On-line monitoring of infected Sf-9 insect cell cultures by scanning permittivity measurements and comparison with off-line biovolume measurements

Two infected Sf-9 cell cultures were monitored on-line by multi-frequency permittivity measurements using the Fogale BIOMASS SYSTEM^® and by applying different off-line methods (CASY^®1, Vi-CELL?, packed cell volume) to measure the biovolume and the mean diameter of the cell population. During the growth phase and the early infection phase the measured permittivity at the working frequency correlated well with the different off-line methods for the biovolume. We found a value of 0.67 pF cm^?1 permittivity per unit of total biovolume (CASY) (μL mL^?1). After the maximum value in the permittivity was reached, i.e. when the viability of the cultures decreased significantly, we observed different time courses for the biovolume depending on the applied method. The differences were compared and could be explained by the underlying measurement principles. Furthermore, the characteristic frequency (f_C) was calculated from the on-line scanning permittivity measurements. The f_C may provide an indication of changes in cell diameter and membrane properties especially after infection and could also be an indicator for the onset of the virus production phase. The changes in f_C were qualitatively explained by the underlying equation that is correlating f_C and the properties of the cell population (cell diameter, intracellular conductivity and capacitance per membrane area).     

Quantitative modeling of viable cell density,cell size,intracellular conductivity,and membrane capacitance in batch and fed‐batch CHO processes using dielectric spectroscopy

Dielectric spectroscopy was used to analyze typical batch and fed‐batch CHO cell culture processes. Three methods of analysis (linear modeling, Cole–Cole modeling, and partial least squares regression), were used to correlate the spectroscopic data with routine biomass measurements [viable packed cell volume, viable cell concentration (VCC), cell size, and oxygen uptake rate (OUR)]. All three models predicted offline biomass measurements accurately during the growth phase of the cultures. However, during the stationary and decline phases of the cultures, the models decreased in accuracy to varying degrees. Offline cell radius measurements were unsuccessfully used to correct for the deviations from the linear model, indicating that physiological changes affecting permittivity were occurring. The β‐dispersion was analyzed using the Cole–Cole distribution parameters Δε (magnitude of the permittivity drop), f_c (critical frequency), and α (Cole–Cole parameter). Furthermore, the dielectric parameters static internal conductivity (σ_i) and membrane capacitance per area (C_m) were calculated for the cultures. Finally, the relationship between permittivity, OUR, and VCC was examined, demonstrating how the definition of viability is critical when analyzing biomass online. The results indicate that the common assumptions of constant size and dielectric properties used in dielectric analysis are not always valid during later phases of cell culture processes. The findings also demonstrate that dielectric spectroscopy, while not a substitute for VCC, is a complementary measurement of viable biomass, providing useful auxiliary information about the physiological state of a culture. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

On-line monitoring of responses to nutrient feed additions by multi-frequency permittivity measurements in fed-batch cultivations of CHO cells

Changes in the nutrient availability of mammalian cell cultures are reflected in the β-dispersion parameter characteristic frequency (f _C ) and the on-line dual frequency permittivity signal. Multi-frequency permittivity measurements were therefore evaluated in fed-batch cultivations of two different CHO cell lines. Similar responses to nutrient depletions and discontinuous feed additions were monitored in different cultivation phases and experimental setups. Sudden increases in permittivity and f _C occurred when feed additions were conducted. A constant or declining permittivity value in combination with a decrease in f _C indicated nutrient limitations. f _C correlated well with changes in oxygen uptake rate when cell diameter remained constant, indicating that metabolic activity is reflected in the value of f _C . When significant cell size changes occurred during the cultivations, the analysis of the β-dispersion parameters was rendered complex. For the application of our findings in other systems it will be hence required to conduct additional off-line measurements. Based on these results, it is hypothesized that multi-frequency permittivity measurements can give information on the intracellular or physiological state in fed-batch mode. Similar observations were made when using different cell lines and feeding strategies, indicating that the findings are transferable to other cell lines and systems. The results should lead to an improved understanding of routine fed-batch processes. Additional studies are, however, required to explore how these observations can be used for fed-batch process development and optimization.     

