Tolerance to and Accumulation of Cadmium by the Mycelium of the Fungi <Emphasis Type="Italic">Scleroderma citrinum</Emphasis> and <Emphasis Type="Italic">Pisolithus tinctorius</Emphasis>

Copper ions are too small to elicit an immune response. Therefore, copper was conjugated to carrier proteins using S-2-(4-isothiocyanatobenzyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid, a bifunctional chelator, to make artificial antigens for copper. Several mice were immunized, and monoclonal antibodies (MAbs) against chelated copper were produced. Spleen cells of immunized mice were fused with myeloma cells. The resulting hybridomas were screened using protein conjugates which were covalently bound to metal-free 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) or Cu-DOTA. Two hybridoma cell lines (F4 and B2) that produced MAbs with high selectivity and sensitivity were expanded for further study. Cross-reactivities with other metals were below 1%. These antibodies were used to construct competitive ELISAs for copper ions. The IC₅₀ for F4 and B2 were 0.39 and 1.66 mg/l, respectively. The detection range and the lowest detection limit for copper using the antibody F4 was 0.019–8.22 and 0.003 mg/l, respectively. Spike recovery studies in tap water showed that the most sensitive antibody could be used for copper detection in drinking water.     

Development of ELISA for Detection of Mercury Based on Specific Monoclonal Antibodies Against Mercury-Chelate

Immunoassays for heavy metals offer an alternative approach to traditional techniques for detection of mercury. In this study, a mercury-chelate was prepared with 1-(4-aminobenzyl) ethylenediamine-N,N,N′,N′-tetraacetic acid (aminobenzyl-EDTA). The resulting complex was linked to keyhole limpet hemocyanin (KLH) or bovine serum albumin via the amino group and used as the immunizing antigen or detection antigen, respectively. BALB/c mice were immunized with KLH-aminobenzyl-EDTA-Hg and spleen cells from BALB/C mice were fused with Sp2/0 cells. One cell line (5F7) produced monoclonal antibodies with preferential selectivity and sensitivity for aminobenzyl-EDTA-Hg. This cell line had an affinity constant of 4.31?×?10⁹ L/mol and its cross-reactivity (CR) with other metals was <2%. The antibody was used for competitive indirect ELISA (CI-ELISA) for Hg²⁺ measurements. The detection range was 0.087–790.4 μg/L and the lower limit of detection was 0.042 μg/L. The concentrations of mercury in environmental water samples obtained by CI-ELISA correlated well with graphite furnace atomic absorption spectrometry (GFAAS), and the mean recovery was 88.82% to 104.64%. These results indicate that this method could be used for monitoring mercury of water.     

Comparison of MRI properties between derivatized DTPA and DOTA gadolinium–dendrimer conjugates

In this report we directly compare the in vivo and in vitro MRI properties of gadolinium–dendrimer conjugates of derivatized acyclic diethylenetriamine-N,N′,N′,N″,N″-pentaacetic acid (1B4M-DTPA) and macrocyclic 1,4,7,10-tetraazacyclododecane-N,N′,N″,N?-tetraacetic acid (C-DOTA). The metal–ligand chelates were pre-formed in alcohol prior to conjugation to the generation 4 PAMAM dendrimer (G4D), and the dendrimer-based agents were purified by Sephadex® G-25 column. The analysis and SE-HPLC data indicated chelate to dendrimer ratios of 30:1 and 28:1, respectively. Molar relaxivity measured at pH 7.4, 22 °C, and 3T are comparable (29.5 vs 26.9 mM^?1 s^?1), and both conjugates are equally viable as MRI contrast agents based on the images obtained. The macrocyclic agent however exhibits a faster rate of clearance in vivo (t_1/2 = 16 vs 29 min). Our conclusion is that the macrocyclic-based agent is the more suitable agent for in vivo use for these reasons combined with kinetic inertness associated with the Gd(III) DOTA complex stability properties.     

