Vision-assisted micromanipulation using closed-loop actuation of multiple microrobots.

Accurate control and precise positioning of opto-thermocapillary flow-addressed bubble microrobots are necessary for micromanipulation. In addition, micromanipulation using the simultaneous actuation of multiple microrobots requires a robust control system to enable independent motion. This paper demonstrates a hybrid closed-loop vision-assisted control system capable of actuating multiple microrobots simultaneously and positioning them at precise locations relative to micro-objects under manipulation. A vision-assisted grasp-planning application was developed and used to calculate the necessary trajectories of the microrobots to form cages around micro-objects. The location of the microrobots and the micro-objects was detected at the caging locations using a particle-tracking application that used image feedback for precise positioning. The closed-loop image feedback information enabled the position update of the microrobots, allowing them to precisely follow the trajectory and caging locations calculated by the grasp-planning application. Four microrobots were assigned to cage a star-shaped micro-object using the closed-loop control system. Once caged, the micro-object was transported to a location within the workspace and uncaged, demonstrating the micromanipulation task. This microrobotic system is well suited for the micromanipulation of single cells.

A novel nicotinoprotein aldehyde dehydrogenase involved in polyethylene glycol degradation.

A gene (pegC) encoding aldehyde dehydrogenase (ALDH) was located 3.4 kb upstream of a gene encoding polyethylene glycol (PEG) dehydrogenase (pegA) in Sphingomonas macrogoltabidus strain 103. ALDH was expressed in Escherichia coli and purified on a Ni-nitrilotriacetic acid agarose column. The recombinant enzyme was a homotetramer consisting of four 46.1-kDa subunits. The alignment of the putative amino acid sequence of the cloned enzyme showed high similarity with a group of NAD(P)-dependent ALDHs (identity 36-52%); NAD-binding domains (Rossmann fold and four glycine residues) and catalytic residues (Glu225 and Cys259) were well conserved. The cofactor, which was extracted from the purified enzyme, was tightly bound to the enzyme and identified as NADP. The enzyme contained 0.94 mol NADP per subunit. The enzyme was activated by Ca(2+), but by no other metals; no metal (Zn, Fe, Mg, or Mn) was detected in the purified recombinant enzyme. Activity was inhibited by p-chloromercuric benzoate, and heavy metals such as Hg, Cu, Pb and Cd, indicating that a cysteine residue is involved in the activity. Enzyme activity was independent of N,N-dimethyl-p-nitrosoaniline as an electron acceptor. Trans-4-(N,N-dimethylamino)-cinnamaldehyde was not oxidized as a substrate, but the compound worked as an inhibitor for the enzyme, as did pyrazole. The enzyme acted on n-aldehydes C(2)-C(14)) and PEG-aldehydes. Thus the enzyme was concluded to be a novel Ca(2+)-activating nicotinoprotein (NADP-containing) PEG-aldehyde dehydrogenase involved in the degradation of PEG in S. macrogoltabidus strain 103.

Linalool suppresses voltage-gated currents in sensory neurons and cerebellar Purkinje cells.

Linalool is a major component of essential oils and possesses various biological effects in sensory or central nervous systems. To investigate the pharmacological and biophysical effects of linalool on voltage-gated currents in sensory neurons, we used the whole-cell patch clamp and the Ca(2+) imaging techniques. Under the voltage clamp, membrane depolarization generated time- and voltage-dependent current responses in newt olfactory receptor cells (ORCs). Linalool significantly and reversibly suppressed the voltage-gated currents in ORCs. The dose-suppression relation of linalool for the voltage-gated Na(+) current could be fitted by the Hill equation with a half-blocking concentration of 0.56 mM and a Hill coefficient of 1.2. To test whether linalool suppresses voltage-gated currents in ORCs specifically or suppresses currents in other neurons generally, we next examined the effects of linalool on voltage-gated currents in newt retinal neurons and rat cerebellar Purkinje cells. Linalool suppressed the voltage-gated currents not only in retinal horizontal cells and ganglion cells but also in Purkinje cells. Furthermore, bath application of linalool inhibited the KCl-induced [Ca(2+)](i) response of ORCs, suggesting that linalool suppresses Ca(2+) currents in ORCs. These results suggest that linalool non-selectively suppresses the voltage-gated currents in newt sensory neurons and rat cerebellar Purkinje cells.

