Carbon Use Efficiency and Cell Expansion of NaCl-Adapted Tobacco Cells

Carbon use efficiencies (gram cell organic dry weight accumulated per gram sugar assimilated from the medium) of unadapted and NaCl-adapted (428 millimolar) cells of tobacco (Nicotiana tabacum L. var Wisconsin 38) were determined to evaluate metabolic costs associated with growth and survival in a saline environment. No net increase in carbon costs was associated with salt adaptation. At low substrate levels, carbon use efficiencies of unadapted and NaCl-adapted cells were not appreciably different (0.495 and 0.422, respectively) and at higher substrate levels carbon use efficiency of NaCl-adapted cells was clearly higher than that of unadapted cells. These results indicate that a homeostasis of metabolic efficiency is established after cells have adapted to NaCl. Altered carbon availability does not cause the reduced cell volume that results from adaptation to NaCl. This does not preclude, however, the possibility that altered intracellular partitioning of carbon affects cell expansion.     

Relationship of Plasmalemma Redox Activity to K+ Uptake by Dunaliella salina Cells

The plasmalemma-bond redox system localized within the plasmalemma of unicellular green alga Dunaliella salina was studied. This system oxidized exogenous NADH, increased O2 consumption to 165 % and increased the pH of the external medium, while K+ influx was inhibited. With no NADH added, ferricyanide stimulated K+ uptake about 3 folds. In the presence of exogenous NADH, ferricyanide was rapidly reduced and the external medium was acidified, generating a greater electrochemical proton gradient across the plasmalemma, thus resulting an 6-fold increase of K+ influx. Typical inhibitors of plasmalemma H+-ATPase and redox system inhibited K+ uptake to different extent. That the inhibition of K+ uptake by vanadate could be resumed partly by addition of NADH and ferricyanide indicated that plasmalemma redox system operated in association with the H+-ATPase to exert an influence on K+ transportation. A model was presented in which the implication of two possible redox chains and H+-ATPase in generating an electrochemical potential gradient for protons (△uH+) was discussed.     

Growth Yields and Maintenance Coefficients of Unadapted and NaCl-Adapted Tobacco Cells Grown in Semicontinuous Culture

Comparison of carbon utilization between unadapted and NaCl (428 millimolar) adapted tobacco (Nicotiana tabacum L.) cells under substrate limited growth conditions was facilitated using semicontinuous culture. Growth yields (Y_g) and maintenance coefficients (m) of unadapted and NaCl adapted cells were similar, indicating that the efficiency of carbon utilization for growth was not altered as a result of salt adaptation and that no additional metabolic costs were associated with growth of adapted cells in the presence of a high concentration (428 millimolar) of NaCl. The Y_g (0.588 grams organic dry weight gain per gram sugar uptake) and m values (0.117 grams sugar uptake per gram organic dry weight per day) were comparable in spite of substantial physiological and biochemical differences that exist between unadapted and NaCl adapted cells. Apparently, a metabolic homeostasis governs biomass production of cells before and after adaptation to salinity.     

Enhanced Cellular Uptake of Albumin-Based Lyophilisomes when Functionalized with Cell-Penetrating Peptide TAT in HeLa Cells

Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS) was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.     

Enhanced Cellular Uptake of G-Rich Oligonucleotides

Abstract

Sequence-dependency of cellular uptake of oligonucleotides into Vero cells has been studied. Cellular uptake of 5′-[³⁵S]-labelled homopolymers decreased in the order (dG)₁₆ >> (dT)₁₆> (dA)₁₆ > (dC)₁₆. The change of two base-pairs (dG → dA) in a dG-rich antisense oligonucleotide with good antiviral activity dramatically decreased cellular uptake and abolished antiviral activity.     

Stimulation of Calcium Uptake in PC12 Cells by the Dihydropyridine Agonist BAY K 8644

Abstract: Methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (BAY K 8644), an analog of dihydropyridine calcium channel antagonists, stimulated ⁴⁵Ca uptake into PC12 pheochromocytoma cells. Half-maximal stimulation occurred at 80 n M BAY K 8644. Enhancement of uptake was inhibited by cationic and organic calcium channel blockers, but not by tetrodotoxin, which is consistent with an effect on voltage-dependent calcium channels. Stimulation of ⁴⁵Ca uptake by BAY K 8644 occurred only at elevated concentrations of extracellular K⁺, suggesting that BAY K 8644 may interact with calcium channels in the open (activated) state.     

