Divalent Cation Chelator Research Articles

Factor VIII assay mimicking in vivo coagulation conditions

Under certain circumstances, the determination of coagulation factor VIII (FVIII) is hampered by assay discrepancies between clotting and chromogenic approaches. These are observed in certain patients' plasma as well as in certain concentrates. We intended to develop a novel assay for the quantification of coagulation FVIII which reflects the physiological situation better than the established assays. It is based on plasma without chelation of divalent cations and simultaneously minimizes the generation of activated factors which could function as uncontrolled triggers of coagulation. FVIII deficient plasma is prepared with the aid of biotinylated antibodies against FVIII from normal plasma in presence of inhibitors of contact activation. To start the assay only tiny amounts of activated FIX serve as trigger. The FVIII determination is performed in a kinetic experiment and is based on the cleavage of a fluorogenic substrate for activated FX. FVIII concentrations between 0.01 and 1 IU mL(-1) are easily determined. Plasma-derived and recombinant FVIII concentrates were compared. All plasma-derived concentrates were found to contain FVIII activities within the specification of the manufacturer. Recombinant concentrates yielded only 35-50% of the claimed potency. The novel in vivo-like assay avoids the undue advantage or disadvantage of certain product characteristics by eliminating unphysiological assay conditions. Its usefulness could turn out in future experiments with plasma from haemophilia A patients.

The Role of Ethylene and Calcium in Programmed Cell Death of Cold-Stored Cucumber Fruit

Programmed cell death (PCD) occurs during plant development and in response to various stimuli, including biotic and abiotic stresses. Characteristics of PCD were detected in cucumber fruit after 9 days of cold storage at 2C, including chromatin condensation and DNA fragmentation. PCD development was accompanied by ethylene emission and cytosolic free calcium ([Ca2+]cyt) release. The role of ethylene production and [Ca2+]cyt in chill-induced PCD were studied through the application of 1-methylcyclopropene (1-MCP; an ethylene perception inhibitor) and ethylene glycol bis (2-aminoethyl) tetraacetic acid (EGTA; a divalent cation chelator). Results indicated that hallmarks of PCD were identified in cucumber fruit suffering from chilling stress and diagnostic events of PCD were alleviated and/or delayed by 1-MCP or EGTA applications. Therefore, ethylene and calcium could play significant roles in the initiation and execution progress of cold-induced PCD. Practical Applications Cucumber fruit is chilling sensitive at temperatures below 10C. It has also been showed that exogenous ethylene could induce PCD features in cucumber fruits and [Ca2+]cyt plays a key role in regulating apoptosis. However, information about roles of endogenous ethylene and calcium in the PCD process of cucumber under cold stress has been rarely reported. The data in this study provided a theoretical basis for understanding the mechanism of chilling injury in cucumber fruit and benefited exploration of strategies to prevent it.

Upregulation of the Host SLC11A1 Gene by Clostridium difficile Toxin B Facilitates Glucosylation of Rho GTPases and Enhances Toxin Lethality

Pseudomembranous enterocolitis associated with Clostridium difficile infection is an important cause of morbidity and mortality in patients being treated with antibiotics. Two closely related large protein toxins produced by C. difficile, TcdA and TcdB, which act identically but at different efficiencies to glucosylate low-molecular-weight Rho GTPases, underlie the microbe's pathogenicity. Using antisense RNA encoded by a library of human expressed sequence tags (ESTs), we randomly inactivated host chromosomal genes in HeLa cells and isolated clones that survived exposure to ordinarily lethal doses of TcdB. This phenotypic screening and subsequent analysis identified solute carrier family 11 member 1 (SLC11A1; formerly NRAMP1), a divalent cation transporter crucial to host defense against certain microbes, as an enhancer of TcdB lethality. Whereas SLC11A1 normally is poorly expressed in human cells of nonmyeloid lineage, TcdB increased SLC11A1 mRNA abundance in such cells through the actions of the RNA-binding protein HuR. We show that short hairpin RNA (shRNA) directed against SLC11A1 reduced TcdB glucosylation of small Rho GTPases and, consequently, toxin lethality. Consistent with the previously known role of SLC11A1 in cation transport, these effects were enhanced by elevation of Mn(2+) in media; conversely, they were decreased by treatment with a chelator of divalent cations. Our findings reveal an unsuspected role for SLC11A1 in determining C. difficile pathogenicity, demonstrate the novel ability of a bacterial toxin to increase its cytotoxicity, establish a mechanistic basis for these effects, and suggest a therapeutic approach to mitigate cell killing by C. difficile toxins A and B.

