Crystal Structure of the VgrG1 Actin Cross-linking Domain of the Vibrio cholerae Type VI Secretion System

Vibrio cholerae is the cause of the diarrheal disease cholera. V. cholerae produces RtxA, a large toxin of the MARTX family, which is targeted to the host cell cytosol, where its actin cross-linking domain (ACD) cross-links G-actin, leading to F-actin depolymerization, cytoskeleton rearrangements, and cell rounding. These effects on the cytoskeleton prevent phagocytosis and bacterial engulfment by macrophages, thus preventing V. cholerae clearance from the gut. The V. cholerae Type VI secretion-associated VgrG1 protein also contains a C-terminal ACD, which shares 61% identity with MARTX ACD and has been shown to covalently cross-link G-actin. Here, we purified the VgrG1 C-terminal domain and determined its crystal structure. The VgrG1 ACD exhibits a V-shaped three-dimensional structure, formed of 12 β-strands and nine α-helices. Its active site comprises five residues that are conserved in MARTX ACD toxin, within a conserved area of ∼10 Å radius. We showed that less than 100 ACD molecules are sufficient to depolymerize the actin filaments of a fibroblast cell in vivo. Mutagenesis studies confirmed that Glu-16 is critical for the F-actin depolymerization function. Co-crystals with divalent cations and ATP reveal the molecular mechanism of the MARTX/VgrG toxins and offer perspectives for their possible inhibition.     

Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae

The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC 2.7.4.2) from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3) and purified on a Ni²⁺ column. The K_M of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 µM at 37°C, the optimal growth temperature for E. coli. The K_M of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V_max was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg²⁺ dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP.     

Magnesium Sulfate Protects Against the Bioenergetic Consequences of Chronic Glutamate Receptor Stimulation

Extracellular glutamate is elevated following brain ischemia or trauma and contributes to neuronal injury. We tested the hypothesis that magnesium sulfate (MgSO₄, 3 mM) protects against metabolic failure caused by excitotoxic glutamate exposure. Rat cortical neuron preparations treated in medium already containing a physiological concentration of Mg²⁺ (1 mM) could be segregated based on their response to glutamate (100 µM). Type I preparations responded with a decrease or small transient increase in oxygen consumption rate (OCR). Type II neurons responded with >50% stimulation in OCR, indicating a robust response to increased energy demand without immediate toxicity. Pre-treatment with MgSO₄ improved the initial bioenergetic response to glutamate and ameliorated subsequent loss of spare respiratory capacity, measured following addition of the uncoupler FCCP, in Type I but not Type II neurons. Spare respiratory capacity in Type I neurons was also improved by incubation with MgSO₄ or NMDA receptor antagonist MK801 in the absence of glutamate treatment. This finding indicates that the major difference between Type I and Type II preparations is the amount of endogenous glutamate receptor activity. Incubation of Type II neurons with 5 µM glutamate prior to excitotoxic (100 µM) glutamate exposure recapitulated a Type I phenotype. MgSO₄ protected against an excitotoxic glutamate-induced drop in neuronal ATP both with and without prior 5 µM glutamate exposure. Results indicate that MgSO₄ protects against chronic moderate glutamate receptor stimulation and preserves cellular ATP following treatment with excitotoxic glutamate.     

Functional Impact of ABCB1 Variants on Interactions between P-Glycoprotein and Methadone

Methadone is a widely used substitution therapy for opioid addiction. Large inter-individual variability has been observed in methadone maintenance dosages and P-glycoprotein (P-gp) was considered to be one of the major contributors. To investigate the mechanism of P-gp’s interaction with methadone, as well as the effect of genetic variants on the interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established in the present study. The RNA and protein expression levels of human P-gp were confirmed by real-time quantitative RT-PCR and western blot, respectively. Utilizing rhodamine 123 efflux assay and calcein-AM uptake study, methadone was demonstrated to be an inhibitor of wild-type human P-gp via non-competitive kinetic (IC₅₀ = 2.17±0.10 µM), while the variant-type human P-gp, P-gp with 1236T-2677T-3435T genotype and P-gp with 1236T-2677A-3435T genotype, showed less inhibition potency (IC₅₀ = 2.97±0.09 µM and 4.43±1.10 µM, respectively) via uncompetitive kinetics. Methadone also stimulated P-gp ATPase and inhibited verapamil-stimulated P-gp ATPase activity under therapeutic concentrations. These results may provide a possible explanation for higher methadone dosage requirements in patients carrying variant-type of P-gp and revealed the possible drug-drug interactions in patients who receive concomitant drugs which are also P-gp substrates.     

