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Ruthenium red (RR) is a widely used inhibitor of Transient Receptor Potential (TRP) cation channels and other types of ion channels. Although RR has been generally accepted to inhibit TRP channels by physically blocking the ion permeation pathway, recent structural evidence suggests that it might also function as an antagonist, inducing conformational changes in the channel upon binding that result in closure of the pore. In a recent manuscript published in EMBO Reports, Ruth A. Pumroy and collaborators solve structures of TRPV2 and TRPV5 channels in the presence and absence of activators and RR. The data sheds light on the mechanism of inhibition by RR, while also opening new questions for further investigation., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Ltd.)

Osteoporosis is a disorder of bone metabolism that is extremely common in elderly patients as well as in postmenopausal women. The main manifestation is that the bone resorption capacity is greater than the bone formation capacity, which eventually leads to a decrease in bone mass, increasing the risk of fracture. There is growing evidence that inhibiting osteoclast formation and resorption ability can be effective in treating and preventing the occurrence of osteoporosis. Our study is the first time to explore the role of the mitochondrial calcium uniporter (MCU) and its inhibitor ruthenium red (RR) in bone metabolism, clarifying the specific mechanism by which it inhibits osteoclast formation in vitro and plays a therapeutic role in osteoporosis in vivo. We verified the suppressive effects of RR on the receptor activator of nuclear factor- κ B ligand (RANKL-)-induced differentiation and bone resorption function of osteoclasts in vitro. The reactive oxygen species (ROS) production stimulated by RANKL and the expression level of P38 MAPK/NFATc1 were also found to be inhibited by RR. Moreover, the promotion of RR on osteogenesis differentiation was investigated by alkaline phosphatase (ALP) and alizarin red S (ARS) staining and the detection of osteogenesis-specific gene expression levels by quantitative polymerase chain reaction (qPCR) and western blotting. Moreover, in ovariectomy (OVX-)-induced osteoporosis models, RR can downregulate the expression and function of the MCU, relieving bone loss and promoting osteogenesis to present a therapeutic effect on osteoporosis. This new finding will provide an important direction for the study of RR and MCU in the study of bone metabolism therapy targets., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Yuxin Wang et al.)

Mitochondrial free calcium is critically linked to the regulation of cellular metabolism. Free ionic calcium concentration within these organelles is determined by the interplay between two processes: exchange across the mitochondrial inner membrane and calcium-buffering within the matrix. During stimulated calcium uptake, calcium is primarily buffered by orthophosphate, preventing calcium toxicity while allowing for well-regulated yet elevated calcium loads. However, if limited to orthophosphates only, this buffering system is expected to lead to the irreversible formation of insoluble precipitates, which are not observed in living cells, under physiological conditions. Here, we demonstrate that the regulation of free mitochondrial calcium requires the presence of free inorganic polyphosphate (polyP) within the organelle. We found that the overexpression of a mitochondrial-targeted enzyme hydrolyzing polyP leads to the loss of the cellular ability to maintain elevated calcium concentrations within the organelle, following stimulated cytoplasmic signal. We hypothesize that the presence of polyP prevents the formation of calcium-phosphate insoluble clusters, allowing for the maintenance of elevated free calcium levels, during stimulated calcium uptake., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The mechanosensitivity of cells depends on the lipid-protein interactions of the plasma membrane. Affectations in the lipid region of the plasma membrane affect the transduction of mechanical forces, and any molecule that modifies the biophysical integrity of the lipid bilayer can alter the mechanical activity of the proteins inside the membrane. To understand whether inhibitors of mechanically activated ion channels affect the mechanical properties of the plasma membrane, we evaluated the rigidity of the membrane of sensory neurons of the DRG of mice using a variant of the scanning ion conductance microscopy method, which allows us to calculate the Young's modulus of individual cells before and after the perfusion of different doses of Gd3+, ruthenium red and GsMTx-4. Our results suggest that these molecules compromise the membrane by increasing the Young's modulus value, which indicates that the membrane becomes more rigid; these compounds act through different mechanisms and by a non-specific manner, each one shows a certain preference for specific cell subpopulations, depending on their cell size and their reactivity to isolectin B4. Our results support the idea that the biophysical properties that result from the interactions that arise in the membranes are part of the mechanotransduction process.

This study aimed to evaluate the antinociceptive effect of oleanolic acid using adult zebrafish models of orofacial pain. Acute nociception was induced by formalin, capsaicin, cinnamaldehyde, menthol, acidified saline or glutamate (cutaneous modes) and hypertonic saline (corneal model). In another set of experiments, animals were pre-treated with naloxone, L-NAME, methylene blue, ketamine, camphor, HC-030031, mefenamic acid, ruthenium red or amiloride to investigate the mechanism of antinociception. The involvement of central afferent C-fibers was also investigated. A molecular docking was performed using the TRPV1 channel. Motor activity was evaluated with the open field test. Pre-treatment with oleanolic acid significantly reduced nociceptive behavior associated with acute pain. Antinociception was effectively inhibited by ruthenium red and capsaicin-induced desensitization. Presence of trpv1 was confirmed by RT-PCR in cerebral tissue of zebrafish. In line with in vivo experiments, docking studies indicated that oleanolic acid may interact with TRPV1. Results confirm the potential pharmacological relevance of oleanolic acid as an inhibitor of orofacial nociception mediated by TRPV1., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Non-invasive imaging techniques like X-ray computed tomography have become very popular in zoology, as they allow for simultaneous imaging of the internal and external morphology of organisms. Nevertheless, the effect of different staining approaches required for this method on samples lacking mineralized tissues, such as soft-bodied invertebrates, remains understudied. Herein, we used synchrotron radiation-based X-ray micro-computed tomography to compare the effects of commonly used contrasting approaches on onychophorans - soft-bodied invertebrates important for studying animal evolution. Representatives of Euperipatoides rowelli were stained with osmium tetroxide (vapour or solution), ruthenium red, phosphotungstic acid, or iodine. Unstained specimens were imaged using both standard attenuation-based and differential phase-contrast setups to simulate analyses with museum material. Our comparative qualitative analyses of several tissue types demonstrate that osmium tetroxide provides the best overall tissue contrast in onychophorans, whereas the remaining staining agents rather favour the visualisation of specific tissues and/or structures. Quantitative analyses using signal-to-noise ratio measurements show that the level of image noise may vary according to the staining agent and scanning medium selected. Furthermore, box-and-whisker plots revealed substantial overlap in grey values among structures in all datasets, suggesting that a combination of semiautomatic and manual segmentation of structures is required for comprehensive 3D reconstructions of Onychophora, irrespective of the approach selected. Our results show that X-ray micro-computed tomography is a promising technique for studying onychophorans and, despite the benefits and disadvantages of different staining agents for specific tissues/structures, this method retrieves informative data that may eventually help address evolutionary questions long associated with Onychophora., (© 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.)