A minocycline derivative reduces nerve injury-induced allodynia, LPS-induced prostaglandin E2 microglial production and signaling via toll-like receptors 2 and 4

Many studies have shown that minocycline, an antibacterial tetracycline, suppresses experimental pain. While minocycline's positive effects on pain resolution suggest that clinical use of such drugs may prove beneficial, minocycline's antibiotic actions and divalent cation (Ca2+; Mg2+) chelating effects detract from its potential utility. Thus, we tested the antiallodynic effect induced by a non-antibacterial, non-chelating minocycline derivative in a model of neuropathic pain and performed an initial investigation of its anti-inflammatory effects in vitro. Intraperitoneal minocycline (100mg/kg) and 12S-hydroxy-1,12-pyrazolinominocycline (PMIN; 23.75mg/kg, 47.50mg/kg or 95.00mg/kg) reduce the mechanical allodynia induced by chronic constriction injury of mouse sciatic nerve. PMIN reduces the LPS-induced production of PGE2 by primary microglial cell cultures. Human embryonic kidney cells were transfected to express human toll-like receptors 2 and 4, and the signaling via both receptors stimulated with PAM3CSK4 or LPS (respectively) was affected either by minocycline or PMIN. Importantly, these treatments did not affect the cell viability, as assessed by MTT test. Altogether, these results reinforce the evidence that the anti-inflammatory and experimental pain suppressive effects induced by tetracyclines are neither necessarily linked to antibacterial nor to Ca2+ chelating activities. This study supports the evaluation of the potential usefulness of PMIN in the management of neuropathic pain, as its lack of antibacterial and Ca2+ chelating activities might confer greater safety over conventional tetracyclines.

Antibacterial Activity of Probiotic Lactobacillus plantarum HK01: Effect of Divalent Metal Cations and Food Additives on Production Efficiency of Antibacterial Compounds

One hundred and sixty lactic acid bacteria, isolated from Iranian traditional dairy products, were screened for antibacterial potential. Among them, an isolate showing remarkable antibacterial activity against both Staphylococcus aureus (PTCC 1112) and Escherichia coli (PTCC 1338) was selected based on minimum inhibitory concentration (AU/mL). The morphological and biochemical characteristics of the isolate matched the literature description about genus Lactobacillus. Partial sequencing of 16S rRNA gene and its alignment with other Lactobacillus strains revealed that the isolate was closely related to the Lactobacillus plantarum. The isolate also exhibited the highest similarity (>99%) to L. plantarum. We thus tentatively classified the bacterial isolate as L. plantarum HK01. The antibacterial active compound from HK01 strain remained stable for 45min at 121°C, and it reached a maximum activity at the end of log phase and the early part of stationary phase. The antibacterial activity of the test isolate, its probiotic properties and production efficacy through addition of some divalent metal cations and food additives were studied as well. The study of bile salt hydrolase (BSH) activity as a function of growth revealed that HK01 strain hydrolysing up to 5% of sodium salt of glycodeoxycholic acid, correlated with the presence of bsh gene in the isolate. HK01 strain showed high resistance to lysozyme, good adaptation to simulated gastric juice and a moderate bile tolerance. Results obtained from simulated gastric juice conditions showed no significant difference occured during the 70min. HK01 strain was classified as a strain with low hydrophobicity (34.2%). Addition of trisodium citrate dehydrates as a food-grade chelator of divalent cations restored antibacterial compound production in MRS broth. Antibacterial compounds of L. plantarum HK01 endured treatment with 10g/L of SDS, Tween 20, Tween 80 and urea. Concerning food additives, the results demonstrated that antibacterial compound production by L. plantarum HK01 was influenced by the presence of surfactants, EDTA, KCl and sodium citrate.

