Anoxia-induced elevation of cytosolic Ca<Superscript>2+</Superscript> concentration depends on different Ca<Superscript>2+</Superscript> sources in rice and wheat protoplasts

The anoxia-dependent elevation of cytosolic Ca²⁺ concentration, [Ca²⁺]_cyt, was investigated in plants differing in tolerance to hypoxia. The [Ca²⁺]_cyt was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca²⁺]_cyt in rice leaf protoplasts. A tenfold drop in the external Ca²⁺ concentration (to 0.1 mM) resulted in considerable decrease of the [Ca²⁺]_cyt shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La³⁺ and EGTA) and intracellular membrane Ca²⁺-channel antagonists (Li⁺, ruthenium red and cyclosporine A) reduced the anoxic Ca²⁺-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca²⁺]_cyt. In wheat leaf protoplasts, the amplitude of the Ca²⁺-shift little depended on the external level of Ca²⁺. Wheat root protoplasts were characterized by a small shift of [Ca²⁺]_cyt under anoxia. Plasmalemma Ca²⁺-channel blockers had little effect on the elevation of cytosolic Ca²⁺ in wheat protoplasts. Intact rice seedlings absorbed Ca²⁺ from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca²⁺. Verapamil abolished the Ca²⁺ influx in rice roots and Ca²⁺ efflux from wheat roots. Anoxia-induced [Ca²⁺]_cyt elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca²⁺ stores are important for anoxic [Ca²⁺]_cyt elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca²⁺]_cyt rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.     

Salt stress-induced programmed cell death via Ca<Superscript>2+</Superscript>-mediated mitochondrial permeability transition in tobacco protoplasts

The change in cytosolic free concentration of calcium ([Ca²⁺]_cyt) plays a key role in regulating apoptosis in animal cells. In our experiment, we tried to investigate the function of Ca²⁺ in programmed cell death (PCD) in tobacco (Nicotiana tobacum, cultivar BY-2) protoplasts induced by salt stress. An obvious increase in [Ca²⁺]_cyt was observed a few minutes after treatment and the onset of a decrease in mitochondrial membrane potential (ΔΨ_m) was also observed before the appearance of PCD, pre-treatment of protoplasts with EGTA or LaCl₃ effectively retarded the increase in [Ca²⁺]_cyt, which was concomitant with the decrease in the percentage of cell death and higher ΔΨ_m, pre-treatment with cyclosporine A (CsA) also effectively retarded the increase in [Ca²⁺]_cyt, the decrease in ΔΨ_m and the onset of PCD. All these results suggest that Ca²⁺ is a necessary element in regulating PCD and the increase in [Ca²⁺]_cyt and the opening of mitochondrial permeability transition pore (MPTP) could promote each other in regulating PCD in tobacco protoplasts induced by salt stress.Jiusheng Lin and Yuan Wang-These authors contributed equally for this work.     

Characterization of whole-cell k<Superscript>+</Superscript> currents across the plasma membrane of pollen grain and tube protoplasts of <Emphasis Type="Italic">Lilium longiflorum</Emphasis>

Outward and inward currents, mainly carried by K⁺, were detected in protoplasts of pollen grains (PG) and pollen tubes (PT) of Lilium longiflorum Thunb. by using the whole-cell configuration of the patch-clamp technique. The outward K⁺ current (I_{K+ out}) was similar in both protoplast types, while the inward K⁺ current (I_{K+ in}) was higher in pollen tube protoplasts. In PT but not in PG protoplasts, inward K⁺ currents were already detectable at negative membrane voltages usually monitored in lily pollen. I_{K+ in} consisted of a slow and a fast current component, as revealed by fitting a sum of two exponential functions to the time-dependent current. The contribution of the fast component to the total inward current was higher in PT than in PG protoplasts, which was even more evident at acidic pH of the external medium. Therefore, based on the measured characteristics, the I_{K+ in} of PT protoplasts may contribute to the endogenous K⁺ currents surrounding a growing pollen tube. Abbreviations: BS, bath solution; BTP, bis-Tris-propane; MES, 2-N-morpholinoethane sulfonic acid; V_act, activation voltage; V_M, membrane voltage; E_rev, reversal potential; I_{K+ in}, inward K⁺ current; I_{K+ out}, outward K⁺ current; PG, pollen grain; PT, pollen tube; PM, pipette medium     