Treatment of the yeast Rhodotorula glutinis with AlCl(3) leads to adaptive acquirement of heritable aluminum resistance.

When aluminum (Al) was added to a culture, growth of Rhodotorula glutinis IFO1125 was temporarily arrested, showing longer lag phases, depending on the Al concentrations (50-300 microM) added, but the growth rates were not affected at all. Resistant strains obtained by one round of plate treatment containing Al reverted the resistance level to the wild-type level when cultivated without Al. Repeated Al treatments, however, induced heritable and stable Al resistance, the level of which was increased up to 4,000 microM by stepwise increments in Al concentrations. Thus, the heritable Al resistance adaptively acquired was due neither to adaptation nor to mutation, but to a mechanism which has yet to be studied. Heritable Al resistance seemed to release the Al inhibition of magnesium uptake.

Direct suppression by odorants of ionotropic glutamate receptors in newt retinal neurons.

Odorants are known to suppress voltage-gated channels not only in olfactory receptor cells but also in neurons of outside of the olfactory system. Here we found that odorants suppress glutamate-gated channels in newt retinal neurons using the Ca(2+) imaging technique. Bath application of 100 microM glutamate rose [Ca(2+)](i) under application of the voltage-gated Ca(2+) channel blocker. Thus, [Ca(2+)](i) rises in the neurons were most likely attributable to Ca(2+) influx via Ca(2+)-permeable glutamate-gated channels rather than voltage-gated Ca(2+) channels. A similar increase of [Ca(2+)](i) was observed by application of 100 microM NMDA and 50 microM kainate, suggesting that both NMDA and AMPA/kainate receptors were expressed in newt retinal neurons. Application of odorants, 1 mM amyl acetate and acetophenone, reversibly reduced [Ca(2+)](i) increased by glutamate, NMDA and kainate. This suggests that odorants can suppress not only voltage-gated channels but also ligand-gated channels such as NMDA and AMPA/kainate receptors.

Microbial degradation of polyethers.

This paper summarizes studies on microbial degradation of polyethers. Polyethers are aerobically metabolized through common mechanisms (oxidation of terminal alcohol groups followed by terminal ether cleavage), well-characterized examples being found with polyethylene glycol (PEG). First the polymer is oxidized to carboxylated PEG by alcohol and aldehyde dehydrogenases and then the terminal ether bond is cleaved to yield the depolymerized PEG by one glycol unit. Most probably PEG is anaerobically metabolized through one step which is catalyzed by PEG acetaldehyde lyase, analogous to diol dehydratase. Whether aerobically or anaerobically, the free OH group is necessary for metabolization of PEG. PEG with a molecular weight of up to 20,000 was metabolized either in the periplasmic space (Pseudomonas stutzeri and sphingomonads) or in the cytoplasm (anaerobic bacteria), which suggests the transport of large PEG through the outer and inner membranes of Gram-negative bacterial cells. Membrane-bound PEG dehydrogenase (PEG-DH) with high activity towards PEG 6,000 and 20,000 was purified from PEG-utilizing sphingomonads. Sequencing of PEG-DH revealed that the enzyme belongs to the group of GMC flavoproteins, FAD being the cofactor for the enzyme. On the other hand, alcohol dehydrogenases purified from other bacteria that cannot grow on PEG oxidized PEG. Cytoplasmic NAD-dependent alcohol dehydrogenases with high specificity towards ether-alcohol compound, either crude or purified, showed appreciable activity towards PEG 400 or 600. Liver alcohol dehydrogenase (equine) also oxidized PEG homologs, which might cause fatal toxic syndrome in vivo by carboxylating PEG together with aldehyde dehydrogenase when PEG was absorbed. An ether bond-cleaving enzyme was detected in PEG-utilizing bacteria and purified as diglycolic acid (DGA) dehydrogenase from a PEG-utilizing consortium. The enzyme oxidized glycolic acid, glyoxylic acid, as well as PEG-carboxylic acid and DGA. Similarly, dehydrogenation on polypropylene glycol (PPG) and polytetramethylene glycol (PTMG) was suggested with cell-free extracts of PPG and PTMG-utilizing bacteria, respectively. PPG commercially available is atactic and includes many structural (primary and secondary alcohol groups) and optical (derived from pendant methyl groups on the carbon backbone) isomers. Whether PPG dehydrogenase (PPG-DH) has wide stereo- and enantioselective substrate specificity towards PPG isomers or not must await further purification. Preliminary research on PPG-DH revealed that the enzyme was inducibly formed by PPG in the periplasmic, membrane and cytoplasm fractions of a PPG-utilizing bacterium Stenotrophomonas maltophilia. This finding indicated the intracellular metabolism of PPG is the same as that of PEG. Besides metabolization of polyethers, a biological Fenton mechanism was proposed for degradation of PEG, which was caused by extracellular oxidants produced by a brown-rot fungus in the presence of a reductant and Fe3+, although the metabolism of fragmented PEG has not yet been well elucidated.

