Descripción: The high-altitude Andean lakes (HAAL) in the Argentinean Puna-high Andes region represent an almost unexplored ecosystem exposed to extreme conditions (high UV irradiation, hypersalinity, drastic temperature changes, desiccation, and high pH). Here we present the first genome sequence, a Sphingomonas sp., isolated from this extreme environment. © 2011, American Society for Microbiology.

Descripción: We studied the transepithelial potential difference (TEPD) and 22Na flux across isolated perfused gills (anterior pair 5 and posterior pairs 6-8) of the crab Chasmagnathus granulatus acclimated to either hypo- or hyper-osmotic conditions. The gills of crabs acclimated to low salinity, perfused and bathed with 10%‰ saline solutions, produced the following TEPDs (hemolymph side with respect to bath side): 0.4±0.7, -10.2±1.6, -10.8±1.3 and -6.7±1.3mV for gills 5, 6, 7 and 8, respectively. Gills 6, 7 and 8 did not differ significantly. Reducing the saline concentration of bath and perfusate from 30%‰ to 20%‰ or 10%‰ increased significantly the TEPDs of these gills. TEPDs of gill 6 (representative of posterior gills) were reduced by 69±5% and 60±5% after perfusion with ouabain or BaCl2 (5mmoll-1 each), respectively. The same gill showed a net ouabain-sensitive Na+ influx of 1150±290 μequiv g-1h-1. Gill 6 of crabs acclimated to high salinity produced TEPDs of -1.5±0.1 and -1.3±0.09mV after perfusion with 30%‰ or 40%‰ salines, respectively. Perfusion with ouabain or BaCl2 reduced TEPDs by 76±7% and 86±4%, respectively. A net ouabain-sensitive Na+ efflux of 2282±337 μequiv g-1h-1 was recorded in gill 6 perfused with 38%‰ saline.

Descripción: With aging, the kidney develops a progressive deterioration of several structures and functions. Proximal tubular acidification is impaired in old rats with a decrease in the activity of brush border Na+/ H+ exchange and a fall of H-ion flux measured with micropuncture experiments. In the present work we evaluate the contribution of 5-N-ethyl-n-isopropyl amiloride- (EIPA) and bafilomycin-sensitive bicarbonate flux (JHCO3-) in proximal convoluted tubules of young and aged rats. We performed micropuncture experiments inhibiting the Na+/H+ exchanger with EIPA (10-4 M) and the V-H+ ATPase with bafilomycin (10-6 M). We used antibodies against the NHE3 isoform of the Na+/H+ exchanger and the subunit E of the V-H+ ATPase for detecting by Western blot the abundance of these proteins in brush border membrane vesicles from proximal convoluted tubules of young and old rats. The abundance of NHE3 and the V-H+ ATPase was similar in 18-month-old and 3-month-old rats. The bicarbonate flux in old rats was 30% lower than in young rats. EIPA reduced JHCO3- by 60% and bafilomycin by 30% in young rats; in contrast, EIPA reduced JHCO3- by ∼40% and bafilomycin by ∼50% in old rats. The JHCO3- inhibited by bafilomycin was the same in young and old rats: 0.62.nmol · cm-2 · s-1 and 0.71.nmol · cm-2 · s-1, respectively. However, the EIPA-sensitive fraction was larger in young than in old rats: 1.26.nmol · cm-2 · s-1 vs. 0.85 nmol · cm-2 · s-1, respectively. These results suggest that the component more affected in bicarbonate reabsorption of proximal convoluted tubules from aged rats is the Na+-H+ exchanger, probably a NHE isoform different from NHE3. © 2006 The Authors Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2006.

Descripción: In this work we studied the effect of NaCl on the thermodynamic and dynamic properties of supercooled water, for salt concentrations between 0.19 and 1.33mol kg-1, using molecular dynamic simulations for TIP5PE water model and ion parameters specially designed to be used in combination with this potential. We studied the isobaric heat capacity (Cp) temperature dependence and observed a maximum in Cp, occurring at Tm, that moves to lower temperature values with increasing salt concentration. Many characteristic changes were observed at scaled temperature TTm ∼ 0.96, namely a minimum in the density of the system, a reduction of the slope of the number of hydrogen bonds vs. temperature, and a crossover from Vogel-Tamman-Fulcher to Arrhenius dynamics. Finally, at low temperatures we observed that water dynamics become heterogeneous with an apparently common relationship between the fraction of immobile molecules and T/Tm for all studied systems. © 2011 American Institute of Physics.