Free-living amoebae of the genus Acanthamoeba are protozoa ubiquitously found in nature. Some species of the genus are potentially pathogenic for humans provoking keratitis in healthy individuals, often in contact lens wearers and opportunistic infections such as pneumonitis, fatal granulomatous encephalitis and skin infections, particularly in immunocompromised individuals. The pathogenic mechanisms of these amoebae are poorly understood, however it had been suggested that contact dependent mechanisms are important during invasion, regardless of the epithelia type, since amoebae penetrate epithelia separating tight junction (TJ). This study was undertaken to determine whether Acanthamoeba sp. (T4) damages the barrier function of the TJ in MDCK epithelial monolayers. Actin cytoskeleton staining and electron microscopy analyses were performed; paracellular permeability and TJ sealing were evaluated by apicobasolateral diffusion of ruthenium red and transepithelial resistance (TER) measurements; immunofluorescence and Western blot assays were performed to locate and estimate expression of TJ protein claudins 2 (Cldn2) and 4 (Cldn4). The results show that Acanthamoeba sp. crosses the MDCK monolayer without altering the actin cytoskeleton or the morphology of the cells. When trophozoites or conditioned medium interact with the monolayer, paracellular diffusion of ruthenium red increases. After 6 h, the amoebae, but not their conditioned medium, increase the TER, and Cldn2 is removed from the TJ, and its overall content in the cells diminishes, while Cldn4 is targeted to the TJ without changing its expression level. In conclusion Acanthamoeba (T4) crosses MDCK monolayer without damaging the cells, increasing permeability and TER through Cldn2 degradation, and redirecting Cldn4 to TJ. These results strongly suggest that contact-dependent mechanisms are relevant during amoebae invasion., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Administration of bolus intravenous fluid is associated with respiratory dysfunction and increased mortality, findings with no clear mechanistic explanation. The objective of this study was to examine whether bolus intravenous (i.v.) fluid administration results in acute lung injury in a rat model and further, to examine whether this injury is associated with transient receptor potential vallinoid (TRPV)4 channel function and endothelial inflammatory response. Healthy male Sprague-Dawley rats were administered 60 ml/kg 0.9% saline i.v. over 30 min. Manifestation of acute lung injury was assessed by lung physiology, morphology, and markers of inflammation. The role of TRPV4 channels in fluid-induced lung injury was subsequently examined by the administration of ruthenium red (RR) in this established rat model and again in TRPV4 KO mice. In endothelial cell culture, permeability and P-selectin expression were measured following TRPV4 agonist with and without antagonist; 0.9% saline resulted in an increase in lung water, lavage protein and phospholipase A 2 , and plasma angiopoietin-2, with worsening in arterial blood oxygen (PaO 2 ), lung elastance, surfactant activity, and lung histological injury score. These effects were ameliorated following i.v. fluid in rats receiving RR. TRPV4 KO mice did not develop lung edema. Expression of P-selectin increased in endothelial cells following administration of a TRPV4 agonist, which was ameliorated by simultaneous addition of RR. Bolus i.v. 0.9% saline resulted in permeability pulmonary edema. Data from ruthenium red, TRPV4 KO mice, and endothelial cell culture suggest activation of TRPV4 and release of angiopoietin 2 and P-selectin as the central mechanism.

Ca 2+ and mitochondria are inextricably linked to cardiac function and dysfunction. Ca 2+ is central to cardiac excitation-contraction coupling and stimulates mitochondrial energy production to fuel contraction. Under pathological conditions of dysregulated Ca 2+ cycling, mitochondrial Ca 2+ overload activates cellular death pathways. Thus, in the cardiomyocyte, the mitochondrial Ca 2+ microdomain is where contraction, energy and death collide. A key component of mitochondrial Ca 2+ signalling is the mitochondrial Ca 2+ uniporter complex (uniplex), an inner membrane Ca 2+ transporter and major pathway of mitochondrial Ca 2+ entry. Once known only as the unidentified target for ruthenium red and related compounds, in recent years, the uniplex has evolved into a complex multiprotein assembly. The identification of the molecular constituents of the uniplex has made possible the generation of targeted genetic models to interrogate uniplex function in vivo. This review will summarize our current understanding of the molecular structure of the uniplex, its impact on mitochondrial energetics and cardiac physiology, its contribution to cardiomyocyte death, and its expanding roles in cardiac biology., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Nanosecond electric pulses (nsEP's) are a well-studied phenomena in biophysics that cause substantial alterations to cellular membrane dynamics, internal biochemistry, and cytoskeletal structure, and induce apoptotic and necrotic cell death. While several studies have attempted to measure the effects of multiple nanosecond pulses, the effect of pulse repetition rate (PRR) has received little attention, especially at frequencies greater than 100 Hz. In this study, uptake of Propidium Iodide, FM 1-43, and YO-PRO-1 fluorescent dyes in CHO-K1 cells was monitored across a wide range of PRRs (5 Hz-500 KHz) using a laser-scanning confocal microscope in order to better understand how high frequency repetition rates impact induced biophysical changes. We show that frequency trends depend on the identity of the dye under study, which could implicate transmembrane protein channels in the uptake response due to their chemical selectivity. Finally, YO-PRO-1 fluorescence was monitored in the presence of Gadolinium (Gd(3+)), Ruthenium Red, and in calcium-free solution to elucidate a mechanism for its unique frequency trend., (Published by Elsevier Inc.)

Non-equilibrium helium atmospheric-pressure plasma (He-APP), which allows for a strong non-equilibrium chemical reaction of O2 and N2 in ambient air, uniquely produces multiple extremely reactive products, such as reactive oxygen species (ROS), in plasma-irradiated solution. We herein show that relatively short-lived unclassified reactive species (i.e., deactivated within approximately 10 min) generated by the He-APP irradiation can trigger physiologically relevant Ca(2+) influx through ruthenium red- and SKF 96365-sensitive Ca(2+)-permeable channel(s), possibly transient receptor potential channel family member(s). Our results provide novel insight into understanding of the interactions between cells and plasmas and the mechanism by which cells detect plasma-induced chemically reactive species, in addition to facilitating development of plasma applications in medicine.

In this study, our aims were to investigate transient receptor potential melastatin-8 channels (TRPM8) involvement in rotundifolone induced relaxation in the mesenteric artery and to increase the understanding of the role of these thermosensitive TRP channels in vascular tissue. Thus, message and protein levels of TRPM8 were measured by semi-quantitative PCR and western blotting in superior mesenteric arteries from 12 week-old Spague-Dawley (SD) rats. Isometric tension recordings evaluated the relaxant response in mesenteric rings were also performed. Additionally, the intracellular Ca2+ changes in mesenteric artery myocytes were measured using confocal microscopy. Using PCR and western blotting, both TRPM8 channel mRNA and protein expression was measured in SD rat mesenteric artery. Rotundifolone and menthol induced relaxation in the isolated superior mesenteric artery from SD rats and improved the relaxant response induced by cool temperatures. Also, this monoterpene induced an increase in transient intracellular Ca2+. These responses were significantly attenuated by pretreatment with capsazepine or BCTC, both TRPM8 channels blockers. The response induced by rotundifolone was not significantly attenuated by ruthenium red, a non-selective TRP channels blocker, or following capsaicin-mediated desensitization of TRPV1. Our findings suggest that rotundifolone induces relaxation by activating TRPM8 channels in rat superior mesenteric artery, more selectively than menthol, the classic TRPM8 agonist, and TRPM8 channels participates in vasodilatory pathways in isolated rat mesenteric arteries.