The first step in polyethylene glycol degradation by sphingomonads proceeds via a flavoprotein alcohol dehydrogenase containing flavin adenine dinucleotide.

Several Sphingomonas spp. utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain. Purification and characterization of PEG-DH from Sphingomonas terrae revealed that the enzyme is membrane bound. The gene encoding this enzyme (pegA) was cloned, sequenced, and expressed in Escherichia coli. The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent. It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zn. PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates. Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from Pseudomonas oleovorans (46%); choline dehydrogenase from E. coli (40%); L-sorbose dehydrogenase from Gluconobacter oxydans (38%); and 4-nitrobenzyl alcohol dehydrogenase from a Pseudomonas species (35%).

Na(+) action potentials in human photoreceptors.

Mammalian photoreceptors are hyperpolarized by a light stimulus and are commonly thought to be nonspiking neurons. We used the whole-cell patch-clamp technique on surgically excised human retina to examine whether human photoreceptors can elicit action potentials. We discovered that human rod photoreceptors express voltage-gated Na(+) channels, and generate Na(+) action potentials, in response to membrane depolarization from membrane potentials of -60 or -70 mV. Na(+) spikes in human rods were elicited at the termination of a light response that hyperpolarized the potential well below -50 mV. This served to amplify the release of a neurotransmitter when a bright light is turned off, and thus selectively amplify the off response to the light signal.

Enhancement by T-type Ca2+ currents of odor sensitivity in olfactory receptor cells.

Mechanisms underlying action potential initiation in olfactory receptor cells (ORCs) during odor stimulation were investigated using conventional and dynamic patch-clamp recording techniques. Under current-clamp conditions, action potentials generated by a least effective odor-induced depolarization were almost completely blocked by 0.1 mm Ni(2+), a T-type Ca(2+) channel blocker, but not by 0.1 mm Cd(2+), a high voltage-activated Ca(2+) channel blocker. Under voltage-clamp conditions, depolarizing voltage steps induced a fast transient inward current, which consisted of Na(+) (I(Na)) and T-type Ca(2+) (I(Ca,T)) currents. The amplitude of I(Ca,T) was approximately one-fourth of that of I(Na) (0.23 +/- 0.03, mean +/- SEM). Because both I(Na) and I(Ca,T) are known to show rapid inactivation, we examined how much I(Na) and I(Ca,T) are activated during the gradually depolarizing initial phase of receptor potentials. The ratio of I(Ca,T)/I(Na) during a ramp depolarization at the slope of 0.5 mV/msec was 0.56 +/- 0.03. Using the dynamic patch-clamp recording technique, we also recorded I(Ca,T) and I(Na) during the generation of odor-induced action potentials. This ratio of I(Ca,T)/I(Na) was 0.54 +/- 0.04. These ratios were more than twice as large as that (0.23) obtained from the experiment using voltage steps, suggesting that I(Ca,T) carries significant amount of current to generate the action potentials. We conclude that I(Ca,T) contributes to enhance odor sensitivity by lowering the threshold of spike generation in ORCs.

