Net Mobility Research Articles

Intrinsic charge ladders of a monoclonal antibody in hydroxypropylcellulose-coated capillaries

Capillary zone electrophoresis (CZE) has been used to resolve the charge heterogeneity of an intact ( approximately 150 kDa) monoclonal IgG antibody (mAb). Although this microheterogeneity can also be observed by isoelectric focusing, CZE allows the net charge of each variant to be measured as a function of pH and other solution conditions. Separation was achieved in both borate and Tris run buffers using capillaries that had been statically coated with hydroxypropylcellulose (HPC). The HPC coating makes inadvertent chromatographic retention of the mAb undetectably small and decreases electroosmotic flow (EOF) to approximately 10(-5) cm(2) V(-1) s(-1), with reasonable stability over dozens of runs under the conditions tested (pH 8.5 and 9.0 for each buffer). We also describe a novel means of measuring small, positive EOF coefficients and larger, negative net mobilities in the same run. This allows determination of accurate electrophoretic mobilities despite variations in EOF. The resolved mAb charge variants (which most likely result from deamidation or partial truncation) constitute what we call an "intrinsic" charge ladder. As with conventional charge ladders formed by deliberate modification of a homogeneous protein, net charge is obtained by extrapolating a plot of electrophoretic mobility versus (assumed) incremental charge difference. At a given pH, the mAb is more negatively charged in borate than in Tris, reflecting specific binding of the B(OH)(4)(-) anion. We also report hydrodynamic radii calculated from the slopes of these plots.

Whole-Body and Hindlimb Protein Breakdown Are Differentially Altered by Feeding in Neonatal Piglets

The high rate of muscle protein accretion in neonates is sustained by the marked increase in muscle protein synthesis in response to feeding. Little is known about the role of proteolysis in the regulation of protein accretion in response to feeding during the neonatal period. To determine the feeding-induced response of protein breakdown at the whole-body level and in the hindlimb of neonates, 10- and 28-d-old piglets that had been food deprived overnight were infused (7 h) with [1-13C]phenylalanine and [ring-2H4]tyrosine during an initial food deprivation period (3 h), followed by a feeding period (4 h). During feeding, endogenous flux of phenylalanine decreased (P < 0.01) in both the whole body and the hindlimb. Feeding reduced (P < 0.01) whole-body proteolysis but increased hindlimb proteolysis (P = 0.04), suggesting that tissues other than the hindlimb are involved in the reduction in whole-body proteolysis during feeding. Overnight food deprivation resulted in a net mobilization of phenylalanine from whole-body proteins (P < 0.01) but not hindlimb proteins. These responses were unaffected by age. The results suggest that the hindlimb requires a continuous supply of free amino acids to sustain the high rate of muscle protein turnover in neonates and that adaptive mechanisms provide free amino acids to sustain skeletal muscle protein accretion in early postnatal life when the amino acid supply is limited.

Analysis of the ups and downs of caries experience among Norwegian children aged five years between 1997 and 2003

Objective. To study caries trends and investigate the possible reasons for changes among 5-year-old Norwegian children in the period 1997–2003. In this cross-sectional analytical time trend study at district, county, and national levels, aggregated data from the Public Dental Services (PDS) and official statistics were used, i.e. number of children and percentage receiving treatment, sale of fluoride tablets, socio-economic background, caries prevalence, and d3mft scores. Results. Caries prevalence increased from 30% in 1997 to just over 40% in 2001, but by 2003 it had dropped to 36%. The corresponding mean number of d3mft were 1.1, 1.6, and 1.4 at national level. Caries prevalence and experience varied considerably between counties and between dental districts throughout the observation period. Multivariate analyses at county level indicated a significant negative association (p<0.05) between caries prevalence, the sale of fluoride tablets, and net mobility. The impacts of the predictor variables education, income, infant mortality, proportion of immigrants, and the percentage of children treated varied but were rarely significant. Conclusions. The increasing trend in the prevalence of caries among 5-year-old Norwegian children in the period 1997 to 2001 has reversed. The deterioration in dental health of 5-year-olds after 1997 was associated with a reduction in the sale of fluoride tablets, whereas increased sales of fluoride tablets after 1998 reflect improved caries prevention among preschool children and may explain the improved caries status of these children in 2003.