Alzheimer's disease (AD) is a devastating neurodegenerative disease most notably characterized by the misfolding of amyloid-β (Aβ) into fibrils and its accumulation into plaques. In this Article, we utilize the affinity of Aβ fibrils to bind metal cations and subsequently imprint their chirality to bound molecules to develop novel imaging compounds for staining Aβ aggregates. Here, we investigate the cationic dye ruthenium red (ammoniated ruthenium oxychloride) that binds calcium-binding proteins, as a labeling agent for Aβ deposits. Ruthenium red stained amyloid plaques red under light microscopy, and exhibited birefringence under crossed polarizers when bound to Aβ plaques in brain tissue sections from the Tg2576 mouse model of AD. Staining of Aβ plaques was confirmed via staining of the same sections with the fluorescent amyloid binding dye Thioflavin S. In addition, it was confirmed that divalent cations such as calcium displace ruthenium red, consistent with a mechanism of binding by electrostatic interaction. We further characterized the interaction of ruthenium red with synthetic Aβ fibrils using independent biophysical techniques. Ruthenium red exhibited birefringence and induced circular dichroic bands at 540 nm upon binding to Aβ fibrils due to induced chirality. Thus, the chirality and cation binding properties of Aβ aggregates could be capitalized for the development of novel amyloid labeling methods, adding to the arsenal of AD imaging techniques and diagnostic tools.

Hair cells at the base of the cochlea appear to be more susceptible to damage by the aminoglycoside gentamicin than those at the apex. However, the mechanism of base-to-apex gradient ototoxicity by gentamicin remains to be elucidated. We report here that gentamicin caused rodent cochlear hair cell damages in a time- and dose-dependent manner. Hair cells at the basal turn were more vulnerable to gentamicin than those at the apical turn. Gentamicin-conjugated Texas Red (GTTR) uptake was predominant in basal turn hair cells in neonatal rats. Transient receptor potential vanilloid 1 (TRPV1) and 4 (TRPV4) expression was confirmed in the cuticular plate, stereocilia and hair cell body of inner hair cells and outer hair cells. The involvement of TRPV1 and TRPV4 in gentamicin trafficking of hair cells was confirmed by exogenous calcium treatment and TRPV inhibitors, including gadolinium and ruthenium red, which resulted in markedly inhibited GTTR uptake and gentamicin-induced hair cell damage in rodent and zebrafish ototoxic model systems. These results indicate that the cytotoxic vulnerability of cochlear hair cells in the basal turn to gentamicin may depend on effective uptake of the drug, which was, in part, mediated by the TRPV1 and TRPV4 proteins.

EF-hand Ca(2+)-binding motif, a structural component of the EF-hand protein, functions as a calcium sensor and/or buffer in the cytosol of the cell. However, in a few exceptional cases, the EF-hand proteins are secreted from cells and play crucial roles extracellularly. We have identified longistatin, an EF-hand Ca(2+)-binding protein, from the salivary glands of the tick, Haemaphysalis longicornis. Longistatin possesses an N-terminal sequence of unknown structure and two EF-hand motifs in the C-terminus, which conserve a calmodulin-like canonical structure. Longistatin shows distinct changes in its migration during electrophoresis through SDS-PAGE gel containing calcium or ethylenediaminetetraacetic acid (EDTA). Both recombinant and endogenous forms of longistatin can be stained with rutheninum red, demonstrating that longistatin is a Ca(2+)-binding protein.

Intervessel pits act as safety valves that prevent the spread of xylem embolism. Pectin-calcium crosslinks within the pit membrane have been proposed to affect xylem vulnerability to cavitation. However, as the chemical composition of pit membranes is poorly understood, this hypothesis has not been verified. Using electron microscopy, immunolabeling, an antimonate precipitation technique, and ruthenium red staining, we studied the distribution of selected polysaccharides and calcium in the pit membranes of four angiosperm tree species. We tested whether shifts in xylem vulnerability resulting from perfusion of stems with a calcium chelating agent corresponded with the distribution of pectic homogalacturonans (HG) and/or calcium within interconduit pit membranes. No HG were detected in the main part of intervessel pit membranes, but were consistently found in the marginal membrane region known as the annulus. Calcium colocalized with HG in the annulus. In contrast to intervessel pits, the membrane of vessel-ray pits showed a high pectin content. The presence of two distinct chemical domains, the annulus and the actual pit membrane, can have substantial implications for pit membrane functioning. We propose that the annulus could affect the observed shift in xylem vulnerability after calcium removal by allowing increased pit membrane deflection., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

We report a new colorimetric assay to quantify endo-polygalacturonase activity, which hydrolyzes polygalacturonic acid to produce smaller chains of galacturonate. Some of the reported polygalacturonase assays measure the activity by detecting the appearance of reducing ends such as the Somogyi-Nelson method. As a result of being general towards reducing groups, the Somogyi-Nelson method is not appropriate when studying polygalacturonase and polygalacturonase inhibitors in plant crude extracts, which often have a strong reducing power. Ruthenium Red is an inorganic dye that binds polygalacturonic acid and causes its precipitation. In the presence of polygalacturonase, polygalacturonic acid is hydrolyzed bringing about a corresponding gain in soluble Ruthenium Red. The described assay utilizes Ruthenium Red as the detection reagent which has been used previously in plate-based assays but not in liquid medium reactions. The new method measures the disappearance of the substrate polygalacturonic acid and is compared to the Somogyi-Nelson assay. The experimental results using lemon peel, a fern fronds and castor leaf crude extracts demonstrate that the new method provides a way to the quickly screening of polygalacturonase activity and polygalacturonase inhibitors in plant crude extracts containing high amounts of reducing power. On the other hand, the Ruthenium Red assay is not able to determine the activity of an exo-polygalacturonase as initial velocity and thus would allow the differentiation between endo- and exo-polygalacturonase activities., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Insect odorant receptors (ORs) function as odorant-gated ion channels consisting of a conventional, odorant-binding OR and the Orco coreceptor. While Orco can function as a homomeric ion channel, the role(s) of the conventional OR in heteromeric OR complexes has largely focused only on odorant recognition., Results: To investigate other roles of odorant-binding ORs, we have employed patch clamp electrophysiology to investigate the properties of the channel pore of several OR complexes formed by a range of different odorant-specific Anopheles gambiae ORs (AgOrs) each paired with AgOrco. These studies reveal significant differences in cation permeability and ruthenium red susceptibility among different AgOr complexes., Conclusions: With observable differences in channel function, the data support a model in which the odorant-binding OR also affects the channel pore. The variable effect contributed by the conventional OR on the conductive properties of odorant-gated sensory channels adds additional complexity to insect olfactory signaling, with differences in odor coding beginning with ORs on the periphery of the olfactory system.

