Characterization of the basic charge variants of a human IgG1

We report a case study of an IgG1 with a unique basic charge variant profile caused by C-terminal proline amidation on either one or two heavy chains. The proline amidation was sensitive to copper ion concentration in the production media during cell culture: the higher the Cu²⁺ ion concentration, the higher the level of proline amidation detected. This conclusion was supported by the analysis of samples that revealed direct correlation between the proline amidation level observed from peptide maps and the level of basic peaks measured by imaged capillary isoelectric focusing and a pH gradient ion-exchange chromatography method. The importance of these observations to therapeutic antibody production is discussed.     

Effects of Dissolved and Complexed Copper on Heterotrophic Bacterial Production in San Diego Bay

Bacterial abundance and production, free (uncomplexed) copper ion concentration, total dissolved copper concentration, dissolved organic carbon (DOC), total suspended solids (TSS), and chlorophyll a were measured over the course of 1 year in a series of 27 sample “Boxes” established within San Diego Bay. Water was collected through a trace metal-clean system so that each Box’s sample was a composite of all the surface water in that Box. Bacterial production, chlorophyll a, TSS, DOC, and dissolved copper all generally increased from Box 1 at the mouth of the Bay to Box 27 in the South or back Bay. Free copper ion concentration generally decreased from Box 1 to Box 27 presumably due to increasing complexation capacity within natural waters. Based on correlations between TSS, chlorophyll a, bacterial production or DOC and the ratio of dissolved to free Cu ion, both DOC and particulate (bacteria and algae) fractions were potentially responsible for copper complexation, each at different times of the year. CuCl₂ was added to bacterial production assays from 0 to 10 μg L⁻¹ to assess acute copper toxicity to the natural microbial assemblage. Interestingly, copper toxicity appeared to increase with decreases in free copper from the mouth of the Bay to the back Bay. This contrasts the free-ion activity model in which higher complexation capacity should afford greater copper protection. When cell-specific growth rates were calculated, faster growing bacteria (i.e. toward the back Bay) appeared to be more susceptible to free copper toxicity. The protecting effect of natural dissolved organic material (DOM) concentrated by tangential flow ultrafiltration (>1 kDa), illite and kaolinite minerals, and glutathione (a metal chelator excreted by algae under copper stress) was assessed in bacterial production assays. Only DOM concentrate offered any significant protection to bacterial production under increased copper concentrations. Although the potential copper protecting agents were allowed to interact with added copper before natural bacteria were added to production assays, there may be a temporal dose–response relationship that accounts for higher toxicity in short production assays. Regardless, it appears that effective natural complexation of copper in the back portions of San Diego Bay limits exposure of native bacterial assemblages to free copper ion, resulting in higher bacterial production.     

Enhanced proline accumulation and salt stress tolerance of transgenic <Emphasis Type="Italic">indica</Emphasis> rice by over-expressing <Emphasis Type="Italic">P5CSF129A</Emphasis> gene

Δ¹-pyrroline-5-carboxylate synthetase (P5CS) is a proline biosynthetic pathway enzyme and is known for conferring enhanced salt and drought stress in transgenics carrying this gene in a variety of plant species; however, the wild-type P5CS is subjected to feedback control. Therefore, in the present study, we used a mutagenized version of this osmoregulatory gene-P5CSF129A, which is not subjected to feedback control, for producing transgenic indica rice plants of cultivar Karjat-3 via Agrobacterium tumefaciens. We have used two types of explants for this purpose, namely mature embryo-derived callus and shoot apices. Various parameters for transformation were optimized including antibiotic concentration for selection, duration of cocultivation, addition of phenolic compound, and bacterial culture density. The resultant primary transgenic plants showed more enhanced proline accumulation than their non-transformed counterparts. This proline level was particularly enhanced in the transgenic plants of next generation (T₁) under 150 mM NaCl stress. The higher proline level shown by transgenic plants was associated with better biomass production and growth performance under salt stress and lower extent of lipid peroxidation, indicating that overproduction of proline may have a role in counteracting the negative effect of salt stress and higher maintenance of cellular integrity and basic physiological processes under stress.     