Accumulation and release of nitrogen and phosphorus in relation to lignin decomposition in leaf litter of 14 tree species

Immobilization and mobilization of nitrogen and phosphorus were investigated in relation to the nitrogen (L/N) ratio and lignin to the phosphorus (L/P) ratio as indicators of the nitrogen and phosphorus dynamics. The present study was carried out on upper and lower parts of a forest slope in a cool temperate forest in Japan. Net immobilization and net mobilization characterized the dynamics of nitrogen and phosphorus in 14 litter types and were related to the changes in the L/N and L/P ratio. The critical values of the L/N and L/P ratio at which the mobilization began were 23–25 and 500–620, respectively. In litter types with the L/N and L/P ratio higher than critical values, nitrogen and phosphorus were immobilized until the ratios reached at the critical values and then nitrogen and phosphorus began decreasing. In litter types with initial L/N and L/P ratios lower than or equal to the critical values, nitrogen and phosphorus were released from litter. The critical values of the L/N and L/P ratios showed convergent trends among litter types as compared to their initial values, and were approached to those of underlying humus layers. These results indicated the usefulness of L/N and L/P ratios as indicators of the nitrogen and phosphorus dynamics in the study site. The general validity of the L/N ratio as an indicator of nitrogen dynamics and the convergent trend of critical L/N ratio at 25–30 were demonstrated by a review of literature on lignin and nitrogen dynamics in 47 litter types in temperate and boreal forests.

Biogeochemistry of the compost bioreactor components of a composite acid mine drainage passive remediation system

The compost bioreactor (“anaerobic cell”) components of three composite passive remediation systems constructed to treat acid mine drainage (AMD) at the former Wheal Jane tin mine, Cornwall, UK were studied over a period of 16 months. While there was some amelioration of the preprocessed AMD in each of the three compost bioreactors, as evidenced by pH increase and decrease in metal concentrations, only one of the cells showed effective removal of the two dominant heavy metals (iron and zinc) present. With two of the compost bioreactors, concentrations of soluble (ferrous) iron draining the cells were significantly greater than those entering the reactors, indicating that there was net mobilisation (by reductive dissolution) of colloidal and/or solid-phase ferric iron compounds within the cells. Soluble sulfide was also detected in waters draining all three compost bioreactors which was rapidly oxidised, in contrast to ferrous iron. Oxidation and hydrolysis of iron, together with sulfide oxidation, resulted in reacidification of processed AMD downstream of the compost bioreactors in two of the passive treatment systems. The dominant cultivatable microorganism in waters draining the compost bioreactors was identified, via analysis of its 16S rRNA gene, as a Thiomonas sp. and was capable of accelerating the dissimilatory oxidation of both ferrous iron and reduced sulfur compounds. Sulfate-reducing bacteria (SRB) were also detected, although only in the bioreactor that was performing well were these present in significant numbers. This particular compost bioreactor had been shut down for 10 months prior to the monitoring period due to operational problems. This unforeseen event appears to have allowed more successful development of AMD-tolerant and other microbial populations with critical roles in AMD bioremediation, including neutrophilic SRB (nSRB), in this compost bioreactor than in the other two, where the throughput of AMD was not interrupted. This study has revealed new insights into the operation of compost bioreactors used to remediate mine waters and has shown that, when operated under appropriate conditions, they can be highly efficient at generating alkalinity and removing metals from extremely acidic, metal-rich AMD.

Novel elution strategy for monitoring DNA counter-migration in the presence of electroosmotic flow

The migration behavior of native (i.e., unlabelled) DNA in the presence of electroosmotic flow (EOF) was investigated in bare fused-silica capillaries. Employing a novel elution strategy, the influence of EOF on the net mobility of DNA was assessed by collecting the DNA that migrated anodically (i.e., against EOF) and out of the capillary inlet. Various conditions of pH and buffer-zone continuity were employed to characterize this phenomenon. Tris acid (TA, pH 5.14) and Tris base (TB, pH 9.36) were used as buffers in continuous systems, in which the capillary and the inlet reservoir contain the same buffer, and discontinuous systems, in which the capillary contains either TA or TB, and the inlet reservoir contains water. DNA that was ejected into the inlet vial was subsequently analyzed by capillary electrophoresis–laser-induced fluorescence. Both ΦX174/ HaeIII DNA and the β-actin product of single-cell reverse transcriptase-polymerase chain reaction were used as DNA samples in this study. The mechanism of elution was found to depend on bulk flow, in the case of continuous solutions. However, with the discontinuous system, a localized decrease in EOF generated in the capillary tip appeared to impact elution. These findings serve to introduce an alternative approach for characterizing the mobility of highly charged species.

