Involvement of photosynthesis in the action of temperature on plasmalemma transport inNitella

Summary At membrane potentials different fromE _K, the temperature effect on membrane potential ofNitella consists of two components. One of them changes its sign atE _K, the other one does not. This leads to the assignment of these components to changes in the K⁺ channel and in the H⁺ pump, respectively. It is shown that the fast time constant (3 to 30 sec) of the temperature effect on the H⁺ pump measured as a change in membrane potential and that of the temperature effect on the K⁺ channel measured as a change in resistance (having about twice the value of that of the pump) are sensitive to light intensity. Both time constants measured inNitella become smaller if light intensity increases from 0 to 15 Wm^–2. This supports the suggestion of Fisahn and Hansen (J. Exp. Bot. 37:440–460, 1986) that temperature acts on plasmalemma transport via photosynthesis via the same mechanism as light does.     

Ca2+ signals: The versatile decoders of environmental cues

Plants are often subjected to various environmental stresses that lead to deleterious effects on growth, production, sustainability, etc. The information of the incoming stress is read by the plants through the mechanism of signal transduction. The plant Ca²⁺ serves as secondary messenger during adaptations to stressful conditions and developmental processes. A plethora of Ca²⁺ sensors and decoders functions to bring about these changes. The cellular concentrations of Ca²⁺, their subcellular localization, and the specific interaction affinities of Ca²⁺ decoder proteins all work together to make this process a complex but synchronized signaling network. In this review, we focus on the versatility of these sensors and decoders in the model plant Arabidopsis as well as plants of economical importance. Here, we have also thrown light on the possible mechanism of action of these important components.     

Evidence for two K+ currents activated upon hyperpolarization ofParamecium tetraurelia

Summary Hyperpolarization of voltage-clampedParamecium tetraurelia in K⁺ solutions elicits a complex of Ca²⁺ and K⁺ currents. The tail current that accompanies a return to holding potential (–40 mV) contains two K⁺ components. The tail current elicited by a step to –110 mV of 50-msec duration contains fast-decaying (3.5 msec) and slow-decaying (20 msec) components. The reversal potential of both components shifts by 55–57 mV/10-fold change in external [K⁺], suggesting that they represent pure K⁺ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K⁺ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca²⁺ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K⁺ currents are inhibited by extracellular TEA⁺ in a concentration-dependent, noncooperative manner, whereas the fast K⁺ current alone is inhibited by 0.7mm quinidine.     

mt-tRNA Components: Synthesis of (2-Thio)Uridines Modified with Blocked Glycine/Taurine Moieties at C-5,1

In this paper, we discuss the usefulness of reductive amination of 5-formyl-2′,3′-O-isopropylidene(-2-thio)uridine with glycine or taurine esters in the presence of sodium triacetoxyborohydride (NaBH(OAc)₃) for the synthesis of the native mitochondrial (mt) tRNA components 5-carboxymethylaminomethyl(-2-thio)uridine (cmnm⁵(s²)U) and 5-taurinomethyl(-2-thio)uridine (τm⁵(s²)U) with a blocked amino acid function. 2-(Trimethylsilyl)ethyl and 2-(p-nitrophenyl)ethyl esters of glycine and 2-(2,4,5-trifluorophenyl)ethyl ester of taurine were selected as protection of carboxylic and sulfonic acid residues, respectively. The first synthesis of 5-formyl-2′,3′-O-isopropylidene-2-thiouridine is also reported.     

Ca2+ and the interaction of pore-formers with membranes

The interaction of pore-forming agents, such as Sendai virus, influenza virus (at pH 5 3), activated complement,Staphylococcus aureus α-toxin, melittin and polylysine, with the surface membrane of cells has been studied. In each case the following changes are initiated: collapse of membrane potential, leakage of ions, and leakage of phosphorylated metabolites. The changes can be inihibited by extracellular Ca²⁺ at physiological concentration; Mg²⁺ is less effective, and Zn²⁺ is more effective, than Ca²⁺ Ca²⁺ appears to act at a stage subsequent to the binding of pore-forming agent to cells. It is concluded that divalent cations are able to protect cells against the damaging effects of certain viruses, toxins or the components of activated complement in a manner that is worthy of further investigation.     