ENHANCEMENT OF INTRACELLULAR PROLINE LEVEL IN CELLS OF ANACYSTIS NIDULANS (CYANOBACTERIA) EXPOSED TO DELETERIOUS CONCENTRATIONS OF COPPER1

The intracellular proline level in Anacystis nidulans cells was enhanced when the cells were exposed to sublethal concentrations of Cu²⁺; the degree of enhancement was positively related to the concentration of Cu²⁺. Analysis by high-performance liquid chromatography confirmed that the enhancement of proline levels was the most pronounced change in the composition of the free amino acid pool during copper treatment. A direct supply of exogenous proline to the cultures lowered the inhibitory influence of Cu²⁺on the growth of cells. Further experiments showed that the supply of exogenous proline lowered the leakage of potassium ions from cells exposed to deleterious concentrations of Cu²⁺. The inhibition of potassium leakage was particularly pronounced when proline was supplied prior to Cu²⁺treatment. The present study suggests that enhanced proline protects cell membranes from being affected by deleterious concentrations of Cu²⁺.     

Effects of ammonium concentration and charge exchange on ammonium recovery from high strength wastewater using a microbial fuel cell

Ammonium recovery using a two chamber microbial fuel cell (MFC) was investigated at high ammonium concentration. Increasing the ammonium concentration (from 0.07 to 4 g ammonium-nitrogen/L) by addition of ammonium chloride did not affect the performance of the MFC. The obtained current densities by DC-voltammetry were higher than 6 A/m² for both operated MFCs. Also continuous operation at lower external resistance (250 Ω) showed an increased current density (0.9 A/m²). Effective ammonium recovery can be achieved by migrational ion flux through the cation exchange membrane to the cathode chamber, driven by the electron production from degradation of organic substrate. The charge transport was proportional to the concentration of ions. Nonetheless, a concentration gradient will influence the charge transport. Furthermore, a charge exchange process can influence the charge transport and therefore the recovery of specific ions.     

Copper(II) interaction with tetrapeptides containing proline and phenylalanine: A potentiometric and spectrophotometric study

The synthesis of four tetrapeptides, L-Phe-L-Pro-Gly-Gly, Gly-L-Pro-L-Phe-Gly, Gly-L-Pro-D-Phe-Gly, and Gly-L-Pro-Gly-L-Phe is described. The hydrogen ion and copper(II) complex formation constants have been measured at 25°C and I = 0.10 mol dm^?3 (KNO₃). Circular dichroism spectra have been recorded for copper(II)-peptide mixtures as a function of pH. The potentiometric and Spectrophotometric studies have been combined to ascertain the complex species over a broad pH range. The results obtained support the earlier suggestion on the specific role of a proline residue as a “break-point” in copper complex formation with peptides: the insertion of a proline residue into the second position of a tetrapeptide sequence leads to a novel coordination mode in Cu(II)-tetrapeptide systems.     

Steady state proline levels in salt-shocked barley leaves

Excised barley (Hordeum vulgare var Larker) leaves were treated with salt solutions or wilted. After the treatment period, the leaves were allowed to recover in a 50 millimolar sucrose and 1 millimolar glutamate solution, and proline, Na⁺, and K⁺ were measured at intervals. Na⁺ and K⁺ concentrations stayed at a constant high level after the salt treatments, and proline increased to a steady state concentration in response. The relationship between the maximum rate of proline accumulation and the Na⁺ concentration reached in each experiment was linear. The final steady state proline concentration reached was also directly proportional to the Na⁺ concentration. For a given Na⁺ concentration in the leaves, the steady state proline level was greater when 410 millimolar NaCl was added to the leaves than when 205 millimolar NaCl was added. These results are consistent with proline acting as a compatible cytoplasmic solute, balancing an accumulation of salts outside of the cytoplasm.