Expression of the zinc importer protein ZIP9/SLC39A9 in glioblastoma cells affects phosphorylation states of p53 and GSK-3β and causes increased cell migration

Zinc importer proteins (ZIPs) have been proven as important molecular regulators in different cancers. As a member of the solute carrier family, ZIP9/SLC39A9 is overexpressed in prostate and breast cancer and affects B-cell receptor signaling. Here, we present data indicating that changes in intracellular zinc levels in glioblastoma cells can cause enhanced cell survival and cell migration, both hallmarks of the disease process. In particular, treatment of human glioblastoma cells with sublethal doses of cell-permeable heavy metal (Zn²⁺ > Fe²⁺ > Mn²⁺) chelator (N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN)) induced ZIP9 expression. Either TPEN treatment or expression of ZIP9 cDNA causes enhanced migration behavior of glioblastoma cells. Compared to untreated glioblastoma cells TPEN treatment or expression of ZIP9 results in activation of the tumor suppressor p53 by phosphorylation at serine residue 46 (Ser46) and in inactivation of the migration relevant glycogen synthase kinase 3 beta (GSK-3β) by phosphorylation at serine residue 9 (Ser9). Whilst p53 activation affects cell survival in response to TPEN, GSK-3β inactivation directly affects glioblastoma cell migration. Therefore, ZIP9 expression could regulate the migratory behavior of glioblastoma cells, so that ZIP9 may be of biological, but not of clinical relevance for glioblastomas, since in GBM tumor tissues, ZIP9 expression is not significantly increased compared to normal brain.     

An osteoclast-targeting agent for imaging and therapy of bone metastasis

A hybrid compound (DO3A-BP) featuring a radiometal bifunctional chelator (1,4,7,10-tetraazacyclotetradecane-N,N′,N″,N-tetraacetic acid, DOTA) and an osteoclast-targeting moiety (bisphosphonate) was designed and synthesized. The ¹¹¹In-labeled complex of DO3A-BP showed significantly elevated uptake in osteoclasts compared to the undifferentiated adherent bone marrow derived cells. Biodistribution studies revealed a favorable tissue distribution profile in normal mice with high bone uptake and long retention, and low or negligible accumulation in non-target organs.     

A proposed mechanism for the thermal denaturation of a recombinant Bacillus halmapalus α-amylase—the effect of calcium ions

The thermal stability of a recombinant α-amylase from Bacillus halmapalus α-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This α-amylase is homologous to other Bacillus α-amylases where crystallographic studies have identified the existence of three calcium binding sites in the structure. Denaturation of BHA is irreversible with a T_m of approximately 89 °C and DSC thermograms can be described using a one-step irreversible model. A 5 °C increase in T_m in the presence of 10-fold excess CaCl₂ was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30–40-fold excess calcium chelator (ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis[β-aminoethyl ether] N,N,N′,N′-tetraacetic acid (EGTA)) results in a large destabilization of BHA, corresponding to about 40 °C lower T_m as determined by both CD and DSC. Ten-fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which probably originate from different populations of BHA/calcium complexes. Combined interpretation of these observations and structural information on homologous α-amylases forms the basis for a suggested mechanism underlying the inactivation mechanism of BHA. The mechanism includes irreversible thermal denaturation of different BHA/calcium complexes and the calcium binding equilibria. Furthermore, the model accounts for a temperature-induced reversible structural change associated with calcium binding.     

Similarity between Cytokinin and Blue Light Inhibition of Cucumber Hypocotyl Elongation

The cytokinin benzyladenine inhibited endogenous hypocotyl elongation in intact etiolated seedlings of cucumber (Cucumis sativus L.). In hypocotyl segments, the inhibitory effect of benzyladenine on growth was clearly detectable in the presence of indoleacetic acid. Fusicoccin-induced elongation was unaffected by the presence of cytokinin. The effect of cytokinin on elongation of the segments was determined by measuring changes in fresh weight, a linear function of extension growth. The effect of benzyladenine on hypocotyl growth was at least as large in segments prepared from red-light-grown seedlings as in those from seedlings grown in total darkness. A comparison was made between the inhibitory effects of cytokinin and blue light. The use of the calcium chelator ethyleneglycol-bis(β-aminoethyl ether)-N, N′-tetraacetic acid indicated that calcium ions are required for manifestation of benzyladenine-induced inhibition.     

