Effects of Salicylic Acid and Its Salts on Electrical Activity of Neurons of <Emphasis Type="Italic">Helix albescens</Emphasis>

We studied changes in the parameters of electrical activity of identified neurons of the parietal ganglion, PPa1 and PPa2, and of non-identified cells of the visceral ganglion (VG) of the snail Helix albescens; these changes were caused by application of salicylic acid and its salts (cobalt and zinc salicylates, CS and ZS, respectively). The above substances began to modify significantly the functional state of the neurons under study when applied in concentrations of 10⁻⁴ to 10⁻³ M. Salicylic acid suppressed the activity of all studied neurons. Application of salicylic acid in the concentration of 10⁻³ M led to a decrease in the impulsation frequency of VG neurons by factors of 1.2 to 1.5 and to an increase in the duration of AP (on average, by 2.8 ± ± 0.6 msec). In PPa1 and PPa2 cells, we observed increases in both the AP duration (by 2.4 ± 0.8 and 3.6 ± ± 1.3 msec, respectively) and that of postactivation hyperpolarization (by 29.8 ± 11 0 and 39.6 ± 9.4 msec). In the concentration of 10⁻² M, salicylic acid completely but relatively reversibly suppressed the impulse activity of all the neurons under study, causing deep hyperpolarization of their membranes. Salts of this acid, CS and ZS, demonstrated significant modulatory effects on the activity of the studied neurons; these substances initiated or enhanced the grouping of APs in bursts and also increased the AP duration. Application of 10⁻³ M CS resulted in an increase in the AP duration by, on average, 2.75 ± 0.4 msec (only in the PPa2 neuron), whereas 10⁻³ M ZS exerted analogous effects on both above neurons (in PPa1, by 2.7 ± 0.4, while in PPa2, by 3.1 ± 0.6 msec). In the case where the tested salicylates were applied in the concentration of 10⁻² M, the AP duration increased in all the cells under study (on average, by 11.8 ± 2.46 msec in VG neurons, and by 7.0 ± ± 0.4 and 7.8 ± 1.2 msec in PPa1 and PPa2 cells, respectively). With application of CS, analogous values determined by application of ZS were 14.6 ± 4.6, 6.8 ± 0.54, and 9.0 ± 0.89 msec. We assume that the modulatory effects of salicylates are mediated by their influence on the intracellular system of cyclic nucleotides. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 142–150, March–April, 2005.     

Mechanisms of Generation of Pacemaker Activity by Identified Helix Neurons

We studied the mechanisms of generation of pacemaker activity in identified neurons of Helix pomatia. For this purpose, we isolated the PPa2 and PPa7 neurons generating spontaneous rhythmic monomodal activity and PPa1 neuron with bursting activity. It was demonstrated that isolated PPa2 and PPa7 cells produce endogenous rhythmic activity that was not considerably modified by external application of 1 mM CdCl₂. Sometimes, only low-amplitude dendritic action potentials (AP) were observed instead of generation of full-amplitude somatic AP. In contrast, isolation of the PPa1 neuron eliminated its bursting activity, but subsequent application of oxytocin on this neuron recovered such activity. This finding shows that the bursting activity of the PPa1 neuron is of an exogenous nature. Application of 1 mM CdCl₂ suppressed this bursting activity, but when Cd²⁺ was applied against the background of superfusion of the neuron with Ringer solution containing a bursting activity-initiating neuropeptide obtained from the molluscan CNS, this blocker was incapable of suppressing the bursting activity. A blocker of the hyperpolarization-activated current (I _h , H current), Cs⁺ (10 mM) exerted no noticeable effect on the activity of the studied neurons. Our findings allow us to conclude that the pacemaker activity is initiated within the dendritic tree of a cell and is then electrotonically spread to the soma, where full-amplitude AP are generated. It seems probable that Ca²⁺ ions and H current are not directly involved in generation of the pacemaker activity in the studied snail neurons.     

Inter-and intraganglionar synaptic connections formed by neurons of the <Emphasis Type="Italic">Helix</Emphasis> visceral ganglion

In experiments on the subpharyngeal complex of the Helix ganglia, we found an excitatory monosynaptic input to the pacemaker PPa2 neuron from an unidentified cell of the visceral ganglion and a polysynaptic inhibitory influence of another unidentified neuron of this ganglion on the PPa1 cell. In addition, we revealed three pairs of neurons synaptically connected with each other (excitatory connections) in the visceral ganglion. In the case where we used high-frequency (11 sec⁻¹) stimulation of presynaptic elements, synaptic transmission to the PPa2 neuron demonstrated the greatest efficiency and stability. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 32–36, January–February, 2007.     