Transient receptor potential vanilloid 3 (TRPV3), a member of the calcium-permeable thermosensitive TRP (thermoTRP) subfamily of receptors, is an important cutaneous sensor that detects thermal and chemical stimuli. TRPV3 is activated by innocuous warm temperature stimuli (>33 degrees C) and a variety of physiologically active substances. While the corneal epithelium is known to respond to such stimuli, it is unknown whether TRPV3 is involved in this phenomenon. We show here that TRPV3 mRNA and protein are abundantly expressed in the epithelial cells of human and mouse cornea. Carvacrol, an agonist of TRPV3, elevated cytosolic Ca2+ concentration in both primary mouse corneal epithelial cells and cultured human corneal epithelial cells (HCE-T cells). The response to carvacrol was inhibited by ruthenium red, a TRPV channel antagonist. Moreover, repetitive agonist stimulation sensitized the response with gradually increasing amplitude, suggesting that the TRPV3 in the cornea has similar physiological and pharmacological characteristics to that in skin keratinocytes. Finally, a wound healing assay revealed that appropriate calcium ion influx via activated TRPV3 in corneal epithelial cells accelerated their proliferation. Thus, functional TRPV3 is present in corneal epithelial cells and may play a role not only in thermosensation, but also in the regulation of cell proliferation.

We present unexpected and novel results revealing that glutamate-dependent oxidative phosphorylation (OXPHOS) of brain mitochondria is exclusively and efficiently activated by extramitochondrial Ca(2+) in physiological concentration ranges (S(0.5) = 360 nM Ca(2+)). This regulation was not affected by RR, an inhibitor of the mitochondrial Ca(2+) uniporter. Active respiration is regulated by glutamate supply to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier with regulatory Ca(2+)-binding sites in the mitochondrial intermembrane space providing full access to cytosolic Ca(2+). At micromolar concentrations, Ca(2+) can also enter the intramitochondrial matrix and activate specific dehydrogenases. However, the latter mechanism is less efficient than extramitochondrial Ca(2+) regulation of respiration/OXPHOS via aralar. These results imply a new mode of glutamate-dependent OXPHOS regulation as a demand-driven regulation of mitochondrial function. This regulation involves the mitochondrial glutamate/aspartate carrier aralar which controls mitochondrial substrate supply according to the level of extramitochondrial Ca(2+).

Heterologous channel expression can be used to control activity in select neuronal populations, thus expanding the tools available to modern neuroscience. However, the secondary effects of exogenous channel expression are often left unexplored. We expressed two transient receptor potential (TRP) channel family members, TRPV1 and TRPM8, in cultured hippocampal neurons. We compared functional expression levels and secondary effects of channel expression and activation on neuronal survival and signaling. We found that activation of both channels with appropriate agonist caused large depolarizing currents in voltage-clamped hippocampal neurons, exceeding the amplitude responses to a calibrating 30 mM KCl stimulation. Both TRPV1 and TRPM8 currents were reduced but not eliminated by 4 hr incubation in saturating agonist concentration. In the case of TRPV1, but not TRPM8, prolonged agonist exposure caused strong calcium-dependent toxicity. In addition, TRPV1 expression depressed synaptic transmission dramatically without overt signs of toxicity, possibly due to low-level TRPV1 activation in the absence of exogenous agonist application. Despite evidence of expression at presynaptic sites, in addition to somatodendritic sites, TRPM8 expression alone exhibited no effects on synaptic transmission. Therefore, by a number of criteria, TRPM8 proved the superior choice for control over neuronal membrane potential. This study also highlights the need to explore potential secondary effects of long-term expression and activation of heterologously introduced channels.

In order to explore the role of mitochondria in proliferation promotion and/or apoptosis induction of lanthanum, the mutual influences between La3+ and Ca2+ on mitochondrial permeability transition pore (PTP) opening were investigated with isolated mitochondria from rat liver. The experimental results revealed that La3+ influence the state of mitochondria in a concentration-dependent biphasic manner. La3+ in nanomolar concentrations, acting as a Ca2+ analog, entered mitochondrial matrix via the RuR sensitive Ca2+ channel and elevated ROS level, leading to opening of PTP indicated by mitochondrial swelling, reduction of ΔΨm and cytochrome c release. Inhibition of PTP with 10 μM CsA attenuated the effects of La3+. However, micromolar concentrations La3+ acted mainly as a Ca2+ antagonist, inhibiting PTP opening induced by Ca2+. We postulated that this action of La3+ on mitochondria through interaction with Ca2+ might be involved in the proliferation-promoting and apoptosis induction by La3?.

The renal distal tubules and collecting ducts play a key role in the control of electrolyte and fluid homeostasis. The discovery of highly calcium selective channels, Transient Receptor Potential Vanilloid 5 (TRPV5) of the TRP superfamily, has clarified the nature of the calcium entry channels. It has been proposed that this channel mediates the critical Ca(2+) entry step in transcellular Ca(2+) re-absorption in the kidney. The regulation of transmembrane Ca(2+) flux through TRPV5 is of particular importance for whole body calcium homeostasis.In this study, we provide evidence that the TRPV5 channel is present in rat cortical collecting duct (RCCD(2)) cells at mRNA and protein levels. We demonstrate that 17beta-estradiol (E(2)) is involved in the regulation of Ca(2+) influx in these cells via the epithelial Ca(2+) channels TRPV5. By combining whole-cell patch-clamp and Ca(2+)-imaging techniques, we have characterized the electrophysiological properties of the TRPV5 channel and showed that treatment with 20-50nM E(2) rapidly (<5min) induced a transient increase in inward whole-cell currents and intracellular Ca(2+) via TRPV5 channels. This rise was significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV5.These data demonstrate for the first time, a novel rapid modulation of endogenously expressed TRPV5 channels by E(2) in kidney cells. Furthermore, the results suggest calcitropic effects of E(2). The results are discussed in relation to present concepts of non-genomic actions of E(2) in Ca(2+) homeostasis.

On bipolar cells are connected to photoreceptors via a sign-inverting synapse. At this synapse, glutamate binds to a metabotropic receptor which couples to the closure of a cation-selective transduction channel. The molecular identity of both the receptor and the G protein are known, but the identity of the transduction channel has remained elusive. Here, we show that the transduction channel in mouse rod bipolar cells, a subtype of On bipolar cell, is likely to be a member of the TRP family of channels. To evoke a transduction current, the metabotropic receptor antagonist LY341495 was applied to the dendrites of cells that were bathed in a solution containing the mGluR6 agonists L-AP4 or glutamate. The transduction current was suppressed by ruthenium red and the TRPV1 antagonists capsazepine and SB-366791. Furthermore, focal application of the TRPV1 agonists capsaicin and anandamide evoked a transduction-like current. The capsaicin-evoked and endogenous transduction current displayed prominent outward rectification, a property of the TRPV1 channel. To test the possibility that the transduction channel is TRPV1, we measured rod bipolar cell function in the TRPV1(-/-) mouse. The ERG b-wave, a measure of On bipolar cell function, as well as the transduction current and the response to TRPV1 agonists were normal, arguing against a role for TRPV1. However, ERG measurements from mice lacking TRPM1 receptors, another TRP channel implicated in retinal function, revealed the absence of a b-wave. Our results suggest that a TRP-like channel, possibly TRPM1, is essential for synaptic function in On bipolar cells.