DCM-Related Tropomyosin Mutants E40K/E54K Over-Inhibit the Actomyosin Interaction and Lead to a Decrease in the Number of Cycling Cross-Bridges

Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu­tion technique. The effects of the Ca²⁺, ATP, phos­phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com­pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T _HC (high Ca²⁺ ten­sion at pCa 4.66) (E40K: 1.21±0.06 T _a, ±SEM, N = 34; E54K: 1.24±0.07 T _a, N = 28), a significantly lower T _LC (low- Ca²⁺ tension at pCa 7.0) (E40K: 0.07±0.02 T _a, N = 34; E54K: 0.06±0.02 T _a, N = 28), and a significantly lower T _act (Ca²⁺ activatable tension) (T _act = T _HC–T_LC, E40K: 1.15±0.08 T _a, N = 34; E54K: 1.18±0.06 T _a, N = 28) than WT (T _HC = 1.53±0.07 T _a, T _LC = 0.12±0.01 T _a, T _act = 1.40±0.07 T _a, N = 25). All tensions were normalized to T _a ( = 13.9±0.8 kPa, N = 57), the ten­sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca²⁺ sensitivity (pCa₅₀) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper­a­tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.     

Actin as target for modification by bacterial protein toxins

Various bacterial protein toxins and effectors target the actin cytoskeleton. At least three groups of toxins/effectors can be identified, which directly modify actin molecules. One group of toxins/effectors causes ADP-ribosylation of actin at arginine-177, thereby inhibiting actin polymerization. Members of this group are numerous binary actin-ADP-ribosylating exotoxins (e.g. Clostridium botulinum C2 toxin) as well as several bacterial ADP-ribosyltransferases (e.g. Salmonella enterica SpvB) which are not binary in structure. The second group includes toxins that modify actin to promote actin polymerization and the formation of actin aggregates. To this group belongs a toxin from the Photorhabdus luminescens Tc toxin complex that ADP-ribosylates actin at threonine-148. A third group of bacterial toxins/effectors (e.g. Vibrio cholerae multifunctional, autoprocessing RTX toxin) catalyses a chemical crosslinking reaction of actin thereby forming oligomers, while blocking the polymerization of actin to functional filaments. Novel findings about members of these toxin groups are discussed in detail.     

Actin as a potential target for decavanadate

ATP prevents G-actin cysteine oxidation and vanadyl formation specifically induced by decavanadate, suggesting that the oxometalate-protein interaction is affected by the nucleotide. The ATP exchange rate is increased by 2-fold due to the presence of decavanadate when compared with control actin (3.1 × 10^− 3 s^− 1), and an apparent dissociation constant (k_dapp) of 227.4 ± 25.7 μM and 112.3 ± 8.7 μM was obtained in absence or presence of 20 μM V₁₀, respectively. Moreover, concentrations as low as 50 μM of decameric vanadate species (V₁₀) increases the relative G-actin intrinsic fluorescence intensity by approximately 80% whereas for a 10-fold concentration of monomeric vanadate (V₁) no effects were observed. Upon decavanadate titration, it was observed a linear increase in G-actin hydrophobic surface (2.6-fold), while no changes were detected for V₁ (0-200 μM). Taken together, three major ideas arise: i) ATP prevents decavanadate-induced G-actin cysteine oxidation and vanadate reduction; ii) decavanadate promotes actin conformational changes resulting on its inactivation, iii) decavanadate has an effect on actin ATP binding site. Once it is demonstrated that actin is a new potential target for decavanadate, being the ATP binding site a suitable site for decavanadate binding, it is proposed that some of the biological effects of vanadate can be, at least in part, explained by decavanadate interactions with actin.     