Modulation by cGMP of the voltage-gated currents in newt olfactory receptor cells.

Effects of cGMP on voltage-gated currents in the somatic membrane of isolated newt olfactory receptor cells were investigated using the whole-cell mode of the patch-clamp technique. Under voltage clamp, membrane depolarization generated time- and voltage-dependent current responses, a transient inward current and a sustained outward current. When cGMP or a membrane permeant analog of cGMP, 8-p-chlorophenylthio-cGMP (CPT-cGMP), was applied to the recorded cell, the amplitude of the transient inward current increased markedly, but that of the sustained outward current did not change significantly. When each current was isolated by pharmacological agents, 0.1 mM CPT-cGMP increased the peak amplitude of a Na(+) current (I(Na)) by approximately 40%, a T-type Ca(2+) current (I(Ca,T)) by approximately 40%, and an L-type Ca(2+)current (I(Ca,L)) by approximately 10%; however it did not change significantly the amplitude of a delayed rectifier K(+) current (I(K)). A selective cGMP-dependent protein kinase inhibitor, KT5823, blocked the enhancement by cGMP of I(Na) and I(Ca,T), suggesting that cGMP increases these currents via cGMP-dependent phosphorylation. Under current-clamp conditions, application of CPT-cGMP lowered the current threshold of action potentials induced by current injection, and increased the maximum spike frequency in response to strong stimuli. We suggest that cGMP may lower the threshold in olfactory perception by decreasing the current threshold to generate spikes, and also prevent the saturation of odor signals by increasing the maximum spike frequency.

Screening and characterization of trehalose-oleate hydrolyzing lipase.

Various soil samples were collected to screen the presence of microorganisms which have ability to degrade TOE. One strain (AKU-883) with good TOE degrading activity was isolated and identified as Burkholderia cepacia and the extracellular enzyme was purified to homogeneity. The purification was achieved by ultrafiltration, Super Q anion-exchange chromatography and Superdex 200HR gel-filtration in the presence of Triton X. The enzyme was purified to 85-fold, and specific activity of 4.910 kU mg protein(-1). The peak preparation on gel filtration showed a single band of 34 kDa on SDS-PAGE and native PAGE which indicate the monomeric nature of the enzyme. The pI of the enzyme was 6.3. The enzyme showed the maximum activity at pH 9 and 65 degrees C, and was stable in the range of pH 5--10 and up to 60 degrees C. Almost all the activity (92%) was kept after incubation for more than 1 week at 50 degrees C (pH 7.3). High activities remained even in water-miscible solvents such as ethanol, dimethyl formamide, diisopropyl ether, and dioxane. The N-terminal 16 amino acid residues were determined as A-N-G-Y-A-A-T-R-Y-P-I-I-L-V-G-G, which showed a consensus sequence for lipases from Burkholderia species. Thus the enzyme was concluded to be a kind of lipase.

Direct suppression by odorants of cyclic nucleotide-gated currents in the newt photoreceptors.