The activity of plasma membrane hyperpolarization-activated Ca<Superscript>2+</Superscript> channels during pollen development of <Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>

Calcium (Ca²⁺) plays crucial roles in regulation of pollen tube growth. The influx of Ca²⁺ into the pollen tube is mediated by ion channels, and the density and activity of Ca²⁺ channels in pollen plasma membranes critically determines their electrical properties. In this report, using whole-cell and single-channel patch-clamping techniques, we investigated developmental changes of hyperpolarization-activated Ca²⁺ channel activity in pear (Pyrus pyrifolia) pollen and its relationship with pollen viability. For both pollen and pollen tubes, hyperpolarization-activated Ca²⁺ channels had the same conductance and cAMP sensitivity, indicating that they were the same channels. However, the Ca²⁺ current density in pollen tube protoplasts was greater than that in pollen protoplasts. Compared with day-3 flowers’ pollen, hyperpolarization-activated Ca²⁺ current density was significantly lower in day 0 and day 3 flowers’ pollen, which was consistent with the pollen germination and pollen tube growth, indicating that pollen protoplasts’ increased Ca²⁺ current density may have enhanced the pollen viability. During pollen tube elongation, pollen tube plasma membrane Ca²⁺ current density increased with increased length pollen tubes up to 300 μm. All of these results indicated that hyperpolarization-activated Ca²⁺ channel activity was associated with in pear pollen development and may have a causal link between Ca²⁺ channel activity and pollen viability.     

Protoplast Formation of the Coccolithophorid <Emphasis Type="Italic">Pleurochrysis haptonemofera</Emphasis> in Hypoosmotic K<Superscript>+</Superscript> Solution: Shedding of the Coccosphere and Regrowth of the Protoplast in Normal Medium

Both coccolith-bearing cells (C-cells) and naked cells (N-cells) of the coccolithophorid Pleurochrysis haptonemofera can grow in salinities of more than 7‰ (about 20% of a “normal” sea water salinity [35‰]), with the highest growth rates in salinities of more than 14‰. Microscopic observations of cells suspended in 100 mM NaCl (7‰) showed that, while N-cells were swelling uniformly all over the cell surface, C-cells were bulging the plasma membrane from the hole of the coccosphere at the apical (flagellar) pole of the cell. Effects of several cations and anions on the morphological change of C-cells under hypoosmotic pressure were investigated. When 100 mM K⁺ was used, protoplasts were released from the coccosphere completely in almost all the cells. This phenomenon was shown with K⁺ most effectively. The protoplasts could grow in the fresh medium and form the first coccolith within 9 h.     

Gating of Cl<Superscript>-</Superscript> Currents in Protoplasts from the Marine Alga Valonia utricularis Depends on the Transmembrane Cl<Superscript>-</Superscript> Gradient and Is Affected by Enzymatic Cell Wall Degradation

The electrical properties of protoplasts of the turgor pressure-regulating giant marine alga Valonia utricularis were investigated by using the patch-clamp technique. In the whole-cell configuration, large inward currents were elicited by negative-going voltage pulses. The time-dependent component was predominantly carried by Cl-, as revealed by 'tail current' analysis. When experiments were performed on protoplasts directly after mechanical release from the 'mother cell', small outward currents were additionally observed at membrane voltages more positive than ECl-. These outward currents disappeared to a large extent after treatment of the protoplasts with a mixture of cell wall-degrading enzymes. Plots of the chord conductance versus the clamped membrane voltage revealed that enzymatic treatment affected the gating properties. By fitting Boltzmann distributions to the data, a midpoint potential of + 5 +/- 5 mV (n = 7) was obtained in symmetrical Cl- solutions for mechanically released protoplasts. In contrast, protoplasts treated additionally with enzymes exhibited a midpoint potential of -13 +/- 5 mV (n = 8). By varying the external and internal Cl- concentration, gating was also shown to depend on the Cl- gradient across the plasmalemma both in enzymatically treated and untreated protoplasts. Plotting of the midpoint potential against the Nernst potential of Cl- rendered a slope less than 1 (0.70 and 0.64, respectively) indicating that gating did not strictly depend on the electrochemical Cl- gradient. The voltage- and Cl--dependence as well as inhibition experiments with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) suggested that the Cl- conductance of the membrane is dominated by the Valonia Anion Channel 1 (VAC1) described by Heidecker, M., Wegner, L.H., Zimmermann, U. 1999: A patch-clamp study of ion channels in proto-plasts prepared from the marine alga Valonia utricularis. J. Membrane Biol. 172:235-247. The relevance of the findings for membrane potential control and turgor regulation in V. utricularis as well as the general implications of the data for electrophysiological work on protoplasts (that are usually obtained by enzymatic digestion of plant tissue) are discussed.     