Inhibition of Leishmania (Leishmania) amazonensis and Rat Arginases by Green Tea EGCG, (+)-Catechin and (?)-Epicatechin: A Comparative Structural Analysis of Enzyme-Inhibitor Interactions

Epigallocatechin-3-gallate (EGCG), a dietary polyphenol (flavanol) from green tea, possesses leishmanicidal and antitrypanosomal activity. Mitochondrial damage was observed in Leishmania treated with EGCG, and it contributed to the lethal effect. However, the molecular target has not been defined. In this study, EGCG, (+)-catechin and (−)-epicatechin were tested against recombinant arginase from Leishmania amazonensis (ARG-L) and rat liver arginase (ARG-1). The compounds inhibit ARG-L and ARG-1 but are more active against the parasite enzyme. Enzyme kinetics reveal that EGCG is a mixed inhibitor of the ARG-L while (+)-catechin and (−)-epicatechin are competitive inhibitors. The most potent arginase inhibitor is (+)-catechin (IC₅₀ = 0.8 µM) followed by (−)-epicatechin (IC₅₀ = 1.8 µM), gallic acid (IC₅₀ = 2.2 µM) and EGCG (IC₅₀ = 3.8 µM). Docking analyses showed different modes of interaction of the compounds with the active sites of ARG-L and ARG-1. Due to the low IC₅₀ values obtained for ARG-L, flavanols can be used as a supplement for leishmaniasis treatment.     

Isoniazid Inhibits the Heme-Based Reactivity of Mycobacterium tuberculosis Truncated Hemoglobin N

Isoniazid represents a first-line anti-tuberculosis medication in prevention and treatment. This prodrug is activated by a mycobacterial catalase-peroxidase enzyme called KatG in Mycobacterium tuberculosis), thereby inhibiting the synthesis of mycolic acid, required for the mycobacterial cell wall. Moreover, isoniazid activation by KatG produces some radical species (e.g., nitrogen monoxide), that display anti-mycobacterial activity. Remarkably, the ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of (pseudo-)enzymatic detoxification systems, including truncated hemoglobins (trHbs). Here, we report that isoniazid binds reversibly to ferric and ferrous M. tuberculosis trHb type N (or group I; Mt-trHbN(III) and Mt-trHbN(II), respectively) with a simple bimolecular process, which perturbs the heme-based spectroscopic properties. Values of thermodynamic and kinetic parameters for isoniazid binding to Mt-trHbN(III) and Mt-trHbN(II) are K = (1.1±0.1)×10⁻⁴ M, k _on = (5.3±0.6)×10³ M⁻¹ s⁻¹ and k _off = (4.6±0.5)×10⁻¹ s⁻¹; and D = (1.2±0.2)×10⁻³ M, d _on = (1.3±0.4)×10³ M⁻¹ s⁻¹, and d _off = 1.5±0.4 s⁻¹, respectively, at pH 7.0 and 20.0°C. Accordingly, isoniazid inhibits competitively azide binding to Mt-trHbN(III) and Mt-trHbN(III)-catalyzed peroxynitrite isomerization. Moreover, isoniazid inhibits Mt-trHbN(II) oxygenation and carbonylation. Although the structure of the Mt-trHbN-isoniazid complex is not available, here we show by docking simulation that isoniazid binding to the heme-Fe atom indeed may take place. These data suggest a direct role of isoniazid to impair fundamental functions of mycobacteria, e.g. scavenging of reactive nitrogen and oxygen species, and metabolism.     

Biochemical and Kinetic Characterization of the Recombinant ADP-Forming Acetyl Coenzyme A Synthetase from the Amitochondriate Protozoan Entamoeba histolytica

Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PP_i-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PP_i were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PP_i displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed.     