Odorants are known to suppress the cyclic nucleotide-gated (CNG) current in olfactory receptor cells. It is unclear, however, whether odorants suppress the olfactory CNG current directly or whether they suppress the current by decreasing the second messenger (cAMP) through the activation of phosphodiesterase. We found that odorants also suppress CNG currents in photoreceptor cells. Under voltage clamp, an odorant puff immediately suppressed the currents induced by the intracellular cGMP in isolated newt rods and cones. Odorants also suppressed the currents induced by another cGMP analog (8-p-chlorophenylthio-cGMP, which strongly resists hydrolysis by phosphodiesterase), suggesting that the second messenger metabolism via phosphodiesterase is not involved in the suppression by odorants. This suggests that odorants suppress the CNG currents directly rather than via the second messenger system in photoreceptors, and also likely in olfactory receptor cells.

Isolation and characterization of acid- and Al-tolerant microorganisms.

Acid- and aluminum (Al)-tolerant microorganisms were isolated from tea fields, from which six strains were selected and identified as Cryptococcus humicola, Rhodotorula glutinis, Aspergillus flavus Link, Penicillium sp., Penicillium janthinellum Biourge and Trichoderma asperellum. They were tolerant to Al up to 100-200 mM and could grow at low pH, 2.5-2.2. In a glucose medium (pH 3.5) the pH of the spent medium decreased to below 3.0. The toxic inorganic monomeric Al in the spent medium decreased with three strains (A. flavus F-6b, Penicillium sp. F-8b and P. janthinellum F-13), but the total Al remained constant for all strains. In a soil extract medium (pH 3.5), the pH of the spent medium of all strains increased to around 6.0-7. 2 and total Al in the spent medium was removed by precipitation due to pH increase. Thus, different tolerance mechanisms were suggested in glucose and soil extract media.

Purification and inactivation by substrate of an allene oxide synthase (CYP74) from corn (Zea mays L.) seeds.

The allene oxide synthase (AOS) was purified from corn (Zea mays) seeds to homogeneity and characterized partially. The corn AOS was a hemoprotein cytochrome P450 with a molecular weight and pI of 53,000 and 6.0, respectively. The corn AOS was found to be irreversibly inactivated by a substrate, 13-hydroperoxyoctadienoic acid. The rate of the enzyme inactivation was higher at low pHs.

Odorants suppress voltage-gated currents in retinal horizontal cells in goldfish.

Odorants are known to suppress non-selectively voltage-gated currents in olfactory receptor cells. We found that odorants also suppress voltage-gated currents in neurons of outside of the olfactory system. Under voltage clamp, odorants such as amyl acetate, limonene, and acetophenone suppressed non-selectively voltage-gated currents (a Ca(2+) current, a delayed rectifier K(+) current, a fast transient K(+) current, and an anomalous rectifier K(+) current) in horizontal cells from the goldfish retina. An amyl acetate puff completely and immediately suppressed the Ca(2+) current (I(Ca)) and the delayed rectifier K(+) current induced by repetitive depolarizations, suggesting that amyl acetate is a closed-channel blocker. Odorants did not change significantly the activation curve of I(Ca), but made the slope of inactivation curve of I(Ca) gentler and shifted its half-inactivation voltage toward a negative voltage. These results are similar to the effects of odorants on voltage-gated currents in olfactory receptor cells. This suggests that odorants may suppress the voltage-gated currents in retinal horizontal cells by the same mechanism described in olfactory receptor cells.

Simulation analysis of effects of adrenaline on spike generation in olfactory receptor cells.

Adrenaline is known to affect action potentials induced by the step current injection in an olfactory receptor cell (ORC). It is unclear, however, whether it also modulates action potentials induced by odor stimuli. In the present study, the effects of adrenaline on action potentials in ORCs were investigated quantitatively using a computer simulation. Adrenaline suppressed simulated action potentials induced by step current injection near threshold, and increased spike frequency to strong stimuli by 8-25%. Similar effects were obtained by applying a pseudo-transduction current to a model cell. Surprisingly, adrenaline markedly increased spike frequency to strong stimuli by 30-140%, and increased the slope of the stimulus-response relation compared with that of the step current injection. This suggests that adrenaline enhances odorant contrast in olfactory perception by modulating signal encoding of ORCs.

Characterization of spontaneous excitatory synaptic currents in newt retinal bipolar cells.