The ABC-transporter AtmA is involved in nickel and cobalt resistance of <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> strain CH34

Cupriavidus metallidurans CH34 genome contains an ortholog of Atm1p named AtmA (Rmet_0391, YP_582546). In Saccharomyces cerevisiae, the ABC-type transport system Atm1p is involved in export of iron–sulfur clusters from mitochondria into the cytoplasm for assembly of cytoplasmic iron–sulfur containing proteins. An ∆atmA mutant of C. metallidurans was sensitive to nickel and cobalt but not iron cations. AtmA increased also resistance to these cations in Escherichia coli strains that carry deletions of the genes for other nickel and cobalt transport systems. In C. metallidurans, atmA expression was not significantly induced by nickel and cobalt, but repressed by zinc. AtmA was purified as a 70 kDa protein after expression in E. coli. ATPase activity of AtmA was stimulated by nickel and cobalt.     

Schema design and implementation of the grasp-related mirror neuron system

 Mirror neurons within a monkey's premotor area F5 fire not only when the monkey performs a certain class of actions but also when the monkey observes another monkey (or the experimenter) perform a similar action. It has thus been argued that these neurons are crucial for understanding of actions by others. We offer the hand-state hypothesis as a new explanation of the evolution of this capability: the basic functionality of the F5 mirror system is to elaborate the appropriate feedback – what we call the hand state– for opposition-space based control of manual grasping of an object. Given this functionality, the social role of the F5 mirror system in understanding the actions of others may be seen as an exaptation gained by generalizing from one's own hand to an other's hand. In other words, mirror neurons first evolved to augment the “canonical” F5 neurons (active during self-movement based on observation of an object) by providing visual feedback on “hand state,” relating the shape of the hand to the shape of the object. We then introduce the MNS1 (mirror neuron system 1) model of F5 and related brain regions. The existing Fagg–Arbib–Rizzolatti–Sakata model represents circuitry for visually guided grasping of objects, linking the anterior intraparietal area (AIP) with F5 canonical neurons. The MNS1 model extends the AIP visual pathway by also modeling pathways, directed toward F5 mirror neurons, which match arm–hand trajectories to the affordances and location of a potential target object. We present the basic schemas for the MNS1 model, then aggregate them into three “grand schemas”– visual analysis of hand state, reach and grasp, and the core mirror circuit – for each of which we present a useful implementation (a non-neural visual processing system, a multijoint 3-D kinematics simulator, and a learning neural network, respectively). With this implementation we show how the mirror system may learnto recognize actions already in the repertoire of the F5 canonical neurons. We show that the connectivity pattern of mirror neuron circuitry can be established through training, and that the resultant network can exhibit a range of novel, physiologically interesting behaviors during the process of action recognition. We train the system on the basis of final grasp but then observe the whole time course of mirror neuron activity, yielding predictions for neurophysiological experiments under conditions of spatial perturbation, altered kinematics, and ambiguous grasp execution which highlight the importance of the timingof mirror neuron activity. Received: 6 August 2001 / Accepted in revised form: 5 February 2002     

Metallopeptide-promoted inactivation of angiotensin-converting enzyme and endothelin-converting enzyme 1: toward dual-action therapeutics