Altered intracellular calcium fluxes in pancreatic cancer induced diabetes mellitus: Relevance of the S100A8 N‐terminal peptide (NT‐S100A8)

After isolating NT‐S100A8 from pancreatic cancer (PC) tissue of diabetic patients, we verified whether this peptide alters PC cell growth and invasion and/or insulin release and [Ca²⁺]_i oscillations of insulin secreting cells and/or insulin signaling. BxPC3, Capan1, MiaPaCa2, Panc1 (PC cell lines) cell growth, and invasion were assessed in the absence or presence of 50, 200, and 500 nM NT‐S100A8. In NT‐S100A8 stimulated β‐TC6 (insulinoma cell line) culture medium, insulin and [Ca²⁺] were measured at 2, 3, 5, 10, 15, 30, and 60 min, and [Ca²⁺]_i oscillations were monitored (epifluorescence) for 3 min. Five hundred nanomolars NT‐S100A8 stimulated BxPC3 cell growth only and dose dependently reduced MiaPaCa2 and Panc1 invasion. Five hundred nanomolars NT‐S100A8 induced a rapid insulin release and enhanced β‐TC6 [Ca²⁺]_i oscillations after both one (F = 6.05, P < 0.01) and 2 min (F = 7.42, P < 0.01). In the presence of NT‐S100A8, [Ca²⁺] in β‐TC6 culture medium significantly decreased with respect to control cells (F = 6.3, P < 0.01). NT‐S100A8 did not counteract insulin induced phosphorylation of the insulin receptor, Akt and IκB‐α, but it independently activated Akt and NF‐κB signaling in PC cells. In conclusion, NT‐S100A8 exerts a mild effect on PC cell growth, while it reduces PC cell invasion, possibly by Akt and NF‐κB signaling, NT‐S100A8 enhances [Ca²⁺]_i oscillations and insulin release, probably by inducing Ca²⁺ influx from the extracellular space, but it does not interfere with insulin signaling. J. Cell. Physiol. 226: 456–468, 2011. © 2010 Wiley‐Liss, Inc.     

CYANOBACTERIAL ACCLIMATION TO RAPIDLY FLUCTUATING LIGHT IS CONSTRAINED BY INORGANIC CARBON STATUS1

Acclimation to rapidly fluctuating light, simulating shallow aquatic habitats, is altered depending on inorganic carbon (C_i) availability. Under steady light of 50 μmol photons·m^?2·s^?1, the growth rate of Synechococcus elongatus PCC7942 was similar in cells grown in high C_i (4 mM) and low C_i (0.02 mM), with induced carbon concentrating mechanisms compensating for low C_i. Growth under fluctuating light of a 1‐s period averaging 50 μmol photons·m^?2·s^?1 caused a drop in growth rate of 28%±6% in high C_i cells and 38%±8% in low C_i cells. In high C_i cells under fluctuating light, the PSI/PSII ratio increased, the PSII absorption cross‐section decreased, and the PSII turnover rate increased in a pattern similar to high‐light acclimation. In low C_i cells under fluctuating light, the PSI/PSII ratio decreased, the PSII absorption cross‐section decreased, and the PSII turnover remained slow. Electron transport rate was similar in high and low C_i cells but in both was lower under fluctuating than under steady light. After acclimation to a 1‐s period fluctuating light, electron transport rate decreased under steady or long‐period fluctuating light. We hypothesize that high C_i cells acclimated to exploit the bright phases of the fluctuating light, whereas low C_i cells enlarged their PSII pool to integrate the fluctuating light and dampen the variation of the electron flux into a rate‐restricted C_i pool. Light response curves measured under steady light, widely used to predict photosynthetic rates, do not properly predict photosynthetic rates achieved under fluctuating light, and exploitation of fluctuating light is altered by C_i status.     

Modelling assimilation and intercellular CO2 from measured conductance: a synthesis of approaches