Suture Pull-Through Insertion Techniques for Descemet Stripping Automated Endothelial Keratoplasty in Chinese Phakic Eyes: Outcomes and Complications

Purpose

To investigate the outcomes and complications of suture pull-through insertion techniques for Descemet stripping automated endothelial keratoplasty (DSAEK) in Chinese phakic eyes.

Patients and Methods

Retrospective case series. Included in the study were all Chinese patients with phakic eyes who underwent DSAEK at Peking University Third Hospital from August 2008 to August 2011. All ocular diseases of the patients were recorded. Distance visual acuity (DVA), near visual acuity (NVA), intraocular pressure (IOP), anterior chamber depth (ACD), central corneal thickness (CCT), and corneal endothelial cell density (ECD) were compared prior to and 12 months after DSAEK. The DSAEK success rate, endothelial cell loss (ECL), complications, and prognosis were analyzed. All patients had at least 12 months of follow up.

Results

Twenty-one eyes of 16 patients were included (11 males and 5 females). Ages ranged from 2 to 47 years with an average age of 29.8 years. The average follow up was 15.4 months (ranging from 12 to 36 months). Diagnoses included 7 eyes (4 patients) with corneal endothelial dystrophy and 14 eyes (12 patients) with bullous keratopathy. Presurgical DVA and NVA (LogMAR) were 1.7±0.7 and 1.2±0.4; postsurgical DVA and NVA were 0.8±0.6 and 0.7±0.5; Z = −3.517, −2.764; P<0.001 and P = 0.006 respectively. Presurgical IOP was 15.8±3.7 mm Hg; postsurgical IOP was 15.2±2.6 mm Hg; Z = −0.505, P = 0.614. Presurgical ACD was 3.00±0.74 mm; postsurgical ACD was 2.72±0.59 mm; Z = −0.524, P = 0.600. Donor ECD was 2992±163 cells/mm², ECD was 1836±412 cells/mm² with a 12-month postsurgical ECL of 39%. Success rate was 86%. Surgery complications included pupillary block-induced hypertension in 5 eyes (24%), graft detachment in 3 eyes (14%), and graft dislocation in 1 eye (5%).

Conclusions

DSAEK on Chinese phakic eyes can significantly improve DVA and NVA by preserving the patient’s own crystalline lens. DSAEK is an optional surgery for patients who need to preserve accommodative function. More attention should be given to postsurgical pupillary block-induced hypertension.     

MACF1与成骨细胞骨架之间的相互关系研究

采用激光共聚焦显微术研究微管微丝交联因子（MACF1）与成骨样细胞（MD63及MC3T3）微丝／微管骨架、黏着斑之间的相互关系．结果表明,MACF1不连续地分布于微管纤维上,与微丝骨架部分共定位于胞质中,在很多的成骨细胞中可见MACF1分布于骨架相关的粘着斑处：细胞松弛素B影响了MACF1在成骨细胞中的分布,并有使其向细胞核周围及核内转位的趋势．秋水仙素对MACF1的分布无明显的影响．转染了siRNA—MACFl的MG．63细胞微丝骨架纤维分布不连续、微管骨架纤维分布紊乱．这些结果提示MACF1不仅起交联微丝及微管细胞骨架的作用．而且还可稳定细胞骨架：成骨细胞MACF1的分布更依赖于微丝骨架的完整性．     

Effect of ATP on the Calcium Efflux in Dialyzed Squid Giant Axons

Dialysis perfusion technique makes it possible to control the internal composition of squid giant axons. Calcium efflux has been studied in the presence and in the virtual absence (<5 µM) of ATP. The mean calcium efflux from axons dialyzed with 0.3 µM ionized calcium, [ATP]_i > 1,000 µM, and bathed in artificial seawater (ASW) was 0.24 ± 0.02 pmol·cm^-2·s^-1 (P/CS) (n = 8) at 22°C. With [ATP]_i < 5 µM the mean efflux was 0.11 ± 0.01 P/CS (n = 15). The curve relating calcium efflux to [ATP]_i shows a constant residual calcium efflux in the range of 1–100 µM [ATP]_i. An increase of the calcium efflux is observed when [ATP]_i is >100 µM and saturates at [ATP]_i > 1,000 µM. The magnitude of the ATP-dependent fraction of the calcium efflux varies with external concentrations of Na⁺, Ca⁺⁺, and Mg⁺⁺. These results suggest that internal ATP changes the affinity of the calcium transport system for external cations.     