The elusive importance of being a mitochondrial Ca uniporter

The molecular components of the mitochondrial Ca²⁺ uptake machinery have been only recently identified. In the last months, in addition to the pore forming subunit and of one regulatory protein (named MCU and MICU1, respectively) other four components of this complex have been described. In addition, a MCU KO mouse model has been generated and a genetic human disease due to missense mutation of MICU1 has been discovered. In this contribution, we will first summarize the recent findings, discussing the roles of the different subunits of the mitochondrial Ca²⁺ uptake complex, pointing to the current contradictions in the published data, as well as possible explanations. Finally we will speculate on the recent, totally unexpected, results obtained in the MCU knock-out (KO) mice.     

Stimulation of lymphocyte receptor capping by the ionophore monensin

Summary The carboxylic ionophore monensin has a biphasic effect on antibody-induced Thy-1 cap formation. At higher concentrations, 5×10^–6–5×10^–5 m monensin causes a significant inhibition of receptor capping similar to that previously found with the Ca²⁺ selective ionophore A23187. At lower concentrations, 5×10^–8–5×10^–7 m capping is stimulated. It is concluded that capping at lower ionophore concentrations is a secific response to the ability of monensin to induce a rise in intracellular Na⁺, which indirectly elevates intracellular Ca²⁺ activity. This in turn activates the contractile machinery required for the aggregation of surface receptors into capped structures. At higher concentrations monensin acts as a nonspecific detergent, which causes detrimental structural alterations in some of the membrane components involved in the capping process.     

Purification and composition of Ca2+/Mg2+ ATPase from rat heart plasma membrane

The Ca²⁺/Mg²⁺ ATPase, which is activated by millimolar concentrations of Ca²⁺ or Mg²⁺, was solubilized from rat heart plasma membrane by employing lysophosphatidylcholine, CHAPS, Nal, EDTA and Tris-HCI at pH 7.4. The enzyme was purified by sucrose density gradient, Affi-Gel Blue column and Sepharose 6B column chromatography. The purified enzyme was seen as a single peptide band in the sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of about 90,000. The apparent molecular weight of the holoenzyme as determined under non-dissociating conditions by gel filtration on Sepharose 6B column was about 180,000 indicating two subunits. The enzyme was insensitive to ouabain, verapamil, vanadate, oligomycin, N,N-dicyclohexylcarbodiimide and NaN₃, but was markedly inhibited by 20 µM gramacidin S and 50 µM trifluoperazine. Analysis of the purified Ca²⁺/Mg²⁺ ATPase revealed the presence of 17 amino acids where leucine, glutamic acid and aspartic acid were the major components and histidine, cysteine and methionine were the minor components. The purified enzyme was associated with 19.7 µmol phospholipid/mg protein which was 60 times higher than the phospholipid content in plasma membrane. The cholesterol content in the purified enzyme preparation was 0.75 µmol/mg protein and this represented an 8-fold enrichment over plasma membrane. The glycoprotein nature of the enzyme was evident from the positive periodic acid-Schiff staining of the purified Cau2+/Mg^ATPase in the sodium dodecyl sulfate polyacrylamide gel. The polysaccharide content of the enzyme was enriched 8-fold over plasma membrane; neurominidase treatment decreased the polysaccharide content. Concanavalin A prevented the ATP-dependent inactivation of the purified Ca²⁺/Mg²⁺ ATPase and was found to bind to the purified enzyme with a K_D of 576 nM and Bmax of 4.52 nmol/mg protein. The results indicate that Ca²⁺/Mg²⁺ ATPase is a glycoprotein and contains a large amount of lipids.     

Three-way decomposition of a complete 3D 15N-NOESY-HSQC

Three-way decomposition is applied for the structural analysis of a complete three-dimensional ¹⁵N-NOESY-HSQC of the 128 residues long protein azurin. The procedure presented includes decomposition using the software MUNIN, providing an initial characterization of the complete spectrum by 355 components. This is followed by post-processing yielding a final list of 149 components, 123 of which characterize 1859 NOE peaks from backbone N-H groups. Components from three-way decomposition are defined as direct products of one-dimensional shapes along the three dimensions. Thus, a complete set of distance constraints from this spectrum is obtained by one-dimensional peak picking of the shapes along the NOE dimension. Correctness and completeness of this set of NOEs are tested for all backbone amide groups against both an independent peak picking algorithm and the three-dimensional crystal structure of azurin, and a coincidence of about 95% is observed. Automated `demixing' of components that are `mixed' in a complex manner due to overlap of the HN and/or ¹⁵N frequencies is illustrated.     

