Triaminopyrimidinium (TAP+) blocks luminal membrane K conductance inNecturus gallbladder epithelium

Summary The effect of triaminopyrimidinium (TAP⁺) on the apical membrane ofNecturus gallbladder epithelial cells was investigated with intracellular microelectrode techniques. TAP⁺, added to the mucosal bathing solution only, produced the following effects (all rapid and reversible): (i) cell depolarization, (ii) increase of apical membrane resistance, and (iii) decrease of the apical membrane potential change produced by K for Na substitution on the mucosal side. These results can be explained by a decrease of apical membrane K conductance. The paracellular effects of TAP⁺ were similar to the ones previously described by Moreno (J.H. Moreno, 1974;Nature (London) 251:150; J.H. Moreno, 1975,J. Gen. Physiol. 66:97). These results indicate that the change of transepithelial potential produced by TAP⁺ cannot be ascribed solely to its effect on the paracellular pathway.     

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.     

Cooperative navigation in robotic swarms

We study cooperative navigation for robotic swarms in the context of a general event-servicing scenario. In the scenario, one or more events need to be serviced at specific locations by robots with the required skills. We focus on the question of how the swarm can inform its members about events, and guide robots to event locations. We propose a solution based on delay-tolerant wireless communications: by forwarding navigation information between them, robots cooperatively guide each other towards event locations. Such a collaborative approach leverages on the swarm’s intrinsic redundancy, distribution, and mobility. At the same time, the forwarding of navigation messages is the only form of cooperation that is required. This means that the robots are free in terms of their movement and location, and they can be involved in other tasks, unrelated to the navigation of the searching robot. This gives the system a high level of flexibility in terms of application scenarios, and a high degree of robustness with respect to robot failures or unexpected events. We study the algorithm in two different scenarios, both in simulation and on real robots. In the first scenario, a single searching robot needs to find a single target, while all other robots are involved in tasks of their own. In the second scenario, we study collective navigation: all robots of the swarm navigate back and forth between two targets, which is a typical scenario in swarm robotics. We show that in this case, the proposed algorithm gives rise to synergies in robot navigation, and it lets the swarm self-organize into a robust dynamic structure. The emergence of this structure improves navigation efficiency and lets the swarm find shortest paths.     

Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning

High-affinity, β2-subunit-containing (β2*) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of β2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from β2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of β2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level β2* nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in β2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation.     

Taxonomic diversity, origin, and conservation status of Bermuda killifishes (Fundulus) based on mitochondrial cytochrome b phylogenies

Sequence variation in the mitochondrialCytochrome b (Cytb) gene was assayed infour of six extant Bermuda killifishpopulations, representing two endemic species,to test taxonomic and phylogenetic hypothesesand reconstruct colonization history. Twodivergent (4.6%) haplotypes were detected; oneis identical to the Georgia 2c haplotype ofF. heteroclitus and is fixed in threeeastern populations: Lover's Lake (F.relictus), Mangrove Lake (F. bermudae),and Walsingham Pond. The second is fixed andrestricted to a western population, Evan'sPond. Likelihood and parsimony cytochrome btrees recognize a Bermuda Fundulus / F. heteroclitus clade in which the Evan's Pondhaplotype is basal. Phylogenies and haplotypedivergence indicate at least two Bermudacolonizations, the more recent involvingtransfer of the Georgia 2c haplotype. Enforcing Bermuda killifish monophyly, aspredicted from a single colonization event,does not increase tree length significantly;i.e., the trees also are consistent with asingle colonization. However, divergencebetween the Evan's Pond haplotype and the F. bermudae / F. relictus / Georgia 2chaplotype (4.6%) far exceeds the maximumdivergence among all F. heteroclitus,F. bermudae, and F. heteroclitushaplotypes (1.2%) and argues for independentcolonizations. Alternatively, recentintroduction of F. heteroclitus couldaccount for occurrence of the GA2c haplotype inBermuda but does not explain the presence ofthe genetically divergent Evans' Pondhaplotype. Cytb sequences areuninformative of the taxonomic status ofBermuda endemics, F. bermudae and F.relictus, but support recognition of theEvan's Pond population as an evolutionarilysignificant unit within the F. heteroclitus group.     

Location of messenger specifying sequences in mammalian chromosomes

In situ hybridization of complementary DNA (cDNA) synthesized from total cytoplasmic polyadenylated RNA isolated from Chinese hamster cells was employed to investigate the distribution of messenger specifying sequences on mammalian chromosomes. The kinetics of cDNA-nuclear DNA annealing indicate that about 85% of the cDNA represents sequences which are transcribed from non-repetitive DNA sequences. When cDNA is hybridized back to its template RNA, the reaction kinetics show that more than 60% of the poly(A) RNA is at least 10⁴ times more complex than rabbit globin mRNA. In situ hybridization of cDNA to Chinese hamster cells fixed on slides shows no significant clustering of silver grains on interphase nuclei. On metaphase chromosomes the majority of silver grains are localized in euchromatic areas. It appears that all euchromatic segments have similar grain densities. Chromosomes 1 and 2, which have relatively little heterochromatin, do not have a higher grain density than the other chromosomes. However, the Y chromosome, which is entirely heterochromatic, contains only about 1/3 the grain density of the chromosomes 1 or 2. — When the cDNA, which anneals only to the high abundancy class of poly(A) RNA was fractionated and hybridized in situ to Chinese hamster chromosomes, the distribution of silver grains is localized in the euchromatic areas. The Y chromosome and the heterochromatic arm of the X chromosome contain less grains; telomeres of some autosomes have higher grain densities. The oligo-(dT) primer in cDNA did not affect the results of this study since no grains are found when ₃H-poly(dT) was used as probe for in situ hybridization. The majority (>90%) of the grains could be blocked by competition with excess repetitive DNA in the hybridization reaction, indicating that the in situ hybridization involved predominantly repetitive sequences.     

Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

ABSTRACT: BACKGROUND: Many growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans (GAGs), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. RESULTS: Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse[trade mark sign]). CONCLUSIONS: A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2 osteogenic activity for in vivo de novo bone generation when delivered together on a scaffold as a single-phase. The use of HS/CS PGs may be useful to augment GF therapeutics, and a plasmid-based approach has been shown here to be highly effective.