Osmotic Pressure in a Bacterial Swarm

Using Escherichia coli as a model organism, we studied how water is recruited by a bacterial swarm. A previous analysis of trajectories of small air bubbles revealed a stream of fluid flowing in a clockwise direction ahead of the swarm. A companion study suggested that water moves out of the agar into the swarm in a narrow region centered ∼30 μm from the leading edge of the swarm and then back into the agar (at a smaller rate) in a region centered ∼120 μm back from the leading edge. Presumably, these flows are driven by changes in osmolarity. Here, we utilized green/red fluorescent liposomes as reporters of osmolarity to verify this hypothesis. The stream of fluid that flows in front of the swarm contains osmolytes. Two distinct regions are observed inside the swarm near its leading edge: an outer high-osmolarity band (∼30 mOsm higher than the agar baseline) and an inner low-osmolarity band (isotonic or slightly hypotonic to the agar baseline). This profile supports the fluid-flow model derived from the drift of air bubbles and provides new (to our knowledge) insights into water maintenance in bacterial swarms. High osmotic pressure at the leading edge of the swarm extracts water from the underlying agar and promotes motility. The osmolyte is of high molecular weight and probably is lipopolysaccharide.     

Oxygen Concentration Inside a Functioning Photosynthetic Cell

The excess oxygen concentration in the photosynthetic membranes of functioning oxygenic photosynthetic cells was estimated using classical diffusion theory combined with experimental data on oxygen production rates of cyanobacterial cells. The excess oxygen concentration within the plesiomorphic cyanobacterium Gloeobactor violaceus is only 0.025 μM, or four orders of magnitude lower than the oxygen concentration in air-saturated water. Such a low concentration suggests that the first oxygenic photosynthetic bacteria in solitary form could have evolved ∼2.8 billion years ago without special mechanisms to protect them against reactive oxygen species. These mechanisms instead could have been developed during the following ∼500 million years while the oxygen level in the Earth’s atmosphere was slowly rising. Excess oxygen concentrations within individual cells of the apomorphic cyanobacteria Synechocystis and Synechococcus are 0.064 and 0.25 μM, respectively. These numbers suggest that intramembrane and intracellular proteins in isolated oxygenic photosynthetic cells are not subjected to excessively high oxygen levels. The situation is different for closely packed colonies of photosynthetic cells. Calculations show that the excess concentration within colonies that are ∼40 μm or larger in diameter can be comparable to the oxygen concentration in air-saturated water, suggesting that species forming colonies require protection against reactive oxygen species even in the absence of oxygen in the surrounding atmosphere.     

Short divalent ethacrynic amides as pro-inhibitors of glutathione S-transferase isozyme Mu and potent sensitisers of cisplatin-resistant ovarian cancers

The linking of ethacrynic acid with ethylenediamine and 1,4-butanediamine gave EDEA and BDEA, respectively, as membrane-permeable divalent pro-inhibitors of glutathione S-transferase (GST). Their divalent glutathione conjugates showed subnanomolar inhibition and divalence-binding to GSTmu (GSTM) (PDB: 5HWL) at ∼0.35 min⁻¹. In cisplatin-resistant SK-OV-3, COC1, SGC7901 and A549 cells, GSTM activities probed by 15 nM BDEA or EDEA revealed 5-fold and 1.0-fold increases in cisplatin-resistant SK-OV-3 and COC1 cells, respectively, in comparison with the susceptible parental cells. Being tolerable by HEK293 and LO2 cells, BDEA at 0.2 μM sensitised resistant SK-OV-3 and COC1 cells by ∼3- and ∼5-folds, respectively, released cytochrome c and increased apoptosis; EDEA at 1.0 μM sensitised resistant SK-OV-3 and A549 cells by ∼5- and ∼7-fold, respectively. EDEA at 1.7 μg/g sensitised resistant SK-OV-3 cells to cisplatin at 3.3 μg/g in nude mouse xenograft model. BDEA and EDEA are promising leads for probing cellular GSTM and sensitising cisplatin-resistant ovarian cancers.     