Moving boundaries in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), moving boundaries I-IV (Table 1) form. They migrate isotachophoretically at displacement rates that increase in the order of I to II to III. Moving boundaries IV and V comprising Pyronin-SDS as a leading constituent are retarded at high gel concentrations in comparison with the isotachophoretically migrating species. Since analytes depending on their net mobilities stack within any of those moving boundaries, previous R(f) values and Ferguson plots may have to be revised.

Regional mobility of economists: An extension

We extend Davis and Patterson’s analysis of the incidence and determinants of regional mobility of Ph.D. economists. By exploiting previously collected data, we identify the patterns of regional mobility not only at the point of entry into the labor market for economists who received their doctoral degree in 1968 but also the net mobility of these economists over a 25-year period. The net effects of regional mobility decisions made between graduation in 1968 and year-25 employment in 1993, and between initial professional employment in 1969 and year-25 employment in 1993, are analyzed. Our results suggest that many economists demonstrate a propensity to“nest“ in their degree and first-job regions of employment and that those employed in government service and those with a greater diversity of work experience are more likely to migrate across regions during their careers.

Ion transport in alkaline and earth alkaline hydrogen fluorides

The impedance of the hydrogen fluorides KHF 2, NH 4HF 2 and BaHF 3 was investigated as a function of temperature and in the presence of HF and H 2O. A comparison of results on different electrode materials (graphite, silver, palladium, Sn∣SnF 2, and Sn|SnF 2|LaF 3) was used to elucidate the contribution of fluoride ions and protons to the net charge transport. All three investigated materials exhibited a considerable and nearly reversible increase of conductivity in the presence of gases that act as proton donors. In the case of BaHF 3, the results indicate both the mobility of fluoride ions ( E A=0.54 eV) as well as a net mobility of protons. The latter is explained by proton exchange due to internal acid–base reactions of the protonated anions. At ambient temperature, KHF 2 only becomes conducting when exposed to HF or H 2O. NH 4HF 2, on the other hand, shows a higher conductivity than BaHF 3 already in dry atmosphere increasing less with humidity than the other two materials. This indicates an intrinsic proton mobility in NH 4HF 2.

Surface Potential Driven Swelling of Polyelectrolyte Multilayers

The adsorption of polyelectrolytes to charged surfaces can be applied to build up multilayers of polyelectrolytes of alternating charge by layer-by-layer assembly. The hydration of such multilayers adsorbed to colloidal particles in dilute aqueous dispersions is investigated by NMR, namely, water 1H spin relaxation rates R2. For the system of alternating layers of poly(diallyl dimethylammonium chloride) (PDADMAC) and poly(sodium 4-styrenesulfate) (PSS) adsorbed to silica particles (PDADMAC/PSS), a linear increase of multilayer thickness and R2 with the number of layers is found, reflecting an increasing amount of hydration water. For the system (PAH)/PSS, involving the weak polyelectrolyte poly(allylamine hydrochloride) (PAH), it is found that while adsorption of a positive layer leads to an increase of R2, a decrease of R2 is observed after the adsorption of a negative layer. This reveals an increase of the net water mobility involving dehydration. Evidence is given that the reversible swelling behavior ...

Application of surface biopassivated disposable poly(dimethylsiloxane)/glass chips to a heterogeneous competitive human serum immunoglobulin G immunoassay with incorporated internal standard.