Simulation of Variation Potential in Higher Plant Cells

Variation potential (VP), a propagating electrical signal unique to plants, induces a number of changes in many physiological processes. However, the mechanisms of its generation and propagation are still under discussion and require experimental and theoretical analysis, including VP simulations. The mathematical model for VP formation in plants has been worked out and is based on our previous description of electrophysiological processes in higher plant cells, including plasma membrane ion transport systems (K(+), Cl(-) and Ca(2+) channels, H(+) and Ca(2+)-ATPase, 2H(+)/Cl(-) symporter and H(+)/K(+) antiporter) and their regulation, ion concentration changes in cells and extracellular spaces and buffers in cytoplasm and apoplast. In addition, the VP model takes into account wound substance diffusion, which is described by a one-dimensional diffusion equation, and ligand-gated Ca(2+) channels, which are activated by this substance. The VP model simulates the experimental dependence of amplitude, velocity and shape of VP on the distance from the wounding site and describes the influence of metabolic inhibitors, divalent cation chelators and anion channel blockers on the generation of this electrical reaction, as shown in experiments. Thus, our model favorably simulates VP in plants and theoretically supports the role of wound substance diffusion and Ca(2+) influx in VP development.

Myeloid-Related Protein-14 Contributes to Protective Immunity in Gram-Negative Pneumonia Derived Sepsis

Klebsiella (K.) pneumoniae is a common cause of pneumonia-derived sepsis. Myeloid related protein 8 (MRP8, S100A8) and MRP14 (S100A9) are the most abundant cytoplasmic proteins in neutrophils. They can form MRP8/14 heterodimers that are released upon cell stress stimuli. MRP8/14 reportedly exerts antimicrobial activity, but in acute fulminant sepsis models MRP8/14 has been found to contribute to organ damage and death. We here determined the role of MRP8/14 in K. pneumoniae sepsis originating from the lungs, using an established model characterized by gradual growth of bacteria with subsequent dissemination. Infection resulted in gradually increasing MRP8/14 levels in lungs and plasma. Mrp14 deficient (mrp14−/−) mice, unable to form MRP8/14 heterodimers, showed enhanced bacterial dissemination accompanied by increased organ damage and a reduced survival. Mrp14−/− macrophages were reduced in their capacity to phagocytose Klebsiella. In addition, recombinant MRP8/14 heterodimers, but not MRP8 or MRP14 alone, prevented growth of Klebsiella in vitro through chelation of divalent cations. Neutrophil extracellular traps (NETs) prepared from wildtype but not from mrp14−/− neutrophils inhibited Klebsiella growth; in accordance, the capacity of human NETs to kill Klebsiella was strongly impaired by an anti-MRP14 antibody or the addition of zinc. These results identify MRP8/14 as key player in protective innate immunity during Klebsiella pneumonia.

Alendronate promotes plasmin-mediated MMP-9 inactivation by exposing cryptic plasmin degradation sites within the MMP-9 catalytic domain

Irreversible MMP-9 inhibition is considered a significant therapeutic goal in inflammatory, vascular and tumour pathology. We report that divalent cation chelators Alendronate and EDTA not only directly inhibited MMP-9 but also promoted irreversible plasmin-mediated MMP-9 inactivation by exposing cryptic plasmin-degradation sites within the MMP-9 catalytic-domain and producing an inhibitory hemopexin–domain fragment. This effect was also observed using MDA-MB-231 breast cancer cells, which activated exogenous plasminogen to degrade endogenous proMMP-9 in the presence of Alendronate or EDTA. Degradation-mediated inactivation of proMMP-9 occurred in the absence of transient activation, attesting to the incapacity of plasmin to directly activate proMMP-9 and direct MMP-9 inhibition by Alendronate and EDTA. Our study provides a novel rational for therapeutic Alendronate use in MMP-9-dependent pathology characterised by plasminogen activation.