The kinetics of glutamate concentration in the synaptic cleft is an important determinant of synaptic function. To elucidate peak concentration of glutamate released from a single vesicle in the cleft, spontaneous excitatory postsynaptic currents (sEPSCs) in Off-bipolar cells from the sliced newt retina were analyzed using whole-cell patch clamp recording and the computer simulation. The sEPSCs were blocked by an AMPA/kainate (KA) antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and prolonged by cyclothiazide. However, an N-methyl-D-aspartate (NMDA) antagonist, D-2-amino-5-phosphonopentanoic acid (D-AP5), was ineffective. These suggest that sEPSCs in Off-bipolar cells are mediated exclusively by AMPA/KA receptors. sEPSCs simulated by a detailed kinetic model of AMPA receptor best approximated the data, when peak glutamate concentration was 10 microM. Therefore, it was concluded that peak concentration of glutamate released from a single vesicle would be elevated to approximately 10 microM at the newt Off-bipolar dendrite.

Hyperechoic renal papillae as a physiological finding in neonates.

AIM: The increased echogenicity of medullary pyramids in neonates and children is poorly understood. Hence we conducted a prospective ultrasound study of the kidneys of neonates and fetuses. Hence we conducted a prospective ultrasound study of the kidneys of neonates and fetuses. PATIENTS AND METHODS: Ultrasound images of kidneys in neonates and fetuses in late pregnancy were analysed. RESULTS: Thirteen percent of the studied neonates showed hyperechogenicity in the renal papillae that disappeared spontaneously within 1 week, although no hyperechoic papillae were seen in any of the fetuses. Urine volume of the neonates with hyperechogenicity was significantly less than that of those without it. CONCLUSION: The fact that no hyperechoic findings appeared before the 34th week of gestation suggests that maturation of renal tubules and ability to concentrate urinary substances were contributing factors. Because only the tips of the pyramids were hyperechogenic, however, the term hyperechoic papillae would seem more appropriate than hyperechoic pyramids, the term generally used today.

Adrenaline enhances odorant contrast by modulating signal encoding in olfactory receptor cells.

Olfactory perception is influenced by hormones. Here we report that adrenaline can directly affect the signal encoding of olfactory receptor cells. Application of adrenaline suppressed action potentials near threshold and increased their frequency in response to strong stimuli, resulting in a narrower dynamic range. Under voltage-clamp conditions, adrenaline enhanced sodium current and reduced T-type calcium current. Because sodium current is the major component of spike generation and T-type calcium current lowers the threshold in olfactory receptor cells, the effects of adrenaline on these currents are consistent with the results obtained under current-clamp conditions. Both effects involved a common cytoplasmic pathway, cAMP-dependent phosphorylation. We suggest that adrenaline may enhance contrast in olfactory perception by this mechanism.

AMPA receptor activates a G-protein that suppresses a cGMP-gated current.

The AMPA receptor, ubiquitous in brain, is termed "ionotropic" because it gates an ion channel directly. We found that an AMPA receptor can also modulate a G-protein to gate an ion channel indirectly. Glutamate applied to a retinal ganglion cell briefly suppresses the inward current through a cGMP-gated channel. AMPA and kainate also suppress the current, an effect that is blocked both by their general antagonist CNQX and also by the relatively specific AMPA receptor antagonist GYKI-52466. Neither NMDA nor agonists of metabotropic glutamate receptors are effective. The AMPA-induced suppression of the cGMP-gated current is blocked when the patch pipette includes GDP-beta-S, whereas the suppression is irreversible when the pipette contains GTP-gamma-S. This suggests a G-protein mediator, and, consistent with this, pertussis toxin blocks the current suppression. Nitric oxide (NO) donors induce the current suppressed by AMPA, and phosphodiesterase inhibitors prevent the suppression. Apparently, the AMPA receptor can exhibit a "metabotropic" activity that allows it to antagonize excitation evoked by NO.