A series of metallopeptides based on the amino terminal copper/nickel (ATCUN) binding motif have been evaluated as classical inhibitors and catalytic inactivators of both rabbit and human angiotensin-converting enzyme (hACE), and human endothelin-converting enzyme 1 (hECE-1). The cobalt complex [KGHK–Co(NH₃)₂]²⁺, where KGHK is lysylglycylhistidyllysine, displayed similar K _I and IC₅₀ values to those found for [KGHK–Cu]⁺, in spite of the enhanced charge, and so either the influence of charge is offset by the steric influence of the axially coordinated ammine ligands, or binding is dominated by contributions from the amino acid side chains, especially the C-terminal lysine that mimics the binding pattern observed for lisinopril. Moreover, the inhibition observed for [KGHK–Co(NH₃)₂]²⁺ contrasts with the activation of hACE by Co²⁺(aq), reflecting the stimulation of enzyme activity following replacement of the catalytic zinc cofactor by cobalt ion at each of the two active sites. Quantitative analysis of the dose-dependent stimulation of activity by Co²⁺(aq) yielded apparent affinities of 1.3 ± 0.2 and 56 ± 8 μM for the two sites in the presence of saturating Zn²⁺ (10 μM). Catalytic inactivation of hACE by [KGHK–Cu] ⁺ at subsaturating concentrations had previously been characterized, with k _obs = 2.9 ± 0.5 × 10⁻² min⁻¹. Under similar conditions, the same complex is found to catalytically inactivate hECE-1, with k _obs = 2.12 ± 0.16 × 10⁻² min⁻¹, demonstrating the potential for dual-action activity against two key drug targets in cardiovascular disease. Irreversible inactivation of a drug target represents a novel mechanism of drug action that complements existing classical inhibitor strategies that underlie current drug discovery efforts.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Zinc treatment affects superoxide dismutase activity in growth retardation

Children with growth dysfunction present complex diagnostic challenges. The purpose of this study was to determine the effects of oral zinc treatment on red cell copper/zinc superoxide dismutase (Cu/Zn-SOD) activity and copper and zinc concentrations in children with “growth retardation.” Twenty-nine patients, average age of 11 yr, whose percentile was under 3% of the National Center of Health Statistics parameters were selected. For the control group, 10 children whose average age was 10 yr were included. Red cell Cu/Zn-SOD activity was determined by spectrophotometer. Red cell copper and zinc concentrations were measured by atomic absorption spectrophotometer. Red cell Cu/Zn-SOD activity was higher than the control group before zinc treatment (p<0.001). There was a decrease in the Cu/Zn-SOD activity after zinc treatment, but the mean value of the Cu/Zn-SOD activity of patients was still higher than the control values (p<0.001). After zinc treatment, there was an increase in red cell zinc concentration (p<0.01) and a decrease in copper concentration (p<0.001), which were statistically significant. The results of this study suggested that Cu/Zn-SOD activity was increased significantly during growth retardation and zinc treatment appeared to ameliorate the enzyme activity. There were also insignificant alterations in red cell copper and zinc concentrations. Presented at the 23rd Congress of Endocrinology and Metabolic Diseases of Turkey Joint Meeting with the European Federation of Endocrine Societies, Bilkent Hotel, Ankara, 7–9 September, 2000.     

The most likely voltage path and large deviations approximations for integrate-and-fire neurons

We develop theory and numerical methods for computing the most likely subthreshold voltage path of a noisy integrate-and-fire (IF) neuron, given observations of the neuron’s superthreshold spiking activity. This optimal voltage path satisfies a second-order ordinary differential (Euler-Lagrange) equation which may be solved analytically in a number of special cases, and which may be solved numerically in general via a simple “shooting” algorithm. Our results are applicable for both linear and nonlinear subthreshold dynamics, and in certain cases may be extended to correlated subthreshold noise sources. We also show how this optimal voltage may be used to obtain approximations to (1) the likelihood that an IF cell with a given set of parameters was responsible for the observed spike train; and (2) the instantaneous firing rate and interspike interval distribution of a given noisy IF cell. The latter probability approximations are based on the classical Freidlin-Wentzell theory of large deviations principles for stochastic differential equations. We close by comparing this most likely voltage path to the true observed subthreshold voltage trace in a case when intracellular voltage recordings are available in vitro. Action Editor: Peter Latham     

Hint of polar distribution in calcium channels under PIXE analysis

Pollen tubes ofLilium longiflorum were treated for 10–30 min with 10⁻⁵ M CoCl₂, which binds to calcium channels in the plasma membrane and blocks them. Cobalt analyses were performed with the Heidelberg proton microprobe, using 3 MeV protons, a beam current of about 200 pA, and a spot size of 3×5 μm². X-ray spectra revealed that coblat has much higher concentrations in the cell than in the surrounding dried medium. The line scans, taken along the longitudinal cell axis in 1-μm steps, showed a cobalt gradient similar to the calcium gradient of the same cell. Based on our findings, we can conclude that neither do the cobalt signals come from the cell wall nor is the cobalt exclusively bound to the intracellular calcium-binding sites. There-fore, the present results suggest a polar distribution in calcium channels in the plasma membrane of pollen tubes.     