Abstract LB-216: Receptor targeted (CD206) Tc99m tilmanocept localization in sentinel nodes in breast cancer and melanoma is concordant with preoperative imaging and a reduction in missed disease

Abstract Purpose: Tilmanocept is wholly synthetic molecule composed of a dextran-10 backbone with both multiple mannose side moieties (for receptor biding) and DTPA (for chelation of divalent cations). Tilmanocept is designed as a high affinity ligand for the human mannose binding receptor (CD206) of reticuloendothelial cells (RECs), and more specifically, those RECs that reside in tumor-draining lymph nodes. Tilmanocept is designed to provide high sensitivity and specificity for intraoperative lymphatic mapping in melanoma, breast cancer, head/neck squamous cell carcinoma, and other solid tumors. This study was designed to evaluate the correlation of preoperative imaging using Tc99m tilmanocept and intraoperative tumor bed-adnexed node findings, along with metatstatic disease finding for Tc99m tilmaocept with its “truth” comparator, Lymphazurin. Results/Conclusions: The results of combined prospective phase 3 clinical trials in breast cancer and melanoma indicate that there is a near 99% correlation between the preoperative imaging (lymphoscintigraphy) based on the use of the CD206 targeted Tc99m tilmanocept and the intraoperative/surgical findings of tumor-adnexed lymph nodes. Additionally, these findings provide exceptional guidance for nodal extraction in contrast to the “truth” mapping agent Lymphazurin, where Tc99m provides a significantly lower failed detection rate leading to a ∼10 percent improved staging rate for breast cancer and mealnoma patients (less missed disease and appropriate post-surgical treatment program). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-216. doi:1538-7445.AM2012-LB-216

Screening and characterization of the alkaline protease isolated from PLI-1, a strain of Brevibacillus sp. collected from Indonesia’s hot springs

A total of 69 strains of thermophilic bacteria were isolated from water, soil and sediment samples from three Indonesia’s hot spring areas (Pantai cermin, Kalianda and Banyu wedang) by using Minimal Synthetic Medium (MSM). The extreme thermophile Brevibacillus sp. PLI-1 was found to produce extracellular thermophilic alkaline protease with optimal activity at 70° and pH 8.0–9.0. The molecular weight of the protease was estimated to be around 56 kD by SDS-PAGE. The maximum activity of the protease was 26.54 U mL−1. The protease activity did not decrease after 30 min and still retained more than 70% of relative activity after 60 min at 70°C and pH 8.0. The ion Mg2+ was found to promote protease activity at both low and high concentrations, whereas Cu2+ and Zn2+ could almost completely inhibit the activity. Divalent cation chelator EDTA inhibited the enzyme activity by 55.06% ± 0.27%, while the inhibition caused by PMSF, Leupeptin, Pepstain A and Benzamidine were 66.78% ± 3.25%, 52.37% ± 0.25%, 62.47% ± 2.96% and 50.99% ± 0.24%, respectively. Based on these observations, the enzyme activity was conspicuously sensitive to the serine and cysteine protease inhibitors. All these results indicated that the protease isolated from the strain PLI-1 was a thermophilic protease and had a high-temperature stability and a pH stability.