A microfluidic platform for a heterogeneous competitive immunoassay of human immunoglobulin G (IgG) employing Cy5-human IgG as tracer and Cy3-mouse IgG as internal standard was developed. The device consisted of microchannels made of poly(dimethylsiloxane) and glass which were patterned with antibodies against human IgG and mouse IgG. Electrokinetic sample transport was employed in order to exploit the small difference between the net mobilities of analyte and tracer, thereby achieving favorable conditions for the performance of the competitive immunoreaction. The overall quality of the disposable chip and performance of the immunoassay were controlled by monitoring the fluorescence of bound tracer and bound internal standard. Analyses with an insufficient internal standard response were discarded, and immunoassay data evaluation was based on the ratio of tracer and internal standard fluorescence. Using synthetic samples in the range from 0 to 80 microg/mL IgG and alkaline running conditions, a concentration-dependent response with reproducible Cy5/Cy3 signal ratios (average relative standard deviation of 6.8%) was obtained. Chips stored with solution in the channels at 4 degrees C over a two-month period were found to perform like freshly prepared chips, whereas chips stored dry at -20 degrees C and rehydrated prior to use could not be employed. The analysis of patient sera showed that the immunoassay platform behaved differently in the presence of serum-based samples. Using the same conditions as for the synthetic samples, no concentration dependence was noted. With a large excess of tracer, however, an IgG concentration dependence was observed, permitting distinction of samples of patients with normal IgG serum levels (8-16 mg/mL) from those with elevated IgG concentrations (>16 mg/mL).

A strain-gradient thermodynamic theory of plasticity based on dislocation density and incompatibility tensors

In this work, we discuss a thermodynamic theory of plasticity for self-organization of collective dislocations in FCC metals. The theory is described by geometrical tensor quantities of crystal defect fields such as dislocation density tensor, representing net mobile dislocation density and geometrically necessary boundaries, and the incompatibility tensor representing immobile dislocation density. Conservation laws for the two kinds of dislocation density are formulated with dislocation products and interactions terms. Based on the second law of thermodynamics, we drive basic constitutive equations for the dislocation flux, production and interaction terms of dislocations. We also derive a set of reaction-diffusion equations for the dislocation density tensor and incompatibility tensor which describes the vein and persistent slip band (PSB) ladder structures. These equations are analyzed using linear stability and bifurcation analysis. An intrinsic mesoscopic length scale is determined which provides an estimate for the wavelength of the PSBs. The basic aspects of the model are motivated and substantiated by analyzing the stress fields of various possible dislocation configurations using discrete dislocation dynamics.

Extended state-space Monte Carlo methods.

In this paper various extensions of the parallel-tempering algorithm are developed and their properties are analyzed. The algorithms are designed to alleviate quasiergodic sampling in systems which have rough energy landscapes by coupling individual Monte Carlo chains to form a composite chain. As with parallel tempering, the procedures are based upon extending the state space to include parameters to encourage sampling mobility. One of the drawbacks of the parallel-tempering method is the stochastic nature of the Monte Carlo dynamics in the auxiliary variables which extend the state space. In this work, the possibility of improving the sampling rate by designing deterministic methods of moving through the parameter space is investigated. The methods developed in this article, which are based upon a statistical quenching and heating procedure similar in spirit to simulated annealing, are tested on a simple two-dimensional spin system (xy model) and on a model in vacuo polypeptide system. In the coupled Monte Carlo chain algorithms, we find that the net mobility of the composite chain is determined by the competition between the characteristic time of coupling between adjacent chains and the degree of overlap of their distributions. Extensive studies of all methods are carried out to obtain optimal sampling conditions. In particular, the most efficient parallel-tempering procedure is to attempt to swap configurations after very few Monte Carlo updates of the composite chains. Furthermore, it is demonstrated that, contrary to expectations, the deterministic procedure does not improve the sampling rate over that of parallel tempering.

Stacking of unlabeled sodium dodecyl sulfate-proteins within a fluorimetrically detected moving boundary, electroelution and mass spectrometric identification.