In contrast to the proline levels in salt-shocked leaves, the concentrations in wilted leaves decreased to near control levels within 24 hours of relief of stress.

     

Effects of NaCl and mannitol induced stress on sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> sp.) callus cultures

The effects of NaCl and mannitol iso-osmotic stresses on calli issued from sugarcane cultivars (cvs.) R570, CP59-73 and NCo310 were investigated in relation to callus growth, water content, ion and proline concentrations. Callus growth and water content decreased under both stresses with the highest reduction under mannitol-induced osmotic stress. The ion concentration was drastically affected after exposure to NaCl and mannitol. Salt stress induced an increase in Na⁺ and Cl⁻ accumulation and a decrease in K⁺ and Ca²⁺ concentrations. Under mannitol-induced osmotic stress, K⁺ and Ca²⁺ concentrations decreased significantly while Na⁺ and Cl⁻ concentrations remained unchanged. Free proline accumulation occurred under both stresses and was more marked in stress-sensitive cv. than in stress-resistant one. Our results indicated that the physiological mechanisms operating at the plant cell level in response to salt- and osmotic-induced stress in sugarcane cvs. are different. Among the cvs., we concluded that the stress resistance is closely related to the maintain of an adequate water status and a high level of K⁺ and Ca²⁺ under both stresses and a low level of Na⁺ concentration in the presence of NaCl. Thus, sugarcane (Saccharum sp.) can be regarded as a Na⁺ excluder. We also provided evidence that proline accumulation is a stress-sensitive trait rather than a stress resistance marker.     

Isolation and characterization of a novel biosurfactant produced by hydrocarbon‐degrading bacterium Alcanivorax dieselolei B‐5

Aims: Our goal was to identify a novel biosurfactant produced by a marine oil‐degrading bacterium. Methods and Results: Biosurfactants were produced by Alcanivorax dieselolei strain B‐5^T growing with diesel oil as the sole carbon and energy source. Culture supernatant was first extracted with chloroform/methanol (1 : 1, v/v), then further purified step by step with a normal phase silica gel column, a Sephadex LH20 gel column and a preparative thin layer plate. The main component was determined to be a lipopeptide; it was chemically characterized with nuclear magnetic resonance, liquid chromatography‐quadrupole ion‐trap mass spectrometry, amino acid analysis and GC–MS and was found to be a mixture of proline lipids. The monomers of the proline lipids were composed of a proline residue and a fatty acid (C_14:0, C_16:0 or C_18:0). The critical micelle concentration of the mixed proline lipids was determined to be 40 mg l^?1. Moreover, activity variations in ranges of pH, temperature and salinity were also detected and showed reasonable stability. Conclusions: Alcanivorax dieselolei B‐5 produced a novel linear lipoamino biosurfactant, characterized as a proline lipid. Significance and Impact of the Study: A proline lipid was characterized for the first time as a bacterial biosurfactant. This product has potential in both environmental and industrial applications.     

Evaluation of heavy chain C‐terminal deletions on productivity and product quality of monoclonal antibodies in Chinese hamster ovary (CHO) cells

Monoclonal antibodies (mAbs) have been well established as potent therapeutic agents and are used to treat many different diseases. During cell culture production, antibody charge variants can be generated by cleavage of heavy chain (HC) C‐terminal lysine and proline amidation. Differences in levels of charge variants during manufacturing process changes make it challenging to demonstrate process comparability. In order to reduce heterogeneity and achieve consistent product quality, we generated and expressed antibodies with deletion of either HC C‐terminal lysine (‐K) or lysine and glycine (‐GK). Interestingly, clones that express antibodies lacking HC C‐terminal lysine (‐K) had considerably lower specific productivities compared to clones that expressed either wild type antibodies (WT) or antibodies lacking HC glycine and lysine (‐GK). While no measurable differences in antibody HC and LC mRNA levels, glycosylation and secretion were observed, our analysis suggests that the lower specific productivity of clones expressing antibody lacking HC C‐terminal lysine was due to slower antibody HC synthesis and faster antibody degradation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:786–794, 2017     