Organized Cell Swimming Motions in Bacillus subtilis Colonies: Patterns of Short-Lived Whirls and Jets

The swimming motions of cells within Bacillus subtilis colonies, as well as the associated fluid flows, were analyzed from video films produced during colony growth and expansion on wet agar surfaces. Individual cells in very wet dense populations moved at rates between 76 and 116 μm/s. Swimming cells were organized into patterns of whirls, each approximately 1,000 μm², and jets of about 95 by 12 μm. Whirls and jets were short-lived, lasting only about 0.25 s. Patterns within given areas constantly repeated with a periodicity of approximately 1 s. Whirls of a given direction became disorganized and then re-formed, usually into whirls moving in the opposite direction. Pattern elements were also organized with respect to one another in the colony. Neighboring whirls usually turned in opposite directions. This correlation decreased as a function of distance between whirls. Fluid flows associated with whirls and jets were measured by observing the movement of marker latex spheres added to colonies. The average velocity of markers traveling in whirls was 19 μm/s, whereas those traveling in jets moved at 27 μm/s. The paths followed by markers were aligned with the direction of cell motion, suggesting that cells create flows moving with them into whirls and along jets. When colonies became dry, swimming motions ceased except in regions close to the periphery and in isolated islands where cells traveled in slow whirls at about 4 μm/s. The addition of water resulted in immediate though transient rapid swimming (> 80 μm/s) in characteristic whirl and jet patterns. The rate of swimming decreased to 13 μm/s within 2 min, however, as the water diffused into the agar. Organized swimming patterns were nevertheless preserved throughout this period. These findings show that cell swimming in colonies is highly organized.     

Water-to-Air Transfer and Enrichment of Bacteria in Drops from Bursting Bubbles

An electrostatic induction technique was used to determine both drop size distribution and concentration of bacteria in the film drops produced by bubbles bursting at the surface of a suspension of Serratia marcescens. Film drops are produced from the collapse of the thin film of water that just before bursting separates the air in the bubble from the atmosphere. Bubbles of 1.7-mm diameter produced from 10 to 20 film drops which ranged from <2 μm to over 30 μm in diameter. Half the drops were <10 μm. For bubbles rising a distance of less than 2 cm through the bacterial suspension, bacterial enrichment factors in the drops were between 10 and 20. Electrostatic methods can be used to determine the enrichment of bacteria in film drops as a function of bubble size and distance of rise through the bacterial suspension.     

Effect of Sarcoplasmic Reticulum (SR) Calcium Content on SR Calcium Release Elicited by Small Voltage-Clamp Depolarizations in Frog Cut Skeletal Muscle Fibers Equilibrated with 20 mM EGTA

Cut muscle fibers from Rana temporaria (sarcomere length, 3.5–3.9 μm; 14–16°C) were mounted in a double Vaseline-gap chamber and equilibrated with an external solution that contained tetraethyl ammonium– gluconate and an internal solution that contained Cs as the principal cation, 20 mM EGTA, and 0 Ca. Fibers were stimulated with a voltage-clamp pulse protocol that consisted of pulses to −70, −65, −60, −45, and −20 mV, each separated by 400-ms periods at −90 mV. The change in total Ca that entered into the myoplasm (Δ[Ca_T]) and the Ca content of the SR ([Ca_SR]) were estimated with the EGTA/phenol red method (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259–336). Fibers were stimulated with the pulse protocol, usually every 5 min, so that the resting value of [Ca_SR] decreased from its initial value of 1,700–2,300 μM to values near or below 100 μM after 18–30 stimulations. Three main findings for the voltage pulses to −70, −65, and −60 mV are: (a) the depletion-corrected rate of Ca release (release permeability) showed little change when [Ca_SR] decreased from its highest level (>1,700 μM) to ∼1,000 μM; (b) as [Ca_SR] decreased below 1,000 μM, the release permeability increased to a maximum level when [Ca_SR] was near 300 μM that was on average about sevenfold larger than the values observed for [Ca_SR] > 1,000 μM; and (c) as [Ca_SR] decreased from ∼300 μM to <100 μM, the release permeability decreased, reaching half its maximum value when [Ca_SR] was ∼110 μM on average. It was concluded that finding b was likely due to a decrease in Ca inactivation, while finding c was likely due to a decrease in Ca-induced Ca release.     

Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (K_s, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (K_d, ≤3 nM) and prothioconazole-desthio the lowest (K_d, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC₅₀s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC₁₀₀, >256 μg ml⁻¹). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC₁₀₀ [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml⁻¹) with the exception of clotrimazole, which was as potent as malachite green (MIC₁₀₀, ∼1 μg ml⁻¹). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC₅₀, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC₁₀₀, ∼1 to 2 μg ml⁻¹) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.     

Microsecond Time-Resolved Absorption Spectroscopy Used to Study CO Compounds of Cytochrome bd from Escherichia coli

Cytochrome bd is a tri-heme (b ₅₅₈, b ₅₉₅, d) respiratory oxygen reductase that is found in many bacteria including pathogenic species. It couples the electron transfer from quinol to O₂ with generation of an electrochemical proton gradient. We examined photolysis and subsequent recombination of CO with isolated cytochrome bd from Escherichia coli in one-electron reduced (MV) and fully reduced (R) states by microsecond time-resolved absorption spectroscopy at 532-nm excitation. Both Soret and visible band regions were examined. CO photodissociation from MV enzyme possibly causes fast (τ<1.5 µs) electron transfer from heme d to heme b ₅₉₅ in a small fraction of the protein, not reported earlier. Then the electron migrates to heme b ₅₅₈ (τ∼16 µs). It returns from the b-hemes to heme d with τ∼180 µs. Unlike cytochrome bd in the R state, in MV enzyme the apparent contribution of absorbance changes associated with CO dissociation from heme d is small, if any. Photodissociation of CO from heme d in MV enzyme is suggested to be accompanied by the binding of an internal ligand (L) at the opposite side of the heme. CO recombines with heme d (τ∼16 µs) yielding a transient hexacoordinate state (CO-Fe²⁺-L). Then the ligand slowly (τ∼30 ms) dissociates from heme d. Recombination of CO with a reduced heme b in a fraction of the MV sample may also contribute to the 30-ms phase. In R enzyme, CO recombines to heme d (τ∼20 µs), some heme b ₅₅₈ (τ∼0.2–3 ms), and finally migrates from heme d to heme b ₅₉₅ (τ∼24 ms) in ∼5% of the enzyme population. Data are consistent with the recent nanosecond study of Rappaport et al. conducted on the membranes at 640-nm excitation but limited to the Soret band. The additional phases were revealed due to differences in excitation and other experimental conditions.     

Single-Channel Kinetics,Inactivation, and Spatial Distribution of Inositol Trisphosphate (IP3) Receptors in Xenopus Oocyte Nucleus

Single-channel properties of the Xenopus inositol trisphosphate receptor (IP₃R) ion channel were examined by patch clamp electrophysiology of the outer nuclear membrane of isolated oocyte nuclei. With 140 mM K⁺ as the charge carrier (cytoplasmic [IP₃] = 10 μM, free [Ca²⁺] = 200 nM), the IP₃R exhibited four and possibly five conductance states. The conductance of the most-frequently observed state M was 113 pS around 0 mV and ∼300 pS at 60 mV. The channel was frequently observed with high open probability (mean P _o = 0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants τ < 5 ms and ∼20 ms; and at least three closed states with τ ∼1 ms, ∼10 ms, and >1 s. Higher cytoplasmic potential increased the relative frequency and τ of the longest closed state. A novel “flicker” kinetic mode was observed, in which the channel alternated rapidly between two new conductance states: F₁ and F₂. The relative occupation probability of the flicker states exhibited voltage dependence described by a Boltzmann distribution corresponding to 1.33 electron charges moving across the entire electric field during F₁ to F₂ transitions. Channel run-down or inactivation (τ ∼ 30 s) was consistently observed in the continuous presence of IP₃ and the absence of change in [Ca²⁺]. Some (∼10%) channel disappearances could be reversed by an increase in voltage before irreversible inactivation. A model for voltage-dependent channel gating is proposed in which one mechanism controls channel opening in both the normal and flicker modes, whereas a separate independent mechanism generates flicker activity and voltage- reversible inactivation. Mapping of functional channels indicates that the IP₃R tends to aggregate into microscopic (<1 μm) as well as macroscopic (∼10 μm) clusters. Ca²⁺-independent inactivation of IP₃R and channel clustering may contribute to complex [Ca²⁺] signals in cells.     