Use of Na-Nuclear Magnetic Resonance To Follow Sodium Uptake and Efflux in NaCl-Adapted and Nonadapted Millet (Panicum miliaceum) Suspensions

Cellular Na⁺ transport was followed in vivo by ²³Na nuclear magnetic resonance (NMR) using anionic dysprosium-based shift reagents to resolve internal and external ²³Na⁺ resonances. Proso millet (Panicum miliaceum) cell suspensions adapted for rapid growth on 130 mm NaCl had biphasic ²³Na efflux kinetics when shifted to low Na⁺ medium, while nonadapted cells had little measurable Na⁺ efflux after preloading with ²³NaCl. Uptake of ²³Na was also observed using ²³Na NMR. The resonance frequency of the external Na⁺-dysprosium (III) triphosphate, relative to that of the ²³Na in the cells, was sensitive to pH, permitting the pH of the external medium to be followed during the course of in vivo experiments.     

Aluminum Uptake by Neuroblastoma Cells

Aluminum uptake studies in viable neuroblastoma cells were performed. Aluminum uptake was largely dependent on the pH of the suspension medium. At physiological pH values, cells were apparently unable to incorporate detectable amounts of aluminum in the absence of proper mediators. Aluminum uptake was enhanced as the pH decreased, attaining a plateau at about pH 6.0. In experiments with 2 x 10(6) cells/ml, pH 6.0, and 25 microM aluminum in the medium, aluminum incorporation reached saturation at 5 nmol of aluminum/mg of cellular protein, accounting for 60-70% of aluminum added. At pH 6.0, cells showed a large capacity for accumulating aluminum; about 70% of intracellular aluminum was associated with the postmitochondrial fraction. At neutral pH, application of apotransferrin seemed to facilitate aluminum translocation into cells via membrane receptors. Fatty acids were also capable of mediating aluminum uptake at neutral pH, probably by forming aluminum-fatty acid complexes. Low molecular weight aluminum chelators, e.g., citrate, inhibited aluminum uptake. Treatment of cells with energy metabolism blockers had virtually no influence on aluminum uptake, indicative of passive mechanisms. The results suggest that aluminum uptake occurs via different modes dependent on growth conditions, such as medium pH.     

Elevated Accumulation of Proline in NaCl-Adapted Tobacco Cells Is Not Due to Altered Delta-Pyrroline-5-Carboxylate Reductase

Tobacco (Nicotiana tabacum L. var Wisconsin 38) cells that are adapted to 428 millimolar NaCl accumulate proline mainly due to increased synthesis from glutamate. These cells were used to evaluate the possible role of Δ¹-pyrroline-5-carboxylate reductase in the regulation of proline biosynthesis. No increase in the specific activity of Δ¹-pyrroline-5-carboxylate reductase in crude extracts throughout the growth cycle was observed in NaCl-adapted cells compared to unadapted cells. The enzyme from both cell types was purified extensively. On the basis of affinity for the substrates NADPH, NADH, and Δ¹-pyrroline-5-carboxylate, pH profiles, chromatographic behavior during purification, and electrophoretic mobility of the native enzyme, the activities of the enzyme from the two sources were similar. These data suggest that the NaCl-dependent regulation of proline synthesis in tobacco cells does not involve induction of pyrroline-5-carboxylate isozymes or changes in its kinetic properties.     

K+ Uptake by Fermenting Escherichia coli Cells: pH Dependent Mode of the TrkA System Operating

Escherichia coli accumulates K⁺ by means of multiple transportsystems, of which TrkA is the most prominent at neutral and alkalinepH while Kup is major at acidic pH. In the present study, K⁺ uptakewas observed with cells grown under fermentative conditions at an initialpH of 9.0 and 7.3 (the medium pH decreased to 8.4 and 6.8, respectively,during the mid-logarithmic growth phase), washed with distilled water andresuspended in a K⁺ containing medium at pH 7.5 in the presence ofglucose. The kinetics for this K⁺ uptake and the amount of K⁺accumulated by the wild type and mutants having a functional TrkA orKup could confirm that K⁺ uptake by E. coli grown either at pH 9.0or pH 7.3 occurs mainly through TrkA. The following results distinguishpH dependent mode of TrkA operating: (1) K⁺ uptake was inhibited byDCCD in cells grown either at pH 9.0 or pH 7.3, although the stoichiometryof K⁺ influx to DCCD-inhibited H⁺ efflux for bacteria grownat pH 9.0 varied with external K⁺ concentration, but remained constantfor cells grown at pH 7.3; (2) K⁺ uptake was observed with an atpDmutant grown at pH 9.0 but not at pH 7.3; (3) The DCCD-inhibited H⁺efflux was increased 8-fold less by 5 mM K⁺ added into a K⁺ freemedium for bacteria grown at pH 9.0 than that for cells grown at pH 7.3;(4) the DCCD-inhibited ATPase activity of membrane vesicles from bacteriagrown at pH 9.0 was reduced a little in the presence of 100 mM K⁺,but stimulated more than 2.4-fold at pH 7.3.     