Descripción: The possible involvement of apoplastic reactive oxygen species produced by the oxidation of free polyamines in the leaf growth of salinized maize has been studied here. Salt treatment increased the apoplastic spermine and spermidine levels, mainly in the leaf blade elongation zone. The total activity of polyamine oxidase was up to 20-fold higher than that of the copper-containing amine oxidase. Measurements of H2O2, ·O2-, and HO· production in the presence or absence of the polyamine oxidase inhibitors 1,19-bis- (ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane suggest that, in salinized plants, the oxidation of free apoplastic polyamines by polyamine oxidase by would be the main source of reactive oxygen species in the elongation zone of maize leaf blades. This effect is probably due to increased substrate availability. Incubation with 200 μM spermine doubled segment elongation, whereas the addition of 1,19-bis-(ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane to 200 μM spermine attenuated and reversed the last effect, respectively. Similarly, the addition of MnCl2 (an ·O2- dismutating agent) or the HO· scavenger sodium benzoate along with spermine, annulled the elongating effect of the polyamine on the salinized segments. As a whole, the results obtained here demonstrated that, under salinity, polyamine oxidase activity provides a significant production of reactive oxygen species in the apoplast which contributes to 25-30% of the maize leaf blade elongation.

Descripción: Split lamellae of posterior gills of Chasmagnathus granulatus adapted to 2.5‰ salinity were mounted in a modified Ussing chamber. With NaCl-saline on both sides of the preparation a transepithelial voltage (Vte) of 4.1±0.5 mV (outside positive) was measured. After voltage-clamping, the negative short-circuit current (Isc) amounted to -142±21 μA cm-2 at a conductance (Gte) of 44±5 mS cm-2. Substitution of either chloride (by nitrate) or sodium (by choline) on both sides of split gill lamellae significantly reduced Isc (by 70-80%) and Gte (by 30-50%). External CsCl (but not BaCl2 or furosemide) inhibited the negative Isc without affecting Gte. Addition of ouabain, BaCl2 or diphenylamine-2-carboxylate to the internal bath inhibited Isc at unchanged Gte. Internal acetazolamide did not affect Isc or Gte across split gill lamellae. Unidirectional Na+ influx across isolated and perfused posterior gills, however, was reduced by internal acetazolamide by approximately 20% at constant Vte. The results suggest that posterior gills of hyperosmoregulating C. granulatus display a high conductance epithelium that actively absorbs NaCl in a coupled way by an electrogenic mechanism similar to that seen in the thick ascending limb of Henle's loop and, to a minor degree, by an electroneutral mechanism, presumably via apical Na+/H+and Cl-/HCO3--antiports.

Descripción: 1. 1. Uroporphyrinogen carboxy-lyase (EC 4.1.1.d), the enzyme catalysing the decaroxylation of uroporphyrinogen to coproporphyrinogen, has been isolated from normal chicken erythrocytes. The enzyme was purified 220-fold with a yield of 24% from haemolysate supernatant by DEAE-cellulose batch treatment, (NH4)2SO4 fractionation and chromatography on DEAE-cellulose. 2. 2. The purified material appears to be homogeneous in polyacrylamide gel disc electrophoresis. 3. 3. The enzyme was heat labile and inhibited by sodium salt; the activity was enhanced by EDTA, GSH and boiled rat-liver extract. 4. 4. The influence of these chemical and physical agents on the removal of the first and second carboxyl groups from uroporphyrinogen was compared; the second group was more susceptible to these agents. 5. 5. The possibility that one or several enzymes were involved in the stepwise decarboxylation of uroporphyrinogen is discussed. 6. 6. The general name of porphyrinogen carboxy-lyase for the enzyme system is proposed because of the different porphyrinogens it can decarboxylate. © 1970.

Descripción: Bath application of compound T-588, a neuroprotective agent, reduced paired-pulse and repetitive-pulse facilitation at mammalian and crustacean neuromuscular junctions. In addition, it reduced voltage-gated sodium and potassium currents in a use-dependent fashion, but had only a small effect on the presynaptic Ca 2+ conductance. By contrast, it blocked FM 1-43 vesicular uptake but not its release, in both species. Postsynaptically, T-588 reduced acetylcholine currents at the mammalian junction in a voltage-independent manner, but had no effect on the crayfish glutamate junction. All of these effects were rapidly reversible and were observed at concentrations close to the compound's acute protective level. We propose that this set of mechanisms, which reduces high-frequency synaptic transmission, is an important contributory factor in the neuroprotective action of T-588.

Descripción: We present results from molecular dynamics simulations performed on reverse micelles immersed in cyclohexane. Three different inner polar phases are considered: water (W), formamide (FM), and an equimolar mixture of the two solvents. In all cases, the surfactant was sodium bis(2-ethylhexyl) sulfosuccinate (usually known as AOT). The initial radii of the micelles were R∼15 Å, while the corresponding polar solvent-to-surfactant molar ratios were intermediate between w0 =4.3 for FM and w0 =7 for W. The resulting overall shapes of the micelles resemble distorted ellipsoids, with average eccentricities of the order of ∼0.75. Moreover, the pattern of the surfactant layer separating the inner pool from the non-polar phase looks highly irregular, with a roughness characterized by length scales comparable to the micelle radii. Solvent dipole orientation polarization along radial directions exhibit steady growths as one moves from central positions toward head group locations. Local density correlations within the micelles indicate preferential solvation of sodium ionic species by water, in contrast to the behavior found in bulk equimolar mixtures. Still, a sizable fraction of ∼90% of Na+ remains associated with the head groups. Compared to bulk results, the translational and rotational modes of the confined solvents exhibit important retardations, most notably those operated in rotational motions where the characteristic time scales may be up to 50 times larger. Modifications of the intramolecular connectivity expressed in terms of the average number of hydrogen bonds and their lifetimes are also discussed. © 2008 American Institute of Physics.

Descripción: The endosperm of the seed of Gleditsia triacanthos L. contains 18.55% of its dry weight as nonreserve, cell-wall carbohydrates. Of this carbohydrate material, comprising mainly mannose, galactose, and glucose, 76.1% was of low-molecular weight or highly hydrophilic. Mannose, galactose, and glucose were also the major sugar components of the polysaccharides extracted with alkali (23.1% of the cell-wall), while the same sugars, with minor amounts of arabinose, form the residues. Methylation analysis of the polysaccharides and the borate-sodium hydroxide residue indicate that the cell walls are built up on a network of galactomannans, with high Man/Gal ratios, reinforced with minor amounts of cellulose.

Descripción: Background. Considering that sunflower production is expanding to arid regions, tolerance to abiotic stresses as drought, low temperatures and salinity arises as one of the main constrains nowadays. Differential organ-specific sunflower ESTs (expressed sequence tags) were previously generated by a subtractive hybridization method that included a considerable number of putative abiotic stress associated sequences. The objective of this work is to analyze concerted gene expression profiles of organ-specific ESTs by fluorescence microarray assay, in response to high sodium chloride concentration and chilling treatments with the aim to identify and follow up candidate genes for early responses to abiotic stress in sunflower. Results. Abiotic-related expressed genes were the target of this characterization through a gene expression analysis using an organ-specific cDNA fluorescence microarray approach in response to high salinity and low temperatures. The experiment included three independent replicates from leaf samples. We analyzed 317 unigenes previously isolated from differential organ-specific cDNA libraries from leaf, stem and flower at R1 and R4 developmental stage. A statistical analysis based on mean comparison by ANOVA and ordination by Principal Component Analysis allowed the detection of 80 candidate genes for either salinity and/or chilling stresses. Out of them, 50 genes were up or down regulated under both stresses, supporting common regulatory mechanisms and general responses to chilling and salinity. Interestingly 15 and 12 sequences were up regulated or down regulated specifically in one stress but not in the other, respectively. These genes are potentially involved in different regulatory mechanisms including transcription/translation/protein degradation/protein folding/ROS production or ROS-scavenging. Differential gene expression patterns were confirmed by qRT-PCR for 12.5% of the microarray candidate sequences. Conclusion. Eighty genes isolated from organ-specific cDNA libraries were identified as candidate genes for sunflower early response to low temperatures and salinity. Microarray profiling of chilling and NaCl-treated sunflower leaves revealed dynamic changes in transcript abundance, including transcription factors, defense/stress related proteins, and effectors of homeostasis, all of which highlight the complexity of both stress responses. This study not only allowed the identification of common transcriptional changes to both stress conditions but also lead to the detection of stress-specific genes not previously reported in sunflower. This is the first organ-specific cDNA fluorescence microarray study addressing a simultaneous evaluation of concerted transcriptional changes in response to chilling and salinity stress in cultivated sunflower.

Descripción: The combined effect of water activity (a(w)) and pH on growth and toxin production by Clostridium botulinum type G strain 89 was investigated. The minimum a(w) at which growth and toxin formation occurred was 0.965, for media in which the pH was adjusted with either sodium chloride or sucrose. The minimum pH (at the optimum a(w)) for growth and toxin production of C. botulinum type G was found to be 5.6. Optimum conditions for toxin activation were a trypsin concentration of 0.1%, a pH of the medium of 6.5, and an incubation for 45 min at 37° C. These data did not show evidence of heat-labile spores, since a heat shock of 75°C for 10 min did not significantly decrease the spore count of strain 89G in media at pH 7.0 or 5.6. It was frequently observed that cells grown at reduced a(w) or pH experienced severe morphological changes.

Descripción: To gain insights into the natural variation of root hydraulics and its molecular components, genotypic differences related to root water transport and plasma membrane intrinsic protein (PIP) aquaporin expression were investigated in 13 natural accessions of Arabidopsis (Arabidopsis thaliana). The hydraulic conductivity of excised root systems (Lpr) showed a 2-fold variation among accessions. The contribution of aquaporins to water uptake was characterized using as inhibitors mercury, propionic acid, and azide. The aquaporin-dependent and -independent paths of water transport made variable contributions to the total hydraulic conductivity in the different accessions. The distinct suberization patterns observed among accessions were not correlated with their root hydraulic properties. Real-time reverse transcription-polymerase chain reaction revealed, by contrast, a positive overall correlation between Lpr and certain highly expressed PIP transcripts. Root hydraulic responses to salt stress were characterized in a subset of five accessions (Bulhary-1, Catania-1, Columbia-0, Dijon-M, and Monte-Tosso-0 [Mr-0]). Lpr was down-regulated in all accessions except Mr-0. In Mr-0 and Catania-1, cortical cell hydraulic conductivity was unresponsive to salt, whereas it was down-regulated in the three other accessions. By contrast, the five accessions showed qualitatively similar aquaporin transcriptional profiles in response to salt. The overall work provides clues on how hydraulic regulation allows plant adaptation to salt stress. It also shows that a wide range of root hydraulic profiles, as previously reported in various species, can be observed in a single model species. This work paves the way for a quantitative genetics analysis of root hydraulics. © 2011 American Society of Plant Biologists.

Descripción: The importance of the content of anionic phospholipids [cardiolipin (CL) and phosphatidylglycerol (PG)] in the osmotic adaptation and in the membrane structure of Bacillus subtilis cultures was investigated. Insertion mutations in the three putative cardiolipin synthase genes (ywiE, ywnE and ywjE) were obtained. Only the ywnE mutation resulted in a complete deficiency in cardiolipin and thus corresponds to a true clsA gene. The osmotolerance of a clsA mutant was impaired: although at NaCl concentrations lower than 1.2 M the growth curves were similar to those of its wild-type control, at 1 .5 M NaCl (LBN medium) the lag period increased and the maximal optical density reached was lower. The membrane of the clsA mutant strain showed an increased PG content, at both exponential and stationary phase, but no trace of CL in either LB or LBN medium. As well as the deficiency in CL synthesis, the clsA mutant showed other differences in lipid and fatty acids content compared to the wild-type, suggesting a cross-regulation in membrane lipid pathways, crucial for the maintenance of membrane functionality and integrity. The biophysical characteristics of membranes and large unilamellar vesicles from the wild-type and clsA mutant strains were studied by Laurdan's steady-state fluorescence spectroscopy. At physiological temperature, the clsA mutant showed a decreased lateral lipid packing in the protein-free vesicles and isolated membranes compared with the wild-type strain. Interestingly, the lateral lipid packing of the membranes of both the wild-type and clsA mutant strains increased when they were grown in LBN. In a conditional IPTG-controlled pgsA mutant, unable to synthesize PG and CL in the absence of IPTG, the osmoresistance of the cultures correlated with their content of anionic phospholipids. The transcriptional activity of the clsA and pgsA genes was similar and increased twofold upon entry to stationary phase or under osmotic upshift. Overall, these results support the involvement of the anionic phospholipids in the growth of B. subtilis in media containing elevated NaCl concentrations. © 2006 SGM.

Descripción: 1. 1. The effect of several metals and reagents on the decarboxylation rate of uroporphyrinogen I by using a 16-fold purified preparation of Uroporphyrinogen Decarboxylase from Rhodopseudomonas palustris, was studied. 2. 2. 1 mM Hg2+ and Cu2+ were strong inhibitors, 1 mM Zn2+ and Fe2+ under certain conditions and 1 mM Fe3+ and Cr3+ also inactivated the enzyme, but Pb2+, Cd2+, and Al3+ did not. Metals inhibition was reversed by 1 mM GSH or CvSH. 3. 3. 0.1 mM DTNB and PCMB, 1 mM pyridoxal phosphate and 100 mM chloral hydrate, as well as 1 mM 2-methoxy-5-nitrotropone and 0.2 mM diethylpyrocarbonate inhibited Uroporphyrinogen Decarboxylase; while GSH, CySH, N-ethylmaleimide, sodium thioglycolate, 1,4-dithioerythritol, EDTA and O-phenantroline did not modify activity. 4. 4. Data obtained would indicate that one cysteine, one or two histidine residues and probably a lysine group are required for enzyme activity. © 1987.

Descripción: To characterize the mechanism of chloroplast fructose‐1,6‐bisphosphatase activation, we have examined kinetic and structural changes elicited by protein perturbants and reductants. At variance with its well‐known capacity for enzyme inactivation, 150 mM sodium trichloroacetate yielded an activatable chloroplast fructose‐1,6‐bisphosphatase in the presence of 1.0 mM fructose 1,6‐bisphosphate and 0.1 mM Ca2+. Other sugar bisphosphates did not replace fructose 1,6‐bisphosphate whereas Mg2+ and Mn2+ were functional in place of Ca2+. Variations of the emission fluorescence of intrinsic fluorophores and a noncovalently bound extrinsic probe [2‐(P‐toluidinyl)naphthalene‐6‐sulfonate] indicated the presence of conformations different from the native form. A similar conclusion was drawn from the analysis of absorption spectra by means of fourth‐derivative spectrophotometry. The effect of these conformational changes on the reductive process was studied by subsequently incubating the enzyme with dithiothreitol. The reaction of chloroplast fructose‐1,6‐bisphosphatase with dithiothreitol was accelerated 13‐fold by the chaotropic anion: second‐order rate constants were 48.1 M−1· min−1 and 3.7 M−1· min−1 in the presence and in the absence of trichloroacetate, respectively. Thus, the enhancement of the reductive activation by compounds devoid of redox activity illustrated that the modification of intramolecular noncovalent interactions of chloroplast fructose‐1,6‐bisphosphatase plays an essential role in the conversion of enzyme disulfide bonds to sulfhydryl groups. In consequence, a conformational change would operate concertedly with the reduction of disulfide bridges in the light‐dependent activation mediated by the ferredoxin–thioredoxin system. Copyright © 1994, Wiley Blackwell. All rights reserved

Descripción: Comparative studies on fatty acid and protein composition of the endosperm and embryo of palmito (Euterpe edulis Martius) were conducted using gas-liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On a dry weight basis, the embryo contained extremely lower amounts of lipids and proteins than did the endosperm, which was associated with the scarce lipid and protein bodies previously reported in axis and cotyledon. The fatty acid composition also exhibited differences between both tissues: (I) the fatty acid diversity was greater in embryo than in endosperm; (II) embryo and endosperm contained predominantly linoleic, palmitic, oleic and stearic acids even though the relative values were different for each tissue. As compared to other palm species, the higher fatty acid unsaturation in Euterpe edulis seed could be involved in the previously reported short longevity and recalcitrant behavior during storage. Proteins of both tissues were heterogeneous in molecular mass. Some proteins were tissue-specific, but other were common, among them a highly glycosylated protein which migrated at about 55 kDa. We hypothesize that the latter, also reported in all previously studied palm species, is one of the proteins characterizing the Arecaceae family.

Descripción: MAP kinases have been linked to guard cell signalling. Arabidopsis thaliana MAP Kinase 3 (MPK3) is known to be activated by abscisic acid (ABA) and hydrogen peroxide (H2O2), which also control stomatal movements. We therefore studied the possible role of MPK3 in guard cell signalling through guard cell-specific antisense inhibition of MPK3 expression. Such transgenic plants contained reduced levels of MPK3 mRNA in the guard cells and displayed partial insensitivity to ABA in inhibition of stomatal opening, but responded normally to this hormone in stomatal closure. However, ABA-induced stomatal closure was reduced compared with controls when cytoplasmic alkalinization was prevented with sodium butyrate. MPK3 antisense plants were less sensitive to exogenous H2O2, both in inhibition of stomatal opening and in promotion of stomatal closure, thus MPK3 is required for the signalling of this compound. ABA-induced H2O2 synthesis was normal in these plants, indicating that MPK3 probably acts in signalling downstream of H2O2. These results provide clear evidence for the important role of MPK3 in the perception of ABA and H 2O2 in guard cells. © The Authors (2007).

Descripción: The 11β-hydroxysteroid dehydrogenase type 2 (11βHSD-2) enzyme is thought to confer aldosterone specificity upon mineralocorticoid target tissues by protecting the mineralocorticoid receptor from binding by the more abundant glucocorticoids, corticosterone and cortisol. We have developed a Chinese hamster ovary cell line stably transfected with a plasmid containing the rat 11βHSD-2 complementary DNA. This cell line has expressed the enzyme consistently for many generations. The 11βHSD-2 was located primarily in the microsomes, but significant amounts also existed in the nuclei and mitochondria. The enzymatic reaction was unidirectional, oxidative, and inhibited by the product, 11-dehydrocorticosterone, with an IC50 of approximately 200 nM. The K(m) for corticosterone was 9.6 ± 3.1 nM, and that for NAD+ was approximately 8 μM. The enzyme did not convert dexamethasone to 11-dehydrodexamethasone. Tunicamycin, an N-glycosylation inhibitor, had no effect on enzyme activity, 11α-Hydroxyprogesterone (11αOH-P) was an order of magnitude more potent a competitive inhibitor of the 11βHSD-2 than was glycyrrhetinic acid (GA) (approximate IC50 0.9 vs. 15 nM). 11βOH-P, progesterone, and GA were almost equipotent (IC50 = 10 and 6 nM, respectively), and 5α-pregnandione and 5β-pregnandione were less potent (IC50 = 100 and 500 nM, respectively) inhibitors of the enzyme. When the inhibitory activities were examined with intact transfected cells, 11αOH-P was more potent than GA (IC50 = 5 and 150 nM, respectively). 11αOH-P was not metabolized by 11βHSD-2. We were unable to demonstrate the presence of 11αOH-P in human urine. In conclusion, a cell line stably transfected with the rat 11βHSD-2 was created, and the enzyme kinetics, including inhibition, were characterized. 11αOH-P was found to be a potent relatively specific inhibitor of the 11βHSD-2 enzyme. Its potential importance is that it is the most specific inhibitor of the 11βHSD-2 so far encountered and would aid in the study of the physiological importance of the isoenzyme.

Descripción: In daily life, memories are intertwined events. Little is known about the mechanisms involved in their interactions. Using two hippocampus-dependent (spatial object recognition and contextual fear conditioning) and one hippocampus-independent (conditioned taste aversion) learning tasks, we show that in rats subjected to weak training protocols that induce solely short term memory (STM), long term memory (LTM) is promoted and formed only if training sessions took place in contingence with a novel, but not familiar, experience occurring during a critical time window around training. This process requires newly synthesized proteins induced by novelty and reveals a general mechanism of LTM formation that begins with the setting of a "learning tag" established by a weak training. These findings represent the first comprehensive set of evidences indicating the existence of a behavioral tagging process that in analogy to the synaptic tagging and capture process, need the creation of a transient, protein synthesis-independent, and input specific tag.