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PubMed2005

Immunoaffinity separation of plasma proteins by IgY microbeads: meeting the needs of proteomic sample preparation and analysis.

Separation of complex protein mixtures that have a wide dynamic range of concentration, such as plasma or serum, is a challenge for proteomic analysis. Sample preparation to remove high-abundant proteins is essential for proteomics analysis. Immunoglobulin yolk (IgY) antibodies have unique and advantageous features that enable specific protein removal to aid in the detection of low-abundant proteins and biomarker discovery. This report describes the efficiency and effectiveness of IgY microbeads in separating 12 abundant proteins from plasma with an immunoaffinity spin column or LC column. The protein separation and sample preparation process was monitored via SDS-PAGE, 2-DE, LC-MS/MS, or clinical protein assays. The data demonstrate the high specificity of the protein separation, with removal of 95-99.5% of the abundant proteins. IgY microbeads against human proteins can also selectively remove orthologous proteins of other mammals such as mouse, rat, etc. Besides the specificity and reproducibility of the IgY microbeads, the report discusses the factors that may cause potential variations in protein separation such as protein-protein interactions (known as "Interactome"), binding and washing conditions of immunoaffinity reagents, etc. A novel concept of Seppromics is introduced to address methodologies and science of protein separation in a context of proteomics.

Albumins↗
PubMed2002

Two-dimensional separations with electrospray ionization ambient pressure high-resolution ion mobility spectrometry/quadrupole mass spectrometry.

The coupling of ion mobility spectrometry (IMS) instruments with mass spectrometers has been described since early in IMS development, most commonly with quadrupole mass analyzers. The recent development of IMS with time-of-flight (TOF) instruments has demonstrated that the time compatibility (IMS milliseconds and TOFMS microseconds) of the two techniques enables rapid two-dimensional separations to be performed, theoretically in the order of seconds for a complete analysis. This study presents a unique way to operate a traditional IMS/QMS system to attain separations similar to those achieved with IMS/TOF. For this new approach, the quadrupole was slowly scanned in the single-ion monitoring mode while IMS spectra were continually embedded in each m/z step. In this way, two-dimensional separations (IMS drift times and m/z) were obtained using the traditional IMS/QMS arrangement. An example of a five amino acid separation (quadrupole scan of 40 m/z values at a rate of approximately 7 steps/min) led to a complete two-dimensional analysis within 6 min, comparable to rapid chromatographic separations with mass spectrometry. Proposed approaches to reduce the analysis time are discussed and a reduction in the analysis time to less than 1 min is feasible when the IMS/QMS separation conditions are optimized.

Algorithms↗
PubMed1995

Quantitative and qualitative comparative analysis of gradient-separated hematopoietic cells from cord blood and chemotherapy-mobilized peripheral blood.

Some of the uncertainty regarding the use of cord blood (CB) in transplant settings includes the suspected relative rarity of hematopoietic stem cells (SC) in CB and the feasibility of incorporating a cell separation protocol to remove red blood cells, which may result in an unacceptable loss of SC. To address this uncertainty, we isolated a SC fraction by Percoll or Ficoll gradients from CB and peripheral blood (PB), which had been mobilized by chemotherapy. The frequencies of mononuclear cells (MNC), CD34+ cells, colony-forming units granulocyte-macrophage (CFU-GM), and the output of colony-forming cells (CFC) after five weeks in long-term culture (LTC) assay were then evaluated. The mean numbers of these cells per ml of CB sample before gradient separation were, respectively, 4.9 x 10(6), 13.8 x 10(4), 3.0 x 10(3) and 681 (n = 37). In the recovery phase of PB, these numbers were, respectively, 2.0 x 10(6), 14.9 x 10(4), 3.5 x 10(3) and 270 per ml of processed blood at apheresis (n = 35). After Percoll separation, the recovery rates of these cells were, respectively, 29%, 92%, 97% and 95% in CB, and 65%, 87%, 123% and 102% in PB. After Ficoll separation of CB, the rates were, respectively, 55%, 107%, 92% and 105%. These results suggest that CB contains an adequate number of more immature progenitors which can be retained after cell separation with Percoll or Ficoll, thereby making it feasible to incorporate a cell processing procedure into a CB transplant protocol. Percoll separation provided a greater enrichment of cells than Ficoll.

Adolescent↗
PubMed1993

Anion exchange separation and pulsed amperometric detection of inositols from flower petals.

