A microfabricated fluorescence-activated cell sorter.

We have demonstrated a disposable microfabricated fluorescence-activated cell sorter (microFACS) for sorting various biological entities. Compared with conventional FACS machines, the microFACS provides higher sensitivity, no cross-contamination, and lower cost. We have used microFACS chips to obtain substantial enrichment of micron-sized fluorescent bead populations of differing colors. Furthermore, we have separated Escherichia coli cells expressing green fluorescent protein from a background of nonfluorescent E. coli cells and shown that the bacteria are viable after extraction from the sorting device. These sorters can function as stand-alone devices or as components of an integrated microanalytical chip.     

Atkinsiella dubia infection in the larvae of Japanese mitten crab,Eriocheir japonicus

Heavy mortalities reaching 100% among larvae of the Japanese mitten crab,Eriocheir japonicus, occurred in Yamaguchi Prefectural Naikai Fisheries Experimental Station, Yamaguchi, Japan. Under the microscope, infected zoeal larvae were seen to be filled with numerous aseptate hyphae. An investigation was carried out to identify the pathogenic fungus and determine its pathogenicity under laboratory conditions. The pathogenic fungus was identified asAtkinsiella dubia. Its optimum growth temperature was 25°C, and it grew only at 2.5% NaCl. Under laboratory conditions, it showed pathogenicity to the larvae of the swimming crab,Portunus trituberculatus. This is the first report of mass mortality in crustaceans due toA. dubia infection in Japan.     

Bactericidal peptidoglycan recognition protein induces oxidative stress in Escherichia coli through a block in respiratory chain and increase in central carbon catabolism

Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill both Gram‐positive and Gram‐negative bacteria through simultaneous induction of oxidative, thiol and metal stress responses in bacteria. However, metabolic pathways through which PGRPs induce these bactericidal stress responses are unknown. We screened Keio collection of Escherichia coli deletion mutants and revealed that deleting genes for respiratory chain flavoproteins or for tricarboxylic acid (TCA) cycle resulted in increased resistance of E. coli to PGRP killing. PGRP‐induced killing depended on the production of hydrogen peroxide, which required increased supply of NADH for respiratory chain oxidoreductases from central carbon catabolism (glycolysis and TCA cycle), and was controlled by cAMP‐Crp. Bactericidal PGRP induced a rapid decrease in respiration, which suggested that the main source of increased production of hydrogen peroxide was a block in respiratory chain and diversion of electrons from NADH oxidoreductases to oxygen. CpxRA two‐component system was a negative regulator of PGRP‐induced oxidative stress. By contrast, PGRP‐induced thiol stress (depletion of thiols) and metal stress (increase in intracellular free Zn²⁺ through influx of extracellular Zn²⁺) were mostly independent of oxidative stress. Thus, manipulating pathways that induce oxidative, thiol and metal stress in bacteria could be a useful strategy to design new approaches to antibacterial therapy.     

Novel diaroylhydrazine ligands as iron chelators: coordination chemistry and biological activity

The search for orally effective drugs for the treatment of iron overload disorders is an important goal in improving the health of patients suffering diseases such as β-thalassemia major. Herein, we report the syntheses and characterization of some new members of a series of N-aroyl-N′-picolinoyl hydrazine chelators (the H₂IPH analogs). Both 1:1 and 1:2 Fe^III:L complexes were isolated and the crystal structures of Fe(HPPH)Cl₂, Fe(4BBPH)Cl₂, Fe(HAPH)(APH) and Fe(H3BBPH)(3BBPH) were determined (H₂PPH=N,N′-bis-picolinoyl hydrazine; H₂APH=N-4-aminobenzoyl-N′-picolinoyl hydrazine, H₂3BBPH=N-3-bromobenzoyl-N′-picolinoylhydrazine and H₂4BBPH=N-(4-bromobenzoyl)-N′-(picolinoyl)hydrazine). In each case, a tridentate N,N,O coordination mode of each chelator with Fe was observed. The Fe^III complexes of these ligands have been synthesized and their structural, spectroscopic and electrochemical characterization are reported. Five of these new chelators, namely H₂BPH (N-(benzoyl)-N′-(picolinoyl)hydrazine), H₂TPH (N-(2-thienyl)-N′-(picolinoyl)-hydrazine), H₂PPH, H₂3BBPH and H₂4BBPH, showed high efficacy at mobilizing ⁵⁹Fe from cells and inhibiting ⁵⁹Fe uptake from the serum Fe transport protein, transferrin (Tf). Indeed, their activity was much greater than that found for the chelator in current clinical use, desferrioxamine (DFO), and similar to that observed for the orally active chelator, pyridoxal isonicotinoyl hydrazone (H₂PIH). The ability of the chelators to inhibit ⁵⁹Fe uptake could not be accounted for by direct chelation of ⁵⁹Fe from ⁵⁹Fe–Tf. The most effective chelators also showed low antiproliferative activity which was similar to or less than that observed with DFO, which is important in terms of their potential use as agents to treat Fe-overload disease.     

Biochemical and spectroscopic studies of human melanotransferrin (MTf): electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.     

Take-off and landing kinetics of a free-ranging gliding mammal, the Malayan colugo (Galeopterus variegatus)

Arboreal animals negotiate a highly three-dimensional world that is discontinuous on many spatial scales. As the scale of substrate discontinuity increases, many arboreal animals rely on leaping or gliding locomotion between distant supports. In order to successfully move through their habitat, gliding animals must actively modulate both propulsive and aerodynamic forces. Here we examined the take-off and landing kinetics of a free-ranging gliding mammal, the Malayan colugo (Galeopterus variegatus) using a custom-designed three-dimensional accelerometry system. We found that colugos increase the propulsive impulse to affect longer glides. However, we also found that landing forces are negatively associated with glide distance. Landing forces decrease rapidly as glide distance increases from the shortest glides, then level off, suggesting that the ability to reorient the aerodynamic forces prior to landing is an important mechanism to reduce velocity and thus landing forces. This ability to substantially alter the aerodynamic forces acting on the patagial wing in order to reorient the body is a key to the transition between leaping and gliding and allows gliding mammals to travel long distances between trees with reduced risk of injury. Longer glides may increase the access to distributed resources and reduce the exposure to predators in the canopy or on the forest floor.     

Crusade for iron: iron uptake in unicellular eukaryotes and its significance for virulence

The effective acquisition of iron is a pre-requisite for survival of all organisms, especially parasites that have a high iron requirement. In mammals, iron homeostasis is meticulously regulated; extracellular free iron is essentially unavailable and host iron availability has a crucial role in the host-pathogen relationship. Therefore, pathogens use specialized and effective mechanisms to acquire iron. In this review, we summarize the iron-uptake systems in eukaryotic unicellular organisms with particular focus on the pathogenic species: Candida albicans, Tritrichomonas foetus, Trypanosoma brucei and Leishmania spp. We describe the diversity of their iron-uptake mechanisms and highlight the importance of the process for virulence.