Polar-localised poplar K<Superscript>+</Superscript> channel capable of controlling electrical properties of wood-forming cells

In previous studies, we have shown that annual expression profiles of cambial and wood tissue with respect to the Shaker K⁺ channel PTORK correlate with cambial activity. To follow PTORK-gene activity on the cellular level, we isolated the respective promoter regions and generated transgenic Arabidopsis plants expressing the GUS gene under the control of the PTORK promoter. Cross-sections of petioles showed PTORK-driven signals predominantly in the xylem parenchyma surrounding the vessels and in the phloem. Antibodies raised against a unique N-terminal region of PTORK in histo-immunochemical analyses recognised this K⁺-release channel in growth-active poplar plants only. PTORK labelling was found in differentiating xylem cells (young fibres) and mature xylem (vessel-associated cells of the ray parenchyma). Patch-clamp measurements on fibre cell protoplasts, derived from young poplar twigs, identified outward-rectifying K⁺ channels as the major K⁺ conductance of this cell type, which resembled the biophysical properties of PTORK when expressed in Xenopus oocytes.Electronic Supplementary Material Supplementary material is available for this article at Matthias Arend and Andrea Stinzing contributed equally to this work     

La<Superscript>3+</Superscript> uptake and its effect on the cytoskeleton in root protoplasts of<Emphasis Type="Italic"> Zea mays</Emphasis> L.

La³⁺ ions are known to antagonize Ca²⁺ and are used as a Ca²⁺ channel blocker but little is known on the direct effects of La³⁺. Micromolar La³⁺ concentrations promoted root growth while higher concentrations were inhibitory. The uptake of La³⁺ in maize root protoplasts revealed a membrane binding component (0.14 and 0.44 pmol min^–1 protoplast^–1 for 100 and 1,000 M La³⁺) followed by a slower concentration and time-dependent uptake. Uptake was reduced by Ca²⁺, but had no substantial effect on other ions. La³⁺ shifted microtubule organization from random to parallel but caused aggregation of microfilaments. Our data suggest that La³⁺ is taken up into plant cells and affects growth via stabilization of the cytoskeleton.     

Mechanisms of Extracellular NO and Ca<Superscript>2+</Superscript> Regulating the Growth of Wheat Seedling Roots

Our previous studies suggested the cross talk of nitric oxide (NO) with Ca²⁺ in regulating stomatal movement. However, its mechanism of action is not well defined in plant roots. In this study, sodium nitroprusside (SNP, a NO donor) showed an inhibitory effect on the growth of wheat seedling roots in a dose-dependent manner, which was alleviated through reducing extracellular Ca²⁺ concentration. Analyzing the content of Ca²⁺ and K⁺ in wheat seedling roots showed that SNP significantly promoted Ca²⁺ accumulation and inhibited K⁺ accumulation at a higher concentration of extracellular Ca²⁺, but SNP promoted K⁺ accumulation in the absence of extracellular Ca²⁺. To gain further insights into Ca²⁺ function in the NO-regulated growth of wheat seedling roots, we conducted the patch-clamped protoplasts of wheat seedling roots in a whole cell configuration. In the absence of extracellular Ca²⁺, NO activated inward-rectifying K⁺ channels, but had little effects on outward-rectifying K⁺ channels. In the presence of 2 mmol L⁻¹ CaCl₂ in the bath solution, NO significantly activated outward-rectifying K⁺ channels, which was partially alleviated by LaCl₃ (a Ca²⁺ channel inhibitor). In contrast, 2 mmol L⁻¹ CaCl₂ alone had little effect on inward or outward-rectifying K⁺ channels. Thus, NO inhibits the growth of wheat seedling roots likely by promoting extracellular Ca²⁺ influx excessively. The increase in cytosolic Ca²⁺ appears to inhibit K⁺ influx, promotes K⁺ outflux across the plasma membrane, and finally reduces the content of K⁺ in root cells.     