A rapid and specific method for the separation of cyclic sugar alcohols by anion exchange HPLC and subsequent detection by pulsed amperometry is presented. The cyclitols include the inositols and O-methyl inositols. Although examples use plant extracts the method has general application and is suitable for detecting as little as 10 pmol of substance. Carbohydrates are weak acids with pKa values typically above 12. While separation of most monosaccharides on high-performance anion exchange columns in aqueous NaOH is adequate, the ionization of inositols and O-methyl inositols is weak and their separation is poor. Use of a high-capacity anion exchange column separated the inositols well but the more ionized sugars were tightly retained. Automatic switching of the inositol fraction eluting from the lower-capacity column onto the high-capacity column with subsequent detection by pulsed amperometry gave specific and rapid separations of the inositols with a turnaround time of 16 min. As an example of application, we chose the separation and identification of cyclic and acyclic sugar alcohols in flower petals from a variety of plants. There was an apparent correlation between the postharvest longevity of flowers and the content of inositols, particularly D-pinitol.

Chromatography, High Pressure Liquid↗
PubMed1993

Separation of the sticky peptides from membrane proteins by high-performance liquid chromatography in a normal-phase system.

The amphiphilic peptides obtained upon cleavage of membrane proteins, including numerous receptors, are recalcitrant to most separation techniques as a consequence of their limited solubility and tendency to aggregate and adsorb to surfaces. This paper describes HPLC systems that can separate these "sticky" peptides on silica and aminopropyl-modified silica columns with a mobile phase consisting of a mixture of chloroform/methanol/isopropylamine. The protocols developed have been applied to synthetic M1 and M2 peptides, which constitute part of the transmembrane domain of glutamate-gated ion-channel proteins. Four of these M1 and M2 peptides were separated from minor synthetic impurities, and a 23-mer was baseline separated from a 28-mer. The HPLC procedures have also led to purification of the 10 peptides resulting from cyanogen bromide cleavage of bacteriorhodopsin, peptides which have so far eluded HPLC separation despite numerous attempts. These HPLC protocols have been used to purify peptides ranging from 4 to 50 amino acids in high yield while the columns continued to resolve sharp peaks after more than 100 separation runs over a 6-month period. These new HPLC systems offer an efficient method for the isolation and analysis of this important albeit troublesome class of peptides.

Amino Acid Sequence↗
PubMed1994

The influence of maternal separation on humoral and cellular immunity in farmed deer.

The effect of maternal separation on in vivo and in vitro immune parameters was studied in young hybrid deer. Groups of fawns separated from their mothers either 2 days prior to or 7 days post immunization with keyhole limpet hemocyanin were compared with a control group of immunized, unseparated fawns. Animals separated prior to antigenic challenge had significantly higher concentrations of antigen-specific IgG in their serum than control animals. There was no influence on the humoral immune response in animals separated following immunization. In contrast, Con A transformations were transiently depressed in separated animals compared to the control group. The time separation was imposed relative to challenge and therefore influenced the subsequent immune response.

Animals↗
PubMed2001

Suppression of lens stalk cell apoptosis by hyaluronic acid leads to faulty separation of the lens vesicle.

How the lens vesicle separates normally from the surface ectoderm has not been adequately explained. Apoptosis is an important mechanism that induces tissue development and morphogenesis by physiological cell death during normal development, and hyaluronic acid has been shown to regulate the apoptotic process in various tissues. To elucidate the normal and abnormal processes of lens vesicle separation, we investigated the distribution of apoptotic cells and hyaluronic acid in normal and abnormal lens vesicle development. The offspring from pregnant C57BL/6NJcl mice were removed by laparotomy on days 9, 10, 11, 12, 13, 14, 15, 16 or 18 of gestation. Tissue blocks of the eyes were fixed in 4% paraformaldehyde solution and embedded in paraffin wax. Serial sections of the eye were cut, and sections containing normal lens tissues and spontaneously occurring faulty separation of the lens vesicle were chosen for subsequent procedures. These sections were stained either with TUNEL method for detection of apoptosis or with biotinylated hyaluronic acid binding protein (HABP) for hyaluronic acid. Then TUNEL-positive and HABP-positive reactions in the lens tissues were examined by light microscopy. In normal eye development, the lens stalk connecting the surface ectoderm with the lens vesicle was observed from day 11 through day 12 of gestation, and the lens stalk completely disappeared on day 13 of gestation in this strain. In normally regressive lens stalk, several TUNEL-positive cells were identified, and no positive staining for HABP was seen. However, in spontaneously occurring persistent lens stalk tissues on days 14, 15, 16 and 18 of gestation, no TUNEL-positive cells were detectable, and evident HABP-positive reactions were seen. In eyes on day 12 of gestation with possibly presumed faulty separation of the lens vesicle, no TUNEL-positive cells were detectable and evident HABP-positive reactions were seen in the lens stalk areas. The results indicated that cell death by apoptosis is anatomically closely associated with, appears to be essential for normal regression of the lens stalk, and its failure to occur leads to faulty separation of the lens vesicle. In addition, excessive hyaluronic acid in the lens stalk area may be a possible cause of the lack of apoptosis, inducing its faulty separation.