Intracellular Zn(2+) toxicity is associated with mitochondrial dysfunction. Zn(2+) depolarizes mitochondria in assays using isolated organelles as well as cultured cells. Some reports suggest that Zn(2+)-induced depolarization results from the opening of the mitochondrial permeability transition pore (mPTP). For a more detailed analysis of this relationship, we compared Zn(2+)-induced depolarization with the effects of Ca(2+) in single isolated rat liver mitochondria monitored with the potentiometric probe rhodamine 123. Consistent with previous work, we found that relatively low levels of Ca(2+) caused rapid, complete and irreversible loss of mitochondrial membrane potential, an effect that was diminished by classic inhibitors of mPT, including high Mg(2+), ADP and cyclosporine A. Zn(2+) also depolarized mitochondria, but only at relatively high concentrations. Furthermore Zn(2+)-induced depolarization was slower, partial and sometimes reversible, and was not affected by inhibitors of mPT. We also compared the effects of Ca(2+) and Zn(2+) in a calcein-retention assay. Consistent with the well-documented ability of Ca(2+) to induce mPT, we found that it caused rapid and substantial loss of matrix calcein. In contrast, calcein remained in Zn(2+)-treated mitochondria. Considered together, our results suggest that Ca(2+) and Zn(2+) depolarize mitochondria by considerably different mechanisms, that opening of the mPTP is not a direct consequence of Zn(2+)-induced depolarization, and that Zn(2+) is not a particularly potent mitochondrial inhibitor.

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress are considered critical components in the development of insulin resistance and Type 2 diabetes. However, understanding the molecular mechanisms underlying these individual disorders and how they are linked has been challenging. Here, we provide evidence that elevated levels of cytosolic free Ca(2+) due to mitochondrial dysfunction and concomitant activation of p38 mitogen activated protein kinase (MAPK) induce ER stress response in human liver sk-HepI cells. Blocking Ca(2+) release from mitochondria or ER using ruthenium red or ryanodine ameliorated the increase in expression of gluconeogenic enzymes due to mitochondrial dysfunction. Disturbance in mitochondrial function results in the activation of p38 MAPK and related transcription factors that are directly responsible for increased phosphoenolpyruvate carboxykinase (PEPCK) expression. In addition, abnormal activation of c-Jun N-terminal kinase (JNK) influences the PEPCK expression by affecting insulin signaling and Forkhead box O (Foxo) 1 activity. Alleviation of ER stress response using a chemical chaperone reduces p38 MAPK activation, as well as PEPCK overexpression, indicating that ER stress response strengthens mitochondrial stress-induced abnormalities. Our results demonstrate that mitochondrial dysfunction is directly linked to the ER stress response, and together, cause aberrant insulin signaling and an abnormal increase of hepatic gluconeogenesis.

We have previously shown that ruthenium red (RuR) binds to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, decreasing channel conductance and protecting against apoptotic cell death. In this report, we define the murine and yeast VDAC1 amino acid residues involved in the interaction with RuR. Binding of RuR to bilayer-reconstituted mVDAC1 and the resulting channel closure was inhibited upon mutation of specific VDAC1 residues. RuR protection against cell death, as induced by overexpression of native or mutated mVDAC1, was also diminished upon mutation of these amino acids. Moreover, RuR-mediated inhibition of cytochrome c release normally induced by staurosporine was not observed in cells expressing mutants VDAC1. We found that four glutamate residues, two each located in the first and third mVDAC1 cytosolic loops, are required for the interaction of VDAC1 with RuR and subsequent protection against cell death. Similar results were obtained with Q72E-yeast VDAC1, except that only three glutamate residues, located in two cytosolic loops were required. As a hexavalent reagent, RuR is expected to bind to more than one negatively charged group. Our results thus clearly indicate that RuR protects against cell death via a direct interaction with VDAC1 to inhibit cytochrome c release and subsequent cell death.

One of the major pathogens of periodontitis, Porphyromonas gingivalis (P. gingivalis), has the ability to aggregate human platelets. To investigate the interaction between P. gingivalis and human platelets in platelet rich plasma (PRP), platelet aggregation was measured by an aggregometer based on laser light scattering (LS) methods, and an ultrastructural study was performed using electron microscopy. A sharp and rapid increase of small-sized platelet aggregates was observed immediately after the addition of P. gingivalis to PRP, followed by the formation of medium- and large-sized aggregates in 2-3 min. In contrast, when Staphylococcus aureus (S. aureus) was used in the control experiment, only a slight increase in small-sized aggregates was detected. By electron microscopy, discoid-shaped platelets were observed prior to adding P. gingivalis. By 5 min after the addition of the bacteria, enormous platelet aggregates were observable. Most of the P. gingivalis were present between the adherent platelets, while some were internalized in platelet engulfment vacuoles. In contrast, when washed platelets were incubated with the bacteria under a non-stirring condition to prevent platelet aggregation, and stained with ruthenium red (RR) as an electron dense tracer of the cell surface including the open canalicular system (OCS), both RR-positive and -negative vacuoles containing P. gingivalis were identified in the activated platelets. Thus, this observation suggests that P. gingivalis residing in the RR-negative vacuoles is incorporated into the platelet cytoplasm by phagocytosis.

This report describes an improvement made to the horizontal cell electrophoresis methodology. It involves using two liquid layers differing in density to produce an interface described as a "density cushion". The electrophoretic system that employed an anti-convective porous matrix to separate red blood cells (RBC) and charged dyes effectively was found to be unsuitable for some other mammalian cells. The "density cushion" method was found to be more versatile and applicable to studies on the separation of a variety of cell types. The experiments described show the differences between the electrophoretic mobilities of a human eosinophilic leukaemia cell line (Eol-1) and RBC, both with and without the modification of the cell surface properties.

Bacterial biofilms, i.e. surface-associated cells covered in hydrated extracellular polymeric substances (EPS), are often studied with high-resolution electron microscopy (EM). However, conventional desiccation and high vacuum EM protocols collapse EPS matrices which, in turn, deform biofilm appearances. Alternatively, wet-mode environmental scanning electron microscopy (ESEM) is performed under a moderate vacuum and without biofilm drying. If completely untreated, however, EPS is not electron dense and thus is not resolved well in ESEM. Therefore, this study was towards adapting several conventional SEM staining protocols for improved resolution of biofilms and EPS using ESEM. Three different biofilm types were used: 1) Pseudomonas aeruginosa unsaturated biofilms cultured on membranes, 2) P. aeruginosa cultured in moist sand, and 3) mixed community biofilms cultured on substrates in an estuary. Working with the first specimen type, a staining protocol using ruthenium red, glutaraldehyde, osmium tetroxide and lysine was optimized for best topographic resolution. A quantitative image analysis tool that maps relief, newly adopted here for studying biofilms, was used to compare micrographs. When the optimized staining and ESEM protocols were applied to moist sand cultures and aquatic biofilms, the smoothening effect that bacterial biofilms have on rough sand, and the roughening that aquatic biofilms impart on initially smooth coupons, were each quantifiable. This study thus provides transferable staining and ESEM imaging protocols suitable for a wide range of biofilms, plus a novel tool for quantifying biofilm image data.