A spectrum of models that estimate assimilation rate A from intercellular carbon dioxide concentration (C_i) and measured stomatal conductance to CO₂ (g_c) were investigated using leaf‐level gas exchange measurements. The gas exchange measurements were performed in a uniform loblolly pine stand (Pinus taeda L.) using the Free Air CO₂ Enrichment (FACE) facility under ambient and elevated atmospheric CO₂ for 3 years. These measurements were also used to test a newly proposed framework that combines basic properties of the A–C_i curve with a Fickian diffusion transport model to predict the relationship between C_i/C_a and g_c, where C_a is atmospheric carbon dioxide concentration. The widely used Ball–Berry model and five other models as well as the biochemical model proposed by Farquhar et al. (1980) were also reformulated to express variations in C_i/C_a as a function of their corresponding driving mechanisms. To assess the predictive capabilities of these approaches, their respective parameters were estimated from independent measurements of long‐term stable carbon isotope determinations (δ¹³C), meteorological variables, and ensemble A–C_i curves. All eight approaches reproduced the measured A reasonably well, in an ensemble sense, from measured water vapour conductance and modeled C_i/C_a. However, the scatter in the instantaneous A estimates was sufficiently large for both ambient and elevated C_a to suggest that other transient processes were not explicitly resolved by all eight parameterizations. An important finding from our analysis is that added physiological complexity in modeling C_i/C_a (when g_c is known) need not always translate to increased accuracy in predicting A. Finally, the broader utility of these approaches to estimate assimilation and net ecosystem exchange is discussed in relation to elevated atmospheric CO₂.     

A genetic link between leaf carbon isotope composition and whole‐plant water use efficiency in the C4 grass Setaria

Genetic selection for whole‐plant water use efficiency (yield per transpiration; WUE_plant) in any crop‐breeding programme requires high‐throughput phenotyping of component traits of WUE_plant such as intrinsic water use efficiency (WUE_i; CO₂ assimilation rate per stomatal conductance). Measuring WUE_i by gas exchange measurements is laborious and time consuming and may not reflect an integrated WUE_i over the life of the leaf. Alternatively, leaf carbon stable isotope composition (δ¹³C_leaf) has been suggested as a potential time‐integrated proxy for WUE_i that may provide a tool to screen for WUE_plant. However, a genetic link between δ¹³C_leaf and WUE_plant in a C₄ species has not been well established. Therefore, to determine if there is a genetic relationship in a C₄ plant between δ¹³C_leaf and WUE_plant under well watered and water‐limited growth conditions, a high‐throughput phenotyping facility was used to measure WUE_plant in a recombinant inbred line (RIL) population created between the C₄ grasses Setaria viridis and S. italica. Three quantitative trait loci (QTL) for δ¹³C_leaf were found and co‐localized with transpiration, biomass accumulation, and WUE_plant. Additionally, WUE_plant for each of the δ¹³C_leaf QTL allele classes was negatively correlated with δ¹³C_leaf, as would be predicted when WUE_i influences WUE_plant. These results demonstrate that δ¹³C_leaf is genetically linked to WUE_plant, likely to be through their relationship with WUE_i, and can be used as a high‐throughput proxy to screen for WUE_plant in these C₄ species.     

Detection of a variable intracellular acid‐labile carbon pool in Thalassiosira weissflogii (Heterokontophyta) and Emiliania huxleyi (Haptophyta) in response to changes in the seawater carbon system

Accumulation of an intracellular pool of carbon (C_i pool) is one strategy by which marine algae overcome the low abundance of dissolved CO₂ (CO_2(aq)) in modern seawater. To identify the environmental conditions under which algae accumulate an acid‐labile C_i pool, we applied a ¹⁴C pulse‐chase method, used originally in dinoflagellates, to two new classes of algae, coccolithophorids and diatoms. This method measures the carbon accumulation inside the cells without altering the medium carbon chemistry or culture cell density. We found that the diatom Thalassiosira weissflogii [(Grunow) G. Fryxell & Hasle] and a calcifying strain of the coccolithophorid Emiliania huxleyi [(Lohmann) W. W. Hay & H. P. Mohler] develop significant acid‐labile C_i pools. Ci pools are measureable in cells cultured in media with 2–30 µmol l^?1 CO_2(aq), corresponding to a medium pH of 8.6–7.9. The absolute C_i pool was greater for the larger celled diatoms. For both algal classes, the C_i pool became a negligible contributor to photosynthesis once CO_2(aq) exceeded 30 µmol l^?1. Combining the ¹⁴C pulse‐chase method and ¹⁴C disequilibrium method enabled us to assess whether E. huxleyi and T. weissflogii exhibited thresholds for foregoing accumulation of DIC or reduced the reliance on bicarbonate uptake with increasing CO_2(aq). We showed that the C_i pool decreases with higher CO₂:HCO₃^? uptake rates.     