Animals↗
PubMed1999

Influence of Component Ratio on Adsorption of Polymer Mixtures under Phase Separation of Solutions.

The influence of the component ratio on polymer mixture adsorption in the two-phase condition is estimated. Transition toward the two-phase condition was attained by increasing the solution concentration up to phase separation. For solutions that have separated into two phases with increasing component concentration, the adsorption from each phase has been studied separately and the total adsorption from two phases has been calculated. The polystyrene-poly(butyl methacrylate)-CCl(4) system was investigated using fumed silica as adsorbent. Simultaneously, the fraction of segments of each type that were immobilized by the surface was determined from NMR spectra. In all cases, poly(butyl methacrylate) is characterized by preferential adsorption. The main features of adsorption are similar in both one-phase and two-phase states; however, the values for adsorption and fraction of immobilized segments are different in the case of adsorption from two separated phases because of the difference between the phases in the ratio of components and their concentration in each phase. The distinctions between adsorption from one-phase solutions before phase separation and from solutions modeling separated phases are connected with the redistribution of components between upper and lower phases. As a result, their ratio does not correspond to the initial ratio, and therefore the thermodynamic conditions of solutions, responsible for adsorption, are changed. Because of this, the shape of isotherms of adsorption and the adsorption values are determined by the different levels of aggregation in solutions with various ratios of components. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

Separation Rate Dependence of Adherence Strength.

The adherence energy measured during the separation of adhesive/substrate assemblies is a complex function of the adhesion energy and also of the dissipative properties of both adhesive and substrate. Dissipative phenomena occurring during an assembly separation are reflected in the dependence of the measured energy on separation rate. Up to now, different relationships have been used to describe the dependence of the adherence energy G on separation speed V. However, these models assume that only the separation speed affects the dissipation function; the adhesion energy W is supposed to remain constant. This paper proposes that the "real" adhesion energy is also rate dependent. Indeed, the number of interfacial interaction points under stress is able to increase with speed. This effect could be a consequence of the influence of separation rate on the adhesive elastic modulus. Copyright 1999 Academic Press.

Journal Article↗
PubMed1992

Complete correction of the tetralogy of Fallot in adults using separated extracorporeal circulation to block intrathoracic collateral circulation and improve perfusion of the renal vasculature.

During the last 10 years, complete correction of the tetralogy of Fallot (TOF) has been performed on 28 adult patients using the "separated extracorporeal circulation" (separated ECC) technique developed by us. In addition to the usual ascending aortic and dual right arterial cannulations for ECC, the separated ECC also involved femoral arterial cannulation and the insertion of a catheter with two balloons via the other femoral artery. Flow via the ascending aorta and femoral artery were maintained separately during ECC. The urine output during separated ECC was significantly better than that during ordinary ECC (P < 0.01) and the operative field for intracardiac correction was clearer as a result of blocking the collateral circulation. Thus we believe separated ECC to be a superior method for total correction of TOF in adults, which may enhance the operative outcome.

Adult↗
PubMed1978

Pattern separability and the effect of the number of connections in a random neural net with inhibitory connections.

It has been claimed that pattern separation in cerebellar cortex plays an important role in controlling movements and balance for vertebrates. A number of the neural models for cerebellar cortex have been proposed and their pattern separability has been analyzed. These results, however, only explain a part of pattern separability in random neural nets. The present paper is intended to study an extended theory of pattern separability in a new model with inhibitory connections. In addition to this, the effect of the number of connections on pattern separability is cleared up. It is also shown that the signal from the inhibitory connections has crucial importance for pattern separability.

Action Potentials↗
PubMed1987

The time of separation of the umbilical cord.

The time of separation of the umbilical cord was studied in 911 neonates. The mean time of separation was 7.4 days (SD 3.3, range 1-29 days). We sought a possible relationship between the time of cord separation and various factors in the perinatal period. Cord separation was delayed when antibiotics needed to be administered to the neonate because of sepsis, when the infant was born prematurely, delivered by Caesarean section or had a low birth weight. The cord separated slightly earlier in female than in male infants. None of the infants studied suffered from omphalitis and it would appear that "delayed" separation of the cord is not always necessarily accompanied by severe leucocyte dysfunction.