Extracellular Ca2+ influx is crucial for the early embryonic development of the sea urchin Echinometra lucunter

The involvement of Ca(2+) in the activation of eggs and in the first steps of the embryonic development of several species is a well-known phenomenon. An association between Ca(2+) sources with the fate of the blastopore during embryonic development has been investigated by several authors. Ca(2+) influx mediated by voltage-gated channels and Ca(2+) mobilization from intracellular stores are the major sources of Ca(2+) to egg activation and succeeding cell divisions. Studies on sea urchins embryonic development show that intracellular Ca(2+) stores are responsible for egg activation and early embryogenesis. In the present work we investigated the involvement of extracellular Ca(2+) in the first stages of the embryonic development of the sea urchin Echinometra lucunter. Divalent cation chelators EDTA and EGTA strongly blocked the early embryonic development. Adding to this, we demonstrated the involvement of voltage-gated Ca(2+) channels in E. lucunter embryogenesis since Ca(2+) channel blockers powerfully inhibited the early embryonic development. Our data also revealed that Ca(2+) influx is crucial for embryonic development during only the first 40 min postfertilization. However, intracellular Ca(2+) remains mandatory to embryonic development 40 min postfertilization, seen that both the intracellular Ca(2+) chelator BAPTA-AM and calmodulin antagonists trifluoperazine and chlorpromazine inhibited the first stages of development when added to embryos culture 50 min postfertilization. Our work highlights the crucial role of extracellular Ca(2+) influx through voltage-gated Ca(2+) channels for the early embryonic development of the sea urchin E. lucunter and characterizes an exception in the phylum Echinodermata.

Structure and elasticity of desmin protofibrils explored with scanning force microscopy

Desmin filaments form the intermediate filament system of muscle cells where they play important role in maintaining mechanical integrity and elasticity. Although the importance of desmin elasticity and assembly-disassembly dynamics in cellular mechanics is being increasingly recognized, the molecular basis of neither desmin's elasticity nor its disassembly pathway is well understood. In the present work, we explored the topographical structure of purified and reconstituted desmin filaments by using scanning force microscopy. With the addition of divalent cation chelators ethyleneglycoltetraacetic acid or ethylenediaminetetraacetic acid, the filaments disassembled on a time scale of hours to days into stable, thin fibrillar components with variable (up to micrometer) length, smooth surface and uniform thickness, which are identified as protofibrils. Desmin protofibrils appear as elastic structures with a persistence length of 51.5 nm, and their Young's modulus (10.6 MPa) far exceeds that of the mature filament (3.7 MPa). Protofibrillar bundling within the desmin filament results in high longitudinal tensile strength at a large bending flexibility. The stability of protofibrils suggests that desmin may disassemble along a pathway quite distinct from its assembly.

Cyanide modulates on the cervical activity in neonatal rat brainstem-spinal cord preparation

Methylmercury (MeHg) is a widespread environmental toxicant with major actions on the central nervous system. Among the neurons reportedly affected in cases of Hg poisoning are motor neurons; however, the direct cellular effects of MeHg on motor neurons have not been reported. Ratiometric fluorescence imaging, using the Ca2+-sensitive fluorophore fura-2, was used to examine the effect of MeHg on Ca2+ homeostasis in primary cultures of mouse spinal motor neurons. In vitro MeHg exposure at concentrations (0.1–2 μM) known to affect other neurons in culture differentially, induced a biphasic rise in fura-2 fluorescence ratio indicating an increase in [Ca2+]i. The time-to-onset of these fura-2 fluorescence ratio changes was inversely correlated with MeHg concentration. TPEN (20 μM), a non-Ca2+, divalent cation chelator, reduced the amplitude of the increase in fura-2 fluorescence induced by MeHg in the first phase, indicating that both Ca2+ and non-Ca2+ divalent cations contribute to the MeHg-induced effect. When examining various Ca2+ entry pathways as possible targets contributing to Ca2+ influx, we found that excitatory amino acid receptor blockers MK-801 (15 μM), and AP-5 (100 μM)—both NMDA receptor-operated ion channel blockers, CNQX (20 μM), a non-NMDA receptor blocker, and the voltage-dependent Ca2+ channel blockers nifedipine (1 μM) and ω-conotoxin-GVIA (1 μM) all significantly delayed the development of increased Ca2+ caused by MeHg. The voltage-dependent Na+ channel blocker tetrodotoxin (TTX, 1 μM) did not alter the MeHg-induced increases in fura-2 fluorescence ratio. Thus, MeHg alters Ca2+ homeostasis in mouse spinal motor neurons through excitatory amino acid receptor-mediated pathways, and nifedipine and ω-conotoxin-GVIA-sensitive pathways. Spinal motor neurons are highly sensitive to this effect of acute exposure to MeHg.