A Nucleotide State-sensing Region on Actin

The nucleotide state of actin (ATP, ADP-P_i, or ADP) is known to impact its interactions with other actin molecules upon polymerization as well as with multiple actin binding proteins both in the monomeric and filamentous states of actin. Recently, molecular dynamics simulations predicted that a sequence located at the interface of subdomains 1 and 3 (W-loop; residues 165–172) changes from an unstructured loop to a β-turn conformation upon ATP hydrolysis (Zheng, X., Diraviyam, K., and Sept, D. (2007) Biophys. J. 93, 1277–1283). This region participates directly in the binding to other subunits in F-actin as well as to cofilin, profilin, and WH2 domain proteins and, therefore, could contribute to the nucleotide sensitivity of these interactions. The present study demonstrates a reciprocal communication between the W-loop region and the nucleotide binding cleft on actin. Point mutagenesis of residues 167, 169, and 170 and their site-specific labeling significantly affect the nucleotide release from the cleft region, whereas the ATP/ADP switch alters the fluorescence of probes located in the W-loop. In the ADP-P_i state, the W-loop adopts a conformation similar to that in the ATP state but different from the ADP state. Binding of latrunculin A to the nucleotide cleft favors the ATP-like conformation of the W-loop, whereas ADP-ribosylation of Arg-177 forces the W-loop into a conformation distinct from those in the ADP and ATP-states. Overall, our experimental data suggest that the W-loop of actin is a nucleotide sensor, which may contribute to the nucleotide state-dependent changes in F-actin and nucleotide state-modulated interactions of both G- and F-actin with actin-binding proteins.     

Higher Prevalence of Metformin-Induced Vitamin B12 Deficiency in Sulfonylurea Combination Compared with Insulin Combination in Patients with Type 2 Diabetes: A Cross-Sectional Study

Long-term and high-dose treatment with metformin is known to be associated with vitamin B₁₂ deficiency in patients with type 2 diabetes. We investigated whether the prevalence of B₁₂ deficiency was different in patients treated with different combination of hypoglycemic agents with metformin during the same time period. A total of 394 patients with type 2 diabetes treated with metformin and sulfonylurea (S+M group, n = 299) or metformin and insulin (I+M group, n = 95) were consecutively recruited. The vitamin B₁₂ and folate levels were quantified using the chemiluminescent enzyme immunoassay. Vitamin B₁₂ deficiency was defined as vitamin B₁₂≤300 pg/mL without folate deficiency (folate>4 ng/mL). The mean age of and duration of diabetes in the subjects were 59.4±10.5 years and 12.2±6.7 years, respectively. The mean vitamin B₁₂ level of the total population was 638.0±279.6 pg/mL. The mean serum B₁₂ levels were significantly lower in the S+M group compared with the I+M group (600.0±266.5 vs. 757.7±287.6 pg/mL, P<0.001). The prevalence of vitamin B₁₂ deficiency in the metformin-treated patients was significantly higher in the S+M group compared with the I+M group (17.4% vs. 4.2%, P = 0.001). After adjustment for various factors, such as age, sex, diabetic duration, duration or daily dose of metformin, diabetic complications, and presence of anemia, sulfonylurea use was a significant independent risk factor for B₁₂ deficiency (OR = 4.74, 95% CI 1.41–15.99, P = 0.012). In conclusion, our study demonstrated that patients with type 2 diabetes who were treated with metformin combined with sulfonylurea require clinical attention for vitamin B₁₂ deficiency and regular monitoring of their vitamin B₁₂ levels.     