Mast cells' hyperplasia and activation are prominent features in Trichinella spiralis infection. Recently, it was shown that TSL-1 antigens from T. spiralis muscle larvae induce IL-4 and TNF release by unsensitized, normal mast cells (MC) involving an Ig-independent mechanism. In this study, we characterized histamine secretion induced by TSL-1 antigens from normal, unsensitized rat peritoneal MC. Maximum histamine secretion (30+/-5.3% SEM, n=13) was achieved with 30 ng/mL TSL-1 antigens. However, TSL-1 did not induce an increase in beta-hexosaminidase release or NADPH oxidase activity by MC. Interestingly, histamine secretion by TSL-1 was completed at 10s, and was inhibited by both Bordetella pertussis toxin and neuraminidase V, characteristics similar to those involved in substance P-induced histamine secretion. However, in contrast to substance P, TSL-1 induced histamine secretion in the absence of detectable changes in intracellular Ca(2+). We are investigating the molecular pathways involved in MC activation by TSL-1.

Transient receptor potential vanilloid 4, the Ca2+-permeable cation channel has been proposed as an osmosensitive and a mechanosensitive channel. We investigated functional expression of transient receptor potential vanilloid 4 in inner hair cells, outer hair cells, and spiral ganglion neurons of the mouse cochlea. Transient receptor potential vanilloid 4 mRNA and protein were expressed in inner hair cells, outer hair cells, and spiral ganglion neurons on the basis of the findings of reverse transcriptase-polymerase chain reaction, single-cell reverse transcriptase-polymerase chain reaction, and immunohistochemistry, whereas they were negative in transient receptor potential vanilloid 4-/- mice cochleae. Hypotonic stimulation and 4-alpha-phorbol 12,13-didecanoate, a transient receptor potential vanilloid 4 synthetic activator, increased the intracellular Ca2+ concentrations in wild-type outer hair cells, whereas in transient receptor potential vanilloid 4-/- mice, outer hair cells failed to exhibit a Ca2+ response to both stimulations. In conclusion, transient receptor potential vanilloid 4 may function as an osmosensory and a mechanosensory receptor in the cochlea.

In colorectal cancer tight junction molecular and morphological alterations are poorly understood. In this study, adenocarcinoma tissues and their paired normal mucosa (n = 12) were analyzed for tight junction alterations molecular. The expression of claudin-1, -3 and -4 was upregulated 5.7-, 1.5- and 2.4-fold, respectively, in colorectal tumor tissues in comparison to the normal ones. Although tight junction remains in the cancerous epithelium, its barrier function was altered. Despite claudins overexpression, paracellular permeability to ruthenium red was increased and a significant disorganization of tight junction strands was observed in freeze fracture replicas. Whereas the functional significance of claudin overexpression in colorectal cancer is unclear, these proteins can become potential markers and targets in colorectal cancer.

The in vitro effect of estradiol on flux of Ca(2+) in the synaptosomal mitochondria from nucleus caudatus and hippocampus of chronically ovariectomized female rats was examined. No effect of estradiol on Ca(2+) influx through ruthenium red-sensitive channels was found. Estradiol, at a concentration of 0.05-5 nmol/L for nucleus caudatus and 0.5-5 nmol/L for the hippocampus, decreased Na-dependent Ca(2+) efflux about 25%.

Childhood absence epilepsy is an idiopathic, generalized, nonconvulsive epilepsy with a multifactorial genetic etiology. The KCNK9 gene coding for the TASK3 (Twik-like acid-sensitive K

For induction of long-term depression (LTD), mechanisms dependent on N-methyl-D-aspartate receptors (NMDARs) and on intracellular calcium stores have been separately known. How these two mechanisms coexist at the same synapses is not clear. Here, induction of LTD at hippocampal Schaffer collateral-to-CA1 pyramidal cell synapses was shown to depend on NMDARs throughout the theoretically predicted activation range for LTD induction. With stimulation at 1 Hz, the largest LTD was induced in a store-independent manner. With stimulation at 0.5 and 2.0 Hz the induced LTD was much smaller, and dependence on calcium stores appeared. Under caffeine application, an enlarged LTD was induced with 0.5 Hz stimulation. Postsynaptic blockade of ryanodine receptors prevented this caffeine-induced enhancement of LTD. It is therefore suggested that calcium release from calcium stores facilitated by caffeine contributed to the LTD enhancement, and that the caffeine effect was exerted on the postsynaptic side. Induction of this enhanced LTD was resistant to NMDAR blockade. We thus propose that the store-dependent mechanism for LTD induction is dormant at the centre of the theoretically predicted activation range for LTD induction, but operates at the fringes of this activation range, with its contribution more emphasized when ample calcium release occurs.

It has been suggested that one key feature of mitochondrial permeability transition (PT) regulation is its control by the proton electrochemical gradient and that depolarization favors pore opening, swelling, and reactive oxygen species (ROS) production. Moreover, ROS have been suggested to facilitate the process of mitochondrial PT pore opening. The aim of this study was to show that collapsing the mitochondrial membrane potential with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), at concentrations of up to 10 microM, does not induce mitochondrial swelling and, in fact, stabilizes mitochondria exposed to oxidant, protecting them from tert-butyl hydroperoxide (TBH)-induced high-amplitude swelling. FCCP decreased polyethylene glycol-induced mitochondrial contraction following exposure to TBH, indicating closing of the PT mega-channel. In the presence of the calcium uniporter inhibitor ruthenium red, FCCP induced PT due to suppression of calcium efflux. Under PT-favorable conditions, ROS production was evaluated in mitochondria following treatments with TBH, inorganic phosphate, or FCCP (with or without ruthenium red). FCCP alone and in combination with ruthenium red attenuated mitochondria-derived ROS production. FCCP also decreased the augmented ROS production induced by inorganic phosphate. It is concluded that mitochondrial depolarization protects and prevents high-amplitude swelling and PT-derived ROS production.

We have purified a prominent 110-kDa protein (p110) from 1.6 M NaCl extracts of rat liver nuclei that appears to bind Ca2+. p110 was originally identified by prominent blue staining with 'Stains-All' in sodium dodecyl sulfate-polyacrylamide gels and was observed to specifically bind ruthenium red and 45Ca2+ in nitrocellulose blot overlays. In spin-dialysis studies, purified p110 saturably bound approximately 75 nmol Ca2+/mg protein at a concentration of 1 mM total Ca2+ with half-maximal binding observed at 105 microM Ca2+. With purification, p110 became increasingly susceptible to proteolytic (likely autolytic) fragmentation, although most intermediary peptides between 40 and 90 kDa retained "Stains-All", ruthenium red, and 45Ca2+ binding. N-terminal sequencing of intact p110 and a 70-kDa autolytic peptide fragment revealed a strong homology to nucleolin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/IEF revealed autolysis produced increasingly acidic peptide fragments ranging in apparent pI's from 5.5 for intact p110 to 3.5 for a 40 kDa peptide fragment. Intact p110 and several peptide fragments were immunostained with a highly specific anti-nucleolin antibody, R2D2, thus confirming the identity of this protein with nucleolin. These annexin-like Ca2+-binding characteristics of nucleolin are likely contributed by its highly acidic argyrophilic N-terminus with autolysis apparently resulting in largely selective removal of its basic C-terminal domain. Although the Ca2+-dependent functions of nucleolin are unknown, we discuss the possibility that like the structurally analogous HMG-1, its Ca2+-dependent actions may regulate chromatin structure, possibly during apoptosis., (Copyright 2002 Wiley-Liss, Inc.)