The previously reported fluorimetric detection of sodium dodecyl sulfate (SDS)-protein in the presence of cascade blue in agarose gel electrophoresis using barbital buffer was found to be equally feasible in the absence of the fluorescent marker and using Tris-Tricinate buffer, provided that SDS was loaded with the sample but not contained in the catholyte. That fluorescent detection is thought to be due to the formation of a moving boundary between leading SDS and trailing barbital, or Tricinate buffer. This hypothesis is supported by the following evidence: (i) The fluorometrically detected band disappears with addition of SDS to the catholyte; (ii) band area is proportional to protein and/or SDS load; (iii) mobility of SDS-proteins differing in mass is the same at agarose concentrations up to 3%; (iv) lowering of protein mobility by increase in gel concentration and/or increase in the size of the SDS-protein leads to band disappearance. Fluorescent detection of the band is like to be nonspecific and due to the light scattering properties of a stack comprising moving boundaries of any analytes with net mobilities intermediate between SDS (or micellar SDS) and the trailing buffer constituent at their regulated very high concentrations. The steady-state stack of SDS-proteins in the size range of 14.4-45.0 kDa, and the transient stack of an SDS-protein of 66.2 kDa have lent themselves to electroelution and characterization by mass of the proteins after removal of SDS and buffer exchange using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. The possibility to form a stack of protein between leading SDS and trailing buffer anions under conditions of weak molecular sieving (open-pore gel and small-sized protein) contributes to the understanding of moving boundaries in gel electrophoresis, but in view of the narrowly defined conditions, under which this stack forms, is of limited practical significance for the gel electrophoresis of SDS-proteins.

Phosphorus mobilization from various sediment pools in response to increased pH and silicate concentration.

Phosphorus (P) release from sediment particles to the interstitial water has been studied extensively, but the contribution of different inorganic P pools in sediment under differing environmental conditions is not fully understood. This study was undertaken to get more detailed information about the chemical mobilization mechanisms. Phosphorus mobilization from reserves bound by Al, Fe, and Ca compounds in response to increased pH and to inorganic silicon (Si) enrichments was investigated using a sequential fractionation analysis and an isotope-labeling technique. The aerobic sediment of Lake Vesijärvi had a high P retention capacity, and Fe-bound P was the largest inorganic P pool as well as the main source of released P. High Si addition (47 mg Si L-1 sediment) released more P to the interstitial water than did the elevation of pH from 6.6 to 9.5, since Si lowered the resorption of released P onto hydrated Al oxides. This finding reveals that P equilibrium between Fe-bound and Al-bound P in sediments regulates P net mobilization to the interstitial water under aerobic conditions. Furthermore, elevated pH combined with high Si enrichment had a positive synergistic effect, resulting in the most substantial P mobilization. This synergism may cause a self-fueled increase in the internal loading of P. It accentuates the effect of diatom sedimentation on P fluxes in eutrophic lakes with high pH and may favor the appearance of bloom-forming cyanobacteria.

Trace analysis of γ-cyclodextrin in a sample of β-cyclodextrin by capillary electrophoresis

A new capillary electrophoretic method for trace analysis of γ-cyclodextrin, γ-CD, in a sample of β-CD has been developed, building on our recent work in which the tetraphenylborate ion, Ph 4B −, was found to bind to γ-CD three orders of magnitude more strongly than to β-CD. The method involves measurement of the change of net electrophoretic mobility of Ph 4B − and its CD complexes in a background electrolyte containing a fixed concentration of β-CD. Good linearity was found between 1/Δ μ and 1/ C γ, where Δ μ is the difference in the mobility of Ph 4B − in the β-CD solution at a given and at zero concentration of γ-CD, and C γ the γ-CD concentration. The limit of detection for γ-CD in a β-CD sample was found to be 0.020% (w/w), and high precision and accuracy were obtained.

Doping profiles of n-type GaAs layers grown on Si by the conformal method

AbstractWe study doping profiles in selectively Si-doped GaAs layers grown by the conformal method. This growth technique allows to obtain GaAs/Si with optoelectronic quality. The samples are laterally grown, and selective doping with Si is carried out in such a way that doped stripes are intercalated with undoped ones. The study of the doping profiles was carried out by cathodoluminescence (CL) and micro-Raman (μR) spectroscopy. Abrupt doping profiles between doped and undoped stripes were demonstrated by monochromatic CL images. Deep level related CL bands can be observed between 1000 and 1400 nm, evidencing the complex mechanism for Si incorporation at the growth temperature (730 °C). Net doping concentrations and mobilities across the layers were determined from the analysis of the phonon-plasmon coupled modes in the μR spectra obtained with a lateral resolution better than 1 μm.