Salt tolerance in rice in vitro: Implication of accumulation of Na+, K+ and proline

The implication of accumulation of both inorganic (Na⁺, K⁺) and organic (proline) solutes were evaluated in unadapted and NaCl-adapted callus of a salt-sensitive (Basmati 370) and a salt-tolerant (SR-26B) cultivar of rice (Oryza sativa L.) after a NaCl shock. Accumulation of Na⁺,K⁺ and/or proline in callus was co relatable and the relative presence of these components in tissues after shock treatment was found to be important factors to support differential regrowth capacities of the shock treated calluses. Presence or retention of K⁺ in rice callus was a key factor for salt tolerance as it was observed to be positively correlated with growth in both the varieties. The results indicated that K⁺ was the first candidate to counteract the negative water potential of outside milieu, while proline was probably the last metabolic device that rice calluses opted for when exposed to salt stress.     

“Aqua-space®”, a New Headspace Method for Isolation of Natural Floral Aromas Using Humidified Air as a Carrier Gas

A new method called “Aqua-space^®” was developed for the isolation of the natural fragrances of plants. Living flowers were enclosed in a space under simulated natural conditions, and humidified air was pumped into the space as a fragrance carrier. In a comparison among three isolation methods, i.e., Aqua-space^®, headspace, and solvent extraction, the Aqua-space^® method proved to be the most effective in retaining natural fragrances with abundant oxygenated components key to floral fragrances.     

Influence of Al3+ on the titer of spiramycin and effective components in fermentor

Spiramycin is a multicomponent antibiotic, and different components have different antibacterial activities. In Streptomyces spiramyceticus 16-10-2, spiramycin II and spiramycin III (SPMII and SPMIII) are the main components, while spiramycin I (SPMI) needs to be controlled below 12%. Based on this, the influences of Al³⁺ on total spiramycin titer and components were investigated in this work. Those experiments were mainly performed in 15?L fermentor and Al³⁺ made a great improvement in spiramycin titer. The optimal adding concentration and adding time of Al³⁺ were 0.32?g/L at 12?hr. Under this condition, spiramycin titer was increased by 19.51% compared with the control. Moreover, the percentage of SPMII and SPMIII was increased by 7.14%. At the same time, the time of mycelia autolysis was lengthened. In addition, the specific activities of acetyl-CoA synthetase, acetate kinase, acetylphosphotransferase, and acylating enzyme were much higher than those of control. The content of acetic acid and succinic acid was beyond 3 and 4.5 times than that of control, respectively.     

Partial purification and characterization of (Na++K+)-ATPase from garfish olfactory nerve axon plasma membrane

Summary The (Na⁺+K⁺)-ATPase of garfish olfactory nerve axon plasma membrane was purified about sixfold by treatment of the membrane with sodium dodecyl sulfate followed by sucrose density gradient centrifugation. The estimated molecular weights of the two major polypeptide components of the enzyme preparation on sodium dodecyl sulfate gels were 110,000 and 42,000 daltons, which were different from those of the corresponding peptides of rabbit kidney (Na⁺+K⁺)-ATPase. No carbohydrate was detected in the 42,000-dalton component either by the periodic acid-Schiff reagent or by the more sensitive concanavalin A-peroxidase staining procedure. The molecular properties of the garfish (Na⁺+K⁺)-ATPase, such as theK _m for ATP, pH optimum, energies of activation, Na and K ion dependence and vanadium inhibition, were, however, similar to those of the kidney enzyme.The partially purified garfish (Na⁺+K⁺)-ATPase was reconstituted into phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted enzyme was found to catalyze a time and ATP dependent²²Na⁺ transport. The ratio of²²Na⁺ pumped to ATP hydrolyzed was about 1; under the same reconstitution and assay conditions, eel electroplax (Na⁺+K⁺)-ATPase, however, gave a²²Na⁺ pumped to ATP hydrolyzed ratio of nearly 3.     