Exclusive cytosolic localization and broad tRNA<Superscript>Ser</Superscript> specificity of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> seryl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) decipher the genetic code, covalently linking amino acids to cognate tRNAs, thus preparing substrates for the process of translation. Although aaRSs funtion primarily in translation and are localized in cytosol, mitochondria and chloroplasts there are many reports on their additional functions and subcellular destinations beyond translation. However, data on plant aaRSs are scarce. Initial analysis of amino acid sequence of Arabidopsis thaliana seryl-tRNA synthetase (SerRS) suggested that protein contains putative nuclear localization signals. GFP-localization experiments in transiently transformed epidermal onion cells and Arabidopsis protoplasts gave ambiguous results because in some cells SerRS appeared to be dually localized to both cytosol and nucleus. However, data obtained on transgenic lines expressing SerRS-TAP and GFP-SerRS revealed exclusive cytosolic location of SerRS. Subcellular distribution of SerRS did not change during stress. Cytosolic Arabidopsis SerRS was expressed and purified. The enzyme efficiently aminoacylated eukaryotic and bacterial tRNAs^Ser, that are structurally very different. Given the fact that the same behavior was previously shown for monocot maize SerRS, it seems that plant SerRSs exhibit unusually broad tRNA^Ser specificity, unlike SerRSs from other organisms. Possible functional implications of this unique characteristic of plant SerRSs are discussed.     

The effects of oligomycin on content of adenylates in mesophyll protoplasts,chloroplasts and mitochondria from Pb<Superscript>2+</Superscript> treated pea and barley leaves

The effects of oligomycin on photosynthesis and respiration in relation to ATP production in chloroplasts and mitochondria were investigated in protoplasts isolated from the detached pea (Pisum sativum L cv. Iłowiecki.) and barley (Hordeum vulgare L. cv. Gunilla) leaves treated 5 mM Pb(NO₃)₂. The oligomycin (OM), an inhibitor of oxidative phosphorylation at 0.1 μM concentration caused the inhibition of photosynthesis rate in the protoplasts from both the control and the Pb-treated pea leaves. The respiration rate and ATP/ADP ratio in the protoplasts and the activity of ATPase in mitochondria, were also diminished in the control protoplasts. These effects were not observed in the protoplasts and mitochondria isolated from the Pb-treated leaves. Oligomycin, an inhibitor of photophosphorylation at 10 μM concentration decreased ATPase activity in chloroplasts from both the control and the Pb- treated leaves. Using the method of rapid fractionation of barley protoplasts it was shown that the ATP/ADP ratio in the mitochondria from Pb-treated leaves was largely suppressed (from 1.8 to 0.4) by OM under nonphotorespiratory conditions (high CO₂), whereas under photorespiratory conditions (low CO₂) this ratio was high (5.3) and under OM decreased less (to 3.1). Our results indicate that oligomycin, in organelle isolated from Pb-treated leaves, had no inhibitory effect on the mitochondrial ATPase, whereas it inhibited chloroplasts ATPase. We suggest that Pb ions affected the catalytic cycle and/or conformational changes of ATPase in pea chloroplasts differently than in mitochondria. The differences in Pb responses may reflect fine mechanisms for the regulation of ATP production in the plant cells under stress conditions.     

<Superscript>13</Superscript>C-<Superscript>13</Superscript>C NOESY: A constructive use of <Superscript>13</Superscript>C-<Superscript>13</Superscript>C spin-diffusion

¹³C-¹³C NOESY experiments were performed under long mixing time conditions on reduced human superoxide dismutase (32 kDa, ¹⁵N, ^13C and 70% ²H labeled). ¹³C-¹³C couplings were successfully eliminated through post-processing of in-phase-anti-phase (IPAP) data. It appears that at mixing time _m of 3.0 s the spin diffusion mechanism allows the detection of 96% of the two-bond correlations involving C and C. The interpretation was confirmed by simulations. This approach broadens the range of applicability of ¹³C-¹³C NOESY spectroscopy.     