Comparison of monovalent and divalent ion distributions around a DNA duplex with molecular dynamics simulation and a Poisson‐Boltzmann approach

The ion atmosphere created by monovalent (Na⁺) or divalent (Mg²⁺) cations surrounding a B‐form DNA duplex were examined using atomistic molecular dynamics (MD) simulations and the nonlinear Poisson‐Boltzmann (PB) equation. The ion distributions predicted by the two methods were compared using plots of radial and two‐dimensional cation concentrations and by calculating the total number of cations and net solution charge surrounding the DNA. Na⁺ ion distributions near the DNA were more diffuse in PB calculations than in corresponding MD simulations, with PB calculations predicting lower concentrations near DNA groove sites and phosphate groups and a higher concentration in the region between these locations. Other than this difference, the Na⁺ distributions generated by the two methods largely agreed, as both predicted similar locations of high Na⁺ concentration and nearly identical values of the number of cations and the net solution charge at all distances from the DNA. In contrast, there was greater disagreement between the two methods for Mg²⁺ cation concentration profiles, as both the locations and magnitudes of peaks in Mg²⁺ concentration were different. Despite experimental and simulation observations that Mg²⁺ typically maintains its first solvation shell when interacting with nucleic acids, modeling Mg²⁺ as an unsolvated ion during PB calculations improved the agreement of the Mg²⁺ ion atmosphere predicted by the two methods and allowed for values of the number of bound ions and net solution charge surrounding the DNA from PB calculations that approached the values observed in MD simulations. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 834–848, 2014.     

Inhibition of mitochondrial electron transport is the prime cause behind proline accumulation during mineral deficiency in Oryza sativa

The concentration of proline in shoots of rice (Oryza sativa) seedlings raised in distilled water was about 3.3 times higher than in the seedlings raised in modified B₅ medium. The shoots of seedlings raised in B₅ medium which was depleted of calcium, iron, magnesium or potassium had a higher concentration of proline than those grown in standard B₅ medium. The shoots of seedlings raised in distilled water with iron had a lower level of proline than those in distilled water. These results suggest that iron deficiency leads to high proline accumulation.The electron transport activity of mitochondria from shoots of etiolated seedlings raised in distilled water or iron-depleted B₅ medium was significantly lower than those from equivalent seedlings raised with an iron source. As suppression in mitochondrial electron transport leads to an increase in NADH/NAD⁺ ratio, we propose that the prime cause of the proline accumulation during iron deficiency is to readily maintain NADH/NAD⁺ ratio.     

Response of two tomato cultivars to field-applied proline under irrigation with saline water: Growth,chlorophyll fluorescence and nutritional aspects

The response of tomato (Solanum lycopersicum L.) to abiotic stress has been widely investigated. Recent physiological studies focus on the use of osmoprotectants to ameliorate stress damage, but experiments at a field level are scarce. Two tomato cultivars were used for an experiment with saline water (6.57 dS m^?1) and subsurface drip irrigation (SDI) in a silty clay soil. Rio Grande is a salinity-tolerant cultivar, while Heinz-2274 is the salt-sensitive cultivar. Exogenous application of proline was done by foliar spray at two concentrations (10 and 20 mg L^?1) during the flowering stage. Control plants were treated with saline water without proline. Proline at the lower concentration (10 mg L^?1) increased dry mass of different plant organs (leaves, stems, and roots) and it improved various chlorophyll a fluorescence parameters compared with controls. Regarding mineral nutrition, K⁺ and P were higher in different organs, while low accumulation of Na⁺ occurred. However, Mg²⁺ was very high in all tissues of Rio Grande at the higher concentration of proline applied. Thus, the foliar spray of proline at 10 mg L^?1 increased the tolerance of both cultivars. The growth of aboveground biomass of Heinz-2274 was enhanced by 63.5%, while Rio Grande improved only by 38.9%.     