N-acetyl-L-cysteine in the presence of Cu<Superscript>2+</Superscript> induces oxidative stress and death of granule neurons in dissociated cultures of rat cerebellum

Addition into the culture medium of the antioxidant N-acetylcysteine (NAC, 1 mM) in the presence of Cu²⁺ (0.0005-0.001 mM) induced intensive death of cultured rat cerebellar granule neurons, which was significantly decreased by the zinc ion chelator TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine). However, the combined action of NAC and Zn²⁺ did not induce destruction of the neurons. Measurement of the relative intracellular concentration of Zn²⁺ with the fluorescent probe FluoZin-3 AM or of free radical production using a CellROX Green showed that incubation of the culture for 4 h with Cu²⁺ and NAC induced an intensive increase in the fluorescence of CellROX Green but not of FluoZin-3. Probably, the protective effect of TPEN in this case could be mediated by its ability to chelate Cu²⁺. Incubation of cultures in a balanced salt solution in the presence of 0.01 mM Cu²⁺ caused neuronal death already after 1 h if the NAC concentration in the solution was within 0.005–0.05 mM. NAC at higher concentrations (0.1–1 mM) together with 0.01mM Cu²⁺ did not cause the death of neurons. These data imply that the antioxidant NAC can be potentially harmful to neurons even in the presence of nanomolar concentrations of variable valence metals.     

Synthesis and initial in vitro biological evaluation of two new zinc-chelating compounds: Comparison with TPEN and PAC-1

The lipophilic, cell-penetrating zinc chelator N,N,N′,N′,-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN, 1) and the zinc chelating procaspase-activating compound PAC-1 (2) both have been reported to induce apoptosis in various cell types. The relationship between apoptosis-inducing ability and zinc affinity (K_d), have been investigated with two new model compounds, ZnA-DPA (3) and ZnA-Pyr (4), and compared to that of TPEN and PAC-1. The zinc-chelating o-hydroxybenzylidene moiety in PAC-1 was replaced with a 2,2′-dipicoylamine (DPA) unit (ZnA-DPA, 3) and a 4-pyridoxyl unit (ZnA-Pyr, 4), rendering an order of zinc affinity TPEN > ZnA-Pyr > ZnA-DPA > PAC-1. The compounds were incubated with the rat pheochromocytoma cell line PC12 and cell death was measured in combination with ZnSO₄, a caspase-3 inhibitor, or a ROS scavenger. The model compounds ZnA-DPA (3) and ZnA-Pyr (4) induced cell death at higher concentrations as compared to PAC-1 and TPEN, reflecting differences in lipophilicity and thereby cell-penetrating ability. Addition of ZnSO₄ reduced cell death induced by ZnA-Pyr (4) more than for ZnA-DPA (3). The ability to induce cell death could be reversed for all compounds using a caspase-3-inhibitor, and most so for TPEN (1) and ZnA-Pyr (4). Reactive oxygen species (ROS), as monitored using dihydro-rhodamine (DHR), were involved in cell death induced by all compounds. These results indicate that the Zn-chelators ZnA-DPA (3) and ZnA-Pyr (4) exercise their apoptosis-inducing effect by mechanisms similar to TPEN (1) and PAC-1 (2), by chelation of zinc, caspase-3 activation, and ROS production.     

A solution thermodynamic study of the Cu(II) and Zn(II) complexes of EBTA: X-ray crystal structure of the dimeric complex [Cu2(EBTA)(H2O)3]2

The copper(II) complex of the acyclic EBTA ligand (H₄EBTA = 1,2-bis(2-aminoethoxy)benzene-N,N,N′,N′-tetraacetic acid) has been prepared and characterized by X-ray analysis. The two copper ions of the dinuclear unit present the same distorted octahedral coordination polyhedra. The EBTA ligand is shared between two copper coordination centres, with the formation of centrosymmetric dimers, which are linked in a supramolecular tridimensional structure via additional interactions through the coordinated waters molecules with adjacent carboxylic oxygen atoms. The stability and protonation constants of EBTA with Cu(II) and Zn(II) ions indicate a higher stability of these complexes with respect to the corresponding complexes with the more flexible EGTA ligand (H₄EGTA = ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid). On the other hand, the lower stability of [Gd(EBTA)]⁻ than [Gd(EGTA)]⁻ results in a decreased overall selectivity (lower K_sel) of EBTA towards Gd(III) and suggests that this complex may undergoes transmetallation reactions under physiological conditions.     