Bacterial Infections↗
PubMedDate not supplied

Myocardial single-photon emission computed tomographic imaging with technetium 99m tetrofosmin: stress-rest imaging with same-day and separate-day rest imaging.

BACKGROUND: Technetium 99m tetrofosmin is a new ethylene diphosphine ligand for myocardial perfusion imaging and has unique properties. We have compared stress-rest single-photon emission computed tomographic (SPECT) imaging with 99mTc tetrofosmin with same-day and separate-day rest imaging to detect myocardial perfusion defects. METHODS AND RESULTS: Myocardial SPECT imaging was performed in 22 patients with coronary artery disease who had undergone planar thallium 201 imaging and coronary angiography. Single-day (stress-rest) and separate-day rest 99mTc tetrofosmin SPECT protocols were compared in the same patient. Images were assessed by a blinded panel to identify myocardial infarction, ischemia, or normal scans. Overall sensitivity for identification of patients with coronary artery disease was 86% (19/22) by both same-day stress-rest and separate-day rest protocols with 99mTc tetrofosmin (p = NS). Of a total of 396 segments studied, 107 abnormal segments were identified at exercise and 76 and 81 at the same-day and separate-day rest tests, respectively (p = NS). Same-day stress-rest and separate-day rest 99mTc tetrofosmin SPECT protocols were also useful for detecting individual coronary stenosis with a greater than 50% lesion: 80% of the left anterior descending, 93% of the right coronary, and 75% of the left circumflex coronary arteries were detected. CONCLUSION: Excellent images were obtained with 99mTc tetrofosmin during both stress and rest. 99mTc tetrofosmin imaging with the same-day stress-rest and separate-day rest imaging protocols have similar diagnostic sensitivities for detection of coronary heart disease.

Aged↗
PubMed1983

Circulating antitriiodothyronine autoantibodies in two euthyroid patients: apparent lack of interference in total T3 radioimmunoassay based on second antibody or solid phase separation techniques.

Two clinically euthyroid patients were noted to have low total T3 levels as assessed by RIA using either dextran-charcoal (DC) or polyethylene glycol (PEG) for separation of bound from unbound T3, in spite of normal free T3, total and free T4 and basal and TRH-stimulated TSH concentrations. The presence of circulating substances binding T3 was suggested by high nonspecific binding in total T3 RIA system using either DC or PEG separation. The presence of anti-T3 autoantibodies was then suspected and confirmed by the presence of [125]-T3 bound to patients' gammaglobulins, precipitated with rabbit anti-human immunoglobulins. Serum T3 concentration determined by extracting T3 from patients' sera with methanol was 166 and 226 ng/dl. Similar or even lower values were unexpectedly obtained in RIA systems with solid phase or second antibody (anti-rabbit) separation and with competitive protein binding assay. To face this paradoxical finding, simulated experiments were carried out by incubating T3- and T4-free sera added with various amounts of stable T3 and T4 in the presence of goat anti-T3 or anti-T4 serum. These samples were then radioimmunoassayed. The DC separation caused a consistent underestimation of the actual T3 and T4 concentration. The second antibody separation caused a T3 and T4 overestimation for actual levels below 200 ng/dl and 10 micrograms/dl, respectively, while at the higher T3 or T4 concentrations, an overlap or, even, an underestimation of actual T3 or T4 levels were found. These data provide evidence that, with second antibody or solid phase separation methods, there could be an apparent lack of interfering effect of endogenously occurring antibodies.

Adult↗
PubMed2005

Effects of early maternal separation on ethanol intake, GABA receptors and metabolizing enzymes in adult rats.

RATIONALE: Maternal separation (MS) in neonatal rats affects ethanol self-administration (SA) in adulthood; however, the conditions and mechanisms need to be clarified. OBJECTIVES: The goal of this study was to determine the effect of MS on ethanol SA in adulthood in different groups of rats, which control for time of separation, handling, and rearing conditions and, for mechanistic assessment, to examine GABA-A receptors in the central nucleus of the amygdala (CeA) and levels of liver metabolizing enzymes. METHODS: Newborn, male Long-Evans rats were randomly assigned to different groups and treated over postnatal days 2-14. The rats were picked up by their tails and put back down with no separation (MS0), separated from their mother for 15 min/day (MS15), separated from their mother for 180 min/day (MS180), handled once for a bedding change (NH), or were animal facility reared (AFR). In adulthood, these rats were allowed 5-day continuous access to ethanol, and GABA-A receptors and liver enzymes were measured. RESULTS: The MS15 group consumed and preferred significantly less ethanol (about one third) than the MS180 group; however, neither group was different from the MS0 or the AFR group. The NH group consumed and preferred significantly more ethanol than all other groups, at least twice that of the MS180s. GABA-A receptors were increased in the CeA in MS15s, which could help explain the effects. Alcohol dehydrogenase may have been altered in the AFRs. CONCLUSIONS: Various treatments in neonates affect ethanol intake and GABA-A receptors, and possibly ethanol metabolism, in adulthood. These changes were not simply related to time of separation but were also due to the degree of handling.