Ca2+ entry pathways in mouse spinal motor neurons in culture following in vitro exposure to methylmercury

Methylmercury (MeHg) is a widespread environmental toxicant with major actions on the central nervous system. Among the neurons reportedly affected in cases of Hg poisoning are motor neurons; however, the direct cellular effects of MeHg on motor neurons have not been reported. Ratiometric fluorescence imaging, using the Ca2+-sensitive fluorophore fura-2, was used to examine the effect of MeHg on Ca2+ homeostasis in primary cultures of mouse spinal motor neurons. In vitro MeHg exposure at concentrations (0.1–2μM) known to affect other neurons in culture differentially, induced a biphasic rise in fura-2 fluorescence ratio indicating an increase in [Ca2+]i. The time-to-onset of these fura-2 fluorescence ratio changes was inversely correlated with MeHg concentration. TPEN (20μM), a non-Ca2+, divalent cation chelator, reduced the amplitude of the increase in fura-2 fluorescence induced by MeHg in the first phase, indicating that both Ca2+ and non-Ca2+ divalent cations contribute to the MeHg-induced effect. When examining various Ca2+ entry pathways as possible targets contributing to Ca2+ influx, we found that excitatory amino acid receptor blockers MK-801 (15μM), and AP-5 (100μM)—both NMDA receptor-operated ion channel blockers, CNQX (20μM), a non-NMDA receptor blocker, and the voltage-dependent Ca2+ channel blockers nifedipine (1μM) and ω-conotoxin-GVIA (1μM) all significantly delayed the development of increased Ca2+ caused by MeHg. The voltage-dependent Na+ channel blocker tetrodotoxin (TTX, 1μM) did not alter the MeHg-induced increases in fura-2 fluorescence ratio. Thus, MeHg alters Ca2+ homeostasis in mouse spinal motor neurons through excitatory amino acid receptor-mediated pathways, and nifedipine and ω-conotoxin-GVIA-sensitive pathways. Spinal motor neurons are highly sensitive to this effect of acute exposure to MeHg.

SNP genotyping through the melting analysis of unlabelled oligonucleotide applied on dilute PCR amplicon

Adaptation of DNA melting analysis for polymorphic single nucleotides (SNPs) genotyping using an unlabeled oligonucleotide probe for polymorphic DNAs under the presence of fluorescent DNA binding dye necessitates a reaction condition where the probe efficiently associates with a target strand that is PCR amplified. We present experimental evidence that application of an unlabeled probe to a dilute PCR amplicon provides a condition such that the fluorescent signals gained subsequently by probe melting are sufficient to discriminate allelic identities. This approach is best exploited by adapting the multiplexing PCR technique in order to cover multiple SNPs for given samples. 3'-end modification of the probe is unnecessary as the amplicon dilution step provides a way of inactivating the polymerase through divalent cation chelation. With the use of low-cost reagents and ordinary laboratory equipment, this method offers a rapid, simple and cost-efficient way of SNP genotyping.