Effects of terbium chelate structure on dipicolinate ligation and the detection of Bacillus spores

Terbium-sensitized luminescence and its applicability towards the detection of Bacillus spores such as anthrax are of significant interest to research in biodefense and medical diagnostics. Accordingly, we have measured the effects of terbium chelation upon the parameters associated with dipicolinate ligation and spore detection. Namely, the dissociation constants, intrinsic brightness, luminescent lifetimes, and biological stabilities for several Tb(chelate)(dipicolinate)_x complexes were determined using linear, cyclic, and aromatic chelators of differing structure and coordination number. This included the chelator array of NTA, BisTris, EGTA, EDTA, BAPTA, DO2A, DTPA, DO3A, and DOTA (respectively, 2,2′,2″-nitrilotriacetic acid; 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol; ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; ethylenediamine-N,N,N′,N′-tetraacetic acid; 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid; diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid; 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid; and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Our study has revealed that the thermodynamic and temporal emission stabilities of the Tb(chelate)(dipicolinate)_x complexes are directly related to chelate rigidity and a ligand stoichiometry of x = 1, and that chelators possessing either aromaticity or low coordination numbers are destabilizing to the complexes when in extracts of an extremotolerant Bacillus spore. Together, our results demonstrate that both Tb(EDTA) and Tb(DO2A) are chemically and biochemically stable and thus applicable as respectively low and high-cost luminescent reporters for spore detection, and thereby of significance to institutions with developing biodefense programs.     

Study on the interaction between a water-soluble dinuclear nickel complex and bovine serum albumin by spectroscopic techniques

The interaction of a water-soluble dinuclear nickel(II) complex, [Ni₂(EGTB)(CH₃CN)₄](ClO₄)₄·4H₂O (EGTB = ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetrakis(2-benzimidazoyl)) (1), and bovine serum albumin (BSA) was investigated under physiological conditions using fluorescence, synchronous fluorescence, UV–vis absorption and circular dichroism (CD). The experimental results suggested that the nickel(II) complex could bind to BSA with binding constant (K) ~ 10⁴ M^?1 and quench the intrinsic fluorescence of BSA through a static quenching mechanism. The thermodynamic parameters, ΔG°, ΔH°, and ΔS°, calculated at different temperatures, indicated that the binding reaction was spontaneous and electrostatic interactions played a major role in this association. Based on the number of binding sites, it was considered that one molecule of complex 1 could bind to a single site or two sites of the BSA molecule or the two binding modes coexisted. In view of the results of site marker competition experiments, the reactive sites of BSA to complex 1 mainly located in subdomain IIA (site I) and subdomain IIIA (site II) of BSA. Moreover, the binding distance, r, between donor (BSA) and acceptor (complex 1) was 5.13 nm according to Förster nonradiation energy transfer theory. Finally, as shown by the UV–vis absorption, synchronous fluorescence and CD, complex 1 could induce conformation and microenvironmental changes of BSA. The results obtained herein will be of biological significance in toxicology investigation and anticancer metallodrug design.     

Facile synthesis and evaluation of C-functionalized benzyl-1-oxa-4,7,10-triazacyclododecane-N,N',N″-triacetic acid as chelating agent for ¹¹¹In-labeled polypeptides

A 12-membered polyazamacrocycle, 1-oxa-4,7,10-triazacyclododecane-N,N′,N″-triacetic acid (ODTA), has been reported to provide an indium chelate of net neutral charge with thermodynamic stability higher than 1,4,7,10-tetraazacyclododecane-N,N′,N″,N?-tetraacetic acid (DOTA). However, neither synthetic procedure for a C-functionalized ODTA (C-ODTA) nor its chelating ability with a trace amount of radioactive indium-111 (¹¹¹In) has been elucidated. We herein present a facile synthetic procedure for C-ODTA, and estimated its ability as a chelating agent for radiolabeling peptides and proteins with ¹¹¹In. The synthetic procedure involves the synthesis of a linear precursor using a para-substituted phenylalanine derivative as a starting material. The following intramolecular cyclization reaction was best performed (>73% yield) when Boc-protected linear compound and the condensation reagent, HATU, were simultaneously added to the reaction vessel at the same flow rate. The cyclic compound was then reduced with BH₃ and alkylated with tert-butyl bromoacetate. The synthetic procedure was straightforward and some optimization would be required. However, most of the intermediate compounds were obtained easily in good yields, suggesting that the present synthetic procedure would be useful to synthesize C-ODTA derivatives. The intramolecular cyclization reaction might also be applicable to synthesize polyazamacrocycles of different ring sizes and cyclic peptides. In ¹¹¹In radiolabeling reactions, C-ODTA provided ¹¹¹In chelates in higher radiochemical yields at low ligand concentrations when compared with C-DOTA. The ¹¹¹In-labeled C-ODTA remained unchanged in the presence of apo-transferrin. The biodistribution studies also showed that the ¹¹¹In-labeled compound was mainly excreted into urine as intact. These findings indicate that C-ODTA would be useful to prepare ¹¹¹In-labeled peptides of high specific activities in high radiochemical yields.     