Alcohol Dehydrogenase↗
PubMed2000

Periodic postpartum separation from the offspring results in long-lasting changes in anxiety-related behaviors and sensitivity to morphine in Long-Evans mother rats.

RATIONALE: According to recent studies daily, 3-h separations from the dam on postnatal days 2-14 produce long-lasting changes in responses to stressors and sensitivity to morphine in Long-Evans offspring. It has not been investigated whether daily dam-litter separations can have long-lasting effects also on the mother beyond weaning of the pups. OBJECTIVE: We tested whether dams that were separated from their litter for 3 h daily (LS) on postpartum days 2-14, like their offspring, exhibit altered anxiety-like behaviors and sensitivity to morphine, compared to dams that experienced only brief (15-min) separations (BS) from the litter or that were left undisturbed (NH) during the same period. METHODS: Four to six weeks after weaning, subjects were tested on the elevated plus-maze, in a novel locomotor activity arena, or were exposed to loud auditory stimuli. Sensitivity to morphine was assessed using hot-plate and tail-flick tests. RESULTS: LS dams spent significantly more time in the open arms of the plus-maze and in the center of the locomotor activity arena, and were more likely to emit ultrasonic vocalizations in response to auditory startle stimuli compared to NH dams. Furthermore, LS dams were less sensitive to morphine, primarily in the tail-flick test. Dams that experienced brief litter separations (BS), like LS dams, exhibited altered performance on the plus-maze and elevated ultrasonic vocalizations. However, BS dams were similar to NH controls in locomotor activity and sensitivity to morphine. CONCLUSIONS: Multiple postpartum separations from the offspring alter the behavior of Long-Evans dams in novel/aversive environments and affect their sensitivity to the antinociceptive effects of morphine.

Analgesics, Opioid↗
PubMed2002

Direct analysis of nine pharmaceuticals in culture media by use of cartridge separation with electrospray mass spectrometric detection.

A 2-cm cartridge has been used for separation before electrospray mass spectrometric analysis of pharmaceutical compounds in cell culture media, alleviating the need for sample extraction and desalting procedures. Nine representative pharmaceuticals listed in the biopharmaceutical classification system (BCS) were chosen as the candidate compounds and Hank's balanced salt solution with Hepes buffer (HBSS-Hepes buffer) was used as the cell-culture medium in an effort to study permeability of chemicals through cell monolayers. Effects of several conditions, e.g. pH and buffer concentration in the mobile phase, flow rate, and temperature on separation efficiency were examined. The nine pharmaceuticals were separated within 2 min by use of a 2-cm C(8) cartridge. Relative standard deviations (RSD) from repeated analysis within the same day or over five days were 0.03-0.2% for retention times and 0.6-5.3% for peak areas; antipyrine was used as internal standard. Calibration curves based on peak-area measurements were linear over the range 0.1-20 micro mol L(-1). The HBSS-Hepes buffer did not interfere with separation and detection; identical separation and peak intensity were obtained when the samples were separately prepared in distilled water or in the culture medium.

Adrenergic beta-Antagonists↗
PubMed2003

Separation of arsenic species by capillary electrophoresis with sample-stacking techniques.

A simple capillary zone electrophoresis procedure was developed for the separation of arsenic species (AsO(2)(2-), AsO(4)(2-), and dimethylarsinic acid, DMA). Both counter-electroosmotic and co-electroosmotic (EOF) modes were investigated for the separation of arsenic species with direct UV detection at 185 nm using 20 mmol L(-1) sodium phosphate as the electrolyte. The separation selectivity mainly depends on the separation modes and electrolyte pH. Inorganic anions (Cl(-), NO(2)(-), NO(3)(-) and SO(4)(2-)) presented in real samples did not interfere with arsenic speciation in either separation mode. To improve the detection limits, sample-stacking techniques, including large-volume sample stacking (LVSS) and field-amplified sample injection (FASI), were investigated for the preconcentration of As species in co-CZE mode. Less than 1 micromol L(-1) of detection limits for As species were achieved using FASI. The proposed method was demonstrated for the separation and detection of As species in water.

Arsenic↗