with the protein A fusion protein (blocked). 4. The Ca2+ signal normally observed on stimulation of SPR with SP in AR42J cells and SP binding to KNRKSPR cells was shown to be diminished in the presence of anti-SPR Fab serum. SPR from both cell lines was immunoprecipitated using the anti-SPR Fab serum. The antiserum itself did not induce intracellular Ca2+ mobilization normally observed when cells were incubated with SP. 5. This specific SPR antiserum will be a useful tool to investigate further the mechanisms of SP/SPR interactions.     

To What Extent Do Food Preferences Explain the Trophic Position of Heterotrophic and Mixotrophic Microbial Consumers in a Sphagnum Peatland?

Although microorganisms are the primary drivers of biogeochemical cycles, the structure and functioning of microbial food webs are poorly studied. This is the case in Sphagnum peatlands, where microbial communities play a key role in the global carbon cycle. Here, we explored the structure of the microbial food web from a Sphagnum peatland by analyzing (1) the density and biomass of different microbial functional groups, (2) the natural stable isotope (δ ¹³C and δ ¹⁵N) signatures of key microbial consumers (testate amoebae), and (3) the digestive vacuole contents of Hyalosphenia papilio, the dominant testate amoeba species in our system. Our results showed that the feeding type of testate amoeba species (bacterivory, algivory, or both) translates into their trophic position as assessed by isotopic signatures. Our study further demonstrates, for H. papilio, the energetic benefits of mixotrophy when the density of its preferential prey is low. Overall, our results show that testate amoebae occupy different trophic levels within the microbial food web, depending on their feeding behavior, the density of their food resources, and their metabolism (i.e., mixotrophy vs. heterotrophy). Combined analyses of predation, community structure, and stable isotopes now allow the structure of microbial food webs to be more completely described, which should lead to improved models of microbial community function.     

Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death.

BACKGROUND: Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS-binding protein, which tethers apoptotic cells to macrophage integrins. METHODS: We utilized fluorescein-labeled lactadherin together with the benchmark PS Probe, annexin V, to detect PS exposure by flow cytometry and confocal microscopy. Immortalized leukemia cells were treated with etoposide, and the kinetics and topology of PS exposure were followed over the course of apoptosis. RESULTS: Costaining etoposide-treated leukemoid cells with lactadherin and annexin V indicated progressive PS exposure with dim, intermediate, and bright staining. Confocal microscopy revealed localized plasma membrane staining, then diffuse dim staining by lactadherin prior to bright generalized staining with both proteins. Annexin V was primarily localized to internal cell bodies at early stages but stained the plasma membrane at the late stage. Calibration studies suggested a PS content less, less than or approximately equal to 2.5%-8% for the membrane domains that stained with lactadherin but not annexin V. CONCLUSIONS: Macrophages may utilize lactadherin to detect PS exposure prior to exposure of sufficient PS to bind annexin V. The methodology enables detection of PS exposure at earlier stages than established methodology.     

A performance-based cost to honest signalling in male green anole lizards (Anolis carolinensis)

Sexual signals are considered costly to produce and maintain under the handicap paradigm, and the reliability of signals is in turn thought to be maintained by these costs. Although previous studies have investigated the costly nature of signal production, few have considered whether honesty might be maintained not by the costliness of the signal itself, but by the costs involved in producing the signalled trait. If such a trait is itself costly to produce, then the burden of energetic investment may fall disproportionately on that trait, in addition to any costs of signal maintenance that may also be operating. Under limited resource conditions, these costs may therefore be great enough to disrupt an otherwise reliable signal-to-trait relationship. We present experimental evidence showing that dietary restriction decouples the otherwise honest relationship between a signal (dewlap size) and a whole-organism performance trait (bite force) in young adult male Anolis carolinensis lizards. Specifically, while investment in dewlap size is sustained under low-resource condition relative to the high-resource treatment, investment in bite force is substantially lower. Disruption of the otherwise honest dewlap size to bite force relationship is therefore driven by costs associated with the expression of performance rather than the costs of signal production in A. carolinensis.     

RIBOSOMAL ACTIVITY IN PRENATAL MOUSE BRAIN

Abstract— Regulation of protein synthesis is important for the proper growth and development of the brain. Our previous work on the regulation of protein synthetic activity in fetal mouse brain cell suspensions showed that the rate of protein synthesis decreased during the prenatal period. In the present study, ribosomal activity of cell-free homogenates and purified ribosomes obtained from fetal neural tissue was measured. The post-mitochondrial supernatant (PMS) fraction actively incorporated amino acids into polypeptides using either endogenous mRNA or polyuridylic acid as template. The protein synthetic activity was dependent upon the age of the fetus. Ribosomes purified from this fraction were also active in protein synthesis. Incorporation of phenylalanine was linear for 20 min, and dependent upon the concentration of ribosomes and the pH 5 enzyme fraction. The age dependent decrease in protein synthetic activity observed with the post-mitochondrial supernatant fractions was not found when these purified ribosomes were employed. Ribosomes obtained from fetal, newborn or adult neural tissue were compared and found equally active in their protein synthetic capacity.     

Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin

While mammals have a limited capacity to repair bone defects, zebrafish can completely regenerate amputated bony structures of their fins. Fin regeneration is dependent on formation of a blastema, a progenitor cell pool accumulating at the amputation plane. It is unclear which cells the blastema is derived from, whether it forms by dedifferentiation of mature cells, and whether blastema cells are multipotent. We show that mature osteoblasts dedifferentiate and form part of the blastema. Osteoblasts downregulate expression of intermediate and late bone differentiation markers and induce genes expressed by bone progenitors. Dedifferentiated osteoblasts proliferate in a FGF-dependent manner and migrate to form part of the blastema. Genetic fate mapping shows that osteoblasts only give rise to osteoblasts in the regenerate, indicating that dedifferentiation is not associated with the attainment of multipotency. Thus, bone can regenerate from mature osteoblasts via dedifferentiation, a finding with potential implications for human bone repair.     

Charge immobilization linked to inactivation in the aggregation model of channel gating

Expected gating currents are derived analytically from a continuous, time-homogenous Markov process formulation of the random behavior of a single aggregation gating site. The concept of aggregation gating involves a voltage-dependent reversible conformational change and a voltage-independent reversible aggregation process. A site is assumed to consist of four hypothetical protein subunits. Based on these assumptions the model is defined by the scheme of transitions between 12 possible site configurations. The model can account for the phenomenon of charge immobilization in asymmetry current data of the voltage-clamped sodium conductance system. It predicts gating currents without a rising phase. A rising phase is obtained, however, if the model is subjected to conventional symmetrical pulse protocols for measuring asymmetry currents in the axon. Novel pulse protocols are given that do not result in a rising phase if applied to the aggregation model. Simplified transition schemes that describe the basic kinetic behavior of the potassium and the sodium conductance system are derived by eliminating transitions of negligible probability from the original scheme.