Inhibition of Platelet Activation and Thrombus Formation by Adenosine and Inosine: Studies on Their Relative Contribution and Molecular Modeling

Background

The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine.

Objectives

The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated.

Results

Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A_2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A_2A is the adenosine receptor present in platelets; it is known that inosine is not an A_2A ligand. Docking of adenosine and inosine inside A_2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH₂ of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A_2A receptor.

Conclusion

Therefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.     

High Sensitivity of Giardia duodenalis to Tetrahydrolipstatin (Orlistat) In Vitro

Giardiasis, a gastrointestinal disease caused by Giardia duodenalis, is currently treated mainly with nitroimidazoles, primarily metronidazole (MTZ). Treatment failure rates of up to 20 percent reflect the compelling need for alternative treatment options. Here, we investigated whether orlistat, a drug approved to treat obesity, represents a potential therapeutic agent against giardiasis. We compared the growth inhibitory effects of orlistat and MTZ on a long-term in vitro culture adapted G. duodenalis strain, WB-C6, and on a new isolate, 14-03/F7, from a patient refractory to MTZ treatment using a resazurin assay. The giardiacidal concentration of the drugs and their combined in vitro efficacy was determined by median-effect analysis. Morphological changes after treatment were analysed by light and electron microscopy. Orlistat inhibited the in vitro growth of G. duodenalis at low micromolar concentrations, with isolate 14-03/F7 (IC50_24h = 2.8 µM) being more sensitive than WB-C6 (IC50_24h = 6.2 µM). The effect was significantly more potent compared to MTZ (IC50_24h = 4.3 µM and 11.0 µM, respectively) and led to specific undulated morphological alterations on the parasite surface. The giardiacidal concentration of orlistat was >14 µM for 14-03/F7 and >43 µM for WB-C6, respectively. Importantly, the combination of both drugs revealed no interaction on their inhibitory effects. We demonstrate that orlistat is a potent inhibitor of G. duodenalis growth in vitro and kills parasites at concentrations achievable in the gut by approved treatment regimens for obesity. We therefore propose to investigate orlistat in controlled clinical studies as a new drug in giardiasis.     

Mammalian Tropomodulins Nucleate Actin Polymerization via Their Actin Monomer Binding and Filament Pointed End-capping Activities

Many actin-binding proteins have been shown to possess multiple activities to regulate filament dynamics. Tropomodulins (Tmod1–4) are a conserved family of actin filament pointed end-capping proteins. Our previous work has demonstrated that Tmod3 binds to monomeric actin in addition to capping pointed ends. Here, we show a novel actin-nucleating activity in mammalian Tmods. Comparison of Tmod isoforms revealed that Tmod1–3 but not Tmod4 nucleate actin filament assembly. All Tmods bind to monomeric actin, and Tmod3 forms a 1:1 complex with actin. By truncation and mutagenesis studies, we demonstrated that the second α-helix in the N-terminal domain of Tmod3 is essential for actin monomer binding. Chemical cross-linking and LC-MS/MS further indicated that residues in this second α-helix interact with actin subdomain 2, whereas Tmod3 N-terminal domain peptides distal to this α-helix interact with actin subdomain 1. Mutagenesis of Leu-73 to Asp, which disrupts the second α-helix of Tmod3, decreases both its actin monomer-binding and -nucleating activities. On the other hand, point mutations of residues in the C-terminal leucine-rich repeat domain of Tmod3 (Lys-317 in the fifth leucine-rich repeat β-sheet and Lys-344 or Arg-345/Arg-346 in the C-terminal α6-helix) significantly reduced pointed end-capping and nucleation without altering actin monomer binding. Taken together, our data indicate that Tmod3 binds actin monomers over an extended interface and that nucleating activity depends on actin monomer binding and pointed end-capping activities, contributed by N- and C-terminal domains of Tmod3, respectively. Tmod3 nucleation of actin assembly may regulate the cytoskeleton in dynamic cellular contexts.     