Atomic Force Microscopy of Connexin40 Gap Junction Hemichannels Reveals Calcium-dependent Three-dimensional Molecular Topography and Open-Closed Conformations of Both the Extracellular and Cytoplasmic Faces

Atomic force microscopy was used to study the three-dimensional molecular topography and calcium-sensitive conformational changes of Connexin40 hemichannels (connexons) reconstituted in 1,2-dioeloyl-sn-glycero-3-phosphatidylcholine lipid bilayers. Two classes of objects were observed that differed in their protrusion heights above the bilayer (2.6 versus 4.2 nm). Comparison to reconstituted connexons containing Connexin40 truncated to eliminate most of its C-terminal cytoplasmic domain showed that the two height classes corresponded to the shorter extracellular and taller cytoplasmic aspects of the hemichannels and that the C-terminal tail of Connexin40 contributes ∼1.6 nm in thickness. Hemichannels imaged in solutions containing < 10 μm Ca(2+) showed 3.1-3.2 nm depressions (openings) in 30% of the cytoplasmic faces and 65% of the extracellular faces, and high-resolution three-dimensional topography of extracellular or cytoplasmic aspects of some connexons was observed. After addition of 3.6 mm Ca(2+), > 75% of the connexons in either orientation adopted closed conformations. In contrast, hemichannels imaged in the presence of 0.1 mm EDTA showed large (5.6- to 5.8-nm diameter) openings in nearly all hemichannels regardless of orientation, and detailed topography was visible in many connexons. Real-time imaging following the addition of 3.6 mm Ca(2+) showed transitions of both extracellular and cytoplasmic orientations from "open" into "closed" conformations within several minutes. These studies provide the first high-resolution topographic information regarding a connexin with a large cytoplasmic domain and suggest that the extramembranous portions of Connexin40 contribute to a channel entrance that is relaxed by chelation of residual divalent cations.

Chromosomal integrity and DNA damage in freeze-dried spermatozoa.

Freeze-drying technology may one day be used to preserve mammalian spermatozoa indefinitely without cryopreservation. Freeze-dried mouse spermatozoa stored below 4°C for up to 1 year have maintained the ability to fertilize oocytes and support normal development. The maximum storage period for spermatozoa increases at lower storage temperatures. Freeze-drying, per se, may reduce the integrity of chromosomes in freeze-dried mouse spermatozoa, but induction of chromosomal damage is suppressed if spermatozoa are incubated with divalent cation chelating agents prior to freeze-drying. Nevertheless, chromosomal damage does accumulate in spermatozoa stored at temperatures above 4°C. Currently, no established methods or strategies can prevent or reduce damage accumulation, and damage accumulation during storage is a serious obstacle to advances in freeze-drying technology. Chromosomal integrity of freeze-dried human spermatozoa have roughly background levels of chromosomal damage after storage at 4°C for 1 month, but whether these spermatozoa can produce healthy newborns is unknown. The safety of using freeze-dried human spermatozoa must be evaluated based on the risks of heritable chromosome and DNA damage that accumulates during storage.

Exposure to an Environmental Neurotoxicant Hastens the Onset of Amyotrophic Lateral Sclerosis-Like Phenotype in Human Cu2+/Zn2+ Superoxide Dismutase 1 G93A Mice: Glutamate-Mediated Excitotoxicity