Effects of ammonium ion removal on growth and MAb production of hybridoma cells

Production of monoclonal antibody against hepatitis B surface antigen was carried out by perfusion culture coupled with a selective removal system for ammonium ion. The removal system is composed of three sub-systems namely, cell separation by cross-flow ceramic filter, dialysis by hollow fiber module and ion-exchange by zeolite A-3 packed bed column. The ammonium ion concentration in the culture broth was effectively maintained below the inhibitory level, and the viable cell density reached 2.5×10⁷ cells ml^–1 which was three times that of conventional perfusion cultures. The monoclonal antibody accumulated to a concentration as high as 26.3×10⁵ mIU^–1. This is already almost half of the amount producedin vivo. The numerical investigation of the ammonium ion removal system showed the possibility to improve much more the performance of this perfusion cultivation system.     

The interaction of proteins with hydroxyapatite: II. Role of acidic and basic groups

The elution behavior from hydroxyapatite columns of the modification products of seven basic and three acidic proteins has been investigated. Three classes of NH₂ derivatives were prepared. These consisted of (1) replacement by a guanidyl group with no change in charge; (2) blocking with loss of charge; and (3) replacement of positive charges by negative ones. Two types of COOH derivatives were prepared: (1) blocking with loss of charge; and (2) replacement of COOH by SO₃H with no change in charge. The elution behavior of the derivatives in PO₄, F^?, Cl^?, ClO₄^?, and Ca²⁺ ion eluants showed that (1) the elution patterns are determined by the isoelectric points of the proteins, there being no symmetry between the binding or elution behavior of acidic and basic proteins; (2) the binding of basic proteins requires the presence of a high density of positively charged groups; (3) the binding of all proteins to hydroxyapatite equilibrated with phosphate buffer is enhanced by a decrease in the number of their negative charges; and (4) calcium ions affect the binding of proteins to hydroxyapatite at the level of carboxyls, since clusters of carboxyls strengthen both the interaction with Ca²⁺ and the binding to hydroxyapatite.     

Coordinate repression of cholesterol biosynthesis and cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase by glucocorticoids in HeLa cells.

The stability constants for the calcium and magnesium complexes of rhodanese are >10⁵m^?1 at both high and low substrate concentrations. The stoichiometry of alkaline earth metal ion binding totals close to 1 per 18,500 molecular weight. The usual assay reagents contain sufficient amounts of these metal ions to maintain added enzyme in its metal-complexed form. When reaction mixtures are treated with oxalate to remove calcium ions, inhibition of rhodanese activity is virtually complete under circumstances such that the contribution of magnesium ion is low.Zinc and a number of transition metal ions are inhibitors of rhodanese activity. Studies of the concentration dependence of these effects with zinc, copper, and nickel showed that: 1) Some cyanide complexes of these metals are competitive with the donor substrate, thiosulfate ion. The binding of the copper and zinc complexes is mutually competitive. 2) Another cyanide species of copper appears to combine with the free enzyme to form a functionally active complex. 3) The zinc cyanide species with a net positive charge is an inhibitor competitive with the acceptor substrate, cyanide ion.All of these observations are consistent with a model in which metal ions serve as the electrophilic site of rhodanese.     