Residual Tumor Cells That Drive Disease Relapse after Chemotherapy Do Not Have Enhanced Tumor Initiating Capacity

Although chemotherapy is used to treat most advanced solid tumors, recurrent disease is still the major cause of cancer-related mortality. Cancer stem cells (CSCs) have been the focus of intense research in recent years because they provide a possible explanation for disease relapse. However, the precise role of CSCs in recurrent disease remains poorly understood and surprisingly little attention has been focused on studying the cells responsible for re-initiating tumor growth within the original host after chemotherapy treatment. We utilized both xenograft and genetically engineered mouse models of non-small cell lung cancer (NSCLC) to characterize the residual tumor cells that survive chemotherapy treatment and go on to cause tumor regrowth, which we refer to as tumor re-initiating cells (TRICs). We set out to determine whether TRICs display characteristics of CSCs, and whether assays used to define CSCs also provide an accurate readout of a cell’s ability to cause tumor recurrence. We did not find consistent enrichment of CSC marker positive cells or enhanced tumor initiating potential in TRICs. However, TRICs from all models do appear to be in EMT, a state that has been linked to chemoresistance in numerous types of cancer. Thus, the standard CSC assays may not accurately reflect a cell’s ability to drive disease recurrence.     

Efficient Uptake of Cesium Ions by Rhodococcus Cells

Bacteria of the genus Rhodococcus were found to be able to accumulate cesium by means of active transport and nonspecific sorption on the cell surface structures. The maximum removal (up to 97%) of cesium from a medium supplemented with ammonium acetate was observed at 28°C, pH 7.8–8.6, and an equimolar content (0.2 mM) of potassium and cesium ions in the medium. The most active cesium-accumulating rhodococcal strains may be useful in biological treatment of industrial wastewaters contaminated with radionuclides.     

Metabolic Factors Affecting Enhanced Phosphorus Uptake by Activated Sludge

Activated sludges obtained from the Rilling Road plant located at San Antonio, Tex., and from the Hyperion treatment plant located at Los Angeles, Calif., have the ability to remove all of the orthophosphate normally present in Tucson sewage within 3 hr after being added to the waste water. Phosphorus removal was independent of externally supplied sources of energy and ions, since orthophosphate and (32)P radioactivity were readily removed from tap water, glass-distilled water, and deionized water. Phosphorus uptake by Rilling sludge in the laboratory appears to be wholly biological, as it has an optimum pH range (7.7 to 9.7) and an optimum temperature range (24 to 37 C). It was inhibited by HgCl(2), iodoacetic acid, p-chloromercuribenzoic acid, NaN(3), and 2, 4-dinitrophenol (compounds that affect bacterial membrane permeability, sulfhydryl enzymes, and adenosine triphosphate synthesis). Uptake was inhibited by 1% NaCl but was not affected by 10(-3)m ethylenediaminetetraacetic acid disodium salt (a chelating agent for many metallic ions).     

Quantitative Determination of Dye Uptake by Bacterial Cells

A colorimetric method is presented for the quantitative determination of dye uptake by bacterial cells. Experiments showed that the dye to cell ratio was of major importance in controlling the amount of dye taken up per weight of bacterial cells. Approximate dye saturation of cells could be obtained (at pH 6.1 to 6.3) although the dye uptake curves did not absolutely level off.     

Induction of Ferric Reductase Activity and of Iron Uptake Capacity in Chlorococcum littorale Cells under Extremely High-CO2 and Iron-Deficient Conditions

The marine green alga, Chlorococcum littorale, accumulated ironin its cells and showed high activity of plasma membrane ferricreductase under high-CO₂ and iron-deficient conditions. Theseactivities disappeared upon exposure to ordinary air and byadding excess FeSO₄. The iron uptake had high affinity for theFe(II) form (K_m of 0.13 µM). Carbonylcyanide m-chlorophenylhydrazoneand N,N-dicyclohexylcarbodiimide significantly suppressed theiron uptake, suggesting that the Fe(II) uptake was driven byATPase. These results indicate that high CO₂ and iron deficiencycooperatively induce the Fe(II) uptake and cell-surface ferricreductase activity. (Received October 20, 1997; Accepted January 29, 1998)     

Alternative Serotype Adenovirus Vaccine Vectors Elicit Memory T Cells with Enhanced Anamnestic Capacity Compared to Ad5 Vectors