Possible mechanisms underlying effects of N-uronoyl derivatives of amino acids on electrical activity in snail neurons

Using intracellular recording, we studied the effects of N-uronoyl derivatives of an amino acid and peptides (1,2:3,4-di-O-isopropylidene-αa-D-galactopyranuronoyl)-β-alanine (DAGU-Ala), DAGU-glycyl-glycine (DAGU-Gly-Gly), DAGU-glycyl-D,L-glutamic acid (DAGU-Gly-Glu), as well as of 1,2:3,4-di-O-isopropylidene-αa-D-galactopyranosyluronic acid (DAGU itself), β-alanine (β-Ala), D,L-glutamic acid (D,L-Glu), and glycyl-glycine (Gly-Gly), which were added to the extracellular milieu, on the electrical activity of PPa1 and PPa2 neurons and unidentified neurons of Helix albescens Rossm. DAGU-Gly-Gly applied in concentrations of 10⁻⁴ to 10⁻² M hyperpolarized the membrane in a dose-dependent manner and decreased insignificantly the amplitude of action potentials (APs). Applications of DAGU-Ala, β-Ala, DAGU-Gly-Glu, D,L-Glu, and Gly-Gly in the same doses resulted in a shift of the membrane potential toward depolarization and in a drop in the amplitude of APs. Measurements of the first AP derivatives showed that all the above-mentioned substances suppressed in a concentration-dependent manner both inward and outward transmembrane ion currents. In this case, DAGU suppressed both inward and outward currents, while DAGU-Ala, β-Ala, DAGU-Glu, D,L-Glu, and Gly-Gly inhibited predominantly the outward potassium ion current; DAGU-Gly-Gly inhibited inward sodium and potassium ion currents. Results of a comparative analysis of the neurotropic action of the tested amino acids and their N-uronoyl derivatives showed that modification of the molecules of neurotransmitter amino acids leads to a decrease in their neurotoxicity and to an increase in their membranotropic properties. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 416–425, September–December, 2006.     

Vegetative survival and reproduction under submerged and air-exposed conditions and vegetative survival as affected by salts,pesticides, and metals in aerial green alga<Emphasis Type="Italic">Trentepohlia aurea</Emphasis>

Trentepohlia aurea vegetative cells do not survive submerged conditions for more than 5 months, but can survive air-exposed conditions for more than 1 year. Disintegration and rapid death of algal cells was observed to a higher extent under submerged than air-exposed condition. Under submerged conditionsT. aurea did not form any sporangium while prolific formation occurred under air-exposed conditions. Under submerged conditions algal cells formed few-celled, filamentous, cytoplasmic type setae. Vegetative cells were resistant to some extent to various levels of salt (NaCl, ≤0.8 mol/L), pesticides (DDT, 2,4-D or captan, 2000 ppm) and ‘heavy’ metals (zinc or nickel, 200 ppm; cobalt, ≤100 ppm.)     

Pretreatment Effect of Resveratrol on Streptozotocin-Induced Diabetes in Rats

This study was planned to investigate the pretreatment effect of resveratrol on streptozotocin-induced diabetic rats. The control group consisted of 10 male albino Sprague–Dawley rats, 10–12 weeks of age, weighing approximately 295 g. The first experimental group consisted of 15 albino Sprague–Dawley rats, 10–12 weeks of age, weighing approximately 305 g. This group was administered streptozotocin (55 mg/kg, intraperitoneally). The second experimental group (n = 15) was administered resveratrol (0.5 ml/day) 10 days before streptozotocin induction. A training period was performed for all groups before the experimental procedure, and systolic arterial blood pressures and heart rates were recorded daily. At the end of the 10th day, blood samples of control and experimental groups were drawn. Total nitrite, nitrite, nitrate, malondialdehyde (MDA), copper, and zinc concentrations in plasma were measured both in control and experimental groups. Additionally, superoxide dismutase, catalase activities, and copper and zinc concentrations in red cell were determined in each group. At the end of the study, increases in catalase activity, nitric oxide level, and zinc concentrations and decreases in lipid peroxidation product MDA and copper concentrations were found in the resveratrol-pretreated diabetic group when compared to the diabetic group. This study was presented at “The 5th International Congress of Pathophysiology (ISP2006)” June 28–July 1, 2006, Beijing, China.     