Physiological responses to NaCl stress in three wild species of potato in vitro

In this study, responses of wild species of potato to NaCl stress were investigated in vitro. In S. stoloniferum and S. bulbosum, length of the shoot, fresh and dry weight, photosynthetic pigments, K⁺ concentration, K⁺/Na⁺ ratio, ascorbate pool, anthocyanin, and phenolic and flavonoid compounds were decreased in response to salinity. In these species, salinity increased the level of Na⁺, lipid peroxidation, proline and ion leakage percentage. In S. acaule, the length of the shoot, and fresh and dry weight were not affected by salinity. Photosynthetic pigments, Na⁺ concentration, proline, flavonoid and phenolic compounds quantities were increased and K⁺/Na⁺ ratio were decreased. K⁺ concentration, lipid peroxidation, ascorbate pool, anthocyanin and ion leakage were not changed by NaCl stress. Superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase and catalase activities were increased in all species. The results suggest that the non-enzymatic antioxidant capacity in S. acaule (salt tolerant) is more important than the enzymatic antioxidant capacity in comparison with the other species.     

Nigericin-induced charge transfer across membranes

Summary The electric properties of the bilayer lecithin membranes have been studied in the presence of the antibiotic nigericin. When the antibiotic concentration is about 10^–6 m the conductivity of the BLM is increased up to 10^–7 ohm^–1 cm^–2. The potassium ion concentration gradient gives rise to a transmembrane potential of the order of 40 mV per 10-fold concentration gradient with the side of the higher potassium concentration negative. The transmembrane potential produced by the hydrogen ion concentration gradient is a function of the potassium ion concentration which is equal on both sides of the membrane. For low potassium ion concentrations the hydrogen potential has the expected polarity with the solution having higher concentration of protons negative. For potassium ion concentrations exceeding 0.03m the hydrogen potential has the reverse polarity. This unexpected result cannot be accounted for in terms of the available simple hypotheses about the charge transport mechanism for nigericin in BLM. In order to account for the experimental results obtained, a theoretical approach has been developed based on the assumption that charge is transported across the membrane by nigericin dimers. The theoretical predictions are in satisfactory agreement with the experimental results. The model also yields some predictions which may be verified in future experiments.     

Ion fluxes and abscisic Acid-induced proline accumulation in barley leaf segments

The increase in proline induced by ABA, a process stimulated by NaCl or KCl in barley leaves, did not occur when Na⁺ (or K⁺) was present in the external medium as the gluconate salt, namely with an anion unable to permeate the plasma membrane. However, proline increase was restored, to different extents, by the addition of various chloride salts but not by ammonium chloride. Moreover, it was shown that the stimulation of the process by NaCl (or KCl) was variously affected by the presence of different salts; all the ammonium salts (10 millimolar NH₄⁺ concentration) inhibited this stimulation almost completely. Inhibition by NH₄⁺ was accompanied by a decreased Na⁺ influx (−40%). Also, in the case of Na-gluconate, Na⁺ uptake was reduced and the addition of Cl⁻ as the calcium or magnesium salt (but not as ammonium salt) restored both the ion influxes and the increase in proline typical of NaCl treatments. Both 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS), an anion transport inhibitor, and tetraethylammonium chloride (TEA), a K⁺ channels-blocking agent, caused, as well as with a reduction of ion influxes, an inhibition of the proline accumulation. The inhibition was practically total with 1 millimolar DIDS and about 80% with 20 millimolar TEA. A possible role of ion influxes in the process leading to the increase in proline induced by ABA is proposed.     

Heavy-metal-induced proline accumulation and its role in ameliorating metal toxicity in Chlorella vulgaris

Exposure of Chlorella vulgaris to elevated concentrations of copper, chromium, nickel and zinc led to intracellular accumulation of high concentrations of these metals. Concomitantly, accumulation of free proline occurred, depending on the concentration of metals in the external medium or in the cell. The greater the toxicity or accumulation of a metal, the greater the amount of intracellular proline in algal cells. However, higher concentrations of copper and chromium were inhibitory to proline accumulation by the test organism. The accumulation of proline was triggered within a few hours of metal treatment. Test metals also induced lipid peroxidation; copper was the most efficient inducer whereas zinc was the least. Pretreatment of C. vulgaris with proline counteracted metal-induced lipid peroxidation and potassium ion efflux. Thus the present work shows a protective effect of proline on metal toxicity through inhibition of lipid peroxidation.