The failure of the adenovirus serotype 5 (Ad5) vector-based human immunodeficiency virus type 1 (HIV-1) vaccine in the STEP study has led to the development of adenovirus vectors derived from alternative serotypes, such as Ad26, Ad35, and Ad48. We have recently demonstrated that vaccines using alternative-serotype Ad vectors confer partial protection against stringent simian immunodeficiency virus (SIV) challenges in rhesus monkeys. However, phenotypic differences between the T cell responses elicited by Ad5 and those of alternative-serotype Ad vectors remain unexplored. Here, we report the magnitude, phenotype, functionality, and recall capacity of memory T cell responses elicited in mice by Ad5, Ad26, Ad35, and Ad48 vectors expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP). Our data demonstrate that memory T cells elicited by Ad5 vectors were high in magnitude but exhibited functional exhaustion and decreased anamnestic potential following secondary antigen challenge compared to Ad26, Ad35, and Ad48 vectors. These data suggest that vaccination with alternative-serotype Ad vectors offers substantial immunological advantages over Ad5 vectors, in addition to circumventing high baseline Ad5-specific neutralizing antibody titers.     

Responses by Coleoptiles of Intact Rice Seedlings to Anoxia: K+ Net Uptake from the External Solution and Translocation from the Caryopses

This study evaluated the effects of anoxia on K⁺ uptake andtranslocation in 3–4-d-old, intact, rice seedlings (Oryzasativa L. cv. Calrose). Rates of net K⁺ uptake from the mediumover 24 h by coleoptiles of anoxic seedlings were inhibitedby 83–91 %, when compared with rates in aerated seedlings.Similar uptake rates, and degree of inhibition due to anoxia,were found for Rb⁺ when supplied over 1·5–2 h,starting 22 h after imposing anoxia. The Rb⁺ uptake indicatedthat intact coleoptiles take up ions directly from the externalsolution. Monovalent cation (K⁺ and Rb⁺) net uptake from thesolution was inhibited by anoxia to the same degree for thecoleoptiles of intact seedlings and for coleoptiles excised,‘aged’, and supplied with exogenous glucose. Transportof endogenous K⁺ from caryopses to coleoptiles was inhibitedless by anoxia than net K⁺ uptake from the solution, the inhibitionbeing 55 % rather than 87 %. Despite these inhibitions,osmotic pressures of sap (_sap) expressed from coleoptiles ofseedlings exposed to 48 h of anoxia, with or without exogenousK⁺, were 0·66 ± 0·03 MPa; however,the contributions of K⁺ to _sap were 23 and 16 %, respectively.After 24 h of anoxia, the K⁺ concentrations in the basal10 mm of the coleoptiles of seedlings with or without exogenousK⁺, were similar to those in aerated seedlings with exogenousK⁺. In contrast, K⁺ concentrations had decreased in aeratedseedlings without exogenous K⁺, presumably due to ‘dilution’by growth; fresh weight gains of the coleoptile being 3·6-to 4·7-fold greater in aerated than in anoxic seedlings.Deposition rates of K⁺ along the axes of the coleoptiles werecalculated for the anoxic seedlings only, for which we assessedthe elongation zone to be only the basal 4 mm. K⁺ depositionin the basal 6 mm was similar for seedlings with or withoutexogenous K⁺, at 0·6–0·87 µmolg^–1 f. wt h^–1. Deposition rates in zones above6 mm from the base were greater for seedlings with, thanwithout, exogenous K⁺; the latter were sometimes negative. Weconclude that for the coleoptiles of rice seedlings, anoxiainhibits net K⁺ uptake from the external solution to a muchlarger extent than K⁺ translocation from the caryopses. Furthermore,K⁺ concentrations in the elongation zone of the coleoptilesof anoxic seedlings were maintained to a remarkable degree,contributing to maintenance of _sap in cells of these elongatingtissues.     

Regulation of K+ Uptake and Transport to the Xylem in Barley Roots; K+ Distribution Determined by Electron Probe X-ray Microanalysis of Frozen-Hydrated Cells

Intact roots of young barley plants (Hordeum vulgare L. cv.Proctor) were induced to transport K⁺ to the xylem at rapidor slow rates. Roots were then rapidly frozen in liquid nitrogenand fractured in the zone 70 mm behind the root tip to givetransverse faces for electron probe microanalysis. With SEMvisualization, analyses were made over the cytoplasm and vacuole(or lumen) of 14 cells types along the root radius between theouter cortex and stele, with particular emphasis on the xylem,xylem parenchyma, and phloem. Data were recorded in the formof colour-coded maps and also quantitatively. For both typesof roots, K⁺ concentration was lower over the xylem and phloemthan in the remainder of the root. The concentration of K⁺ wasgreater in the vacuole than in the cytoplasm, while for P itwas the reverse. Significantly, in roots induced to transportK⁺ rapidly the concentration of K⁺ was low in the early maturingmetaxylem and protoxylem, and in the sieve tubes of the metaphloemand protophloem. The concentrations of K⁺ in various cell typesare discussed in relation to regulation of K⁺ loading of thexylem in long-distance ion transport. Key words: Ion transport, nutrient deficiency