Synthesis, characterization and equilibrium study of the dinuclear adducts formation between nickel(II) Salen-type complexes with diorganotin(IV) dichlorides in chloroform

Adduct formation between R₂SnCl₂ (R = methyl and n-butyl) as acceptors, and nickel(II) complexes of tetradentate Schiff base ligands ([NiL]) where L = [3-methoxysalen, N,N′-bis(3-methoxysalicylidene)ethylenediamine], [4-methoxysalen, N,N′-bis(4-methoxysalicylidene)ethylenediamine], [5-methoxysalen, N,N′-bis(5-methoxysalicylidene)ethylenediamine], [salen, N,N′-bis(salicylaldehydo)ethylenediamine], [5-chlorosalen, N,N′-bis(5-chlorosalicylidene)ethylenediamine] and [5-bromosalen, N,N′-bis(5-bromosalicylidene)ethylenediamine] as donors have been investigated in chloroform as a solvent by means of UV-Vis spectrophotometeric analysis. Adducts have been characterized by ¹H NMR, IR and electronic spectroscopy. The formation constants and the thermodynamic free energies were measured using UV-Vis spectrophotometry titration for 1:1 adduct formation at various temperatures (T = 278-308 K). The trend of the adduct formation of the nickel Schiff base complexes with a given tin acceptor decreases as follow:

Ni(3-MeOSalen)>Ni(5-MeOSalen)>Ni(4-MeOSalen)     

Production and Characterization of Two Monoclonal Antibodies Specific for Plasmopara halstedii

Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races of P. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.     

Zinc flexes its muscle: Correcting a novel analysis of calcium for zinc interference uncovers a method to measure zinc

The divalent cation chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), often used to buffer physiological changes in cytosolic Ca²⁺, also binds Zn²⁺ with high affinity. In a recently published method (Lamboley et al. 2015. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201411250), the absorbance shift of BAPTA at 292 nm was successfully used to determine the total calcium concentrations of various skeletal muscle tissues. In the present study, we show that endogenous Zn²⁺ in rat skeletal muscle tissue can be unknowingly measured as “Ca²⁺,” unless appropriate measures are taken to eliminate Zn²⁺ interference. We analyzed two rat skeletal muscle tissues, soleus and plantaris, for total calcium and zinc using either inductively coupled plasma mass spectrometry (ICP-MS) or the BAPTA method described above. ICP-MS analysis showed that total zinc contents in soleus and plantaris were large enough to affect the determination of total calcium by the BAPTA method (calcium = 1.72 ± 0.31 and 1.96 ± 0.14, and zinc = 0.528 ± 0.04 and 0.192 ± 0.01; mean ± standard error of the mean [SEM]; n = 5; mmole/kg, respectively). We next analyzed total calcium using BAPTA but included the Zn²⁺-specific chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) that buffers Zn²⁺ without affecting Ca²⁺/BAPTA binding. We found that estimated concentrations of total calcium ([Ca_T]_WM) in soleus and plantaris were reduced after TPEN addition ([Ca_T]_WM = 3.71 ± 0.62 and 3.57 ± 0.64 without TPEN and 3.39 ± 0.64 and 3.42 ± 0.62 with TPEN; mean ± SEM; n = 3; mmole/kg, respectively). Thus, we show that a straightforward correction can be applied to the BAPTA method to improve the accuracy of the determination of total calcium that should be applicable to most any tissue studied. In addition, we show that using TPEN in combination with the BAPTA method allows one to make reasonable estimates of total zinc concentration that are in agreement with the direct determination of zinc concentration by ICP-MS.     

Integrated role of ROS and Ca<Superscript>+2</Superscript> in blue light-induced chloroplast avoidance movement in leaves of <Emphasis Type="Italic">Hydrilla verticillata</Emphasis> (L.f.) Royle