Desmocytes scattered over the surface of the corallum of the scleractinian Mycetophyllia reesi attach the calicoblastic tissue to the skeleton. The structure of the desmocyte is generally consistent with that of other scleractinians except for their more rectangular profiles and greater size. However, the extent of attachment is distinctive, and the mode of attachment to mineral is described for the first time. The skeleton contains dual rows of interconnected pits between the septa, within and among which desmocytes form virtually uninterrupted sheets. Desmocytes terminate with hemidesmosomes that attach the epithelium to a fibrillar basal lamina. Fibrils extend from the basal lamina into the skeletal matrix anchoring tissue firmly to the skeleton. In addition, the basal lamina itself appears to be incorporated within the organic matrix during growth, partitioning the skeleton into compartments. Because the skeletal organic matrix is physicochemically labile during demineralization, these intraskeletal details cannot be observed unless polycationic dyes such as Ruthenium red or other glycan precipitating agents are employed in the fixative sequence.

Previous studies proposed that N-ethylmaleimide (NEM) alkylates 3 classes of thiols on skeletal muscle ryanodine receptors (RyRs) producing 3 phases of channel modification, as function of time and concentration. NEM (5 mm) decreased, increased, and then decreased the open probability (P(o)) of the channel by thiol alkylation, a reaction not reversed by reducing agents. We now show that low NEM concentrations (20-200 microm) elicit Ca(2+) release from sarcoplasmic reticulum (SR) vesicles, but contrary to expectations, the effect was fully reversed by reducing agents or by washing SR vesicles. In bilayers, NEM (0.2 mm) increased P(o) of RyRs within seconds when added to the cis (not trans) side, and dithiothreitol (DTT; 1 mm) decreased P(o) in seconds. High (5 mm) NEM concentrations elicited SR Ca(2+) release that was not reversed by DTT, as expected for an alkylation reaction. A non-sulfhydryl reagent structurally related to NEM, N-ethylsuccinimide (0.1-0.5 mm), also elicited SR Ca(2+) release that was not reversed by DTT (1 mm). Other alkylating agents elicited SR Ca(2+) release, which was fully (N-methylmaleimide) or partially (iodoacetic acid) reversed by DTT and inhibited by ruthenium red. Nitric oxide (NO) donors at concentrations that did not activate RyRs inhibited NEM-induced Ca(2+) release, most likely by an interaction of NO with NEM rather than an inactivation of RyRs by NO. Thus, at low concentrations, NEM does not act as a selective thiol reagent and activates RyRs without alkylating critical thiols indicating that the multiple phases of ryanodine binding are unrelated to RyR activity or to NEM alkylation of RyRs.

The imidazoline-1 receptor (IR1) is considered a novel target for drug discovery. Toward cloning an IR1, a truncated cDNA clone was isolated from a human hippocampal lambda gt11 cDNA expression library by relying on the selectivity of two antisera directed against candidate IR proteins. Amplification reactions were performed to extend the 5' and 3' ends of this cDNA, followed by end-to-end PCR and conventional cloning. The resultant 5131-basepair molecule, designated imidazoline receptor-antisera-selected (IRAS) cDNA, was shown to encode a 1504-amino acid protein (IRAS-1). No relation exists between the amino acid sequence of IRAS-1 and proteins known to bind imidazolines (e.g., it is not an alpha2-adrenoceptor or monoamine oxidase subtype). However, certain sequences within IRAS-1 are consistent with signaling motifs found in cytokine receptors, as previously suggested for an IR1. An acidic region in IRAS-1 having an amino acid sequence nearly identical to that of ryanodine receptors led to the demonstration that ruthenium red, a dye that binds the acidic region in ryanodine receptors, also stained IRAS-1 as a 167-kD band on SDS gels and inhibited radioligand binding of native I1 sites in untransfected PC-12 cells (a source of authentic I1 binding sites). Two epitope-selective antisera were also generated against IRAS-1, and both reacted with the same 167-kD band on Western blots. In a host-cell-specific manner, transfection of IRAS cDNA into Chinese hamster ovary cells led to high-affinity I1 binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS-1 is the first protein discovered with characteristics of an IR1.

The effects of gonadal steroid hormone, 17beta-estradiol (E2), in vitro on rat brain mitochondria Ca2+ movement were investigated. Intrasynaptosomal mitochondria Ca2+ uptake via an energy-driven Ca2+ uniporter have Km = 112.73 +/- 7.3 micromol x l(-1) and Vmax = 21.97 +/- 1.7 nmol 45Ca2+ mg(-1). Ca2+ release trough a Na+/Ca2+ antiporter was measured with a Km for Na+ of 43.7 +/- 2.6 mmol x l(-1), and Vmax of 1.5 +/- 0.3 nmol 45Ca2+ mg(-1). Addition of estradiol in preincubation mixture did not affect the uptake of Ca2+ mediated by the ruthenium red-sensitive uniporter, while it produced biphasic effect on Na-dependent Ca2+ efflux. Estradiol at concentrations up to 1 nmol x l(-1) decreased the efflux significantly (63% inhibition with respect to the control), and at concentrations above 10 nmol x l(-1) increased it exponentially. The maximum inhibiting concentration of estradiol (0.5 nmol x l(-1)) increased the affinity of the uniporter (Km reduced by about 30%), without affecting significantly the capacity (Vmax) for Na+. The results presented suggest that estradiol inhibits Na-dependent Ca2+ efflux from mitochondria and acts on mitochondrial retention of Ca2+, which may modulate mitochondrial and consequently synaptosomal content of Ca2+, and in this way exerts its role in the homeostasis of calcium in nerve terminals.

Effects of ruthenium red (RR) on the slow Ca(2+)-activated Ca(2+)-permeable vacuolar channel have been studied by patch-clamp technique. Applied to the cytosolic side of isolated membrane patches, RR at concentrations of 0.1-5 microM produced two distinct effects on single channel kinetics, long lasting closures and a flickering block of the open state. The first effect was largely irreversible, whereas the second one could be washed out. The extent of flickering block steeply increased (zdelta = approximately 1.35) with the increase of cytosol-positive voltage, dragging RR into the channel pore. At least two RR ions are involved in the block according to Hill coefficient n = approximately 1.30 for the dose response curves. The on-rate rate of the drug binding linearly depended on the RR concentration, implying that one RR ion already plugged the pore. The blocked state was further stabilized by binding of the second RR. This stabilization was in excess of that predicted by independent binding as the dependence of unblocking rate on RR concentration revealed. A cooperative model was therefore employed to describe the kinetic behavior of RR binding. At zero voltage the half-blocking RR concentration of 36 microM and the bimolecular on-rate constant of 1.8 x 10(8) M(-1) s(-1) were estimated.

Extracellular material molecules play a key role in the regulation of morphogenesis and differentiation of a large number of organs including the central nervous system. However, the role of the neural basement membrane in the growth of different parts of the neural tube has yet to been delineated. Here, the structural and compositional modifications of the basement membrane (BM) of rhombencephalic tectoria lamina anlage (RTLA) have been examined during the process of RTLA epithelial attenuation. Between stages 10 to 11-the presumptive RTLA epithelium showed a structure, thickness and cell-proliferating capacity similar to those observed in other zones of the rhombencephalic walls. Moreover, the rhombencephalic vesicles were surrounded by a continuous BM that was heterogeneous both ultrastructurally and with regard to ruthenium red, laminin and tenascin distribution. After stage 11, the RTLA epithelium underwent a rapid process of attenuation and change to a stratified flattened epithelium. During this remodelling process, apoptosis and inhibition of both PCNA expression and 3H-thymidine uptake occurred in the RTLA epithelium. The BM of the RTLA underwent a process of degration at the beginning of the remodelling, and apoptosis and cell proliferation inhibition of RTLA epithelium were also observed. The loss of the biochemical signals encoded within the BM could lead to cell shape changes, cell proliferation inhibition and to the anoikis type of cell death. Our findings support the idea that the BM surrounding the neural tube plays a key role in controlling both the structure and growth of the CNS during the early developmental stages.