Electrophoresis of Composite Molecular Objects. 1. Relation between Friction, Charge, and Ionic Strength in Free Solution

We present a study of the mobility of streptavidin end-labeled ssDNA fragments in free solution capillary electrophoresis. Our data show that the contribution of the uncharged streptavidin label to the net electrophoretic mobility of the complex depends on the buffer ionic strength because this frictional contribution is actually related to the persistence length of the ssDNA fragment. Therefore, the ionic strength of the buffer affects both the free-solution mobility of naked ssDNA and the effective slowing down (or “drag”) due to the streptavidin label. We further show that our experimental results can be interpreted using recently published polyelectrolyte electrophoresis theories. Our results thus confirm that the so-called free-draining picture of local ssDNA electrophoretic friction and charge is not valid.

Protein nutrition in late pregnancy, maternal protein reserves and lactation performance in dairy cows.

Empirical evidence suggests that prolonged underfeeding of protein to late-pregnant dry cows can have modest negative carry-over effects on milk volume and/or protein yield during early lactation, and may also cause increased incidence of metabolic diseases associated with fatty liver. However, assessment of requirements is hampered by lack of information on relationships between dietary intake of crude protein (N x 6.25) and metabolizable protein supply during late pregnancy, and by incomplete understanding of the quantitative metabolism of amino acids in maternal and conceptus tissues. Inability of the postparturient cow to consume sufficient protein to meet mammary and extra-mammary amino acid requirements, including a significant demand for hepatic gluconeogenesis, necessitates a substantial, albeit transient, mobilization of tissue protein during the first 2 weeks of lactation. Ultimately, much of this mobilized protein appears to be derived from peripheral tissues, especially skeletal muscle and, to a lesser extent, skin, through suppression of tissue protein synthesis, and possibly increased proteolysis. In the shorter term, soon after calving, it is likely that amino acids required for hepatic glucose synthesis are diverted from high rates of synthesis of splanchnic tissue and export proteins, including serum albumin. The prevailing endocrine milieu of the periparturient cow, including major reductions in plasma levels of insulin and insulin-like growth factor-I, together with insulin resistance in peripheral tissues, must permissively facilitate, if not actively promote, net mobilization of amino acids from these tissues.

Response of nitrogen metabolism in preparturient dairy cows to methionine supplementation.

Three multiparous Holstein cows (607 kg of BW) were surgically prepared with an elevated carotid artery and indwelling catheters in the hepatic, portal, and two mesenteric veins to study the effects of methionine supplementation on amino acid metabolism during the last 2 wk of pregnancy. The study began 15 d before the expected calving date. Dietary treatments were Control (1.53 Mcal NE(l)/kg, 15.6% CP, and 40% ruminally undegradable protein) and Control supplemented with 60 g/d of ruminally protected methionine (MET, supplying 39 g/d of DL-methionine and approximately 18 g/d of methionine available for intestinal absorption). Each cow received both dietary treatments in a crossover design. Cows were fed once daily. After 5 d on treatment, a blood flow marker (para-aminohippurate) was infused into a mesenteric vein, and arterial, portal, and hepatic blood samples were obtained at 0, 2, 6, 12, and 18 h after feeding. Net flux of methionine was calculated as the plasma arteriovenous difference multiplied by plasma flow. Dry matter intake (10.8 kg/d) and portal (824 L/h) and hepatic (995 L/h) plasma flows were not affected (P > .10) by treatment. Arterial plasma concentration of methionine was greater (P = .10) with MET (27.67 microM) than with Control (16.42 microM). Net portal absorption of methionine increased (P = .10) with MET (26.2 g/d) compared with Control (9.5 g/d). The net portal methionine flux was negatively correlated (r = -.59; P < .001) with arterial urea concentrations. Net flux of methionine across splanchnic tissues shifted (P = .06) from a net uptake with Control (4 g/d) to a net output with MET (11 g/d). Therefore, MET increased by 15 g/d the methionine supply to the rest of the body. The net uptake of methionine by splanchnic tissues observed with Control indicated a net mobilization of methionine by peripheral tissues. Results indicate that methionine was the limiting amino acid with Control and that MET was beneficial because it increased methionine supply to peripheral tissues and reduced arterial urea concentrations.