Neuronal Chemokines: Versatile Messengers In Central Nervous System Cell Interaction

Whereas chemokines are well known for their ability to induce cell migration, only recently it became evident that chemokines also control a variety of other cell functions and are versatile messengers in the interaction between a diversity of cell types. In the central nervous system (CNS), chemokines are generally found under both physiological and pathological conditions. Whereas many reports describe chemokine expression in astrocytes and microglia and their role in the migration of leukocytes into the CNS, only few studies describe chemokine expression in neurons. Nevertheless, the expression of neuronal chemokines and the corresponding chemokine receptors in CNS cells under physiological and pathological conditions indicates that neuronal chemokines contribute to CNS cell interaction. In this study, we review recent studies describing neuronal chemokine expression and discuss potential roles of neuronal chemokines in neuron–astrocyte, neuron–microglia, and neuron–neuron interaction.     

Studies on the survival of corn stalk rot bacteria

Summary Erwinia carotovora andPseudomonas lapsa which cause stalk rot of corn survived in soil and host tissues under different conditions.E. carotovora survived for 150 days and 90 days in sterile and unsterile soils, respectively, whileP. lapsa survived for 135 days and 75 days under similar conditions when they were added as cell suspensions.E. carotovora survived in diseased tissues for 22, 8, and 7 months at 0–5°C (refrigerator), 22–28°C (room), and 20–37°C (field) temperatures whileP. lapsa survived for 26, 10, and 9 months, respectively, under similar conditions. Seeds of Ganga 3 were found to carryP. lapsa for 14 months, when they were stored at room (22–28°C) temperature.     

Taurine and Zinc Modulate Outgrowth from Goldfish Retinal Explants

Taurine and zinc, highly concentrated in the retina, possess similar properties in this structure, such as neuro-protection, membrane stabilization, influencing regeneration, and modulating development, maybe by acting in parallel or as interacting agents. We previously demonstrated that there are some correlations between taurine and zinc levels in hippocampus, dentate gyrus and retina of the developing rat. In the present study we evaluate the possible effects of taurine and zinc on outgrowth from goldfish retinal explants. The optic nerve was crushed 10 days before plating and culturing retinal explants in Leibovitz medium with 10% fetal calf serum and gentamicin. Neurites were measured with SigmaScanPro after 5 days in culture. Taurine (HPLC) and zinc (ICP) concentrations were determined in the retina between 1 and 180 days after crushing the optic nerve. Zinc sulfate (0.01–100 μM), N,N, N′,N′-tetrakis (pyridylmethyl) ethylenediamine (TPEN, 0.1–5 nM) and diethylenetriamine penta-acetic acid (DTPA, 10–300 μM), intracellular and extracellular zinc chelators, respectively, were added to the medium. TPEN was also injected intraocular (0.1 nM). Combinations of them were added with taurine (1–16 mM). Taurine concentrations were elevated in the retina 72 h after the crush, but were normalized by 180 days, those of zinc increased at 24 h, preceding the increase of taurine. The axonal transport of [³H]taurine from the optic tectum to the retina was not affected in fish with or without crush of the optic nerve at early periods after the injection, indicating an increase of it post-lesion. Zinc sulfate produced a bell-shaped concentration dependency on in vitro outgrowth, with stimulation at 0.05 μM, and inhibition at higher levels, also increased the effect of 4 mM taurine at 0.02 μM, but diminished it at higher concentrations in the medium. TPEN decreased outgrowth at 1 nM, but not at 0.5 nM, although the simultaneous presence of 4 mM taurine and 0.5 nM TPEN decreased outgrowth respecting the stimulation by taurine alone. The intraocular administration of TPEN decreased outgrowth in vitro, an effect counteracted by the addition of 4 mM taurine to the culture medium. DTPA decreased outgrowth from 10 μM in the medium. The present results indicate that an optimal zinc concentration is necessary for outgrowth of goldfish retinal explants and that, in zinc deficient retina, taurine could stimulate outgrowth. In addition, the observations of variations in tissue concentrations and of the effects of intraocular administration of TPEN indicate that these effects could occur in vivo. Special issue dedicated to Dr. Simo S. Oja     

Effects of zinc on the mineralization of bone nodules from human osteoblast-like cells

