Tissue factor expression in newt iris coincides with thrombin activation and lens regeneration

Lens regeneration in adult salamanders occurs at the pupillary margin of the mid-dorsal iris where pigmented epithelial cells (PEC) re-enter the cell cycle and transdifferentiate into lens. It is not understood how the injury caused by removal of the lens (lentectomy) in one location is linked to initiating the response in a different spatial location (dorsal iris) and to this particular sector. We propose that the blood provides a link between the localised coagulation and signal transduction pathways that lead to regeneration. A transmembrane protein (tissue factor) is expressed in a striking patch-like domain in the dorsal iris of the newt that localises coagulation specifically to this location, but is not expressed in the axolotl, a related species that does not show thrombin activation after lentectomy and cannot regenerate its lens. Our hypothesis is that tissue factor expression localises the initiation of regeneration through the activation of thrombin and the recruitment of blood cells, leading to local growth factor release. This is the first example of gene expression in a patch of cells that prefigures the location of a regenerative response, and links the immune system with the initiation of a regenerative program.     

Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable beta-sandwich

Actinoporins are eukaryotic pore-forming proteins that create 2-nm pores in natural and model lipid membranes by the self-association of four monomers. The regions that undergo conformational change and form part of the transmembrane pore are currently being defined. It was shown recently that the N-terminal region (residues 10-28) of equinatoxin, an actinoporin from Actinia equina, participates in building of the final pore wall. Assuming that the pore is formed solely by a polypeptide chain, other parts of the toxin should constitute the conductive channel and here we searched for these regions by disulfide scanning mutagenesis. Only double cysteine mutants where the N-terminal segment 1-30 was attached to the beta-sandwich exhibited reduced hemolytic activity upon disulfide formation, showing that other parts of equinatoxin, particularly the beta-sandwich and importantly the C-terminal alpha-helix, do not undergo large conformational rearrangements during the pore formation. The role of the beta-sandwich stability was independently assessed via destabilization of a part of its hydrophobic core by mutations of the buried Trp117. These mutants were considerably less stable than the wild-type but exhibited similar or slightly lower permeabilizing activity. Collectively these results show that a flexible N-terminal region and stable beta-sandwich are pre-requisite for proper pore formation by the actinoporin family.     

A Fundamental Trade-off in Covalent Switching and Its Circumvention by Enzyme Bifunctionality in Glucose Homeostasis

Covalent modification provides a mechanism for modulating molecular state and regulating physiology. A cycle of competing enzymes that add and remove a single modification can act as a molecular switch between “on” and “off” and has been widely studied as a core motif in systems biology. Here, we exploit the recently developed “linear framework” for time scale separation to determine the general principles of such switches. These methods are not limited to Michaelis-Menten assumptions, and our conclusions hold for enzymes whose mechanisms may be arbitrarily complicated. We show that switching efficiency improves with increasing irreversibility of the enzymes and that the on/off transition occurs when the ratio of enzyme levels reaches a value that depends only on the rate constants. Fluctuations in enzyme levels, which habitually occur due to cellular heterogeneity, can cause flipping back and forth between on and off, leading to incoherent mosaic behavior in tissues, that worsens as switching becomes sharper. This trade-off can be circumvented if enzyme levels are correlated. In particular, if the competing catalytic domains are on the same protein but do not influence each other, the resulting bifunctional enzyme can switch sharply while remaining coherent. In the mammalian liver, the switch between glycolysis and gluconeogenesis is regulated by the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). We suggest that bifunctionality of PFK-2/FBPase-2 complements the metabolic zonation of the liver by ensuring coherent switching in response to insulin and glucagon.     

Investigating the activity of quinine analogues versus chloroquine resistant Plasmodium falciparum

Plasmodium falciparum, the deadliest malarial parasite species, has developed resistance against nearly all man-made antimalarial drugs within the past century. However, quinine (QN), the first antimalarial drug, remains efficacious worldwide. Some chloroquine resistant (CQR) P. falciparum strains or isolates show mild cross resistance to QN, but many do not. Further optimization of QN may provide a well-tolerated therapy with improved activity versus CQR malaria. Thus, using the Heck reaction, we have pursued a structure-activity relationship study, including vinyl group modifications of QN. Certain derivatives show good antiplasmodial activity in QN-resistant and QN-sensitive strains, with lower IC(50) values relative to QN.     