Asparagine and glutamine differ in their propensities to form specific side chain-backbone hydrogen bonded motifs in proteins

Short range side chain‐backbone hydrogen bonded motifs involving Asn and Gln residues have been identified from a data set of 1370 protein crystal structures (resolution ≤ 1.5 Å). Hydrogen bonds involving residues i ? 5 to i + 5 have been considered. Out of 12,901 Asn residues, 3403 residues (26.4%) participate in such interactions, while out of 10,934 Gln residues, 1780 Gln residues (16.3%) are involved in these motifs. Hydrogen bonded ring sizes (C_n, where n is the number of atoms involved), directionality and internal torsion angles are used to classify motifs. The occurrence of the various motifs in the contexts of protein structure is illustrated. Distinct differences are established between the nature of motifs formed by Asn and Gln residues. For Asn, the most highly populated motifs are the C₁₀ (CO^δ_i …NH_{i + 2}), C₁₃ (CO^δ_i …NH_{i + 3}) and C₁₇ (N^δH_i …CO_{i ? 4}) structures. In contrast, Gln predominantly forms C₁₆ (CO^ε_i …NH_{i ? 3}), C₁₂ (N^εH_i …CO_{i ? 2}), C₁₅ (N^εH_i …CO_{i ? 3}) and C₁₈ (N^εH_i …CO_{i ? 4}) motifs, with only the C₁₈motif being analogous to the Asn C₁₇structure. Specific conformational types are established for the Asn containing motifs, which mimic backbone β‐turns and α‐turns. Histidine residues are shown to serve as a mimic for Asn residues in side chain‐backbone hydrogen bonded ring motifs. Illustrative examples from protein structures are considered. Proteins 2012; © 2011 Wiley Periodicals, Inc.     

Higher electron transport rate observed at low intercellular CO2 concentration in long-term drought-acclimated leaves of Japanese mountain birch (Betula ermanii)

The influence of long‐term drought stress on photosynthesis of Japanese mountain birch (Betula ermanii Cham.) was examined using chlorophyll fluorescence and gas exchange measurements. Drought stress was imposed in potted plants by reducing irrigation frequency from daily (control) to twice‐weekly and once‐weekly. Thirty‐day‐old leaves, which had developed under fully stressed conditions, were used for the measurements. The decline in net CO₂ assimilation rate (A) observed in situ in drought‐stressed plants resulted from a lower intercellular CO₂ concentration (C_i) due to stomatal closure but the carboxylation efficiency was not affected as there was no difference in the initial slope of the A/C_i response after watering. Although there were no treatment differences in A at C_i below 270 μmol mol^?1 (with ambient air at 360 μmol mol^?1 CO₂), higher electron transport rate (ETR), photochemical quenching (q_P) and the efficiency of energy conversion of open PSII (F_v′/F_m′), and similar or even lower non‐photochemical quenching (NPQ) were observed at a given C_i in drought‐stressed plants (of both twice‐ and once‐weekly irrigation), suggesting a higher fraction of open PSII resulting from energy dissipation achieved through higher electron flow rather than through thermal dissipation in PSII antennae. The once‐weekly watered plants showed a lower ratio of gross carbon assimilation rate to ETR (A*/ETR), suggesting an enhanced alternative pathway of electron flow probably involving the Mehler‐peroxidase (MP) reaction as indicated by a higher Φ_PSII at a given Φ_CO2 under non‐photorespiratory conditions. On the other hand, plants of twice‐weekly watering exhibited almost the same A*/ETR and Φ_PSII–Φ_CO2 relationship as control plants, indicating no enhanced alternative pathways under mild drought stress.     

Multiple source pools for Galápagos plant species richness: a critical analysis of the line of sight connectivity index

An index (C_i*E) combining the number of line‐of‐sight islands (C_i) within a radius i and target island elevation (E) has been proposed as an improved predictive model of plant species richness (S_t) in the Galápagos Archipelago. We examined this index critically and found that several major flaws preclude it from being a useful predictive tool for the archipelago. Although the number of collecting trips to an island was reported over 20 years ago to have substantial predictive value for reported plant species richness in the Galápagos Islands, this relationship was ignored in multiple regression analyses of the index. When we included the number of collecting trips in different multiple regression analyses of the index, C_i*E had less predictive power than collecting trips or ceased to be significant at all. Additionally, the strong significant relationship between elevation and area in the Galápagos Archipelago results in area having a major confounding influence on the C_i*E index. When elevation is removed from the C_i*E index, the predictive power of C_i is far less than area alone. Finally, the data used to construct and correlate the C_i*E index with (S_t) were based only on a subset of the islands and species lists that were incomplete or out of date. Species richness on islands can be related to the interaction of different factors, so development and testing of indices like C_i*E is not inappropriate. However, it is important to examine the interrelationships among the components of these indices thoroughly, and not ignore the effect of factors already known to have high predictive power. We propose several ways in which more meaningful indices of source pool(s) capacity can be constructed.     