Zinc is an important mineral that is required for normal bone development. However, the direct effects of zinc on the mineralization of bone cells of human origin are not clear. The objective of this study was to determine the effects of zinc on the differentiation of SaOS-2 human osteoblast-like cells and the formation of mineralized bone nodules. Cells were cultured for 8 d and then transferred to zinc-free medium and treated with varying concentrations (0–50 μM) of zinc. Alkaline phosphatase (ALP) activity was used as a measure of osteoblast differentiation, and bone nodules were detected by von Kossa staining. After 4, 6, and 8 d of treatment, zinc increased ALP activity at 1 and 10 μM, but decreased activity at 50 μM. After 9 d of treatment, zinc increased both the number and area of mineralized bone nodules at low concentrations (1 and 10 μM), but decreased both at higher concentrations (25 and 50 μM). These findings demonstrate that zinc has biphasic effects on the differentiation and mineralization of human osteoblast-like cells.     

Isolation and characterization of a nitrile hydrolysing acidotolerant black yeast—<Emphasis Type="Italic">Exophiala oligosperma</Emphasis> R1

Different nitriles were used as sole sources of nitrogen in a series of enrichments under acidic conditions to isolate acidotolerant nitriles hydrolysing microorganisms. From an enrichment in Na–citrate–phosphate buffer at pH 4 with glucose as carbon source and phenylacetonitrile as sole source of nitrogen, a black yeast (strain R1) was obtained which was identified by subsequent 18S rRNA gene sequencing as Exophiala oligosperma. The growth conditions of the organism were optimized for the production of cell material and the induction of the nitrile converting activity. Resting cell experiments demonstrated that phenylacetonitrile was converted via phenylacetic acid and 2-hydroxyphenylacetic acid. The organism could grow at pH 4 with phenylacetonitrile as sole source of carbon, nitrogen, and energy. The nitriles hydrolysing activity was also detected in cell-free extracts and indications for a nitrilase activity were found. The cell-free extracts converted, in addition to phenylacetonitrile, also different substituted phenylacetonitriles. Whole cells of E. oligosperma R1 converted phenylacetonitrile with almost the same reaction rates in the pH range from pH 1.5–pH 9.     

Induction of xylanases by sugar cane bagasse at different cell densities of Cellulomonas flavigena

The effect of cell density on xylanolytic activity and productivity of Cellulomonas flavigena was evaluated under two different culturing conditions: fed-batch culture with discontinuous feed of sugar cane bagasse (SCB; condition 1) and glycerol fed-batch culture followed by addition of SBC as xylanases inducer (condition 2). The enzymatic profile of xylanases was similar in both systems, regardless of the initial cell density at time of induction. However, the xylanolytic activity changed with initial cell density at the time of induction (condition 2). The maximum volumetric xylanase activity increased with increased initial cell density from 4 to 34 g l⁻¹ but decreased above this value. The largest total volumetric xylanase productivity under condition 2 (1.3 IU ml⁻¹ h⁻¹) was significantly greater compared to condition 1 (maximum 0.6 IU ml⁻¹ h⁻¹). Consequently, induction of xylanase activity by SCB after growing of C. flavigena on glycerol at intermediate cell density can be a feasible alternative to improve activity and productivity of xylanolytic enzymes.     

Inhibition of proteasomal degradation of Mcl-1 by cobalt chloride suppresses cobalt chloride-induced apoptosis in HCT116 colorectal cancer cells

Cobalt promotes apoptosis in multiple cell systems, however, the molecular mechanisms that influence cobalt-induced apoptosis are not fully understood. We investigated mechanisms of cobalt chloride induced apoptosis in HCT116 colorectal cancer cells. Cobalt chloride induced dose dependent apoptosis in HCT116 cells (250–750 μM) which, at higher concentrations (500–750 μM), was associated with an increase in the expression of the Bcl-2-related Mcl-1 survival protein. Cobalt chloride caused the accumulation of higher molecular weight ubiquitin-conjugates of Mcl-1 in intact HCT116 cells and inhibited the activity of the trypsin-like site of the 20S proteasome in an in vitro assay. Although siRNA-mediated knockdown of Mcl-1 increased apoptosis in HCT116 cells, the combination of Mcl-1 siRNA and cobalt chloride induced very high levels of cell killing. Therefore, inhibition of the proteasome by cobalt chloride leads to the accumulation of Mcl-1 which acts to limit cobalt chloride induced apoptosis.