Amiodarone Inhibits Apamin-Sensitive Potassium Currents

Background

Apamin sensitive potassium current (I _KAS), carried by the type 2 small conductance Ca²⁺-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles.

Objective

To test the hypothesis that amiodarone inhibits I _KAS in human embryonic kidney 293 (HEK-293) cells.

Methods

We used the patch-clamp technique to study I _KAS in HEK-293 cells transiently expressing human SK2 before and after amiodarone administration.

Results

Amiodarone inhibited I_KAS in a dose-dependent manner (IC₅₀, 2.67±0.25 µM with 1 µM intrapipette Ca²⁺). Maximal inhibition was observed with 50 µM amiodarone which inhibited 85.6±3.1% of I_KAS induced with 1 µM intrapipette Ca²⁺ (n = 3). I_KAS inhibition by amiodarone was not voltage-dependent, but was Ca²⁺-dependent: 30 µM amiodarone inhibited 81.5±1.9% of I _KAS induced with 1 µM Ca²⁺ (n = 4), and 16.4±4.9% with 250 nM Ca²⁺ (n = 5). Desethylamiodarone, a major metabolite of amiodarone, also exerts voltage-independent but Ca²⁺ dependent inhibition of I _KAS.

Conclusion

Both amiodarone and desethylamiodarone inhibit I _KAS at therapeutic concentrations. The inhibition is independent of time and voltage, but is dependent on the intracellular Ca²⁺ concentration. SK2 current inhibition may in part underlie amiodarone''s effects in preventing electrical storm in failing ventricles.     

Calpain Inhibition Reduces Amplitude and Accelerates Decay of the Late Sodium Current in Ventricular Myocytes from Dogs with Chronic Heart Failure

Calpain is an intracellular Ca²⁺ -activated protease that is involved in numerous Ca²⁺ dependent regulation of protein function in many cell types. This paper tests a hypothesis that calpains are involved in Ca²⁺ -dependent increase of the late sodium current (I_NaL) in failing heart. Chronic heart failure (HF) was induced in 2 dogs by multiple coronary artery embolization. Using a conventional patch-clamp technique, the whole-cell I_NaL was recorded in enzymatically isolated ventricular cardiomyocytes (VCMs) in which I_NaL was activated by the presence of a higher (1μM) intracellular [Ca²⁺] in the patch pipette. Cell suspensions were exposed to a cell- permeant calpain inhibitor MDL-28170 for 1–2 h before I_NaL recordings. The numerical excitation-contraction coupling (ECC) model was used to evaluate electrophysiological effects of calpain inhibition in silico. MDL caused acceleration of I_NaL decay evaluated by the two-exponential fit (τ₁ = 42±3.0 ms τ₂ = 435±27 ms, n = 6, in MDL vs. τ₁ = 52±2.1 ms τ₂ = 605±26 control no vehicle, n = 11, and vs. τ₁ = 52±2.8 ms τ₂ = 583±37 ms n = 7, control with vehicle, P<0.05 ANOVA). MDL significantly reduced I_NaL density recorded at –30 mV (0.488±0.03, n = 12, in control no vehicle, 0.4502±0.0210, n = 9 in vehicle vs. 0.166±0.05pA/pF, n = 5, in MDL). Our measurements of current-voltage relationships demonstrated that the I_NaL density was decreased by MDL in a wide range of potentials, including that for the action potential plateau. At the same time the membrane potential dependency of the steady-state activation and inactivation remained unchanged in the MDL-treated VCMs. Our ECC model predicted that calpain inhibition greatly improves myocyte function by reducing the action potential duration and intracellular diastolic Ca²⁺ accumulation in the pulse train.

Conclusions

Calpain inhibition reverses I_NaL changes in failing dog ventricular cardiomyocytes in the presence of high intracellular Ca²⁺. Specifically it decreases I_NaL density and accelerates I_NaL kinetics resulting in improvement of myocyte electrical response and Ca²⁺ handling as predicted by our in silico simulations.