Structure and function of LCI1: a plasma membrane CO2 channel in the Chlamydomonas CO2 concentrating mechanism

Microalgae and cyanobacteria contribute roughly half of the global photosynthetic carbon assimilation. Faced with limited access to CO₂ in aquatic environments, which can vary daily or hourly, these microorganisms have evolved use of an efficient CO₂ concentrating mechanism (CCM) to accumulate high internal concentrations of inorganic carbon (C_i) to maintain photosynthetic performance. For eukaryotic algae, a combination of molecular, genetic and physiological studies using the model organism Chlamydomonas reinhardtii, have revealed the function and molecular characteristics of many CCM components, including active C_i uptake systems. Fundamental to eukaryotic C_i uptake systems are C_i transporters/channels located in membranes of various cell compartments, which together facilitate the movement of C_i from the environment into the chloroplast, where primary CO₂ assimilation occurs. Two putative plasma membrane C_i transporters, HLA3 and LCI1, are reportedly involved in active C_i uptake. Based on previous studies, HLA3 clearly plays a meaningful role in HCO₃^? transport, but the function of LCI1 has not yet been thoroughly investigated so remains somewhat obscure. Here we report a crystal structure of the full‐length LCI1 membrane protein to reveal LCI1 structural characteristics, as well as in vivo physiological studies in an LCI1 loss‐of‐function mutant to reveal the C_i species preference for LCI1. Together, these new studies demonstrate LCI1 plays an important role in active CO₂ uptake and that LCI1 likely functions as a plasma membrane CO₂ channel, possibly a gated channel.     

Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm

The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (C_m) to monitor membrane changes in protoplasts from Viciafaba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 μM) at constant ambient osmotic pressure (π_o) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (f_FM,i) increased with reference to the total fluorescence (f_FM,t) by 7.4 × 10⁻⁴ min⁻¹. This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of π_o by 200 mosmol kg⁻¹ caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time f_FM,i/f_FM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of C_m and total fluorescence of a protoplast (f_FM,p) showed that an increase in π_o evoked a decrease in C_m but no change in f_FM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 μm) with FM1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area. Received: 4 May 1999 / Accepted: 19 August 1999     

A dominant‐negative form of Arabidopsis AP‐3 β‐adaptin improves intracellular pH homeostasis

Intracellular pH (pH_i) is a crucial parameter in cellular physiology but its mechanisms of homeostasis are only partially understood. To uncover novel roles and participants of the pH_i regulatory system, we have screened an Arabidopsis mutant collection for resistance of seed germination to intracellular acidification induced by weak organic acids (acetic, propionic, sorbic). The phenotypes of one identified mutant, weak acid‐tolerant 1‐1D (wat1‐1D) are due to the expression of a truncated form of AP‐3 β‐adaptin (encoded by the PAT2 gene) that behaves as a as dominant‐negative. During acetic acid treatment the root epidermal cells of the mutant maintain a higher pH_i and a more depolarized plasma membrane electrical potential than wild‐type cells. Additional phenotypes of wat1‐1D roots include increased rates of acetate efflux, K⁺ uptake and H⁺ efflux, the latter reflecting the in vivo activity of the plasma membrane H⁺‐ATPase. The in vitro activity of the enzyme was not increased but, as the H⁺‐ATPase is electrogenic, the increased ion permeability would allow a higher rate of H⁺ efflux. The AP‐3 adaptor complex is involved in traffic from Golgi to vacuoles but its function in plants is not much known. The phenotypes of the wat1‐1D mutant can be explained if loss of function of the AP‐3 β‐adaptin causes activation of channels or transporters for organic anions (acetate) and for K⁺ at the plasma membrane, perhaps through miss‐localization of tonoplast proteins. This suggests a role of this adaptin in trafficking of ion channels or transporters to the tonoplast.     