Mice expressing the human Cu(2+)/Zn(2+) superoxide dismutase 1 (hSOD1) gene mutation (hSOD1(G93A); G93A) were exposed to methylmercury (MeHg) at concentrations that did not cause overt motor dysfunction. We hypothesized that low concentrations of MeHg could hasten development of the amyotrophic lateral sclerosis (ALS)-like phenotype in G93A mice. MeHg (1 or 3 ppm/day in drinking water) concentration-dependently accelerated the onset of rotarod failure in G93A, but not wild-type, mice. At the time of rotarod failure, MeHg increased Fluo-4 fluorescence (free intracellular calcium concentration [Ca(2+)](i)) in soma of brainstem-hypoglossal nucleus. These motor neurons control intrinsic and some extrinsic tongue function and exhibit vulnerability in bulbar-onset ALS. The α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainic acid receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione reduced [Ca(2+)](i) in all G93A mice, irrespective of MeHg treatment. N-acetyl spermine, which antagonizes Ca(2+)-permeable AMPA receptors, further reduced [Ca(2+)](i) more effectively in MeHg-treated than untreated G93A mice, suggesting that MeHg-treated mice have a greater Ca(2+)-permeable AMPA receptor contribution. The non-Ca(2+) divalent cation chelator N,N,N',N'-tetrakis(pyridylmethyl)ethylenediamine reduced Fluo-4 fluorescence in all G93A mice; FluoZin-(Zn(2+) indicator) fluorescence was increased in all MeHg-treated mice. Thus in G93A mice Zn(2+) apparently contributed measurably to the MeHg-induced effect. This is the initial demonstration of accelerated onset of ALS-like phenotype in a genetically susceptible organism by exposure to low concentrations of an environmental neurotoxicant. Increased [Ca(2+)](i) induced by the G93A-MeHg interaction apparently was associated with Ca(2+)-permeable AMPA receptors and may contribute to the hastened development of ALS-like phenotypes by subjecting motor neurons to excessive elevation of [Ca(2+)](i), leading to excitotoxic cell death.

TRPM Channels Modulate Epileptic-like Convulsions via Systemic Ion Homeostasis

Neuronal networks operate over a wide range of activity levels, with both neuronal and nonneuronal cells contributing to the balance of excitation and inhibition. Activity imbalance within neuronal networks underlies many neurological diseases, such as epilepsy. The Caenorhabditis elegans locomotor circuit operates via coordinated activity of cholinergic excitatory and GABAergic inhibitory transmission. We have previously shown that a gain-of-function mutation in a neuronal acetylcholine receptor, acr-2(gf), causes an epileptic-like convulsion behavior. Here we report that the behavioral and physiological effects of acr-2(gf) require the activity of the TRPM channel GTL-2 in nonneuronal tissues. Loss of gtl-2 function does not affect baseline synaptic transmission but can compensate for the excitation-inhibition imbalance caused by acr-2(gf). The compensatory effects of removing gtl-2 are counterbalanced by another TRPM channel, GTL-1, and can be recapitulated by acute treatment with divalent cation chelators, including those specific for Zn(2+). Together, these data reveal an important role for ion homeostasis in the balance of neuronal network activity and a novel function of nonneuronal TRPM channels in the fine-tuning of this network activity.

Elicitation of diosgenin production in Trigonella foenum-graecum L. seedlings by heavy metals and signaling molecules

Seedlings of Trigonella foenum-graecum were treated with four heavy metal salts (CdCl2, CoCl2, K2Cr2O7 and NiCl2) to study the effect of heavy metals on growth and diosgenin production. It was found that CdCl2 increased diosgenin production up to 40-fold and CoCl2 increased diosgenin production up to 41-fold at concentrations which did not affect growth significantly. But K2Cr2O7 and NiCl2 were toxic to growth and inhibited diosgenin production. Effect of exogenously applied methyl jasmonate (MeJa) and calcium (Ca2+) on diosgenin production in seedlings of T. foenum-graecum was also investigated. MeJa enhanced the production of diosgenin. Maximum increase (10.5-fold) was found at 100 µL L−l concentration of MeJa. To study the role of Ca2+ on diosgenin production, seedlings of T. foenum-graecum were treated with a promoter of Ca2+ influx (calcium ionophore A23187), calcium depleted medium, Ca2+ channel blocker (verapamil) and antagonist (LaCl3), a divalent cation chelator (EGTA) and modulator of calcium release (caffeine). All the treatments were compared with a control containing 220 mg L−l concentration of CaCl2. The results suggest that the increase in cytosolic Ca2+ has an inhibitory role on diosgenin production. However, a calcium chelator or Ca2+ channel inhibitors could be used to elicit diosgenin production in this plant.