Simultaneous <Superscript>1</Superscript>H- or <Superscript>2</Superscript>H-, <Superscript>15</Superscript>N- and multiple-band-selective <Superscript>13</Superscript>C-decoupling during acquisition in <Superscript>13</Superscript>C-detected experiments with proteins and oligonucleotides

Significant resolution improvement in ¹³C,¹³C-TOCSY spectra of uniformly deuterated and ¹³C, ¹⁵N-labeled protein and ¹³C,¹⁵N-labeled RNA samples is achieved by introduction of multiple-band-selective ¹³C-homodecoupling applied simultaneously with ¹H- or ²H- and ¹⁵N-decoupling at all stages of multidimensional experiments including signal acquisition period. The application of single, double or triple band-selective ¹³C-decoupling in 2D-[¹³C,¹³C]-TOCSY experiments during acquisition strongly simplifies the homonuclear splitting pattern. The technical aspects of complex multiple-band homonuclear decoupling and hardware requirements are discussed. The use of this technique (i) facilitates the resonance assignment process as it reduces signal overlap in homonuclear ¹³C-spectra and (ii) possibly improves the signal-to-noise ratio through multiplet collapse. It can be applied in any ¹³C-detected experiment.     

Development of an optimal culture system for callogenesis of <Emphasis Type="Italic">Chrysanthemum indicum</Emphasis> protoplasts

This study is a comparison of four methods to induce calli formation in a protoplast culture of Chrysanthemum indicum. Culture in liquid medium (17.6 calli/10⁵ protoplasts) was preferable to culture in solid agarose beads, although the efficiency of the latter could be improved by layering them on glass beads (12.5 vs. 0.83 calli/10⁵ protoplasts). Culture of protoplasts on moistened filter paper was unsuccessful. In the liquid media, microcalli and calli were induced efficiently and easily after 6 weeks. These effects may be explained by reduced toxicity due to cell breakdown and improved aeration.     

An efficient protocol for plant regeneration from protoplasts of the moss <Emphasis Type="Italic">Atrichum undulatum P. Beauv in vitro</Emphasis>

A system for plant regeneration from protoplasts of the moss, Atrichum undulatum (Hedw.) P. Beauv. in vitro, is first reported. Viable protoplasts were isolated at about 9 × 10⁵ protoplasts g⁻¹ fresh weight from 10 to 18 days protonemata. For regeneration of protoplasts, viable protoplasts were cultured in liquid–solid medium containing surface liquid medium MS (0.4 M mannitol) and subnatant solid medium Benecke (0.3 M mannitol) at 20 °C under a 16-h photoperiod white light after 12 h preculture in darkness at 20 °C. The great majority of protoplasts follow a regenerative sequence: formation of asymmetric cells in 2–3 days; division of the asymmetric cells to 2–3 cells in 4–5 days, and further develop to produce a new chloronemal filament in 15 days. Juvenile gametophyte can be visible in 20 days. The plating ratio of cell cluster regenerated from protoplasts reaches up to 45%. Transient expression experiments indicate the electroporation uptake of DNA is possible.     

Competitive binding of Fe<Superscript>3+</Superscript>, Cr<Superscript>3+</Superscript>, and Ni<Superscript>2+</Superscript> to transferrin

Competitive binding of Fe³⁺, Cr³⁺, and Ni²⁺ to transferrin (Tf) was investigated at various physiological iron to Tf concentration ratios. Loading percentages for these metal ions are based on a two M ⁿ⁺ to one Tf (i.e., 100% loading) stoichiometry and were determined using a particle beam/hollow cathode–optical emission spectroscopy (PB/HC-OES) method. Serum iron concentrations typically found in normal, iron-deficient, iron-deficient from chronic disease, iron-deficient from inflammation, and iron-overload conditions were used to determine the effects of iron concentration on iron loading into Tf. The PB/HC-OES method allows the monitoring of metal ions in competition with Fe³⁺ for Tf binding. Iron-overload concentrations impeded the ability of chromium (15.0 μM) or nickel (10.3 μM) to load completely into Tf. Low Fe³⁺ uptake by Tf under iron-deficient or chronic disease iron concentrations limited Ni²⁺ loading into Tf. Competitive binding kinetic studies were performed with Fe³⁺, Cr³⁺, and Ni²⁺ to determine percentages of metal ion uptake into Tf as a function of time. The initial rates of Fe³⁺ loading increased in the presence of nickel or chromium, with maximal Fe³⁺ loading into Tf in all cases reaching approximately 24%. Addition of Cr³⁺ to 50% preloaded Fe³⁺–Tf showed that excess chromium (15.0 μM) displaced roughly 13% of Fe³⁺ from Tf, resulting in 7.6 ± 1.3% Cr³⁺ loading of Tf. The PB/HC-OES method provides the ability to monitor multiple metal ions competing for Tf binding and will help to understand metal competition for Tf binding.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis of sialyl Lewis<Superscript>a</Superscript> (sLe<Superscript>a</Superscript>, CA19-9) and construction of an immunogenic sLe<Superscript>a</Superscript> vaccine