Inhibition of Photosynthesis in Certain Algae by Extreme Red Light

This paper shows that in Porphyridium cruentum and in Chlorella pyrenoidosa (but apparently not in Anacystis nidulans) “extreme red” light (> 720 mμ) can inhibit photosynthesis produced by “far red” light (up to 720 mμ). From the action spectrum of this phenomenon, it appears that an unknown pigment with an absorption band around 745 mμ must be responsible for it.     

NMR solution structures of Runella slithyformis RNA 2′-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity

Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO₄ to NAD⁺ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD⁺. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD⁺-bound states. ITC measurements of RslTpt1 binding to NAD⁺ (K_D ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD⁺-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP⁺, which contains an adenylate 2′-PO₄ (mimicking the substrate RNA 2′-PO₄), binds with lower affinity (K_D ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD⁺ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD⁺ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.     

Quantification of Transmembrane Currents during Action Potential Propagation in the Heart

The measurement, quantitative analysis, theory, and mathematical modeling of transmembrane potential and currents have been an integral part of the field of electrophysiology since its inception. Biophysical modeling of action potential propagation begins with detailed ionic current models for a patch of membrane within a distributed cable model. Voltage-clamp techniques have revolutionized clinical electrophysiology via the characterization of the transmembrane current gating variables; however, this kinetic information alone is insufficient to accurately represent propagation. Other factors, including channel density, membrane area, surface/volume ratio, axial conductivities, etc., are also crucial determinants of transmembrane currents in multicellular tissue but are extremely difficult to measure. Here, we provide, to our knowledge, a novel analytical approach to compute transmembrane currents directly from experimental data, which involves high-temporal (200 kHz) recordings of intra- and extracellular potential with glass microelectrodes from the epicardial surface of isolated rabbit hearts during propagation. We show for the first time, to our knowledge, that during stable planar propagation the biphasic total transmembrane current (I_m) dipole density during depolarization was ∼0.25 ms in duration and asymmetric in amplitude (peak outward current was ∼95 μA/cm² and peak inward current was ∼140 μA/cm²), and the peak inward ionic current (I_ion) during depolarization was ∼260 μA/cm² with duration of ∼1.0 ms. Simulations of stable propagation using the ionic current versus transmembrane potential relationship fit from the experimental data reproduced these values better than traditional ionic models. During ventricular fibrillation, peak I_m was decreased by 50% and peak I_ion was decreased by 70%. Our results provide, to our knowledge, novel quantitative information that complements voltage- and patch-clamp data.     

Action Spectrum for the Appearance of the 520 mμ Difference Band in Illuminated Chlorella Cells

An action spectrum of the 520 mμ difference band in Chlorella is determined using dim illumination. Pigment (or pigments) absorbing most strongly at and above 680 mμ, probably the so-called “long-wave forms” of chlorophyll a appear to be the primary sensitizer of the 520 mμ effect.     

Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na⁺-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H⁺-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.     

Kinesin-2 KIF3AC and KIF3AB Can Drive Long-Range Transport along Microtubules

Mammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 μm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 μm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 μm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter k_cat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons.     

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (K_D = 267 μm) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D–F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20–50 μm Ca²⁺. At 20 μm Ca²⁺, the dissociation rate was substantially lower, indicating increased binding (K_D = ∼10⁻⁹) compared with 0 μm Ca²⁺ (K_D = ∼10⁻⁸), suggesting a calcium-mediated stabilization of the C2 domain·t-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 μm and with reduction at 0 μm Ca²⁺, a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a “closed” tertiary structure at low calcium that “opens” as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.     