Phosphoregulation of K+‐Cl− cotransporter 4 during changes in intracellular Cl− and cell volume

It has long been stated that the K⁺‐Cl^? cotransporters (KCCs) are activated during cell swelling through dephosphorylation of their cytoplasmic domains by a protein phosphatase (PP) but that other enzymes are involved by targeting this PP or the KCCs directly. To date, however, the role of signaling intermediates in KCC regulation has been deduced from indirect evidence rather than in vitro phosphorylation studies, and examined after simulation of ion transport through cell swelling or N‐ethylmaleimide treatment. In this study, the oocyte expression system was used to examine the effects of changes in cell volume (C_VOL) and intracellular [Cl^?] ([Cl^?]_i) on the activity and phosphorylation levels (P_LEV) of KCC4, and determine whether these effects are mediated by PP1 or phorbol myristate acetate (PMA)‐sensitive effectors. We found that (1) low [Cl^?]_i or low C_VOL leads to decreased activity but increased P_LEV, (2) high C_VOL leads to increased activity but no decrease in P_LEV and (3) calyculin A (Cal A) or PMA treatment leads to decreased activity but no increase in P_LEV. Thus, we have shown for the first time that one of the KCCs can be regulated through direct phosphorylation, that changes in [Cl^?]_i or C_VOL modify the activity of signaling enzymes at carrier sites, and that the effectors directly involved do not include a Cal A‐sensitive PP in contrast to the widely held view. J. Cell. Physiol. 219: 787–796, 2009. © 2009 Wiley‐Liss, Inc.     

Mechanism and role of furosemide-sensitive K+ transport in L cells: A genetic approach

Summary As part of a genetic study of the mechanisms for cation transport in cultured mammalian cells, two mouse fibroblastic cell lines have been compared with respect to unidirectional⁴²K⁺ influx. The cell lines areLM(TK ^–) andLTK-5, a mutant selected fromLM(TK ^–) by the ability to grow in medium containing 0.2mm K⁺. In both cell lines, the overall influx can be resolved into three components: (i) a ouabain- and vanadate-sensitive component ( ⁱ M_K ^f), presumably the Na/K pump, which is a saturable function of extracellular K⁺ with aK _1/2 of 1.3mm; (ii) a furosemide-sensitive component ( ⁱ M_k ^fx), also a saturable function of extracellular K⁺, with aK _1/2 of 6mm; and (iii) a diffusional component ( ⁱ M_k ^d); which is a linear function of extracellular K⁺.By several independent criteria, ⁱ M_k ^o and ⁱ M_k ^f appear to be distinct transport processes. First, as indicated above, they can be separated with the use of inhibitors. In addition, they can be separated genetically, since theLTK-5 mutant shows a threefold elevation in ⁱ M_k ^f with no change in ⁱ M_k ^o. And finally, extracellular Na⁺ has no effect on ⁱ M_k ^o, but stimulates ⁱ M_k ^f, a result consistent with the notion that ⁱ M_k ^f influx occurs by Na–K cotransport.Further experiments were directed towards understanding the nature of theLTK-5 mutation and the physiological role of ⁱ M_k ^f. LTK-5 differs from the parental cell line, not only in having an increased ⁱ M_k ^f, but also in having a large cell volume, a slow maximal growth rate, and an ability to grow at 0.2mm K⁺. The most straightforward interpretation — that the increased ⁱ M_k ^f is itself responsible—is unlikely since the addition of furosemide to the growth medium had no effect upon the growth rate or cell volume of the mutant at either normal or low extracellular K⁺ concentrations. It did, however, render the parent capable of growth at 0.2mm K⁺. Possible interpretations are discussed.     

Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N‐terminal Fic domain and a C‐terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS‐mediated translocation into host cells. A proteolysis resistant fragment (residues 10–302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α‐[³²P]‐ATP. Its crystal structure, determined to 2.9‐Å resolution by the SeMet‐SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional β‐rich domain at the C‐terminus. On crystal soaking with ATP/Mg²⁺, additional electron density indicated the presence of a PP_i/Mg²⁺ moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg²⁺ and target tyrosine. The model is consistent with an in‐line nucleophilic attack of the deprotonated side‐chain hydroxyl group onto the α‐phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence‐independent mechanism of target positioning through antiparallel β‐strand interactions between enzyme and target is suggested.