Sialyl Lewis^a (sLe^a), also termed CA19-9 antigen, is recognized by murine mAb19-9 and is expressed on the cancer cell surface as a glycolipid and as an O-linked glycoprotein. It is highly expressed in a variety of gastrointestinal epithelial malignancies including colon cancer and pancreatic cancer, and in breast cancer and small cell lung cancer, but has a limited expression on normal tissues. sLe^a is known to be the ligand for endothelial cell selectins suggesting a role for sLe^a in cancer metastases and adhesion. For these reasons, sLe^a may be a good target for antibody mediated immunotherapy including monoclonal antibodies and tumor vaccines. However, sLe^a is structurally similar to sLe^x and other blood group related carbohydrates which are widely expressed on polymorphonucleocytes and other circulating cells, raising concern that immunization against sLe^a will induce antibodies reactive with these more widely expressed autoantigens. We have shown previously both in mice and in patients that conjugation of a variety of carbohydrate cancer antigen to keyhole limpet hemocyanin (KLH) and administration of this conjugate mixed with saponin adjuvants QS-21 or GPI-0100 are the most effective methods for induction of antibodies against these cancer antigens. We describe here for the first time the total synthesis of pentenyl glycoside of sLe^a hexasaccharide and its conjugation to KLH to construct a sLe^a-KLH conjugate. Groups of five mice were vaccinated subcutaneously four times over 6 weeks. Sera were tested against sLe^a-HSA by ELISA and against sLe^a positive human cell lines adenocarcinoma SW626 and small cell lung cancer (SCLC) DMS79 by FACS. As expected, mice immunized with unconjugated sLe^a plus GPI-0100 or unconjugated sLe^a mixed with KLH plus GPI-0100 failed to produce antibodies against sLe^a. However, mice immunized with sLe^a-KLH conjugate without GPI-0100 produced low levels of antibodies and mice immunized with sLe^a-KLH plus GPI-0100 produced significantly higher titer IgG and IgM antibodies against sLe^a by ELISA. These antibodies were highly reactive by FACS and mediated potent complement mediated cytotoxicity against sLe^a positive SW626 and DMS79 cells. They showed no detectable cross reactivity against a series of other blood group-related antigens, including Le^y, Le^x, and sLe^x by dot blot immune staining. This vaccine is ready for testing as an active immunotherapy for treating sLe^a positive cancer in clinical settings. Govind Ragupathi and Philip O. Livingston are paid consultants and shareholders in MabVax Therapeutics, Inc., San Diego, CA 92121. The sLe^a vaccine is licensed to MabVax.     

Modulation of the reaction cycle of the Na<Superscript>+</Superscript>:Ca<Superscript>2+</Superscript>, K<Superscript>+</Superscript> exchanger

Ca²⁺ concentration in retinal photoreceptor rod outer segment (OS) strongly affects the generator potential kinetics and the receptor light adaptation. The response to intense light stimuli delivered in the dark produce potential changes exceeding 40 mV: since the Ca²⁺ extrusion in the OS is entirely controlled by the Na⁺:Ca²⁺, K⁺ exchanger, it is important to assess how the exchanger ion transport rate is affected by the voltage and, in general, by intracellular factors. It is indeed known that the cardiac Na⁺:Ca²⁺ exchanger is regulated by Mg-ATP via a still unknown metabolic pathway. In the present work, the Na⁺:Ca²⁺, K⁺ exchanger regulation was investigated in isolated OS, recorded in whole-cell configuration, using ionic conditions that activated maximally the exchanger in both forward and reverse mode. In all species examined (amphibia: Rana esculenta and Ambystoma mexicanum; reptilia: Gecko gecko), the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. Since hyperpolarisation increases Ca²⁺ extrusion rate, the recovery of the dark level of Ca²⁺ (and, in turn, of the generator potential) after intense light stimuli results accelerated. Mg-ATP increased the size of forward and reverse exchange current by a factor of ∼2.3 and ∼2.6, respectively, without modifying their voltage dependence. This indicates that Mg-ATP regulates the number of active exchanger sites and/or the exchanger turnover number, although via an unknown mechanism. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.