Ruthenium red, a promising cationic reagent for electron microscopy (EM), has long been an important tool in histology. The reagent was initially used by botanists as a semispecific stain for pectic substances, but it has gradually been embraced by investigators in microbiology and the animal sciences as a stain for anionic glycosylated polymeric substances. Luft developed a reliable method and demonstrated that ruthenium red was a useful reagent for visualizing ultrastructural detail. Many investigators, using modifications of Luft's approach, have identified numerous applications for this important reagent. Ruthenium red has been used to show the ultrastructural detail of bacterial glycocalyces. Strong, sharp and consistent observations of this ultrastructural component of the bacterial cell have given a better understanding its fibrous anionic matrix. Any variations in staining owing to artifactual alteration of the fine delicate ultrastructural features have been overcome by incorporation of diamine lysine into ruthenium red methods, thus providing flexible processing times under less than ideal laboratory sampling conditions. Ruthenium red has broad utility in the biological sciences, and in combination with lysine, it is an excellent EM stain for enhanced visualization of bacterial glycocalyx from culture or from clinical specimens.

Polysaccharides produced in technical scale from red algae serve in a variety of industrial applications. Ruthenium red (RR) is a cytochemical stain which has been used to visualize these mostly acidic polysaccharides in light microscopy. The binding of RR to algal polysaccharides is highly dependent on the ionic strength of the surrounding medium. The dye is firmly bound at low ionic strength, but is released at increasing salt concentrations. These properties were exploited to develop a new method to quantify the amount of cell wall material synthesized by algal cells under different physiological conditions. Soluble or extracted polysaccharides are quantified by a dot-blot procedure with subsequent image analysis, while cell-bound polysaccharides are determined in situ by a dye-binding assay followed by photometry. In comparison to the current methods of quantification, the new assay is quick, reliable, and avoids extensive use of hazardous chemicals., (Copyright 1999 Academic Press.)

Tumor necrosis factor-alpha (TNF) induces apoptosis in confluent LLC-PK1 epithelial cells, but also activates NF-kappaB, a negative regulator of apoptosis. The presence of increased TNF-induced apoptosis causes a transient increase in epithelial permeability, but the epithelial barrier function recovers, as assessed by measuring the transepithelial electrical resistance, the paracellular flux of mannitol and by the electron microscopic evaluation of the penetration of the electron-dense dye ruthenium red across the tight junctions. The integrity of the epithelial cell layer is maintained by rearrangement of non-apoptotic cells in the monolayer and by the phagocytosis of apoptotic fragments. To study the role of NF-kappaB in an epithelium exposed to TNF, NF-kappaB was inhibited in LLC-PK1 epithelial cells with either the dietary compound, curcumin, or by transfection with a dominant negative mutant inhibitor I kappaB alpha. Replacement of serine 32 and 36 by alanine has been shown to prevent its phosphorylation and degradation, blocking NF-kappaB activation. Inhibition of NF-kappaB altered the morphology of TNF-induced apoptotic cells, which showed lack of fragmentation and membrane blebbings, and absence of phagocytosis by neighboring cells. TNF treatment of NF-kappaB-inhibited cells also caused altered distribution of the tight junction-associated protein ZO-1, increased epithelial leakiness, and impaired the recovery of the epithelial barrier function, which normally occurs 6 hours after TNF treatment of LLC-PK1 cells. These data demonstrate that NF-kappaB activation is required for the maintenance of the barrier function of an epithelium undergoing TNF-induced apoptosis.

Increased binding of ruthenium red to pectin as the number of methyl esters attached to the pectin decreases was used as the basis for a gel diffusion assay for pectin methylesterase (PME, EC 3.1.1.11) activity. The stained zone diameters resulting from the hydrolysis of 0.1% (w/v) 90% esterified pectin in an agarose gel by diffused, commercial PME were log-linear over 4 orders of magnitude, with a minimum detection limit of 3.6 pkatals. Pectin deesterification as the cause for a stained zone after PME incubation was confirmed when only 1 N NaOH, which will chemically deesterify the pectin, and not methanol or acid, the two products formed when PME acts on a methyl ester, resulted in the characteristic stained zone. The stained zone diameters decreased with increasing percentage of substrate esterification, were independent of pH, and were insensitive to simultaneous incubation with two forms of pectin lyase (EC 4.2.2.10), polygalacturonase (EC 3.2.1.15), or all combinations. PME extracted from tomato seeds, cotton fibers, and melon fruit showed pH optima of 6, 6, and 8, respectively. Using individual tomato seed parts, the assay was adapted to quantify diffusate activity and to localize activity in tissue prints. The sensitivity, specificity, and simplicity of this PME assay are superior to all others.

The sonic muscle of the oyster toadfish, Opsanus tau, can produce unfused contractions at 300 Hz. Electron microscopy shows a great abundance of the Sarcoplasmic reticulum (SR) in this muscle, but no functional characterization of the capabilities of the SR has been reported. We measured the oxalate-supported Ca2+ uptake rate and capacities of homogenates of toadfish sonic muscle and rat extensor digitorum longus (EDL) muscle, and estimated the number of pump units by titration with thapsigargin, a high-affinity, specific inhibitor of the SR Ca-ATPase. The Ca2+ uptake rate averaged 70.9 +/- 9.5 mumol min -1 per g tissue for the toad fish sonic muscle, and 73.5 +/- 3.7 mumol min -1 g-1 for rat EDL. The capacity for Ca2+ -oxalate uptake was 161 +/- 20 mumol g -1 and 33 +/- 2 mumol g -1 for toadfish sonic muscle and rat EDL, respectively. Thus, the rates of Ca2+ uptake were similar in the two muscles, but the toadfish sonic muscle had about five times the capacity of the rat EDL. The number of pumps as estimated by thapsigargin titration was 68 +/- 4 nmol of Ca-ATPase per g tissue in the toadfish, and 42 +/- 5 nmol Ca-ATPase per g tissue in the rat EDL. The turnover number, defined as the Ca2+ uptake divided by the number of pumps, was 1065 +/- 150 min -1 for toadfish and 1786 +/- 230 min -1 for rat EDL (p < 0.05) at 37 degrees C. The Ca2+ uptake rate of toadfish sonic muscle at 22 degree C, a typical temperature for calling toadfish, averaged 42 +/- 1% of its rate at 37 degree C. At these operating temperatures, the toadfish SR is likely to be slower than the rat fast-twitch SR, yet the toadfish sonic muscle supports more rapid contractions. One explanation for this is that the voluminous SR provides activator Ca2+ for contraction, but the abundant parvalbumin plays a major role in relaxation.