A comparative study between a brain Na+,K(+)-ATPase inhibitor (endobain E) and ascorbic acid

In the search of Na⁺,K⁺-ATPase modulators, we have reported the isolation by gel filtration and HPLC of a brain fraction, termed endobain E, which highly inhibits Na⁺,K⁺-ATPase activity. In the present study we compared some properties of endobain E with those of ascorbic acid. Kinetic experiments assaying synaptosomal membrane K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activity in the presence of endobain E or ascorbic acid showed that in neither case did enzyme inhibition prove competitive in nature versus K⁺ or p-NPP concentration. At pH 5.0, endobain E and ascorbic acid maximal UV absorbance was 266 and 258 nm, respectively; alkalinization to pH 14.0 led to absorption drop and shift for endobain E but to absorbance disappearance for ascorbic acid. After cysteine treatment, endobain E absorbance decreased, whereas that of ascorbic acid remained unaltered; iodine treatment led to absorbance drop and shift for endobain E but to absorbance disappearance for ascorbic acid. HPLC analysis of endobain E disclosed the presence of two components: one eluting with retention time and UV spectrum indistinguishable from those of ascorbic acid and a second, as yet unidentified, both exerting Na⁺,K⁺-ATPase inhibition.     

Picosecond fluorescence kinetics in spinach chloroplasts at room temperature. Effects of Mg

Single-photon timing with picosecond resolution is used to investigate the effect of Mg²⁺ on the room-temperature fluorescence decay kinetics in broken spinach chloroplasts. In agreement with an earlier paper (Haehnel, W., Nairn, J.A., Reisberg, P. and Sauer, K. (1982) Biochim. Biophys. Acta 680, 161–173), we find three components in the fluorescence decay both in the presence and in the absence of Mg²⁺. The behavior of these components is examined as a function of Mg²⁺ concentration at both the F₀ and the F_max fluorescence levels, and as a function of the excitation intensity for thylakoids from spinach chloroplasts isolated in the absence of added Mg²⁺. Analysis of the results indicates that the subsequent addition of Mg²⁺ has effects which occur at different levels of added cation. At low levels of Mg²⁺ (less than 0.75 mM), there appears to be a decrease in communication between Photosystem (PS) II and PS I, which amounts to a decrease in the spillover rate between PS II and PS I. At higher levels of Mg²⁺ (about 2 mM), there appears to be an increase in communication between PS II units and an increase in the effective absorption cross-section of PS II, probably both of these involving the chlorophyll light-harvesting antenna.     

Remodeling of calcium signaling in tumor progression

Intracellular Ca²⁺ is one of the crucial signalings that modulate various cellular functions. The dysregulation of Ca²⁺ homeostasis has been suggested as an important event in driving the expression of the malignant phenotypes, such as proliferation, migration, invasion, and metastasis. Cell migration is an early prerequisite for tumor metastasis that has a significant impact on patient prognosis. During cell migration, the exquisite spatial and temporal organization of intracellular Ca²⁺ provides a rapid and robust way for the selective activation of signaling components that play a central role in cytoskeletal reorganization, traction force generation, and focal adhesion dynamics. A number of known molecular components involved in Ca²⁺ influx pathways, including stromal interaction molecule (STIM)/Orai-mediated store-operated Ca²⁺ entry (SOCE) and the Ca²⁺-permeable transient receptor potential (TRP) channels, have been implicated in cancer cell migration and tumor metastasis. The clinical significance of these molecules, such as STIM proteins and the TRPM7 channel, in tumor progression and their diagnostic and prognostic potentials have also been demonstrated in specific cancer types. In this review, we summarize the recent advances in understanding the important roles and regulatory mechanisms of these Ca²⁺ influx pathways on malignant behaviors of tumor cells. The clinical implications in facilitating current diagnostic and therapeutic procedures are also discussed.     

A CAUTIONARY NOTE ON THE USE OF THE 31P NMR SPECTROSCOPY IN STEREOCHEMICAL CORRELATION ANALYSIS

Stereoselectivity in condensation of protected ribonucleoside 3′-H-phosphonates with hydroxylic components was investigated using ³¹P NMR spectroscopy. The correlation between absolute configuration at the phosphorus center and the chemical shifts of the produced H-phosphonate diesters and the corresponding phosphorothioates, was studied.