Improving protein extraction yield in reversed micellar systems through surface charge engineering

The mechanism of extraction of rat cytochrome b(5) from water into a sodium dioctylsulfosuccinate (AOT) micellar organic phase was studied using protein engineering of surface charged residues. The extraction behavior of native cytochrome b(5) and modified proteins with substitutions of the type glutamic acid --> lysine at positions 44 (E44K), 56 (E56K), and 92 (E92K), was studied as a function of pH. The results indicate that an important mechanism of extraction is an electrostatic interaction of this protein with the negatively charged surfactant. We demonstrate that it is possible to improve extraction by engineering the protein surface charge, increasing the driving force responsible for the protein transfer to the micellar phase. (c) 1994 John Wiley & Sons, Inc.     

Re-Directing an Alkylating Agent to Mitochondria Alters Drug Target and Cell Death Mechanism

We have successfully delivered a reactive alkylating agent, chlorambucil (Cbl), to the mitochondria of mammalian cells. Here, we characterize the mechanism of cell death for mitochondria-targeted chlorambucil (mt-Cbl) in vitro and assess its efficacy in a xenograft mouse model of leukemia. Using a ρ° cell model, we show that mt-Cbl toxicity is not dependent on mitochondrial DNA damage. We also illustrate that re-targeting Cbl to mitochondria results in a shift in the cell death mechanism from apoptosis to necrosis, and that this behavior is a general feature of mitochondria-targeted Cbl. Despite the change in cell death mechanisms, we show that mt-Cbl is still effective in vivo and has an improved pharmacokinetic profile compared to the parent drug. These findings illustrate that mitochondrial rerouting changes the site of action of Cbl and also alters the cell death mechanism drastically without compromising in vivo efficacy. Thus, mitochondrial delivery allows the exploitation of Cbl as a promiscuous mitochondrial protein inhibitor with promising therapeutic potential.     

Ecomorphology of eye shape and retinal topography in waterfowl (Aves: Anseriformes: Anatidae) with different foraging modes

Despite the large body of literature on ecomorphological adaptations to foraging in waterfowl, little attention has been paid to their sensory systems, especially vision. Here, we compare eye shape and retinal topography across 12 species representing 4 different foraging modes. Eye shape was significantly different among foraging modes, with diving and pursuit-diving species having relatively smaller corneal diameters compared to non-diving species. This may be associated with differences in ambient light intensity while foraging or an ability to tightly constrict the pupil in divers in order to facilitate underwater vision. Retinal topography was similar across all species, consisting of an oblique visual streak, a central area of peak cell density, and no discernible fovea. Because the bill faces downwards when the head is held in the normal posture in waterfowl, the visual streak will be held horizontally, allowing the horizon to be sampled with higher visual acuity. Estimates of spatial resolving power were similar among species with only the Canada goose having a higher spatial resolution. Overall, we found no evidence of ecomorphological adaptations to different foraging modes in the retinal ganglion cell layer in waterfowl. Rather, retinal topography in these birds seems to reflect the ‘openness’ of their habitats.     

Using unmanned aerial vehicle (UAV) surveys and image analysis in the study of large surface‐associated marine species: a case study on reef sharks Carcharhinus melanopterus shoaling behaviour

A novel image analysis‐based technique applied to unmanned aerial vehicle (UAV) survey data is described to detect and locate individual free‐ranging sharks within aggregations. The method allows rapid collection of data and quantification of fine‐scale swimming and collective patterns of sharks. We demonstrate the usefulness of this technique in a small‐scale case study exploring the shoaling tendencies of blacktip reef sharks Carcharhinus melanopterus in a large lagoon within Moorea, French Polynesia. Using our approach, we found that C. melanopterus displayed increased alignment with shoal companions when distributed over a sandflat where they are regularly fed for ecotourism purposes as compared with when they shoaled in a deeper adjacent channel. Our case study highlights the potential of a relatively low‐cost method that combines UAV survey data and image analysis to detect differences in shoaling patterns of free‐ranging sharks in shallow habitats. This approach offers an alternative to current techniques commonly used in controlled settings that require time‐consuming post‐processing effort.     

Metal chelate affinity precipitation of RNA and purification of plasmid DNA

The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine `tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.     

Sequential and compartmentalized action of Rabs,SNAREs, and MAL in the apical delivery of fusiform vesicles in urothelial umbrella cells

Uroplakins (UPs) are major differentiation products of urothelial umbrella cells and play important roles in forming the permeability barrier and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic Escherichia coli receptor. Although it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immunomicroscopy of normal and mutant mouse urothelia show that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently a Rab27b/Slp2-a complex mediated FV–membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also show that keratin 20 (K20), which forms a chicken-wire network ∼200 nm below the apical membrane and has hole sizes allowing FV passage, defines a subapical compartment containing FVs primed and strategically located for fusion. Finally, we show that Rab8/11 and Rab27b function in the same pathway, Rab27b knockout leads to uroplakin and Slp2-a destabilization, and Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking.