Real-Time Insights into Biological Events: In-Cell Processes and Protein-Ligand Interactions

FlowNMR has the aim of continuously monitoring processes that occur in conditions that are not compatible with being carried out within a closed tube. However, it is sample intensive and not suitable for samples, such as proteins or living cells, that are often available in limited volumes and possibly low concentrations. We here propose a dialysis-based modification of a commercial flowNMR setup that allows for recycling the medium while confining the sample (proteins and cells) within the active volume of the tube. This approach is demonstrated in the specific cases of in-cell NMR and protein-based ligand studies.     

Interactions between Root Temperature and Nitrogen Deficiency Influence Preferential Uptake of NH+4 and NO-3 by Oilseed Rape

Oilseed rape (Brassica napus L. cv. Bien venu) plants were grownin a system of flowing solution culture and pre-treated at roottemperatures of 3 ?C or 13 ?C for 7 d with or without N suppliedas 10 mmol m^–3 NH₄NO₃. Subsequently, N was re-suppliedand root temperatures were reversed for 7 d. Shoot temperatureswere common at 20/15 ?C day/night. Net uptake of , and K⁺, leaf area, root length, and transpirationwere measured and compared with control plants having root temperaturesof 3 ?C and 13 ?C throughout. Plants that were continuouslysupplied with N and pre-treated at 3 ?C showed a 50% increasein total uptake of , and measured at 13 ?C over 7 d compared with control plants at 3?C,but uptake of was 28% lower and uptake of was 43% higher than that shown by control plants at 13 ?C. Pre-treatment at 3 ?C did not enhance the subsequentuptake of total N or of K⁺ at 13 ?C relative to the 13 ?C control.Transpiration rates at 3 ?C were on average 70% of those at13 ?C. The concentration of total N in plants was halved after7 d without a supply of N, but total dry matter production wasnot significantly affected. N starvation also increased thetemperature sensitivity of subsequent uptake relative to that of uptake. After N starvation at 13 ?C the uptake of and measured at 13 ?C was 50% higher over 7 d than that measuredunder continuous N supply. In contrast, after N starvation at3 ?C the uptake of at 3 ?C was 70% less, whilst uptake was 50% more than the respective totals absorbed by plants that were continuously supplied withN at 3 ?C. After N starvation the uptake of was generally 40–60% of the daily total N uptake, comparedwith 60–80% in plants continuously supplied with N. Key words: Brassica napus, oilseed rape, root temperature, nitrate, ammonium, potassium, N-deficiency, ion uptake rate, transpiration     

Ammonium Uptake by Rice Roots (II. Kinetics of 13NH4+ Influx across the Plasmalemma)

Short-term influxes of 13NH4+ were measured in intact roots of 3-week-old rice (Oryza sativa L. cv M202) seedlings that were hydroponically grown at 2, 100, or 1000 [mu]M NH4+. Below 1 mM external concentration ([NH4+]0), influx was saturable and due to a high-affinity transport system (HATS). For the HATS, Vmax values were negatively correlated and Km values were positively correlated with NH4+ provision during growth and root [NH4+]. Between 1 and 40 mM [NH4+]0, 13NH4+ influx showed a linear response due to a low-affinity transport system (LATS). The 13NH4+ influxes by the HATS, and to a lesser extent the LATS, are energy-dependent processes. Selected metabolic inhibitors reduced influx of the HATS by 50 to 80%, but of the LATS by only 31 to 51%. Estimated values for Q10 (the ratio of rates at temperatures differing by 10[deg]C) for HATS were greater than 2.4 at root temperatures from 5 to 10[deg]C and were constant at approximately 1.5 between 5 and 30[deg]C for the LATS. Influx of 13NH4+ by the HATS was insensitive to external pH in the range from 4.5 to 9.0, but influx by the LATS declined significantly beyond pH 6.0. The data presented are discussed in the context of the kinetics, energy dependence, and the regulation of ammonium influx.     

COVID-19 Pandemic: Insights into Interactions between SARS-CoV-2 Infection and MAFLD

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become an ongoing global health pandemic. Since 2019, the pandemic continues to cast a long shadow on all aspects of our lives, bringing huge health and economic burdens to all societies. With our in-depth understanding of COVID-19, from the initial respiratory tract to the later gastrointestinal tract and cardiovascular systems, the multiorgan involvement of this infectious disease has been discovered. Metabolic dysfunction-associated fatty liver disease (MAFLD), formerly named nonalcoholic fatty liver disease (NAFLD), is a major health issue closely related to metabolic dysfunctions, affecting a quarter of the world''s adult population. The association of COVID-19 with MAFLD has received increasing attention, as MAFLD is a potential risk factor for SARS-CoV-2 infection and severe COVID-19 symptoms. In this review, we provide an update on the interactions between COVID-19 and MAFLD and its underlying mechanisms.     

Structural Insights into the Interactions between Platelet Receptors and Fibrillar Collagen

Collagen peptides have been used to identify binding sites for several important collagen receptors, including integrin α₂β₁, glycoprotein VI, and von Willebrand factor. In parallel, the structures of these collagen receptors have been reported, and their interactions with collagen peptides have been studied. Recently, the three-dimensional structure of the intact type I collagen fiber from rat tail tendon has been resolved by fiber diffraction. It is now possible to map the binding sites of platelet collagen receptors onto the intact collagen fiber in three dimensions. This minireview will discuss these recent findings and their implications for platelet activation by collagen.     

Regulation of K+ Influx in Barley: Evidence for a Direct Control of Influx by K+ Concentration of Root Cells

Siddiqi, M. Y. and Glass, A. D. M. 1987. Regulation of K⁺ influxin barley: Evidence for a direct control of influx by K⁺ concentrationof root cells.—J. exp. Bot. 38: 935–947. The kinetics of K⁺ (⁸⁶Rb⁺) influx into intact roots of barley(Hordeum vulgare L. cv. Fergus) seedlings having different combinationsof root and shoot [K⁺], different growth rates and differentroot:shoot weight ratios were studied. K⁺ influx was stronglycorrelated with root [K⁺]; shoot [K⁺], growth rates, and root:shoot ratios appeared to have little effect on K⁺ influx. Adetailed study showed that both V_max and K_m for K⁺ influx wereaffected by root [K⁺] but not by shoot [K⁺]. We have suggestedthat factors such as growth rates and root: shoot ratio mayaffect K⁺ influx indirectly primarily via their influence onroot factors such as root [K⁺]. We have reiterated that othertypes of kinetic control, e.g. increased or decreased synthesisof ‘carrier systems’, may operate in addition todirect (allosteric?) control of K⁺ influx by root [K⁺]. Thenegative feedback signal from root [K⁺] appeared to be the primeeffector in the regulation of K⁺ influx. Key words: Barley, K⁺ influx     

Root growth inhibition by NH4+ in Arabidopsis is mediated by the root tip and is linked to NH4+ efflux and GMPase activity

Root growth in higher plants is sensitive to excess ammonium (NH₄⁺). Our study shows that contact of NH₄⁺ with the primary root tip is both necessary and sufficient to the development of arrested root growth under NH₄⁺ nutrition in Arabidopsis. We show that cell elongation and not cell division is the principal target in the NH₄⁺ inhibition of primary root growth. Mutant and expression analyses using DR5:GUS revealed that the growth inhibition is furthermore independent of auxin and ethylene signalling. NH₄⁺ fluxes along the primary root, measured using the Scanning Ion‐selective Electrode Technique, revealed a significant stimulation of NH₄⁺ efflux at the elongation zone following treatment with elevated NH₄⁺, coincident with the inhibition of root elongation. Stimulation of NH₄⁺ efflux and inhibition of cell expansion were significantly more pronounced in the NH₄⁺‐hypersensitive mutant vtc1‐1, deficient in the enzyme GDP‐mannose pyrophosphorylase (GMPase). We conclude that both restricted transmembrane NH₄⁺ fluxes and proper functioning of GMPase in roots are critical to minimizing the severity of the NH₄⁺ toxicity response in Arabidopsis.     

发酵体系中氨态氮含量的测定及影响因素的探讨

靛酚蓝-分光光度法是一种灵敏度高、设备要求简单、线性相关性好、重现性高的氨态氮检测方法。本文对该方法的检测波长,反应温度、时间以及催化剂浓度等条件进行了优化,确定了最佳的检测波长为637nm,最佳的反应条件为37℃、25 mg.L-1的催化剂和20 m in的反应时间。并由此得到一条线性相关系数为0.9996的氨态氮检测标准曲线。同时,对靛酚蓝法测定氨态氮用于普通发酵体系进行了探讨。对常用发酵基质如炭源、氮源、金属离子以及消泡剂等进行了考察,结果发现这些常用基质基本不影响本法用于氨态氮的测定。最后,将这种氨氮测定方法用于泰乐菌素和阿维菌素的发酵实践,取得了良好效果,由此证明该方法是一种切实可行的发酵中氨态氮的检测方法。     

Interactions between N application rate, CH4 oxidation and N2O production in soil

Here we report on a controlled environment experiment in which we applied ¹³C- and ¹⁵N-enrichment approaches to quantify methane oxidation rates and source partition N₂O production in a silt loam soil following application of NH₄NO₃, enabling us to look for potential interactions between methane oxidation and nitrifier-N₂O production. ¹⁵N-N₂O, ¹⁴⁺¹⁵N-N₂O and CO₂ fluxes and mineral N concentrations were measured over a 23-day period after application of NH₄NO₃ (5 at.% excess ¹⁵N) at rates of 0, 5, 10, 20, 30 and 40 g N m^?2 to a silt loam soil. Change in ^12/13C-CH₄ concentrations (as indicative of ¹³C-CH₄ oxidation rates) and production of ¹³C-CO₂ were monitored over the first 72 h after addition of 1.7 ??l ¹³C-CH₄ l^?1 (10 at.% excess ¹³C) to these N treatments. Oxidation of applied ¹³C-CH₄ was slower in the 5, 10, 20 and 30 g N m^?2 (5 at.% excess ¹⁵N) treatments (0.24?C0.32 ??g ¹³C-CH₄ l^?1 day^?1) than in the control (0.40 ??g ¹³C-CH₄ l^?1 day^?1), suggesting that these N loadings inhibited oxidation. N₂O production was raised after N addition, and in the 10, 20 and 30 g N m^?2 treatments nitrification was the predominant source of N₂O accounting for 61, 83 and 57% of the total ¹⁵N-N₂O produced, respectively. Our results point towards the possibility of methylotrophs switching function to oxidise ammonia in the presence of N, which may result in greater atmospheric loading of both CH₄ and N₂O.     

Interactions between Multiple Phosphorylation Sites in the Inactivation Particle of a K+ Channel : Insights into the Molecular Mechanism of Protein Kinase C Action

Protein kinase C inhibits inactivation gating of Kv3.4 K⁺ channels, and at least two NH₂-terminal serines (S15 and S21) appeared involved in this interaction (Covarrubias et al. 1994. Neuron. 13:1403–1412). Here we have investigated the molecular mechanism of this regulatory process. Site-directed mutagenesis (serine → alanine) revealed two additional sites at S8 and S9. The mutation S9A inhibited the action of PKC by ∼85%, whereas S8A, S15A, and S21A exhibited smaller reductions (41, 35, and 50%, respectively). In spite of the relatively large effects of individual S → A mutations, simultaneous mutation of the four sites was necessary to completely abolish inhibition of inactivation by PKC. Accordingly, a peptide corresponding to the inactivation domain of Kv3.4 was phosphorylated by specific PKC isoforms, but the mutant peptide (S[8,9,15,21]A) was not. Substitutions of negatively charged aspartate (D) for serine at positions 8, 9, 15, and 21 closely mimicked the effect of phosphorylation on channel inactivation. S → D mutations slowed the rate of inactivation and accelerated the rate of recovery from inactivation. Thus, the negative charge of the phosphoserines is an important incentive to inhibit inactivation. Consistent with this interpretation, the effects of S8D and S8E (E = Glu) were very similar, yet S8N (N = Asn) had little effect on the onset of inactivation but accelerated the recovery from inactivation. Interestingly, the effects of single S → D mutations were unequal and the effects of combined mutations were greater than expected assuming a simple additive effect of the free energies that the single mutations contribute to impair inactivation. These observations demonstrate that the inactivation particle of Kv3.4 does not behave as a point charge and suggest that the NH₂-terminal phosphoserines interact in a cooperative manner to disrupt inactivation. Inspection of the tertiary structure of the inactivation domain of Kv3.4 revealed the topography of the phosphorylation sites and possible interactions that can explain the action of PKC on inactivation gating.     

Detection of Transient In Vivo Interactions between Substrate and Transporter during Protein Translocation into the Endoplasmic Reticulum

The split-ubiquitin technique was used to detect transient protein interactions in living cells. N_ub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum of Saccharomyces cerevisiae. C_ub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of C_ub, serve as a gauge of proximity between the two test proteins linked to N_ub and C_ub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-α-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. C_ub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with N_ub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-α-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.     

SASH1: A Novel Eph Receptor Partner and Insights into SAM-SAM Interactions

The Eph (erythropoietin-producing human hepatocellular) receptor family, the largest subclass of receptor tyrosine kinases (RTKs), plays essential roles in embryonic development and neurogenesis. The intracellular Sterile Alpha Motif (SAM) domain presents a critical structural feature that distinguishes Eph receptors from other RTKs and participates in recruiting and binding downstream molecules. This study identified SASH1 (SAM and SH3 domain containing 1) as a novel Eph receptor-binding partner through SAM-SAM domain interactions. Our comprehensive biochemical analyses revealed that SASH1 selectively interacts with Eph receptors via its SAM1 domain, displaying the highest affinity for EphA8. The high-resolution crystal structure of the EphA8-SASH1 complex provided insights into the specific intermolecular interactions between these proteins. Cellular assays confirmed that EphA8 and SASH1 co-localize and co-precipitate in mammalian cells, with cancer mutations (EphA8 R942H or G978D) impairing this interaction. We demonstrated that SAM-SAM interaction is critical for SASH1-mediated regulation of EphA8 kinase activity, shedding new light on the Eph signaling pathway and expanding our understanding of the molecular basis of the tumor suppressor gene SASH1.     

N2 fixation and NH 4 + assimilation in the thermophilic anaerobes Clostridium thermosaccharolyticum and Clostridium thermoautotrophicum

Inorganic nitrogen metabolism in the obligate anaerobic thermophiles Chlostridium thermosaccharolyticum and Clostridium thermoautotrophicum differs in several respects. C. thermosaccharolyticum contains a nitrogenase as inferred from NH ₄ ⁺ repressible C₂H₂ reduction, a glutamine synthetase which is partially repressed by ammonium, very labile glutamate synthase activities with both NADH and NADPH, NADPH-dependent glutamate dehydrogenase, and NH ₄ ⁺ -dependent asparagine synthetase. C. thermoautotrophicum contains no nitrogenase, but glutamine synthetase, no glutamate synthase, no glutamate dehydrogenase, but a NADH-dependent alanine dehydrogenase and a NH ₄ ⁺ -dependent asparagine synthetase.Abbreviation GOGAT glutamine-oxoglutarate amidotransferase amidotransferase (glutamate synthase)     

Identification of a Transport Mechanism for NH4+ in the Symbiosome Membrane of Pea Root Nodules

Symbiosome membrane vesicles, facing bacteroid-side-out, were purified from pea (Pisum sativum L.) root nodules and used to study NH4+ transport across the membrane by recording vesicle uptake of the NH4+ analog [14C]methylamine (MA). Membrane potentials ([delta][psi]) were imposed on the vesicles using K+ concentration gradients and valinomycin, and the size of the imposed [delta][psi] was determined by measuring vesicle uptake of [14C]tetraphenylphosphonium. Vesicle uptake of MA was driven by a negative [delta][psi] and was stimulated by a low extravesicular pH. Protonophore-induced collapse of the pH gradient indicated that uptake of MA was not related to the presence of a pH gradient. The MA-uptake mechanism appeared to have a large capacity for transport, and saturation was not observed at MA concentrations in the range of 25 [mu]M to 150 mM. MA uptake could be inhibited by NH4+, which indicates that NH4+ and MA compete for the same uptake mechanism. The observed fluxes suggest that voltage-driven channels are operating in the symbiosome membrane and that these are capable of transporting NH4+ at high rates from the bacteroid side of the membrane to the plant cytosol. The pH of the symbiosome space is likely to be involved in regulation of the flux.     

Role of NH3 and NH4+ transporters in renal acid-base transport

Renal ammonia excretion is the predominant component of renal net acid excretion. The majority of ammonia excretion is produced in the kidney and then undergoes regulated transport in a number of renal epithelial segments. Recent findings have substantially altered our understanding of renal ammonia transport. In particular, the classic model of passive, diffusive NH3 movement coupled with NH4+ "trapping" is being replaced by a model in which specific proteins mediate regulated transport of NH3 and NH4+ across plasma membranes. In the proximal tubule, the apical Na+/H+ exchanger, NHE-3, is a major mechanism of preferential NH4+ secretion. In the thick ascending limb of Henle's loop, the apical Na+-K+-2Cl- cotransporter, NKCC2, is a major contributor to ammonia reabsorption and the basolateral Na+/H+ exchanger, NHE-4, appears to be important for basolateral NH4+ exit. The collecting duct is a major site for renal ammonia secretion, involving parallel H+ secretion and NH3 secretion. The Rhesus glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), are recently recognized ammonia transporters in the distal tubule and collecting duct. Rhcg is present in both the apical and basolateral plasma membrane, is expressed in parallel with renal ammonia excretion, and mediates a critical role in renal ammonia excretion and collecting duct ammonia transport. Rhbg is expressed specifically in the basolateral plasma membrane, and its role in renal acid-base homeostasis is controversial. In the inner medullary collecting duct (IMCD), basolateral Na+-K+-ATPase enables active basolateral NH4+ uptake. In addition to these proteins, several other proteins also contribute to renal NH3/NH4+ transport. The role and mechanisms of these proteins are discussed in depth in this review.     

Orthophosphate Relations of Root: NO-3 Effects on Orthophosphate Influx, Accumulation and Secretion into the Xylem

Lamaze, T., Sentenac, H. and Grignon, C. 1987. Orthophosphaterelations of root: NO^–₃effects on orthophosphate influx,accumulation and secretion into the xylem.—J. exp. Bot.38: 923–934. Orthophosphate (Pi) accumulation by barley (Hordeum vulgareL.) roots was specifically inhibited by NO^–₃ as comparedto Cl^– and SO₄^{2 –}, and Pi secretion into the xylemwas stimulated. The inhibition of Pi accumulation by NO^–₃was also observed in roots of intact photosynthesizing horsebean(Vicia faba L.), rice (Oryza sativa L.) and soybean (Glycinemax L.) plants. NO^–₃ effects on Pi transport by rootswere more thoroughly investigated with corn (Zea mays L.). Theywere due to intracellular NO^–₃. Pi secretion was stillstimulated by NO^–₃ after Pi withdrawal from the absorptionsolution. ³²Pi influx decreased during Pi accumulation, supportingthe hypothesis that this ion allosterically regulated its owntransport system by feedback control. This control was modulatedby other anions: the decrease was more pronounced in the presenceof nitrate. Chronologically, the depressive effect of NO^–₃on ³²Pi influx appeared after the inhibition of Pi accumulation.Furthermore, under conditions where Pi accumulation was notaffected by NO^–₃, ³²Pi influx and Pi secretion into thexylem became insensitive to the presence of nitrate. Our hypothesisis that the stimulative effect of NO^–₃ on Pi secretionand the depressive one on ³²Pi influx are the repercussionsof an increase in the Pi cytosolic concentration due to an NO^–₃-induced decrease in Pi uptake by the vacuoles. Key words: Root, orthophosphate fluxes, orthophosphate accumulation, nitrate, ionic interaction     

Affinity and Specificity of Levamlodipine-Human Serum Albumin Interactions: Insights into Its Carrier Function

The affinity and specificity of drugs with human serum albumin (HSA) are crucial factors influencing the bioactivity of drugs. To gain insight into the carrier function of HSA, the binding of levamlodipine with HSA has been investigated as a model system by a combined experimental and theoretical/computational approach. The fluorescence properties of HSA and the binding parameters of levamlodipine indicate that the binding is characterized by one binding site with static quenching mechanism, which is related to the energy transfer. As indicated by the thermodynamic analysis, hydrophobic interaction is the predominant force in levamlodipine-HSA complex, which is in agreement with the computational results. And the hydrogen bonds can be confirmed by computational approach between levamlodipine and HSA. Compared to predicted binding energies and binding energy spectra at seven sites on HSA, levamlodipine binding HSA at site I has a high affinity regime and the highest specificity characterized by the largest intrinsic specificity ratio (ISR). The binding characteristics at site I guarantee that drugs can be carried and released from HSA to carry out their specific bioactivity. Our concept and quantification of specificity is general and can be applied to other drug-target binding as well as molecular recognition of peptide-protein, protein-protein, and protein-DNA interactions.