Directional chloroplast photorelocation is a major physio-biochemical mechanism that allows these organelles to realign themselves intracellularly in response to the intensity of the incident light as an adaptive response. Signaling processes involved in blue light (BL)-dependent chloroplast movements were investigated in Hydrilla verticillata (L.f.) Royle leaves. Treatments with antagonists of actin filaments [2,3,5-triiodobenzoic acid (TIBA)] and microtubules (oryzalin) revealed that actin filaments, but not microtubules, play a pivotal role in chloroplast movement. Involvement of reactive oxygen species (ROS) in controlling chloroplast avoidance movement has been demonstrated, as exogenous H₂O₂ not only accelerated chloroplast avoidance but also could induce chloroplast avoidance even in weak blue light (WBL). Further support came from experiments with different ROS scavengers, i.e., dimethylthiourea (DMTU), KI, and CuCl₂, which inhibited chloroplast avoidance, and from ROS localization using specific stains. Such avoidance was also partially inhibited by ZnCl₂, an inhibitor of NADPH oxidase (NOX) as well as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a photosynthetic electron transport chain (ETC) inhibitor at PS II. However, methyl viologen (MV), a PS I ETC inhibitor, rather accelerated avoidance response. Exogenous calcium (Ca⁺²) induced avoidance even in WBL while inhibited chloroplast accumulation partially. On the other hand, chloroplast movements (both accumulation and avoidance) were blocked by Ca⁺² antagonists, La³⁺ (inhibitor of plasma membrane Ca⁺² channel) and ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, Ca⁺² chelator) while LiCl that affects Ca⁺² release from endosomal compartments did not show any effect. A model on integrated role of ROS and Ca⁺² (influx from apolastic space) in actin-mediated chloroplast avoidance has been proposed.     

The inhibitory effect of Ca2+ on the flavonoid production of Tetrastigma hemsleyanum suspension cells induced by metal elicitors

Tetrastigma hemsleyanum suspension cells were treated with four metal salts to screen suitable elicitors for the promotion of plant cell biomass and flavonoid production. The effects of calcium ions (Ca²⁺) on induction were also studied. It was found that the most effective elicitors were 50 μM of the heavy metal ion copper (Cu²⁺) and 100 μM of the rare earth element cerium (Ce³⁺). The maximal biomass levels under respective treatments over a 16-d culture period increased by 1.3- and 1.6-fold, and the total flavonoid content was 1.8- and 1.6-fold greater than the control, respectively. Reducing the exogenous Ca²⁺ concentration or adding Ca²⁺ antagonists (1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N-tetraacetc acid (EGTA) or 1 mM verapamil) strengthened inductive effects of metal elicitors and enhanced flavonoid production. However, 0.5 μM of the calcium ionophore A23187 showed contrary results. The increase in exogenous Ca²⁺ concentration in the presence of A23187 suppressed H₂O₂ bursts and peroxidase activity caused by metal elicitors. The results suggest that Ca²⁺ plays an inhibitory role in the plant cell response to metal elicitors. This suppression could have been caused by Ca²⁺ preventing the cells from absorbing metal ions and then easing the induction, or because the decrease of Ca²⁺ concentration worked as an induction signal. Therefore, reducing the Ca²⁺ concentration in culture medium, or adding Ca²⁺ antagonists could be used to improve flavonoid production and cell growth in combination with induction by metal elicitors during in vitro culture of T. hemsleyanum suspension cells.     

The use of diaminodithiol for labeling small molecules with technetium-99m

To investigate the labeling of small molecules with ^99mTc by the bifunctional chelate approach, we have synthesized both a fatty acid and an estrone derivative containing a chelator of the N₂S₂ type. In the case of the fatty acid, this was a diaminodithiol (DADT) while for the estrone, a diaminodisulfide (DADS) was attached. The estrone derivative (5-(2-methylene estrone 3-methyl ether)-3,3,10,10-tetramethyl-1, 2-dithia-5,8-diazacyclodecane hydrochloride, DADS-E) was prepared by alkylation of DADS while the fatty acid derivative (N-(11-undecanoic acid)-N,N′-bis(2-methyl-2-mercaptopropyl) ethylenediamine hydrochloride, DADT-FA) was synthesized by alkylation of DADS followed by reduction. DADS-E was labeled in ethanol at elevated temperatures while DADT-FA was labeled at room temperature, both by stannous reduction. Paper chromatography showed both to be labeled and reverse-phase HPLC showed multiple peaks for both. Serum stability studies were performed by incubation at 37 °C with aliquots removed at 1 min and 1 day for analysis by size-exclusion HPLC. Initially, little pertechnetate or binding to serum proteins was observed whereas after 1 day the majority of activity in both cases was protein bound with 20 and 38% pertechnetate appearing for DADT-FA and DADS-E respectively. In conclusion, small biologically active molecules may be labeled with ^99mTc through an attached diaminodithiol or diaminodisulfide group.