More than Motility: Salmonella Flagella Contribute to Overriding Friction and Facilitating Colony Hydration during Swarming

We show in this study that Salmonella cells, which do not upregulate flagellar gene expression during swarming, also do not increase flagellar numbers per μm of cell length as determined by systematic counting of both flagellar filaments and hooks. Instead, doubling of the average length of a swarmer cell by suppression of cell division effectively doubles the number of flagella per cell. The highest agar concentration at which Salmonella cells swarmed increased from the normal 0.5% to 1%, either when flagella were overproduced or when expression of the FliL protein was enhanced in conjunction with stator proteins MotAB. We surmise that bacteria use the resulting increase in motor power to overcome the higher friction associated with harder agar. Higher flagellar numbers also suppress the swarming defect of mutants with changes in the chemotaxis pathway that were previously shown to be defective in hydrating their colonies. Here we show that the swarming defect of these mutants can also be suppressed by application of osmolytes to the surface of swarm agar. The “dry” colony morphology displayed by che mutants was also observed with other mutants that do not actively rotate their flagella. The flagellum/motor thus participates in two functions critical for swarming, enabling hydration and overriding surface friction. We consider some ideas for how the flagellum might help attract water to the agar surface, where there is no free water.     

Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy

In multicellular organisms, single-fluorophore imaging is obstructed by high background. To achieve a signal/noise ratio conducive to single-molecule imaging, we adapted reflected light-sheet microscopy (RLSM) to image highly opaque late-stage Drosophila embryos. Alignment steps were modified by means of commercially available microprisms attached to standard coverslips. We imaged a member of the septate-junction complex that was used to outline the three-dimensional epidermal structures of Drosophila embryos. Furthermore, we show freely diffusing single 10 kDa Dextran molecules conjugated to one to two Alexa647 dyes inside living embryos. We demonstrate that Dextran diffuses quickly (∼6.4 μm²/s) in free space and obeys directional movement within the epidermal tissue (∼0.1 μm²/s). Our single-particle-tracking results are supplemented by imaging the endosomal marker Rab5-GFP and by earlier reports on the spreading of morphogens and vesicles in multicellular organisms. The single-molecule results suggest that RLSM will be helpful in studying single molecules or complexes in multicellular organisms.     

Estrogen Modification of Human Glutamate Dehydrogenases Is Linked to Enzyme Activation State

Mammalian glutamate dehydrogenase (GDH) is a housekeeping enzyme central to the metabolism of glutamate. Its activity is potently inhibited by GTP (IC₅₀ = 0.1–0.3 μm) and thought to be controlled by the need of the cell in ATP. Estrogens are also known to inhibit mammalian GDH, but at relatively high concentrations. Because, in addition to this housekeeping human (h) GDH1, humans have acquired via a duplication event an hGDH2 isoform expressed in human cortical astrocytes, we tested here the interaction of estrogens with the two human isoenzymes. The results showed that, under base-line conditions, diethylstilbestrol potently inhibited hGDH2 (IC₅₀ = 0.08 ± 0.01 μm) and with ∼18-fold lower affinity hGDH1 (IC₅₀ = 1.67 ± 0.06 μm; p < 0.001). Similarly, 17β-estradiol showed a ∼18-fold higher affinity for hGDH2 (IC₅₀ = 1.53 ± 0.24 μm) than for hGDH1 (IC₅₀ = 26.94 ± 1.07 μm; p < 0.001). Also, estriol and progesterone were more potent inhibitors of hGDH2 than hGDH1. Structure/function analyses revealed that the evolutionary R443S substitution, which confers low basal activity, was largely responsible for sensitivity of hGDH2 to estrogens. Inhibition of both human GDHs by estrogens was inversely related to their state of activation induced by ADP, with the slope of this correlation being steeper for hGDH2 than for hGDH1. Also, the study of hGDH1 and hGDH2 mutants displaying different states of activation revealed that the affinity of estrogen for these enzymes correlated inversely (R = 0.99; p = 0.0001) with basal catalytic activity. Because astrocytes are known to synthesize estrogens, these hormones, by interacting potently with hGDH2 in its closed state, may contribute to regulation of glutamate metabolism in brain.